nepal j biotechnol. 2 0 2 2 d e c ; 1 0 (2): 5 2 5 6 research article doi: https://doi.org/10.54796/njb.v10i2.240 ©njb, bsn 52 comparative study on antioxidant activity of propolis of apis mellifera from different regions of nepal rajendra gyawali , aparna paudel, babita lamsal, pranisha bhatta, albina maharjan, nina khaitu, rajan shrestha department of pharmacy, herbal research laboratory, school of science, kathmandu university, dhulikhel, kavre, nepal received: 16 aug 2022; revised: 20 dec 2022; accepted: 23 dec 2022; published online: 31 dec 2022 abstract propolis is a waxy material obtained from honey bee hives. the physical and chemical property of this product is variable based on the source of hive, plant biodiversity where honeybee feed, season of collection, geographical origin etc. propolis has several useful chemical compounds, and among them polyphenols are mainly contributing for their broad spectrum of medicinal quality such as antimicrobial, antifungal, antibacterial, and anti-inflammatory activities as well as antioxidant properties. the present study aims to analyze the ethanolic extract of propolis for their phenol and flavonoid content as well as its antioxidant characteristics. the samples (spls) were collected from farmers of six different locations of nepal i.e. jhapa, lalitpur, kathmandu, banke and chitwan districts. total phenolic content (tpc) and total flavonoid content (tfc) were measured by folin ciocalteu method and the aluminum chloride method respectively expressed as the gallic acid (gae) and quercetin (qe) and equivalent (gae) per gram. the diphenyl-picrylhydrazyl (dpph) assay method was used to evaluate the free radical scavenging activity. the antioxidant effect of propolis was reported in ascorbic acid equivalent antioxidant capacity per gram of propolis. the highest content of phenolic and flavonoid content was found in sample spl 2. the range of these compounds' concentrations were from 127.36±5.50 mg gae/gram to 242.7±4.50 mg gae/gram. similarly, total flavonoid content ranged from 1.3197±0.0261 qe mg/ grams to 5.3921±0.0261 qe mg/ grams. whereas samples from spl 2, and spl 5 showed highest antioxidant properties. all samples were found to have strong antioxidant capacity which was greater than standard. it is concluded that there is no direct correlation on total antioxidant property of propolis with their total phenolic and total flavonoid content among collected samples. the phenolic characteristics of the samples were variable to the geographical location, and plant diversity of their origin. keywords: nepalese bee propolis, quality, phenolics, antioxidant, dpph assay corresponding author, email: ragyawali@gmail.com introduction propolis is a resinous material, also known as bee glue, that bees collect from various plant sources for their hives. bees create propolis, by combining saliva, beeswax, and exudate obtained from tree buds, sap flowers, or other botanical sources. it has a variety of biological activities and is an well-known natural remedy. propolis is used to fill minor gaps, and burr comb is used to cover bigger gaps [1]. it possesses the antibacterial capabilities provide protection against infections [2, 3] . the caffeic acid phenethyl ester is considered as a main constituent to inhibit the nuclear factor κ-b, inhibition of cell proliferation, induction of cell cycle arrest and apoptosis. the different climatic circumstances in which propolis is produced affect its chemical components and overall nature. geographic origin is associated with variations in the chemical composition and consequently in the medicinal property of propolis. propolis contains phenolic compounds, esters, flavonoids, aromatic aldehydes, resin, wax, and oil [4]. numerous studies on propolis have revealed that it contains a variety of biologically active constituents as well as unique properties such as anti-inflammatory, antimicrobial, antioxidant, wound healing, and others [5]. the biological activity of several beneficial chemicals derived from propolis for antibacterial, antifungal, antiviral, antiinflammation, anticancer, antioxidant, and other properties can be applied in the pharmaceutical and health sectors [6–8]. propolis can be employed as a free radical scavenger due to its significant oxidation inhibitory action [9]. the significant biological potency of propolis is demonstrated by its diverse composition [10]. many researchers are working in the propolis because of its broad range of medicinal value and availability in different part of the world. there is an escalating scientific concern in the impact of geographical origin of propolis on their chemical constituents, physical characteristics and biological nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-5745-0702 mailto:ragyawali@gmail.com mailto:ragyawali@gmail.com nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :5 2 5 6 gyawali et al. ©njb, bsn 53 activities. therefore the primary objective of this study was to calculate the flavonoids and phenolics, and evaluate the antioxidant activities of propolis collected from various locations of nepal. materials and methods collection of propolis bee propolis were collected from jhapa, lalitpur, kathmandu, banke and chitwan during june-july 2021. the propolis was collected as chunks in propolis traps every 15 days. the collected propolis chunks were stored in an air-tight container under refrigerated condition at 40c for subsequent research purposes. sample preparation total 10 grams of raw pulverized propolis was dissolved in 100 ml of 70% ethanol and warmed in a water bath. the mixture was transferred to conical flasks and set on a rotary shaker with suitable rpm for 72 hours. thereafter, whatman no 1 filter was used to obtain filtrate in conical flask. the extract was concentrated in rotavapor (buchi r-215, switzerland), 75-90 rpm, under 100 mbar pressure maintaining 40℃ temperature of water bath (figure 1). figure 1. propolis sample preparation total phenolic content total phenolic content (tpc) of developed propolis powder was estimated with slight modification in previously described method [11] . for each sample, 1 ml of folin-ciocalteu reagent was mixed with 0.2 ml of extract. after 3 minutes, 1 ml of 10% sodium carbonate was added, sodium carbonate helped speed up the oxidation reaction of phenol. the resulting mixture was incubated at room temperature for 30 minutes. furthermore, the absorbance at 280 nm was measured in triplicates for each sample of propolis. the phenolic content of propolis was recorded in mg of gallic acid equivalent per gram. the standard curve was generated with concentration of 0.8mg/ml of gallic acid. all samples were calculated in triplicate. total flavonoid content total flavonoid content was measured with slight modification in the procedure of previously described method [12]. for each sample, approximately 1.5 ml of ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 mol/l potassium acetate and 2.8 ml of water was added to 0.5 ml of extract. for about 30 minutes, the mixture was incubated at room temperature. after that, the absorbance at 415 nm was measured in uv-visible spectrophotometer. each sample of propolis was measured in triplicates expressing the flavonoid content in mg of quercetin equivalent per gram of propolis. the standard curve was generated with concentration of 0.8 mg/ml of quercetin. dpph radical scavenging activity assay dpph (2, 2′diphenyl-1-picrylhydrazyl) assay was used to measure the free radical scavenging activity of the fractions as per the method described earlier [13]. the stock solution in 100 ml methanol was prepared by dissolving 3.94 mg dpph. ethanolic extracts of the propolis samples, and ascorbic acid of different concentrations (i.e., 60, 120, 180, 240 and 300 ppm) were prepared and reacted with aliquot solution of dpph in the ratio 1:3. the resulting mixture was incubated at room temperature for 30 minutes. it was kept in a dark place to protect from light. after that, the absorbance at 517 nm was measured in triplicate. based on the percentage of dpph radical scavenged, the scavenging activity was calculated using the following equation: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100% where acontrol is the mean absorbance reading of 0 ppm solution against methanol as blank asample is the mean absorbance reading of different concentrations of the solutions results and discussions propolis from various locations of nepal were subjected for their quality analysis with the prospective of phenolics and antioxidant property. the electron donating capacity of natural products can be estimated by using 2, 2′-diphenyl-1picrylhydrazyl radical (dpph) reagent. this method is based on scavenging of dpph radical through the addition of antioxidant which reduces the free radical of dpph leading to its decolorization. according to the study's findings, propolis extract contains phytoconstituents which can donate hydrogen to a free radical to scavenge possible damage. nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :5 2 5 6 gyawali et al. ©njb, bsn 54 total phenolic content gallic acid was used to perform the total phenolic content assay, and the standard curve equation was y = 0.001x + 0.5883, where r2 = 0.9894. the absorbances were put in the equation obtained from the gallic acid standard, and tpc were determined (figure 2). the range of these compounds' concentrations was 127.36±5.50 mg gae/gram to 242.7±4.50 mg gae/gram. spl 2 had the highest concentration of phenols, followed by spl 6. the lowest concentration was found in spl 3 and spl 4. others had average total phenolic content. in propolis sample, phenolic acids such as benzoic acid, cinnamic acid, and their derivatives are present. p-hydroxybenzoic acid, anisic acid, and gallic acid are some of the most prominent benzoic acid derivatives. additionally, there is vanillic acid, salicylic acid, gentisic acid, 3,4dimethoxybenzoic acid, protocatechuic acid, and 2amino-3-methoxybenzoic acid present. [14]. figure 2: total phenolic content of propolis collected from different area of nepal. total flavonoid content quercetin used as the standard to estimate tfc in the samples comparing the standard curve equation. quercetin equivalent was calculated in milligram quercetin equivalent (qe) per gram. the samples were evaluated based on the quercetin equivalent. total flavonoid content ranged from 1.3197±0.0261 qe mg/ grams to 5.3921±0.0261 qe mg/ grams. spl 2 was found to have the highest content of flavonoids 5.3921±0.0261 mg qe/gram (figure 3). phenolic compounds such as; odoratin, 7,3',4'-trihydroxy-5'-methoxyisoflavonoid, 6,7,3'-trihydroxy-4'-methoxyisoflavonoid, 7,3'dihydroxy-6,5'methoxyisoflavonoid, neoflavonoid 1 to neoflavonoid 10, (s)-3'-hydroxy-4-methoxydalbergione, (s)-3',4'-dihydroxy-4-methoxydalbergione were also reported from nepalese propolis [15-16]. figure 3. bar graph comparing total flavonoid content in given samples antioxidant activity the results from our experimental studies shows that the antioxidant capacity of the nepalese originated propolis samples are very strong (figure 4). the ic50 range of the sample were from 35 to 218 as compared with ascorbic acid standard. the sample's antioxidant property has an inverse relationship with the ic50 value. in other words, if the ic50 value is lower, the sample will need less to scavenge the free radical, and vice versa. free radical scavenging activity in the sample is caused by the presence of bioactive components known as antioxidants. additionally, based on the above total phenolic content and total flavonoid content, spl 2 contains the highest tfc and tpc, resulting in the strongest ic50 value. dpph• is a stable purple radical that turns pale yellow when it absorbs free radicals. the antioxidant effect of propolis was reported in ascorbic acid equivalent antioxidant capacity (aeac) per gram of propolis. all propolis showed an antioxidant capacity greater than 35 aeac; 5 samples exceeded 100 aeac, and sample number 6 exceeded 200 aeac. spl2 has a lower ic50 value, indicating higher antioxidant activity, as less extract is required to inhibit 50% of the dpph radical. propolis comprises components from several different chemical families, including flavonoids, four aromatic carboxylic acids, and eleven phenolic acid esters, which are responsible for its antioxidant properties, according to a prior study. numerous studies show that the overall number of phenolic compounds is related to antioxidant activity [17]. the current investigation has also reinforced the antioxidant property of nepalese originated propolis samples. the results also supported the assumption that an increase in the flavonoid concentration can lead to higher antioxidant activity. 0 1 2 3 4 5 6 spl1 spl2 spl3 spl4 spl5 spl6 q u e rc e ti n e q u iv a le n t (m g q e / g ) propolis samples total flavonoid content 0 50 100 150 200 250 300 spl1 spl2 spl3 spl4 spl5 spl6 g a ll ic a ci d e q u iv a le n t (m g g a e / g ) propolis samples total phenolic content nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :5 2 5 6 gyawali et al. ©njb, bsn 55 it was observed that bee propolis chemistry and antioxidant properties need to be compared together with their geographical factors and plant species found around the beehive and bee feeding area having prominent nepalese antioxidant herbs [18-20]. the future research on the propolis, can be focused on feeding experiments, together with local biodiversity to identify the species and behavior on propolis. the chemical composition of the propolis and its biological activities can be correlated with their geographical origin of the samples. figure 4. comparison of ic50 value of samples. conclusion it is concluded that the propolis of apis mellifera from different regions of nepal possesses various strengths of antioxidant property. spl 2 exhibited the highest levels of tfc and tpc, but antioxidant property was stronger in another sample, spl 5. the concentrations of phenolic compounds in sample might be influenced by the vegetation of bee farming area. author’s contribution project supervisors are dr. rajendra gyawali, dr. rajan shrestha. lab, fieldwork and writing manuscript were done by aparna paudel, albina maharjan, babita lamsal, nina khaitu and pranisha bhatta. review and final editing were done by rajendra gyawali, aparna paudel and babita lamsal. all authors read and approved the final manuscript. competing interest no competing interests were disclosed. ethical approval and consent not applicable. acknowledgement the authors would like to express the gratitude to the department of pharmacy at kathmandu university for supplying the essential equipment, resources, and facility for this research project. furthermore, we thank to the kathmandu university integrated rural development project (ku-irdp) funded by korean international cooperation agency (koica) nepal for the research and business development (r&bd) program. we 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2022 abstract the present research work was conducted to evaluate the efficacy of eight fresh botanicals namely azadirachta indica (neem leaf), allium cepa (onion bulb), allium sativum (garlic), curcuma longa (turmeric), zingiber officinale (ginger), allamanda cathartica (allamanda leaf), nigella sativa (black cumin) and aloe vera (aloe vera) against magnaporthe oryzae triticum (mot) and solvent, ethanol (95%) was used for the phytochemical extraction of various plant parts. three concentrations namely 1:1 (w/v), 1:0.50 (w/v) and 1:0.25 (w/v) of plant parts and ethanol were used for botanical extraction. the antifungal activity of botanicals against a virulent mot isolate chmot07 was evaluated in-vitro using poison food technique. the lowest mycelia growth was recorded with aloe vera (aloe vera leaf) extracts and nigella sativa (black cumin seeds) extracts @ 1:1 w/v and @ 1:0.25 w/v with growth rate of 3.00 mm and 3.33 mm respectively at 7 days after inoculation, whereas the highest mycelia growth rate of mot isolates was recorded in control plates both at 7 dai and 14 dai under in-vitro condition. keywords: wheat blast, magnaporthe oryzae triticum, botanical extracts, in vitro corresponding author, email: aminsaupp@yahoo.com introduction wheat (triticum aestivum) is recognized as one of the most important cereal crops within the world [1]. on the national economy, it is typically a human food grain and created optimistic impact globally. wiese (1987) [2] mentioned that, wheat provides about 20% of the world food calories and nearly 40% of the total world population consumed it as staple food. origin of wheat is from the levant region of the near east however currently cultivated worldwide. thus, being larger than any other crop, wheat is grown on more than 701.5 million hectares [3]. in 2017, wheat ranked as the third most-produced cereal in the world with the production of 771.7 million tons [4] through china (131.4 million tons), india (99.7 million tons), russia (72.1million tons) and usa (51.3 million tons) were the four largest wheat producers in 2018 [5]. according to the department of agricultural extension, bangladesh in 2016, total wheat cultivated area in bangladesh is about 498,000 ha. in the year of 2016 an outbreak of wheat blast was reported for the first time in bangladesh particularly in the districts of kushtia, meherpur, chuadanga, jhenaidah, jessore, barisal, bhola, magura, narail, and faridpur [6]. it was estimated that 15% area were affected around 101,660 ha of cultivated wheat area by a devastating wheat blast in the same year. the incidence and severity of wheat blast associated with yield losses among completely different districts varied considerably. the prevalence of wheat blast was higher in meherpur (70%) although the very best average yield loss (51%) was recorded in jhenaidah. the average yield loss was lower than 51% across districts and in several cases,100% yield losses were recorded in individual wheat fields [7]. the area beneath wheat cultivation currently extends to concerning 1.78 lakh ha and the annual production is about 10 lakh metric tons [8]. some severely infected fields were burned due to approximately 15% of bangladesh’s total wheat-affected area, which reduced 15% wheat production in nine infected districts [6, 7, 9]. in spite of such a decrease, in 2016 compared to that of 2015, 2.7% of total wheat production in bangladeshis increased (35,000 metric ton [mt]). a rise in total harvested areas (420,000-425,000 ha) and increasing yields (3.10-3.14 mt/ha) contributed to the overall wheat production in 2016 [10]. use of fungicides against wheat blast wasn’t therefore effective and chemical application conjointly caused environmental pollution and toxicity to beneficial soil microbes, higher plants furthermore animals and human nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:aminsaupp@yahoo.com https://orcid.org/0000-0003-4804-0100 mailto:aminsaupp@yahoo.com mailto:aminsaupp@yahoo.com nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 77-84 rehena et al. ©njb, bsn 78 beings. it has also enhanced production costs of farmers [11] and natural biological systems have disrupted over decades because of their continual use and sometimes resulted in the development of fungal resistance besides producing undesirable effects on non-target organisms, and also fostered environmental and human health considerations [12]. rout and tewari (2012) [13] stated in their study the potentiality of integrating in the management of economically important diseases, the products prepared from green plants should be preferred as they are environmentally safe, non-pollutive, nontoxic and non-hazardous to beneficial microorganisms. allamanda (allamanda cathertica) leaves are the promising source of many antifungal compounds with medicinal properties [14]. the antifungal effect of the extracts of neem (azadirachta indica l.), garlic (allium sativum l.) and calatropis (calotropis procera l.) against magnapothe oryzae triticum (mot) was evaluated by khanzada and shah [15] following poisoned food technique. they found complete growth inhibition of the test fungus in a higher dose of garlic extract in in vitro bioassay. development of fungicidal resistance into the pathogens and residual toxicity in soil and in the crop plants were owing to frequent use of fungicides. on the other hand, some botanical pesticides and bio-control agents have tested to be reliable and don’t have any adverse effect on environment [16, 17]. mondol et al. 2018 [18] studied that chitosan (0.4%), salicylic acid (9mm) and benzoic acid (9mm) are effective in suppressing the growth of mot. chitosan, salicylic acid and benzoic acid are bio-polymers and not harmful for ecosystem and are completely safe for human health. therefore, these biopolymers can be utilized as alternative to chemical fungicides for wheat blast management. therefore, to control this devastating pathogen mot ecofriendly management with non hazardous botanical extracts are going to be most effective until crop improvement with desired resistance gene against this pathogen in bangladesh. the present research work was thus undertaken to find out the antifungal effect of some botanical extracts against mot in vitro. materials and methods the present investigations were carried out under laboratory conditions from january to july, 2019 to ascertain the incidence, severity of wheat blast and invitro evaluation of botanicals against mot in the department of plant pathology, sher-e-bangla agricultural university, dhaka. figure 1. flow chart of isolation, identification, and culture of magnaporthe oryzae triticum on oma media diseased plant samples were collected from infected wheat fields and preserved at 4° c temperature in the laboratory of sau for isolation. standard blotter method [19] was followed for the isolation of mot pathogen from infected wheat spikes. the water agar was prepared by mixing 20 g agar with 1000 ml distilled water, and potato dextrose agar media was made consisting of 200 g peeled potatoes, 20 g dextrose, and 20 g agar combined with 1000 ml distilled water were used for the isolation of blast pathogen. appropriate size (15-20 cm in size) of diseased spikes infected with pathogen of wheat cultivars were cut around the area showing the blast lesion and were surface sterilized with 1% sodium hypochlorite (naocl) for 1 minute followed by 3 times washes with sterile distilled water. to encourage sporulation, the plant pieces were placed in petri dishes lined with moist filter papers and incubated at 26±1°c for 24 hours. after incubation, these infected spike pieces were examined under stereo-dissecting microscope (motic, china). abundant sporulation with grey, dense and bushy appearance was observed in and around the lesions. single conidium was picked out using a sterile moistened needle across the sporulating lesion observing under the stereo microscope. the conidia were placed on water agar for further growth experimentation. after 12 hours, mycelium was visible in petri dish under the stereo microscope and then the hyphal tip was cut and placed in potato dextrose agar media plates containing streptomycin (40 mg/l) and pure culture of mot were prepared by incubating there in 26±1°c. the marginal mycelial growth that developed subsequently was picked-up aseptically for sub-culturing until a pure culture of mot was obtained. the culture was kept under 12 hrs light and 12 hrs darkness conditions for sporulation. mot isolate was identified by three-celled, pyriform, light-colored conidia (figure 1). conidia of mot(×100) bleached panicle pure culture of mot on oma nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 77-84 rehena et al. ©njb, bsn 79 the pure culture was maintained by subculturing at an interval every 15 days and preserved at low temperature (4⁰c) in refrigerator. fresh plant parts namely azadirachta indica (neem leaf), allium cepa (onion bulb), allium sativum (garlic), curcuma longa (turmeric), zingiber officinale (ginger), allamanda cathartica (allamanda leaf), nigella sativa (black cumin) and aloe vera (aloe vera) were used as treatments (table 1 & figure 2). 95% ethanol solvent was used for the phytochemical extraction of various plant parts [20]. three concentration 1:1 (w/v), 1:0.50 (w/v) and 1:0.25 (w/v) of ethanol was used for botanical extraction. for 1:1 (w/v) concentration extraction with ethanol, 100g of plant materials was dissolved in 100 ml ethanol. to avoid evaporation and subjected to filtration through sterilized whatman no.1 filter paper the mixture was kept undisturbed at room temperature in a sterile flask covered with aluminum foil for 24 hrs. figure 2. botanicals used in controlling mycelial growth of magnaporthe oryzae triticum in-vitro. extracts of a. allium cepa, b. allium sativum, c. curcuma longa, d. zingiber officinale, e. azadirachta indica, f. nigella sativa, g. allamanda cathartica, h. aloe vera figure 3. botanical extracts used in controlling mycelial growth of magnaporthe oryzae triticum in-vitro. extracts of a. allium cepa, b. allium sativum, c. curcuma longa, d. zingiber officinale, e. azadirachta indica, f. nigella sativa, g. allamanda cathartica, h. aloe vera nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 77-84 rehena et al. ©njb, bsn 80 after filtration, the extract was evaporated in water bath until 100 ml extract was left in the container (figure 3). for 1:0.50 (w/v) and 1:0.25 (w/v) 100 g of plant materials were dissolved in 50 ml and 25 ml ethanol, respectively [20]. ethanolic extract thus obtained were immediately evaluated for antifungal activities using the poisoned food technique [21]. pda plates were amended with different concentration (1:1 w/v, 1:0.50 w/v, 1:0.25 w/v) of botanical extracts separately. the plates were inoculated with 5 mm fungal blocks with the help of sterilized needle, mot and these blocks were transferred to the center of the petri plates. the mycelial growth of mot was recorded at seven days after inoculation by measuring the average of two diameters at right angles (90 degrees) to one another. three replications were maintained for each treatment and the mean radial mycelial growth was considered for measuring each treatment. the effect of plant extract was calculated as percent growth inhibition using the following formula as adopted by [22, 23]. % inhibition = (dc – dt)/dc × 100 where dc = average increase in mycelial growth in control, dt = average increase in mycelial growth in treatment. statistical analysis a completely randomized design (crd) with three replications was applied in this experiment. analysis of data of different parameters was subjected to perform by statistical analysis using r software version 3.6.0 [24]. results all botanicals performed significantly effective against mot isolate in lessening mycelial growth compare to control (table 2). the result revealed that aloe vera (aloe vera leaf) extracts was found most effective in reducing the mycelial growth at 7 days but in 14 days nigella sativa (black cumin seed) extracts and aloe vera (aloe vera leaf) extracts both botanicals significantly reduced the mycelial growth of mot. table 2. efficacy of botanicals on mycelial growth of m. oryzae triticum treatments radial mycelial growth (mm) % growth reduced over control 7 dai 14 dai 7 dai 14 dai allium cepa 35.22b 42.00d 28.93 38.93 allium sativum 21.67e 52.56b 56.27 23.58 curcuma longa 26.78d 47.11c 45.94 31.50 zingiber officinale 26.78d 36.78f 49.96 46.52 azadirachta indica 31.56c 52.00b 36.31 24.39 nigella sativa 4.11g 10.00g 91.70 85.46 allamanda cathartica 26.22d 40.33e 47.09 41.36 aloe vera 5.22f 8.89g 89.46 87.07 control 49.56a 68.78a cv (%) 3.68 3.02 in a column treatment means with the same letter are not significantly different. dai= days after inoculation table 3. effect of different concentration levels of botanicals on mycelial growth of m. oryzae triticum concentration of botanicals radial mycelial growth (mm) 7 dai 14 dai 1:0.25 w/v @0.1% 30.37a 47.59a 1:0.50 w/v @0.2% 25.89b 40.74b 1:1 w/v @0.4% 19.44c 31.15c cv (%) 3.68 3.02 in a column treatment means with the same letter are not significantly different. dai=days after inoculation table 1. botanicals used in controlling mycelia growth of magnaporthe oryzae triticum in-vitro name of botanicals concentration used scientific name english name plant parts used allium cepa onion bulb 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) allium sativum garlic clove 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) curcuma longa turmeric rhizome 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) zingiber officinale ginger rhizome 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) azadirachta indica neem leaf 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) nigella sativa black cumin seed 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) allamanda cathartica allamanda leaf 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) aloe vera aloe vera leaf 1:0.25(w/v) 1:0.50 (w/v) 1:1 (w/v) control no botanical extracts nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 77-84 rehena et al. ©njb, bsn 81 three concentration level of botanicals (1:0.25 w/v @ 0.1%, 1:0.50 w/v @ 0.2%, 1:1 w/v @ 0.4%) was tested against the pathogen and (1:1) found the best concentration in both 7 dai and 14 dai (table 3). with the concentration increase of all tested plant extracts, the mycelial growth of mot decreases. the growth characteristics like media color, colony color and shape of colony of mot on pda media were noticed supplemented with different plant extracts. in case of pda media, the colony color of mot was grey ash centre and black margin. in contrast, pda media supplemented with azadirachta indica (neem leaf) extract was showed white with grey, allamanda cathartica (allamanda leaf) extract, aloe vera (aloe vera) leaf extract, and nigella sativa (black cumin) seed extracts showed white colony color. allium cepa (onion bulb) extracts, allium sativum(garlic) extracts and curcuma longa (turmeric rhizome) extracts and zingiber officinale (ginger) showed rhizome extract showed grey with white color colony. this result showed that pda media supplemented with totally different plant extracts have an effect on the colony color of m. oryzae triticum (figure 4 and table 4). antimicrobial activities of eight botanicals with specific concentration were assayed and results on presented in table 4. the data revealed that botanicals were found significant in suppression of mycelia growth at higher concentration over untreated check in the fungal pathogens. antimicrobial activities of eight botanicals with specific concentration were assayed and results on presented in table 4. the result reveals that the minimum radial mycelial growth was recorded from nigella sativa: 1:1 figure 4. mycelial growth, color and appearance of m. oryzae triticum on pda media supplemented with ethanol extracts of different botanicals (7 dai). a. allium cepa, b. allium sativum, c. curcuma longa, d. zingiber officinale, e. azadirachta indica, f. nigella sativa, g. allamanda cathartica, h. aloe vera, i. control nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 77-84 rehena et al. ©njb, bsn 82 w/v @ 0.4% (3.00 mm) combination at 7 days culture age which is statistically similar with aloe vera: 1:1 w/v @ 0.4% (3.33mm) and nigella sativa: 1:0.50 w/v @ 0.2% (4.33 mm) combination. control plates (only pda: 1:0.50 w/v @ 0.2% and only pda: 1:1 w/v @ 0.4%) were always observed the highest growth of mycelium. at 14 days culture age, lowest mycelial growth was observed in aloe vera: 1:1 w/v @0.4% combination (6.00 mm). in 2nd lower growth recorder from aloe vera: 1:0.50 w/v @0.2% (9.00 mm) and nigella sativa: 1:1 w/v @0.4% (9.00) combination, which were statistically identical to each other. control plates (only water amended pda: (50.00), (49.00) and (49.67)) were observed highest growth of mycelium both at 7 days culture age and 14 days culture age. the highest concentration of botanical extracts was more effective compared to low concentration in reducing the radial mycelia growth of the fungus as observed in case of blast pathogen. with the concentration increase of all tested botanical extracts, the mycelial growth of mot reduced compared to control. discussion in bangladesh about 80 % of people lean on directly on agriculture for their food and livelihood, with wheat being the second most important crop after rice. wheat blast is caused by mot is a new disease in bangladesh which may cause up to 100% yield loss of wheat. in another study significant losses in grain yield due to wheat blast were observed in all the survey sites of the two south-western districts of bangladesh (tanjina et al., 2019) [25]. the result evaluated that the lowest radial mycelial growth was recorded from nigella sativa: 1:1 w/v @ 0.4% (3.00 mm) combination at 7 days culture age which is statistically similar with aloe vera: 1:1 w/v @ 0.4% (3.33mm) and nigella sativa: 1:0.25 w/v @ 0.2% (4.33 mm) combination. control plates (only pda) were always observed highest growth of mycelium. at 14 days culture age, lowest mycelial growth was observed in aloe vera: 1:1 w/v @ 0.4% combination (6.00 mm). in 2nd lower growth recorder from aloe vera: 1:0.25 w/v @0.2% (9.00 mm) and nigella sativa: 1:1 w/v @0.4% (9.00) combination, which were statistically identical each other. control plates (only pda (50.00), (49.00) and (49.67)) were observed highest growth of mycelium both at 7 days culture age and 14 days culture age. the findings are similar to zohura et al. (2018) [26] who reported the effectiveness of 12 plant extracts namely neem leaf extract, bishkatali leaf extract, nishinda leaf extract, allamonda leaf extract, acasia leaf extract, tulsi leaf extract, mehendi leaf extract, datura leaf extract, bishkochu leaf, black cumin seed extract, garlic clove extract and mehogoni seed extracts against mot in vitro. among these twelve plant extracts, four plant extracts viz. tulsi leaf extract, mehendi leaf extract, datura leaf extract and garlic clove extract impede the highest percentage (93.75%) of mycelial growth followed by black cumin seed extracts (90%) at 10 dai where lowest percentage of mycelial growth inhibition (7.5%) were recorded in allamonda leaf extract over control. the highest concentration of botanicals extracts was more effective compare to low concentration in reducing the radial growth of the fungus as observed in case of blast pathogen. the results are similar with the findings of table 4. effects of ethanol extracts of botanicals on mycelia growth and colony characters of magnaporthe oryzae triticum treatments ethanol botanicals ratio (w/v) radial mycelia growth (mm) colony character 7 dai 14 dai colony color shape allium cepa 1:0.25 38.33b 58.33c gray ash regular 1:0.50 36.00c 41.67hi 1:1 31.33f 26.00l allium sativum 1:0.25 31.00f 60.33bc gray ash regular 1:0.50 23.00i 56.33d 1:1 11.00m 41.00i curcuma longa 1:0.25 33.33e 59.00bc gray with white margin regular 1:0.50 29.00g 49.00f 1:1 18.00k 33.33j zingiber officinale 1:0.25 34.00de 46.33g gray with white margin regular 1:0.50 26.67h 40.33i 1:1 19.67j 23.67m azadiracht a indica 1:0.25 39.00b 61.00b grey with white regular 1:0.50 30.33fg 51.67e 1:1 25.33h 43.33h nigella sativa 1:0.25 5.00o 11.33n white regular 1:0.50 4.33opq 9.67no 1:1 3.00q 9.00o allamanda cathartica 1:0.25 35.00cd 51.00e white regular 1:0.50 30.00fg 40.33i 1:1 13.67l 29.67k aloe vera 1:0.25 7.67n 11.67n white regular 1:0.50 4.67op 9.00o 1:1 3.33pq 6.00p control only pda 50.00a 69.33a grey with white regular only pda 49.00a 68.67a only pda 49.67a 68.33a cv% 3.68 3.02 in a column treatment means with the same letter are not significantly different dai= days after inoculation nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 77-84 rehena et al. ©njb, bsn 83 zohura et al., (2018) [26] where no growth inhibition had taken placed on control plate. allamonda leaf extract performed with the minimum percent growth inhibition (7.5%) compared to control while the maximum inhibition was observed in the plates containing garlic clove, tulsi leaf, mehendi leaf (93.75%) and black cumin seed extract (90%) respectively after 10 days of inoculation. this result is also in conformity with the findings where they stated that h. anthelminthicus fruit extracts exhibited antifungal potential to growth inhibition at the 10,000-ppm concentration and 100% growth inhibition against pyricularia oryzae, p. palmivora and r. solani were noted followed by s. rolfsii at 96.33% when compared with water control [27]. for controlling the rice blast disease in-vitro and in-vivo experiment were carried out by hubert et al. (2015) [28] where the effects of aloe vera, allium sativum, annonamuricata, azadirachta indica, bidenspilosa, camellia sinensis, chrysanthemum coccineum, processed coffee arabica, datura stramonium, nicotiana tabacum and zingiber officinalis extracts for control of rice blast disease (magnaporthe oryzae) both were assessed. at 10% and 25% (v/v) the highest (81.12%) and (89.40%) inhibitory effect was observed in processed c. arabica against p. grisea, respectively. to manage rice blast disease these plant extracts can be used. conclusions wheat blast caused by magnaporthe oryzae triticum (mot) recognized as a devastating disease and caused up to 100% yield loss first time in some wheat growing areas of bangladesh in the year 2016. as there are no appropriate control measures have been developed till now so the effective management of this pathogen is the environment friendly management with botanical extracts until the establishment of resistance cultivars against this notorious pathogen in bangladesh. the highest radial mycelial growth inhibition aloe vera (aloe vera leaf) extracts and nigella sativa (black cumin seeds) extracts 1:1 w/v @ 0.4% (3.00mm and 3.33mm) concentration at 7 days after inoculation, whereas the lowest inhibition allium cepa (onion) extracts 1:0.25 w/v @ 0.1% (38.33mm) concentration of mot under in-vitro condition. however, this experiment with plant extracts urgent to be invented out to assess the field efficacy of these botanical extracts with different concentrations and frequencies in controlling blast of wheat. acknowledgements we acknowledge shere-bangla agricultural university research system (saures), dhaka, bangladesh for their support. conflict of interest the authors declare no conflict of interest. ethical issues there are no ethical issues involved in this research work. funding this research work was supported by shere-bangla agricultural university research system (saures), no. sau/saures/2019/1847(35), dhaka, bangladesh. author contributions m. k. rehena collected wheat blast samples from the field, conducted the research and wrote the article; f. m. aminuzzaman designed and supervised the study and edited the manuscript; m. l. ashrafi and u. a. habiba collected the blast samples from field and analyze the data; m. s. m. chowdhury, z. nazifa and m. ahmed read the manuscript contributed to the conceptualization, and methodology of the study. references 1. lu, m., cao, x., pan, j., gurajala, h. k., he, z., yang, x., and khan, m. b. 2020. genotypic variations in zinc accumulation and bioaccessibility among wheat (triticum aestivum l.) genotypes under two different field conditions. journal of cereal science, 93, 102953. https://doi.org/10.1016/j.jcs.2020.102953. 2. wiese, m. v. 1987. “compendium of wheat diseases”.2nd ed. american phytopathol. soci. st. paul. minnesota. 112. 3. bbs. 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http://doi.org/10.1146/annurev.phyto.41.121702.103726 https://dx.doi.org/10.5423%2fppj.rw.05.2012.0072 https://doi.org/10.1128/9781555816636.ch37 nepal j biotechnol. 2 0 2 2 d e c ; 1 0 (2): 45-51 research article doi: https://doi.org/10.54796/njb.v10i2.239 ©njb, bsn 45 assessment of phytochemicals, antioxidant activity, total phenolic and flavonoid contents of selected nepalese medicinal plants deepak kumar shrestha , basanta kumar sapkota , krishna prasad sharma tribhuvan university, department of chemistry, butwal multiple campus, butwal received: 31 aug 2022; revised: 24 dec 2022; accepted: 25 dec 2022; published online: 31 dec 2022 abstract several medicinal plants have been used from the traditional period of times to cure different diseases but there is little scientific evidence. the phytochemicals of plants can reduce cardiovascular and other diseases. the present study analyzed the five different medicinal plants of the gulmi and rupandehi districts of nepal using in vitro studies. they were crateva unilocularis, aegle marmelos, nyctanthes arbor-tristis, urtica dioica and justicia adhatoda. the antioxidant potential of the methanolic extract of plants was evaluated by dpph radical scavenging assay, total phenolic content was determined by using the folin-ciocalteu method and total flavonoid content was determined by using the aluminium chloride colorimetric method. results revealed that the methanolic extract of plants contained phytochemicals such as alkaloids, flavonoids, polyphenols, saponins, quinones, terpenoids, etc. the extract of nyctanthes arbor-tristi showed the highest % of radical scavenging activity up to 64.931±0.032% with an ic50 value of 70.506±1.55µg/ml followed by aegle marmelos and the lowest in urtica dioica. nyctanthes arbor-tristis revealed the highest tpc (97.647±7.01mggae/g) and lowest in urtica dioica. crateva unilocularis had the highest tfc 31.99±2.345mgqe/g and followed by nyctanthes arbortristis and lowest in justicia adhatoda. these parameters were analyzed from the period 5 september 2021 to 10 october 2021. keywords: antioxidant, flavonoid, phenolic compound, 2,2-diphenyl-1-picrylhydrazyl. corresponding author, email: shresthadeepak854@gmail.com introduction nepal is situated in south asia. it is a land-locked country that occupies 0.03 % and 0.3% land area of the world and asia respectively and has total area 1,47,181 sq. km [1,2]. nepal is ranked 25th and 11th positions in biodiversity richness in the world and asia, respectively. nepal is enriched with several climatic conditions, geographical variations, and an immense variety of medicinal plants has contributed about 10% medicinal plants of the expected 7000 species of flowering plants [2,3]. gulmi district, a part of province no. 5 of nepal, which altitude ranges from 465 m to 2690 m [4]. rupandehi district is also part of lumbini province, its altitude ranges from 100m to 1229m from sea level. natural products are the major sources of new drug discovery. about 90% of nepalese populations, residing in rural areas are still using medicinal plants for their primary health care. despite the widespread use of medicinal plants in nepal, there are limited studies on phytoconstituents and their antioxidant activity [2]. synthetic drugs in the long run, show harmful side effects and they are expensive too. hence, drug development from the natural product is a promising field. therefore, it is urgent to identify phytochemicals and explore the antioxidant with a quantitative estimation of flavonoid and phenolic content in the natural resources of nepal. hence, the present study mainly focused on medicinal plants found in gulmi and rupandehi districts. the oxidative stress inside body produces chronic diseases such as diabetes, heart disease, and cancer [5]. in oxidative stress, the balance between the formation of reactive oxygen species (ros) and the amount of antioxidants in the body is destroyed which causes damage to cell components such as proteins, lipids, and nucleic acid and eventually leads to cell death [6]. ros and reactive nitrogen species (rns) are the main sources of free radicals which lead to serious disorders such as alzheimer's disease, parkinson's disease, and strokes [7]. although no and superoxide radicals are involved in host defense, overproduction of these two radicals contributes to the pathogenesis of inflammatory diseases [8]. antioxidants are compounds that hinder oxidative processes and thereby delay or prevent oxidative stress [9]. increasing intake of antioxidants can prevent diseases and lower health problems. in the present context, many antioxidants are manufactured synthetically such as butylated hydroxyanisole (bha), butylated hydroxytoluene (bht), tertiary butylated hydroxyquinone (tbhq), and gallic acid ester. these synthetically produced antioxidants possess certain side effects and toxic effects. natural products, mainly obtained from dietary sources provide a large number of nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-4185-0103 mailto:shresthadeepak854@gmail.com https://orcid.org/0000-0002-9085-539x https://orcid.org/0000-0003-3071-4648 mailto:shresthadeepak854@gmail.com nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :4 5 5 1 shrestha et al. ©njb, bsn 46 antioxidants that decreases oxidative injury [1]. plants contain many phytochemicals that are useful sources of natural antioxidants, such as phenolic diterpenes, flavonoids, tannins, alkaloids and phenolic acids etc [10]. polyphenols are the strong antioxidant in plant extracts [11]. flavonoids are highly effective scavengers of most oxidizing molecules, including singlet oxygen and various free radicals. as per [12] by gc-ms analysis for different extract of bark of crateva religosa suggest that this plant has vast variety of photochemical which can be used for various ailments and drug formulations in future. the ethnic claims of this plant were also verified by the present study. as per [13] the phytochemical analysis of methanolic extracts of all nine medicinal plants of kavre district, displayed the presence of various secondary metabolites such as alkaloids, flavonoids, polyphenols, saponins, and quinones [13]. the methanolic extract of s. pinnata showed the highest percentage of radical scavenging activity up to 87.94±1.88 with 50% inhibitory concentration (ic50) 17.51±1.27 μg/ml [13]. moreover, s. pinnata displayed the highest total phenolic content (tpc) 48.26±1.23 mg gae/g while the highest flavonoid content was displayed by melia azedarach 41.07±1.53 mg qe/g [13],[14]. as per [15] bioactive properties were found strongly on 4 selected traditionally used medicinal plants with strong enzyme inhibition potential where α-glucosidase and α-amylase inhibitory activities were investigated using in vitro model followed up by antioxidant and antimicrobial activities. the study showed that ethyl acetate fraction of melastoma melabathrium (ic50 9.1 ± 0.3 µg/ml) and water fraction acacia catechu (ic50 9.0 ± 0.6 µg/ml) exhibit strong α-glucosidase inhibition. furthermore, to identify the metabolites within the fractions, they employed highresolution mass spectrometry (lc-hrms) and annotated 17 known metabolites [15]. as per [16] hydro-alcoholic extract of urtica dioica shows positive results for antioxidant activity with ic50 value of 88.33 ± 2.88 µg/ml while standard ascorbic acid showed ic50 value of 2.8 ± 0.62 µg/ml [13]. extract of nyctanthes arbor-tristis exhibited various pharmacological activities like hepatoprotective, antileishmanial, antipyretic, antihistaminic, antimalarial, antibacterial, anti-inflammatory, antioxidant, antiviral, and antifungal etc. because of the presence of glycosides and phenolic compounds [17]. gnidia glauca and d. bulbifera contained significant amounts of phytochemicals with antioxidative properties [18]. similarly, the highest radical scavenging activity of dioscorea bulbifera fruit powder was found in vacuum drying ranged from 65.36% to 81.33% of the concentration of 200 μg/ml to1000 μg/ml, [19]. in vitro antioxidant activity of the aegle marmelos plant extract revealed that both the extracts showed good antioxidant power with ic50 value ranges of 37.11±3.50 to 158.99±59.46 µg/ml for aqueous extract and 35.02±8.10 to 283.06 ± 135.80µg/ml and 35.02±8.10 to 283.06 ± 135.80µg/ml for alcoholic extract [20]. polyphenols, especially flavonoids are the strong antioxidant in plant extracts [21]. different extract of justicia adhatoda by ultra uhplc analysis revealed the presence of polyphenolic compounds and flavonoids which might be responsible for bioprotective activity. among the five fractions (hexane, chloroform, ethyl acetate, n-butanol and aqueous), n-butanol and ethyl acetate exhibited significant antioxidant activity with minimum ic50 value (< 105.33 µg/ml) [22]. the present study was to analyze secondary metabolites, total phenolic, flavonoid contents, and antioxidant activity of five different medicinal plants. it helps to find scientific pieces of evidence of the medicinal value of plants and also help to further investigation. methods and materials assembling and identifying plant samples different parts of five different selected medicinal plants crateva unilocularis from gulmi district and other four plants viz aegle marmelos, nyctanthes arbotristis ,urticadiocia, justicia adhatoda were collected from different region of rupandehi (table 1). they were collected in the period of 1 march 2021 to 2 april 2021. botanist dr. ananta gopal singh of bmc identified the plants. statistical evaluation there were three copies of each experiment run. the mean ± standard deviation (sd) was used to express the results. microsoft excel 2016 was used for all statistical analysis. the ic50 values was calculated by using microsoft excel 2016. extract preparation after drying the plant's raw fruits and leaves were gathered locally and processed. the components of the gathered plant were cleaned, then dried at room temperature in the table 1: chosen medicinal plants list name of plants local name utilized parts family aegle marmelos bel raw fruits rutaceae nyctanthes arbor-tristis parijat leaves oleaceae urtica dioica sisnu leaves urticaceae crateva unilocularis siplikan buds and leaves capparaceae justicia adhatoda asuro leaves acanthaceae nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :4 5 5 1 shrestha et al. ©njb, bsn 47 shade. they were ground into a powder using a mechanical grinder, stored at a low temperature in a clean plastic bag. by using the cold percolation process, extract was created. 400 ml of methanol and 150 g of dried powdered components were combined in separate, clean, dry conical flasks. extraction was carried out in a flask that was firmly sealed for 72 hours while being frequently shaken and filtered. the resulting residue was once more steeped in methanol. until the sample's methanol became colorless, the procedure was repeated. the rotary evaporator was used to concentrate the thusly produced filtrate. different plants' solid methanolic extracts were prepared and kept in a refrigerator at 4°c until analysis [13]. table 2. methods used for data analysis parameters methods employed antioxidant activity dpph radical scavenging assay total phenolic content folin-ciocalteu method total flavonoid content aluminium chloride colorimetric method uv visible spetrophotometer aczel pvt. ltd, model no: auv-8s id: 210702206 chemicals and reagents needed analytical-grade chemicals were employed throughout. fehling's solution, alpha-naphthol, fecl3, nahco3, bi(no3)3, ki, hgcl2, picric acid, dimethyl sulfide, sodium hydrogen phosphate, sodium carbonate, sodium chloride, sodium hydroxide, sodium nitroprusside, and distilled water are a few examples of substances that fall under this category. the 2,2-diphenyl-1-picrylhydrazyl (dpph), gallic acid, quercetin, and the folin-ciocalteu reagent were bought from qualigens fine chemicals in india. meyer's reagent, dragendorff's reagent, molisch's reagent, and other reagents and solvents used in phytochemical analysis were made in the lab using chemicals of the laboratory reagent grade. required apparatus hot air oven, mechanical grinder, digital weighing balance, cuvettes, burettes, pipettes, micropipettes, thermometer, condensers, beakers, conical flasks, test tubes, reagents bottles, stands, vial tubes, round bottom flasks, rotary evaporator with water bath was used for the evaporation of solvents. using a uv-visible spectrophotometer, absorbance for the dpph assay and absorbance for total phenolic and flavonoid content were determined. phytochemical analysis the method used for phytochemical screening was based on protocol put forward as per [23]. basically phytochemical screening helps to identify secondary metabolites (bioactive compounds) present in plants. the analysis was done by the color reaction using different specific reagents [24]. the qualitative results are expressed as (+) for the presence and (−) for the absence of phytochemicals. the phytochemical screening was carried out to test for alkaloids (meyer’s test, dragendorff’s test), coumarins, flavonoids, quinones, polyphenols, glycosides, reducing sugar, saponins, tannins, and carbohydrates (molish’s test) etc. respectively antioxidant activity measurement dpph radical scavenging activity the protocol developed as per [25] and [26] was used to conduct the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging experiment. this test is a straightforward, popular, and widely accepted method to assess the antioxidant potential of plant extracts. each plant sample's extract solution was diluted to a different concentration (10 μg/ml to 100 μg/ml) and mixed with 2 ml of dpph solution (60 μm). for a 30-minute reaction, the mixture was left to stand in full darkness. finally, a uv spectrophotometer was used to assess each plant sample's absorbance at 517 nm. each sample's radical scavenging activity was determined using the formula below: radical scavenging (%) = [(a0 a1 / a0) × 100%] where a0 is the absorbance of the control and a1 is the sample extract's absorbance. the test solution without the sample is the control. the standard was ascorbic acid. a similar process was used with ascorbic acid solutions that ranged in concentration from 10 to 100 μg/ml. each sample's antioxidant activity was quantified using its ic50 (the concentration needed to suppress the production of dpph radicals by 50%) value. the sample's effective concentration was defined as the 50% inhibitory table 3. phytochemical screening of methanolic extract of different plant samples. phytochemicals a.m n.a u.d c.u j.a alkaloids + + + + ++ terpenoids + + + + + coumarins + + + + + flavonoids + + + ++ quinones + + + + + polyphenols + + + + glycosides + + + + reducing sugar + + saponins + + tannins + + + + carbohydrates + + + a.m= aegle marmelos, n.a= nyctanthes arbortristis, u.d= urtica dioica, c.u= crateva unilocularis, j.a= justicia adhatoda, and (-) for absence & (+) for presence. nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :4 5 5 1 shrestha et al. ©njb, bsn 48 concentration (ic50) value, which was needed to effectively scavenge 50% of the dpph free radicals. by graphing the extract concentration versus the corresponding scavenging action, the inhibition curve was used to determine the ic50 values. total phenolic content determination using gallic acid as a reference and the oxidationreduction reaction as a basis, the total phenolic content of the extract was calculated using the folin-ciocalteu technique. the method described as per [13] was used to determine the total phenolic content, with a few changes [5]. 5 ml of 10% folin-ciocalteu reagent were mixed with 1 ml of crude extract. 4 ml of 7% (w/v) sodium carbonate was mixed and shook after standing for 5 minutes. after 40 minutes of incubation, the mixture's absorbance at 760 nm was measured. the entire experiment was run in triplicate. gallic acid was used as the standard in the creation of the calibration curve. the calibration curve was used to quantify the total phenolic content, and the results were represented as mg of gallic acid equivalent (gae) per gram dry weight of extract using formula, tpc= c×v/m where v= volume of extract in ml, m=weight of plant extract in mg, and c=concentration of galic acid obtained from calibration curve in mg/ml. the gallic acid calibration curve was used to derive the linear correlation coefficient (r2) value and regression equation. each extract's concentration was determined using the regression equation. the tpc was determined using the calculated value of each extract's concentration. total flavonoid content determination with a small modification of protocol as per [11] and [27], the aluminum chloride colorimetric method was employed to determine the flavonoid concentration. 4 ml of distilled water and readily added 0.3 ml of 5% sodium nitrite were combined with 1 ml of each extract solution. 0.3 ml of 10% aluminum chloride was added after 5 minutes, and the mixture was let to stand for 6 minutes. after shaking well, 2 ml of 1 m sodium hydroxide and 2.4 ml of distilled water were added, bringing the total volume to 10 ml. the absorbance was then measured at 510 nm using a uv spectrophotometer. the standard used to create the calibration curve was quercetin. from the calibration curve, the total flavonoid concentration was determined and results were represented as mg of quercetin equivalent (qe) per gram dry extract weight. results extraction yield value the extract of the selected plant samples was prepared in methanolic solvent by cold percolation method. the yield percent of the plant extract is given in table 4. table 4: methanolic extract of different plant samples' yield percentages. name of plants yield% of extract (in methanol) aegle marmelos 12.42% nyctanthes arbo-tristis 10.28% urtica dioica 11.42% crateva unilocularis 10.14% justicia adhatoda 12.4% note: extract yield percent is equal to [(weight of dry extract/weight of crude plant sample) 100%]. variations in the scavenging of dpph radicals figure 1 displays the percentage of dpph radical scavenging activity of various samples at various concentrations in methanolic solvent, whereas table 4 displays the ic50 value for the dpph radical scavenging activity. with the least amount of inhibitory concentration and the highest dpph radical scavenging activity (ic50) value was found nyctanthes arbor-tristis (ic50 value 70.506±1.55 µg/ml), aegle marmelos has 99.872±1.27, justicia adhatoda has103.146±1.33, crateva unilocularis has 127.672±2.61 whereas urtica dioica (ic50 value 179.103±3.58 µg/ml) has relatively little dpph radical scavenging efficacy in comparison to ascorbic acid standard (ic50 value 55.40±0.003 µg/ml).the antioxidants are the chemical elements found in plants that can effectively quench the stable purple dpph radical, converting it to the more visible yellow dpph. table 5. antioxidant activity of ascorbic acid s.n concentration (ppm) absorbance mean ± sd % scavenging a1 a2 a3 1 10 0.445 0.441 0.452 0.446 ± 0.005 9.712 ± 0.132 2 20 0.397 0.401 0.403 0.400 ± 0.003 19.01 ± 0.013 3 40 0.312 0.322 0.323 0.319 ± 0.006 35.38 ± 0.002 4 60 0.209 0.208 0.216 0.211 ± 0.004 57.12 ± 0.003 5 80 0.140 0.148 0.144 0.144 ± 0.004 71.42 ± 0.012 6 100 0.054 0.057 0.057 0.056 ± 0.001 88.47 ± 0.013 ic50 55.40 blank 0.494±0.001 nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :4 5 5 1 shrestha et al. ©njb, bsn 49 table 6. percentage scavenging (antioxidant) activity of methanolic extract of different plants {result expressed as the mean ±sd (n=3) plants concentration (ppm) 10 20 40 60 80 100 nyctanthes arbortristis mean% scavenging 5.697±.089 26.031 ±0.12 32.514 ±0.33 44.007 ±0.45 57.563 ±0.33 64.931±0.032 aegle marmelos mean % scavenging 1.305±2.3 3.343±1.023 6.534±0.45 14.021±0.12 30.931±0.22 63.033±0.14 crateva unilocularis mean % scavenging 1.089±1.8 2.008±1.4 4.144±0.45 9.509±0.34 21.034±0.01 48.310±0.22 urtica dioica mean% scavenging 0.495±2.4 1.450±1.44 3.432±0.89 9.098±0.76 19.231±0.33 40.789±0.02 justicia adhatoda mean % scavenging 2.024±1.34 5.048±0.98 9.09±0.42 14.56±0.56 29.123±0.32 60.756±0.13 figure 1. percentage scavenging of dpph free radicals by methanolic extract of plants with reference to ascorbic acid, results expressed as the mean ± standard deviation (n=3) at a concentration of 10, 20, 40, 60, 80, and 100 µg/ml. table 7. ascorbic acid and methanolic extract of several plant samples' dpph radical scavenging activity, expressed in terms of ic50 value. name of plant extracts ic50(µg/ml), mean ± sd 1. ascorbic acid (standard) 55.40 ±0.003 2. aegle marmelos 99.872 ±1.27 5. nyctanthes arbortristis 70.506 ±1.55 6. urtica dioica 179.103 ±3.58 7. crateva unilocularis 127.673 ±2.61 8. justicia adhatoda 103.146 ±1.33 variation of total phenolic content by applying the folin-ciocalteu method and using gallic acid as a reference, the total phenolic content of methanolic extracts was calculated [13,5]. at 760 nm, the highest absorption was noted. concentrated gallic acid solution (10-100 µg/ml) confirmed at 760 nm with a regression coefficient (r2) of 0.9698 to beer's law (figure 2). with the aid of a calibration curve using gallic acid as the reference, the total phenolic content was determined and expressed as mg gae/g dry extract weight, total phenolic content was found highest in nyctanthes arbortristis (97.647±7.01) mg gae/g followed by aegle marmelos (88.559.647±6.71), crateva unilocularis (83.333±2.73),and lowest in justicia adhatoda (64.126±1.368) respectively and was found lowest in urtica dioica (24.36±1.33 mg gae/g). table 8. total phenolic content of different plant extract name of plants absorbance tpc (mggae/g) a1 a2 a3 c1 c2 c3 mean±sd a.m 1.645 1.646 1.624 88.919 88.973 87.78 88.559±.6.715 n.a 1.846 1.664 1.915 99.7831 89.8459 103.514 97.647±7.01 u.d 0.473 0.424 0.455 25.5676 22.9189 24.5946 24.36±1.33 c.u j.a 1.533 1.215 1.496 1.167 1.596 1.177 82.8649 65.67 80.8649 63.081 83.333 63.621 83.333±2.73 64.126±1.368 gae: gallic acid equivalent, sd: standard deviation,a.m: aegle marmelos, n.a: nyctanthes arbortristis,u.d: urtica dioica, c.u= crateva unilocularis,j.a= justicia adhatoda, tpc: total phenolic content, a1 a2, a3= absorbances triplicate, c1, c2, c3= tpc triplicates figure 2: calibration curve of gallic acid. y = 0.0376x r² = 0.9923 0 0.5 1 1.5 2 2.5 3 3.5 0 20 40 60 80 100 a b so rb a n ce concentration(µg/ml) series1 linear (series1) 0 20 40 60 80 100 120 p e rc e n ta g e i n h ib it io n plant samples dpph assay series1 series2 series3 series4 series5 series6 nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :4 5 5 1 shrestha et al. ©njb, bsn 50 variation in total amount of flavonoids using quercetin as a reference, the total flavonoid content of methanolic extracts was determined using the aluminum chloride colorimetric technique. with a regression coefficient (r2) of 0.9729, the quercetin solution (10–100 µg/ml) obeyed beer's law at 510 nm (figure 3). total flavonoid content was found highest in crateva unilocularis (31.99±2.345) qe/g, followed by nyctanthes arbortritis (17.62±3.225) then aegle marmelos (10.998±0.134), urtica dioica (10.518±5.231) and lowest in justicia adhatoda (9.724±0.103) mg qe/g. figure 3. calibration curve for quercetin conclusion the dpph radical scavenging activities and subsequently the ic50 values of chosen plants' methanolic extracts revealed various levels of antioxidant activity. nyctanthes arbortristis showed highest percent scavenging, it has ic50 value 70.505±1.55 μg/ml while the standard, ascorbic acid has 55.40±0.003 μg/ml, followed by aegle marmelos. the greater antioxidant property on them might be due to the presences of bioactive compounds such as phenolic ,terpenoids, flavonoid, saponins, tannin, alkaloids etc .the highest tpc was found in nyctanthes arbortristis (97.647±7.01) followed by a.marmelos and lowest in u.dioica.while highest tfc is found in crateva unilocularis(31.99±2.345) and lowest in justicia adhatoda. hence, these plants contain appreacible amount of antioxidants, tpc and tfc. these results gave some scientific proof for these indigenous medicinal plants. even while some medical plants have substantial antioxidant properties, they cannot be used directly as drugs. to make these therapeutic plants a possible source of natural antioxidants, more thorough phytochemical and pharmacological research must be done. author’s contribution d. k. shrestha and b.k sapkota designed research and performed the experiments. b.k.sapkota, d. k.shresth and k.p. sharma analysed the data and k. p. sharma reviewed the literature and edited the manuscript. all the authors contributed equally in drafting and revising the manuscript. competing interests we declare that authors have no conflict of interests of any kind. funding no financial support for this work was provided by any institution, organisation or agency.all financial resources was managed by the authors themselves. few solvents, reagents and glasswares and instruments were supported by butwal multiple campus, butwal. acknowledgement the authors are grateful to the department of chemistry, butwal multiple campus for providing laboratory and instrumental facilities, the campus chief of bmc, botanist dr. anant gopal singh, and research management cell of bmc for providing support to conduct research successfully. ethical approval and consent: all the ethical guidlines from erb and nhrc are strictly followed. we ensure that there was no use of animal models and hazardous materials in this study.the plants used in this study were not endangered species or banned by the government. no people other than the table 9: different plant extracts' total flavonoid content plants absorbance tfc (mg qe/g) a1 a2 a3 c1 c2 c3 mean ± sd aegle marmelos 0.172 0.171 0.168 11.0968 11.0323 10.838 10.989 ± 0.134 nyctanthes arbortristis 0.273 0.323 0.223 17.612 20.838 14.387 17.62 ± 3.225 urtica dioica 0.172 0.171 0.168 11.0468 11.032 10.438 10.518 ± 5.231 crateva unilocularis 0.435 0.505 0.487 28.064 32.580 31.419 31.99 ± 2.345 justicia adhatoda 0.157 0.156 0.143 10.1290 9.936 9.108 9.724 ± 0.103 tfc=total flavonoid content (n=3), sd= standard deviation, q.e=quercetin equivalent y = 0.0314x r² = 0.9903 0 0.5 1 1.5 2 2.5 3 0 20 40 60 80 a b so rb a n ce concentration(µg/ml) series1 linear (series1) nepal j biotechnol. 2 0 2 2 d e c ; 1 0 :4 5 5 1 shrestha et al. ©njb, bsn 51 authors are involved in this research. the harm or discomfort for others during this research was nill. references 1. elmastaş m, gülçin i, işildak ö, küfrevioğlu öi̇, i̇baoğlu k, aboulenein hy. radical scavenging activity and antioxidant capacity of bay leaf extracts. journal of the iranian 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(2012). screening of in vitro antioxidant activity of methanolic leaf and root extracts of hypochaeris radicata l.(asteraceae). journal of applied pharmaceutical science, 2(7), 149154. 26. shackelford l, mentreddy sr, cedric s. determination of total phenolics, flavonoids and antioxidant and chemopreventive potential of basil (ocimum basilicum l. and ocimum tenuiflorum l.). international journal of cancer research. 2009;5(4):130-43. 27. chang cc, yang mh, wen hm, chern jc. estimation of total flavonoid content in propolis by two complementary colorimetric methods. journal of food and drug analysis. 2002 jul 1;10(3). https://doi.org/10.4236/cm.2012.33021 nepal j biotechnol. 2 0 2 2 d e c ; 1 0 (2): 85-90 research article doi: https://doi.org/10.54796/njb.v10i2.242 ©njb, bsn 85 molecular genetic diversity in bambara groundnut [vigna subterranea (l.) verdc] assessed by microsatellite markers. nwakuche chinenye onwubiko1 , michael ifeanyi uguru2 , grace ovute chimdi3 1department of crop science and technology, federal university of technology, owerri 1526, nigeria 2department of crop science, university of nigeria, nsukka 410001, nigeria 3department of agricultural technology, federal polytechnic bauchi, bauchi 0231, nigeria received: 17 mar 2022; revised: 26 nov 2022; accepted: 03 dec 2022; published online: 31 dec 2022 abstract bambara groundnut is a valuable leguminous crop with many landraces. a study was carried out to establish genetic diversity and phylogenetic relationship, among 33 bambara groundnut accessions based on simple sequence repeat (ssr) markers. the nine microsatellite markers amplified a total of 27 alleles with a mean of 6.00 alleles per locus. marker p 36 had the highest number of polymorphic bands while makers p131 and p68 were monomorphic. genetic distance among the accessions based on jaccard’s similarity coefficient ranged from 0.84 to 1.00. cluster analysis resolved the accessions into five major groups with subgroups. each group had a combination of distinct accessions from different geographical origin. a substantial level of intra-accession polymorphism was obtained among the evaluated collection of bambara groundnut. the significant genetic diversity observed can support the selection of appropriate parental genotypes for the improvement of bambara groundnut through various breeding programmes. keywords: accessions, bambara groundnut, genetic diversity, microsatellite markers. corresponding author, email: nwakuche.onwubiko@futo.edu.ng introduction variation exists in crops; it can be interspecific or intraspecific. naturally, variation occur due to mutation and it is maintained over the years by processes of evolution and natural selection. the reality of natural variation in both wild and domesticated crops species is evident on existence of recognisable diverse forms of crop species, which is the basis for selection and for crop improvement. however not all recognisable variation in crops is genetic. variations triggered by the influence of the environment occurs, in which differences observed in the expression of some characters among crop species were not intrinsic. on the other hand, variation in crops because of the action of gene(s) is genetic. genes regulate structure (size, shape, colour), physiological processes and functions, adaptability, phenology, and expression of characters (1). conventionally assessment of genetic variability in crops is achieved through field screening in which morphological descriptors are used to discriminate between and within species. this approach is popular among workers because it is easy to carry out and is relatively cost-effective (2-4). however, it has some limitations because the expression of some characters (especially polygenic) is environment specific. therefore, the obtained result may not be reliable. molecular characterisation on the other hand is the most reliable and effective technique to establish variations that exist in crop species. it uses molecular descriptors or markers that are not affected by the environment to discriminate among species at dna level and can be used to screen the entire population at any stage of crop development. the use of molecular markers to establish genetic diversity has enabled breeders to identify the presence of allelic variations in the genes controlling different agronomic characters in crops (5), to differentiate between homozygote and heterozygote genotypes (6), and to manipulate important agronomic traits in crops (7). bambara groundnut is a leguminous food security crop. its seed is a complete food and many forms of this crop has been reported by workers who made collections from major areas in west africa eco-regions where this crop is grown (25). unfortunately, the diversity reported by these workers where based on field morphological characterization of accessions. (8-11), there are few documented reports on assessment of diversity on bambara groundnut based on molecular markers (12 13, 3, 14. 15), hence the need for this study whose primary target was to assess genetic diversity in bambara groundnut based on microsatellite markers with a view to establishing phylogenetic relationship among the accessions. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0003-3654-0861 mailto:nwakuche.onwubiko@futo.edu.ng mailto:nwakuche.onwubiko@futo.edu.ng nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 85-90 onwubiko et al. ©njb, bsn 86 materials and method the plant materials were 33 accessions of bambara groundnut obtained from the germplasm collection maintained at the gene bank of the international institute of tropical agriculture (iita), ibadan (table 1). dna extraction genomic dna was extracted using the modified minipreparation protocol described by dellaporta et al (16). approximately 200 mg (0.2 gm) of lyophilized leaf sample was ground into fine powder with the aid of genogrinder 2000. table 1: passport data of the 33 bambara groundnut accessions used for the study. s/n accession name country of origin 1 tvsu-1483 ghana 2 tvsu-1503 nigeria 3 tvsu-1504 nigeria 4 tvsu-1509 nigeria 5 tvsu-1510 nigeria 6 tvsu-1512 nigeria 7 tvsu-1513 nigeria 8 tvsu-1552 nigeria 9 tvsu-1554 nigeria 10 tvsu-1555 nigeria 11 tvsu-1559 nigeria 12 tvsu-1563 nigeria 13 tvsu-1584 nigeria 14 tvsu-1591 togo 15 tvsu-1604 togo 16 tvsu-1605 togo 17 tvsu-1610 togo 18 tvsu-1614 togo 19 tvsu-1620 togo 20 tvsu-1625 togo 21 tvsu-1627 togo 22 tvsu-1631 togo 23 tvsu-1638 mali 24 tvsu-1639 mali 25 tvsu-1688 togo 26 tvsu-1697 togo 27 tvsu-1702 togo 28 tvsu-1713 zambia 29 tvsu-1766 malawi 30 tvsu-1769 malawi 31 tvsu-1788 malawi 32 tvsu-1819 cameroon 33 tvsu-1917 cameroon to each tube 700 µl of hot (65oc) plant extraction buffer(peb) [which contains 637.5 ml of double distilled water (ddh20), 100 ml of 1m tris-hcl (ph 8.0), 100 ml of 0.5 m ethylenediaminetetraacetic acid (edta) (ph 8.0), 100 ml of 5m nacl2 and 62.5ml of 20% sodium dodecylsulphate (sds)] was added. one percent bmercaptoethanol was added to the prewarmed peb just before use. the tubes were capped and inverted gently 67 times to mix the sample with buffer. the solution was incubated at 65°c in water bath for 20 minutes with occasional mixing to homogenize the samples. after 20 minutes, samples were removed from the water bath and uncapped. the tubes were allowed to cool at room temperature for 2 minutes. after which 500 µl of 5m of potassium acetate (ch3cook) was added to each tube and recapped. the tubes were mix inverted 6-7 times and incubated on ice for 20 minutes. after 20 minutes of incubation on ice, tubes were spun at 12,000 rpm for10 minutes at 4°c. the supernatant was transferred into new 1.5 ml eppendorf tubes using wider bore pipette tips (1000 µl) and making sure debris were not taken along with the supernatant. 700-µl chloroform isoamylalcohol was added to the supernatant and spun at 10,000 rpm for 10 minutes. the supernatant was transferred again to a new correspondingly labeled tubes and 700-µl ice-cold isopropanol was added to each tube and mixed by gently inverting the tubes 6-10 times. the tubes were allowed to stand undisturbed in a rack and stored in a freezer (-20°c) for at least 1 hour to precipitate the dna. after 1-hour precipitation in the tubes, the dna were centrifuged at 12,00 rpm for 10 minutes at 4°c. the supernatant in the freezer, was carefully discarded with great care to disallow the pellet from dislodging from the bottom of the tube. the tubes were allowed to drain inverted on clean paper towels for 1 hour. the dna pellets were washed twice in 100µl, cold 70% ethanol for 20 minutes and air dried completely. after drying, 60µl of 1×te [10mm tris-hcl (ph 8.0), 1mm edta (ph 8.0)] was added to the pellets, followed by 2µl of 10 ng/ml rnase to remove the rna. the dna was then diluted to 10 ng/ul and then used for polymerase chain reaction (pcr) pcr amplification nine ssr markers (inqaba biotech, south africa) for bambara groundnut was used to perform the pcr reactions and analysis for genetic diversity among the bambara groundnut accessions. the amplification was performed in a 25 µl reaction volume containing 10x buffer, 1.6 µl of 25 mm of mgcl2, 2.0 of 5µ/µl tag, 8.0µl sterile distilled water and 4.0 µl sample dna using a ptc-200 thermal cycle. the pcr reaction was carried out with the following protocol: initial denaturation at 94oc for 5mins followed by 45 cycles of 30 secs at 94oc, then 30mins at 65oc and continues in that order. the resulting amplicons were loaded on 1.5% agarose. agarose gel electrophoresis of the pcr product 1.5% agarose was prepared, which was microwaved to dissolve the agarose and cooled down (560c). the gel nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 85-90 onwubiko et al. ©njb, bsn 87 was poured into the gel tray that was prefixed with comb. the gel tray was immersed into an electrophoresis tank containing 0.5 xtbe buffer. the comb in the gel was removed to expose the wells formed. the amplified dna (10 µl) was loaded into the wells of the gel with the aid of a pipette. a standard dna molecular size marker (1 kb dna lambda) was also loaded as a check. the gel ran for 2 hours at 150v and 0.5mamp. after electrophoresis, the gel was silver stained. thereafter the gel was destained in distilled water for 10 minutes. and the dna bands in the gel was observed under uv (ultra violet) lamp and photographed using a digital camera. each accession was scored (1) for presence and (0) for absence of polymorphic band for each primer. the band scoring data was used to calculate genetic similarity based on jaccard’s similarity coefficients (17) as follows: gsij= a/(a+b+c), where, gsij is the similarity between two accession i and j; a is the number of bands present in both i and j; b is the number of bands present in i but absent in j; and c is the number of bands present in j and absent in i. in addition, the jaccard’s similarity coefficient was used to perform cluster analysis based on the unweighted pair group with arithmetic mean (upgma) and in constructing a dendrogram. the computer program ntsys pc version 2.1 (18) was used for these analyses. furthermore, the genetic diversity, allele frequency and polymorphic information content (pic) were computed using powermarker (version 3.25). results and discussion polymorphism in several genes controls all phenotypic variations within a species. in this respect, the assessment of genetic variability within crop species using molecular markers is of great importance to plant breeders (15, 19). the molecular analysis of genetic diversity in the evaluated accessions of bambara groundnut as determined by the ssr markers amplified a total of 27 alleles. the pic values, which is a measure of the allelic diversity of ssrs, ranged from a minimum of 0.001 to a maximum of 0.617 with a mean of 0.419. seemingly the obtained pic mean was relatively high when compared to the report from basu et al (20), but however relatively lower than the report of mohammed (21). apparently the ssr makers used in this study were not vigna subterranea specific but vigna unguiculata. concisely maker p36 had the highest marker index (mi) which is a measure of efficiency to detect polymorphism. invariably maker p36 was more genetically efficient in distinguishing the phenotypical similarity that exist between the bambara groundnut accessions. on the other hand, markers p131 and p68 had monomorphic phenotype. these two markers recorded the least number of alleles per locus. a similar result has been reported by other workers (14, 15, 22, 19). generally, the 9 ssr markers (table 2) showed the availability of a substantial level of polymorphism among the bambara groundnut lines as revealed by both genetic distance and cluster analysis. the accessions were grouped into five groups based on jaccard neighborjoining dendrogram. each group had subgroups comprised of distinct genotypes from different ecoregions. this implies that variations among individual genotypes was mainly responsible for the observed genetic variation among the bambara groundnut lines and not variations established between specific accession groups. generally, reasonable intra-accessions polymorphism was observed in the cluster analysis of the accessions. in previous studies some workers reported extensive genetic diversity (12, 15), while others observed considerable genetic diversity (13, 3), yet some others reported a low range of genetic diversity (23) in bambara groundnut. further in this study, divergent genotype was revealed by the cluster analysis using unweighted pair-group method with arithmetic average (upgma). tvsu 1554 from nigeria was the only accession found in group 5 among the five groups of the cluster analysis. invariably this accession was dissimilar from other accessions evaluated. divergent genotype(s) usually have good breeding value which can be used for crop improvement, in both direct selection and as parents for making crosses. table 2. description of the ssrs markers used in this study marker forward primer reverse primer gene bank id p36 51-aaaattggagaaaggggttttt-31 51-gatttcgccatatccccatc-31 gq411715.1 p56 51-gcaatgggttcgtcgatact-31 51-gctcgatgctttttgtttcc-31 eu717407.1 p57 51-gggaaacaaaaagcatcgag-31 51-cgctaccccaaaataccaaa-31 eu717407.1 p61 51-gtcagaggcgaattgaaagc-31 51-aggtcttcccgttccttcat-31 eu717373.1 p63 51-atgaaggaacgggaagacct-31 51-cctaagggcatatcggttga-31 eu717373.1 p68 51-caagtccctctatccccaaa-31 51-caagtccctctatccccaaa-31 eu717348.1 p71 51-gtgttgggttcaaagctggt-31 51-catcggtccacacagttgtc-31 eu717266.1 p131 51-caaagccattgctgaagaca-31 51-ggatgctacaccgttcgatt-31 hb823749.1 p184 51-gccagagactctcacgttcc-31 51-tgcatggtccctgttgtaga-31 hb465729.1 nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 85-90 onwubiko et al. ©njb, bsn 88 figure.1: molecular dendrogram analysis of 33 accession of bambara groundnut with unweighted pair group method with arithmetic method (upgma). the genetic distance from the molecular dendrogram analysis based on jaccard’s similarity coefficient ranged from 0.84 to 1.00 (figure 1). this implies that at 100% level of similarity, all the accessions were distinct from each other, while at 84% level of similarity all the accessions clustered to form a single accession. invariably it means that each accession had at least one neighbour with more than 84% similarity. however, at 94% level of similarity, the accessions were grouped into five clusters. the pattern of clustering of the bambara groundnut accessions in groups with the unweighted pair group with arithmetic mean (upgma) and jaccard’s neighbour-joining dendrogram was similar (figure 2). accessions were clustered in the same group based on genetic similarity and not on sources of collection or ecoregion of origin. there were five heterogenic groups in the two-cluster analysis. the reason why accessions from the same geographical area could not form a distinct cluster was because they were genetically dissimilar. each cluster, therefore, contained accessions with similar genetic characters. for example, group one of the unweighted pair group with arithmetic mean (upgma) grouping was the smallest group. it had two accessions, and both were collected from different geographical areas as can be seen in their identification numbers; tvsu 1483 was from ghana, while tvsu 1631 from togo. however, both were clustered together in group one, implying that the two accessions were duplicates or closely similar, and not two different accessions from two different localities as indicated in their identification numbers and places of collections. another outstanding example was observed in group four. the accessions clustered in this group were tvsu 1584, tvsu 1591 and tvsu 1604. accession tvsu 1584 was collected from nigeria, while tvsu 1591 and tsvu 1604 were from togo. a detailed analysis of this result surprisingly showed that these three accessions, that were collected from different geographical areas, were the most genetically similar lines among the evaluated bambara groundnut collections. similarly, in group 1 of jaccard neighbour-joining (jnj) dendrogram, a comparable association was substantiated between tvsu 1697 and tvsu 1627 (from togo) and tvsu 1503 (from nigeria). these complex linkages suggest the possibility that these accessions were related. they either had similar genes or where from a common origin but were given different identification numbers. apparently, these results showed the potentials of the ssr markers in detecting differences and establishing the extent of genetic relatedness in existence among the evaluated genetic materials. it also emphasizes the superiority of ssr markers in classifying collections of bambara groundnut more precisely, as against the use of morphological markers in germplasm characterization (19). nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 85-90 onwubiko et al. ©njb, bsn 89 figure 2: jaccard neighbour-joining dendrogram illustrating genetic diversity and relationships among 33 bambara groundnut accessions. clustering of accessions of bambara groundnut collected from different geographical areas in the same group may have arisen due to duplication of genotypes caused by high rate of seed exchange between farmers from diverse ethnic and agro-geographical areas. in fact, there was a report from a previous study on a similar trend of association between bambara groundnut accessions collected from different geographical areas, and in that study, it was concluded that these accessions were either related, or the genotypes were the same (22). a detailed examination of the result from the dendrogram showed that accessions from nigeria were more dispersed among the clusters of accessions than accessions from other african countries. these accessions were found in four out of the five genetic groups. this observation may have some implication on the origin of bambara groundnut, in that it supports the report of earlier studies on the origin of this crop; nigeria may have been a regional centre of diversity of bambara groundnut (24), which other studies confirmed by the existence of genuinely wild state of the crop in this area (25, 26). conclusion molecular analysis of genetic diversity of 33 accessions of bambara groundnut based on ssr maker was reported. genetic distances result and the cluster analysis revealed high level of polymorphism, indicating the existence of a wide range of genetic diversity among the accessions. this result can facilitate selection of appropriate genotypes for the development of improved lines of bambara groundnut through various breeding programs. this study has contributed to broadening the genetic base of bambara groundnut. the usefulness of this report in the effective utilisation, management, and conservation of bambara groundnut germplasm is undoubtable. author’s contribution miu conceptualized the research proposal and supervised the research activities. nco performed the lab works, scoring, data analysis and interpretation. nco and goc wrote the first draft of the paper. all authors read and approved the final manuscript. competing interests no competing interests were disclosed. funding this research work was not funded by any organization. acknowledgment the authors thank the staff of the international institute of tropical agriculture (iita), ibadan for providing us with seeds of the accessions of bambara groundnut used for this study. nepal j biotechnol. 2 0 2 2 d e c ; 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1 0 (2): 57-76 review article doi: https://doi.org/10.54796/njb.v10i2.243 ©njb, bsn 57 detoxification and removal of hexavalent chromium in aquatic systems: applications of bioremediation a.m.k.c.b. aththanayake1 , i.v.n. rathnayake1 , m.p. deeyamulla2 1department of microbiology, faculty of science, university of kelaniya, kelaniya, gq 11600, sri lanka. 2department of chemistry, faculty of science, university of kelaniya, kelaniya, gq 11600, sri lanka. received: 01 oct 2022; revised: 01 nov 2022; accepted: 09 nov 2022; published online: 31 dec 2022 abstract. chromium is a transition metal with a wide range of applications in leather tanning, textile, electroplating, stainless steel production, inorganic chemical production and wood preservation industries due to yellow colouration, corrosion resistance, higher melting-point and crystalline structure with raging of oxidation states from 0 to +6. trivalent and hexavalent chromium are the most abundant forms of chromium discharged into the aquatic environment by industries. it has been reported that hexavalent chromium is highly toxic than trivalent chromium due to the higher solubility, mobility and tendency to accumulate in higher trophic levels, which, therefore, become bioavailable and causes carcinogenic, mutagenic and teratogenic effects on most microorganisms and animals, growth inhibition, morphological and physiological changes and yield reductions in plants. therefore, it is essential to detoxify the above hazardous pollutants up to permissible limits, which local and international authorities have legislated concerning its threat towards biotic components. hexavalent chromium detoxification is possible to achieve using three methods i.e. physical, chemical and biological methods. these remediation processes can eliminate highly toxic hexavalent chromium or transform it into a less toxic form of trivalent chromium, completely or partially by adsorption and reduction. biological remediation is considered a cost-effective and ecofriendly method compared to physical and chemical remediation. further, many biological agents have been identified as agents that can tolerate the hexavalent chromium toxicity up to certain higher levels depending on the internal and external environmental factors, indicating different metal tolerance mechanisms that are assumed to be applied in metal remediation aspects. according to the testimonies of novel bioremediation studies, some hexavalent chromium tolerant organisms such as plants, bacteria, unicellular and multicellular fungi and algae are promising eco-friendly alternatives in detoxification and hexavalent chromium removal perspective. this article reviews the bioremediation approaches available for hexavalent chromium detoxification and removal and highlights the strengths and weaknesses of current bioremediation methods. keywords: hexavalent chromium, toxicity, biological remediation, chemical remediation, physical remediation, biofilms corresponding author, email: vayanga@kln.ac.lk introduction chromium is a highly valued industrial raw material with a wide range of industrial applications such as pigment production for paints, inks and plastics, anticorrosion coating production, stainless steel production, wood preservation and leather tannins which are discharged both trivalent chromium (cr(iii)) and hexavalent chromium (cr(vi)) in higher quantities [1,2]. according to toxicological studies, cr(vi) is 100 times more toxic than cr(iii) due to solubility, mobility and permeability in biota [3–7]. from the view of toxicology, prolonged exposure to cr(vi) can lead to carcinogenic, mutagenic and teratogenic effects on animals which has been clinically proved, and morphological and physiological effects on plants, algae and other microorganisms [8–12]. the working community in chromium based industries, including chrome mining, has the highest potential for chromium poisoning [8,13– 16]. therefore, international authorities for the occupational community, such as occupational safety and health administration (osha), has set the maximum limit for cr(vi) exposures as similar to the conventional public health concerning local and international authorities; world health organization (who), united states environmental protection agency (us epa). the minimization and detoxification of cr(vi) in industrial effluents can be achieved by physical, chemical, and biological methods. among the above methods, biological remediation is considered the most cost-effective and environmentally friendly method. [17– 20]. chemical nature and uses of chromium. chromium is the 21st most abundant element in earth’s crust, which belongs to d-block in the periodic table with a molar mass of 51.9961 g mol-1 with a wide range of industrial applications based on chemical and physical properties such as inert nature, hardness, strength, hightemperature resistance and corrosion resistance etc. [21]. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0001-7743-0074 https://orcid.org/0000-0003-3476-7018 mailto:vayanga@kln.ac.lk https://orcid.org/0000-0002-3085-4280 nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 58 cr(iii) and cr(vi) are the most stable and domain oxidation forms of chromium that exists in nature among the other oxidation forms of metallic chromium (cr(0)), divalent chromium (cr(ii)), tetravalent chromium (cr(iv)) and pentavalent chromium (cr(v)). the majority of chromium is used for metallurgical (67%) and refractories (18%), while the rest of 15 % are used for chromium induced chemical production, which is used for wood preservation, leather tanning, metal finishing, pigment production and textile industry as a raw material [2] (table 1). table 1 – industrial applications of cr(0), cr(iii) and cr(vi). oxidation state industrial application reference cr (0) stainless steel production alloy production metal manufacturing [2] cr (iii) metal and alloy production textile and leather tanning copy machine toners brick lining chrome plating catalysts production paint production [2,22] cr (vi) chrome plating leather tanning textile industry copy machine toners dye/paint pigment production wood preservation high temperature battery production metal finishing catalyst production stainless steel production plastic production [2,22,23] toxicity of chromium. among the most stable oxidation states of chromium in nature, cr(iii) is considered an essential micronutrient of higher organisms with less toxic effects due to lower solubility and impermeability [6,24]. further, it has been reported that cr(iii) can assist in regulating the glucose level of the human body [25]. in contrast, cr(vi) has been categorized as a carcinogenic agent by the usepa and international agency for research on cancer (iarc) due to high water solubility and mobility [26]. toxicity of cr(vi) to humans. prolonged cr(vi) exposes through breathing, ingesting, and skin contacts can cause nasal irritations, nasal perforations, skin irritations, skin ulcerations, skin allergies, lung cancers, stomach upsets, convulsions, kidney and liver damages [2]. the working community in chromium-based industries (leather tanning, electroplating, mining and pigment production, etc.) has a high tendency to be affected by cr(vi) toxicity. cr(vi) can produce highly reactive hydroxyl radicals in blood vessels during the reduction into cr(iii), which can cause blood cell damages with organ degradations and cellular activity interruption by metal-dna bindings [27]. further it believes that cr(vi) is responsible for causing teratogenic effects in human as it has proven with animal model trials [28]. toxicity of cr(vi) to plants. chromium being a non-essential element it does not have a specific mechanism of uptake into plants. it is believed that, the plants use a passive process to uptake cr(iii) and an active process for uptake cr(vi) with carriers competing with iron, sulphur and phosphorus [29]. part of the cr(vi) is taken up into plants after reducing into cr (iii) on the root surface, and the rest of the cr(vi) is taken up by plants by dissolving in water and without reducing [29,30]. in the toxicological point of view, cr(vi) affect plants both morphologically and physiologically. it has been found that, high concentrations of cr(vi) affect the seed germination negatively due to the depressive effects on enzyme activity and sugar transport to embryo axes [31,32], it also reduces the root growth due to inhibition of water absorption [33], and shoot growth due to chromium transportation in aerial parts [31]. toxicological studies done else ware using oryza sativa, acacia holosericea, leucaena leucocephala and albizia lebbek and phaseolus vulgaris reported that leaf area and biomass can be adversely affected by cr(vi) [31,34]. plant physiological studies revealed that cr(vi) can lead to yield reduction by decreasing chlorophyll a, chlorophyll b and carotenoid pigments and affecting water and mineral transportation due to high oxidative potential [31,35]. toxicity of cr(vi) to microorganisms. microorganisms are commonly exposed to many pollutants, including toxic metals, as they are widely dispersed in the environment, causing many toxic effects. cr(vi) can become toxic to most bacterial strains causing cell enlargements, cell elongations and cell division inhibitions [23]. cr(vi) can rapidly enter into the bacterial cytoplasm and reduces to lower oxidation states which are free radicals such as cr(v), which leads to genotoxic effects by causing oxidative damages to dna. moreover, it has been found 400 – 800 𝜇g of cr(vi) can directly nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 59 interact with bacterial dna causing frameshift mutations and base-pair replacements [36]. the cr(vi) tolerance limits of bacteria have not clearly been defined as it can depend on several factors, including the type of the strain and physio-chemical conditions of the habitat, nature of the waste etc. providing evidence to the above assumption a study of reported that 10 – 12 mg/l of cr(vi) was adequate to inhibit most soil bacteria [22], while some strains in activated sludge can tolerate up to 80 mg/l of cr(vi) [37]. further, they have reported that cr (vi) was able to stimulate bacterial growth up to 25 mg/l of cr(vi). compered to bacteria, fungi are less sensitive to cr(vi) due to the decreased uptake and production of antioxidants [38,39,39–41]. however, some studies describe that, cr(vi) can cause genotoxic and mutagenic effects on several strains of fungi, including saccharomyces cerevisiae, sclerotium rolfsii and pycnoporus sanguineus leading to complex physiological changes and functional changes such as inhibition of oxygen uptake, induction of petite mutations and inducing mitochondrial functional damages [24,42–44]. further studies on fungi have shown that effects of chromium toxicity vary on the nature of carbon substrate [45]. cr(vi) can affect ps ii reaction centers of algae, which leads to inhibition of photosynthesis and cause significant morphological changes in some genera, including chlorella, scenedesmas, ulva, isochrysis, micrasterias, and chlamydomonas [36,46–51]. disposal and remediation process of chromium wastes. the chromium-based industries i.e., electroplating, tanning, water cooling, textile, wood preservation, alloy manufacturing, dye and pigment production discharge large quantities of contaminated chromium containing waste to soil, air and water annually. considerable proportions of used chromium as a raw material and / or a reagent for industries including tannery (40%), chrome plating (35%), academic, research and industry laboratories (100%) discharge cr(iii) and cr(vi) as effluents [52]. these chromium contaminated effluents should be remediated before discharging into the environment, due to toxicity of chromium to the environment and public health. therefore, rules and regulations have been legislated and implemented by national and international authorized bodies such as who, us epa, and national environmental acts of host countries for industrial wastewater and drinking water. according to the us epa and who standards maximum permissible level of cr(vi) in drinking water and industrial wastewater have been legislated to 0.05 mg/l and 0.10 mg/l, respectively. considering the health hazard to the occupational community in chromiumbased industries, occupational safety and health administration (osha) has set the maximum limit for cr(vi) compounds for 8-hour work shifts and 40-hour workweeks as 0.052 mg/l [23,53]. based on these regulations, cr(vi) contaminated wastes should be remediated before being discharged into the environment. remediation of chromium containing waste can be carried out using three (03) methods i.e. chemical, physical and biological which are summarized in the table 2. chemical methods of cr(vi) remediation. chemical reduction and photocatalysis are the most common chemical remediation methods that have been applied in chemical remediation processes. the chemical reduction uses reducing agents such as sulfur dioxide (so2), calcium polysulfide (cas5), ferrous sulfate (feso4), sodium metabisulfite (nahso3), sodium sulfite (na2so3), barium sulfite (baso3), hydrazine hydrate (n2h4), hydrogen peroxide (h2o2) and, calcium carbonate (na2co3) [19,54–56]. redox reactions of above mentioned reducing agents are kinetically slow at low cr(vi) concentrations [57]. therefore, it may require different methods to remediate residual cr(vi), which are even higher than the discharge limits. further, it has been found that this reduction process is also influenced by physical and chemical characteristics of the discharging sites (ph, conductivity, soil type and texture, presence of transition metals) [58,59]. semiconductor based photocatalysis is a developing technology for toxic metal remediation such as cr(vi), hg(ii), as(v), cu(ii), and pb(ii) [62]. this technology is more advantageous as there are no requirements for secondary disposal methods. titania based photocatalysts such as tio2 and la2ti2o7 are extensively used for photocatalytic reduction of cr(vi) in specific values [19,61]. but these titania based photocatalysts cannot be applied practically to mass-scale commercial reactor systems due to high cost and operational disturbances due to sunlight irradiation and highly acidic conditions [62]. physical methods of cr(vi) removal. physical remediation is achieved by techniques such as adsorption, electrolysis, ion exchange, membrane filtration and capping [19,63,64]. adsorption is widely used for chromium removal in wastewater, consisting of nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 60 significant advantages such as low cost, profitability, availability, high efficiency, and minimum effort operation than other physio-chemical methods. a range of synthetic and natural adsorbents, including activated carbon, zeolite, chitosan, treated wastes, biological materials of coconut shell, wood husk, orange peel, hazelnut shell, sawdust, are used for cr(vi) removal with a wide range of removal percentages under different ph values which are mostly laid on the extreme acidic range [65–69]. as some of the above adsorbents are freely available in nature, so that, adsorption is considered as one of the cost-effective methods of physical remediation. membrane filtration technology is implemented with reverse osmosis, which is considered as one of the best available technology for removing all forms of chromium [70–72]. literature shows that different membrane technology modifications to enhance cr(vi) removal effectiveness, including micellar enhanced ultrafiltration, polymer inclusion membranes, ion exchange membranes and nanofiltration [73,74]. however, membrane technology is considered as a costly method with generating a large volume of concentrated liquid toxic wastes [72]. the acidic and basic ion exchange resins have also reported as effective cr(vi) removal methods from chromium contaminated wastewater. a study of [75] have developed a complete cr (vi) removal process from real wastewater by using a strongly basic synthetic dowex 2-x4 resin without affecting ph. further studies of [57] indicate 99.5% of cr(vi) removal from “synthetic wastewater” using solvent impregnated resins which are acidic. biological methods of cr(vi) removal. remediation of chromium contaminated sources using biological agents including bacteria, fungi, algae, and plants play an important role in remediation approaches. bacteria and fungi have shown efficient remediation agents than other agents. it has shown that organisms that can survive in a contaminated site may have the ability to remediate the contaminated site by themselves up to a certain level by transforming toxic pollutants into nontoxic forms [19,76–81]. this detoxification is achieved through biosorption, bioaccumulation and biotransformation. bioremediation is affected by several physio-chemical factors including, energy source (electron donors), electron acceptors, nutrients, ph, temperature and inhibitory substrates or metabolites [82]. bioremediation of chromium is implemented in both in situ and ex situ depending on the nature and requirements of the contaminated site [83,84]. biological methods are considered as more advantageous from the economic and environmental point of view as they are cost-effective due to low installation and operational cost, eco-friendly with generating much less secondary pollutants, convenient and straightforward operation compared to physiochemical methods [63,85– 87]. bioremediation of cr(vi) by bacteria. bacterial bioremediation of cr(vi) is explained in terms of chromium tolerance mechanisms such as biosorption and biotransformation/ bioreduction in both grampositive and gram-negative strains [19]. during the bioreduction, highly toxic cr(vi) is reduced into lesser toxic cr (iii) inside the bacterial cytoplasm, cell wall, or in both. the bacterial strains that can reduce cr (vi) are usually named chromium reducing bacteria (crb). it is believed that gram-positive crb have a significant high tolerance to high cr(vi) concentrations than gramnegative crb [88]. according to previous studies, bacterial genera such as pseudomonas, bacillus, enterobacter, deinococcus, shewanella, agrobacterium, escherichia, thermus, microbacterium, desulfovibrio, deinococcus, brucella, and staphylococcus have the potential to reduce cr(vi) “directly” with enzymes and “indirectly” with metabolic end products [88–92]. it has also been reported that chromium tolerance and reduction are independent properties of bacteria, which means not all cr(vi) resistant bacteria can reduce cr(vi) into cr(iii) [88,93]. bacterial cr(vi) reduction is achieved under aerobic, anaerobic and both conditions [94]. aerobic reduction is associated with soluble proteins and nadh as electron donors to enhance the reduction process, while anaerobic cr(vi) reduction is associated with cell membrane bound reductase (flavin reductase, cytochromase, hydrogenases) and soluble reductase or both [95,96]. bacterial bioreduction rate of cr(vi) is influenced by initial cell density/ concentration, initial chromium concentration, initial ph, temperature, electron donors, oxyanions, salt concentration, presence of other heavy metals, metabolic inhibitors and oxidation-reduction potential of culture [96,97]. further, the bacterial strains in the same species have different cr(vi) tolerance and removal potentials depending on the level of the contaminants in the environment. this phenomena was evidenced in a comparative study carried out between uncontaminated and cr(vi) polluted environments [98]. nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 61 table 2. chemical, physical and biological method of cr(vi) removal method technique mechanism type of contaminated source (tested) cr, cr (vi) removal percentag e time ph temp. (c) reference chemical reduction cr(vi) reduction and adsorption by edrgo. synthetic wastewater 100% 24 hrs. 2.0 n/a [120] reduction cr(vi) reduction by calcium polysulfide (casx). contaminated ground water 90% 4 days 8 12.5 n/a [121] reduction and biosorption cr(vi) reduction and biosorption by chemically treated brown seaweed (ecklonia sp.). synthetic wastewater 100% 12 hrs. 2.0 25 [122] reduction cr(vi) removal by chemically and electrochemically. synthetic wastewater 99.99% 10 min. 8.5 – 10.0 n/a [123] reduction cr(vi) reduction by sodium corboxymethyl stabilized nanoscale zero valent iron. synthetic waste soil sample 80% 72 hrs. 4.73 – 7.36 n/a [124] reduction and coagulation cr(vi) removal by ferrous sulfate (feso4). spiked ground water 95% 46 hrs. > 7.5 n/a [125] physical adsorption chromium removal by fly ash. industrial waste 97.86% 12 hrs. n/a 25 [126] adsorption cr(vi) removal by ragi husk powder. synthetic wastewater 81.34% 2 hrs. 1.75 n/a [127] adsorption cr(vi) removal by green algae and activated carbon. waste water 99.52% 2 hrs. 1.0 25 [110] adsorption cr(vi) removal by treated waste newspaper (twnp). synthetic wastewater 64% 1 hrs. 3.0 25 [65] adsorption cr(vi) removal by green coconut shell. synthetic wastewater 95% 30 min. 6.5 28 [66] adsorption cr(vi) removal by agriculture wastes. maize corncob. cane bagasse. jatrophica oil cake. synthetic wastewater 62% 92% 97% 1 hrs. 2.0 30 [128] adsorption cr(vi) removal by mangifera indica leaves. synthetic wastewater 91% 2 hrs. 2.0 30 [129] retention/ filtration cr(vi) removal by aromatic polymide thin film membrane. synthetic wastewater 77% n/a 8.0 25 [73] adsorption cr(vi) removal by anion exchange resins. synthetic wastewater 99.4% 30 min. 3.0 – 5.0 25 60 [130] adsorption cr(vi) removal by hydrophobic resin. synthetic wastewater 99.5% 92% 24 hrs. 3.0 25 [131] adsorption cr(vi) removal by boiled rice husk. synthetic wastewater 71% 3 hrs. 2.0 27 [132] adsorption cr(vi) removal by formaldehyde treated rice husk. synthetic wastewater 76.5% 3 hrs. 2.0 27 [132] nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 62 physical adsorption cr(vi) removal by modified montmorillonite clay nanocomposite. synthetic wastewater 99.9% 24 hrs. 2.0 6.6 25 [133] adsorption cr(vi) removal by fe2o3/ graphene adsorbents. synthetic wastewater 70.33% n/a 3 4 25 [134] adsorption cr(vi) removal by synthesized hydroxyapatite microfibrillated cellulose (cha/mfc) synthetic wastewater 94% 5 min. 7 5 25 [135] adsorption cr(vi) removal by magnetite nanoparticles synthetic wastewater 66% 2 hrs. 3.0 25 [136] adsorption cr(vi) removal by mixed waste tea and coffee ground synthetic wastewater 95% 3 hrs. 2.0 50 65 [137] adsorption cr(vi) removal by natural adsorbents. wool olive cake sawdust pine needles almond coal cactus synthetic wastewater 69.3% 47.1% 53.5% 42.9% 23.5% 23.6% 19.8% 2 hrs. 2.0 30 [138] adsorption polypyrrole – montmorillonite clay composite synthetic wastewater 100% 24 hrs. 2.0 25 [139] adsorption nanocomposite of zno with cotton stalks biochar synthetic wastewater 96.19% 1 hr. 2-4 25 [140] filtration green emulsion liquid membrane synthetic wastewater 97-99% 0.5 hrs. 0.45 30 [71] filtration green synthesized cuo nanoparticles synthetic wastewater 88.08% 2 hrs. 6.9 25 [70] biological reduction cr (vi) bioreduction by effluent bacteria staphylococcus cohnii synthetic wastewater 90% 96 hrs. 7.2 37 [141] reduction cr (vi) bioreduction by pseudomonas umsongensis synthetic wastewater 93.9% 72 hrs. 7.0 30 [142] reduction adsorption cr (vi) bioreduction and biosorption by bacillus sp. synthetic wastewater 97.04% 96 hrs. 7.0 37 [143] reduction cr (vi) bioreduction by aeromonas hydrophila synthetic wastewater 88% 72 hrs. 7.2 30 [144] reduction cr(vi) reduction by bacillus thuringiensis synthetic wastewater 86.42% 96 hrs. 7.0 35 [91] reduction cr(vi) reduction by staphylococcus capitis synthetic wastewater 97.34% 96 hrs. 7.0 35 [91] reduction cr(vi) reduction by bacillus cereus synthetic wastewater 98.5% 72 hrs. 7.1 26 [98] nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 63 reduction sorption cr(vi) reduction and sorption by enterobacter sp. synthetic wastewater 99.1% 25 hrs. 6.0 45 [145] reduction cr(vi) reduction by morganella morganii synthetic wastewater raw tannery effluent 92% 90% 48 hrs. 48 hrs. 7.0 7.0 37 37 [146] reduction sorption cr(vi) reduction and sorption by stenotrophomonas rhizophila synthetic wastewater 100% 28 hrs. 7.5 30 [147] reduction cr(vi) reduction by cellulosimicrobium sp. synthetic wastewater 100% 48 hrs. 7.0 30 [148] reduction cr(vi) reduction by geobacter sulfurreducens synthetic wastewater 99% 2hrs. n/a 30 [149] reduction cr(vi) reduction by pseudomonas aeruginosa synthetic wastewater 93% 96 hrs. 7-8 30 [150] sorption cr(vi) biosorption by shewanella putrefaciens synthetic wastewater 85.68% 17 hrs. 8.0 38.44 [151] reduction cr(vi) bioreduction by mixed bacterial consortium. synthetic wastewater 100% 120 hrs. 8.0 30 [152] reduction adsorption cr(vi) bioreduction and biodorption by corynebacterium paurometabolum, synthetic wastewater 55% 2 hrs. 3.0 30 [153] reduction cr(vi) bioreduction by cellulosimicrobium funkei synthetic wastewater 80.43% 120 hrs. 7.0 35 [154] biological reduction cr(vi) bioreduction by pseudomonas stutzeri synthetic wastewater 97% 24 hrs. 7.0 37 [155] reduction cr(vi) bioreduction by acinetobacter baumannii synthetic wastewater 99.58% 24 hrs. 8.0 37 [155] reduction cr(vi) bioreduction by ochrobactrum sp. synthetic wastewater 96.5% n/a 7.0 30 [156] adsorption cr(vi) biosorption by trichoderma sp. synthetic wastewater 97.39% 2 hrs. 5.5 25 [103] reduction adsorption cr(vi) biosorption and bioreduction by paecilomyces lilacinus synthetic wastewater 100% 120 hrs. 5.5 25 [157] adsorption cr(vi) biosorption by phanerochaete chrysosporium synthetic wastewater 99.7% 72 hrs. 7.0 40 [158] adsorption cr(vi) biosorption by pleurotus ostreatus synthetic wastewater 80% 12 hrs. 2.0 – 11.0 65 [159] adsorption cr(vi) adsorption by cationic surfactantmodified, kazachstania yasuniensis kodamaea transpacifica saturnispora quitensis saccharomyces cerevisiae synthetic wastewater 80.70% 85.80% 85.40% 75.80% 4 hrs. 4.5 25 [105] nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 64 biological adsorption cr(vi) adsorption by a hydroxylfunctionalized magnetic aspergillus niger nanocomposite synthetic wastewater 64.91% 4 hrs. 5.0 50 [160] adsorption cr (vi) adsorption by chlorella sorokiniana. synthetic waste water 99.68% 72 hrs. 7.0 40 [161] reduction adsorption cr (vi) removal by green algal strain cladophora albida synthetic waste water industrial waste water 100% 120 hrs. 0.5 25 [162] adsorption cr(vi) removal by marine algae turbinaria ornata synthetic wastewater 95.25% 3.5 hrs. 4.7 33.6 [163] n/a – not applicable table 3. microorganisms and substrates used in biofilm formation for bioremediation of cr(vi) organism adhesion substrate cr (vi) removal percentage (%) ph temp. (℃) time reference arthrobacter viscosus granular activated carbon 99.9% 5 – 5.5 28 30 days [190] pseudomonas sp. bacillus sp. azotobacter sp. acremonium sp. glass wool 90% 5.6-6.1 30 10 days [172] streptomyces strain cg252 glass bead 100% n/a 30 48 – 72 hrs. [191] arthrobacter sp. gravel packed bed reactors 100% n/a 30 26 hrs. [192] morganella morganii stb5 polystyrene polysulfone 99.47% 90.78% 7.0 30 72 hrs. [193] arthrobacter sp. suk 1205 glass beads 100% 7.0 37 96 hrs. [194] halomonas sp. pumic particle stones 94.5% 6.5 28 48 hrs. [195] bacillus subtilis escherichia coil acinetobacter junii alginate bead 97.84% 7.0 25 7 hrs. [196] wickerhamomyces anomalus wood husk 92.5% 3.72 30 n/a [171] acinetobacter haemolyticus wood husk 97% 7.0 25 72 hrs. [69] streptococcus salivarius stainless steel aisi 316l 42% n/a 37 72 hrs. [197] pseudomonas fluorescens lb 300 glass beads 100% 6.8-7.0 30 8 days [198] cellulosimicrobium sp. pvc rubber tubing sand small stone 99.5% 90.0% 96% 88.4% n/a 25 11 days [199] escherichia coli kaolin 100% 4.6-5.1 37 10 days [200] nostoc sp. polystyrene 86.49% 7.0 25 7 days [201] shewanella xiamenensis zeolite 100% 3.0 22-25 35 days [202] cunninghamella elegans stainless steel compression springs 98.6% 7.0-3.0 28 40 hrs. [203] arthrobacter sp. suk 1201 glass beads 100% 7.0 37 3 days [204] lysinibacillus sphaericus rta-01 glass slide 82.8% 5.2 37 72 hrs. [205] ochrobactrum pseudintermedium adv31 polyurethane foam 82% 7.0 45 5 days [206] n/a – not applicable nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 65 bioremediation of cr(vi) by fungi. similar to the bacterial remediation of toxic metals, some fungal strains have been investigated for bioremediation with the same metal removal techniques used with bacteria, i.e. biosorption, bioaccumulation and biotransformation/ bioreduction. fungi aspergillus sp. was reported to remove chromium through bioreduction from contaminated effluents [99]. this study also indicates 65% of chromium removal from tannery effluent and 85% of cr(vi) removal from the synthetic medium at ph 6 within 07 days. the similar bioreduction of cr(vi) has been also reported by [19,100– 102] with hypocrea tawa, trichoderma inhamatum and isolated yeast strains. however, the cr(vi) reduction capability of fungal strains can be changed with the initial cr(vi) concentration and initial biomass of the strain. fusarium oxysporum and trichoderma sp. have shown to adsorb cr(vi) on to their cell surface by forming chemical bonds with cell surface proteins with analytically verified evidence using ft-ir spectrum [19,103]. comparison of cr(vi) removal in synthetic and raw wastes was found to be 77% and 85% of cr(vi) removal respectively, with 200-1000 mg/l of initial cr(vi) concentrations using immobilized baker’s yeast strain (saccharomyces cerevisiae) in biomass/polymer matrices beads (bpmm) through biosorption [104]. a study comparing cr(vi) biosorption by native ecuadorian yeast species, reported that kazachstania yasuniensis, kodamaea transpacifica, and saturnispora quitensis have the ability to remove cr(vi). furthermore, they have reported that efficient cr(vi) removal can be achieved by inducing belzalkonium chloride (bzk) to cell surface as a chemical modification to the applying bio agent [105]. bioremediation of cr(vi) by algae. it is evident that both freshwater and marine algal species such as cladophora sp., selenastrum sp., spirogyra sp., ceramium sp, chlorella sp. and ulva sp. can be used to remediate chromium contaminated wastewater by applying as cultures or incorporating with other physiochemical methods following biosorption and bioreduction [19,106–109]. unlike other organisms, algae have been used in both living and non-living forms for cr(vi) remediation. introducing an efficient cr(vi) removal method [110] reports that dried ulva lactuca incorporated into activated carbon can be used to remediate highly acidic and halophilic wastewater. chlorella sp. has been used in most cr(vi) algal remediation bioreactors as it is widely dispersed in the aquatic environment with higher cr(vi) tolerance [109,111–114]. constructing a hybrid remediation system [115] has introduced efficient and reusable alumina hollow fibers immobilized with tio2 and chlorella vulgaris cells. additionally, this hybrid system has been achieved greater than 90% of cr(vi) removal after five sequential reuses. however, similar to bacterial bioremediation, algal cr(vi) bioremediation is influenced by physio-chemical parameters including ph, temperature, initial biomass, initial cr(vi) concentration, light intensity, contact time of cells and wastes of the treatment process bioremediation [19,116]. bioremediation of cr(vi) by plants. limited studies have reported that the cr(vi) detoxification and removal potential of plants compared to the other biological agents. green plants detoxify many pollutants using various mechanisms followed by uptake, known as phytoremediation [117]. plants can either store heavy metals in roots or partially translocate to shoot through the xylem after getting diffused into the root system. further, it has been reported that upward translocation of heavy metals is retarded by the cation exchange process in plant tissues and leads to considerable heavy metal accumulation in roots compared to axial parts of plants. in the point of view of cr(vi) and other chromium forms, this phenomenon has been reported else ware using phragmitus australis, ailantus altissima and salix viminalis [118]. this may be due to the encapsulation in vacuoles of root cells based on natural counteraction of plants against chromium toxicity [119]. according to observations over 360 days, a study suggests that salix viminalis used for large scale phytoremediation application for removal of cr(vi) and other chromium forms from contaminated sources as s.viminalis were removed 70% of total chromium and 90% of cr(vi) removal with indicating higher translocation capacity [118]. in vitro study of nopalea cochenillifera found that it has the potential to accumulate a wide range of cr(vi) (600 – 26,000 mg/ kg) from the growth medium. as n. cochenillifera is a non-consuming plant for diets, the risk of bioaccumulation can be avoided in the ecosystem. furthermore, the above study has also reported plant species with different chromium accumulation potentials including gynura pseudochina, brassica napus, prospis juliflora, leersia hexandra, urtica dioica, salix matsudana, brassica napus, helianthus annuus, lycopersicon lycopersicum and saponaria officinalis perhaps considered for the phytoremediation [117]. nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 66 biofilms an aggregated community of prokaryotic and eukaryotic microorganisms adhering to substance/matrix surface and submerged/embedded in self-produced extracellular polymeric substances (eps) is termed a "biofilm" [164,165]. these aggregates are omnipresent in the biosphere, including soil, water, plant and animal tissues, abiotic substances like pipelines, ship hulls and filters. these biofilms can be developed in solid–water, water–air and solid–air interfaces with composing eps, multivalent cations, biogenic particles, colloidal and dissolved compounds [166]. biofilms are comprised of both single and multiple microbial species. among them, multiple species biofilms are the most dispersed biofilm type in the environment [167]. biofilm formation is a subsequent process that consists of 03 main steps; surface attachment, biofilm maturation and dispersal [165]. in surface attachment, microbial cells undergo reversible attachment at cell poles by involving cell appendages (flagella, pilli and fimbriae) followed by irreversible attachment. after the reversible attachment stage, microbial cells can be adapted to biofilm lifestyle or left the matrix. during the irreversible attachment, stage cells adhere to the matrix by eps and surface proteins (sad b and lap a). biofilm maturation starts after the irreversible attachment with developing microcolonies. at this stage, previous microbial cells are assembled and proliferated along with producing eps. further studies explain that biofilm structure, including thickness and cell density, is dynamically changed according to environmental conditions such as temperature, presence of oxygen, ph and amounts of nutrients [164,165]. the immobilized microbial cells are transferred back to planktonic growth as the final stage of the biofilm lifecycle and as an initial step of a naval forming biofilm. this dispersal can be happened “actively” by cell motility and eps degradation or “passively” by external physical forces. environmental applications of biofilms even though free-living microorganisms are capable of bioremediating polluted environments, the remediation process can be disrupted due to high concentrated toxic compounds, availability of nutrients and environmental stress. applying sessile or floating biofilms for remediation is highly advantageous as biofilm communities have higher tolerance towards environmental stress, including lack of nutrient availability, high concentrated chemical exposures, ph and temperature fluctuations, lack of moisture content than free-living microorganisms [168]. when selecting biofilms for remediation purposes, several factors must be considered: the capability to tolerate environmental stress, exchange of genetic materials, growth rates, metabolic diversity, and symbiotic relationships. based on above factors bacterial, algal and fungal biofilms are used in bioreactors to remediate contaminated sources by a wide range of pollutants including, organic pollutants (polyaromatic hydrocarbons, chlorinated aromatic compounds, aromatic amine compounds, polyethylene and polythene), heavy metals (cu, zn, cd, ni, as, fe, hg, mn), inorganic pollutants (nitrate ions and synthetic dyes) contaminates which can adversely affect the ecosystems [168–170]. cr(vi) remediation by biofilms cr(vi) bioremediation is achieved using bacterial, algal and fungal single species and multi-species biofilms growing on either natural or artificial substrates through bioremediation and biosorption techniques [171–174]. biofilms are more effective for cr(vi) remediation than planktonic cells. the study investigating streptomyces sp. strain cg252 and pseudomonas aeruginosa a2chr, respectively, reported evidence for the above phenomena [102,175]. another study using three (03) different biosorbents including lyophilized escherichia coli aus 7 cells, granulated activated carbon (gac) and biofilm of escherichia coli aus 7 on gac exhibited that biofilms are able to achieve a higher adsorption of cr(vi) than gac and lyophilized cells in according to langmuir and freundlich isotherm models from aqueous solutions under acidic conditions [176]. however, contradictory results were also reported else ware, that the planktonic cells have more significant potential for cr(vi) reduction than biofilms based on their study of bacillus subtilis atcc-6633. furthermore above study revealed that, biofilm debris are susceptible to immobilized reduced cr(iii) ions completely [177]. compared to bacteria, reports on cr(vi) remediation by fungal biofilms are scarce in the literature. immobilized cells of aspergillus niger, coriolus versicolor, saccharomyces cerevisiae, and lentinus sajorcaju have been used in sorption and reduction techniques [99,178–180]. moreover, immobilized algal cells on different matrixes have been used for cr(vi) remediation by sorption of metal ions to the cell wall components [181–185]. algal-bacterial biofilms/consortia have also been used for cr(vi) bioremediation. further, it is reported that these consortia have symbiotic effects on each other by nepal j biotechnol. 2 0 2 2 d e c ; 1 0 : 57-76 aththanayake et al. ©njb, bsn 67 supplying each other’s nutritional needs such as o2 for aerobic bacteria by algae and co2 for algae by bacteria during the remediation process. therefore, algalbacterial consortia are termed as a self-sustaining system [186–188]. the algal-bacterial system has the potential to remove higher cr(vi) contents such as 100 mg/l, 75 mg/l and 50 mg/l providing a carbon source to the mixed consortium of chromium reducing bacterial (crb) cultures of escherichia coli, bacillus thermoamylovorans and citrobacter sedakii from the algal strain of chlamdomonas reinhartii. furthermore, the above study suggests that the algal-biofilm consortia as a cost-effective method that prevents cost of carbon sources as it fulfils by algae even though algal-bacterial consortia takes a longer time duration for the cr(vi) removal [189]. table 3 illustrates some of the microorganisms and substrates used in bioremediation of cr(vi). limitations and remedial actions of the current bioremediation methods in cr(vi) removal the notable limitations of biological methods available for cr(vi) removal have been identified including, varying cr(vi) tolerance and removal levels environmental conditions and nutritional requirements of biological components used, toxic substances present in wastewater which can interfere with the biological components, disposal of the accumulated cr in the biological component, and practical difficulties in extrapolating bench/pilot-scale to full-scale field application [207–209]. customized solutions need to be sought by assessing the remediation requirements at individual level, because the environmental conditions and the nutritional requirements of the biological component vary depending on the contaminated site. moreover, to overcome the cr disposal after bioremediation, it is possible to percolate the biotransformed cr(iii) through reduction at low ph conditions and tend to be reoxidised to cr(vi) in the presence of manganese oxide and chlorine in treated effluent or discharging environment [210,211]. in order to prevent the discharge of higher amounts of chromium and to enhance the sorption capacity of biomasses, the metal desorption process should be followed. this desorption can be done by acid digestions [212–215] and alkaline treatments [216–220] as a hybrid cr(vi) remediation process, which has many benefits such as reduction of generation of secondary pollutants and recovery of valuable metals. these recovered cr(vi) and cr(iii) can be applied for tannery and chromium-based chemical production as raw materials [221]. conclusion the wide industrial and research application of chromium followed by emitting considerable amounts of cr(vi), coupled with the fact that it leads to serious problems to all components of the ecosystem. therefore, it has been legislated to remediate cr(vi) contaminated effluents by national and international authorities before it being discharged to the environment. this remediation is carried out by chemical, physical and biological methods. biological remediation is considered as the most environment-friendly and cost-effective method rather than chemical and physical remediation. however, considering the limitations of the current bioremediation processes, hybrid remediation processes combining the bioremediation with other chemical and physical methods are being used for the effective remediation of cr(vi) in aquatic systems. authors 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study on cr(iii) removal from aqueous solution using bacillus subtilis biomass. clean technol environ policy [internet]. 2012 aug [cited 2021 jul 19];14(4):727–35. available from: http://link.springer.com/10.1007/s10098-011-0440-7 https://linkinghub.elsevier.com/retrieve/pii/s0304389420302454 https://linkinghub.elsevier.com/retrieve/pii/s0304389420302454 https://linkinghub.elsevier.com/retrieve/pii/s0960852419312945 https://linkinghub.elsevier.com/retrieve/pii/s0960852419312945 http://link.springer.com/10.1007/s10098-013-0636-0 https://linkinghub.elsevier.com/retrieve/pii/s221334371630238x https://linkinghub.elsevier.com/retrieve/pii/s221334371630238x https://linkinghub.elsevier.com/retrieve/pii/s221334371630238x http://link.springer.com/10.1007/s10098-011-0440-7 http://link.springer.com/10.1007/s10098-011-0440-7 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 16-19 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 16 nepjol.info/index.php/njb cyclospora cayetanensis: an infestation among diarrheal children in kathmandu valley, nepal balkrishna bhattachan1, 2*, ganesh rai1, nabin narayan munankarmi 3, jeevan bahadur sherchand2 1shi-gan international college of science and technology (sicost), kathmandu, nepal 2department of microbiology of tribhuvan university teaching hospital, kathmandu, nepal 3biotechnology society nepal (bsn) kausaltar-3, bhaktapur, nepal abstract cyclospora cayetanensis, a coccidian parasites which is responsible for recurring diarrhea and gastroenteritis especially among children living under poor hygienic condition in developing country like nepal. aimed of this research is to find out the prevalence of intestinal parasites and c. cayetanensis among diarrheal children in a pediatric hospital in kathmandu valley, nepal. cross-sectional type of study was done. altogether 196 stool specimens were collected from june to september 2013 among outpatient diarrheal children in kanti children’s hospital. modified ziehl neelson staining method was applied for detection of oocysts of cyclospora after formal-ether sedimentation. parasites were detected in 13.7% (27/196) of stool samples from ≤ 15 year old diarrheal patients. c. cayetanensis was detected in 4.8% (8/196). in genderwise, infection rate of c. cayetanensis 4.5 % (5/112), in male were higher than 3.6% (3/84) in female. in agewise, infection rate of c. cayetanensis in 5.7 % (3/52) 11-15 year old were highest followed by 4.1% (3/78) in 0-5 year old and 3.0% (2/66) in 6-10 year old. in seasonwise, infection rate of cyclospora was highest in the month of august 7.4% (4/54) followed by 3.9% (2/51) in july, 2.3% (1/44) september and 2.1% (1/47) in june. altogether five different type of parasites were detected. infection rate of giardia lamblia were highest 5.1 % (10/196) whereas lowest was cryptosporidium parvum 1.0 % (2/196). prevalence of c. cayetanensis is highly probable to infant, neonate, toddler and diarrheal children. therefore, attention should be made in laboratory investigation of c. cayetanensis while suspecting the diarrheal patients infected with other parasites. keywords: cyclospora, parasites, modified ziehl neelson, children *corresponding author email: balkrishna_bhattachan@hotmail.com introduction cyclospora cayetanensis, is a coccidian parasite which is responsible for prolonged diarrhea in both immune-competent and immunecompromised patients. the pathogenesis and virulence factors phenomenon of c. cayetanensis are yet to be defined, but inflammation and jejunitis had been found out [1]. c. cayetanensis infect human and cause of acute and chronic gastroenteritis [2]. it has been reported from various parts of the world in southeast asia [3,4]. the important vehicle for infection is source of water either by ingestion of parasite through contaminated water directly or through contaminated vegetable. moreover, it has been implicated in outbreaks developing countries (e.g. like nepal) [5]. in the study of sherchand and cross, between 2001-2004 in nepal showed that contaminated drinking water, raw green vegetables, infected animals were the possible sources of infection with cyclospora [6]. the patients suffering from cyclosporisis show the symptoms of an abdominal cramps, diarrhea, fatigue, nausea, and vomiting, chronic watery, anorexia, and weight loss. however, the cause of disease is still unrevealed. this study was carried out at kanti children’s hosptial (kch), kathmandu valley, aiming to find out the prevalence of parasites and c. cayetanensis especially diarrheal children. material and methods study site: cross-sectional type of study was undertaken at kch in kathmandu valley from june to september 2013. collection of data and samples: demographic data were collected by using a standard questionnaire and with proper instruction ≤ 15 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 16-19 bhattachan et al. ©njb, biotechnology society of nepal 17 nepjol.info/index.php/njb year old outpatient diarrheal children were requested for collection of stool sample. for the nearly 30 ml of fresh stool) a clean, dry and screw capped container were given. during collection of samples were kept in icebox by maintaining cold chain before transportation to public health research laboratory, institute of medicine, and kathmandu, nepal. microscopic examination formal-ether sedimentation technique: three ml of stool samples was taken in test-tube and shaken well then filtered through cotton gauge. after that, 3 ml of ether was added and shaken well then centrifuged. the sediment portion was examined by microscopy after addition of saline solution. microscopic detection of parasites cyst were carried out under 10x followed by 40x objectives [7]. modified acid fast staining method: the smears were flooded with carbol fuchsin and heated to steaming. it was shaken with tap water, decolorized with 5% aqueous sulfuric acid, and shake again. flooded with methylene blue counterstain was added on smear formed stain, then rinsed with tap water. smear was drained, and air dried [8]. oocysts of c. cayetanensis were identified having size (8-10 µm), circle shape and pink color. ethics: written informed consent was taken from all participants and/or their parents before enrolling into the study. institutional review board, institute of medicine, research department, kathmandu, nepal was approved this research. win-pepi statistical software was used for data calculation where p value of data <0.05 considered as statistically significant. results parasites were detected in 13.7% (27/196) of stool samples from ≤ 15 year diarrheal patients. c. cayetanensis was detected in 4.8% (8/196). infection rate of c. cayetanensis in 5.7 % (3/52) 1115 years old were highest followed by 4.1% (3/78) in 0-5 years old and 3.0% (2/66) in 6-10 years old [table 1]. in gender, ratio between male and female were 1.3:1, infection rate of c. cayetanensis 4.5 % (5/112) in male were higher than c. cayetanensis 3.6% (3/84) in female [table 2]. table 1. distribution of age and intestinal parasites among diarrheal children from outpatients in kanti children’s hospital characteristics total no. of children any parasite detected p-value c. cayetanensis pvalue no no.(%) no. (%) age (year) 0-5 78 11 (14.1) 0.902 3 (4.1) 0.768 6-10 66 8 (12.1) 2 (3.0) 11-15 52 8 (15.4) 3 (5.7) total 196 27(13.8) 8 (4.1) table 2. distribution of sex and intestinal parasites among diarrheal children from outpatients in kanti children’s hospital characteristics total no. of children any parasite detected p-value c. cayetanensis pvalue no no.(%) no. (%) gender male 112 16 (31.3) 0.835 5 (4.5) 0.764 female 84 11 (13.1) 3 (3.6) total 196 27 (13.8) 8 (4.1) table 4: number of positive and percentage rate of intestinal parasites among diarrheal children were shown in table 4. name of parasites number (%) giardia lamblia 10 (5.1) cyclospora cayetanensis 8 (4.8) entamoeba histolytica 4(2.5) entamoeba coli 3(1.5) cryptosporidium parvum 2(1.0) *none of multiparasites was detected. table 3: monthwisedistribution of cyclospora cayetanensis and total parasites in proportion in stool samples cyclospora cayetanensis (%) total parasites (%) month june 2.1 12.9 july 3.9 9.8 august 7.4 18.8 september 2.3 15 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 16-19 bhattachan et al. ©njb, biotechnology society of nepal 18 nepjol.info/index.php/njb in seasonwiser, infection rate of c. cayetanensis was highest in the month of august 7.4% (4/54) followed by 3.9% (2/51) in july, 2.3% (1/44) september and 2.1% (1/47) in june [figure 1]. altogether 6 different types of parasites were detected. infection rate of giardia lamblia were highest 5.1 % (10/196) whereas lowest was cryptosporidium parvum 1.0 % (2/196) [figure 2]. photograph 1: oocyst of cyclospora cayetanensis (ziehl neelsen, 100x) discussion prevalence of c. cayetanensis infections among ≤15 year outpatient diarrheal children in kch, kathmandu, nepal was conducted in this study. intestinal parasitosis is detected to be highly prevalent in nepal, small developing country located in south asia [9]. infection rate of c. cayetanensis and other parasites was higher between 1115 years old in diarrheal children. altogether five species of parasites were detected in 13.7% of stool samples from ≤ 15 year old diarrheal patients. among them, most of prevalence in g. lamblia was detected. in nepal, c. cayetanensis was detected in 4.8% of patients. in previous study, 7.9 % were found to be positive for c. cayetanensis in diarrheal children in nepal [10]. in this study, we could not detect significant differences in prevalence of c. cayetanensis among < 15 year age groups and in gender. in gender, prevalence of c. cayetanensis 4.5% in male was higher than in female 3.6%. in previous study, prevalence of c. cayetanensis in female (53.4%) was higher than male (46.6%) in nepal [10]. total of the 60 patients infected with cyclospora identified in this study, 63.4% were male and 36.6% were female in mexico [11]. in age, prevalence of c. cayetanensis in 5.7 % 1115 years were highest. in previous study, the most of the infection rate of c. cayetanensis infection (50.7 %) was found between 2-5 year old of age group whereas the lowest prevalence (4.1%) were above 28 years of age in nepal [10]. cyclospora was most frequently identified in boys of school age (36.7%) in mexico [11]. the highest attack rates occur among children older than 18 months where as in our study, however, all age groups may acquire the disease, the highest attack rates was detected among children age between 2 to 5 years [12, 13]. there is no apparent immunity to infection, and reinfection can occur at all ages [14, 15]. prevalence of c. cayetanensis was most in the month of august. the higher distribution of cyclospora infection in nepal occurs during summer and rainy season [10]. their infections were relevantly identified in the rainy season (june–august) in children of mexico [11]. cyclosporiasis occurs with high incidence during the rainy seasons from april to june in peru and may to september in nepal [1, 2]. in kanti children’s hospital, c. cayetanensis infection with diarrheal illness patients is not routinely diagnosed. thus, adequate diagnosis and treatment are not always conducted promptly. in addition, cyclospora makes it difficult to include its differential diagnosis due to lack of epidemiologic information. therefore, our study suggests that there is need of highly specific assays to diagnose cyclospora infections. conclusions prevalence of c. cayetanensis is highly probable in infant, neonate, toddler, and children and is one of the important etiologic agents of diarrheal illness among children. it is also equally probable infection in male and female as well as in rainy season. therefore, attention should be made for laboratory investigation of c. cayetanensis while suspecting the diarrheal patients infected with other parasites. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 16-19 bhattachan et al. ©njb, biotechnology society of nepal 19 nepjol.info/index.php/njb author contributions bb and gr design the proposal and format of research. bb collects the stool sample from hospital and proceeding in lab of tuth. jbs and bb and nnm write the article. all authors revised and finalized the draft. acknowledgements the authors would express sincere thanks to mrs. sarmila tandukar mr. kalyan subedi, ms. anisha shrestha. we are indebted to hospital’s staffs and guardian of children who support continually. conflict of interest: none declared references 1. ortega yr, roxas cr, gilman rh, miller nj, cabrera l, taquiri c, sterling cr: isolation of cryptosporidium parvum and cyclospora cayetanensis from vegetables collected in markets of an endemic region in peru. american journal of tropical medicine and hygiene. 1997 57:683-686. 2. ortega yr., sterling cr, gilman rh, cama va, diaz f.: cyclospora species—a new protozoan pathogen of humans. n engl j med. 1993 328:1308–1312. 3. soave r: cyclospora: an overview. clin infect dis. 1996,23: 329-378. 4. hart as, ridinger mt, soundarajan r, peters cs, swiatlo al, kocka fe: novel organism associated with diarrhoea in aids. lancet. 1990 335: 69. 5. rabold jg, hoge cw, shlim dr: cyclospora outbreak associated with chlorinated drinking water (letter). lancet. 1994 344: 13601361. 6. sherchand jb, cross jh, jimba m, sherchand s, shrestha mp: study of cyclospora cayetanensis in health care facilities, sewage water and green leafy vegetables in nepal. southeast asian journal trop med public health. 1999; 30: 58-63 7. bhattachan b, panta yb, tiwari s, et al: intestinal parasitic infection among school children in chitwan district of nepal. j inst med. 2015 37(2): 79-84. 8. garcia ls, bruckner da, brewer tc, et al: techniques for the recovery and identification of cryptosporidium oocysts from stool specimens. j clin microbiol. 1983; 18:185-190. 9. rai sk, hirai k, abe a, et al: intestinal parasitosis among school children in a rural hilly area of dhading district, nepal. nepal med coll j. 2002 4: 54-58 10. sherchand jb, sherchand jb, cross jh. an epidemiological study of cyclospora cayetanensis in nepalese people. j inst med. 2007 29 (1) :8–13. 11. orozco-mosqueda ge, martı´nez-loya oa, and ortega yr: cyclospora cayetanensis in a pediatric hospital in morelia, mexico. am j trop med hyg. 2014 91(3): 537–540, doi:10.4269/ajtmh.13-0535. 12. hoge cw, shlim dr, ghimire m, rabold jg, pandey p, walch a rajah jg, guadio p & echeverria p: placebo controlled trial of cotrimoxazole for cyclospora infection among travelers and foreign residents in nepal. lancet. 1995, 345: 691-693. 13. schubach tm, neves es, leite ac, araujo aqc & de mouta h: cyclospora cayetaensis in an asymptomatic patient infected with hiv and htlv1. transaction of the royal society of tropical medicine. 1997, 91: 175. 14. connor ba & shlim dr: food borne transmission of cyclospora. lancet. 1995 346: 1634. 15. caramello p, brancale t, forno b, lucchini a, macor a, mazzucco g, tettoni c & ullio a: clinical and diagnostic aspects of travelers diarrhea due to cyclospora organisms. journal of travel medicine. 1995 2: 232-234. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 issn 2091-1130(print)/issn 2467-9319 (online) case study ©njb, biotechnology society of nepal 54 nepjol.info/index.php/njb pattern of cancer in nepal from 2003 to 2011 sunil kumar sah1, naval kishor yadav2, roshan kurmi3, ramanuj rauniyar3, krishna das manandhar4, birendra prasad gupta4* 1department of pathology, bp koirala memorial cancer hospital , bharatpur, chitwan nepal 2department of biochemistry, manipal medical college, nepal 3bhawani hospital, birgunj, parsa, nepal 4central department of biotechnology, tribhuvan university, kirtipur, kathmandu, nepal abstract cancer is global burden of disease in developed and developing countries. it is one of the main causes of death. the environmental factor and life styles are major causes of cancer. this hospital based retrospective study was carried out using data retrieved from the register maintained at seven cancer centers. the most common basis of diagnosis were microscopic (histopathological and cytopathological examination). the diagnosis was also based on clinical examination, radiological examination, endoscopy, biochemical and immunological tests. most of the cancer cases were diagnosed at bpkmch (23908) followed by bpkihs (9668) and bh (5959) and few cases from kch (518) in 2003 to 2011. the total number of cancer cases were increasing from 2003 to 2011 and it become double in 2011.. out of 75 district of nepal, more number of cancer cases was found in kathmandu, sunsari, morang, chitwan, lalitpur, jhapa, kaski, nawalparasi, rupendehi and kavrepalchowk in 2010. similarly, in 2011 more number of cancer cases was found in kathmandu, morang, jhapa, sunsari, chitwan, lalitpur, rupendehi, kaski, saptari, bhaktapur. lung cancer was the common cancer and similarly, other prevalent cancers were cervical, breast, stomach, ovarian and colo-rectum cancer in 2003 to 2011. the common cancers were lung, cervical, breast, stomach, ovarian and colo-rectum. the number of patients is increasing, which may be due to change in life style and lack of education. key words: cancer, nepal, 2003-2011, kathmandu, *corresponding author email:birendraphd@gmail.com introduction cancer is characterized by uncontrolled growth and spread of abnormal cells with multi-factorial etiology [1]. it is one of the most dreaded noncommunicable diseases that have become the important contributor to the global burden of disease [2]. the causes of cancer is not only genetic factor, which contribute 5-10%, while environmental factor and life styles cover 90-95% [3,4]. the lifestyle factors include cigarette smoking, diet (fried foods, red meat), alcohol, sun exposure, environmental pollutants, infections, stress, obesity, and physical inactivity [5,6] in 2012, new cancer cases were 14.1 million and with 8.2 million deaths. the lung cancer was the most common cancer 16.7% of all new cases in men and breast cancer 25.2% in women [7]. according to estimates from the international agency for research on cancer (iarc), the global burden is expected to grow to 21.4 million new cancer cases and 13.2 million cancer deaths by 2030. [8] in developed countries, most common diagnosed cancers were prostate, lung and bronchus, and colorectal among men while breast, colorectal, and lung among women. similarly, lung, stomach, and liver cancer in men while breast, cervix uteri, and lung in women in developing countries [9]. nepal is a developing country where cancer cases are increasing day by day. the aim of this study is to see the pattern of cancer cases from 2003 to 2011 in nepalese people. materials and methods this hospital based retrospective study was carried out using data retrieved from the register maintained at seven cancer centers of nepal from 2003 to 2011. the cancer centers were b.p. koirala memorial cancer hospital (bpkmch) bharatpur, chitwan, bhaktapur cancer hospital (bch) bhaktpur, bir hospital (bh) kathmandu, tu teaching hospital (tuth) kathmandu, kanti children’s hospital (kch) kathmandu, b.p.koirala institute of health sciences (bpkihs) dharan and manipal teaching hospital (mth), pokhara, nepal. the most common basis of diagnosis were microscopic (histopathological and cytopathological examination). the diagnosis was also based on clinical examination, radiological nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 kurmi et al. ©njb, biotechnology society of nepal 55 nepjol.info/index.php/njb examination, endoscopy, biochemical and immunological tests. the collected variables are age, sex, occupation, religion, ethnicity, region of residence and type of cancer. the data were analysed using excel 2007. results most of the cancer cases were diagnosed at bpkmch (23908) followed by bpkihs (9668) and bh (5959) and few cases from kch (518) in 2003 to 2011 (figure 1). figure 1: the number of total cancer between 2003 to 2011 from seven cancer reporting institute of nepal. the total number of cancer cases were increasing from 2003 to 2011 and it became double in 2011 (figure 2). figure 2: this data represents total number of cancer cases and sex wise distribution of cancer from 2003 to 2011. cancer cases were more in female than male in all the years (table 2). most of the cancer patients were married (table 3) and hindu religion followed by buddhist religion (table 4). more cancer patients were illiterate followed by literate (table 5). agriculture was main occupation of most cancer patients followed by housework (table 6). most of cancer cases were prevalent in age group 40-75 years. less number of cancer cases was found in age group below 15 years (table 7). biopsy and histology were the major method for the diagnosis of cancer cases followed by cytology and haematology. the clinical examination and biochemical/immunological test were help to identified only few cancer cases. (table 8). out of 75 district of nepal, more number of cancer cases was found in kathmandu, sunsari, morang, chitwan, lalitpur, jhapa, kaski, nawalparasi, rupendehi and kavrepalchowk in 2010 (figure 4). similarly, in 2011 more number of cancer cases was found in kathmandu, morang, jhapa, sunsari, chitwan, lalitpur, rupendehi, kaski, saptari, bhaktapur (figure 5). lung cancer was the common cancer and similarly, other prevalent cancers were cervical, breast, stomach, ovarian and colo-rectum cancer in 2003 to 2011(figure 5). discussion nepal is a developing country and divided into beautiful three region terai, mountain and himalayan. people of different region have their own language, religion, festival and source of income to run the family. this study reveals, diagnosed cancer cases were mostly from terai region and most of them were working in field. the second occupation was housework as cancer cases diagnosed high in female compare to male. more than half of the cancer cases were illiterate. most of cancer cases were prevalent in age group 40-75 years. less number of cancer cases was found in age group below 15 years. the study conducted in india in 1994 and 1955 showed prevalence of cancer in same age group [11]. it may be due to decreases in immune system, iliteracy and exposure to cance causing agents in environmet. the most diagnosed cancer cases were hindus followed by buddhist, islam and christians. this is due to high number of population were hindus in nepal as we know nepal were hindu country in world. in nepal, among all diagnosed cancer cases, the common cancer were lung, cervical, breast, stomach, ovarian and colo-rectum [12]. the report by who showed common cancers was lung, breast and colo-rectum in the world [13]. some studies showed similar finding for these common cancers 0 2000 4000 6000 8000 n u m b e r years total cancer cases male female 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 kurmi et al. ©njb, biotechnology society of nepal 56 nepjol.info/index.php/njb [13, 14, 15]. the lung cancer was estimated a total of 239,320 new cases in us in 2010 [16]. the lung cancer is due to maximum use of tobacco which can be seen mainly in 25-60 yrs. age group in nepal. tobacco smoking [16] and tobacco uses were higher in mid-western, rural, far western and mountainous areas of nepal [17]. conclusion the cancer is becoming a major problem in nepal with the number rising continuously. all this may be due to environmental factors and life style changes i.e. tobacco use, food habits, alcohol use and physical inactivity. references 1. peppas lb, blanchette jo: nanoparticle and targeted systems for cancer therapy. adv. drug. delivery rev. 2003 56: 1649-59. 2. binu v, chandrashekhar t s, subba s h, jacob s, kakria a, gangadharan p, menezes r g: cancer pattern in western nepal: a hospital based retrospective study. asian pac. j. cancer. prev. 2007 8:183-186. 3. loeb kr, and loeb la: significance of multiple mutations in cancer. carcinogen. 2000 21:379–85. 4. hahn wc, and weinberg ra: modelling the molecular circuitry of cancer. nat. rev. cancer 2002 2:331–41. 5. anand p, kunnumakara ak, sundaram c et al: cancer is a preventable disease that requires major lifestyle changes. pharma. res. 2008 25 (9): 2097-2116. 6. cogliano v, baan r, straif k, et al: preventable exposures associated with human cancers. j. natl. cancer inst. 2011 103 :1827-39. 7. ferlay j, soerjomataram i, ervik m, dikshit r, eser s, mathers c, rebelo m, parkin dm, forman d, bray f: globocan 2012: cancer incidence and mortality worldwide: iarc cancer base no. 11 [internet]. lyon, france: international agency for research on cancer; 2013 8. ferlay j, shin hr, bray f, forman d, mathers cd, parkin d: globocan 2008: cancer incidence and mortality worldwide: iarc cancerbase no.10 [internet]. lyon, france: international agency for research on cancer. 2010 9. irigaray ja, newby r, clapp l, hardell v, howard, l. montagnier, s. epstein, and d. belpomme: lifestyle-related factors and environmental agents causing cancer: an overview. biomed. pharmacother 2007 61:640–58. 10. iarc, globocan 2008 (2010): international agency for research on cancer (iarc). http://globocan.iarc.fr/ factsheet.asp accessed on 25.7.2012. 11. mitra s and gupta ad: an estimate of the prevalence of cancer in india. bull. org. inond. sante. 1960 22: 485-492. 12. iarc: globocan 2012, international agency for research on cancer (iarc). http://globocan.iarc.fr/ factsheet.asp accessed on 12.12.2013 13. jemal a, bray f, center mm, et al: global cancer statistics. ca: cancer j. clin. 2011 61(2): 69–90. 14. behera d, balamugesh t: lung cancer in india. indian j. chest dis. allied sci. 2004 46(4): 269-81. 15. noronha v, dikshit r, raut n, joshi a, pramesh cs, george k, agarwal jp, munshi a, prabhash k: epidemiology of lung cancer in india: focus on the differences between non-smokers and smokers: a single-centre experience. indian j. cancer 2012 49(1): 74-81 16. jemal a, ward e, hao y, et al: trends in the leading causes of death in the united states 1970– 2002. jama. 2005 294(10): 1255–1259. 17. chandrashekhar t, sreeramareddy n , ramakrishnareddy hn, kumar h, sathian b, arokiasamy jt: prevalence, distribution and correlates of tobacco smoking and chewing in nepal: a secondary data analysis of nepal demographic and health survey-2006. subst. abuse treat prev. policy 2011 6: 33. http://www.ncbi.nlm.nih.gov/pubmed/?term=behera%20d%5bauthor%5d&cauthor=true&cauthor_uid=15515828 http://www.ncbi.nlm.nih.gov/pubmed/?term=balamugesh%20t%5bauthor%5d&cauthor=true&cauthor_uid=15515828 http://www.ncbi.nlm.nih.gov/pubmed/15515828 http://www.ncbi.nlm.nih.gov/pubmed/?term=noronha%20v%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=dikshit%20r%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=raut%20n%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=joshi%20a%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=pramesh%20cs%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=pramesh%20cs%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=george%20k%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=agarwal%20jp%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=munshi%20a%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=prabhash%20k%5bauthor%5d&cauthor=true&cauthor_uid=22842172 http://www.ncbi.nlm.nih.gov/pubmed/?term=sreeramareddy%20ct%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=ramakrishnareddy%20n%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=ramakrishnareddy%20n%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=harsha%20kumar%20h%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=sathian%20b%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=arokiasamy%20jt%5bauth%5d nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 kurmi et al. ©njb, biotechnology society of nepal 57 nepjol.info/index.php/njb table 1: total cancer cases from seven reporting institute in 2003 to 2011 name of hospital 2003 2004 2005 2006 2007 2008 2009 2010 2011 bpkmch 1869 (57.5%) 1815 (43.2%) 2154 (49.0%) 2564 (52.2%) 3261 (54.03%) 2936 (49.4%) 2772 (44.1%) 3216 (47.5%) 3320 (46.8%) bch 328 (10.0%) 952 (22.7%) 1008 (22.9%) 1086 (22.1%) 1138 (18.85%) 1253 (21.1%) 1289 (20.8%) 1253 (18.5%) 1361 (19.2%) bpkihs 418 (12.9%) 457 (10.9%) 702 (16.0%) 613 (12.5%) 640 (10.6%) 655 (11.0%) 660 (10.6%) 553 (8.2%) 512 (7.2%) bh 127 (4.0%) 385 (9.2%) 206 (4.7%) 350 (7.1%) 696 (11.53%) 798 (13.4%) 1228 (19.8%) 1173 (17.3%) 1005 (14.2%) mth 215 (6.6%) 307 (7.3%) 162 (3.7%0 172 (3.5%) 192 (3.18%) 153 (2.6%) 79 (1.3%) 77 (1.1%) 160 (2.1%) tuth 248 (7.6%) 190 (4.5%) 134 (3.0%) 89 (1.8%) 68 (1.12%) 91 (1.5%) 188 (3.0%) 392 (5.9%) 648 (9.1%) kch 46 (1.4%) 95 (2.3%) 31 (0.7%) 34 (0.7%) 40 (0.6%) 63 (1.1%) 23 (0.4%) 104 (1.1%) 82 (1.2%) total 3251 4201 4397 4908 6035 5949 6199 6773 7088 table 2: distribution of cancer cases based on marital status in 2010 to 2011 marital status 2010 2011 male female total male female total unmarried 132 111 243 127 92 219 married 2678 3084 5762 2773 3243 6016 widow 68 172 240 70 199 269 divorced 3 4 7 1 4 5 separated 5 1 6 4 3 7 not available 106 107 213 131 135 266 not applicable 1 0 1 189 117 306 total 2993 3479 6472 3295 3793 7088 table 3: distribution of cancer cases based on the religion in 2010 to 2011 religion 2010 2011 male female total male female total hindu 2735 3057 5792 2683 3093 5776 buddhist 217 254 471 301 358 659 islam 51 53 104 57 59 116 christian 16 32 48 24 26 50 others 63 85 148 160 191 351 not available 110 100 210 70 66 136 total 3192 3581 6773 3295 3793 7088 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 kurmi et al. ©njb, biotechnology society of nepal 58 nepjol.info/index.php/njb table 4: distribution of cancer cases based on the education status in 2010 to 2011 education 2010 2011 male female total male female total literate 1346 1118 2464 1342 1068 2410 illiterate 1291 1936 3227 1494 2233 3727 not applicable 25 17 42 397 466 863 not available 476 479 955 62 26 88 total 3138 3550 6688 3295 3793 7088 table 5: distribution of cancer cases based on occupational status in 2010 to 2011 occupation 2010 2011 male female total male female total agriculture 1769 1154 2923 1757 1082 2839 business 172 83 255 202 182 384 housework 206 1790 1996 265 1895 2160 office work 256 77 333 251 105 356 others 291 158 449 328 150 478 not applicable 8 5 13 303 262 565 not available 441 363 804 189 117 306 total 2993 3479 6773 3295 3793 7088 table 6: distribution of cancer cases based on the age group in 2010 to 2011 age group 2010 2011 (years) male female total male female total 0-4 54 31 85 62 26 88 5-9 66 39 105 52 41 93 10-14 79 32 111 75 50 125 15-19 70 75 145 74 63 137 20-24 74 96 170 93 84 177 25-29 77 117 194 78 120 198 30-34 91 159 250 103 155 258 35-39 128 241 369 141 251 392 40-44 154 328 482 205 380 585 45-49 228 439 667 234 439 673 50-54 293 491 784 295 464 759 55-59 319 362 681 404 474 878 60-64 459 414 873 426 449 875 65-69 427 304 731 381 359 740 70-74 351 228 579 333 232 565 75-79 193 132 325 210 138 348 80 + 129 93 222 129 68 197 total 3192 3581 6773 3295 3793 7088 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 kurmi et al. ©njb, biotechnology society of nepal 59 nepjol.info/index.php/njb figure 3: showed the sex wise distribution of cancer cases in different district of nepal in 2010 table 7: distribution of cancer cases based on type of diagnosis in 2010 to 2011 2010 2011 male female total male female total clinical examination 3 1 4 23 25 48 endoscopy 36 12 48 37 17 54 biopsy/histology 1699 2151 3850 1648 2296 3944 cytology/haematology 812 834 1646 860 816 1676 exploratory surgery 2 2 4 0 0 0 biochemical/immunological test 0 7 7 1 15 16 radiology 239 225 464 300 274 574 death certificate 2 3 5 3 6 9 not available 399 346 423 344 767 total 3192 3581 6773 3295 3793 7088 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:54-60 kurmi et al. ©njb, biotechnology society of nepal 60 nepjol.info/index.php/njb figure 4: showed the sex wise distribution of cancer cases in different district of nepal in 2011 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 75-78 research article doi: https://doi.org/10.3126/njb.v9i1.38670 ©njb, bsn 75 thyroid function and thyroglobulin level in iodine-deficient children of eastern nepal saroj kunwar1, saroj khatiwada2 , basanta gelal3, saroj thapa3, gaurishankar shah4, nirmal baral3, madhab lamsal3 1department of biochemistry, modern technical college, lalitpur, nepal *2school of medical sciences, university of new south wales sydney, australia 3department of biochemistry, b p koirala institute of health sciences, ghopa, dharan, nepal 4department of obstetrics and gynaecology, b p koirala institute of health sciences, ghopa, dharan, nepal received: 13 oct 2020; revised: 04 apr 2021; accepted: 16 apr 2021; published online: 31 jul 2021 abstract iodine deficiency during childhood affects physical and mental development. iodine deficiency or excess both can negatively impact thyroid function. we conducted this study to assess iodine nutrition and thyroid function in children with insufficient urinary iodine concentration. a community-based cross-sectional study was conducted among the selected schools of udayapur district. urinary iodine concentration (uic) was measured in 1012 school children (6-14 years). based on uic data, 83 blood samples were collected to measure serum thyroglobulin (tg), thyroid-stimulating hormone (tsh), free triiodothyronine (ft3), and free thyroxine (ft4). uic was measured by ammonium persulfate digestion method, and tg, tsh, ft4, and ft3 were measured using elisa kits. the median uie was 236 µg/l, and 11.1% of the children had insufficient uic. the mean ft3, ft4, and tsh in children with insufficient uic were 2.55±0.43 pg/ml, 0.96±0.28 ng/dl, and 3.60±1.44 miu/l respectively. among children with low uic levels, the median tg was 17.5 ng/ml. overt hypothyroidism was seen in 6%, and subclinical hypothyroidism in 3.6%. the children had sufficient iodine nutrition, and the frequency of thyroid dysfunction was low among the children with insufficient uic. keywords: children; iodine deficiency; nepal; thyroid dysfunction; urinary iodine concentration (uic) corresponding author, email: khatiwadasaroj22@gmail.com introduction iodine is an essential micronutrient required for the production of thyroid hormones [1, 2]. iodine deficiency affects 2 billion people, with 36.4% being school-age children [3]. iodine deficiency during pregnancy and childhood has severe consequences, including developmental disorders, poor cognition, and intellectual disability [4]. supplementation of iodine in food or salt has led to a significant reduction in iodine deficiency disorders worldwide [5]. nepal has been continuously improving in iodine nutrition after starting the universal salt iodization program [6-8]. several studies, particularly in eastern nepal show promising results in the improvement of iodine deficiency as revealed by an increase in median urinary iodine concentration (uic) of school children [68]. however, there are also concerns about excess iodine intake in such a population [9]. iodine deficiency or excess both affects thyroid function; therefore, it is important to maintain thyroid function in such a population [1]. currently, the impact of iodine deficiency on thyroid function and thyroglobulin level in children living in the eastern part of nepal is unknown. therefore, in this study, we specifically aimed to find the recent iodine status in a large cohort of school children, and then measure thyroid function in the children with low uic. materials and methods study design this community-based study was conducted among school children from the udayapur district of eastern nepal. the study was conducted by the department of biochemistry in collaboration with the department of pediatrics of b. p. koirala institute of health sciences (bpkihs), dharan, nepal. a multistage random sampling technique was used to enroll school children aged 6 to 14 years in the study. out of 46 village development committees (vdc) in the udayapur district, four vdcs (beltar, basaha, rampur, and chaudandi) were randomly selected for study purposes. in those four vdcs, six schools (four schools from 2 vdcs and two schools from the other 2 vdcs) were chosen for sample collection. all the students from those schools in the age range, and fitting into the inclusion criteria were enrolled. a total of 1012 children participated, and in the first stage, 1012 urine samples nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:sarojkunwar00@gmail.com mailto:gelalbasanta@gmail.com mailto:drsarojthapa@gmail.com mailto:gaurishankarshah@live.com mailto:nirmalbaral@hotmail.com mailto:madhablamsal@yahoo.co.uk mailto:khatiwadasaroj22@gmail.com mailto:khatiwadasaroj22@gmail.com mailto:khatiwadasaroj22@gmail.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 5 7 8 kunwar et al. ©njb, bsn 76 were collected from the participants. after uic measurement, only those children with insufficient uic (uic<100 µg/l) were followed up for blood sampling. blood samples were taken from 83 children out of 112 children with insufficient uic (uic<100 µg/l), as 29 children dropped out for blood sampling. blood samples were collected to assess thyroid function and thyroglobulin level. this study was approved by the institutional review committee of bpkihs and conducted under the helsinki declaration of 1975. sample collection and analysis written informed consent was obtained from the guardian of children prior to participation in the study. all the stakeholders, including participants, were informed about the purpose of the study. all healthy school-going children (aged 6-14 years of age) were included. children taking drugs that interfere with thyroid function, taking iodine supplements, or with any severe illness were excluded. in the first stage of the study, anthropometric measurements (height and weight) were taken, and spot urine samples were collected from all 1012 participants. urine samples were collected in a clean, tightly screw-capped plastic vial and transported to the biochemistry laboratory for analysis. in the second phase of the study, blood samples (3ml) were collected in the plain vial by venipuncture following the standard protocol. clotted blood was centrifuged at 3000 rpm, and serum was separated. serum and urine samples were refrigerated at -20 ºc until analysis. urinary iodine concentration was estimated by ammonium persulfate digestion microplate (apdm) method using sandell-kolthoff’s reaction [10]. serum-free triiodothyronine (t3), free tetraiodothyronine (t4), tsh, and thyroglobulin (tg) were measured by the elisa method using commercial kits from diametra company. based on the reference ranges of thyroid hormones (ft3, ft4, and tsh), thyroid function was classified as normal thyroid function (ft3, ft4, and tsh within the reference range), overt hypothyroidism (tsh above the reference range and ft3 and ft4 below the reference range) and subclinical hypothyroidism (tsh above the reference range, and ft3 and ft4 within the reference range). the reference range followed for ft3, ft4 and tsh were 1.24.2 pg/ml, 0.8-2.2 ng/dl, and 0.39-6.16 miu/l respectively. statistical analysis the data was analyzed using spss version 20.0. the data are presented in the form of mean ± sd (for normally distributed variables), median with inter quartiles (iqr) for non-normally distributed variables and frequency (percentage). age, height, weight, serum ft3, serum ft4 and serum tsh concentration showed normal distribution, so they were expressed as mean±sd. the uic and serum thyroglobulin levels were expressed as median with iqr as they were not normally distributed. chi-square test was applied to assess statistical difference among categorical variables (gender versus iodine status, gender versus thyroid function). spearman’s analysis was used to find a relationship between quantitative variables (uic with thyroid hormones and tg, tg with thyroid hormones). a ‘p’ value of ≤0.05 was considered statistically significant at a 95% confidence interval. results a total of 1012 school children (482 males and 530 females) from 4 vdc of the udayapur district participated in the study. the mean±sd age, weight, and height were 10.81±2.32 years, 29.04±8.79 kg, and 131.64±14.61 cm respectively. the median uic with iqr was 236 µg/l (156, 331) indicating adequate iodine intake. the median uic (iqr) among male and female children were 219 µg/l (149, 335) and 248 µg/l (165, 330) respectively. table 1 shows the classification of iodine nutrition status in the study participants according to modified who assessment criteria with adequate and more than adequate group merged as sufficient category [3]. the overall prevalence of iodine deficiency based on individual uic cutoffs was 11.1% (severe, moderate, and mild deficiency). about one-third, (33.9%) of the children had excessive uic (uic>300 µg/l). the mean ft3, ft4, tsh, and median tg (iqr) in children with insufficient uic were 2.55±0.43 pg/ml, 0.96±0.28 ng/dl, 3.60±1.44 miu/l, and 17.5 (12, 29.4) ng/ml respectively. out of 83 participants with low uic, 75 (90.3%) had normal thyroid function, 5 (6.0%) table 1. iodine status according to gender. the data are expressed as frequency (percentage). a chi-square test was used between gender and iodine status at a 95% confidence interval. id= iodine deficiency severe id (<20 µg/l) moderate id (20-49 µg/l) mild id (50-99 µg/l) sufficient (100-299 µg/l) excessive (>300 µg/l) p value gender male (n=482) 5 (0.5%) 13 (1.2%) 40 (3.9%) 266 (26.3%) 158 (15.6%) 0.5 female (n=530) 4 (0.4%) 19 (1.8%) 31 (3.0%) 291 (28.7%) 185 (18.3%) total (n=1012) 9 (0.9%) 32 (3.2%) 71 (7.0%) 557 (55.0%) 343 (33.9%) nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 5 7 8 kunwar et al. ©njb, bsn 77 had overt hypothyroidism, and 3 (3.6%) had subclinical hypothyroidism. among males with low uic (n=40), 36 (90%) had normal thyroid function, 2 (5%) had overt hypothyroidism, and 2 (5%) had subclinical hypothyroidism. among females with low uic (n=43), normal thyroid function, overt hypothyroidism, and subclinical hypothyroidism were seen in 39 (91%), 3 (7%), and 1 (2%) children respectively. no significant differences (p=0.761) in thyroid function was seen between male and female with insufficient uic. in the children with insufficient uic, tg has positive correlation with ft3 (r=0.273, p=0.013). however, tg had no significant correlation with ft4 (r=0.012, p=0.916) and tsh (r=0.056, p=0.615). in the same group, there was no significant correlation of uic with ft3 (r=0.075, p=0.503), ft4 (r=-0.139, p=0.212), tsh (r=0.175, p=0.114) and tg (r=-0.075, p=0.503). discussion iodine deficiency has remained the most common cause of preventable brain damage in children worldwide [11]. supplementation of iodine in salt through universal salt iodization has become effective in nepal as shown by improving median uic in the previous studies from eastern nepal [6-8]. median uic indicates recent iodine intake and is used as a marker of iodine status in the community settings. in the present study, the median uic was 236 µg/l, which indicates sufficient iodine nutrition among children of this district. the previous report showed a median uic of 268 µg/l among children of this district [7]. in this study, 11.1% of the children had uic< 100 µg/l, which is considered iodine deficiency. in the previous study, iodine deficiency in this district was 12.7% [7]. national surveys conducted in nepal in the years 1998, 2005, and 2007 depicted iodine deficiency in 43.6%, 27.4%, and 19.4% school-age children respectively [12]. in another study in eastern regions, dhankuta and dharan, iodine deficiency was 26.6% and 15.6% respectively among school children [13, 14]. thyroglobulin, a thyroid-specific protein, and precursor in the synthesis of thyroid hormones is considered a sensitive marker of the iodine status than uic [15]. the median thyroglobulin level in the children with insufficient uic was 17.5 ng/ml. our previous study among pregnant women of eastern nepal reported a median tg of 6.5 ng/ml [16]. thyroglobulin increment in plasma is also seen with a thyroid mass, inflammation, and hyperactivity of tsh [17]. we observed normal thyroid function in most of the children with insufficient uic. out of 83 children with insufficient uic, 6% (n=5) had overt hypothyroidism, and 3.6% (n=3) had subclinical hypothyroidism. previous studies by shakya et al., chaudhari et al., and khatiwada et al. found subclinical hypothyroidism in 19.5% and 16.7 % (in morang and sunsari respectively), 31.8% and 25.59% (in sunsari and dhankuta respectively) and 17.6 % respectively (in hilly regions) [18, 14, 19]. low prevalence of thyroid dysfunction in the current study than previous studies may be due to differences in the geographic location, genetic factor, and small sample size of the current study. in the current study, one-third (33.9%) of the children had excessive uic, which suggests the risk of iodineinduced hyperthyroidism and autoimmune thyroid disease in those children. future studies need to investigate the effect of excessive iodine consumption on thyroid function. furthermore, the salt iodine fortification level may need to be readjusted to lower iodine intake in the community. interestingly, our finding of a low rate of thyroid abnormalities in children with insufficient uic suggests that the thyroid gland can adapt to low iodine intake. moreover, uic is an indicator of recent iodine intake, and it does not reflect long-term iodine nutrition in those children with insufficient uic. in addition, the coexistence of autoimmune thyroiditis also contributes to abnormal thyroid function in the community. overall, thyroid function can be impaired both in the condition of iodine deficiency and excess, and therefore, maintaining optimal iodine intake by continuous monitoring of dietary iodine intake and uic is crucial [1]. we observed a non-significant negative correlation of uic with tg level and thyroid hormones in children with insufficient uic, and a positive correlation between thyroglobulin and ft3 (r= 0.273, p=0.013). in iodinedeficient cases, thyroglobulin tends to rise due to hyperactivity of the thyroid gland and tsh; however, the thyroid gland may be able to adapt to short-term iodine deficiency maintaining normal thyroid hormones [1]. the present study has several limitations. individual uic was used as a marker of iodine deficiency, and thyroid function was not assessed in all the participants. thyroglobulin was estimated in the children with low uic, and other common causes of thyroid dysfunction such as autoimmune thyroiditis were not investigated. future studies need to consider several of these factors. conclusion sufficient iodine nutrition occurs among the school children of the udayapur district, with one-third of the children having excessive uic, and a small proportion nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 5 7 8 kunwar et al. ©njb, bsn 78 having insufficient uic. the incidence of thyroid dysfunction is low in children with insufficient uic. future studies need to be undertaken to measure thyroid function in children with excessive uic, and other potential causes of hypothyroidism in children with insufficient uic need to be investigated. abbreviations apdm: ammonium persulphate digestion method free t4: free thyroxine free t3: free triiodothyronine tg: thyroglobulin tsh: thyroid-stimulating hormone uic: urinary iodine concentration author contributions sk1, sk2, bg, nb, and ml designed the study. sk1 and st collected the samples. sk1 analyzed the samples. sk2 performed data analysis and drafted the manuscript. sk1 helped in drafting the manuscript. all authors read and approved the final version of the manuscript. competing interests none funding none acknowledgments we kindly acknowledge all the participants of the study. we kindly acknowledge the department of biochemistry of b p koirala institute of health sciences, dharan for supporting the study. ethics approval and consent the study was approved by the institutional ethical review committee (irc) of bpkihs (code no: irc/422/014). written consent was taken from children’s parents before participation. availability of data and material data are available on reasonable request. references 1. chung hr. iodine and thyroid function. ann pediatr endocrinol metab. 2014;19(1):8-12. doi: 10.6065/apem.2014.19.1.8. 2. mullur r, liu yy and brent ga. thyroid hormone regulation of metabolism. physiol rev. 2014;94(2):355-82. doi: 10.1152/physrev.00030.2013. 3. zimmermann mb, jooste pl, pandav cs. iodine-deficiency disorders. lancet. 2008;372(9645):1251-62. doi: 10.1016/s0140-6736(08)61005-3. 4. zimmermann mb. the effects of iodine deficiency in pregnancy and infancy. paediatr perinat epidemiol. 2012;26(1):108-17. doi: 10.1111/j.1365-3016.2012.01275.x. 5. ershow ag, skeaff sa, merkel jm, pehrsson pr. development of databases on iodine in foods and dietary supplements. nutrients. 2018;10(1). pii: e100. doi: 10.3390/nu10010100. 6. khatiwada s, gelal b, gautam s, lamsal m, baral n. iodine status among school children of remote hilly regions of nepal. indian pediatr. 2015;52(5):436-7. 7. khatiwada s, lamsal m, gelal b, gautam s, nepal ak, brodie d, et al. anemia, iron deficiency and iodine deficiency among nepalese school children. indian j pediatr. 2016;83(7):617-21. doi: 10.1007/s12098-015-1924-y. 8. khatiwada s, gelal b, shakya pr, lamsal m, baral n. urinary iodine excretion among nepalese school children in terai region. indian j pediatr. 2016;83(1):15-7. doi: 10.1007/s12098-015-1755-x. 9. shakya pr, gelal b, baral n. high iodine intakes in school children in eastern nepal. idd newsletter. 2011;39:10-3. 10. ohashi t, yamaki m, pandav cs, karmarkar mg, irie m. simple microplate method for determination of urinary iodine. clin chem. 2000;46(4):529-36. 11. vincenzo t, emilio t, angelo vg, carlo s, francesco r, brunella l et al. role of iodine, selenium and other micronutrients in thyroid function and disorders. endocr metab and immune disord drug targets. 2009;9(3):277-94. 12. ministry of health and population, department of health services, government of india and alliance nepal. national survey and impact study for iodine deficiency disorders (idd) and availability of iodized salt in nepal; 2007. 13. gelal b, chaudhari rk, nepal ak, sah gs, lamsal m, brodie da, et al. iodine deficiency disorders among primary school children in eastern nepal. indian j pediatr. 2011;78(1):45-8. doi: 10.1007/s12098-010-0239-2. 14. chaudhari rk, gelal b, brodie d, and baral n. thyroid function and urinary iodine status in primary school age children of the hills and plains of eastern nepal. indian pediatr. 2012;49:332-333. 15. ma zf, skeaff sa. thyroglobulin as a biomarker of iodine deficiency: a review. thyroid. 2014;24(8):1195-209. doi: 10.1089/thy.2014.0052. 16. chaudhary ln, khatiwada s, gelal b, gautam s, lamsal m, pokharel h, et al. iodine and thyroid function status, and anti-thyroid peroxidase antibody among pregnant women in eastern nepal. j nepal health res counc. 2017;15(2):114-119. 17. vejbjerg p, knudsen n, perrild h, laurberg p, carlé a, pedersen ib, et al. thyroglobulin as a marker of iodine nutrition status in the general population. eur j endocrinol. 2009;161(3):475-81. doi: 10.1530/eje-09-0262. 18. shakya pr, gelal b, das bkl, lamsal m, pokharel p k, nepal ak, et al. urinary iodine excretion and thyroid function status in school age children of hilly and plain regions of eastern nepal. bmc res notes. 2015; 8: 374. 19. khatiwada s, gelal b, baral n, lamsal m. association between iron status and thyroid function in nepalese children. thyroid res. 2016;9:2. doi: 10.1186/s13044-0160031-0. nepal j biotechnol. 2020 dec; 8 (3): 82-86 doi: https://doi.org/10.3126/njb.v8i3.30080 research article ©njb, bsn 82 antibiogram of escherichia coli and staphylococcus aureus isolated from milk sold in kathmandu district shyamala rai, barsha karki, sujita humagain, sandesh rimal, sandhya adhikari, shilpa adhikari, suchitra thapa . amrit campus, tribhuvan university, department of microbiology, thamel, kathmandu article history:received: 15 jun 2020; revised: 17 dec 2020; accepted: 18 dec 2020; published online: 30 dec 2020 abstract the emergence of antibiotic resistance in microorganisms and the presence of such isolates in milk pose a great risk to public health. therefore, this study aims to determine the antibiotic susceptibility pattern of escherichia coli and staphylococcus aureus isolated from milk and assess the microbial quality of milk. for this, a total of 70 milk samples were collected and the total bacterial count (tbc) was determined. e. coli and s. aureus were isolated using their respective selective media while antibiotic susceptibility testing was carried out by kirby bauer disc diffusion method. the tbc showed that the raw milk samples contained two-fold higher microbial load while the pasteurized milk samples contained four-fold higher microbial loads than the standard guidelines. a total of 62 isolates were identified from culture-positive milk samples of which 32 were e. coli and 30 were s. aureus. a significant correlation was observed between microbial load and the organism isolated (r = 0.339, p<0.01). all s. aureus isolates were susceptible to chloramphenicol while 40% were resistant to cefoxitin, indicating the presence of methicillin resistant s. aureus (mrsa). also, 12 multidrug resistant (mdr) s. aureus were identified. while for e. coli, all were susceptible to chloramphenicol but resistant to ampicillin. also, 9 mdr e. coli were detected. higher resistance was observed among isolates from the raw milk samples than the pasteurized milk. it can be concluded that the milk produced by smallscale farms and dairy industries of kathmandu district are of poor quality. hence, routine microbial quality assessment and antimicrobial resistance monitoring should be followed to safeguard public health. keywords: antibiotic resistance, milk-borne infection, multidrug resistance, e. coli, s. aureus, total bacterial count, mrsa corresponding author, email: suchitrathapa69@gmail.com introduction milk, a daily diet requirement of people, can become microbiologically hazardous to consumers when the principles of hygiene and sanitation are not met. such conditions may become a vehicle for transmission of food-borne infections [1]. among all microorganism, escherichia coli and staphylococcus aureus are the most common food contaminants [1]; and in recent years, both are observed to cause a number of significant illnesses in animals and humans [2]. s. aureus is specifically a versatile pathogen capable of causing numerous diseases in humans [2]. in addition, it is also a major causative pathogen of clinical or subclinical mastitis of dairy animals [1]. further, mrsa together with extended spectrum β-lactamase (esbl) producing e. coli, are considered as serious threats to human health [3]. therefore, the relative importance of these pathogens along with other pathogenic microorganisms in milk is inevitable. but the lack of awareness about milk-borne infections in many developing countries and consumption of contaminated milk predisposes consumers at risk of contracting infections with these pathogens [4]. antibiotics are essential to treat infections caused by pathogenic bacteria, both in humans and animals. however, their overuse and misuse in veterinary and human medicine has been linked to the emergence and spread of resistant bacteria, rendering the treatment of infectious diseases ineffective in animals and humans. and, now antimicrobial resistance is one of the main threats to modern medicine [5]. further, the escalating prevalence of antimicrobial resistance among foodborne pathogens [6, 7] has exaggerated the public health hazards including milk-borne infections. available studies around the world have reported about the presence of multidrug resistant e. coli [8], esbl [9, 10], toxin-producing s. aureus [11] along with mrsa strains [7, 11] in milk samples but in the case of nepal very less studies are found on antibiotic resistance of milk isolates. therefore, it is necessary to know the microbial quality of marketed milk and understand the recent trend of antimicrobial resistance among milk pathogens so as to properly diagnose and treat the infection. this study was conducted to determine the current trend of antibiotic resistance of e. coli and s. aureus isolated from milk and assess the microbial quality of milk sold in kathmandu district. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:suchitrathapa69@gmail.com https://orcid.org/0000-0001-5995-1909 mailto:suchitrathapa69@gmail.com nepal j biotechnol. 2020 dec; 8 (3): 82-86 rai et al. ©njb, bsn 83 materials and methods sampling site and sample a total of 70 milk samples were tested in this study. forty raw milk samples (30 ml each) from 4 different municipalities of kathmandu district (kageshworimanohara, chandragiri, tarkeshwor and dakshinkali municipality) were collected in a sterile screw capped bottle and transported to laboratory in an icebox within 2 hours. further, 30 pasteurized milk pack (500ml) was bought from local vendors from the same municipalities. the collection of the sample was done from december 2018 to march 2019 in compliance with the guidelines stated in bacteriological analytical manual [12]. total bacterial count of milk samples the collected milk samples were serially diluted up to 10-8 dilution. the bacterial count was determined by culturing the serially diluted milk samples. conventional pour plate technique was employed for culturing the diluted milk samples [13]. isolation and identification of e. coli and s. aureus isolation of e. coli and s. aureus was done by enrichment in buffered peptone water and cultured in emb (eosin methylene blue) and msa (mannitol salt agar) media respectively [14]. the distinct colonies were identified and confirmed by following their respective biochemical characteristics [13]. antibiotic susceptibility testing of milk isolates isolates of e. coli and s. aureus were subjected to antibiotic susceptibility testing using kirby bauer disk diffusion method as recommended by clsi [15]. the antibiotics used for e. coli were ampicillin (10µg), chloramphenicol (30µg), ciprofloxacin (5µg), nalidixic acid (30µg), tetracycline (30µg) and ceftriaxone (5µg); and for s. aureus, cefoxitin (30µg), tetracycline (3µg), chloramphenicol (30µg), ceftriaxone (30µg) and ciprofloxacin (5µg) were used. data analysis the data was initially entered in ms excel and exported to spss. the frequency distribution, normal distribution testing, variance analysis and correlation were done using spss (version 20). the significance was measured at both 95% and 99% confidence interval. the resistance profile was analyzed using whonet2019 (32-bit version 19.13.21) and the outcome was interpreted accordingly. results microbial load of milk samples the tbc of raw milk ranged from 0.31×105 cfu/ml to 1000×105 cfu/ml with mean tbc of 8.13×106 cfu/ml (s.d = 18.9x 106). likewise, the tbc of pasteurized milk ranged from 0.35×103 cfu/ml to 1600×103 cfu/ml with mean tbc of 14.25×104 cfu/ml (s.d = 29.71 x 104). figure 1. quality of milk samples according to bis standard guideline. for raw milk, the tbc exceeding 50×105 cfu/ml is graded as poor while below it is graded either fair, good and very good according to bureau of indian standards (bis) [16] and are considered within safe limit. following this standard, among the total raw milk samples 14 samples were observed to fall under "very good" grading, 6 under "good" grading, 10 under "fair" grading and rest 10 under "poor" grading. in case of pasteurized milk, 9 samples had tbc below 3×104 cfu/ml and 21 samples had higher than that. the distribution of milk samples in terms of their standard limit of tbc is given in figure 1. statistically, a highly significant difference was observed in the distribution of tbc across the raw and pasteurized milk sample (p<0.01). prevalence of e. coli and s. aureus in milk e. coli and s. aureus were isolated from raw and pasteurized milk samples using selective media (figure 3a and 3b). out of 70 samples, 46 (65.71%) samples showed culture positivity towards e. coli or s. aureus or to both. the percentage distribution of organisms among the raw and pasteurized milk sample is given in figure 2. it was observed that the distribution of microbial count is not the same across culture positivity (p<0.05) and isolated organism (p<0.01). a total of 32 e. coli isolates were subjected to antibiotic susceptibility test using ciprofloxacin, ceftriaxone, chloramphenicol, nalidixic acid and ampicillin antibiotic disc. 30% 75% 70% 25% 0% 10% 20% 30% 40% 50% 60% 70% 80% pasteurised milk raw milk p e rc e n ta g e o f sa m p le s type of milk sample within limit above limit nepal j biotechnol. 2020 dec; 8 (3): 82-86 rai et al. ©njb, bsn 84 figure 2. distribution of e. coli and s. aureus in the milk sample (n=46) figure 3a: e. coli culture on emb media plate and 3b: s. aureus culture on msa media plate antibiogram of e. coli all the isolates showed 100% susceptibility towards chloramphenicol while none of the isolates showed susceptibility towards ampicillin. also, the susceptibility for ciprofloxacin, ceftriaxone, tetracycline and nalidixic acid were 96.88%, 87.5%, 81.25% and 78.13%, respectively. of the 9 mdr e. coli identified, 4 isolates of important antibiotic resistance was recognized through antibiogram analysis. a detail resistance profile of these isolates is provided in table 1. antibiogram of s. aureus a total of 30 s. aureus isolates were subjected to antibiotic susceptibility test using cefoxitin, ciprofloxacin, ceftriaxone, chloramphenicol and nalidixic acid antibiotic disc. all the isolates showed 100% susceptibility towards chloramphenicol, while the susceptibility for tetracycline, ciprofloxacin, cefoxitin and ceftriaxone were 93.33%, 70%, 60% and 30%, respectively. a total of 12 mdr s. aureus (40%) were identified and they were confirmed as mrsa due to their resistance towards cefoxitin. a detail resistance profile of important isolates of s. aureus is provided in table 2. table 2 antibiotic resistance profile of s. aureus (n=12) milk sample type resistance antibiotics no of s. aureus importance resistance priority raw milk cipr cxr 2 yes* medium* raw milk ctrr cx r 6 yes* medium* raw milk ctrr cx r ter 1 yes* medium* raw milk ctrr cx r cipr 1 yes* medium* pasteurized milk ctrr cx r cipr 2 yes* medium* * according to the whonet 2020 software interpretation legends: cx-cefoxitin, ctrceftriaxone, te-tetracycline, cip-ciprofloxacin discussion this study, which examined milk samples from four different municipality of kathmandu districts, showed high significance (p<0.01) in the distribution of microbial load among the raw and pasteurized milk samples. this significance may comply with the fact that pasteurized milks are heat treated and ought to have lower microbial load. however, the majority of pasteurized milk samples (70%) were below the standard recommended guidelines of indian standards [16] compared to raw milk (25%) (figure 1). also, the raw milk samples showed two-fold higher microbial load than the recommended value of bis while pasteurized milk showed four-fold higher microbial load than the standard recommended value of bis. this indicates that the pasteurized milk samples in our study were of bad quality. such results of high microbial load may be due to inefficient pasteurization, poor packaging material and pipeline, post pasteurization contaminants, presence of heat resistant bacteria, poor air and storage condition, etc. however, as both harmful and beneficial microbes can reside in milk and higher microbial load does not necessarily indicate the exact type of microbes present in the milk, it may not be appropriate to gauge the quality of milk solely based on microbial load unless each of the microbial strain in the milk are identified. the quality issue aside, apparently similar prevalence of higher microbial load in pasteurized milk in kathmandu was documented in a table 1 antibiotic resistance profile of e. coli (n=4) milk sample type resistance antibiotics number of e. coli importance resistance priority inference raw milk ampr narctrr 1 yes* medium* possible esbl pasteurized milk ampr ter narctrr 2 yes* medium* possible esbl raw milk ampr ter ciprctrr 1 yes* medium* possible esbl * according to the whonet 2020 software interpretation legends: amp-ampicillin, na-nalidixic acid, ctrceftriaxone, te-tetracycline, cip-ciprofloxacin 9 1 2 7 13 14 0 2 4 6 8 10 12 14 16 e. coli s. aureus both n u m b e r o f m il k sa m p le organism isolated from milk samples pasteurized milk raw milk a b nepal j biotechnol. 2020 dec; 8 (3): 82-86 rai et al. ©njb, bsn 85 study conducted by acharya et al. (2017) [17]. even the dftqc, nepal reported the higher rate of noncompliance among milk and milk product in the annual bulletin of 2019 [18]. the presence of microbes in high number in treated milk sample is a risk to the consumers as the microbes present may be pathogenic strains. in this study, almost half of the milk sample showed the presence of e. coli (45.71%) and similar results were reported in other studies [8, 19]. also, a study conducted by arjyal et al. (2004), has reported e. coli prevalence rate as high as 92% [20]. understandably, e. coli is a commensal enteric microorganism; yet, as their pathogenic strains are associated with a range of illness in animal and humans especially the toxin producers (shiga toxin-producing e.coli) and thus their presence in milk should not be overlooked. in case of s.aureus, their presence in milk is of greater concern as they also produce heat-stable toxin which causes food poisoning [1]. the finding in this study related to s. aureus – almost half of the milk sample (42.86%) had the presence of s. aureus –is comparable to the results of several studies conducted in nepal [20, 21] as well as in the rest of the world [22, 23, 24]. statistically, a significant positive correlation (p<0.05) was observed in the distribution pattern of tbc across the culture; it signifies that the microbial load affects the presence of e. coli or s. aureus and vice versa. similar correlation but with higher significance (p<0.01) was observed among microbial load and the type of organism which suggests that the microbial load affects the type of organism present in the sample and vice versa. this justifies the existence of higher prevalence of s. aureus (90%) and e. coli (65.63%) in raw milk as they have higher microbial load (figure 2). regarding the antibiotic susceptibility test of e. coli and s. aureus, the result showed an emerging antibiotic resistance among both isolates. all the e. coli isolates in this study were resistant to ampicillin which was in compliance with the findings of badri et al. (2017) [9] and singh et al. (2018) [25]. besides ampicillin resistance, the results revealed higher resistance among e. coli towards nalidixic acid, tetracycline, ceftriaxone and ciprofloxacin in descending order. even though xdr was not detected, 9 mdr was present and their antibiogram analysis indicated that 4 out of 9 isolates were important resistance of medium priority (table 1). such presence of mdr in milk samples were also reported in similar studies [8, 10]. this presence of mdr is of greater concern to public health and indicates an alarming situation. while in case of s. aureus, the resistance was higher for most of the antibiotics except for chloramphenicol. such full susceptibility was also reported in various other studies of milk samples [22, 17]. besides chloramphenicol susceptibility, the results revealed higher resistance among s. aureus towards ceftriaxone, cefoxitin, ciprofloxacin and tetracycline in descending order. further, 12 mdr (40%) were detected which were mrsa as well, and their antibiogram analysis indicated that these are important resistance of medium priority (table 2). several other studies have also reported the higher prevalence of mdr [7, 11, 26] and mrsa [7, 11, 17]. simultaneous presence of mdr in mrsa reported in this study also resembled the study of aliyu et al. (2020) [27]. this result indicates an emerging trend of antimicrobial resistance among s. aureus. this emerging antibiotic resistance among both e. coli and s. aureus isolates was observed in higher number in raw milk sample compared to pasteurized milk sample. also, the multidrug resistant isolate was found to be higher in raw milk than pasteurized milk. since the exposure to environment is more in raw milk compared to pasteurized milk, the chances of resistant isolates finding its way to raw milk is more likely. further the extensive misuse of antibiotics for the treatment of farm animals may have created selective pressure and resulted in the survival and persistence of resistant isolates. this emerging resistance may lead to treatment failure of the last resort drug. thus, routine monitoring of resistant profile of milk pathogens should be implemented in order to properly diagnose and treat milk-borne infections effectively, along with the assessment of microbial quality of milk with the purpose of safeguarding the public health. conclusion to conclude, the resistance towards common antimicrobials is emerging among milk isolates and infections by these isolates pose a serious threat to animal and public health. therefore, regular monitoring programs, good farming practice training, improved standard guidelines, antibiotic surveillance program on food isolates and rational use of antibiotics are needed to improve sustainable food production and avoid the emergence of antibiotic-resistant strains. authors' contribution the development of concept, preliminary work and laboratory analysis, was done by sr, bk, sh, sr and saa under the guidance of sut and sha. further, sut performed data analysis, prepared, reviewed and edited the manuscript. all authors read and approved the final manuscript. nepal j biotechnol. 2020 dec; 8 (3): 82-86 rai et al. ©njb, bsn 86 competing interests the authors declare that they have no conflicts of interest. funding this study was not funded by any agency or institution. acknowledgments all authors are grateful to the faculty and laboratory staff of microbiology department of amrit campus for their continuous support in this research work, and extent especial mention to the farm owners of kathmandu district who supported us by providing the milk sample. ethical approval and consent a brief detail of this research study was provided to the farm owner, and a verbal consent was obtained before sampling. this study was carried out with the approval from the concerned authorities. data availability the data can be made available upon request. references 1. tamime ay, editor. milk processing and quality management. john wiley & sons; 2009 jan 30. http://www.foodtechnologist.yolasite.com/resources/milk%20p rocessing%20 and%20quality%20management.pdf 2. food and drug administration. the bad bug book: foodborne pathogenic microorganisms and natural toxins handbook, 2nd edn. 2012. https://www.fda.gov/downloads/food/ foodborneillnesscontaminants/ucm297627.pdf 3. centres for disease control and prevention (us). antibiotic resistance threats in the united states, 2019. centres for disease control and prevention, us department of health and human services; 2019. http://www.cdc.gov/drugresistance/threatreport-2019. 4. mosalagae d, pfukenyi dm, matope g. milk producers’ awareness of milk-borne zoonoses in selected smallholder and commercial dairy farms of zimbabwe. tropical animal health and production. 2011 mar 1;43(3):733-9. https://doi.org/10.1007/s11250-010-9761-5 5. world health organization. antimicrobial resistance: global report on surveillance. world health organization;2014. https://apps.who.int/iris/bitstream/handle/10665/112642/9789 241564748eng.pdf 6. threlfall ej, ward lr, frost ja, willshaw ga. the emergence and spread of antibiotic resistance in food-borne bacteria. international journal of food microbiology. 2000 dec 5;62(1-2):1-5. https://doi.org/10.1016/s0168-1605(00)00351-2 7. joshi lr, tiwari a, devkota sp, khatiwada s, paudyal s, pande kr. prevalence of methicillin-resistant staphylococcus aureus (mrsa) in dairy farms of pokhara, nepal. international journal of veterinary science. 2014;3(2):87-90. http://www.ijvets.com/pdffiles/volume-3-no-2-2014/87-90.pdf 8. rasheed mu, thajuddin n, ahamed p, teklemariam z, jamil k. antimicrobial drug resistance in strains of escherichia coli isolated from food sources. revista do instituto de medicina tropical de são paulo. 2014 aug;56(4):341-6. https://10.1590/s003646652014000400012 9. badri am, ibrahim it, mohamed sg, garbi mi, kabbashi as, arbab mh. prevalence of extended-spectrum beta-lactamase (esbl) producing escherichia coli and klebsiella pneumoniae isolated from raw milk samples in al jazirah state, sudan. mol. biol. 2017;7(1):201.https://10.4172/2168-9547.1000201 10. batabyal k, banerjee a, pal s, dey s, joardar sn, samanta i, isore dp, singh ad. detection, characterization, and antibiogram of extended-spectrum beta-lactamase escherichia coli isolated from bovine milk samples in west bengal, india. veterinary world. 2018 nov;11(10):1423. https://10.14202/vetworld.2018.1423-1427 11. riva a, borghi e, cirasola d, colmegna s, borgo f, amato e, pontello m, morace g. methicillin-resistant staphylococcus aureus in raw milk: prevalence, sccmec typing, enterotoxin characterization, and antimicrobial resistance patterns. journal of food protection. 2015 jun 1;78(6):1142-6. https://doi.org/10.4315/0362-028x.jfp-14-531 12. andrews wh, hammack ts. bam: food sampling/preparation of sample homogenate. retrieved july. 2003 apr;19:2016. https://www.fda.gov/food/laboratory-methods-food/bamchapter-1-food-samplingpreparation-sample-homogenate 13. cheesbrough m. district laboratory practice in tropical countries, part 2. cambridge university press; 2006 mar 2. 14. singh p, prakash a. isolation of escherichia coli, staphylococcus aureus and listeria monocytogenes from milk products sold under market conditions at agra region. acta agriculturae slovenica. 2008 nov;92(1):83-8. http://aas.bf.uni-lj.si 15. clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing. clsi supplement m100. 2013. 16. bis dw. 10500: 1991. first revision, bureau of indian standards, india. 1992. 17. acharya s, bimali nk, shrestha s, lekhak b. bacterial analysis of different types of milk (pasteurized, unpasteurized and raw milk) consumed in kathmandu valley. tribhuvan university journal of microbiology. 2017;4:32-8. https://doi.org/10.3126/tujm.v4i0.21674 18. dftqc . annual bulletin. department of food technology and quality control, babar mahal, kathmandu, nepal,8-9. 2019 http://www.dftqc.gov.np/downloadfile/dftqc_english_annu al_buletin_book_1591495040.pdf 19. parajuli a, rimal p, maharjan r, chaudhary r, chaturwedi sb. quality analysis of milk in kathmandu valley. tribhuvan university journal of microbiology. 2018 sep 26;5:7-10. https://doi.org/10.3126/tujm.v5i0.22295 20. arjyal c, dahal bn, khadka b. microbial quality of milk available in kathmandu valley. journal of the nepal medical association. 2004 may 1;43(153).https://doi.org/10.31729/jnma.475 21. shrestha s, bindari yr. prevalence of sub-clinical mastitis among dairy cattle in bhaktapur district, nepal. international journal of agriculture and biosciences. 2012;1(1):16-9. http://www.ijagbio.com/wp-content/uploads/pdf-files/ volume1-issue-1-2012/16-19.pdf 22. uddin ma, motazzim-ul-haque hm, noor r. isolation and identification of pathogenic escherichia coli, klebsiella spp. and staphylococcus spp. in raw milk samples collected from different areas of dhaka city, bangladesh. stamford journal of microbiology. 2011;1(1):19-23. https://doi.org/10.3329/sjm.v1i1.9098 23. titouche y, hakem a, salmi d, yabrir b, chenouf n, chergui a, chenouf a, houali k. assessment of microbiological quality of raw milk produced at tizi ouzou area (algeria). https://10.3923/ajava.2016.854.860 24. bano s, hayat m, samreen t, asif m, habiba u, uzair b. detection of pathogenic bacteria staphylococcus aureus and salmonella sp. from raw milk samples of different cities of pakistan. natural science. 2020; 12, 295-306. https://doi.org/10.4236/ns.2020.125026 25. singh a, chhabra d, sikrodia r, shukla s, sharda r, audarya s. isolation of e. coli from bovine mastitis and their antibiotic sensitivity pattern. international journal of current microbiology and applied sciences. 2018; 7(10): 11-18. https://doi.org/10.20546/ijcmas.2018.710.002 26. massawe hf, mdegela rh, kurwijila lr. antibiotic resistance of staphylococcus aureus isolates from milk produced by smallholder dairy farmers in mbeya region, tanzania. int. j. one health. 2019;5:31-7. https://doi:10.14202/ijoh.2019.31-37 27. aliyu y, abdullahi io, whong cz, olalekan bo, reuben rc. occurrence and antibiotic susceptibility of methicillin-resistant staphylococcus aureus in fresh milk and milk products in nasarawa state, north-central nigeria. journal of microbiology and antimicrobials. 2020 jan 31;12(1):32-41. https://doi.org/10.5897/jma2020.0424 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:90-95 doi: https://doi.org/10.3126/njb.v7i1.26958 ©njb, biotechnology society of nepal 90 nepjol.info/index.php/njb. original research article vancomycin intermediate mrsa isolates obtained from retail chicken meat and eggs collected at pokhara, nepal surya prasad devkota 1, 2, 3*, ashmita paudel 3, krishna gurung 1, 4 1pokhara bigyan tatha prabidhi campus, nayabazzar, pokhara 2 school of health and allied sciences, pokhara university 3regional college of health science and technology, nayabazzar, pokhara 4prithvi narayan campus, pokhara abstract antimicrobial resistance among food animal isolates is increasing as a result of their uncontrolled uses. the monitoring of antibiotic resistance among these isolates is very necessary. s aureus was isolated from eggshells and chicken meat samples collected from different retail outlets of the pokhara metropolitan. samples were inoculated on mannitol salt agar aseptically and inoculated overnight. isolated yellow colonies were further examined by gram-staining, catalase, and coagulase test to detect s aureus. methicillin resistance was screened using cefoxitin disc. vancomycin minimum inhibitory concentration (mic) of methicillin-resistant s aureus (mrsa) isolates were determined by the agar dilution method following clsi guidelines. 139 s aureus were isolated from 205 samples. among them, 89 were from egg samples (out of 125 samples) and 50 from chicken (out of 80 samples). the overall prevalence of mrsa was 12.94%. antibiotic resistance was significantly higher in mrsa isolates compared to methicillin sensitive s aureus (mssa) isolates. the highest rate of resistance was noted for ampicillin, amoxicillin, and erythromycin while the least resistance was noted for gentamicin and amikacin. vancomycin minimum inhibitory concentration (mic) range of the mrsa isolates was 0.25-8µg/ml indicating the detection of both vancomycin-intermediate and sensitive isolates from the samples. this is the first study reporting vancomycin-intermediate s aureus (visa) isolates from nepal and indicates the increasing drug resistance among animal isolates. further surveillance studies about the transmission of these pathogens to humans as well as detail molecular analyses are imminent. keywords: mrsa, vancomycin-intermediate, s aureus, eggs, meat *corresponding author email: devkotasp1@gmail.com introduction staphylococcus aureus is a significant pathogen that causes diseases both in humans and animals and their treatment is gradually becoming difficult due to their increasing resistance to antimicrobial agents [1]. dry resistance properties of the organism assist them to thrive in adverse environmental conditions including various food items, human skin, nose and other inanimate objects for a longer period [2]. the presence of these organisms in animals has started gaining importance these days as they are the cause of increasing livestock infections and their ability to cause zoonotic infections [3]. staphylococcus associated with animals may be hazardous to humans not only due to their antibiotic resistance but also as the causative agents of food-related infections [4]. the presence of drug-resistant bacteria in meat is a significant public health challenge and the incidence of resistance has risen in this decade among pathogens that are transmitted via food [5]. methicillin-resistant s. aureus (mrsa) has been detected from various animals used for food production and from animal-related food items including milk, meat, and dairy products which necessitate the targeted researches to determine the prevalence of these isolates in food to assess the probable risk of these foods as sources for human infection [6]. in addition to two common types of mrsa i.e community-associated mrsa and hospital-associated mrsa, a third form of mrsa known as livestock-associated mrsa has now emerged which infects various animals [7]. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 90-95 devkota et al. 2019 ©njb, biotechnology society of nepal 91 nepjol.info/index.php/njb. there is the possibility of transmission of mrsa isolates associated with livestock not only between animals and humans but also between humans [8]. there are reports of decreasing vancomycin sensitivity among s aureus isolated in nepal in recent times [9]. the study of adhikari et al have reported that vancomycin mic range for s aureus isolated from pus samples was 0.016µg/ml to 1µg/ml [10]. very recently, vancomycin-resistant and vancomycin-intermediate s aureus have been isolated from clinical samples [11]. many studies have reported that all clinical s aureus isolates from nepal were absolutely sensitive to vancomycin [12, 13]. however, there have not been any studies that focus on vancomycin mic value of s aureus isolated from non-clinical samples including food animals, food samples, and environmental samples in nepal. there is a huge and uncontrolled use of antibiotics in animal farms in nepal. this might have led to the development of resistance among animal isolates. in this study, we particularly focused on s. aureus isolates as they are one of the frequent isolates from eggs and meat samples. however, there are no studies about the effects of this unscientific use of antibiotics on these animal isolates. this is the first study focusing on vancomycin mic values of mrsa isolates obtained from these samples in this region and the country as well. materials and methods samples chicken eggs and chicken meat samples were collected in sterile zipped plastic bags from different retail outlets of pokhara metropolitan from january to february and july to august 2019. the number of samples collected was 125 and 80 respectively for eggs and chicken meat. sample processing eggshells were swabbed thoroughly using the sterile cotton swabs moistened with sterile nutrient broth and inoculated on mannitol salt agar using aseptic technique. similarly, 5 grams of each meat sample was transferred to 50 ml of sterile nutrient broth in a sterile environment and incubated overnight. then one loopful of the broth was inoculated on mannitol salt agar aseptically. sterile forceps, mask, and gloves were used during sample processing to prevent contamination during the procedure. the inoculated plates were then incubated at 37˚c for 18-24 hrs. identification of s aureus after incubation, the mannitol salt agar plates were observed for yellow colonies. isolated yellow colonies on mannitol salt agar were further tested using gram-staining, catalase test, oxidase test, and coagulase test. gram-positive cocci in a cluster that was catalase-positive, coagulase-positive were confirmed as s aureus as reported earlier [4]. ast of s. aureus isolates and mrsa screening kirby bauer disc diffusion method was used to study the antimicrobial susceptibility pattern of the s. aureus isolates as per clsi guidelines [14]. antibiotics used were amikacin (30µg), ciprofloxacin (5µg), ampicillin (10µg), amoxycillin (10µg), gentamicin (10µg), erythromycin (15µg), and cefoxitin (30µg). cefoxitin was used for the screening of mrsa isolates. isolates with a zone of inhibition 21 mm or less were confirmed as mrsa isolates as per clsi guidelines [14]. mrsa isolates were preserved using glycerol stock preparation and by stabbing on semi-solid media for vancomycin mic detection. vancomycin mic detection the agar dilution method was used to detect mic of the isolates. 526 mg of vancomycin powder (hi-media, india with potency 950 µg/mg) was dissolved in 10 ml sterile distilled water to prepare the stock solution (50 mg/ml). then tenfold dilutions (two times) of the stock were prepared to prepare working solutions having a concentration of 0.5 mg/ml. from the working solution, further dilutions were made on brain heart infusion agar. various dilutions of vancomycin prepared were 0.25µg/ml, 0.5µg/ml, 1µg/ml, 2µg/ml, 4µg/ml, 8µg/ml, 16µg/ml, and 32µg/ml as reported earlier [15]. staphylococcus aureus atcc 25923 was used as a negative control. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 90-95 devkota et al. 2019 ©njb, biotechnology society of nepal 92 nepjol.info/index.php/njb. mac-farland standard (0.5) suspension of each test isolates was prepared by mixing one or two isolated colonies on blood agar. each isolate was then inoculated on brain heart infusion agar containing various dilutions of vancomycin and incubated at 37°c for 24 hours and observed for presence or absence of growth as indicated earlier [16]. lowest dilution of vancomycin preventing notable growth was considered as the mic value for that isolate. isolates were classified as vancomycin sensitive, intermediate, and resistant if the mic value were ≤2µg/ml, 48µg/ml, and ≥16µg/ml respectively based on clsi guidelines [14]. results a total of 125 chicken eggs were processed during the study period. among them, 50 were analyzed during winter and 75 during the summer season. similarly, 80 meat samples were included for the study (35 in winter and 45 in summer). in winter isolation rate of s aureus from eggs was 68 % (34 from 50 samples). only 2 isolates (5.88%) were confirmed as mrsa. likewise, in the same season, 21 s aureus was isolated from the meat sample. among them, 5 (23.8%) were confirmed as mrsa (table 1). the prevalence of both s aureus and mrsa was slightly higher in summer. during this period 73.33 % of egg samples were positive for s aureus. only four s aureus isolates (out of 55 total isolates) from egg were mrsa. similarly, 64.44 % of chicken samples yielded s aureus. the incidence of mrsa was 24.13% in chicken s aureus isolates (table 1). both vancomycin sensitive and vancomycinintermediate mrsa isolates were detected both from egg and chicken samples. however, the mic value was higher for meat isolates. mic range of vancomycin for egg isolates was 0.25 to 4µg/ml. similarly, the mic range was 0.25 to 8µg/ml for chicken isolates (table 3). the majority of egg mrsa isolates were vancomycin sensitive while only 1 isolate (16.6%) isolates were vancomycin-intermediate. 8 (66.66%) of the chicken meat isolates were sensitive to vancomycin and the remaining 4(33.33%) isolates were vancomycin-intermediate. this result indicates that there is reduced vancomycin susceptibility among mrsa isolates obtained from meat and egg. this increase in resistance may be either due to the direct use of vancomycin in animal farms or due to the use of similar antibiotics for various purposes. if we consume these food products without proper cooking they may cause infections that are difficult to treat. in addition to consumers, the workers working in slaughterhouse and retail meat shops are at high risk of acquiring these pathogens as they work there without proper safety measures. if the current practices of animal farming, processing, and distribution of meat and egg samples continue, vancomycin resistance may be observed in s aureus isolates from these food items soon. hence, all the concerned sectors should be converged to minimize the increasing drug-resistance among various food isolates. discussion antibiotic resistance among animal and food animal isolates has been increasing throughout the world. this problem is more severe in developing countries due to the lack of proper monitoring of antibiotics use and the lack of awareness about the adverse effects of antibiotic table 1. prevalence of mrsa isolates from meat and egg samples study period sample number s aureus mrsa no (%) janfeb 2019 chicken eggs 50 34 2 (5.88%) chicken 35 21 5 (23.8%) julaug 2019 chicken eggs 75 55 4 (7.27%) chicken 45 29 7 (24.13%) table 2: resistance pattern of mrsa and mssa isolates antibiotics mrsa (n=18) mssa (n=121) ampicillin 11(61.1%) 21(17.3%) amoxycillin 9(50%) 19(15.7%) amikacin 7 (38.8%) 12(9.9%) gentamicin 8 (44.4%) 11 (9%) ciprofloxacin 10 (55.5%) 13 (10.7%) erythromycin 10 (55.5%) 15 (12.3%) co-trimoxazole 9 (50%) 14 (11.5%) cefoxitin 18 (100%) 0 (0%) nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 90-95 devkota et al. 2019 ©njb, biotechnology society of nepal 93 nepjol.info/index.php/njb. resistance. this problem is further complicated by the lack of proper hygiene during animal slaughter and the use of contaminated equipment and water during the processing of meat and eggs before sending it to the retail market. hence this study was designed to monitor the antibiotic resistance pattern of mrsa and mssa isolates obtained from meat and egg samples as well as to access the vancomycin mic value of the mrsa isolates obtained from these samples. the overall prevalence of s aureus among egg and chicken samples was 59.02% (121 out of 205 samples). among the isolates, 89 were from eggs and the remaining 50 isolates were from meat samples. this result indicates gross contamination of these food products with s aureus and is the indication of poor hygiene practices. the very similar isolation rate of s aureus was reported in nigeria from meat and other food items [17]. while the prevalence was significantly different in other studies [18, 19]. the prevalence of this bacteria varies based on geographic location and sanitation status. the average incidence of mrsa from both samples was 12.94% (6.74% among egg isolates and 24% among chicken isolates). almost identical incidence of mrsa was noted among chicken samples from nigeria [20]. on the other hand, a low prevalence of mrsa was also noted from meat and other food items in china [21], and no mrsa isolates at all from meat, egg, and other food isolates in china [22] as indicated by the absence of meca gene in all isolates. the prevalence of mrsa from various meat and other food samples was comparatively high in algeria compared to this study [23]. antibiotic resistance was quite high among mrsa isolates compared to mssa isolates as the resistance range for the tested antibiotics was 917.3% for mssa while the range was 38-61% for mrsa isolates. resistance was high for ampicillin (61.1%), ciprofloxacin (55.5%), and erythromycin (55.5%) while low for gentamicin (44.4%) and amikacin (38.8%) for mrsa isolates. one study from china [22] has shown increased drug resistance of s aureus isolated from food items as these isolates were resistant to penicillin (92.47%), erythromycin (58.06%), and kanamycin (25.8%). another study has also reported elevated antibiotic resistance among s aureus isolated from raw meat samples collected from various provinces of china where the resistance pattern was 83.7 % for penicillin, 52.1% for erythromycin, and 17.4 % for ciprofloxacin [24]. these findings are comparable with the resistance pattern of isolates obtained in the current study. to the best of our knowledge, this is the first study reporting vancomycin mic value of mrsa isolates obtained from food samples in nepal. similarly, this is the first report indicating the detection of vancomycin intermediate methicillin resistant s aureus isolated from retail chicken meat and egg samples. similar to the finding of this study various authors also have reported vancomycin-intermediate s aureus from various food samples obtained from animals. vancomycin intermediate isolates (mic 8µg/ml) have been detected in india from milk samples [25]. not only this, several reports are indicating the presence of vancomycin-resistant s aureus (vrsa) isolates from various food samples. the study of bhattacharyya et al [25] reported vancomycin-resistant staphylococcus from milk samples. likewise, vrsa isolates were reported from egypt from diverse samples including minced meat, mastitis milk, and sausage samples with vancomycin mic range of 64-1024µg/ml [26]. vrsa also has been detected from camel meat and slaughterhouse workers in egypt with a prevalence of 35% among s aureus isolates [27]. though vancomycin resistance was not detected in this study further studies are very necessary throughout the nation including more sample size and diverse samples as only large scale study can provide an actual picture of this problem. timely surveillance of these pathogens is also table 3: vancomycin minimum inhibitory concentration (mic) values of mrsa isolates sample total isolates 0.25µg/ml no (%) 0.5µg/ml no (%) 1µg/ml no (%) 2µg/ml no (%) 4µg/ml no (%) 8µg/ml no (%) 16µg/ml no (%) 32µg/ml no (%) egg 6 1 (16.6) 1(16.6) 2 (33.3) 1(16.6) 1(16.6) 0 (0) 0 (0) 0 (0) meat 12 1 (8.33) 2 (16.6) 3 (25) 2 (16.6) 2 (16.6) 2 (16.6) 0 (0) 0 (0) nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 90-95 devkota et al. 2019 ©njb, biotechnology society of nepal 94 nepjol.info/index.php/njb. necessary. physician and microbiology labs should not completely rely on vancomycin disc test for the susceptibility of mrsa isolates and must determine the vancomycin mic values before treatment. they should be updated with the recent susceptibility trend of these isolates of the particular locality as well. conclusion this study reported the existence of vancomycinintermediate s aureus isolates in nepal and typically in pokhara from meat and egg samples. rise of vancomycin-intermediate s aureus isolates among various food isolates is a serious concern as there is a possibility of transmission of these isolates to the consumers. hence, further epidemiological and molecular analyses are needed to prevent their spread to humans. similarly, the prudent use of antibiotics in animal farms is urgent. limitations of the study vancomycin mic of the mssa isolates couldn’t be performed. similarly, molecular analysis (meca gene detection) of the mrsa isolates was not done due to limited laboratory facilities and economical constrain. acknowledgment we are very thankful to pokhara bigyan tatha prabdhi campus, pokhara for allowing us to conduct this research in their laboratory and all the 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and meat products in china: incidence, antibiotic resistance and genetic diversity. front microbiol. 2018 9: 2767. 19. higenyi j, kabasa jd: microbial contamination load of hatching eggs in butaleja, eastern uganda. anim vet sci. 2014 2: 22-30. 20. igbinosa eo, beshiru a, akporehe lu, oviasogie fe, igbinosa oo: prevalence of methicillinresistant staphylococcus aureus and other staphylococcus species in raw meat samples intended for human consumption in benin city, nigeria: implications for public health. int j environ res public health. 2016 13(10): 949. 21. wu s, huang j, zhang f, wu q, zhang j, pang r et al: prevalence and characterization of food related methicillin resistant staphylococcus aureus (mrsa) in china. front microbiol. 2019 10:1-13. 22. ma y, zhao y, tang j, et al: antimicrobial susceptibility and presence of resistance & enterotoxins/enterotoxin-likes genes in staphylococcus aureus from food. cyta journal of food. 2018, 16(1): 76-84. 23. chaalal w, chaalal n, bourafa n, et al: characterization of staphylococcus aureus isolated from food products in western algeria. foodborne pathog. dis. 2018, 15: 353– 360. 24. wang w, baloch z, jiang t, et al: enterotoxigenicity and antimicrobial resistance of staphylococcus aureus isolated from retail food in china. front. microbiol. 2017, 8:2256. 25. bhattacharyya d, banerjee j, bandyopadhyay s, et al: first report on vancomycin-resistant staphylococcus in bovine and caprine milk. microb drug resist. 2016, 22(8): 675-681. 26. abd el-aziz nk, abd el-hamid mi, bendary mm, et al: existence of vancomycin resistance among methicillin resistant s aurues recovered from animal and human sources in egypt. slov vet res. 2018, 55 (suppl 20): 221-30. 27. al-amery k, elhariri m, elsayed a, et al: vancomycin-resistant staphyloocus aureus isolated from camel meat and slaughterhouse workers in egypt. antimicrob resist infect control. 2019 5(8):129 https://www.ncbi.nlm.nih.gov/pubmed/30740152 https://www.ncbi.nlm.nih.gov/pubmed/?term=wu%20s%5bauthor%5d&cauthor=true&cauthor_uid=30498486 https://www.ncbi.nlm.nih.gov/pubmed/?term=huang%20j%5bauthor%5d&cauthor=true&cauthor_uid=30498486 https://www.ncbi.nlm.nih.gov/pubmed/?term=wu%20q%5bauthor%5d&cauthor=true&cauthor_uid=30498486 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc6249422/ https://www.ncbi.nlm.nih.gov/pubmed/?term=igbinosa%20eo%5bauthor%5d&cauthor=true&cauthor_uid=27669285 https://www.ncbi.nlm.nih.gov/pubmed/?term=beshiru%20a%5bauthor%5d&cauthor=true&cauthor_uid=27669285 https://www.ncbi.nlm.nih.gov/pubmed/?term=akporehe%20lu%5bauthor%5d&cauthor=true&cauthor_uid=27669285 https://www.ncbi.nlm.nih.gov/pubmed/27669285 https://www.ncbi.nlm.nih.gov/pubmed/?term=bhattacharyya%20d%5bauthor%5d&cauthor=true&cauthor_uid=26990514 https://www.ncbi.nlm.nih.gov/pubmed/?term=banerjee%20j%5bauthor%5d&cauthor=true&cauthor_uid=26990514 https://www.ncbi.nlm.nih.gov/pubmed/?term=bandyopadhyay%20s%5bauthor%5d&cauthor=true&cauthor_uid=26990514 https://www.ncbi.nlm.nih.gov/pubmed/26990514 https://www.ncbi.nlm.nih.gov/pubmed/?term=al-amery%20k%5bauthor%5d&cauthor=true&cauthor_uid=31404199 https://www.ncbi.nlm.nih.gov/pubmed/?term=elhariri%20m%5bauthor%5d&cauthor=true&cauthor_uid=31404199 https://www.ncbi.nlm.nih.gov/pubmed/?term=elsayed%20a%5bauthor%5d&cauthor=true&cauthor_uid=31404199 https://www.ncbi.nlm.nih.gov/pubmed/31404199 https://www.ncbi.nlm.nih.gov/pubmed/31404199 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 43-46 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 43 nepjol.info/index.php/njb prevalence of non-albicans candida among the patients attending a tertiary care hospital in kathmandu, nepal manisha sharma1, narayan dutt pant2*, pratikshya pandey3 1department of microbiology, kathmandu medical college, kathmandu, nepal. 2medical microbiologist, department of microbiology, grande international hospital, dhapasi, kathmandu, nepal. 3quality control officer, department of microbiology, mark formulations pvt. ltd., kathmandu, nepal. abstract the main objective of this study was to determine the prevalence of non-albicans candida among the patients attending a tertiary care hospital in kathmandu, nepal. candida spp. isolated from different clinical samples (sputum, urine, vaginal swab, blood, endotracheal (et) secretion, pus) from 250 patients between the period of february 2013 and december 2015 were included in the study. of those 250 patients, 20% were immunocompromised. sabouraud dextrose agar was used for the isolation of candida spp. and the identification was performed on the basis of colony morphology, gram’s stain, india ink preparation, germ tube test, temperature tolerance test, characteristic color change in chromagar, chlamydospore production, sugar fermentation test and sugar assimilation test. out of total 300 candida spp., majority were isolated from sputum (43.33%) followed by urine (40%) and vaginal swab (6.67%). of total 151 (50.33%) non-albicans candida, the most common species isolated were c. tropicalis (62.25%) followed by c. glabrata (23.84%). high prevalence of non-albicans candida among the patients attending a hospital in kathmandu, nepal was noted. key words: candida, non-albicans candida, nepal *corresponding author email: ndpant1987@gmail.com introduction incidence of fungal infection has increased significantly over the past few years, causing considerable morbidity and mortality, mainly affecting immunocompromised patients. the emergence of fungal infections as the worldwide health care problems may be attributed to the extensive use of broad spectrum antibiotics and immune-suppressive agents, as well as an increase in the population of immunocompromised people [1]. candida is one of the commonest fungal pathogens and may cause infection in immunocompetent as well as immunocompromised person [2]. due to presence of candida spp. as normal flora of mucosal surfaces, they mostly cause endogenous infections [3]. candida albicans is considered as the commonest species of candida responsible for causing various infections [4]. but in recent years there have been significant increase in infections caused by the species of the non-albicans candida [5]. only small numbers of species among the 150 species of candida are well established as human pathogens [6]. among them, candida glabrata, candida parapsilosis and candida tropicalis are non-albicans candida of increasing clinical significance [5]. most of non-albicans candida like c. glabrata, c. krusei, c. tropicalis and c. parapsilosis, which are of clinical significance are known to show resistance toward certain commonly used antifungal agents [7, 8]. so, for optimizing the treatment of the infections caused by candida spp., it is necessary to identify the candida spp. up to species level, even if it is not possible to perform antifungal susceptibility testing [9]. further the isolation of the candida spp. from the clinical samples like sputum, urine, vaginal swab etc. does not necessarily suggest the infection and clinical correlation or confirmation of infection by alternative methods is necessary. however, the high rate of isolation of candida spp. from clinical specimens (mainly from debilitated patients) suggests the possibility of high rate of endogenous infections by these organisms. in nepal there are limited data regarding the rate of isolation of non-albicans candida from different clinical specimens. so, in this study we determined the nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 43-46 sharma et al. ©njb, biotechnology society of nepal 44 nepjol.info/index.php/njb prevalence of non-albicans candida among the patients attending a tertiary care hospital. materials and methods candida spp. isolated from different clinical samples (sputum, urine, vaginal swab, blood, endotracheal secretion, pus) from 250 patients between the period of february 2013 and december 2015, were included in the study. of those 250 patients, 20% were immunocompromised. the clinical specimens were collected using standard techniques [10]. for the isolation of the candida spp. the samples were inoculated on sabouraud dextrose agar and were incubated aerobically at 35oc for 48 hrs. easily emulsifiable, white, opaque, dome or flat shaped colonies were subjected to further identification by gram’s stain and india ink preparation. the candida spp. were identified up to species level by using the following methods: germ tube test: this test was used for the preliminary identification of the candida albicans. a small inoculum of the yeast cells from pure culture was suspended in 0.5 ml of sheep serum and was incubated at 37oc for three hours. then a drop of the incubated serum was observed under microscope using 40x objective. the isolates were identified as germ tube producing or germ tube non-producing. temperature tolerance: this test was also used for the presumptive identification for candida albicans. the isolates were cultured on sabouraud dextrose agar and incubated aerobically at 45°c for 72 hours and observed for any growth if present. chromagar candida: chromagar candida (himedia, mumbai, india) was used for presumptive identification of various candida species. the pure culture was seeded into chromagar media and incubated at 35°c for 48 hours. the media was observed for characteristic color change. chlamydospore production: it was used for the preliminary confirmatory identification of candida albicans. the isolates were inoculated on corn meal agar by slide culture technique and incubated at 25°c for 72 hours and observed for chlamydospore production using lactophenol cotton blue stain. sugar fermentation test: six percent solution of dextrose, maltose, lactose and sucrose with basal media were used for the test. sugar assimilation test: sugars used for this test were glucose, lactose, maltose, sucrose and galactose. ethics statement: our study was in compliance with helsinki declaration. results between february 2013 and december 2015, 300 candida spp. were isolated from various clinical samples. majority of the candida spp. were isolated from sputum (43.33%) followed by urine (40%) and vaginal swab (6.67%) (figure 1). figure 1: sample wise distribution of candida spp. 0 20 40 60 80 100 120 140 no. of samples 130 120 20 15 10 5 table 1: candida spp. isolated from different clinical samples. candida spp. sputum urine vaginal swab et secretion blood pus total c. albicans 65 56 10 8 6 4 149 c. tropicalis 43 40 6 2 2 1 94 c. glabrata 10 16 3 5 2 _ 36 c. kefyr 7 6 _ _ _ _ 13 c. krusei 3 _ 1 _ _ _ 4 c. dubliniensis 2 2 _ _ _ _ 4 total 130 120 20 15 10 5 300 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 43-46 sharma et al. ©njb, biotechnology society of nepal 45 nepjol.info/index.php/njb among total 300 candida spp. isolated, 149 (49.67%) were candida albicans and 151 (50.33%) were nonalbicans candida. out of 151 non-albicans candida spp., c. tropicalis (62.25%) was the most common nonalbicans candida followed by c. glabrata (23.84%) (table 1). discussion in spite of their ubiquitous presence, only a few fungi are considered pathogenic. skin, nail and hair are the commonest sites of fungal infections. but recently there have been increased numbers of systemic fungal infections due to the use of broad spectrum antibiotics, increase in the numbers of immunocompromised persons (due to conditions like lymphomas, leukemia, organ transplantation, human immunodeficiency virus (hiv) infection and use of immunosuppressive drugs) [11]. among the fungal pathogens, candida is a leading cause of a variety of human infections [2]. in our study the rate of isolation of non-albicans candida (among all candida spp.) was > 50%; which was in accordance to the result reported by deorukhkar et al. (63.3%) [2]. in the present study, among total 130 candida spp. isolated from sputum, the rate of isolation of c. albicans was 50% followed by c. tropicalis (33.076%) and c. glabrata (7.69%). similar to our observation, in a study by jha et al., the commonest species of candida isolated from sputum was found to be c. albicans (70%) followed by candida tropicalis (13.33%) [3]. however, the isolation of the candida spp. from sputum in our study did not necessarily suggest the infection and clinical correlation or confirmation of infection by alternative method was necessary. but the isolation of the candida spp. from the immunocompromised people (having predisposing risk factors) suggests the higher risk of endogenous infection. the main risk factors responsible for increased numbers of respiratory tract infections by candida spp. are smoking, chronic obstructive pulmonary disease, tuberculosis, malnutrition, malignancy, diabetes mellitus, human immunodeficiency virus infection and prolonged use of antibiotics [3]. in urine samples, the majority of candida spp. isolated were c. albicans (46.67%) followed by c. tropicalis (33.3%) and c. glabrata (19.2%). among all candida spp. the rate of isolation of non-albicans candida from urine was 53.33%. our finding was in accordance with the findings of deorukhkar et al. [2] and kauffmann [12], in which >50% of the candida spp. isolated from urine samples were non-albicans candida. along with the well adaptability of the nonalbicans candida spp. for urinary tract infection, it is more difficult to treat the infection caused by them in comparison to that caused by c. albicans [2]. old age, diabetes mellitus, pregnancy and urinary catheterization are the predisposing factor for urinary tract infection by candida spp. in a study by helmy, 14 % of the cases of vulvovaginal candidiasis were due to non-albicans candida [8] but in our study 50% of the candida spp. isolated from vaginal swab were non-albicans candida. however, in both studies the c. tropicalis followed by c. glabrata were the most common species of candida isolated from vaginal swab. in the study done by deorukhkar et al. the predominant candida spp. responsible for causing vulvovaginal candidiasis were found to be c. glabrata followed by c. tropicalis [2]. although, the isolation of the candida spp. from urine and vaginal swab does not necessarily suggest the infection; the high rate of isolation of these organisms from patients having risk factors for infection by candida spp. could not be neglected. in our study 20 % of the patients were immunocompromised. all the patients from whom candida spp. were isolated from endotracheal secretion were from intensive care unit and were intubated. and nonalbicans candida isolated from their endotracheal secretion were 43.75% of all the candida spp. isolated from endotracheal secretion. this indicates that the intubation is a very important risk factor for acquiring yeast infection. in our study, 40% of the non-albicans candida were isolated from the cases of candidemia. the predominant non-albicans candida isolated from blood were c. glabrata followed c. tropicalis. among all candida spp., we found c. albicans to be the most predominant cause of candidemia. but in the study by deorukhkar et al. c. glabrata was the nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 43-46 sharma et al. ©njb, biotechnology society of nepal 46 nepjol.info/index.php/njb commonest species isolated from the cases of candidemia [2]. despite the isolation of c. glabrata (mortality rate due to infection by which is higher) from only 20% of the cases of the candidemia, in our study the mortality rate for the cases of candidemia was 80% [2]. in our study rate of isolation of candida spp. was higher among patients from intensive care unit and those having history of treatment with broad spectrum antibiotics (for long period of time). the use of broad spectrum antibiotics upsets the balance of the normal bacterial flora and results in infection by candida spp. [2]. further the patients from intensive care unit are debilitated. due to adaptation of the candida spp. to various habitats including medical devices, the incidence of hospital acquired infection due to candida spp. has increased [2]. candida spp. have ability to form biofilm and in the study by deorukhkar et al. the biofilm forming capability has been found to be higher in c. tropicalis as compared to c. albicans. this attribute can help non-albicans candida like c. tropicalis to show higher drug resistance [2]. further, some non-albicans candida are intrinsically resistant to some antifungals mainly in context of opportunistic infections in immunocompromised [4]. conclusion high prevalence of non-albicans candida among the patients attending a hospital in kathmandu, nepal was noted. from our study, it can be concluded that non-albicans candida may be of high clinical significance mainly in case of the patients with predisposing risk factors. author’s contribution ndp and ms designed the study, performed the research work, analysed the data and prepared the final manuscript. pp contributed in analyzing of the data. competing interests the authors declare that they have no competing interests. acknowledgement the authors would like to thank all who contributed directly or indirectly in carrying out of this research. references 1. agrawal v, bhagwat am, vishalakshi v, gode v, sawant cs: exploring the potential of chromogenic medium for the identification of medically important yeast species other than candida. int j pharm pharm sci. 2014, 6(3):291-294. 2. deorukhkar sc, saini s, mathew s. non-albicans candida infection: an emerging threat. interdiscip perspect infect dis. 2014; 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8(1):12-16 doi: https://doi.org/10.3126/njb.v8i1.30205research article ©njb, bsn 12 phenolic compounds from the aerial parts of adenophora triphylla (thunb.) a. dc. var. triphylla and their free radical scavenging activity kengo hori1, hari prasad devkota 1,2 1graduate school of pharmaceutical sciences, kumamoto university, 5-1 oe-honmachi, chuo-ku, kumamoto 862-0973, japan 2program for leading graduate schools, health life sciences: interdisciplinary and glocal oriented (higo) program, 5-1 oe-honmachi, chuo-ku, kumamoto 862-0973, japan article history:-received: 17 may 2020; revised: 19 jun 2020; accepted: 25 jun 2020; published online: 31 jul 2020 abstract adenophora triphylla (thunb.) a. dc. var. triphylla (family: campanulaceae) is distributed in japan, korea, and china. it is locally known as “saiyousyajin” in japan and the roots are used in traditional medicine to treat chronic bronchitis and whooping cough, and also as anti-inflammatory and antitussive agents. till now, there is no report on the chemical constituents of aerial parts. thus, the main aim of this study was to isolate and identify major chemical constituents of aerial parts of a. triphylla var. triphylla, and to evaluate their free radical scavenging activity. the 70% methanol extract of the aerial parts was subjected to repeated column chromatography using mci gel chp-20p, sephadex lh-20, ods and silica gel columns to isolate the five phenolic components (1-5). free radical scavenging activity of the extract and compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (dpph) free radical scavenging activity method. the structures of the isolated compounds were elucidated as luteolin (1), luteolin 4’-o-β-glucopyranoside (2), luteolin 7-o-βglucopyranoside (3), luteolin 7-o-neohesperidoside (4) and chlorogenic acid (5) based on their nuclear magnetic resonance (nmr) spectral data and comparison with literature values. all these compounds were isolated for the first time from a. triphylla var. triphylla. the extract showed weak free radical scavenging activity. among isolated compounds, luteolin (1), luteolin 7-o-β-glucopyranoside (3), luteolin 7-oneohesperidoside (4) and chlorogenic acid (5) showed potent free radical scavenging activity. results from this study suggest that the aerial parts of a. triphylla var. triphylla might be a potential plant source for the development of functional foods, however further detailed research is necessary. keywords: adenophora triphylla; saiyousyajin; phenolic compounds; free radical scavenging corresponding author, email: devkotah@kumamoto-u.ac.jp introduction medicinal plants and their phytochemicals have played a vital role in human healthcare as an important source of traditional medicines, drug discovery and development of nutritional and functional foods [1–3]. however, many plant species are yet to be explored for their chemical constituents and potential biological activities and healthpromoting effects. the genus adenophora belonging to belonging to campanulaceae family consists of about 62 species distributed in east asia and europe, among which, 12 species are distributed in japan [4]. adenophora triphylla (thunb.) a. dc. var. triphylla (“saiyousyajin” in japanese) and a. triphylla (thunb.) a. dc. var. japonica (regel) h. hara (“tsuriganeninjin” in japanese) are among many varieties of plant a. triphylla (thunb.) a. dc. and are distributed in japan, korea, and china [4, 5]. roots of both of these plants are used in traditional medicine to treat chronic bronchitis and whooping cough and as an anti-inflammatory and antitussive agents [5–8]. there have been many studies on the chemical constituents/biological activities of roots and leaves of a. triphylla var. japonica [5, 7, 9, 10]. kim et al. [5] reported the high phenolic and flavonoid contents and potent free radical scavenging activities of leaves of a. triphylla var. japonica, however, no nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:devkotah@kumamoto-u.ac.jp https://orcid.org/0000-0002-0509-1621 nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):12-16 hori and devkota ©njb, bsn 13 such studies are reported on the aerial parts of a. triphylla var. triphylla for the best of our knowledge. thus, in this study, the main aim was to isolate and identify major chemical constituents of aerial parts of a. triphylla var. triphylla and to evaluate their free radical scavenging activity. materials and methods general experimental procedure 1hand 13cnmr spectra were measured on bruker avance 600 nmr spectrometer (bruker, billerica, ma, usa) (1h-nmr: 600 hz and 13c-nmr: 150 hz). chemical shift values (δh and δc) are given in ppm with reference to tetramethyl silane (tms). column chromatography (cc) was carried out with mci gel chp20p (75 ~ 150 μm, mitsubishi chemical industries co. ltd., tokyo, japan), sephadex lh-20 (amersham pharmacia biotech, tokyo, japan) and silica gel 60 (0.040-0.063 mm, merck kgaa, darmstadt, germany). thin layer chromatography (tlc) was performed on a pre-coated silica gel 60 f254 (aluminum sheet, merck kgaa, darmstadt, germany). plant material the aerial parts (stems and leaves) of a. triphylla var. triphylla were collected from mt. tawarayama kumamoto, japan in august 2015 and shade dried for two weeks. plant material was identified by mr. masato watanabe, technical officer, school of pharmacy, kumamoto university. chemicals 1,1-diphenyl-2-picrylhydrazyl (dpph) was purchased from sigma aldrich, co. (tokyo, japan). 6–hydroxy2, 5, 7, 8– tetramethylchroman–2carboxylic acid (trolox) was from wako pure chemical industries, ltd. (tokyo, japan) and 2morpholinoethanesulfonic acid monohydrate (mes) was purchased from dojindo chemical research (kumamoto, japan). extraction and isolation the dried aerial parts (2600 g) were then extracted twice with 70% meoh (18 l). the combined extract was evaporated under reduced pressure to give 414.0 g of extract. the extract was suspended in water and subjected on mci gel chp20p column chromatography (cc) and eluted successively with water, 40%, 70% and 100% meoh to give eight fractions (1~8). fraction 2 (109.4 g, h2o eluate) was subjected on sephadex lh-20 cc (h2o) to obtain four subfractions (2-1~2-4). subfraction 2-3 was subjected on mci gel cc (h2o) to afford compound 5 (153.8 mg). fraction 7 (3.05 g, 80% meoh eluate) was subjected on sephadex lh-20 cc (meoh) to afford compound 1 (174.4 mg). fraction 6 (4.0 g, 60% meoh eluate) was subjected to sephadex lh-20 cc (h2o-meoh; 1:1) to afford compound 2 (211.4 mg). table 1. proton nmr spectroscopic data of compound 1 4 (h, mult. (j in hz)) position 1a 2 b 3 a 4 b 3 6.65, s 6.56, s 6.74, s 6.57, s 6 6.17, d (2.1) 6.18, d (2.0) 6.43, d (2.1) 6.38, d (2.1) 8 6.43, d (2.1) 6.41, d (2.0) 6.78, d (2.1) 6.73, d (2.1) 2’ 7.40, d (2.3) 7.41, d (2.3) 7.40, d (2.1) 7.38, d (2.5) 5’ 6.87, d (8.2) 7.28, d (8.5) ) 6.89, d (8.4) ) 6.89, d (8.5) )6’ 7.38, dd (8.2, 2.3) 7.39, dd (8.5, 2.3) 7.43, dd (8.4, 2.1) 7.40, dd (8.5, 2.5) glc-1 4.93, d (7.5) 5.05, d (7.5) 5.18, d (6.7) glc-2 3.40-4.00 3.40-4.00 3.40-4.00 glc-3 3.40-4.00 3.40-4.00 3.40-4.00 glc-4 3.40-4.00 3.40-4.00 3.40-4.00 glc-5 3.40-4.00 3.40-4.00 3.40-4.00 glc-6 3.40-4.00 3.40-4.00 3.40-4.00 rha-1 5.28, d (1.2) rha-2 3.40-4.00 rha-3 3.40-4.00 rha-4 3.40-4.00 rha-5 3.40-4.00 rha-6 1.33, d (6.2) a in dmso-d6, b in cd3od nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):12-16 hori and devkota ©njb, bsn 14 fraction 3 (14.3 g) and fraction 4 (2.5 g) were combined and subjected to sephadex lh-20 cc (50% meoh) followed by ods cc (20%, 30%, 40% meoh) and silica gel cc (ch2cl2: meoh: h2o =8:2:0.1) to afford compound 3 (1610.7 mg) and 4 (7.0 mg). measurement of dpph free radical scavenging activity the antioxidant potential was determined using the dpph free radical scavenging method as described previously [3]. results and discussion the 70% methanol extract of aerial parts of a. triphylla var. triphylla was subjected to various column chromatographic methods including mci gel chp20p, sephadex lh-20, ods and silica gel to afford five compounds. the structures of these compounds were elucidated as luteolin (1) [11], luteolin 4’-oβ -glucopyranoside (2) [12], luteolin 7oβ -glucopyranoside (3) [11, 12], luteolin 7-oneohesperidoside (4) [13] and chlorogenic acid (5) [14] (figure 1) based on their nmr spectral data and comparison with literature values. proton and 13c nmr data for compounds 1-4 are provided in table 1 and table 2, respectively. all of these compounds were isolated for the first time from a. triphylla var. triphylla. previously, hashiba et al. [10] had reported flavonoids including luteolin (1), luteolin 7-oβ glucopyranoside (3), luteolin 4’7-di-o-βglucopyranoside, quercetin, quercetin 3-o-βglucopyranoside from the leaves of a. triphylla var. japonica. characterization of similar flavonoids in the aerial parts of a. triphylla var. triphylla in this study suggests their chemotaxonomic similarity and these compounds can be used as the chemotaxonomic markers for these varieties. further studies on other varieties of a. triphylla or other species of adenophora may help explore their further similarity. the 70% extract and all isolated compounds were evaluated for their dpph free radical scavenging activity (table 3). the extract showed weak free table 2. 13c nmr spectroscopic data of compound 1 4 position 1a 2 b 3 a 4 b 2 146.8 165.4 164.5 166.9 3 135.7 105.1 103.2 105.6 4 175.8 183.8 182.0 184.0 5 160.7 163.2 161.1 163.0 6 98.1 100.3 99.6 102.5 7 163.8 166.1 163.0 164.4 8 93.3 95.1 94.8 97.3 9 156.1 159.4 156.7 159.0 10 102.9 105.5 105.4 107.1 1’ 121.9 127.2 121.4 123.5 2’ 115.4 118.0 113.6 114.3 3’ 145.0 148.6 145.8 147.1 4’ 147.6 150.0 150.1 151.2 5’ 115.5 114.9 116.0 117.0 6’ 119.9 120.0 119.2 120.6 glc-1 103.3 99.9 99.6 glc-2 74.8 73.1 78.3 glc-3 77.5 76.4 79.0 glc-4 71.3 69.6 71.4 glc-5 78.5 77.2 79.1 glc-6 62.3 60.7 62.5 rha-1 101.0 rha-2 72.2 rha-3 72.2 rha-4 74.0 rha-5 70.0 rha-6 18.3 a in dmso-d6, b in cd3od. figure 1. chemical structures of compounds isolated from a. triphylla var. triphylla nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):12-16 hori and devkota ©njb, bsn 15 radical scavenging activity with ic50 value of 248.3 µg/ml. compounds 1, 3, 4, and 5 showed potent free radical scavenging activity with ic50 values of 10.6, 14.3, 20.4 and 19.3 µg/ml, respectively as compared to positive control trolox (ic50=12.8 µg/ml). table 3. ic50 (µg/ml) values of extract and isolated compounds of a. triphylla var. triphylla for dpph free radical scavenging activity compounds ic50 value (µg/ml) 70% methanol extract 248.3±2.4 luteolin (1) 10.6±0.7 luteolin 4’-oβ glucopyranoside (2) 62.9±0.9 luteolin 7-oβ glucopyranoside (3) 14.3±1.5 luteolin 7-oneohesperidoside (4) 20.4±2.4 chlorogenic acid (5) 19.3±2.1 trolox (positive control) 12.8± 1.1 in recent years, there is growing attention on the plant-based functional foods. previously, kim et al. [5] reported the high phenolic and flavonoid contents and free radical scavenging activities of leaves of a. triphylla var. japonica but the active compounds were not reported. in our study, the extract of aerial parts of a. triphylla var. triphylla showed weak activity but the isolated compounds showed strong free radical scavenging activity. hashiba et al. [10] had also reported similar flavonoids from a. triphylla var. japonica, hence, luteolin derivatives can be regarded as the active antioxidant compounds in both of these verities. flavonoids including luteolin derivatives are well reported as strong antioxidant phytochemicals with various health-promoting and disease prevention activities [15–20]. further detailed research on these plants may help in the development of functional foods. conclusion five bioactive phenolic compounds; luteolin (1), luteolin 4’-oβ -glucopyranoside (2), luteolin 7-oβ -glucopyranoside (3), luteolin 7-o-neohesperidoside (4) and chlorogenic acid (5) were isolated for the first time from the leaves a. triphylla var. triphylla. some of the isolated compounds showed potent free radical scavenging activity. the aerial parts of a. triphylla var. triphylla might be an important plant source for the development of functional foods. however, detailed research related to pharmacological activity and safety are necessary in future. author’s contribution hpd conceived the idea and designed the study. both authors contributed to experiments and analysis, and wrote the manuscript. competing interest no competing interests were disclosed. funding this work was supported partially by program for leading graduate schools, health life sciences: interdisciplinary and glocal oriented (higo) program, mext, japan acknowledgments we are grateful to ms. teruo tanaka of institute of resource development and analysis, kumamoto university for the measurement of nmr. ethical approval and consent not applicable. references 1. belwal t, devkota hp, hassan ha, ahluwalia s, ramadan mf, mocan a, atanasov ag. phytopharmacology of acerola (malpighia spp.) and its potential as functional food. trends in food science & technology. 2018 apr 1;74:99-106. https://doi.org/10.1016/j.tifs.2018.01.014 2. atanasov ag, waltenberger b, pferschy-wenzig em, linder t, wawrosch c, uhrin p, temml v, wang l, schwaiger s, heiss eh, rollinger jm. discovery and resupply of pharmacologically active plant-derived natural products: a review. biotechnology advances. 2015 dec 1;33(8):1582-614. https://doi.org /10.1016/j.biotechadv.2015.08.001 3. dirar ai, alsaadi dh, wada m, mohamed ma, watanabe t, devkota hp. effects of extraction solvents on total phenolic and flavonoid contents and biological activities of extracts from sudanese medicinal plants. south african journal of botany. 2019 jan 1;120:261-7. https://doi.org/ 10.1016/j.sajb.2018.07.003 4. ohashi h, monda y, murata j, yonekura k, kihara h. wild flowers of japan. tokyo: heibonsha. 2017. 5. kim jh, hong jy, shin sr, yoon ky. comparison of antioxidant activity in wild plant (adenophora triphylla) leaves and roots as a potential source of functional foods. international journal of food sciences and nutrition. 2009 jan 1;60(sup2):150-61. https://doi.org/10.1080/09637480902956594 6. suzuki h. encyclopedia of traditional oriental medicine. ishiyaku publishers. 2005. 7. ahn ek, oh js. lupenone isolated from adenophora triphylla var. japonica extract inhibits adipogenic differentiation through the downregulation of pparγ in 3t3‐ l1 cells. phytotherapy research. 2013 may;2 7(5):7 61-6. https:// doi.org/10.1002/ptr.4779 nepal j biotechnol. j u l y 2 0 2 0 ; 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revised: 21 dec 2020; accepted: 22 dec 2020; published online: 30 dec 2020 abstract barley diseases are the major yield limiting factors for barley cultivation in nepal. stripe/yellow rust (p. striformis f.sp. hordei and p. striformis f.sp. tritici), leaf rust (puccinia hordei), and crown rust (p. coronata) are the major rust diseases in nepal. pyramiding resistance genes against all these rust diseases are possible through molecular marker assisted breeding. sweden originated barley variety ‘bonus’ is found resistant to stripe rust and having linked microsatellite markers for stripe rust and crown rust resistance. similarly, nepalese hull-less barley variety ‘solu uwa’ and nepalese awn-less barley landrace npgr acc# 2478 have linked microsatellite markers for leaf rust resistance. therefore, one polymorphic sequence tagged sites (sts) marker (abg054) for stripe rust resistance, two polymorphic simple sequence repeats (ssr) markers (bmac0144h and hvm049) for leaf rust and one polymorphic ssr marker (bmag0006) for crown rust resistance were used to select the advanced barley lines (at f8 stage) from above parents. field screening of stripe rust resistance was also conducted. among 51 advanced and field disease resistance lines from bonus/solu uwa cross, 10 pyramided lines for all three types of barley rust resistance were selected. similarly, among 39 advanced and field disease resistance lines from bonus/npgr acc#2478 cross, three pyramided lines were selected and advanced for further yield testing for general cultivation purpose. the chances of losing the desired gene are higher in late generation selection using molecular marker assisted selection (mas), but the chances of getting agronomically superior varietal output is expected to increase. keywords: rust, pyramiding, advanced lines, barley, marker assisted selection (mas) corresponding author, email: reshamamgain@yahoo.com introduction barley diseases are the major yield limiting factors in nepal. among many barley diseases, rust diseases are critical from the crop production view. strip/yellow rust (caused by puccinia striformis f.sp. hordei and p. striformis f.sp. tritici) is prevalent rust in the nepalese barley field. prasad et al. [1] also observed it as a major disease causing a problem in the nepalese barley field. however, leaf rust (caused by p. hordei) can be observed in some warm barley cultivating areas. crown rust of barley (caused by p. coronata) can be observed very sporadically only. any barley variety having resistance gene for all three types of rust pathogen is highly sought in nepalese barley breeding program. since, nepalese barley germplasm has a high grain yielding capacity for hill and mountain regions of the country [2], adding rust resistance characteristics to them may improve their yield and stability. selection, identification and incorporation of rust resistance genes is the only option for the development of rust resistance barley varieties for nepal. therefore, pyramiding major rust resistance genes for barley will be beneficial to farmers. molecular markers are highly preferred for gene pyramiding program like this. the sporadic nature of the crown rust occurrence in the nepalese barley field and overlapping of leaf rust and stripe rust in the disease screening field further pushed molecular marker assisted selection (mas) as the most viable option for gene pyramiding for rust resistance varietal development. materials and methods parent and advanced lines selection a swedish introduced variety ‘bonus’ is the two-rowed stripe rust resistance barley variety for nepal [2] and also have linked microsatellite markers for stripe rust and crown rust resistance. the polymorphic linked microsatellites are described in ‘identification of polymorphism in parents’ sub-heading. similarly, nepalese hull-less barley variety ‘solu uwa’ and nepalese awn-less barley landrace ‘npgr acc# 2478’ has linked microsatellite markers for leaf rust resistance. therefore, crosses between bonus with solu uwa and acc #2478 will have a lot of chances of having pyramided lines. use of marker assisted selection at early stage of barley breeding such as in f2 and f3 is practically not feasible in our context due to cost, time and manpower shortage. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:reshamamgain@yahoo.com https://orcid.org/0000-0002-3651-4359 mailto:reshamamgain@yahoo.com nepal j biotechnol. 2020 dec; 8 (3): 111-115 amgai et al. ©njb, bsn 112 so, we have selected 51 advanced barley lines at f8 stage from ‘bonus’ and ‘solu uwa’ crosses. similarly, we have also selected 39 advanced barley lines at f8 stage from ‘bonus’ and ‘acc#2478’ crosses to detect the pyramided lines (table 1). all the selected lines showed the field disease resistance. similarly, they are forwarded to f8 based on their superior agronomic characteristics comparing to their parents. field rust evaluation two rows per line were sown at khumatar, lalitpur during normal barley growing season (november to april) in 2017. spacing between each row was 20 cm and the length of the row was 1.5m. resistance and susceptible parental lines were sown repeatedly after every 15 advanced lines. a susceptible landrace ‘local jau’ was used in two spreader rows around the disease screening plots. modified cobb scale [3] was used for rust scoring at the heading stage for all three types of rust. identification of polymorphism among parents a series of microsatellite markers linked with rust resistance genes were screened to identify the polymorphic markers among the parents. sequence tagged sites (sts) marker abg54, and simple sequence repeats (ssr) markers bmac144h, hvm49 and bmag6 were found polymorphic among the parents (table 2). these markers were used for the selection of advanced barley lines. dna extraction and pcr reaction modified ctab method as described by sul and korban [4] was used to extract the genomic dna of selected barley advanced lines. pcr reaction mixture of 15 µl table 1. list of barley advanced breeding lines used for molecular marker assisted selection. bonus/solu uwa-3 bonus/solu uwa-51 bonus/solu uwa99 bonus/solu uwa153 bonus/acc#2478201 bonus/acc#2478231 bonus/solu uwa-6 bonus/solu uwa-54 bonus/solu uwa102 bonus/solu uwa156 bonus/acc#2478202 bonus/acc#2478235 bonus/solu uwa-9 bonus/solu uwa-57 bonus/solu uwa105 bonus/solu uwa159 bonus/acc#2478204 bonus/acc#2478238 bonus/solu uwa-12 bonus/solu uwa-60 bonus/solu uwa108 bonus/acc#2478162 bonus/acc#2478205 bonus/acc#2478244 bonus/solu uwa-15 bonus/solu uwa-63 bonus/solu uwa111 bonus/acc#2478165 bonus/acc#2478206 bonus/acc#2478246 bonus/solu uwa-18 bonus/solu uwa-66 bonus/solu uwa114 bonus/acc#2478168 bonus/acc#2478209 bonus/acc#2478248 bonus/solu uwa-21 bonus/solu uwa-69 bonus/solu uwa117 bonus/acc#2478171 bonus/acc#2478210 bonus/acc#2478254 bonus/solu uwa-24 bonus/solu uwa-72 bonus/solu uwa126 bonus/acc#2478174 bonus/acc#2478213 bonus/acc#2478257 bonus/solu uwa-27 bonus/solu uwa-75 bonus/solu uwa129 bonus/acc#2478177 bonus/acc#2478216 bonus/acc#2478259 bonus/solu uwa-30 bonus/solu uwa-78 bonus/solu uwa132 bonus/acc#2478180 bonus/acc#2478218 bonus/acc#2478268 bonus/solu uwa-33 bonus/solu uwa-81 bonus/solu uwa135 bonus/acc#2478183 bonus/acc#2478222 bonus/acc#2478278 bonus/solu uwa-36 bonus/solu uwa-84 bonus/solu uwa138 bonus/acc#2478186 bonus/acc#2478225 bonus/solu uwa-39 bonus/solu uwa-87 bonus/solu uwa141 bonus/acc#2478189 bonus/acc#2478227 bonus/solu uwa-42 bonus/solu uwa-90 bonus/solu uwa144 bonus/acc#2478192 bonus/acc#2478228 bonus/solu uwa-45 bonus/solu uwa-93 bonus/solu uwa147 bonus/acc# 2478195 bonus/acc#2478229 bonus/solu uwa-48 bonus/solu uwa-96 bonus/solu uwa150 bonus/acc#2478198 bonus/acc#2478230 table 2. polymorphism observed in parental lines for different molecular markers and disease characteristics parent field stripe rust abg054 (stripe rust qtl) bmac0144h (leaf rust-r gene) hvm049 (leaf rustrph19) bmag0006 (crown rust-rpc1) bonus 0 1 0 0 1 solu uwa 10s 0 1 1 0 acc#2478 60s 0 0 1 1 note: number in bracket is the linked resistance gene. acc# = npgr accession number nepal j biotechnol. 2020 dec; 8 (3): 111-115 amgai et al. ©njb, bsn 113 volume was prepared using 1.5 µl (1 µm) for each primer, 7.5 µl of pcr master mix (promega corporation, usa), 2.5 µl water and 2 µl (100ng) dna template. this pcr mixture was amplified as per the following protocol. for marker abg54, bmag6 and bmac144h thirty cycles: denaturation 30 sec at 95oc, annealing 1 min with temperature as per table 3 and extension 2 min at 72oc. for marker hvm49 touch down pcr protocol was followed as 18 cycles of 1 min denaturation at 94oc, 30 sec of touchdown protocol with decreasing 1oc per 2 cycle from 64oc until 55oc as annealing and 1 min at 72oc for the extension. this touchdown cycle was followed by another 30 cycles of 1 min denaturation at 94oc, 1 min annealing at 55oc and 1 min extension at 72oc. the final extension was 7 min at 72oc and the final holding is at 4oc. the pcr products were separated in 2% agarose gel in 1xtae buffer at 100v for one hour. gels were stained with ethidium bromide (0.1 µg/ml) and visualized under uv rays. the presence of a particular band size (table 3) was considered the presence of a particular linked gene. results we have observed stripe rust in susceptible parents; however, we had not observed leaf rust and crown rust in khumaltar conditions (table 2). this suggests that the use of mas techniques is very essential for pyramiding any resistance gene that cannot be screened in field condition at that time. many breeding lines showed the presence of one or more genes for the rust resistance based on the particular marker band (figure 1-4). however, the pyramided lines were very limited than our expectation for both types of crosses (table 4 and table 5). figure 1. amplification of ssr marker hvm049 (105bp) in barley advanced lines. (note: parents are underlined and bold; and selected lines are bold with asterisk mark) figure 2. amplification of ssr marker abg054 (180bp) in barley advanced lines from bonus/solu uwa. (note: parents are underlined and bold; and selected lines are bold with asterisk mark) table 3. list of molecular markers showing polymorphism and used in selection process marker name forward primer [5' … 3'] reverse primer [5'… 3'] annealing temperature resistance gene pcr product size chromosome no. reference abg054 gtgcttgg cggtcga ccagt gatgtccaac ggtggcttga 55 stripe rust (qtl) 180bp* 4h [5] bmag0006 ttaaaccc cccccctc tag tgcagttact atcgctgatt tagc 58 crown rust (rpc1) 174 3h [6] bmac0144h tacgtgta catactct acgatttg acttattctg catcctgggt 55 leaf rust (r-gene) 179 1h [7] hvm049 ctctatag gcacgaa aaattcc ttgcacatat ctctctgtca ca 55 leaf rust (rph19) 105 7h [8] note: *=field disease resistance data is used to identify the product size nepal j biotechnol. 2020 dec; 8 (3): 111-115 amgai et al. ©njb, bsn 114 figure 3. amplification of ssr marker hvm049 (105bp) in barley advanced lines. (note: parents are underlined and bold; and selected lines are bold with asterisk mark) figure 4. amplification of ssr marker abg054 (180bp) in barley advanced lines from bonus/acc#2478. (note: parents are underlined and bold; and selected lines are bold with asterisk mark) discussion selection on the late stage of the breeding may lead to eroding many useful lines with the important genes that showed the neutral effect in the previous season of field disease screening. the leaf rust and crown rust could not be screened in khumaltar condition for all previous seasons, which ultimately lead us a few lines with leaf rust and crown rust resistance along with stripe rust resistance. but, the agronomic characteristics of our selected lines are superior and always safe from ending with disease resistance but poor yielding varieties. due to the less polymorphism between the parent bonus and acc#2478; we can select the lines with pyramided stripe rust and leaf rust resistance linked markers only. the linked marker for crown rust resistance found in ‘bonus’ is also found in nepalese landrace ‘acc#1478’ (table 2). we identified linked markers for the leaf rust resistance gene in nepalese local variety ‘solu uwa’ and landrace ‘acc#1478’ which support our observation of barley field at khumaltar and surroundings with negligible infection from leaf rust. higher leaf rust resistance in nepalese barley is also supported by the observation of tyrshkin [9] and henderson [10]. tyrshkin [9] also concluded that nepalese barley germplasm nb-3002 has one dominant gene for leaf rust resistance. table 4. list of selected advanced breeding lines from crosses between bonus and solu uwa with corresponding molecular marker polymorphism. line field stripe rust abg054 bmac0144h hvm049 bmag0006 bonus 0 1 0 0 1 solu uwa 10s 0 1 1 0 bonus/solu uwa-30 0 1 0 1 1 bonus/solu uwa-33 0 1 0 1 1 bonus/solu uwa-45 0 1 0 1 1 bonus/solu uwa-48 0 1 0 1 1 bonus/solu uwa-60 0 1 1 1 1 bonus/solu uwa-63 0 1 1 1 1 bonus/solu uwa-81 0 1 1 0 1 bonus/solu uwa-90 0 1 1 1 1 bonus/solu uwa-135 0 1 1 1 1 bonus/solu uwa-138 0 1 0 1 1 note: 1 = present, 0 = absent table 5. list of selected advanced breeding lines from bonus and acc#2478 cross with corresponding molecular marker polymorphism. line field stripe rust abg054 hvm049 bonus 0 1 0 acc#2478 60s 0 1 bonus/acc#2478-186 0 1 1 bonus/acc#2478-189 0 1 1 bonus/acc#2478-209 0 1 1 note: 1 = present, 0 = absent nepal j biotechnol. 2020 dec; 8 (3): 111-115 amgai et al. ©njb, bsn 115 similarly, we also observed that hull-less parent (‘solu uwa’) is less stripe rust susceptible than the hulled parent (‘acc#2478’) (table 2) as baniya et al. [11] already concluded for covered (hulled) barley and naked (hullless) barley collection for nepal. conclusion selection in an early generation for marker assisted selection (mas) program is considered a thumb rule; however, in the condition where laboratory resource is poor and costly than growing crops in the field, late generation selection for pyramided lines using molecular techniques will be still competitive. on one hand, the chances of losing the desired gene (or marker) are high; but in another hand, the chances of getting agronomical superior varietal output will also increase by late use of mas techniques since early generation selection on mas largely depends on particular gene/marker rather than crop performance itself. author’s contribution rba selected parents and made the crosses. sup, ap, ss advanced the lines and maintain them. rba, sup did disease scoring. rba, shp, ss did dna extraction, pcr and gel electrophoresis. rba did data analysis, wrote and finalized the manuscript. all the authors read and approved the final manuscript. competing interests no competing interests were disclosed. funding part of this research is conducted under ng-narc-fund # 411. acknowledgements not applicable. ethical approval and consent not applicable. references 1. prasad rc, karki cb, sharma s. pathological report on barley. hill crops proceedings. national hill crops research program, kabre dolakha, nepal; 1993. pp 52-78. 2. riley kw, singh km. diversity and stability of barley in nepal. 1980 [cited 2020 nov 5]. available from: https://idl-bncidrc.dspacedirect.org/bitstream/handle/10625/6009/40369.pdf 3. peterson rf, campbell ab, hannah ae. a diagrammatic scale for estimating rust intensity of leaves and stem of cereals. can j res sci. 1948; 26:496-500. doi: https://doi.org/10.1139/cjr48c-033 4. sul iw, korban ss. a highly efficient method for isolating genomic dna from plant tissues. plant tiss. cult. biotech. 1996; 2: 113-116. 5. rossi c. testing the effectiveness of barley stripe rust resistance qtl detected in mexico and the usa against a possible new race in peru and mapping of genes conferring resistance to leaf rust and mildew in the same population [ms thesis]. [oregon]: oregon state university; 2005. 91p. 6. agrama ha, dahleen l, wentz m, jin y, steffenson b: 2004. molecular mapping of the crown rust resistance gene rpc1 in barley. phytopathology. 2004; 94(8):858-861. doi: 10.1094/phyto.2004.94.8.858 7. jafary h, albertazi g, marcel tc, niks re. high diversity of genes for non-host resistance of barley to heterologous rust fungi. genetics. 2008; 178: 2327-2339. doi: 10.1534/genetics.107.077552 8. park rf, poulsen d, barr ar, cakir m, moody db, raman h and read bj. mapping genes for resistance to puccinia hordei in barley. australian j agricultural research. 2003; 54: 1323-1333. https://doi.org/10.1071/ar02244 9. tyryshkin lg: genetic control of effective leaf rust resistance in collection accessions of barley (hordeum vulgare l). russian journal of genetics 2009, 45 (3): 376-378. https://doi.org/10.1134/s1022795409030181 10. henderson, mt. studies of sources of resistance and inheritance of reaction to leaf rust (puccinia anomala rostr.) in barley [ph.d. thesis]. [minneapolis]: university of minnesota, minneapolis, 1945. 11. baniya bk, riley rw, dongol dms, sherchand kk. characterization of nepalese hill crop landraces (barley, buckwheat, finger millet, grain amaranth, foxtail, proso and barnyard millets). national hill crops research program, nepal agriculture research council, dolakha, nepal; 1992. pp 7-17. nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 1-6 research article doi: https://www.doi.org/10.54796/njb.v9i2.41892 ©njb, bsn 1 isolation, identification and screening of bacillus species with antimicrobial activity from different soil samples of kathmandu valley aishwarya thapa , anupa budhathoki, anupama sapkota, muskan sainju, prativa shrestha, shyam prasad pant department of microbiology, st. xavier’s college, maitighar, kathmandu, nepal received: 21st oct 2020; revised: 24th jun 2021; accepted: 26th dec 2021; published online: 31st dec 2021 abstract bacillus species are one of the predominant soil bacteria that are able to produce essential secondary metabolites that have antagonistic effects on other microorganisms. they are gram-positive, endospore-forming, chemoheterotrophic, aerobic or facultative anaerobic rods usually consisting of peritrichous flagella for motility. the major aim of this study was to isolate the antimicrobials producing bacillus spp. from soil samples of different parts of the kathmandu valley, identify them and to assess their antimicrobial activity against different pathogenic bacteria. the test organisms used were staphylococcus aureus (atcc 25923), e. coli (atcc 25922), pseudomonas spp., salmonella spp., methicillin-resistant staphylococcus aureus (mrsa) and extended spectrum beta-lactamase (esbl) producing e. coli. twenty four isolates from 9 soil samples identified as bacillus spp. showed the zone of inhibition around their growth on nutrient agar during isolation. these 24 isolates were chosen for primary screening of production of antimicrobial by perpendicular streaking method using four test organisms. . of these 24 isolates, six isolates showing a significant zone of inhibition (≥1mm) against two or more test organisms from the primary screening were chosen for secondary screening which was further tested with six test organisms including esbl e.coli and mrsa. they were further characterized through different physiological and biochemical tests. all 6 isolates showed inhibitory action against mrsa and the largest zone of inhibition (30mm) was shown by isolate u6. isolate u3 was found to have broad spectrum antimicrobial activity with inhibitory effect against gram negative organismspseudomonas and salmonella and gram positive organism s. aureus (atcc 25923). isolate u5 showed a zone of inhibition of about 25mm against s. aureus which was comparable to that of erythromycin. hence, this study determines the soil in kathmandu valley as a potential source of antimicrobial producing bacillus spp. and recommends isolation and further characterization of bacillus isolates as a possible source of novel drug to combat with the emergence of multidrug resistant strains. keywords: antimicrobials, kathmandu, bacillus spp., staphylococcus aureus, mrsa corresponding author, email: aaishu.thapa01@gmail.com introduction members of the bacillus genus are the predominant soil bacteria with resistant-endospore forming ability and play a major role in organic matter decomposition, biotransformation, biogas production and nitrogen fixation by associating with the plant roots which ultimately promote the growth of the plants [1,2]. they exhibit a wide range of physiological abilities and produce several metabolites with antagonistic effects as a strategy to survive, eliminate competition with other existing organisms and colonize their natural habitat. most of the bacillus species accompany actinomycetes and other antibiotic producers in the ecosystem. therefore, they might have acquired resistance to antibiotics from such sources [3,4]. they produce a wide range of antimicrobial compounds that have different chemical structures and stability through a broad range of ph and temperature and are resistant to enzyme treatments to some extent, therefore, can be used as antibacterial, antifungal and antiviral agents [5]. a bacillus strain is singly capable to produce different antimicrobial compounds and each compound can be active only against the same or closely related species i.e. other gram positive bacteria or may have broad spectrum activity, for example, bacteriocins usually show action against closely related bacteria [4, 6]. most of the species from the genus bacillus are considered as safe microorganisms and they are easier to handle in the lab with a low incubation period i.e. only 24-48hrs thereby, making bacillus spp. a preferable microorganism to investigate antimicrobial properties [6]. the multidrug resistant pathogens are emerging at an alarming rate, and this situation can be linked to inappropriate usage and shortage on the part of the manufacturers causing a steady decline of efficient antibiotics. moreover, it has created a consequential issue in the treatment of infectious disease, so, the exploration on this topic is yet important for the discovery of novel antibiotics with new metabolites from thus far unscathed nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-2571-1197 mailto:aaishu.thapa01@gmail.com nepal j biotechnol. 2021 dec.9 (2): 1-6 thapa et al. ©njb, bsn 2 habitats of potential sources that help in controlling the problem [7]. kathmandu valley lies in an alluvial plain where melting snow of four different mountain ranges in the himalayas feeds rivers and streams that flow down from the mountain. based on the variation in different locations of the valley, soil type and their composition, there is a possibility of different diversified habitat. therefore, the quest of discovering novel microflora and finding antimicrobial producing bacillus spp. can be fulfilled [8]. thus, the project will help in analysis of distribution and antimicrobial activities of bacillus spp. collected from the sampling sites. materials and methods sample collection nine soil samples were collected from different areas of the kathmandu valley (organically cultivated fields, rhizospheric area and river banks) which were processed during the study. the samples were collected from the depth of 10-12.5 cm in the sterile polythene bags and taken to the laboratory. the sample collection sites were sundarijal, balaju, bhaktapur, sitapaila and dillibazar and the samples were designated as s1, s2, s3, s4, s5, s6, s7, s8 and s9. the samples s1 and s8 were from bhaktapur (nagarkot latitude: 27°4254°n, longitude: 85°3114°e and 27°4015.7°n, 85°2621.3°e), s2 from dillibazar (27.7054° n, 85.3267° e), s3 from sundarijal (27.7909° n, 85.4272° e),s4 from shivapuri (27.8129° n, 85.3859° e) ,s5 from gokarneshwor ( 27° 44' 2.688" n 85° 22' 55.38" e), s6 and s7 from balaju (27.73192° n, 85.29945° e and 27.7309° n, 85.2955° e) and s9 from sitapaila (27.7170° n, 85.2735° e). isolation and screening of antimicrobial producing species the bacteria were isolated using a spread plate technique after heat treatment of dilutions (10-4 and 10-5) for 10 minutes in a water bath at 80℃ [9]. the zone of inhibition producing colonies were observed and selected preliminarily on nutrient agar. initially, the isolated colonies were identified by gram staining and spore staining and then sub-cultured. primary screening was done by ‘perpendicular streaking method’ in which a vertical line of isolates was streaked on nutrient agar (with 2% agar to prevent spreading colonies of bacillus spp.) and incubated at 37˚c for 24 hrs. the test organisms are s. aureus, e. coli, pseudomonas spp. and salmonella spp. (standardized with 0.5 mcfarland, with cell suspension 108 cfu/ml) were then streaked perpendicular to the line of growth of isolate. these plates were then incubated overnight at 37˚c .the zone of inhibition was measured respectively after the complete incubation [10]. characterization of antimicrobial producing isolates characterization of bacillus spp. was done on the basis of morphological properties, biochemical tests and growth at a temperature at 55℃ and salt concentration of 6.5% nacl according to bergey's manual of determinative bacteriology [11]. production of crude extract six isolates with high antimicrobial activity through primary screening were selected and inoculated a loopful of sample in about 25 ml of nutrient broth (nb) in a conical flask and incubated for 48 hrs at 37˚c in the shaker incubator. the broth culture was then transferred in tubes and centrifuged at 2012 g for 20 min. after the centrifugation, the pellets were discarded and the supernatant was mixed with ethyl acetate (1:1 ratio), collected in screw cap tubes using sterile dropper and then centrifuged at 6000 rpm for 20 min. the upper layer was collected and transferred to vials. this extract was labeled as crude extract [12]. agar well diffusion method six test organisms, mrsa, esbl e. coli (available at the research laboratory of st. xavier’s college, maitighar, kathmandu, nepal) and four same test organism as used in the primary screening and were taken and swabbed on muller hinton agar plates after standardization with 0.5 mcfarland solution. on the agar plate, 5 wells of 8 mm diameter using sterile cork borer and 150 µl of crude extract was poured in each well. they were then incubated at 37oc without inverting for 24 hrs. the ethyl acetate was used as negative control and antibiotic discs erythromycin (15 mcg), nitrofurantoin (300 mcg), vancomycin (30 mcg) and gentamicin (30 mcg) (himedia laboratories) were used as positive control. [12] results isolation and identification of the bacillus spp. from the 9 soil samples collected from different parts of the kathmandu valley, 24 isolates that produced a zone of inhibition around their growth on the isolation media i.e. nutrient agar selected for identification and screening. the zone of inhibition around them is suggestive of antimicrobial(s) produced by them that diffuse through the media and inhibit growth of other microorganisms around them. these isolates were identified as bacillus spp. on the basis of gram staining (gram positive rods) and spore staining (sporulating). nepal j biotechnol. 2021 dec.9 (2): 1-6 thapa et al. ©njb, bsn 3 primary screening of the antimicrobial producing isolates the 24 isolates of bacillus spp. were chosen for primary screening to detect their ability to produce antimicrobial substances by perpendicular streaking method. all of the isolates were found to show the zone of inhibition around their growth in primary screening (perpendicular streaking method) against four test organisms viz. s. aureus, e. coli, pseudomonas spp. and salmonella s pp. of these 24 isolates, eight isolates produced a significant zone of inhibition (≥1mm) against any one of the test organisms (table1). characterization of antimicrobial producing isolates among these eight isolates six isolates showing a significant zone of inhibition (≥1mm) against two or more test organisms during primary screening were chosen for secondary screening and were further characterized through different morphological, physiological and biochemical tests (table 2). secondary screening of the antimicrobial producing isolates the six isolates showing a significant zone of inhibition (≥1mm) against two or more test organisms during table 1. zone of inhibition demonstrated by antimicrobial metabolite producing isolates against test organisms sample isolate number name of the isolate s. aureus e. coli pseudomonas spp. salmonella spp. s2 5 u1 1 mm 6 mm s3 1 u2 22 mm 1 mm 8 mm s4 1 u3 11 mm s5 3 u4 1 mm 1 mm 2 mm s6 1 u5 6 mm 9 mm s1 2 u6 15 mm s3 1 u7 5 mm s6 2 u8 5 mm : not inhibited; u1-8: bacillus spp. isolates table 2. characterization of the isolates based on morphology and different biochemical tests characteristics u1 u2 u3 u4 u5 u6 spore spore-former spore-former spore-former non spore-former spore-former spore-former motility motile motile non-motile non-motile motile motile cell diameter >1mm <1mm >1mm <1mm <1mm <1mm catalase + + + + + + mr + + + + vp + + + citrate + + + + starch hydrolysis + + + + nitrate reduction + + acid from glucose + + acid from mannitol + + + acid from arabinose growth at 55˚c growth in 6.5%nacl + + + + + identified as: bacillus spp. + : positive, : negative table 3. zone of inhibition demonstrated by crude antimicrobial metabolite extract against test organisms test organisms isolates mrsa s. aureus esbl producing e. coli e. coli pseudomonas spp. salmonella spp. u1 11 mm 12 mm 20 mm u2 12 mm 12 mm 15 mm u3 12 mm 12 mm 15 mm 15 mm u4 20 mm 15 mm u5 20 mm 25 mm u6 30 mm 18 mm : not inhibited nepal j biotechnol. 2021 dec.9 (2): 1-6 thapa et al. ©njb, bsn 4 primary screening were chosen for secondary screening by agar well diffusion method and tested against test organisms including mrsa and esbl producing e. coli as tabulated in table 3. the results obtained in this study showed that the isolates u3, u5 and u6 have the potential for producing antimicrobial substances inhibiting the gram positive. similarly, u1, u2 and u3 have inhibited gram negatives salmonella spp. as well. the most prominent finding of the study was isolate u5, which produced a zone of inhibition of about 25 mm against staphylococcus aureus (atcc 25923) which was comparable to the zone of inhibition produced by erythromycin against the organism. discussion the study was carried out to isolate and identify antimicrobial metabolites producing bacillus spp. from various sites of kathmandu valley in nepal. the initial selection of the antimicrobial producing strains was based on the clear zone around their colony shown by different bacterial colonies in primary culture on na plates (figure 1). in a similar study done by rai et al, the colonies with a clear halo zone were considered as antibiotic producers. the competition for the growth of a type of organism present in the sample was inhibited by the other organisms marking a boundary where no organism grew giving rise to the halo zone [12]. the identification tests performed as per bergey’s manual of determinative bacteriology showed that the isolates u1, u3 and u4 were close to those of bacillus macquariensis, b. sphaericus and b. inosolitus respectively whereas the results of isolates u2, u5 and u6 were close to that of b. subtilis (figure 2). nb used as a culture medium during production of crude extract in this study supported the production of antimicrobial compounds which was observed in the form of a zone of inhibition against the test organisms (figure 3). in contrast, a study showed that culture of b. subtilis b38 strain in nb resulted in sufficient growth, but did not exhibit any antibacterial activity [6]. extract from trypticase soy broth (tsb) showed better results than nb in another study [13]. although majority of the antimicrobial compounds do not require metal ions to perform their biological activities, there are some compounds that need metal ions to maintain their structures and physiological roles properly which may not be provided by nutrient broth and the difference in inhibitory effect of the extracts might have differed due to the type of the compound produced [14]. figure 1. isolates with antimicrobial activity on nutrient agar. figure 2. grams staining under 100x figure 3. agar-well diffusion method with test organism s. aureus (central wellnegative control). nepal j biotechnol. 2021 dec.9 (2): 1-6 thapa et al. ©njb, bsn 5 further, extracts of all 6 isolates demonstrated a zone of inhibition against mrsa with the highest zone of inhibition of 30 mm being shown by the isolate u6. it can be inferred that the antimicrobials produced by the isolates have greater potency as a novel remedy for the alarming rate of antibiotic resistance and in particular, the methicillin resistant staphylococcus aureus. the extract from the isolates showed more inhibition against gram positive bacteria than gram negative bacteria, however, e. coli and esbl producing e. coli were not inhibited by any of them. in a similar study, 63.63% colonies demonstrated inhibitory effects against gram positive bacteria and 27.27% colonies were inhibitory to gram negative bacteria. 9.09% of the colonies had a broad spectrum activity [12]. it has been reported that antimicrobial properties of genus bacillus show greater inhibition to the gram positive bacteria than gram negative bacteria in comparison. the resistance of gramnegative test strains to certain antimicrobial metabolites is due to the barrier created for the hydrophobic compounds because of the presence of outer lipopolysaccharide layer which is less permeable. it is also suggested that the produced antimicrobial substances produced by a gram-positive bacterium are restricted to other gram-positive bacteria [15,16]. in contrast, the antimicrobials produced by b. subtilis mir 15 in a study have shown inhibitory action mostly against gram-negative bacteria including e. coli and p. aeruginosa [4]. among all 6 isolates, crude obtained from isolate u3 was found to have broad spectrum activity with inhibitory effect even against gram negative organisms like pseudomonas spp. and salmonella spp. one of the important findings that this isolate was active against pseudomonas spp. is of great importance as gram negative bacterium such as pseudomonas spp. is usually resistant to a wide range of antibiotics [17]. the extracellular secretion of antimicrobial metabolites in soluble form are significant from an industrial point of view as disruption of the bacterial cells and solubilization processes can be avoided during downstream processing. the reason that we could not characterize bacillus spp. was due to unavailability of molecular tools (detection of marker genes) in the routine laboratory of st. xavier’s college, nepal and limited capital (nonfunded) for outsourcing. the increase in antibiotic resistance has been attributed to inappropriate use, inadequacies on the part of the manufacturers and leads to the steady decline of effective antibiotics annually worldwide. antibiotic resistance is present in every country. the patients with drug resistant infections consuming more healthcare resources are high risk to clinical outcome and eventually death than the patients with nonresistant infections. this situation is a serious challenge to drug manufacturers, public health practitioners worldwide [18]. reducing the spread of resistant pathogens and the rate of evolution of resistance is complex. antibiotic resistance is forcing scientists to search for these antibiotic-producing bacteria in hopes of finding new ways of killing pathogens. therefore, this study is an attempt to identify bacillus species with potential of antibiotic production that could be used to stem the scourge of drug resistance which suggest that the soil of kathmandu valley is inhabited by different antimicrobials producing bacillus spp and its proper study may create a way towards the development of novel antibiotics. conclusion this study demonstrated that the isolated strains of antimicrobial producing bacillus spp. can be used as a potent source of antibiotics as isolates u3, u5 and u6 showed an appreciable zone of inhibition against the test organisms especially with the gram positive ones including mrsa, than the gram negative ones. however, isolates u1, u2 and u3 were effective antimicrobials for gram negative organisms such as salmonella spp. and pseudomonas spp. authors' contributions at designed and conceptualized the project. at, ab, as ms and ps carried out the experiments, lab works and participated in the data analysis. sp helped to design the study, amended the methodology, managed necessary arrangements during laboratory investigations and supervised the complete study. all authors have read, edited and approved the final manuscript. competing interests no competing interests were disclosed. funding the authors declared that there were no grants were involved in supporting this work. acknowledgements our sincere gratitude to the department of microbiology, st. xavier’s college, maitighar, kathmandu, nepal for providing the laboratory facilities and all the support for completing this study. nepal j biotechnol. 2021 dec.9 (2): 1-6 thapa et al. ©njb, bsn 6 ethical approval and consent humans or human samples were not used in this study. so, ethical approval from concerned authority was not needed. references 1. mackie tj, mccartney je. mackie, mccartney practical medical microbiology. 1st ed. singapore: longman singapore publishers ltd. 1996. 2. wafula en, kinyua j, karuiki d, muigui a, mwirichia r. isolation and characterization of bacillus species from soil in ngere tea catchment area of murang’a county, kenya. international journal of life sciences research. 2014;2(3):27-35. 3. mansour a, zeinab r, amanollah za. isolation and identification of bacillus species from soil and evaluation of their antibacterial properties. avicenna j clin microb infec. 2015;2(1):e23233, doi: 10.17795/ajcmi-23233 4. aslim b, saglam n, beyatli y. determination of some properties of bacillus isolated from soil. turk j biol. 2002;26:41–8. 5. baruzzi f, quintieri l, morea m, caputo l. antimicrobial compounds produced by bacillus spp. and applications in food. science against microbial pathogens: communicating current research and technological advances. 2011;1102-1111. 6. tabbene o, slimene ib, bouabdallah f, mangoni ml, urdaci mc, limam f. production of anti-methicillin-resistant staphylococcus activity from bacillus subtilis sp. strain b38 newly isolated from soil. appl biochem biotechnol. 2009;157:407–419. 7. blomberg b. antimicrobial resistance in developing countries. tidsskr nor laegeforen. 2008;128(21):2462-2466. 8. vinod as, more sm. isolation, identification and characterization of bacillus species from lonar lake for production of cyclodextrin glycosyltransferase. int.j .curr. microbiol. app. sci. 2013;2(12):1423. 9. sharma m, manandhar m. practical approach to microbiology. 3rd ed. kathmandu: national book centre; 2017. 200 p. 10. iqbal s, qasim m, begum f, rahman h, sajid i. screening, characterization and optimization of antibacterial peptides, produced by bacillus safensis strain mk-12 isolated from waste dump soil kp, pakistan .biorxiv. 2018. doi: https://doi.org/10.1101/308205 11. goodfellow m, kampfer p, dusse hj, trujillo me, suzuki ki, ludwig w, whitman wb bergey’s manual of systematic bacteriology. 2nd ed. new york: springer; 2012. 12. rai m, aryal s, parajuli p. characterization and activity of antimicrobial polypeptide of bacillus spp from wastelands of kathmandu valley. annals of applied bio-sciences. 2017;4(1):5057. 13. kumar a, saini p, shrivastav jn. production of peptide antifungal antibiotic and biocontrol activity of bacillus subtilis. indian journal of experimental biology. 2009;47:57-62. 14. jamil b, hasan f, hameed a, ahmed s. isolation of bacillus subtilismh4 and its potential of polypeptide antibiotic production. pak. j. pharm. sci. 2007;20(1):26-31. 15. altarawni ah, alzereini wa, tarawneh ka. bacillus sp. 1a1 as a producer of antibacterial crude extract: taxonomy, cultivation and partial purification. current research in bacteriology. 2015;8:18-25. 16. motta as, olivera fc, brandelli a. screening for antimicrobial activity among bacteria isolated from the amazon basin. braz. j. microbiol. 2004;35(4):307-310 17. pang z, raudonis r, glick br, lin tj, cheng z. antibiotic resistance in pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. biotechnology advances.2019;37(1):177192. 18. antimicrobial resistance [inernet]. world health organization; 2020 [ cited 2021 march, 4]. available from: https://www.who.int /news-room/fact-sheets/detail/ antimicrobial-resistance http://dx.doi.org/10.17795/ajcmi-23233 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 79-84 research article doi: https://doi.org/10.3126/njb.v9i1.38672 special issue: icbsn 2021 ©njb, bsn 79 screening of potential plant growth promoting properties of bacillus species isolated from different regions of nepal enish pathak1, arjun sanjyal1, chhatra raj regmi1, saroj paudel2, anima shrestha1 1 department of microbiology, tri-chandra multiple campus, kathmandu, nepal 2 nepalese farming institute, kathmandu, nepal received: 29 apr 2021; revised: 28 jun 2021; accepted: 28 jul 2021; published online: 31 jul 2021 abstract the deleterious effects of intensive use of chemical fertilizers and pesticides in agriculture has led to the substantial research efforts on finding the alternatives to these agrochemicals. this study was aimed to isolate bacillus species from soil of different regions of nepal and screen for their ability to promote plant growth directly or indirectly by testing their ability to produce plant growth hormone indole acetic acid, hydrogen cyanide, ammonia and protease as well as phosphate solubilization. thirty nine bacillus strains were isolated from 25 soil samples of different regions of kathmandu and chitwan districts of nepal. these isolates were tested for plant growth promoting traits in vitro. among the total isolates, about 48.7% were indole acetic acid producers, 38.4% of the isolates showed the ability to solubilize the phosphate, 71.8% were able to produce ammonia and all the isolates had the ability to produce hydrogen cyanide and protease. the isolated strains showed positive results to maximum pgpr traits and exhibited a potential to be used as alternatives to chemical fertilizers and pesticides and could be used as low-cost bio-based technology to promote plant growth in the agricultural sector. keywords: pgpr, biocontrol agents, plant growth promotion, bacillus, biofertilizers corresponding author, email: animashrestha77@gmail.com introduction the application of chemical fertilizers has long been used in conventional agriculture. while chemical fertilizers have aided farmers in increasing crop production there are also several harmful effects of chemical fertilizers which may include water pollution, chemical burn to crops, increased air pollution, acidification of the soil and many other direct and indirect effects to the human health ecosystem itself [1]. so, substantial research efforts are now focused on finding new alternatives to supplement the use of chemicals in agriculture. the use of beneficial rhizobacteria to increase the productivity and growth of plants could be one of the substitutes to agrochemicals. the strains of bacteria available in the rhizosphere that stimulate plant growth are termed as plant growth promoting rhizobacteria (pgpr) [2]. the mechanism by which pgpr promotes plant growth can be direct or indirect [3]. pgpr can promote plant growth directly by facilitating resource acquisition i.e., fixation of atmospheric nitrogen, solubilization and mineralization of soil phosphorus, sequestering iron producing phytohormone and modulating phytohormones level by producing phytohormones like cytokinin, gibberellins, ethylene, indole acetic acid etc. biocontrol bacteria produce antibiotics, siderophores and lytic enzymes including chitinases, cellulases, proteases that cause deleterious effects to phytopathogens and indirectly promote plant growth. competition between pathogens and plant growth promoting bacteria can also check the disease incidence and severity [3]. bacillus species are abundant in the rhizosphere, so they can be one of the major aspects of bio-based products to supplant agrochemicals. bacillus spp are gram positive common rhizobacteria and widely considered as a major aspect of plant growth promoting rhizobacteria [4]. the ability to replicate rapidly and resistant to adverse environmental conditions provide a unique feature to bacillus species [5]. their ability to produce hard, resistant endospores and antibiotics that limit wide ranges of phytopathogens make bacillus spp an attractive option for biocontrol agents [6]. various researches done worldwide identified bacillus spp as pgpr [2,4-6]. but studies regarding pgpr bacillus spp in nepal are limited [7,8]. this study was thus aimed to isolate bacillus spp from soil samples of different areas of kathmandu and chitwan of nepal and screen the isolates for some direct and indirect plant growth nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-9919-476x mailto:animashrestha77@gmail.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 9 8 4 pathak et al. ©njb, bsn 80 promoting traits. this includes the test for the production of indole acetic acid (iaa), solubilization of phosphate and production of hydrogen cyanide (hcn), ammonia and protease. methodology sample collection and processing soil samples (10 g each) were collected from rhizospheric soils of different regions namely khairahani and fasera in the chitwan district and hanumate khola, jagati in the bhaktapur district of nepal. the soil samples were collected from a depth of 5-10 cm in sterile plastic bags and carried to the laboratory, nepalese farming industry, kathmandu for further processing and analysis. the study was conducted from january to march 2019. one gram of soil sample was dispensed into 99 ml of sterile distilled water and homogenized. one ml of homogenized soil sample was transferred into 9 ml sterile distilled water and serial dilution was carried out up to 10-8 dilution. serially diluted bacterial cultures (100 µl) were spread on nutrient agar media and incubated at 37°c for 24 h and examined for the appearance of colonies. identification of the isolated colonies was done on the basis of colony characteristics, gram reaction, spore staining and catalase test [5]. screening of isolates was done for plant growth promoting properties – iaa production, phosphate solubilization activity, hcn production, ammonia production and protease enzyme production. iaa production iaa production was qualitatively estimated [9). all the isolates were incubated in nutrient broth containing 5µg/ml l-tryptophan for 48 h at 28°c. following incubation, culture was centrifuged at 5,000 rpm for 20 min and 1 ml of supernatant was mixed with 2 ml of salkowski reagent and kept in a dark room for 20 min. appearance of pink color indicated the iaa production. phosphate solubilization activity screening for phosphate solubilization ability of isolate was done using pikovskaya’s agar medium [10]. the isolates were spot inoculated on pikovskaya’s agar plates and incubated at 28°c for 48 h. clear zones around the colonies indicated the positive test. the diameter of the halo zone was measured. hcn production qualitative estimation of hcn was done by using the methods described by lorck (as cited by [11]). each isolate was streaked on nutrient agar plate supplemented with 4% glycine. a whatmann filter paper soaked in a solution of 2% na2co3 in 0.5% picric acid was placed between base and lid of petriplate and incubated at 28 °c in inverted position for 48 h and observed for color change from yellow to brown. ammonia production all the bacterial isolates were tested for the production of ammonia [12]. for ammonia production strain was inoculated into 5 ml peptone medium and incubated for 48 h at 28 °c. after the bacterial growth, nessler’s reagent (0.5 ml) was added to the tube in 2:1 ratio. development of brown to yellow color indicated positive test for ammonia production. protease test for protease production test, isolated bacillus spp were spot inoculated on skim milk agar plate and kept for incubation for 24h at 28 °c. appearance of halo zone around the colonies was considered as positive for protease production [10]. the diameter of the halo zone was measured. results thirty nine isolates of bacillus were obtained from 25 soil samples. the isolates were gram positive rods (figure 1), endospore forming, and catalase enzyme producers. the colonies on nutrient agar were rough, creamy white, dry and folded, opaque and irregular edged. figure 1. gram stained bacillus species under oil immersion objective plant growth promoting properties the plant growth promoting properties of the isolated were evaluated based on the ability to produce iaa, solubilization of phosphate, ability to produce hcn, ammonia and protease. iaa production out of 39 isolates of bacillus, 19 isolates (48.7%) showed the ability to produce iaa. development of pink color after 20 min of addition of 2 drops of salkowski reagent nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 9 8 4 pathak et al. ©njb, bsn 81 figure 2. appearance of pink color after the addition of salkowski reagent in iaa test in 2ml of the cell free supernatant indicated the positive test for iaa (figure 2). the iaa producing strains were further classified into three groups as +++ (strong), ++ (moderate) and + (weak) based on the intensity of color visible. the strains showing deep pink, pink and light pink were placed in the group +++, ++ and + respectively. four isolates showed deep pink color after 20 min of addition of salkowski reagent into the cell free supernatant liquid whereas 7 isolates showed pink color and 8 showed light pink color (table 1). phosphate solubilization the isolates showing a halo zone around the colonies after 48 h of incubation following spot inoculation in pikovskaya’s agar plate were taken as positive tests for phosphate solubilization (figure 3). out of 39 isolates, 15 (38.4%) isolates exhibited the halo zone. the width of the halo zone was also measured (table 1). the width of the table 1. properties of 39 isolates tested for different plant growth promoting traits. bacillus strain (bs) iaa production p-solubilization width of halo zone (mm) hcn production ammonia production protease production width of halo zone (mm) bs00 + + 1 bs01 ++ + ++ 2 bs02 + + 3 bs03 + + 1 bs04 + + + 2.5 bs05 +++ 1.5 +++ 1 bs08 + 1 ++ 1.5 bs09 + 1 + +++ 1 bs10 + 1.5 +++ + 1 bs12 + +++ 1 bs13 ++ 1 +++ ++ 2.5 bs14 ++ + 1.5 bs15 +++ 2 + 3 bs16 + 1.5 + ++ 2.5 bs17 1 + ++ 3 bs18 + 1 bs19 ++ + 3 bs20 1 + +++ 1 bs21 + ++ 1 bs22 + ++ 2 bs23 + + 1 bs24 3 + 1.5 bs25 +++ +++ + 3 bs26 ++ ++ + 2 bs27 1.5 + 2.5 bs28 + ++ 2 bs29 ++ 3 +++ +++ 2 bs30 +++ +++ 1 bs31 +++ ++ 2 bs32 + +++ 1 bs33 +++ + 1 bs34 + 1.5 + 1.5 bs35 1 + + 1.5 bs36 + ++ 1 bs37 ++ 0.5 + + 1.5 bs38 +++ 1.5 bs39 ++ ++ + 1 bs40 ++ + + 1 bs41 +++ +++ 1 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 9 8 4 pathak et al. ©njb, bsn 82 halo zone was as high as 3mm (bs24 and bs29) to most of the bacillus spp showing the clear zone of 1mm diameter. figure 3. appearance of halo zone around colony in phosphate solubilization test hcn production all 39 isolates of bacillus produced hcn as evidenced by the change in color of the whatmann filter paper from yellow to brown. in the presence of glycine, the brown color of filter paper was observed giving a clear indication of hcn production by bacillus strains (figure 4). however, different strains produced the different intensity of brown color as light brown, orange brown to reddish brown. based on the distinction in the color of filter paper the strains are grouped into +++ (strong), ++ (moderate) and + (weak) for those producing reddish brown, orange brown and light brown respectively (table 1). ten strains changed the color of filter paper to reddish brown, 6 changed the color to orange brown while 23 strains changed the color to only light brown. figure 4. change is color of filter paper to reddish brown following incubation in hcn production test ammonia production for ammonia production, the development of brown to yellow color after the addition of 0.5 ml of nessler’s reagent was observed as a positive test (figure 5). among 39 isolates, 28 isolates developed the color of the medium to brown or yellow following the addition of nessler’s reagent. however, different strains produced different intensity of color as yellow, light brown and deep brown. based on the distinction in the color of media the strains are grouped into +++ (strong), ++ (moderate) and + (weak) for those producing deep brown, light brown and yellow respectively (table 1). six strains changed the color of media to deep brown, 8 changed the color to light brown while 14 strains changed the color to yellow. figure 5. change in color of peptone water in ammonia test figure 6. appearance of halo zone in protease test protease production the isolates showing the halo zone around the colonies after 28h of incubation following spot inoculation on skim milk agar were taken as positive tests for protease production (figure 6). from the 39 isolates taken for experiment, all the isolates produced the halo zone. the nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 9 8 4 pathak et al. ©njb, bsn 83 width of the halo zone was also measured (table 1). the width of the halo zone was as high as 3 mm (5 isolates) to most of the bacillus spp showing the clear zone of 1mm diameter. discussion certain strains of bacillus spp have gained worldwide attention in recent years due to their abilities in promoting plant growth. therefore, in this study, isolates of bacillus spp. obtained from soil of different regions of kathmandu valley and chitwan district were primarily tested for the plant growth promoting traits in vitro. the capability to increase plant growth parameters is highly related to the iaa level, which was produced by bacillus spp isolates. iaa, the major auxin in plants, plays a major role in both the shoot and root development [13]. among the 39 isolates, 19 isolates gave pink color on incubating the supernatant liquid with salkowski’s reagent. salkowski’s reagent when reacted with iaa, tris-(indole-3-acetato) iron (iii) complex is formed which displays pink coloration due to the formation of iaa complex and reduction of fe3+ [14].. in a similar study, 7 of 12 test strains of bacillus produced iaa [15]. similar observation of iaa production by bacillus strains has been reported in a study [16]. in their study, 76.3% (n=90) were able to produce iaa. similar method applied for iaa production in pseudomonas exhibited all the isolates were able to produce iaa [10]. the availability of different level of precursors affects the ability of bacteria to produce iaa. despite phosphorus being abundant in soil it is insoluble and cannot contribute to the plant growth [3]. so, the solubilization and mineralization of insoluble phosphate in soil by rhizobacteria makes an important property of plant growth promoting bacteria. rodriguez and fraga studied that bacillus and other phosphate solubilizing bacteria (psb) like pseudomonas and rhizobium were capable of converting insoluble phosphate available in the soil into soluble form [17] . phosphate solubilizing ability of isolated strains was tested using pikovskaya’s agar medium. fifteen out of the total isolates produced halo zone on pikovskaya’s agar medium after incubation following the spot inoculation on the plates as a result of phosphate solubilization. the halo zone around the colony was due to the polysaccharides, organic acids or phosphatase produced by the phosphate solubilizing bacillus strains (18). the bigger diameter of halo zones may be due to their greater ability to solubilize phosphate. in a similar study of phosphate solubilization by bacillus strains out of 12 isolates that promoted soybean seedling significantly, 11 isolates showed phosphate solubilization (16). another study documented only 11.5 % of bacillus spp. isolated from the samples obtained from various sites such as vineyard soil, fig orchard soil, forest soil, sewage soil, coastal area soil, compost of mushroom and paddy field were able to solubilize phosphate (19). the study indicates that the phosphate solubilization capacity of bacillus differs according to their site of existence. production of hcn by rhizobacteria is believed to promote plant growth by indirect mechanism. hydrogen cyanide is supposed to act synergistically with bacterially encoded antibiotics [3]. rijavec and lapanje in their study concluded that hcn increases the availability of phosphate for rhizobacteria and plant hosts, especially in oligotrophic alpine environments and thus indirectly contributing to plant growth [20]. picric acid present in the filter paper reacts with free cyanide produced by the bacteria to produce colored iso purpuric acid, thus the change in color of filter paper is visible. the color developed is directly proportional to free cyanide. plants can only utilize the reduced forms of the nitrogen; hence, nitrogen first must be fixed and converted to a combined form (either ammonia/nitrate) and then trapped by the plants [21]. twenty eight isolates (71.8%) were able to produce ammonia. these rates of ammonia production are lower than 95% and 80% demonstrated by other researchers [11, 22]. the enzyme protease, produced by most of the microorganisms, causes the hydrolysis of the peptide bonds that link amino acids in the polypeptide chain [23]. the production of protease by beneficial rhizobacteria helps to lyse a portion of pathogenic fungi and act as a biocontrol agent [24]. screening of protease producing ability of bacillus isolates was carried using skim milk agar medium. the casein present in the skim milk agar medium is hydrolyzed by the proteolytic bacteria, bacillus which is indicated by the formation of clear zones around the colonies [25]. all the isolates tested for protease enzyme production produced the halo zone around the colonies on skim milk agar medium following the incubation displaying the ability of bacillus to produce protease enzymes. the greater the ability to produce protease, the bigger is the diameter of halo zones. six isolates bs09, bs10, bs13, bs16, bs29 and bs37 showed positive results for pgpr traits assessed in the study. among these, bs29 showed maximum iaa production, phosphate solubilization, hcn production, ammonia production and proteolytic activity. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):7 9 8 4 pathak et al. ©njb, bsn 84 conclusion it is evident from the present studies that the bacillus species tested were able to demonstrate multiple plant growth promoting traits. the results concluded that bacillus strains have a huge potential to be used as alternatives to chemical fertilizers and pesticides for the promotion in growth and production 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availability of phosphate. front microbiol. 2016;7(nov):1–14. 21. smil v. nitrogen cycle and world food production. world agric. 2011;2(smil):9–13. 22. joseph b, patra rr, lawrence r. characterization of plant growth promoting rhizobacteria associated with chickpea ( cicer arietinum l .). plant prod. 2007;1(september). 23. singh p, rani a, chaudhary n. isolation and characterization of protease producing bacillus strain fs-1. agric eng int cigr j. 2007;6(04):633–9. 24. kundan r, pant g, jadon n, agrawal pk. plant growth promoting rhizobacteria: mechanism and current prospective. j fertil pestic. 2015;06(02). 25. yasmin f, othman r, saad ms, sijam k. screening for beneficial properties of rhizobacteria isolated from sweetpotato rhizosphere. biotechnology. 2007;6(1):49–52. http://dx.doi.org/10.3923/biotech.2007.49.52 special issue nepal j biotechnol. 2 0 2 0 o c t ; 8(2): 76-81 doi: https://doi.org/10.3126/njb.v8i2.31889 research article ©njb, bsn 76 study of the impact of organic manures and biofertilizers on growth of phaseolus aureus roxb. chhaya bhalshankar new arts commerce and science college, shevgaon, dist.-ahmednagar, m.s. (india) article history:received: 15 jun 2020; revised: 20 sep 2020; accepted: 1 oct 2020; published online: 22 oct 2020 abstract weeds are wild plants growing where they are not wanted, and they compete with the cultivated crop for nutrition. though they are seen as agricultural waste throughout the year, they are rich sources of nutrients. they grow in abundance during the rainy season, but as the season ends these biomasses get wasted. in the present investigation, tephrosia hamiltonii drumm belonging to family fabaceae, and achyranthes aspera l. belonging to the family amaranthaceae were used as a nutrient source to develop crop phaseolus aureus roxb. weed manures, vermicompost and compost, were prepared by using weeds t. hamiltonii drumm and a. aspera l. in 1:1 proportion. chemical analysis of weed and weed manures were done before administering it into the soil. neem cake was also used as one of the organic manures. in the experiment, a single dose of biofertilizers azotobacter and phosphate solubilizing bacteria were mixed with weed vermicompost, weed compost and neem cake; and in one of the treatments, only biofertilizers were used indouble dose. treatments were given to the crop as atvb, atcb, ncb, biod, npk, and control in a randomized block design of experimental plot size 1.5x 1.5 m. the use of chemical pesticides or fertilizers was completely avoided except for npk treatment plots. single plant analysis of pulse crop p. aureus roxb. was done. observations were recorded in the forms of fresh weight and dry weight of root, stem, leaves, leaf (4th number), and legumes. total fresh yield (kg ha-1), dm (kg ha-1) increase over control, and nitrogen efficiency ratio were recorded. results showed that %dm (an increase over control) and dm kg ha-1 recorded highest in atvb treatment and the highest n efficiency ratio was in biod. the present investigation emphasized reducing the input cost of the farm products along with protection of the environment and natural resources. keywords: biofertilizer, neemcake, organic agriculture, p. aureus, weed compost, weed vermicompost. corresponding author, email: bhalshankarchhaya@gmail.com introduction recent agricultural trends are focused on both reducing the usage of inorganic fertilizers by using organic manure and applying biofertilizers such as vermicompost and phosphatic biofertilizers [1]. microbial activities play a key role in agriculture because they are significant in the movement and availability of minerals required for plant growth and ultimately lower the use of chemical fertilizers [2]. the maintenance of nutrients in the soil is most important for healthy plant growth [3]. biofertilizers enhance soil health and crop yield. they improve fertility of soil, nutrient uptake, decomposition of crop residue, and microbial diversity of soil. they also reduce the requirement of chemical fertilizers [4]. the use of excessive chemical fertilizer, however, causes hazardous effects on the soil, leading to serious problems; thus, biofertilizers are important alternative sources of nutrients. they are biologically active microorganisms, like bacteria, algae, fungi; they can provide nutrients to crops [5, 6]. among biofertilizers, beneficial bacteria are azotobacter, azospirillum, rhizobium, symbiotic fungi mycorrhizae; they are essential in crop production. biofertilizers improve plants’ resistance to an unfavorable environment [7]. the biological manure helps to increase crop yields, and also plays a vital role in the nutrient accessibility in soil by improving the physical, chemical, and biological structure of soil, and it enhances the utilization of applied fertilizers [8]. in developing countries, residue management is very important as the amount of nutrients in crop residue is several times higher than the quantities of these nutrients applied as high cost fertilizer [9]. weed plants compete with the agricultural crops; they cause a tremendous reduction in crop yields and increase their production costs. several scientists have estimated such losses in crop yields in different parts of india. a very broad-based nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-7518-5754 mailto:bhalshankarchhaya@gmail.com mailto:bhalshankarchhaya@gmail.com nepal j biotechnol. 2 0 2 0 o c t ; 8(2) [special issue]: 76-81 bhalshankar ©njb, bsn 77 average of these estimates show that weeds reduced productivity of wheat by 15-30%, rice by 30-35%, and maize, sorghum, pulses and oilseeds by 18-85% each. many cases of complete crop failure due to weeds particularly in upland rice and vegetable crops were recorded [10, 11, 12]. t. hamiltonii drumm. and a. aspera l. are the weed plants used in the present study. the present study emphasized conversion and utilization of weeds beneficially by using them for the preparation of compost and vermicompost. neem cake is a residue left after the extraction of neem oil and used as an organic fertilizer. with the utilization of these organic manuresalong with biofertilizers like azotobacter and phosphate solubilizing bacteria for the cultivation of pulse crop p. aureus roxb. belonging to the family fabaceae. we can minimize the cost of production, increase output per hectare by using organic manures like compost, vermicompost prepared from weed biomass, neem cake, and biofertilizers for the production of crops and for sustainable agriculture. material and methods the experiment was conducted during march 2008. a summer variety of p. aureus roxb. was cultivated at college campus of new arts, commerce and science college, shevgaon district ahmednagar, (maharashtra), india. shevgaon extends between 19013 north latitude to 19035 north latitude and between 75001 east longitudes to 75037 east longitude. weed collection and preparation of manures the fresh vegetation of weeds i.e. aghada (a. aspera l.) and unhali (t. hamiltonii drumm.) were collected from different localities and chopped into small pieces (2-3 cm) by locally available iron cutter. equal amount (6944+6944 kg ha-1) 1:1 proportion mixture of weed pieces were used for the preparation of compost and vermicompost. to prepare compost this material was placed into pit (90x90x90 cm) and then added cow dung, soil and weed plant material layer by layer and sprinkled with water per requirement. finally, the compost pit was sealed with dung-mud mixture to prevent loss of heat and moisture. after partial decomposition first turning was given after 15 days for homogeneous decomposition, subsequent turnings were given after every 15 days interval. sufficient water was sprinkled to maintain moisture. finally, amorphous, dark brown, well fermented compost was obtained within 70 days. fresh weight of compost obtained from pit was 33 kg. same procedure was applied for vermicomposting, only with the addition of the worms in the pits after 15 days (worms’ variety eudriluseugeniae and iceniafoetida). identification of earthworms was done by the method prescribed in fauna of india and adjacent countries [13]. the prepared vermicompost was used for field trials. fresh weight of vermicompost obtained from pit was 32 kg. the uniformly mixed samples (100 g) were collected immediately from the pit for nutrient analyses. chemical analyses of weeds and weed manures and neem cake were done using oven dried and pulverized powder of samples. all the manures compost, vermicompost and neemcake (1000 kg ha-1)were mixed with biofertilizer azotobacter and phosphate solubilizing bacteria at the rate 25 kg ha-1 (recommended dose); and only biofertilizer double dose treatment 50 kg ha-1 in two split doses were applied to appropriate plots except chemical fertilizer (npk) plots. the mung (p. aureus roxb.) variety “raj biotech” balwan r.j. biotech, pvt ltd. siddharth arcade, station road, aurangabad was sown in the research plots of size 1.5 x1.5 m. at the rate of 20 kg ha-1. application of inorganic fertilizers the inorganic fertilizers were supplied to the experimental plots as nitrogen (n), phosphorus (p) and potassium (k) through urea, single super phosphate (ssp) and muriate of potash at the rate of 25 kg n, 50 kg p and ‘0’ k kg ha-1 (25:50:0) only for fertilizer treatment plots. entire amount of p2o5 and k2o and n was applied at the time of sowing. the crop supplemented with irrigation during periods of growth and whenever necessary weeding was done. use of insecticides and pesticides was completely avoided. seeds were planted in rows at a distance 30 cm x 10 cm. soil was murum so the crop was grown under frequent irrigation after each 8-10 days. sample from each plot was brought into laboratory chopped into 3-4 cm pieces. measured amount of biomass was kept in digital electrical oven separately in preweighted tray at 95±50c for 48 hours or more till constant weight. weight of dried samples were reported as dm. results were used to calculate %dm, dm kgha-1, increase over control and nitrogen efficiency ratio of crop. nepal j biotechnol. 2 0 2 0 o c t ; 8(2) [special issue]: 76-81 bhalshankar ©njb, bsn 78 results table 1. analyses of weeds administered in experimental plots through compost and vermicompost weed manures. here,weeds used were a. aspera l. and t. hamiltonii drumm. kg plot-1 (plot size 1.5 m x 1.5m) fresh weight dm nitrogen % c:n weed name kg plot-1 kg ha-1 % dm kg ha-1 % n kg ha-1 ash p k c achyranthes 1.56 6944 19.29 1339.50 2.03 27.19 17.43 0.123 0.43 10.11 4.99 tephrosia 1.56 6944 22.40 1555.46 1.94 30.18 18.57 0.115 0.51 10.77 5.54 table 2.analyses of weed manure and neem cake amendment along with biofertilizer. here, atvb=achyranthes, tephrosia vermicompost mixed with biofertilizer single dose, atcb=achyranthes, tephrosia compost along with biofertilizer single dose, nc=neem cake along with biofertilizer single dose treat ments fresh weight kg plot-1 fresh weight kg ha-1 dm n % % kg hect-1 % n kg hect-1 p k ca atvb 2.00 8889 67.21 5974.30 0.42 25+4.485 0.13 0.14 3.6 atcb 2.06 9169 65.07 5966.27 0.50 30+4.485 0.12 0.16 4.3 ncb 0.23 1000 97.94 0979.40 1.96 19+4.485 0.81 0.48 0.9 (amount of nitrogen fixed by single dose of biofertilizer is 4.485 kg ha-1 as according to n balance method[14]). these values added in n kg ha-1 of other treatments and amount of n kg ha-1 fixed by azotobacter biofertilizer double dose was 8.97 kg ha-1). table 3. c:n ratio of organic amendments. here, atv=achyranthes, tephrosia vermicompost, atc=achyranthes, tephrosia compost, nc=neem cake treatments % c:n ash c n atv 32.00 18.56 0.42 44.56 atc 36.50 21.17 0.50 42.36 nc 74.93 43.46 1.96 22.17 table 4. fresh wt and dm analyses of single plant of phaseolus (at 56 das). here, atvb=achyranthes, tephrosia vermicompost mixed with biofertilizer single dose, atcb=achyranthes, tephrosia compost mixed with biofertilizer single dose, nc=neem cake along with biofertilizer single dose. biod=biofertilizer double dose, npk=inorganic fertilizer, con=control. (das=days after sowing) treatment plant fresh wt in gm dm in gm root stem leaves 4th leaf total plant legume root stem leaves 4th leaf total plant legume atvb 0.49 4.89 11.29 3.59 22.55 5.83 0.21 1.44 2.99 0.82 7.99 2.71 atcb 0.48 3.93 09.79 2.57 18.53 4.21 0.19 1.17 2.77 0.60 6.47 3.03 biod 0.60 5.29 12.87 3.92 24.37 5.44 0.24 1.40 3.25 0.90 8.30 3.49 ncb 0.39 2.75 06.79 1.66 12.71 2.65 0.13 0.74 1.80 0.43 4.20 1.67 npk 0.51 3.70 08.38 2.15 15.09 2.63 0.20 1.08 2.21 0.53 4.76 1.60 con 0.19 1.82 03.49 1.14 07.26 1.72 0.08 0.47 1.02 0.27 2.33 1.41 s.e 0.06 0.53 1.37 0.44 2.61 0.68 0.02 0.16 0.34 0.10 0.95 0.36 c.d. 0.12 1.13 2.92 0.94 5.56 1.45 0.05 0.33 0.73 0.21 2.02 0.76 table 5. increase over control of total biomass and nitrogen efficiency ratio (73das). it is the ratio of the crop nitrogen uptake to the total input of nitrogen fertilizer. treatment fresh wt % dry wt % n efficiency ratio fw kg ha-1 increase over con increase over con dm kg ha-1 increase over con increase over con input n fresh dry atvb 4685 1676 55.71 1660 695 72.02 29.485 56.85 23.57 atcb 4312 1303 43.31 1505 540 55.96 34.485 37.79 15.66 biod 4136 1127 37.44 1409 444 46.01 8.970 125.60 49.50 ncb 3480 0471 15.64 1150 185 19.17 23.485 20.04 7.88 npk 3380 0371 12.33 1066 101 10.47 25.000 14.84 4.04 con 3009 0000 00.00 965 000 00.00 0.00 00.00 00.00 nepal j biotechnol. 2 0 2 0 o c t ; 8(2) [special issue]: 76-81 bhalshankar ©njb, bsn 79 analyses of weeds were done on dry matter basis. observations of weed analyses are recorded in the table 1. fresh weightswere used, 1.56 kg plot -1 (6944 kg ha-1) of each weed, for preparations of manures. % dm of tephrosia weed was higher (22.4), followed by achyranthes (19.29). the dm kg ha-1of tephrosia weed was higher (1555.46) followed by achyranthes weed (1339.50). % n was higher in achyranthes (2.03) followed by tephrosia (1.94) (tephrosia weed was collected from comparatively non fertile land and achyranthes from fertile land with ample domestic waste nearby. so, nitrogen percent of tephrosia was less than achyranthes though it is leguminous weed). n kg ha-1of tephrosia weed was higher (30.18) it was followed by achyranthes (27.19). % ash of tephrosia weed was higher (18.57); it was followed by achyranthes (17.43). % pwas higher in achyranthes (0.123) followed by tephrosia (0.115). % k was higher in tephrosia (0.51) followed by achyranthes (0.43). % cof tephrosia weed was higher (10.77); it was followed by achyranthes (10.11). c:n ratio of tephrosia weed was higher (5.54), and it was followed by achyranthes (4.99). analyses of achyranthes and tephrosia weed manure and neemcake were done; it is presented in table 2. fresh weight of weed compost (atc) was administered at the rate of 2.06 kg plot-1 ( 9169 kg ha1) and weed vermicompost (atv) was added at the rate of 2.00 kg plot-1 (8889 kg ha-1). fresh weight of neem cake (nc) was used at the rate of 0.23 kg plot1 (1000 kg ha-1). all the manures treatment was mixed with single dose of biofertilizer i.e. 25 kg ha-1. double dose of biofertilizer 50kg ha-1was given to the biofertilizer treatment (biod). % dm of atv (on 211th day) was 67.21 %; it was followed by atc (on 211th day) was 65.07% and neemcake 97.94%. dm kg ha-1 was highest in vermicompost (5974.30) followed by compost (5966.27) and lowest in neemcake (979.40). % n and n kg ha-1 was highest in atcb (0.5%, 30 kg) followed by atvb (0.42%, 25 kg) and ncb (1.96%, 19 kg). single dose of biofertilizer fixed 4.485 nkg ha-1; so, input of n was 29.485 for atvb, 34.485 for atcb and 23.485 for ncb. %phosphorus recorded highest in neem cake (0.81) and % potassium in neemcake (0.48); and %ca was highest in atc (4.3). amount of nitrogen fixed by single dose (recommended dose) of biofertilizer was 4.485 kg ha1 and amount of n kg ha-1 fixed by azotobacter biofertilizer double dose was 8.97 kg ha-1 in according to n balance method [14]. as per table 3, % ash % c and % n were highest in nc (74.93, 43.46 and 1.96, respectively) followed by atc (36.5, 21.17, & 0.5) and lowest in atv (32, 18.56 & 0.42). c:n ratio was highest in atv (44.56), followed by atc (42.36) lowest in neemcake (22.17). in table 4, analyses of fresh weight and dry weight of single plantare presented. fresh weight of root was highest in biod (0.60) followed by npk, atvb, atcb, ncb and lowest in the con (0.19), statistically significant in all the treatments. the fresh weight of stem leaves and 4th leaf and total plant was highest in biod followed by atvb, atcb, npk, ncb and lowest in the con, fw of stem and total plant statistically not significant in ncb. fw of legume was highest in atvb (5.83) followed by biod, atcb, ncb, npk and lowest in con (1.72), statistically significant in all the treatments except in ncb and npk. the dm of root was highest in biod (0.24) followed by atvb, npk, atcb, ncb and lowest in the con (0.08), statistically significant in all the treatments. dm of stemwas highest in atvb (1.44) followed by biod, atcb, npk, ncb and lowest in con (0.47), statistically significant in all the treatments except in ncb. dm of leaves, 4th leaf and total plant was highest in biod followed by atvb, atcb, npk, ncb and lowest in con treatments, statistically not significant in ncb for 4th leaf and total plant. dm of legume was highest in biod (3.49) followed by atcb, atvb, ncb, npk and lowest in con (1.41), statistically not significant in ncb and npk treatments. in table 5, percent increase over control and nitrogen efficiency ratio is presented. the percent increase over control in phaseolus for fresh weight was found highest in atvb (55.71) followed by atcb (43.31), biod (37.44), ncb (15.64), and minimum in npk (12.33). similarly, dry matter percentage (dm%) was found maximum with the treatment atvb (72.02) followed by atcb (55.96), biod (46.01), ncb (19.17) and minimum in npk (10.47).dm kg ha-1 recorded highest in atvb (1660) followed by atcb, biod, ncb, npk and lowest in con (965), statistically significant in atvb, atcb, biod, but statistically not significant in npk and ncb. the nitrogen efficiency ratio for fresh weight was found highest in biod (125.60) followed by atvb (56.85), atcb (37.79), ncb (20.04) and lowest nepal j biotechnol. 2 0 2 0 o c t ; 8(2) [special issue]: 76-81 bhalshankar ©njb, bsn 80 in npk (14.84). similarly, the nitrogen efficiency ratio for dry matter (dm) was found highest in biod (49.50) followed by atvb (23.57), atcb (15.66), ncb (7.88) and lowest in npk (4.04). highest fresh weight and dm kg ha-1 was recorded in treatment atvb. discussion azotobacter treated seedlings of knolkhol showed the highest whole plant weight [15]. biofertilizers such as azotobacter, azospirillum, psb, and a mixture of aza + azo + psb were administered to crops which showed the increased plant fresh weight, dry weight [16]. similar results showing fresh weight and dry weight of biod treatment was recorded highest at 56 das. combined inoculation of soybean by symbiotic bacteria improved the dry weight of soybean [17]. vermicompost and phosphate biofertilizer showed improved growth and yield in anise (pimpinella anisum l) [1]. vermicompost and psb when applied together was found helpful in developing production and yield in anise [18]. azotobacter increases the production of agriculture crop plants by 10-12%. azotobacter can also improve growth and grain yield in wheat crops. azotobacter act as one of the vital biofertilizers in the case of rice and some cereals could be applied by seed dipping and seedling root dipping methods [19]. maize hybrid seed priming with azotobacter showed the highest grain yield (7.01 ton/ha) and dm accumulation (2019 gr /m2) in treatment compound sc-434 [20]. panchgavyawas found to contribute to better growth and yield of pisum sativum as compared to npk [21]. in biochemical analyses of the total biomass of plant, nitrogen, and total crude protein was recorded highest in atvb [22]. the findings of the present experiment showed that fresh weight and dry weight was recorded highest in biofertilizer double dose at 56 das. but at harvesting 73 das maximum fresh and dry yield was recorded highest in weed vermicompost + biofertilizer azotobacter and phosphate solubilizing bacteria treatment (atvb). conclusion the results of this investigation concluded that weed vermicompost, weed compost along with a single dose of biofertilizer and biofertilizer double dose can effectively be used as a nutrient source to increase crop yield and soil fertility. weed manures and neem cake with biofertilizers worked more efficiently as compared to the chemical fertilizers (npk) to improving the quality of the crop; it could reduce the input cost of the farm produce as well in addition to protecting the environment and natural resources. author’s contribution cb is responsible for all the data collection, conceptualization, writing – original draft preparation, review &editing the final draft of the manuscript. cb read and approved the final manuscript. competing interests no competing interests funding the author(s) declared that no grants supported this work. acknowledgments i express my deep sense of gratitude to my respected teacher and research guide late capt. dr. bharati jadhav (retired professor), department of botany; dr. babasaheb ambedkar marathwada university, aurangabad. i express my deep sense of gratitude to my respected institute ahmednagar jilha maratha vidya prasarak samaj’s management for their continuous encouragement. ethical approval and consent not applicable. references: 1. darzi mt, hadi mh, rejali f. effects of vermicompost and phosphate biofertilizer application on yield and yield components in anise (pimpinella anisum l.). iranian journal of medicinal and aromatic plants. 2011;26(4). 2. verma s, singh a, pradhan ss, singh rk, singh jp. bioefficacy of organic formulations on crop production-a review. international journal of current microbiology and applied sciences. 2017;6(5):648-65. https:// doi.org/ 10.20546/ ijcmas. 2017.605.075 3. devi v, sumathy vj. production of biofertilizer from fruit waste. european journal of pharmaceutical and medical research. 2017;4(9):436-43. 4. singh m, dotaniya ml, mishra a, dotaniya ck, regar kl, lata m. role of biofertilizers in conservation agriculture. inconservation agriculture 2016 (pp. 113-134). springer, singapore.https://doi.org/10.1007/978-981-10-2558-7_4 5. javoreková s, maková j, medo j, kovácsová s, charousová i, horák j. effect of bio-fertilizers application on microbial diversity and physiological profiling of microorganisms in arable soil. eurasian journal of soil science. 2015;4(1):54. https://doi.org/10.18393/ejss.07093 6. rao dl, balachandar d, thakuria d. soil biotechnology and sustainable agricultural intensification. indian journal of fertilisers. 2015 oct;11:87-105. nepal j biotechnol. 2 0 2 0 o c t ; 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2016 [cited 2020 jan 2]. available from: http://irmjcr. scholarsworld.net/index.html#archive?archiveid=62317036 09671680 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):18-23 research article doi: https://doi.org/10.3126/njb.v9i1.38646 ©njb, bsn 18 assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of tinospora cordifolia (gurjo) tara shrestha , janardan lamichhane department of biotechnology, kathmandu university, dhulikhel, kavre, nepal. received: 23 feb 2021; revised: 14 jul 2021; accepted: 19 jul 2021; published online: 31 jul 2021 abstract traditionally used medicinal plants are the major resources of biologically active metabolites which are widely used for the cure of numerous diseases especially in developing countries where health facilities are rare. many plants are in use for centuries but there is not enough scientific evidence and exploration. this research is focused on phytochemicals, antibacterial, antioxidant and cytotoxicity activity analysis of one of the most commonly used ethnomedicine tinospora cordifolia collected from the kavrepalanchok district of nepal. phytochemicals analysis of methanol extract of t. cordifolia showed the presence of alkaloids, coumarin saponins, glycosides, reducing sugar, and triterpenes. antibacterial activity performed by disc diffusion method exhibited the highest activity against streptococcus with a zone of inhibition are 10.3mm, 8.5mm, 6.5mm, and 6mm at 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml of concentration respectively. dpph radical scavenging activity increased with increasing concentration of extract. when compared with ascorbic acid at equivalent concentration, the extract shows a lower scavenging profile (56.07% for the extract and 98.01% for ascorbic acid at 320 ppm). cytotoxicity was evaluated in terms of lc50 (lethality concentration). the result showed that the extract of t. cordifolia was found to be toxic with an lc50 value of 232.64μg/ml. the bioactive component present in the plants could be the result of its pharmacological effects that support the traditional use of plants. keywords: tinospora cordifolia, antioxidant activity, antibacterial activity, phytochemicals, dpph, brine shrimp bioassay. corresponding author, email: tarashrestha2@gmail.com introduction: herbal medicines are the biological resource of medicines that have been used in different cultures around the world as safe therapeutic drugs. in addition, they are also the enormous resource of dietary supplements, popular drugs, pharmaceutical intermediaries, nutraceutical drugs, and chemical entities for synthesized drugs [1]. the activity of medicinal plants is focused on the rich experiences of countless healers over centuries, whether inherited from ancestors, passed down from healer to healer, or established over time through personal experiences [2]. secondary metabolites are non-nutrient plant chemical compounds or bioactive components that protect the plant from microbial infections or insect infestations. they are also known as phytochemicals or phytoconstituents [3]. t. cordifolia, also known as "guduchi," is well-known in traditional ayurvedic literature for its extensive use in the treatment of various diseases like jaundice, urinary diseases, rheumatism, anemia, fever, vomiting, diabetes, skin disease, etc. it is found mostly throughout it is a big deciduous climbing shrub with a variety of coiling branches that spreads widely. the plant's stem is succulent, with long, fleshy, and climbing tendencies [4, 5]. the discovery of active components of the plant and their biological role in disease control has generated intense interest in the plant [6]. a wide number of chemical compounds like aporphine alkaloids, diterpenes, berberine, palmatine, tembertarine, magniflorine, choline, and tinosporin, etc. have been isolated from this plant. [7]. t. cordifolia methanol extracts of stem [8] are effective against microbial infections. it is reported that aqueous, ethanol, and acetone extract of leaves and stem of t. cordifolia showed inhibitory activity against urinary pathogens such as klebsiella pneumoniae and pseudomonas aeruginosa [9]. methanolic, ethanolic and water extracts [10] of t. cordifolia has found to be significant nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-4191-2052 mailto:tarashrestha2@gmail.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 18-23 shrestha & lamichhane . ©njb, bsn 19 antioxidant compared to other solvents. it is suggested that t. cordifolia methanol extract when taken orally has increased the erythrocytes membrane lipid peroxide and catalase activity [11] t. cordifolia is a well-known medicinal plant in traditional medicine, and several recent scientific investigations have highlighted its potential value in modern medicine. there have been several studies are carried on, but t. cordifolia research in nepal is limited. the purpose of this research is to document t. cordifolia medicinal characteristics as well as its potential for further scientific inquiry in the production of effective therapeutic molecules. isolation of phytochemicals, antibacterial activity, antioxidant activity, and cytotoxicity analysis of phytoconstituents derived from methanol extract stem of t. cordifolia all are addressed in this research. the decision of solvent is made due to the idea of solvent to separate wide assortments of hydrophilic and lipophilic substance constituents. materials and methods sample collection the sample of t. cordifolia stems was collected from bhakundebesi, kavrepalanchok district of nepal situated at an altitude of 1120 msl with latitude 27.560677° and longitude 85.6409178° shown in figure 1. the identification of the plant was done by tirtha maya shrestha, taxonomist, department of pharmacy, kathmandu university. figure 1: the sample collection site, kavrepalanchok district, nepal. sample preparation and extraction: the collected plant was shade dried at 25ºc and finely powdered using a grinder. the sample extract was prepared using a cold methanol maceration process [12] in which 50 g of finely ground powder was infused with 200 ml of methanol. the mixture was then stirred for approximately half an hour and stored for 48 hours. the solution was filtered using the whatman no. 1 filter paper after 48 hours. a rotary evaporator was used to evaporate the filtrate sample. the extraction yield was calculated using the formula: 𝐸𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑(%) = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 𝑡𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 × 100 phytochemical screening phytochemical screenings were performed for basic alkaloids, coumarins, saponins, glycosides, reducing sugars, sterols, triterpenes, and flavonoids [13]. antimicrobial screening antimicrobial analysis was done by the disc diffusion method [14]. test solution of 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml concentration of plant was prepared for antimicrobial test against the bacterial pathogens namely staphylococcus aureus, klebsiella pneumoniae, bacillus subtilis, and streptococcus in muller hinton agar. the human pathogenic strain was obtained from dhulikhel hospital, kavrepalanchok, nepal. the plant extract was prepared in the no.1 whatman filter. cip30: ciprofloxacin-30 µg/disc, t30: tetracyclin-30 µg/disc, and a10: ampicillin-10 µg/disc were used to equate the zone of inhibition with regular antibiotic discs. dpph free radical scavenging assay the dpph (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging method was used to test antioxidant activity [15, 16]. ascorbic acid was used as a standard scavenger. dimethyl sulfoxide (dmso) was used as a solvent to prepare a solution of ascorbic acid and plant extracts at concentrations of 0.5, 2, 4, 8, 16, and 32 µg/ml. then, using a uv spectrophotometer set to 517nm, the ic50 (the sample needed to scavenge 50% of the dpph free radical) was measured for each concentration of the sample. the equation for ic50 calculation: nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 18-23 shrestha & lamichhane . ©njb, bsn 20 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔(𝐼𝐶50) = 𝐴0 − 𝐴1 𝐴0 × 100 where, a0 = dpph solution absorbance, a1= dpph absorbance in relation to various extract concentrations. the ic50 was determined by plotting a graph of concentration versus percent inhibition and using a linear trend line equation. higher free radical scavenging activity was demonstrated by a lower absorbance of the reaction mixture. brine shrimp lethality assay the toxic property of t. cordifolia methanolic extract was determined using the brine shrimp lethality [17] technique. to obtain various concentrations, the extracts were dissolved in dmso and diluted with artificial sea saltwater. the total volume was adjusted to 10 ml with aerated seawater (1000, 100, and 10ppm). every tube held ten nauplii, which were incubated for 24 hours. after that, the tubes were examined under a magnifying glass, and the number of survivor’s nauplii in each tube was counted. experiments were carried out in a series of three tubes per dose, with a monitor (dmso in seawater) and various concentrations of the test substances. the logarithm of the extract concentration was used (to make dealing with a wide variety of values simpler). death percentage was calculated by: 𝐷𝑒𝑎𝑡ℎ 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 = 𝐷𝑒𝑎𝑡ℎ 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 × 100. % death corrected vs. log extract conc (ppm) was plotted and the lethal dose 50 (ld50) from the graph from the equation of straight-line. lethal concentration was determined from the equation of the straight line. results phytochemical screening the term "phytochemical investigation" refers to the process of identifying and characterizing crude pharmaceuticals in terms of their phytochemical ingredients. chemical elements of the plant were assessed. the results for the different types of phytochemicals presence are shown in table 1. the presence of these phytochemicals explains why plants are used to treat ailments such as diabetes, anticancer drugs, jaundice, etc. antibacterial activity the antibacterial susceptibility test showed that bacterial strains are susceptible to standard antibiotics. the zone of inhibition (zoi) shown by standard antibiotics cip30: ciprofloxacin-30mcg/disc, t30: tetracycline30mcg/disc, and a10: ampicillin-10mcg/disc are shown in table 2. triplicates of the experiment were carried out, and the zoi values are the average in millimeters (mm). the activity was carried out against four disease-causing bacteria, including, klebsiella pneumoniae, bacillus subtilis, staphylococcus aureus, and streptococcus. in comparison to standard antibiotics. streptococcus was found to be a more susceptible comparison to other bacterial strains showing zone to inhibition 10.3mm, 8.5mm, 6.5mm, and 6mm at 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml table 1: qualitative phytochemical screening of t. cordifolia from methanol extract shows the presence of alkaloids, coumarin, saponins, glycosides, reducing sugar and triterpenes phytochemicals alkaloids coumarins saponins glycosides reducing sugar sterols triterpenes flavonoids tannins and polyphenols + + + + + + present(+) or absent(-) table 2: antibacterial activity shown by the standard antibiotics, dmso and plant extract against b. subtilis, k. pneumonia, s. aureus and streptococcus species. standard antibiotics ampicillin, ciprofloxacin and tetracycline were taken as positive control while dmso was taken as negative control. four different concentration of t. cordifolia sample (tc-200mg/ml, 100mg/ml. 50mg/ml and 25mg/ml) was tested for its bactericidal activity and corresponding values represent the respective zone of inhibition. bacterial strain test sample zone of inhibition(mm) bacillus subtilis klebsiella pneumoniae staphylococcus aureus streptococcus ampicillin(10µg) 11±1 14±0.5 12 10±1 ciproflaxin(30µg) 26±1 27 26 24 tetracycline(30µg) 19±1 22±1 19±1.08 19 dmso 0 0 0 0 tc 200mg/ml 8 6.5 6 10.3 tc100mg/ml 7 6.5 6 8.5 tc 50mg/ml 6.5 6 6 6.5 tc 25mg/ml 6 6 6 6 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 18-23 shrestha & lamichhane . ©njb, bsn 21 of concentration respectively. similarly, t. cordifolia was found to be mildly inhibitory bacillus subtilis showing a zone of inhibition 8mm, 7mm, 6.6mm and 6mm at 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml respectively. t. cordifolia did not show a prominent zone of inhibition for klebsiella pneumoniae and staphylococcus aureus. the zone of inhibition against all the microorganisms is shown in figure 2, figure 3, and table 2. assay for scavenging dpph free radicals dpph scavenging percentage was found to be concentration-dependent. the absorbance was determined using a spectrophotometer at 517nm for the calculation of %scavenging of t. cordifolia. ascorbic acid was taken as a standard analyte. the scavenging percentage of ascorbic acid and extract of t. cordifolia is compared in figure 4. from, the linear regression analysis the ic50 value of t. cordifolia and ascorbic acid was observed to be 238µg/ml and 4.76µg/ml respectively. figure 4. the graph shows the dpph scavenging activity of methanol extract of t. cordifolia. the percent scavenging activity is compared with ascorbic acid. figure 5. toxicity effects of t. cordifolia shown by brine shrimp bioassay. the trend line equation gives lethal concentration (lc50) of 232.64 ppm. brine shrimp bioassay the methanol extract of t. cordifolia showed good brine shrimp larvicidal activity. the regression trend line graph was plotted using the concentration and death figure 2. comparision of zoi of t.cordifolia with standard antbiotic discs amp10, cip30 and t30. figure 3. t. cordifolia showing zoi against streptococcus spp. at 200mg/ml, 100mg/ml, 50mg/ml and 25mg/ml. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 18-23 shrestha & lamichhane . ©njb, bsn 22 percentage. the lethality concentration (lc50) obtained from regression analysis (figure 5)was found to be 232.64 ppm. discussion the presence of phytochemicals alkaloids, coumarin, saponins, glycosides, reducing sugar and triterpenes gives evidence of its anti-diabetic, anticancer, antiinflammatory infections, anti-viral infections. many of these active compounds have various immunomodulatory and physiological functions [18]. the presence of these compounds may be the reason for the antibacterial, antioxidant, and cytotoxic activity of plants. the antibacterial activity was found to be susceptible mostly to gram-positive bacteria. the presence of triterpenes shows that it has antibacterial activity. a previous study suggests that ethanol extract t. cordifolia showed good inhibition against both grampositive and gram-negative bacteria. present findings support the applicability of t. cordifolia in traditional systems for its claimed uses like fever inflammations, urinary and skin diseases [19]. methanol was employed to extract antioxidant-rich fractions from the stem of t. cordifolia in this investigation. methanol is a better solvent for the extraction of chemicals than other solvents, according to previous research. the presence of polyphenols and tannins in plant extracts can contribute to their antioxidant activity [20]. according to meyer’s toxicity index [21], extracts with lc50 < 1000 μg/ml are considered as toxic, while extracts with lc50 > 1000 μg/ml are considered as non-toxic . the lethal concentration of the extract was found to be 232.64 which indicates the extract is toxic and worthy of further investigation. the early toxicity information acquired from the brine shrimp lethality assay provides lc50 values, which can be used as a starting point for further toxicity research. [22]. conclusion herbal medicines are critical in the restoration of modern civilization's health. t. cordifolia has been utilized pharmacologically in the treatment of a variety of diseases, according to the literature review. the findings of the phytochemicals study back up the common use of t. cordifolia to treat several ailments like jaundice, rheumatism, urinary tract infections, dermal diseases, anemia, inflammation, diabetes, etc. [7]. the main constituents of t. cordifolia are tinosporine, tinosporide, tinosporaside, cordifolide, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin, and b-sitosterol. berberine, palmatine, tembertarine, magniflorine, choline, and tinosporin are reported from its stem. this chemical constituent plays a major role in the antibacterial, antioxidant, and cytotoxicity role of t. cordifolia [23]. t. cordifolia has gain attention due to its immense application in traditional as well as modern medicine. tinospora cordifolia is seasonal and geographically dependent. at the same time, the organic and aqueous extracts of tinospora cordifolia could be used in the pharmaceutical sector in the future as a source of important phytochemical substances. author’s contributions janardan lamichhane contributed to designing the experiment. tara shrestha contributed to all the laboratory works and data analysis work. jl and ts contributed equally in drafting and reviewing the manuscript. competing interests no competing interests were disclosed. funding this research is funded by the koica 1-1 project entitled ‘establishment of ks b&w center for industrialization of natural resources in nepal’. acknowledgment tara shrestha is thankful to janardan lamichhane, for the guidance during the project and department of biotechnology, kathmandu university for the equipment and laboratory facilities. ethical approval and consent not applicable. references 1. handa ss. an overview of extraction techniques for 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ferrigni, n.r., putnam, j.e., jacobsen, l.b.,nichols, d.e., mclaughlin, j.l., 1982. brine shrimp: a convenient general bioassay for active plant constituents.planta medica. 45, 31-34. 22. apu as, muhit ma, tareq sm, pathan ah, jamaluddin at, ahmed m. antimicrobial activity and brine shrimp lethality bioassay of the leaves extract of dillenia indica linn. journal of young pharmacists. 2010 jan 1; 2(1):50-3. https://doi.org/10.4103/0975-1483.62213 23. devprakash sk, subburaju t, gurav s, singh s. tinospora cordifolia:-a review on its ethnobotany, phytochemical & pharmacological profile. asian journal of biochemical and pharmaceutical research. 2011 sep;4(1):291-302. https://dx.doi.org/10.4103%2f0257-7941.107344 https://dx.doi.org/10.4103%2f0257-7941.107344 https://doi.org/10.1016/s0378-8741(03)00006-0 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 69-73 issn 2091-1130 (print)/issn 2467-9319 (online) review article ©njb, biotechnology society of nepal 69 nepjol.info/index.php/njb nipah virus (niv) infection: is nepal prepared for the possible outbreak? dhiraj shrestha1, 2*, balkrishna bhattachan3 1department of microbiology, tri-chandra multiple college, kathmandu, nepal 2department of microbiology, shi-gan international college of science and technology, kathmandu, nepal 3siddhi memorial hospital, bhaktapur, nepal abstract after 20 years of the first nipah virus (niv) outbreak in the world, it re-emerged as the outbreak in india. who has recognized niv as a potent epidemic threat to human health. both animal-to-human and human-to-human transmission of zoonotic niv has been documented. fruit bat of pteropodidae family is the natural reservoir of the virus. thus, the territorial habitat of these bats is the high risk zone of niv outbreak. the symptoms are very nonspecific and the pathogenicity of niv is yet to be fully understood. diagnosis of niv infection still relies on molecular techniques. till date, no drugs or vaccines against niv has been approved. some research have presented arrays of the possible treatment and prevention option, but without sure shot implications. so, appropriate precautions are the only currently available prevention option. nepal is yet to experience a niv outbreak but that does not undermine the risk posed to the general population. high risk countries including nepal should be well prepared to tackle the possible outbreak in future. keywords: nipah virus, niv, outbreak, nepal *corresponding author email: hiraj.diamond@gmail.com introduction outbreaks in glance in the past few years, the outbreak of ebola virus was on high rise throughout the world. in counter response against the virus, vaccine was developed and was being tested in high risk zones of democratic republic of the congo on 19 may, 2018 [1]. coincidentally on the same day, this was overshadowed by the news of the outbreak of the nipah virus (niv) in india. initially three deaths were reported due to niv infection. since then 15 people have been tested positive for niv, of which 13 are already dead. other 16 suspected cases identified through contact tracing are under observation and at least 753 additional people are quarantined [2]. niv is featured in the who list of blueprint priority diseases 2018 with potent epidemic threat demanding urgent research and development (r&d) action [3]. the first recorded outbreak of niv occurred in 1998 in malaysia following in singapore in 1999. the outbreak involved severe respiratory illness in pigs and encephalitis in humans. later outbreak involving human infections was reported from bangladesh and india in 2001 [4,5]. till date, more than 600 cases of niv human infections has been documented. the outbreaks in indian subcontinent have been recurrent [6]. higher mortality of around 70% has been observed in bangladesh and india, compared to mortality in malaysia and singapore outbreak [5]. this is probably due to higher engagement of the respiratory tract in the bangladesh and india outbreaks, differences in pathogenicity of the viral strains and lack of advanced healthcare facilities [5]. nipah virus (niv) niv is a member of the family paramyxoviridae, genus henipavirus [4,7]. the name nipah virus originated from sungai nipah, a malaysian village reporting the onset of an outbreak in 1998 [7]. niv is a zoonotic virus. both animal-tohuman transmission (from infected bats/pigs) and human-to-human transmission have been documented. during malaysian and singaporean outbreaks transmission occurred from infected pigs, an intermediate host. however during bangladeshi and indian outbreak, transmission occurred through consumption of fruits/sap contaminated with infectious bat secretions and also through nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 69-73 shrestha and bhattachan et al. ©njb, biotechnology society of nepal 70 nepjol.info/index.php/njb human-to-human transmission. during this outbreak, no any intermediate host was reported owing to the lack of pig farms in the region [4,7]. figure 1. adapted from “nipah virus distribution map,” by centers for disease control and prevention (cdc) 2018, available at: https://www.cdc.gov /vhf/nipah/outbreaks/distribution-map.html. copyright 2018 by the cdc. adapted with permission. natural host the natural hosts of niv are fruit bats belonging to the pteropodidae family, pteropus spp. in particular [4,8]. geographical distribution of niv and pteropus overlaps, ranging from madagascar to australia; covering south asia, south-east asia and oceania region (figure 1). besides, african fruit bats of pteropodidae family were also tested positive for niv antibodies indicating further geographical distribution of niv to african territory. niv has also been tested positive in other animals including pigs, horses, goats, sheep, cats and dogs but only during the outbreaks [4]. distribution map of these bats published by who includes nepal however exact distribution and population of these bats are still to be studied and reported from nepal. pathogenesis niv contains a negative-strand rna. the nonsegmented rna contain six genes which encodes structural proteins of virus namely, fusion protein (f), glycoprotein (g), polymerase (l), matrix protein (m), nucleocapsid (n) and phosphoprotein (p). the p gene encodes additional three accessory proteins namely, c, v and w proteins [9,10]. v protein was the key determinants for pathogenesis in hamster and ferret infection model [11-13]. v proteins target multiple host proteins and thus suppress the host antiviral response. in addition, v proteins interact and suppress different signaling pathways. v protein suppress the dephosphorylation of melanoma differentiation-associated protein 5 (mda5) thus inhibiting the activation of the interferon (ifn) β promoter [14-16]. v proteins also suppress the retinoic acid-inducible gene-i (rig-i) dependent induction of ifn [17]. v proteins also block signaling through toll-like receptors 7/9 (tlrs 7/9) and suppress ifn induction [18,19]. v proteins interact with ifn-responsive signaling pathway preventing activation and nuclear accumulation [20,21]. similarly, c proteins regulate early host proinflammatory response thereby contributing virulence [22]. c protein was responsible for respiratory diseases in a ferret infection model [11,12]. the exact molecular mechanism of the pathogenicity of niv is still to be revealed. signs and symptoms the incubation time of niv ranges between 4-14 days, and upto 45 days in some cases. infections in human ranges from asymptomatic infection to acute respiratory infection, and fatal encephalitis in severe cases. initial symptoms include headaches, influenza-like fever, myalgia (muscle pain), sore throat and vomiting. these symptoms are followed by acute encephalitis. finally in severe cases, encephalitis and seizures occurs, leading to coma within 24-48 hrs. fatality varies from 40% to 75%. surviving cases are reported to demonstrate long-term sequel including persistent convulsions, personality change, seizure disorder, relapse and delayed onset encephalitis [4,7]. diagnosis initial signs and symptoms of niv infection are nonspecific, thus accurate diagnosis is challenging especially during an outbreaks. igm elisa for niv, real-time polymerase chain reaction (rt-pcr) and viral isolation are the tests employed for diagnosis [4]. these tests demands higher technical expertise and resources challenging the effectiveness of immediate counter measures during an outbreak. treatment currently, no drugs or vaccines are approved for niv infection. supportive care with intensive care is the only recommended treatment [4,7]. https://www.cdc.gov/ nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 69-73 shrestha and bhattachan et al. ©njb, biotechnology society of nepal 71 nepjol.info/index.php/njb favipiravir (t-705) has been demonstrated to inhibit niv replication and transcription in syrian hamster model, with twice daily oral administration for 14 days [23]. ribavirin, which has broad spectrum anti-dna and anti-rna virus activity and can transverse the blood-brain barrier, was also reported to reduce mortality in an open-label trial [24]. prevention vaccines have not been developed against niv. however, routine and thorough disinfection of pig farms can prevent the infection. also, avoiding the consumption of the bat eaten fruits or saliva/urine contaminated fruits/sap can prevent the infection. thus, public awareness of the associated risk factors is the only measure to reduce risk of niv infection. during outbreaks, suspected animal premises should be quarantined and infected animals should be culled. also, healthcare workers caring suspected or confirmed niv infected patients should adhere to standard infection control precautions [4]. personal protection, such as masks, goggles, gloves, gowns, and boots, is advocated for field and farm workers of niv risk zones. personal protection should be accompanied by hand-washing and disinfection of equipments for better prevention [5]. future prospects respiratory route administration of lipopeptides prevented niv infection in both hamsters and non-human primates. also, retention of peptides in respiratory tract avoided systemic delivery of niv thus increasing safety and enhancing interventions [25]. vaccines using a vesicular stomatitis virus vector have demonstrated protection against niv in hamsters, ferrets and african green monkeys [26]. recently in australia, subunit vaccines using hendra g protein have demonstrated protection against niv in horses by producing cross-protective antibodies. this vaccine has potential for niv protection in humans as well [7]. we could be on verge of testing vaccine against niv soon. nepalese prospects till date no outbreak of niv or niv confirmed mortality has been documented within the territory of nepal. no research has been done on surveilling antibodies against niv in nepalese human and/or bat population. however, nepal harbors well distribution of pteropus bat. pig farms are widespread throughout the country. nepal also has significant pork eating ethnic communities. this clearly outlines the possibility of surveilling niv in bats and/or pigs of nepal. furthermore, nepal and india share open border without medical surveillance and population mobility is higher in the border region. this further adds possible risk of viral transmission in nepal. thus, nepal is at high risk of potential niv outbreak. conclusion twenty years ago, niv unveiled itself to the world causing significant morbidity and mortality. initially niv shattered the pigfarming industry in malaysia, and is continually causing outbreaks in indian subcontinent. the natural reservoir pteropus bat is widespread, thus outbreaks can occur in any risk zones. the recent outbreak in india has again increased the concern of niv as potential threat to human health. governments and universities should fund research to develop vaccine or drug against niv to neutralize the potential threat. all high risk countries including nepal, should be well prepared to tackle the possible future catastrophe. conflict of interest the authors declare no any conflict of interest. funding none authors contribution both authors ds and bb contributed to the work. references 1 world health organization (who): ebola virus disease-democratic republic of the congo: update on ring vaccination. 2018, available at: http://www.who.int/csr/don/21-may-2018ebola-drc/en/ [accessed 23/05/2018]. 2 world health organization (who): nipah virus india. 2018, available at: http://www.who.int/csr/don/31-may-2018nipah-virus-india/en/ [accessed15/06/2018]. 3 world health organization (who): r&d blueprint. list of blueprint priority diseases. 2018, available at: nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 69-73 shrestha and bhattachan et al. ©njb, biotechnology society of nepal 72 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shrestha and bhattachan et al. ©njb, biotechnology society of nepal 73 nepjol.info/index.php/njb 24 chong ht, kamarulzaman a, tan ct, goh kj, thayaparan t, kunjapan sr, chew nk, chua kb, lam sk: treatment of acute nipah encephalitis with ribavirin. ann neurol 2001, 49:810-813. doi: 10.1002/ana.1062pmid: 11409437. 25 mathieu c, porotto m, figueira t, horvat b, moscona a: fusion inhibitory lipopeptides engineered for prophylaxis of nipah virus in primates. j infect dis 2018, 218(2):218-227. doi: 10.1093/infdis/jiy152. pmid: 29566184. 26 satterfield ba, dawes be, milligan gn: status of vaccine research and development of vaccines for nipah virus. vaccine 2016, 34(26):2971-2975. doi: 10.1016/j.vaccine.2015.12.075 pmid: 26973068. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 research article doi: https://doi.org/10.3126/njb.v9i1.38648 ©njb, bsn 42 comparative study of nutritional profile of rice varieties in nepal evance pakuwal , prakash manandhar department of microbiology, st. xavier’s college, kathmandu, nepal received: 16 sep 2020; revised: 04 may 2021; accepted: 15 may 2021; published online: 31 jul 2021 abstract the purpose of this study was to evaluate and compare the nutritional quality of different rice varieties (taichung-176, khumal4 rice, and black rice) with jumli marsi rice. the highest nutritional factors and phytochemical components were found in the marsi rice (rr) and black rice (br). the highest amount of antioxidant property and phenolic content was found in black rice which was 61.58 ± 0.02% and 22.75 ± 0.02gae/100g respectively. the reducing sugar was found to be highest in the tr rice variety, which was 2.74±0.01%. the results also highlight the cooking and physicochemical properties of rice depending on the amylose content of rice varieties. the qualitative analysis of the phytochemical content in different rice varieties showed the presence of tannin, flavonoid, alkaloid, and terpenoid in marsi and br. while anthraquinone and saponin were negative for all the rice varieties, protein and glycoside were found to be present in all the rice varieties. also, the pigmented rice varieties were found to have high nutritional components compared to the non-pigmented rice varieties. all the data observed in the study was found to be statistically significant (p < 0.05). keywords: pigmented rice, antioxidant, phytochemicals, jumli marsi corresponding author, email: pakuwalevance@gmail.com introduction cereal grains are consumed worldwide and are considered an important dietary source of proteins, carbohydrates, vitamins, minerals, and fiber [1]. rice is the cereal grain that is consumed as a major staple food around the world. rice (oryza sativa l) belongs to the family poaceae. the contribution of rice as a percentage of total dietary energy supplied in developing countries, which is 715 kcal/day, 27% of total dietary energy supply, 20% of dietary protein, and 3% of dietary fat [2,3, 4]. there are different varieties of rice available around the world. among those, pigmented rice varieties such as red, black, purple, and brown rice are known to be consumed for a long time in southeast asian countries. they are also known to be originating in southeast asia [1]. in the case of nepal, there are three different regions (tarai, mountains, and hilly) classified according to their geographic location. different varieties of rice are cultivated from tarai (lowland plains) to high hilly regions. jumla (2300 msl) is the key site for growing cold-tolerant rice genotypes for the hilly regions of nepal. the major area for the production of jumli marsi is tila and sinja valleys in jumla, which are the world heritage sites present in nepal [5]. marsi (rr), sometimes spelled as marsee is a variety of rice cultivated in the highest altitude in the world, chhumchaur, jumla [6]. it is commonly known as nutritionally rich in compounds such as protein, polyphenol, flavonoids, and antioxidants. the marsi rice is classified by the farmers in different groups namely, kali marsi, raato marsi (can be seen in photograph 4), seto marsi, daarime marsi, mehele marsi, etc. marsi is also known to have medicinal value [7,8]. black rice (br) is a type of pigmented rice that has black bran covering the endosperm of rice grain. they are black (shown in photograph 1) and turn purple when cooked. the color intensities of pigmented rice are known to be related to the value of lightness, redness, and yellowness. it takes a longer time to cook and has stickier consistency compared to white rice [9]. the supplementation of diets with black rice and the anthocyanin-rich extract of the black rice is found to have various health benefits [10]. the composition of rice variety is important for healthconscious consumers as well as useful to reduce fuel consumption during the process of cooking. amylose content can be a factor that can influence the cooking time of rice. these are different in different varieties of rice [11]. amylose is a branched carbohydrate mainly based on α (1–4) bonds. amylose content is an important factor in determining the cooking properties of a rice variety [12,13]. the amylose content in different rice varieties is classified into waxy, low, intermediate, and high according to the percentage of amylose present in the rice, which is classified as waxy (0-2% amylose), low (10 20% amylose), intermediate (20-25% amylose), and high (>25% amylose) [14]. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0001-5333-5291 mailto:pakuwalevance@gmail.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 pakuwal and manandhar. ©njb, bsn 43 the cooking quality of rice depends on the components of the rice variety such as proteins and amylopectin [15]. the antioxidant activities of black and red rice and their crude extracts have given results that show the addition of the pigmented rice could increase antioxidant property in daily meals. the pigment in rice is the indicator of bioactive compounds present in the rice [9]. these varieties are studied and found to have high anticancer, anti-inflammatory properties as well as antioxidant content. some phytochemicals such as phenolic compounds, bioflavonoids, terpenoids, and alkaloids are also found in pigmented rice [8,16]. the grains with red and black (darker) colored pericarp grains have higher molecular weight compared to the light brown pericarp color [16]. carotenoids, vitamin e, terpenoids, and phenolics are some important groups of phytochemicals found in whole grains [17]. phenolics are produced by the process of secondary metabolism in plants, which are also found to have a positive impact on human health [18]. the anthocyanins and pro-anthocyanidins/condensed tannins are some of the commonly found pigments in black rice and red rice, respectively. the flavonoids like luteolin, apigenin, quercetin, isorhamnetin, kaempferol, and myricetin are found in rice. black rice has high phenolic and antioxidant compounds, which contribute to multiple biological activities leading to an increase in today’s market demand [19,20]. the taichung-176 (tr) rice variety (shown in photograph 3) is one of the commonly used rice varieties in nepal and it is used to prepare traditional fermented food varieties such as chyang, selroti, etc. marsi and black rice varieties are expensive compared to the other two rice varieties. khumal-4 (kr) (shown in photograph 2) rice is affordable and is consumed by the locals for a daily meal [21]. there have been various research studies performed to study different rice varieties, however, no researches have been performed to compare red rice (rr), also known as marsi, which is mainly found in the altitudes of nepal, with other rice varieties. therefore, in this study, the main objective was to comparatively study the nutritional characteristics of marsi rice with other rice varieties available in nepal. materials and methods sample collection and identification four different varieties of rice (taichung-176 rice, khumal4 rice, black rice, and red rice/ jumli marsi rice) were collected from the local market of khumaltar and kathmandu organics (as suggested by food research division, narc, khumaltar, nepal). determination of physical properties of rice determination of length, breadth was done by randomly selecting 10 grains from each sample using a slide caliper [22]. length = main scale reading + vernier scale reading × vernier constant breadth = main scale reading + vernier scale reading × vernier constant weight of kernels kernel grains (1000 grains counted manually) from each variety were randomly selected and weight was recorded in grams [23]. determination of cooking characteristics of rice varieties swelling property/ water uptake for the determination of swelling property, finely ground rice was mixed in distilled water (0.35 g in 12.5 ml). the mixture was then heated in the water bath for 30 minutes at a temperature of 60°c. it was then centrifuged at 3500 rpm for 20 minutes. the supernatant was decanted in a pre-weighed dish. the temperature was maintained at 100 °c for drying (20 minutes) [24]. the residue was weighed. the swelling power was calculated by: swelling property = weight of the residue /original weight cooking time rice (2 g) of each variety was cooked in distilled water (20 ml) separately, and the temperature was maintained at 90°c in a water bath. two glass slides were used to press cooked rice samples in between (to find out the minimum cooking time) [23,24]. proximate analysis of rice varieties determination of moisture and ash was done based on the standard method. the reducing sugar content was determined by the anthrone method. the carbohydrate content was determined using the anthrone method for carbohydrate estimation. spectrophotometric analysis was done at 630 nm [24,25,26]. calculation: amount of carbohydrate present in 100mg of the sample = glucose (in mg)/ volume of test sample × 100 % carbohydrates = (concentration sample * dilution factor)/ mass sample x 100 amylose content the rice flour was mixed with ethanol (95%) and 1 n naoh. a mixture was prepared by thoroughly mixing it nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 pakuwal and manandhar. ©njb, bsn 44 in a volumetric flask. the mixture was heated in a boiling water bath for 10 minutes. it was then cooled to room temperature, to gelatinize the starch present in the rice sample. five milliliters of the solution was then transferred to another volumetric flask. one ml of 1n acetic acid and iodine solution was added to the solution. the distilled water was used to adjust the volume to 100 ml. the vortex mixing method was used to mix it again. it was then allowed to stand for the time duration of 20 minutes. the absorbance of the sample solution was measured at 620 nm using a uv spectrophotometer. finally, the determination of amylose content in samples was done by using the amylase standard curve [23]. determination of reducing sugar by using 3, 5dinitrosalicylic acid / dns method for the rice sample, 100 mg of the sample was weighed and centrifuged with the hot 80% ethanol twice (5 ml each time). the supernatant was collected and evaporated by keeping it in a water bath at 80°c. 10 ml water was added to dissolve the sugars. 0.5 to 3 ml was pipette out of the extract in test tubes and volume was equalized to 3 ml with water in all the tubes. the standard sugar solution was prepared in the range of 0 to 3 ml in different test tubes. the final volume was made up to 3 ml with distilled water, which made the concentrations ranging from 0 to 750 mg. 3 ml of dns reagent was added. the contents were heated in a boiling water bath for 5 minutes. it was then cooled and the intensity of dark color was read at 540nm. reducing sugar in rice varieties was determined by using the 3, 5dinitrosalicylic acid / dns method by spectrophotometric analysis at 540 nm [27]. ● antioxidant and phenolic content of rice varieties the antioxidant content was determined by using the dpph assay method. the phenolic content was determined by the folin-ciocalteu method using spectrophotometric analysis (gallic acid as the standard). antioxidant activity (dpph assay method) the antioxidant activity of rice samples was evaluated spectrophotometrically, as a measure of radical scavenging activity by using dpph [24,25]. the capability to scavenge the dpph radical (% inhibition) was calculated using the following equation: scavenging ability % = [absorbance of control (515 nm) absorbance of sample (515 nm)/absorbance 515 nm of control] x 100 phenolic content (folin-ciocalteu method) total phenolic content was determined by the standard graph of gallic acid, which was prepared by dissolving 1 mg of dry gallic acid, and absorbance was measured at 760 nm [26]. phytochemical properties of rice varieties the qualitative analysis of different rice varieties was done for the phytochemicals: terpenoid, flavonoid, tannin, anthraquinone, saponin, and glycoside [9]. extraction each variety of rice was milled into flour and then the rice flour was kept in 20 ml of 80% methanol for 24 hrs at room temperature and then centrifuged at 4000 g for 15 minutes. terpenoid: rice extract (5 ml) was mixed with 2 ml of chloroform. the concentrated h2so4 (3 ml) was added to the solution. reddish-brown coloration formed in between two liquid phases indicated the presence of terpenoids. flavonoid: crude rice sample (2 g) was mixed with 10 ml of ethyl acetate heated in a water bath (5 minutes). the whatman paper no.1 was used to filter the solution. then, the filtrate (4 ml) and the dilute ammonia solution (10%) were mixed well and observed for color change (yellowish coloration), which indicated the presence of flavonoids. tannin: a few drops of 0.1% fecl3 solution were mixed with the (10 ml) rice extract filtrate. the formation of bluish-black precipitation indicated the presence of tannin. phlobatannin: the concentrated hcl was added to the rice extract filtrate (10 ml) in a test tube, and boiled for 1 minute. it was then observed for the formation of a red precipitate, which indicated the presence of phlobatannins. protein: rice extract (2 ml) was taken in a test tube was taken. one ml of 40% naoh solution was added to it. the solution was then mixed properly. one to two drops of cuso4 solution were added to the solution. the change of color to violet indicated the presence of proteins. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 pakuwal and manandhar. ©njb, bsn 45 saponin: the crude sample (0.5 g) was mixed in distilled water, and boiled by using a water bath. after that, it was shaken well. the sample was then observed for the formation of froth, which indicated the presence of saponin. anthraquinone: the crude sample and benzene were mixed in a conical flask. a magnetic stirrer was used to mix it properly for 4 hrs. the filtrate (10 ml) was taken and mixed with 0.5 ml ammonia solution (10%). the change in color of the solution indicated the presence of anthraquinone. glycoside: the rice extract (5 ml) was mixed with a mixture of glacial acetic acid (2ml), 2% fecl3 solution and 1 ml concentrated h2so4 (added slowly when mixed). the mixture was observed for the formation of a brown ring, which indicated the presence of glycoside. data analysis all the tests (except length, breadth of rice, and amylose content) were performed on triplicates (n =3). the length and breadth of rice were measured in n=10 by randomly selecting grains from each sample. the determination of the level of significance of various parameters of different rice varieties was performed by using minitab version 18. one factor analysis of variance was performed for a completely randomized design to check the level of significance. results four different varieties of rice tr, kr, rr, and br were collected from four different places. table 1 shows that the antioxidant was highest for br (61.58±0.02%), lowest for tr (8.13%) and the phenolic content was highest for br (22.75 ±0.02gae/100g), lowest for tr (9.35±0.01gae/100g). the physical characteristics and cooking characteristics of different rice varieties were studied. the rice kernel weight (g) (in table 2) was 19.21±0.23, 17.12±0.05, 18.04±0.03, and 17.03±0.02 for tr, kr, rr, and br respectively. the results showed that the length and breadth of rice varieties ranged between 4 mm to 6 mm and 2 mm to 4 mm. the swelling capacity ranged from 1.80 w/w to 3.83 w/w. the cooking time ranged minimum for kr (20 minutes) and maximum for br (40 minutes). the moisture content, ash content, and amylose content, as well as amylopectin, which was determined for each variety of rice (in table 3). the moisture content of tr, kr, rr, and br 9.05± 0.01%, 7.9± 0.00%, 10.2± 0.03%, and 12.15± 0.01% respectively. table 1. bioactive compounds in different rice varieties s.n. type of rice antioxidant (%inhibition) phenolic content (gae/100g) 1. taichung-176 (tr) 8.13±0.01 9.35±0.01 2. khumal-4 (kr) 16.38±0.00 11.74±0.01 3. marsi/ red rice (rr) 53.06±0.01 15.47±0.03 4. black rice (br) 61.58±0.02 22.75±0.02 note: phenolic content was expressed in the unit of mg gae/100g. all the values are mean ± standard deviation of triplicate analysis. the data presented were found to be significant (p < 0.05). table 2.physical characteristics of different rice varieties s. n. rice type kernel weight (g) length (mm) breadth (mm) cooking time (min) amylose content (%) amylose type amylopectin (%) swelling property 1. tr 19.21±0.23 4±0.01 3±0.2 23±2.0 27.62 high 72.40 3.83±0.25 2. kr 17.12±0.05 5±0.20 3±0.1 20±0.5 24.12 medium 75.88 3.25±0.02 3. rr 18.04±0.03 6±0.25 4±0.25 35±1.0 20.67 medium 79.33 2.32±0.18 4. br 17.03±0.02 6±0.25 3±0.5 40±2.0 5.28 low 94.72 1.80±0.15 note: all the tests (except length and breadth of rice) were performed in triplicate and the data listed are the mean ± standard deviation of triplicate readings. the data presented were found to be significant (p < 0.05). table 3. proximate analysis of different rice varieties s. n. tests performed types of rice tr kr rr br 1. ash content (%) 1.62±0.01 0.74±0.01 1.56±0.01 1.70±0.01 2. moisture (%) 9.05±0.01 7.91± 0.00 10.2± 0.03 12.15± 0.01 3. carbohydrate (%) 82.5±0.02 65.3± 0.01 74.5± 0.01 73.2±0.01 4. reducing sugar (%) 2.74±0.01 1.42± 0.01 1.12± 0.02 1.87± 0.03 note: x±y= mean ± s.d of triplicate values. all the experimental values were generated three times and the mean was then calculated. the data presented were found to be statistically significant (p < 0.05). nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 pakuwal and manandhar. ©njb, bsn 46 the ash was highest in br 1.70±0.01%. amylose content was highest in tr (27.62±0.01%) and lowest in br (5.28±0.01%). the reducing sugar was found highest in tr (2.74±0.01%), and lowest in rr (1.12±0.03%). the phytochemical properties of different varieties of rice samples (table 4). tannin, alkaloids, flavonoids, and terpenoids were present only in red rice and black rice. anthraquinone and saponin were absent in all the samples. glycoside and protein were present in all the rice samples. discussion the antioxidant activity was determined by the dpph assay method by determining the percentage inhibition for every rice variety. the antioxidant activity of marsi was compared with tr, kr, and br. br had the highest antioxidant property and the phenolic content which was 61.58± 0.02 % and 22.75± 0.02 gae/100 g respectively. the lowest antioxidant activity was found in tr, which was 8.13± 0.01%. the dpph radical scavenging activity was found higher in br variety (59.02 to 75.52%) with the highest observed in aromatic black rice poireiton chakhao (75.52%) [26]. another study reported that the dpph scavenging activity of red rice, parboiled rice, and sona masuri were 57.06%, 9.13%, and 16.38%, respectively [18,29]. photograph 1. black rice variety table 4. qualitative analysis of phytochemical properties in rice sample s. n. tests performed types of rice taichung-176 (tr) khumal-4 (kr) red rice/ marsi (rr) black rice (br) 1. tannin + + 2. flavonoid + + 3. alkaloids + + 4. terpenoid + + 5. protein + + + + 6. glycoside + + + + 7. anthraquinone 8. saponin note: (+) denotes the presence of the phytochemical and (–) denotes the absence of phytochemical flowchart 1. schematic representation of study of different rice varieties nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 pakuwal and manandhar. ©njb, bsn 47 photograph 2. khumal-4 rice variety photograph 3. taichin rice (taichung-176) photograph 4. red rice variety (jumli rato marsi) the phenolic content of marsi was compared to that of other rice varieties. only br had a higher phenolic content than that of marsi rice variety, which was 22.75± 0.02 gae/100 g. the lowest phenolic content was found in tr, which was 9.36± 0.01 gae/100 g. similar findings of the phenolic content and antioxidant were found in another study on pigmented rice varieties [27] [29]. all the results from this study were found to be similar to the result of previous experiments, and also were found to be statistically significant (p < 0.05). the moisture content of marsi rice was compared to that of tr, kr, and br. the moisture content of marsi was 10.2% and for tr, kr, and br, it was 9.05%, 7.9%, and 12.15% respectively. only br was found to have more moisture than marsi. only br was found to have the highest ash content compared to that of marsi, which was found to be 1.70%. in a similar study, the ash value was higher in black rice varieties compared to the other varieties of rice, which is the result of a similar trend as in our study [28]. in another study, black rice was compared to white, brown, glutinous, and basmati rice, black rice was found to have higher ash content than other varieties [18][30]. carbohydrate content was found to be 74.5± 0.01% in marsi rice variety and only tr had carbohydrate content higher than that of marsi which was 82.5±0.02%. similar findings were reported in a previous study for the jumli marsi rice [7]. amylose content plays an important role in determining the cooking and pasting properties of a rice variety [13,15]. the cooking quality of rice depends on the components of the rice variety such as proteins and amylopectin varieties [31][32]. amylose content in tr, kr, marsi rice, and br were found to be 27.6± 0.01%, 24.12± 0.01 %, 20.67± 0.01%, and 5.28± 0.01% respectively. in this study, the relation was found to be positive as the rice varieties with a high amount of amylose had shorter cooking time duration (table 2). similar trends in results were found in another study varieties [14,24,31]. in this study, a qualitative analysis of phytochemical properties was also performed in all the different varieties of rice. tannin, alkaloids, flavonoids, and terpenoids were present only in red rice and black rice. anthraquinone and saponin were absent in all the samples. glycoside and protein were present in all the samples (pigmented and non-pigmented rice). similar results were observed in a study performed on different rice varieties [9]. the purpose of the study was to compare different characteristics of varieties of rice available in nepal. marsi rice and br are expensive when compared to the other two varieties, and they are mainly consumed for their nutritional benefits. the reason for choosing these four rice varieties is that they are of different price range and also have different colors as well as characteristics. the pigmented and non-pigmented range of rice varieties were taken for the study because of how they are promoted in the food market these days. pigmented rice varieties were found to be more nutritious as they nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 4 2 4 9 pakuwal and manandhar. ©njb, bsn 48 were found to have more amount of antioxidant content as well as phenolic content. this could be because the pigments of the rice varieties are directly linked to the amount of nutrition. marsi and black rice get their pigment from anthocyanin, which is known to have antioxidant properties. conclusion in conclusion, jumli marsi rice variety is highly valued in nepal for its unique color and high nutritional content. from the result of this study, marsi rice was found to be rich in antioxidants, phenolics when compared to the other two non-pigmented rice varieties but the black rice variety was found to be richest in the bioactive compounds. for a detailed comparison of these rice varieties, the determination of anthocyanin, tannin content, and flavonoid could be done. since methanol is used as an extracting solvent for most of the polar components, there may be the presence of non-polar components which may not have been measured by using this method. different methods could be used to quantify the non-polar content present in different rice varieties. author’s contribution the study was performed by ms. evance pakuwal under the supervision of mr. prakash manandhar. competing interest there are no competing interests involved in this study. funding there was no funding provided by any internal or external sources. ethical approval and consent not applicable. acknowledgment i would like to acknowledge my supervisor, lecturer mr. prakash manandhar for his constant encouragement, and valuable supervision at every stage of this work. i would like to thank our head of the department of microbiology, mr. sudhakar pant, all the faculty members and staff of the department of microbiology (st. xavier’s college) for their constant technical and academic support. references 1. the rice department moac. thailand rice cultivation areas. 2018. available from: www. ricethailand.go.th/rkb3/eb_024.pdf 2. faostat. food and agriculture organization of the united nations. 2001. 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qualities of locally grown and imported rice varieties marketed in penang, malaysia. int. food res. j. 2013; 20(3): 1345-1351. 32. singh n, kaur l, singh sn and sekhon ks. physicochemical, cooking and textural properties of milled rice from different indian rice cultivars. food chemistry. 2005; 89: 253–259. doi: 10.1016/j.foodchem.2010.05.115 http://dx.doi.org/10.1016/j.foodchem.2011.07.127 https://www.sciencedirect.com/science/article/pii/s1026309811001945#! https://www.sciencedirect.com/science/article/pii/s1026309811001945#! https://www.sciencedirect.com/science/article/pii/s1026309811001945#! https://www.sciencedirect.com/science/journal/10263098 https://doi.org/10.1016/j.scient.2011.09.014 special issue nepal j biotechnol. 2 0 2 0 o c t ; 8 (2): 69-75 doi: https://doi.org/10.3126/njb.v8i2.31893 research article ©njb, bsn 69 comparative assessment of antibiotic resistance in lactic acid bacteria isolated from healthy human adult and infant feces rasika pawar1 , vasudeo zambare2 , bela nabar1 1department of microbiology, smt chandibai himathmal mansukhani college, ulhasnagar, thane, maharashtra, india 2department of life sciences, school of science, sandip university, nashik, maharashtra, india article history:received: 20 jun 2020; revised: 23 sep 2020; accepted: 30 sep 2020; published online: 22 oct 2020 abstract lactic acid bacteria are normal inhabitants of the gastrointestinal tract of humans. their occurrence in infant and adult feces is abundant. the current study assesses and compares the antibiotic resistance in lactic acid bacteria isolated from healthy human adult and healthy infant fecal samples. a total of 255 lactic acid bacteria isolates (126 from adult feces and 129 from infant feces) were isolated and characterized from 60 fecal samples. lactobacillus spp., pediococcus spp. and enterococcus spp. were included in the study. the study was done using the whonet software for the analysis of antibiotic susceptibility data of lactic acid bacteria. most of the lactobacillus and pediococcus strains were sensitive to vancomycin. enterococcus strains showed resistance against vancomycin. ampicillin, ciprofloxacin and cefuroxime resistance were significantly (p<0.05) higher in lactobacillus strains isolated from adult fecal samples than those isolated from infant fecal samples. a similar pattern was observed in enterococcus strains with erythromycin, gentamycin and tobramycin resistance. pediococcal isolates from adult feces showed significantly higher resistance against tobramycin, ciprofloxacin, gentamycin, cefotaxime and cefuroxime in comparison with infant fecal isolates. antibiotic resistance was exhibited by lactic acid bacteria against most commonly used antibiotics and it was higher in strains isolated from adult fecal samples than in the strains isolated from infant fecal samples. the increasing trend in antibiotic resistance from infant to adult might be due to food habits and antibiotic intakes. thus, the widespread antibiotic resistance in different lactic acid bacteriamay pose a food safety concern as well. keywords: lactic acid bacteria, antibiotic resistance, lactobacillus, feces, fecal microbes corresponding author, email: belanabar23@gmail.com introduction the lactic acid bacteria (lab) originate from a taxonomically diverse group of microorganisms, which are non-sporing rods and cocci, usually nonmotile that ferment carbohydrates and form lactic acid. lactic acid bacteria contain the genera namely lactobacillus, lactococcus, pediococcus, streptococcus, enterococcus, oenococcus, leuconostoc, carnobacterium, vagococcus, tetragenococcus, and weissella [1]. the microflora of humans and animal gut is complex and it is primarily dominated by lactic acid bacteria. there is high density and rich diversity of microorganisms in the gut, and the microflora complexity increases from the upper gastrointestinal tract to the colon [2]. the human gut contains more than a thousand bacterial species and some of them start to colonize the gut during infancy [3]. soon after the birth of a newborn infant, the gut flora begins to develop and microbes start to colonize the small intestine and large intestine. aerobic and facultative anaerobic bacteria (enterobacteria, enterococci and streptococci) are the early colonizers in the human gut. after they colonize, they create anaerobic environment in the gut. this helps anaerobic bacteria (bifidobacteria, bacteroides and clostridia) to start with their colonization majorly in the large intestine [4]. the development of complex, diverse and stable microflora continues from infancy to one year of age. after a year it is similar to adults and it is stable [4]. many factors are governing the development, diversity, composition and colonization gut microflora of infants, out of which mother’s gut microflora, food and environment are the deciding ones [5]. during birth, an infant is exposed to the mother’s vaginal microflora and also to fecal microflora, and with this exposure colonization of the gut in infants begins [6]. infant gut microflora is affected by colostrum and later by breast milk. after the introduction of formula and solid foods, nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:belanabar23@gmail.com https://orcid.org/0000-0003-3739-3752 https://orcid.org/0000-0001-9064-8289 https://orcid.org/0000-0002-4370-3510 mailto:belanabar23@gmail.com mailto:belanabar23@gmail.com nepal j biotechnol. 2 0 2 0 o c t ; 8 (2) [special issue]: 69-75 pawar et al. ©njb, bsn 70 complexity and diversity is generated in the gut microflora of infants. microbes present in the environment and those present directly on the skin of the infant also enter the gut and create a complex niche [7]. colonization of the gut with diverse microflora creates continuous impacts on the immune system; and in this process, it strengthens the immune system [8]. over the past few decades, there has been a huge interest developed in lab physiology and genetics, involving their increasing importance as starter cultures in different industrial fermentation processes and also as probiotics. since probiotics are directly administered in humans and animals it is very necessary to determine the level of antibiotic resistance. this is a part of the assessment of the safety of the probiotic cultures which are administered as therapeutics. in the past 60 years, approximately 10 million tons of antibiotics have been utilized and released into the environment. as presented in the reports of european commission there is a huge probability of the spread of antibiotic resistance in the biosphere [9]. hence,there is a very strong selective pressure in the development of antibiotic resistance in bacterial strains [10]. lactic acid bacteria dominate the gastrointestinal tract of humans. they are present in large amounts in the gut and are also added or sometimes additionally consumed along with the regular diet. hence, it is speculated that the presence of antibiotic resistance in lactic acid bacteria used as probiotics can be dangerous. probiotics are generally administered to maintain microbial balance during gastrointestinal tract infections such as diarrhea. they are administered as therapeutic agents along with antibiotics. if probiotics harbor antibioticresistant genes, it could be beneficial in sustaining the antibiotics during the treatment; however, there is a risk of antibiotic-resistant probiotic strains to transfer the resistance genes to the pathogenic bacteria. this could complicate the treatment of a patient with an antibiotic-resistant bacterial infection or disease. additionally, there is the possibility of the transfer of antibiotic resistance from beneficial lactic acid bacteria, in the food chain. therapy with any antibiotic, particularly long term and especially oral administration is liable to alter the balance of antibiotic-resistant to sensitive organisms in the intestine [11]. certain strains of these genera are more commonly used in the food and especially dairy industries or as probiotics [12]. the world health organization (who) has established a program known as the antimicrobial resistance monitoring (arm) program for monitoring antimicrobial resistance. who has also devised an electronic format whonet, freely available to download. a special focus of antimicrobial susceptibility test results is available on windows-based database software, developed for the management and analysis of microbiology data [13]. this study aimed to determine the antibiotic susceptibility of lactic acid bacteria (using whonet software) isolated from adult and infant feces to various groups of antibacterial agents that are mainly isolated from the feces of breastfed infants. also, the comparative assessment was done to determine the isolates that are more resistant to antibiotics. materials and methods sample collection and ethics statement thirty healthy adult human volunteers (from mumbai and suburbs, india) aged between 25 and 30, who were not suffering from any chronic disease, had not taken antibiotics, proton pump inhibitors, bismuth compounds, histamine h2-receptor, nonsteroidal anti-inflammatory drugs within the previous 6 months, were selected for the study. similarly, fecal samples were also collected from thirty healthy infants aged between 3 months to 9 months. infants who were exclusively breast-fed, healthy and free from acute or chronic disease were selected in the study. the study protocol was approved by an independent ethical committee and performed in compliance with the us code of federal regulations on good clinical practices (21 cfr 10.90, 50, 56 and 812) and the world medical association declaration of helsinki (1996 amendment) [14]. all adult volunteers and parents of infants signed informed consent before samples were collected. isolation of lactic acid bacteria from the fecal sample fecal samples were collected in sterile polypropylene containers and processed immediately as follows. a 0.5 g portion of feces was taken from mid sample, added in 4.5 ml of sterile nepal j biotechnol. 2 0 2 0 o c t ; 8 (2) [special issue]: 69-75 pawar et al. ©njb, bsn 71 saline solution, and completely homogenized. a dilution series (10–1 to 10–7) was made and 100 µl aliquots of each dilution were inoculated on the agar plates by spread plating. rogosa sl agar (himedia, mumbai, india) was used to isolate lab and the plates were incubated micro-aerobically for 3 days at 37°c. kenner fecal (kf) agar was used for the isolation of enterococcus and incubated aerobically at 37°c for 24 h [15]. enumeration and selection of bacterial isolates after incubation, the plates that showed discrete colonies were selected and the colonies were counted. the total count of lactic acid bacteria in feces was expressed as colony-forming units/g (wet weight). from each fecal sample, 10-20 colonies of lab were randomly selected. a provisional identification of genera was made based on gram’s staining, and catalase reaction using 3% (v/v) h2o2 on single colonies. putative lactobacilli colonies (gram-positive, catalase test-negative, rod-shaped) were chosen and further purified using mrs agar. similarly, putative colonies of enterococci and pediococci (gram-positive, catalase test-negative, cocci, able to grow at 10oc and 45oc, and in 18% nacl and at ph 4.4) from kf agar plates were purified by re-streaking on the mrs agar. the cultures were stored in mrs broth with 15% glycerol at –20°c [15]. antibiotic resistance the antibiotic resistance/susceptibility patterns of isolated strains of lactic acid bacteria were studied using the kirby-bauer disk diffusion method (according to the clsi document m2-a9 suggestions) [16]. the antibiotics used in this study were penicillin (10 µg), ampicillin (10 µg), vancomycin (30 µg), cefuroxime (30 µg), cefotaxime (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), tobramycin (10 µg), erythromycin (15 µg) and chloramphenicol (30 µg). the culture densities were adjusted to mcfarland 1.5; they were spread on mrs agar plates. antibiotic discs (hi-media, mumbai, india) were placed on the surface of the agar plates, which were incubated at 37°c for 24 h. the diameters of the clearance zones around the discs were measured and the result (the average of 2 readings) was expressed as susceptible, intermediate, or resistant according to the standard disc diffusion method [16]. the experiment was done in triplicates. microsoft excel (2013) was used to obtain data in the appropriate format for baclink 2019, used to format data to be used in whonet 2019, which automatically calculates the % resistance using a data analysis tool. statistical analysis the data was analyzed to check the significant difference between groups using student’s t-test with a probability level of 0.05 (p < 0.05) using microsoft excel (2013). results isolation of lactic acid bacteria from the fecal sample a total of 255 lab isolates were isolated from 30 human adult and 30 human infant fecal samples. out of the 255 isolates, 126 isolates were from the adult fecal sample, and 129 from the infant fecal sample, the results are presented in table 1. the isolates were identified phenotypically and characterized. based on the characters, the lab isolates were characterized as mesophilic homofermentative cocci, able to grow at 10oc and 45oc as enterococcus (81 isolates). homofermentative cocci in tetrads, unable to grow in 18% nacl, and showing growth at ph 4.4 were characterized as pediococcus (84 isolates). lactobacilli (90 isolates) were represented as catalase-negative, slender gram-positive rods. all strains grew at 4oc and 6.5% nacl concentration. antibiotic resistance of lactic acid bacteria data of diameter of zone of clearance in mm of lab isolated from adult and infant feces was entered in microsoft excel and via baclink software incorporated into whonet software (table 2). table 1. count of lab isolates in the adult and infant fecal samples. sample source number of samples (n = 60) lab isolates (n = 255) lactobacillus spp. (n=90) pediococcus spp. (n=84) enterococcus spp. (n=81) adult feces 30 126 46 41 39 infant feces 30 129 44 43 42 *lab= lactic acid bacteria nepal j biotechnol. 2 0 2 0 o c t ; 8 (2) [special issue]: 69-75 pawar et al. ©njb, bsn 72 figure 1. antibiotic resistance pattern of pediococcus spp. (a), lactobacillus spp. (b) and enterococcus spp. (c) isolated from adults and infant feces, respectively [ampampicillin, chl-chloramphenicol, cip-ciprofloxacin, ctx-cefotaxime, cxm-cefuroxime, ery-erythromycin, gen-gentamicin, pen-penicillin g, tob-tobramycin, van-vancomycin]. all experiments were performed in triplicates and the error bar represents the standard deviation of independent performs experiments (n=3). pediococcus spp. isolated from adult feces was comparatively more resistant to antibiotics than those isolated from infant feces. significantly higher resistances (p < 0.05) were found against ampicillin (7.3%), cefotaxime (22.0%), cefuroxime (36.6%), penicillin (12.2%) gentamycin (26.8%), erythromycin (19.5%), tobramycin (29.3%) and ciprofloxacin (26.8%) from isolates from adult feces than those isolated from infant feces, 7.0%, 9.3%, 0.0%, 11.6%, 9.3%, 0.0%, 2.3% and 20.9% respectively (figure 1a). all the isolates from both adult and infant samples were sensitive to vancomycin and chloramphenicol. pediococcus spp. were intrinsically resistant to high levels of glycopeptides and penicillin. resistance to erythromycin was also reported and was due to a plasmid with an erythromycin resistance methylase b [erm(b)] gene [17]. discussion to develop probiotics for human or animal consumption, it is necessary to distinguish strains harboring antibiotic resistance genes from other strains because of potential risk for the dissemination of resistance genes. in this study, it was demonstrated that strains isolated from infants were more sensitive than those isolated from adult feces. lactobacilli and pediococciare widely used as probiotics and promoters for biological growth. lactobacilli are reported to be resistant to several antibiotics [18]. in the present study, lactobacillus spp. isolated from adult feces were more resistant to antibiotics than those isolated from infant feces. significantly higher resistance was found against cefuroxime (26.1%) and ciprofloxacin (32.6%) from isolates from adult feces than those isolated from infant feces, 4.5%, and 2.3% respectively. lactobacillus spp. isolated from feces also showed moderate resistance to cefotaxime (13.0%), penicillin (10.9%), chloramphenicol, (10.9%), gentamycin (23.9%), erythromycin (13.0%) and tobramycin (26.1%). whereas those isolated from infant feces showed comparatively lesser resistance 4.5%, 11.4%, 9.1%, 20.5% and 9.1%, respectively (figure 1b). resistance to gentamycin and ciprofloxacin was earlier documented [19, 20]. concerning cell wall synthesis inhibitors, lactobacilli are reported to be resistant to oxacillin and cephalosporins (cefoxitin and ceftriaxone) [21]. they were also found to show resistance to aminoglycosides (neomycin, kanamycin, 0 10 20 30 40 50 % r e si st a n t antibiotics [a] pediococcus spp. adult 0 10 20 30 40 50 % r e si st a n t antibiotics [b] lactobacillus spp. adult infant 0 10 20 30 40 50 % r e si s ta n t antibiotics [c] enterococcus spp. adul t nepal j biotechnol. 2 0 2 0 o c t ; 8 (2) [special issue]: 69-75 pawar et al. ©njb, bsn 73 streptomycin, and gentamicin) [22]. there are many species of lactobacilli which contain intrinsic resistance to vancomycin, erythromycin and tetracycline. the matter of concern is that since lactobacilli are added to infant food, they can act as reservoirs of antibiotic resistance genes, which could be transferable [23]. enterococcus spp. also followed a similar pattern where the antibiotic resistance associated with adult fecal samples was higher than those isolated from infant feces. adult fecal isolates were 30.8% resistant to erythromycin, 20.5% resistant to tobramycin, and 41% resistant to gentamycin. this was significantly higher (p < 0.05) than infant fecal isolates, which were sensitive to erythromycin, 9.5% resistant to tobramycin, and 4.8% to gentamycin. higher resistance was also found against vancomycin (30.8%), ciprofloxacin (33.3%), ampicillin (28.2%), cefuroxime (20.5%) and cefotaxime (28.2%); however, it was not statistically significant in comparison to infant fecal isolates which showed 11.9%, 9.5%, 21.4%, 9.8% and 26.2% resistance against above-mentioned antibiotics, respectively. all the isolates (fecal and adult) were susceptible to chloramphenicol. infant isolates were 11.9% resistant to penicillin; this was higher than adult isolates, which showed 10.3% resistance (figure 1c). enterococci showed intrinsic and acquired resistance against many antibiotics [24, 25]. such intrinsic resistance was reported inlincosamides, nalidixic acid penicillin, polymyxins, quinupristin– dalfopristin, monobactams, and low levels of aminoglycosides. resistance to high levels of aminoglycosides, high levels of trimethoprim, and high levels of clindamycin, chloramphenicol, tetracyclines, penicillins (due to βlactamase), fluoroquinolones, macrolides (e.g. erythromycin), glycopeptides and oxazolidinones (linezolid) were acquired [26-27]. acquired resistance is a major threat in treatment, such a trait was found to be transferred to other enterococci in the gut [28]. vancomycin resistance is especially important as vancomycin is the last drug option for treating diseases caused by multidrug resistance enterococci [29]. apart from probiotic use, pediococci are also widely used for the fermentation of meat and vegetables and also in cheese production [30]. according to the efsa’s feedap panel [31] (european food safety authority panel on additives and products or substances used in animal feed), the bacterial cultures which are used for the production of animal feed should be susceptible to antibiotics used in treating humans bacterial infections. hence, it is extremely necessary to distinguish antibiotic susceptible and resistant strains. this also emphasizes the importance of safe source or niche of a selection of strains used as probiotics. the results of the study indicate that infant feces could be a better source for isolation of lab cultures intended to be used as probiotics. apart from being used traditionally as starter cultures in dairy products, lab are also used for the production of animal feed. they also belong to normal flora of the human gut and confer health benefits to the host. during the process of food manufacturing and passage of food through gut, there is a possibility of antibiotic resistance, carried by lab getting transferred to human pathogenic bacteria [32]. hence, it is imperative to select strains table 2. percent antibiotic resistance in target microorganisms isolated from adult and infant fecal samples. expressed in percentage (%) mechanism of action antibiotic lactobacillus spp. pediococcus spp. enterococcus spp. adult infant adult infant adult infant cell wall inhibitors ampicillin 10.9 11.4 7.3 7.0 28.2 21.4 cefotaxime 13.0 4.5 22.0 9.3 28.2 26.2 cefuroxime 26.1 4.5 36.6 0.0 20.5 9.8 penicillin 10.9 11.4 12.2 11.6 10.3 11.9 vancomycin 0.0 0.0 0.0 0.0 30.8 11.9 protein synthesis inhibitor chloramphenico l 10.9 9.1 0.0 0.0 0.0 0.0 erythromycin 13.0 9.1 19.5 0.0 30.8 0.0 gentamycin 23.9 20.5 26.8 9.3 41.0 4.8 tobramycin 26.1 6.8 29.3 2.3 20.5 9.5 dna synthesis inhibitor ciprofloxacin 32.6 2.3 26.8 20.9 33.3 9.5 nepal j biotechnol. 2 0 2 0 o c t ; 8 (2) [special issue]: 69-75 pawar et al. ©njb, bsn 74 that have low resistance against antibiotics for human and animal use. from the results of the antibiotic susceptibility in the current study, obtained from a broad range of antibiotics, it was found that the isolated strains of lactobacillus, pediococcus and enterococcus were resistant to various antibiotics. however, antibiotic resistance was lesser in strains obtained from infant fecal samples than adult fecal samples. conclusion lactobacillus, pediococcus and enterococcus as lab were isolated from the human fecal samples exhibiting more antibiotic resistance from adult fecal isolate than the infant. the development of antibiotic resistancein lab can be attributed to the long term exposure of antibiotic as therapeutic agents as well as food habits which pose food safety concerns. thus, it is essential to see safety measure during antibiotic uptake in day to day life. in addition to this, the low antibiotic-resistant strains from infant could be the choice of strain to avoid the risk of transferof lab linked antibiotic resistance to human pathogenic bacteria. authors contribution rp has made a substantial contribution to data analysis and its interpretation. vz contributed in designing the experiments. bn contributed to data interpretation and all authors rp, vz and bn contributed equally to drafting and reviewing of the manuscript followed by final approval from bn. competing interests no competing interests were disclosed. funding the author(s) declared that no grants were involved in supporting this work. acknowledgements rsp is thankful to principal, smt chm college and vz is thankful to sandip university for providing the laboratory facilities and chemicals. ethical approval and consent the study protocol was approved by an independent ethical committee and performed in compliance with the us code of federal regulations on good clinical practices (21 cfr 10.90, 50, 56, and 812) and the world medical association declaration of helsinki (1996 amendment). all adult volunteers and parents of infants signed informed consent before sample collection. references 1. holzapfel wh, haberer p, geisen r, 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https://doi.org/10.1089/mdr.2017.0147 30. reuter g. present and future of probiotics in germany and in central europe. bioscience and microflora. 1997;16(2):43-51. https://doi.org/10.12938/bifidus1996.16.43 31. efsa panel on additives and products or substances used in animal feed (feedap). guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. efsa journal. 2012 jun;10(6):2740. https://doi.org/10.2903/j.efsa.2012.2740 32. schjørring s, krogfelt ka. assessment of bacterial antibiotic resistance transfer in the gut. international journal of microbiology. 2011 oct;2011. https:// doi.org/10.1155/2011/312956 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 1-7 research article doi: https://doi.org/10.3126/njb.v9i1.38644 ©njb, bsn 1 antibiogram and phytochemical analysis of cinnamon, clove, and sichuan pepper extracts. bibek adhikari 1,2 , pradeep kumar shah1, roman karki2 1 department of microbiology, tri-chandra multiple campus, ghantaghar, kathmandu, nepal 2 national food research centre (nfrc), nepal agricultural research council (narc), khumaltar, lalitpur, nepal received: 07 aug 2020; revised: 08 apr 2021; accepted: 16 apr 2021; published online: 31 dec 2021 abstract a wide range of medicinal plant extracts has phytochemicals that possess antimicrobial properties and these plants are used to treat several infections. the study aimed to assess the antimicrobial activities of some spices extracts and to evaluate the phytochemicals present in them. the extracts of spices were prepared using soxhlet apparatus refluxing with methanol and ethanol. the well diffusion technique was implemented for the evaluation of antimicrobial activities of the extracts and the zone of inhibitions was recorded in millimeters. the antimicrobial test was done against five bacterial isolates: escherichia coli, proteus mirabilis, pseudomonas aeruginosa, salmonella enterica serotype typhi, and staphylococcus aureus and a fungal isolate: candida albicans. the extracts were concentrated by rotary vacuum evaporator and a stock solution of 200 mg/ml was prepared by dissolving in 10 % dmso. concentrations of 40, 60, 80 and 100 mg/ml extracts were used for antimicrobial activity. the result of this study showed that clove extracts had the highest antimicrobial property against all the test microorganisms. methanolic extract of clove had the highest inhibitory effect against proteus mirabilis (24.21±0.15 mm), pseudomonas aeruginosa (19.78±0.23 mm), and candida albicans (20.07±0.08 mm) whereas ethanolic extract was effective against escherichia coli (20.44±0.16 mm), salmonella typhi (21.66±0.31 mm) and candida albicans (21.11±0.09 mm). cinnamon and pepper extracts, leaving some exceptions, also had antimicrobial properties. the presence of phytochemicals: polyphenols, flavonoids, and tannins are the major components responsible for antimicrobial activity. thereby, this study successfully demonstrated the possibilities of using spices extracts in the treatment of microbial infections. keywords: antimicrobial activity, dmso, ethanol, methanol, phytochemicals. corresponding author, email: a.bibek52@gmail.com introduction herbal medicine or phytomedicine is the use of plants for medicinal and therapeutic purposes for the curing of diseases and improving human health [1]. a large portion of the world population, especially in developing countries, relies on the traditional systems of medicine to treat a variety of diseases [2]. presently, more than twothirds of the world’s population leans on plant-based medicines relying on the fact that they are harmless and efficient against various afflictions [3, 4]. abundant molecules with antimicrobial properties are present in medicinal herbs. several infectious diseases are treated using a wide range of plant extracts since they possess antimicrobial potentials. noticing the side effects on human health due to synthetic drugs, professionals are on the way to getting advantages from medicinal plants. a wide variety of screened plant molecules are traded as raw materials for several herbal preparations in the market. out of reported 422,127 worldwide plant species, approximately 35,000 to 70,000 plant genera are utilized for medicinal purposes [5, 6]. plants have secondary metabolites called phytochemicals (phyto from greek meaning plant) that protect plants against microbial infections or pests infestations. phytochemicals are active ingredients that possess therapeutic properties that are considered as a medicine or drug [1]. alkaloids, flavonoids, phenolic compounds, and tannins are the essential phytochemicals present in the plants with possible therapeutic activities and these phytochemicals obtained in plant extracts have demonstrated antimicrobial potential against a wide range of infectious microorganisms [7, 8]. microbial infection is a prevailing health problem around the world. plants remain one of the potential sources of effective agents against microbes, including the deadly infection like tuberculosis (mycobacterium tuberculosis), syphilis (treponema pallidum), gonorrhea (neisseria gonorrhoeae), skin and wound infections [9], diarrhea [10], typhoid fever (salmonella typhi), and pseudomonas aeruginosa which directly infects the urinary tract, the pulmonary tract, wounds, burns and also causes other blood infections [8]. microbial infection has been a major cause of death globally. the rapid increase in the development of resistance to antimicrobial agents by the microorganisms has led to the new incidence and re-exposure of disease nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0003-1622-1783 mailto:a.bibek52@gmail.com https://orcid.org/0000-0002-9897-2486 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 1 7 adhikari et al. ©njb, bsn 2 making them difficult and expensive to treat. to overcome these problems pharmaceutical industries are in search of alternative antimicrobial agents. spices possessing a wide range of bioactive compounds such as alkaloids, polyphenols, flavonoids, tannins, saponins, and various antioxidants have great potential as antimicrobial agents that can counteract pathogenic microorganisms. the spices extract solely or in combination with other antibiotics have the potential to work effectively against several infectious microorganisms [9]. cinnamomum verum (syn. c. zeylanicum) also known as cinnamon is a small evergreen tree belonging to the family lauraceae. the volatile oil produced from its leaf and barks are used as a flavoring agent in the food and beverage industry [11,12]. it is also used to treat abdominal pain, impotence, frigidity, dyspnoea, inflammation of the eye, leukorrhoea, vaginitis, rheumatism, neuralgia, wounds, toothache, and diabetes [11]. the principal constituents of leaf, bark and root oils are eugenol, cinnamaldehyde, and camphor, respectively [12,13]. eugenol has been reported to inhibit the growth of escherichia coli o157:h7 and listeria monocytogenes [14]. cinnamaldehyde has been reported to inhibit the growth of staphylococcus aureus, e. coli o157:h7, and salmonella typhimurium [15,16]. syzygium aromaticum also known as clove is the aromatic dried flower buds of a perennial tree belonging to the family myrtaceae. essential oil of clove is used as anodyne for dental emergencies, and against acne, warts, scars, and parasites. also, the clove has antimutagenic, anti-inflammatory, antioxidant, antiulcerogenic, antithrombotic, and antiparasitic, antibacterial, and antiinflammatory properties [3]. research has shown that clove oil is an effective mosquito repellent [17]. the major constituents of clove’s essential oil are carvacrol, thymol, eugenol, and cinnamaldehyde. several studies have demonstrated potent antifungal, antiviral and antibacterial effects of clove [18]. aqueous clove infusion was found to inhibit the growth of germinated spores of bacillus subtilis, and inhibit the pathogens campylobacter jejuni, salmonella enteritidis, and escherichia coli [19]. clove oil exhibited antibacterial activity against s. epidermidis, salmonella typhi, klebsiella pneumoniae, listeria monocytogenes, staphylococcus aureus, and bacillus cereus [20]. zanthoxylum armatum, commonly known as sichuan pepper, belongs to the rutaceae family. the bark, fruits, and seeds of pepper are extensively used in the indigenous system of medicine as a carminative, stomachic, and anthelmintic. the seed and bark are also used as an aromatic tonic in fever, dyspepsia. because of their deodorant, disinfectant, and antiseptic properties, the fruits are used in dental troubles, their lotion for scabies, and also used to ward off houseflies. besides this, it is also used as a flavoring agent in the confectionery industry, and the manufacturing of soft drinks [21]. pepper consists of several phytochemicals such as chlorogenic acid, cinnamic acid, epicatechin, rutin, trifolin, quercitrin, etc. which have anthelmintic, antifungal, and anti-insecticidal activities [22,23]. different solvent extracts of pepper demonstrated antimicrobial properties against several pathogenic such as escherichia coli, staphylococcus aureus, salmonella typhi, proteus vulgaris, pseudomonas aeruginosa, and klebsiella pneumoniae [24]. materials and methods plant samples collection plant materials used in the study consisted cinnamomum verum (cinnamon), syzygium aromaticum (clove), and zanthoxylum armatum (sichuan pepper) which were collected from different parts of nepal (table 1). since these three spices are most commonly found in the nepalese kitchen, the authors selected these spices for antimicrobial and phytochemical analyses. table 1. nomenclature of the spices and the locality from which they were obtained for this study. common name scientific name part used collected from cinnamon cinnamomum verum bark kathmandu clove syzygium aromaticum bud (fruit) kathmandu sichuan pepper zanthoxylum armatum fruit salyan chemicals barium chloride, dimethyl sulfoxide, folin-ciocalteu reagent, folin-denis reagent, methanol, and sodium chloride were purchased from thermo fisher scientific india pvt. ltd. similarly, ethanol was purchased from jiangyin tenghua co. ltd., china, and gallic acid and tannic acid were purchased from loba chemie pvt. ltd., india. additionally, gentamicin, muller hinton agar (mha), nutrient agar (na), potato dextrose agar (pda), and sterile swabs were purchased from himedia laboratories pvt. ltd., india. microorganisms the pure cultures of candida albicans, esherichia coli, pseudomonas aeruginosa, salmonella enterica serotype typhi, and staphylococcus aureus isolated from patients were obtained from the department of microbiology, tribhuvan university teaching hospital (tuth), nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 1 7 adhikari et al. ©njb, bsn 3 maharajgunj, and the culture of proteus mirabilis were obtained from nepal academy of science and technology (nast), khumaltar. the bacterial samples were maintained on na and fungal samples on pda at 4ºc for further experiments. gentamicin 10 µg disc of gentamicin was used as a positive control against bacterial cultures. extract preparation air-dried, 30 g spices powder was taken in 120 ml of absolute methanol and absolute ethanol for 48 hours in soxhlet apparatus (borosil, india) separately and then whatman no. 1 filter paper was used to filter the extract and allowed to evaporate using rotary vacuum evaporator (buchi type) (victo lab, india) at 65ºc for methanolic extract and 50ºc for ethanolic extract [7, 10]. the concentrated extracts were stored in small containers in a refrigerator (4ºc) for further use. standard concentration preparation two grams of each alcoholic extract were taken in a vial and 1ml, 100% dimethyl sulphoxide (dmso) was added and dissolved in 9 ml of sterile double distilled water. thus, 200 mg/ml of stock was obtained as a standard concentration. the extracts were then autoclaved (victo lab, india) for 20 minutes at 121ºc and 15 pounds per square inch pressure [23]. inoculum preparation a colony of the organism from the stored agar plate was taken, transferred to a test tube containing 2.5ml sterilized nb and incubated for about 4 hours at 37ºc in the incubator (sanjeev scientific udhyog, india) and standardized the turbidity to 0.5 mcfarland solution, microbial load equivalent to 1.5x108 cfu/ml [24]. antimicrobial assay by well diffusion method mha plates were prepared and test organisms were inoculated by spreading the bacterial inoculum on the surface of the media with the help of a sterile swab. wells of 6 mm diameter were punched in the agar by using a cork borer. 50 μl of extracts with the concentration of 40, 60, 80, and 100 mg/ml were poured into the well. 50μl of absolute methanol, ethanol, and dmso (10%) were used as controls. the plates were incubated at 37°c for 24 hours. measurement of the diameter of the inhibition zone was done to evaluate the antimicrobial property with the help of a vernier caliper and recorded in mm [25]. phytochemical analysis the extracts of the spices were prepared as described by dimitrijević et al (2014) [26] and some modifications made by bhattarai et al (2019) [27]. 5 g powdered spice was ground with 80 % methanol (30 ml) and was kept in an orbital shaker (accumax, india) shaking continuously for about 20 minutes, and filtered through whatman no. 1 filter paper in a 100 ml volumetric flask. the residue was again subjected to two more extractions with 30 ml each of 80 % methanol for a total time frame of an hour. the volume was made up to 100 ml using 80% methanol and the extracts were stored in a refrigerator at 4ºc until further use. polyphenol content the polyphenol content of sample extracts was measured by using the folin-ciocalteu method, as described by mahdavi et al (2010) [28]. 1 ml of extract was decanted in a volumetric flask of 25 ml containing 9 ml of distilled water. 1 ml of folin-ciocalteu reagent was added and shaken. after 5 minutes, 10 ml of 7 % na2co3 solution was added and the volume was made up with distilled water and mixed. the absorbance was measured at a wavelength of 765 nm using a uv-vis spectrophotometer (genesys, usa) against a prepared reagent blank (distilled water), after incubation for 90 min at room temperature. the polyphenol content was reported as mg gallic acid equivalent per 100g sample (mg gae/100g). a calibration curve was created from the standard and used to determine the corresponding gallic acid concentration of the samples. flavonoid content aluminum trichloride (alcl3) assay as described by samatha et al (2012) [29] with some modification done by faleye et al (2018) [30] was used for the determination of flavonoid content. 0.5 ml of 80 % methanol extract was taken in different test tubes followed by the addition of 2 ml of distilled water and 0.15 ml of sodium nitrite (5 % nano2, w/v). after 6 minutes, 0.15 ml of aluminum trichloride (10 % alcl3) was added and incubated for 6 min, followed by the addition of 2 ml of sodium hydroxide (naoh, 4 % w/v), and volume was made up to 5 ml with distilled water. after 15 min of incubation, absorbance at 510 nm was measured against a reagent blank of distilled water. the calibration standard curve was prepared by preparing gallic acid solutions and the result was expressed as mg of gallic acid equivalents per 100g (mg gae/100g) of the sample. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 1 7 adhikari et al. ©njb, bsn 4 tannin content the folin-denis method was used for the determination of tannin content [31]. in a test tube containing 7.5 ml of distilled water, 0.1 ml of the sample extract was added. 0.5 ml of folin-denis phenol reagent and 1 ml of 0.5 % na2co3 solution were also added and diluted to 10 ml with distilled water. the mixture was shaken well and kept at room temperature for 30 min. absorbance at 775 nm was measured against the reagent blank and a set of reference standard solutions of gallic acid. the tannin content was expressed in terms of mg tannic acid equivalent per 100g (mg tae/100g) of extract. statistical analysis experimental analyses were performed in triplicates. ibm spss (statistical package for social sciences) statistics version 20 was used for the statistical analysis. one-way analysis of variance (anova) was analyzed and the significant differences among them were studied by using tukey hsd at a 5 % level of significance. results antimicrobial activity by well diffusion method agar well-diffusion method was employed to evaluate the antibacterial and antifungal activities of different concentrations (40, 60, 80, and 100 mg/ml) of the spice extracts. the result obtained by the well diffusion method has been summarized in table 2 and table 3. the zone of inhibition greater than 6.5 mm has been taken into account. from table 2 the results obtained in this study revealed that the bacterial strains: e. coli, p. mirabilis, p. aeruginosa, s. typhi, and s. aureus, and a fungal strain: c. albicans to be susceptible against the methanolic and ethanolic extracts of the three distinct spices. both the methanolic and ethanolic extracts of cinnamon had a table 2. antibiogram of methanolic extracts spices conc (mg/ml) zone of inhibition (zoi) mm e. coli p. mirabilis p. aeruginosa s. typhi s. aureus c. albicans cinnamon 40 0 0 0 0 0 0 60 0 0 0 9.02±0.08b 0 7.16±0.07a 80 6.60±0.10a 7.89±0.10a 0 10.19±0.07c 6.52±0.11a 8.02±0.06b 100 7.20±0.11ab 9.02±0.06b 0 11.12±0.05d 7.87±0.09b 10.00±0.07c clove 40 10.62±0.18d 14.90±0.42d 11.61±0.22d 10.22±0.05c 11.69±0.16c 16.50±0.21d 60 14.53±0.23f 18.63±0.30e 13.99±0.14e 11.60±0.26de 13.60±0.23d 18.48±0.12e 80 17.19±0.34g 21.60±0.26f 16.15±0.22f 15.66±0.11g 16.68±0.11e 19.52±0.07f 100 18.43±0.19h 24.21±0.15g 19.78±0.23g 18.95±0.26h 17.41±0.16f 20.07±0.08g pepper 40 7.54±0.14b 7.99±0.10a 0 9.35±0.15b 10.99±0.14c 0 60 8.73±0.20c 12.36±0.21c 7.07±0.20a 12.06±0.22e 11.43±0.21c 0 80 12.97±0.08e 14.4±0.12d 8.46±0.16b 13.46±0.06f 16.65±0.18e 0 100 15.29±0.24f 15.06±0.09d 9.74±0.10c 15.12±0.21g 16.99±0.08ef 0 controls absolute methanol 7.15±0.02bc 7.66±0.24a 7.14±0.27a 7.24±0.07a 7.19±0.14ab 7.45±0.13a gentamicin 20.83±0.02i 18.70±0.08e 29.50±0.03h 36.63±0.11i 21.84±0.08g nd nd= not determined the measurements are the mean values (n, 3) ± standard error of the mean, whereas different alphabets vertically indicate a significant difference at 0.05 probability level. table 3. antibiogram of ethanolic extracts spices conc (mg/ml) zone of inhibition (zoi) mm e. coli p. mirabilis p. aeruginosa s. typhi s. aureus c. albicans cinnamon 40 7.60±0.11abc 0 0 0 0 7.42±0.03a 60 7.94±0.50bc 8.99±0.10a 0 0 0 9.11±0.45b 80 8.12±0.08c 10.06±0.05abc 0 9.57±0.30a 7.78±0.08bc 11.14±0.36c 100 10.28±0.08d 11.97±0.09de 0 14.41±0.30b 8.14±0.15c 12.59±0.18d clove 40 10.25±0.12d 10.41±0.06bc 6.97±0.13a 19.13±0.08c 12.5±0.06d 17.64±0.48e 60 13.12±0.19e 11.15±0.37cd 9.20±0.10b 19.51±0.18cd 14.42±0.16e 18.75±0.39ef 80 15.45±0.29f 12.38±0.68e 10.84±0.17c 20.25±0.12d 15.10±0.11f 19.28±0.35f 100 20.44±0.16g 13.90±0.05f 11.78±0.08d 21.66±0.31e 15.72±0.08f 21.11±0.09g pepper 40 0 0 0 0 0 0 60 0 0 0 0 0 0 80 6.75±0.02a 0 0 0 7.48±0.12ab 0 100 7.08±0.07ab 0 0 0 8.02±0.12c 0 controls absolute ethanol 8.19±0.26c 9.98±0.09ab 6.75±0.13a 8.87±0.27a 7.06±0.01a 7.28±0.14a gentamicin 20.83±0.02g 18.70±0.08g 29.50±0.03e 36.63±0.11f 21.84±0.08h nd nd= not determined the measurements are the mean values (n, 3) ± standard error of the mean, whereas different alphabets vertically indicate a significant difference at 0.05 probability level. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 1 7 adhikari et al. ©njb, bsn 5 similar effect against test microorganisms, with little to no effect at lower concentrations and distinctive effects at higher doses. however, p. aeruginosa was found to be resistant to both extracts of cinnamon. at 100 mg/ml concentration, the zoi measurement of methanolic extract of cinnamon against e. coli, p. mirabilis, s. typhi, s. aureus, and c. albicans were 7.20±0.11, 9.02±0.06, 11.12±0.05, 7.87±0.09, and 10.00±0.07 mm; whereas 10.28±0.08, 11.97±0.09, 14.41±0.30, 8.14±0.15, and 12.59±0.18 mm by the ethanolic extracts of the same concentration. the methanolic extract of clove demonstrated higher antimicrobial activity against p. mirabilis, p. aeruginosa, and s. aureus with the zoi measurement of 24.21±0.15 mm, 19.78±0.23 mm, and 17.41±0.16 mm at 100 mg/ml. whereas ethanolic extract was comparatively found to be most effective against e. coli, s. typhi, and c. albicans with 20.44±0.16 mm, 21.66±0.31 mm, and 21.11±0.09 mm inhibition zone at the same concentration. the methanolic extract of pepper was effective against all the bacterial strains: e. coli, p. mirabilis, p. aeruginosa, s. typhi, and s. aureus with the zoi of 15.29±0.24 mm, 15.06±0.09 mm, 9.74±0.10 mm, 15.12±0.21 mm, and 16.99±0.08 mm at 100 mg/ml and had no any effect against fungal strain i.e., c. albicans. however, the ethanolic extract had a selective activity against e. coli, and s. aureus with the zoi measurement of 7.08±0.07 mm, and 8.02±0.12 mm respectively at the highest concentration i.e., 100 mg/ml. also, absolute methanol and absolute ethanol used as controls demonstrated antimicrobial property to some extent. however, 10% dmso (data not shown) had no significant actions against the tested microorganisms. phytochemical properties the spices used for the experiment demonstrated a decent amount of phytochemical properties which have been tabulated in table 4. among all the spices used, clove had the highest amount of phytochemical properties and pepper showed the lowest. the polyphenol content in spices ranged from 188.48±7.65 to 455.86±9.91 mg gae/100g (y = 0.0178x, r² = 0.9356; where x, y, and r2 represent concentration, absorbance, and correlation coefficient respectively). the flavonoids varied from 117.52±2.68 to 399.70±5.34 mg gae/100g (y = 0.0088x, r² = 0.9924) whereas the range of tannin contents were found to be in between 64.82±1.89 to 326.86±7.96 mg tae/100g (y = 0.0022x + 0.0055, r² = 0.9767). the phytochemicals followed a similar trend: polyphenol> flavonoids> tannins, only the exception being clove where tannin contents were slightly higher than the flavonoid content. discussions dose-reliant antimicrobial activity of extracts was observed, which means with the increase in extract’s concentration (from 40 to 100 mg/ml) the lethal effect was more pronounced. the methanolic and ethanolic extracts of cinnamon were found to be equally effective against most of the experimental microorganisms, the only exception being pseudomonas aeruginosa. both extracts showed the highest inhibitory action against salmonella typhi. vyas et al (2015) [12], recorded the antibacterial activity of methanolic extract of cinnamon against s. aureus, and e. coli and antifungal activity against c. albicans which is in harmony with the result obtained in this study. similarly, tomar et al (2015) [32] reported ethanolic extract of cinnamon to be effective against e. coli, s. aureus, and c. albicans, al-mariri et al (2014) [33] noted decent antibacterial property of the ethanolic extract and the essential oil of cinnamon against proteus species. according to the study performed by abdelfadel et al (2016) [34], water extract of cinnamon has potential antibacterial property against salmonella species. similarly, the methanolic and ethanolic extracts of clove were found to be effective against all of the microorganisms used in the experiment. methanolic extract of clove showed higher inhibition against proteus mirabilis, whereas the ethanolic extract was found to be effective against salmonella typhi. the one-way anova (p<0.05) of methanolic extract demonstrated it to be more efficient than gentamicin against pseudomonas aeruginosa. anova (p<0.05) argued that there is a difference between the treatment but the post hoc test shows there is no difference between ethanolic extract and gentamicin. so, it has the same lethal effect as gentamicin. lopez et al (2005) [35] reported clove oil to be efficacious against foodborne bacteria viz s. aureus, e. coli, p. aeruginosa, and salmonella species. also, karuppiah et al (2012) [36], found that the alcoholic extract of clove has an antimicrobial effect against p. mirabilis. the study performed by pinto et al (2009) [37], table 4. phytochemicals present in spices spices polyphenol (mg gae/100g) flavonoid (mggae/100g) tannin (mg tae/100g) cinnamon 362.95±19.49b 329.29±1.90b 326.86±7.96b clove 455.86±9.91c 399.70±5.34c 402.24±4.25c sichuan pepper 188.48±7.65a 117.52±2.68a 64.82±1.89a gae= gallic acid equivalent, tae= tannic acid equivalent the measurements are the mean values (n, 3) ± standard error of the mean, whereas different alphabets vertically indicate a significant difference at 0.05 probability level. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 1 7 adhikari et al. ©njb, bsn 6 noticed the essential oil of the clove has an antifungal effect against c. albicans. additionally, the methanolic extract of pepper was found to be inhibitory against all of the bacterial strains but not to the fungal strain (c. albicans), showing the highest activity against s. aureus. on the contrary, the ethanolic extract was found to be effective only against e. coli and s. aureus at higher concentrations. the study performed by joshi et al (2009) [38] and dahal et al (2017) [24] reported the antimicrobial effect of pepper methanolic extracts against e. coli, s. aureus, s. typhi, p. aeruginosa, and proteus species which coincides with the result obtained by our study. the phytochemical analyses (table 4) of the spices confirm the presence of bioactive compounds as polyphenols, flavonoids, and tannins which in terms are responsible for antimicrobial, anti-inflammatory, and anti-cancer activities. the phytochemicals of the test spices correspond to that reported by abdelfadel et al (2016) [34], yang et al (2012) [39], al-numair et al (2007) [40], gupta (2013) [41], mishra et al (2014) [42], and zhang et al (2014) [23]. the phytochemicals and their derivatives are responsible for the antibacterial and antifungal activities of the plant extracts. methanol (polarity index = 6.6) being highly polar than ethanol (polarity index = 5.2) is responsible for a higher yield of phytochemicals from the spices [43]. therefore, the methanolic extracts of the spices demonstrated higher antimicrobial properties with the experimental microorganisms than that of ethanolic extracts. cinnamaldehyde, eugenol, linalool, eugenyl acetate, and cinnamyl acetate present in cinnamon is responsible for its antimicrobial property [35]. according to gupta et al (2015) [44], a higher concentration of eugenol, β-caryophyllene, and eugenyl acetate are responsible for antibacterial and antifungal activities of clove. vashist et al (2016) [45] have documented, volatile components as linalool-8-mono terpentriol-3, 7-dimethyl 1-octane-3,6,7-triol, transcinnamic acid, etc are the major chemicals responsible for the antimicrobial effect of pepper. conclusion the spices sample used for investigation constituted phytochemicals. the spices with a high amount of phytochemicals were found to be more effective against various pathogenic microorganisms whereas those spices with little phytochemicals were not as effective as those with higher phytochemicals. this result reveals that phytochemicals are the major constituents of medicinal plants that are responsible for their antimicrobial property. author’s contribution ba conceptualized the research proposal, performed lab works, scoring, and data analyses. pks and rk supervised the research activities and supported data analyses and interpretations. all authors read and approved the final manuscript. competing interests the authors declare that there is no conflict of interest regarding the publication of this paper. funding national food research centre (nfrc) partially funded this research works. acknowledgements the authors would like to express high lexis of thanks to mr. pravin ojha, scientist and mr. ujjwol subedi, technical officer, and other supportive staffs of national food research centre (nfrc), nepal agricultural research council (narc) for their invaluable support and cooperation. ethical approval and consent not applicable references 1. shakya ak. medicinal plants: future source of new drugs. int. j. herb. med. 2016; 4 (4): 59-64. https://doi.org/10.13140/rg.2.1.1395.6085 2. mcgaw lj, jäger ak, van staden j. antibacterial, anthelmintic and anti-amoebic activity in south african medicinal plants. j ethnopharmacol. 2000;72(1-2): 247-263. doi:10.1016/s03788741(00)00269-5 3. kumar y, agarwal s, srivastava a, kumar s, agarwal g, khan mza. antibacterial activity of clove (syzygium aromaticum) and 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analysis of dystrophin gene in nepalese patients with duchenne muscular dystrophy kushal shrestha1, smita shrestha2, mr. saroj khatiwada3, bishnu acharya4, sulochana manandhar5, rohit kumar pokharel6,7 * 1central department of biotechnology, tribhuvan university, kirtipur, nepal. 2central department of biotechnology, tribhuvan university, kirtipur, nepal. 3department of biochemistry, modern technical college, lalitpur, nepal. 4muscular dystrophy foundation-nepal (mdf-nepal), lalitpur, nepal. 5center for molecular dynamics-nepal (cmdn), nepal. 6muscular dystrophy foundation-nepal (mdf-nepal), lalitpur. 7department of orthopedics and trauma surgery, iom, tribhuvan university, kathmandu, nepal. abstract duchenne muscular dystrophy (dmd) is x-linked recessive neuromuscular disorders caused due to mutation in dystrophin gene, leading to progressive muscle weakness. this study was done to identify mutation in dystrophin gene in nepalese patients with dmd using multiplex ligation dependent probe amplification (mlpa) assay in nepal. twenty one patients from different regions of nepal, who were clinically diagnosed as dmd were enrolled in the study. peripheral blood samples were collected in edta vials, gdna was extracted, and deletion mutation in the dystrophin gene was analysed using multiplex ligation dependent probe amplification (mlpa) assay. exon deletion mutation in the dystrophin gene was observed in 14 (66.6%) out of 21 dmd cases. the most common exon deletion was observed and confined in exon 7-14 and 45-53 of dystrophin gene. the location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions. use of mlpa is useful in detecting copy number changes in dmd proband and suspected carriers in nepal. keywords: duchene muscular dystrophy, multiplex ligation dependent probe amplification, mutation, nepal. *corresponding author email: pokharel.rohit@gmail.com introduction duchenne and becker muscular dystrophies (dmd & bmd) are x-linked recessive neuromuscular disorders with incidence of 1 in 3,500 and 1 in 30,000 live male births, respectively [1]. dmd is characterized by progressive muscle weakness with onset at 3-5 years of age, leading to loss of ambulation by 10-12 years of age without treatment. respiratory, orthopaedic, and cardiac complications emerge with age, and without intervention, the mean age at death is around 19 years [2]. both dmd and bmd are caused by mutations in the dystrophin gene (mim 300377), one of the largest known human genes, spanning about 2.4 mb of genomic dna [3]. the most common mutations in the dmd gene are the deletion or duplication of one or more exons [3]. mutations lead to an absence of or defect in the dystrophin protein, which results in progressive muscle degeneration leading to loss of independent ambulation by the age of 13 years. variable phenotypic expression relates mainly to the type of mutation and its effect on the production of dystrophin [4]. muscular dystrophy is diagnosed on the basis of clinical examination, raised creatine phosphokinase (cpk) level, muscle biopsy and dystrophin gene mutation analysis. the genetic tests commonly used to identify dystrophin mutations are multiplex polymerase chain reaction (mpcr), multiplex ligation dependent probe amplification (mlpa), single condition amplification/internal primer, and multiplex amplifiable probe hybridization [5]. mutation analysis of dystrophin gene in dmd/bmd has been performed in several countries [2, 3]. however, no such studies have been performed in nepal. mlpa, a simple and cheaper genetic test to detect duplication/ deletion mutation, could be a better tool for genetic diagnosis of dmd in nepalese patients; thereby assisting in timely diagnosis, treatment and management. we conducted present study among clinically diagnosed dmd patients to find the mutation pattern in dystrophin gene using mlpa and test the efficacy of mlpa in identifying mutation in dmd patients in the nepalese context. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 37-42 shrestha et al. ©njb, biotechnology society of nepal 38 nepjol.info/index.php/njb materials and methods the study was conducted among 21 clinically diagnosed dmd patients from different regions of nepal, who were referred to muscular dystrophy foundation-nepal (mdf-nepal). diagnosis of dmd was done on the basis of clinical presentations and markedly elevated serum creatine phosphokinase (cpk) levels. proper genetic counselling was done, and consent was taken from each patient and their parents. peripheral blood samples (5 ml) were collected from each patient in edta vials and transported to laboratory of central department of biotechnology, tribhuvan university, kirtipur maintaining cold chain. the samples were stored at -200 c until analysis. genomic dna was isolated from blood, as recommended by the mrc-holland, by using the extraction method of the qiaamp dna mini kit [6]. the absorbance of dna samples were measured at 230 nm, 260 nm and 280 nm using the uv spectrophotometer to check for purity of dna samples required for mlpa assay. a commercial mlpa kit (mrc holland) with probes of p034 (dmd exons 1-10, 21-30, 41-50, and 61-70) and p035 (dmd exons 11-20, 31-40, 51-60, and 71-79) was used to detect dmd deletion/duplication in dmd patients according to the manufacturer’s recommended protocol (amsterdam, netherlands). 50-250 ng of genomic dna, in a volume of 5 µl trisedta, was denatured at 980 c for 5 min, cooled down, and then mixed with mlpa p034 or p035 probemix. the mixture was then heated to 950 c for 5 min and incubated at 600 c overnight for probe hybridization. after 16 hours, ligation was performed with ligase-65 enzyme at 540 c for 15 min and ligase-65 enzyme was inactivated at 980 c for 5 min. then, pcr amplification was performed with specific salsa fam pcr primers (5’gggttccctaagggttgga-3’). after that, the mixture was separated by capillary electrophoresis and then analyzed using abi-310 genetic analyzer [7]. the genescan analysis software in the abi-310 genetic analyzer analyzed the raw data to quantify the dna fragments and determine the size of the fragments by comparing them to fragments in a size standard. the electropherograms of test samples were analyzed by comparing with the peak pattern of the male and female reference samples. the novel software coffalyser.net was used to analyze the data obtained after the capillary electrophoresis run which was further confirmed by visual assessment by overlaying two fragment profiles and comparing the relative intensities of fragments. the relative peak ratio (rpr) of every single exon was plotted to its corresponding bar chart [8]. absence of peaks corresponding to two or more contiguous exons was taken to represent a genuine deletion. the absence of only one peak in males, corresponding to a single exon, were also recorded which are yet to be investigated further by applying the novel methods as pcr primer flanking the exons in question or by sequencing. the framedness of the mutation in probands were also estimated by using the dystrophin exonic deletions/duplications reading frame checker 1.9 as recommended by mrc holland. the data from the study was entered into ms excel and analyzed using spss version 11.0. results the mean age of dmd patients at the time of study was 11.4 years (age range from 8-18 years). all the patients had very high serum cpk level (median: 6245 u/l, range: 2100-32890 u/l). the genomic dna extracted from samples had concentration range within 27-59 μg/ml. the ratio of the optical density (od) of dna samples at 260 nm and 280 nm ranged from 1.5-2.0 with mean od 1.76. among the 21 samples of dmd analyzed by mlpa assay, deletions were observed at various exons of dystrophin gene in 14 samples. seven samples however did not have any deletion or duplication in the exons. thus, mlpa tool could detect mutations in about 66.6% (14 of 21 samples). the most prevalent exonic deletion regions were found to be confined in the exon 7-14 and 45-53. deletion mutations at different exons observed in samples are shown in table 1. similarly, electropherograms of control sample and test sample after mlpa assay with salsa probe mix p034 and p035 are shown in figure 1. as indicated by the dystrophin exonic deletions/ duplications reading frame checker 1.9, all the 14 samples with detection of deletion mutation were detected by mlpa assay have out of frame mutations suggesting that the samples were those of dmd patients. but the confirmation of the framedness is yet to be made by gene sequencing, and the 7 samples with negative mlpa result need further genetic testing like sequencing. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 37-42 shrestha et al. ©njb, biotechnology society of nepal 39 nepjol.info/index.php/njb table 1: exon deletions detected by mlpa assay in dmd patients case age (years) serum creatine kinase level (u/l) exon deletions phenotype 1 5 12122 48-50 dmd 2 7 3412 52 dmd 3 6 14467 10-43 dmd 4 8 1287 52 dmd 5 9 11580 45 dmd 6 5 24200 51-53 dmd 7 14 22000 45-50 dmd 8 5 21887 46-49 dmd 9 12 2700 52 dmd 10 11 2100 51 dmd 11 8 4328 12-14 dmd 12 9 3220 51 dmd 13 6 9123 45 dmd 14 7 6245 7-9 dmd discussion in nepal, dmd/bmd patients are generally diagnosed late due to the lack of complete understanding of the disease, poor screening of paediatric group for muscular dystrophy and lack of genetic diagnostic facilities in the country. most of the cases are diagnosed on the basis of history given by the parents, positive clinical signs of dmd and very high ck level in the blood. for the diagnosis at the gene level clients usually go to or the sample is send outside nepal, which is not accessible and affordable to many of the parents. this study to detect deletion mutation in dmd using mlpa is first genetic pilot study in nepal. among the 21 samples of clinically diagnosed dmd patients, deletion of dystrophin exon was found in 14 samples (66.6%). however, 7 samples (33.3%) did not show any deletion or duplication. thus, mlpa was efficient in accurately confirming mutations in about 67% of all the cases. a number of studies have found mlpa assay to be highly sensitive diagnostic assay to identify mutation in dmd and bmd patients [9]. in the study to evaluate the efficacy of mlpa technique in comparison with the traditional multiplex pcr assay in detection of exon deletions and duplications of the dmd gene by lai et al., mlpa was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex pcr [10]. in a study in china amongst 70 dmd/bmd patients, mlpa detected exonic deletions in 42 patients (60%), exonic duplications in 7 patients (10%) and 21 patients (30%) showed normal results [11]. in the present study, only exons deletion was observed. no exon duplication was found. we found most prevalent exonic deletion regions to be confined in the exons 7-14 and 45-53. one, 3, 4, 6 and 44 exons deletion was observed in 7, 4, 1, 1 and 1 patients respectively. no novel mutations were identified in this study. in a study in iranian dmd/bmd patients, 30.9 % of patients had single exon deletion, while group and contiguous exon deletions were identified in 41% of the patients [12]. the most numerous exon deletions included exons 45-50, and two exons 3-5 and 41-43 duplications (1.4 %) was observed in a bmd and a dmd patient, respectively [12]. in a study in polish dmd/bmd patients, zimowski et al. identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. deletions involved mainly exons 45-54 and 3-21, whereas most duplication involved exons 3-18 [13]. it has been found that deletions account for approximately 60-65% of mutations and duplications for 5-10% in dmd/bmd. the remaining cases are mainly point mutations (30-35% of the cases). although deletions encompass all 79 exons, two deletion hotspots (exons 45-55 and exons 2-19) are recognized [14]. identification of exon deletion is important to design gene therapy by exon skipping method that is an emerging therapy for dmd that can transform dmd into bmd is based on the recovery of reading frame induced by alternative splicing of antisense oligonucleotides [15]. more patients may benefit from individual exon skipping therapies following a comprehensive understanding of the correlation between genotypes and phenotypes under the guidance of large-scale genetic epidemiological studies [16]. similarly identifying disease at earlier stage could help in better management of patients and thus better life quality. this was the first genetic study on dmd in nepal. the location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions in nepalese population as well. use of mlpa is useful in detecting copy number changes in dmd proband and suspected carriers. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 37-42 shrestha et al. ©njb, biotechnology society of nepal 40 nepjol.info/index.php/njb a b a c a nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 37-42 shrestha et al. ©njb, biotechnology society of nepal 41 nepjol.info/index.php/njb figure 1. electropherogram of control sample and test sample after mlpa. fig. 1a and fig. 1b are for normal control sample with p034 and p035 respectively. fig. 1c and fig. 1d are for dmd patient with deletion in dystrophin gene with p034 and p035 respectively. competing interest none acknowledgement we appreciate the parents and dmd boys who gave consent to take part in this study. we acknowledge the support of mdf-nepal for counseling parents and probands, providing the detail information about the clients and also bearing the cost of some chemicals for gdna extraction and charge of genetic analyzer. we express our gratitude to prof. masafumi matsuo, m.d; ph.d. from kobe, japan for providing us the mlpa kits, which pioneered the genetic testing of dmd cases in nepal for the first time. we thank prof. tribikram bhattarai, former head of department of biotechnology, tribhuvan university and current head prof. dr. rajani malla for encouraging and providing the laboratory for the study. we also thank intrepid nepal pvt ltd.; centre for molecular dynamics (cmdn), thapathali-11, kathmandu, nepal, for allowing us to use the genetic analyzer at the center. author’s contribution ks, ss and rkp designed the study and collected samples. ks, ss, sk, ba and sm engaged in sample collection and conducted laboratory analysis. sk performed statistical analysis and wrote the manuscript. ks, ss and rkp revised the draft. all authors read and approved the final version of the manuscript. references 1. hegde mr, chin el, mulle jg, okou dt, warren st, zwick me: microarray-based mutation detection in the dystrophin gene. hum mutat. 2008, 29(9):1091-99. 2. li x, zhao l, zhou s, hu c, shi y, shi w, et al: a comprehensive database of duchenne and becker muscular dystrophy patients (0-18 years old) in east china. orphanet j rare dis. 2015; 10:5. doi: 10.1186/s13023-014-0220-7. 3. santos r, gonçalves a, oliveira j, vieira e, vieira jp, evangelista t, et al: new variants, challenges and pitfalls in dmd genotyping: implications in diagnosis, prognosis and therapy. j hum genet. 2014, 59(8):454-64. 4. bushby k, finkel r, birnkrant dj, case le, clemens pr, cripe l, et al: diagnosis and management of duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. lancet neurol. 2010, 9(1):77-93. 5. flanigan km, dunn dm, von niederhausern a, soltanzadeh p, gappmaier e, howard mt, et al: mutational spectrum of dmd mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. hum mutat. 2009, 30(12):1657-66. 6. mlpa, general protocol, mrc-holland, mlpa, protocol version mdp-v002, last update 23-012012. d a nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 37-42 shrestha et al. ©njb, biotechnology society of nepal 42 nepjol.info/index.php/njb 7. chen c, ma h, zhang f, chen l, xing x, wang s, et al: screening of duchenne muscular dystrophy (dmd) mutations and investigating its mutational mechanism in chinese patients. plos one. 2014, 9(9):e108038. 8. wang x, wang z, yan m, huang s, chen tj, zhong n: similarity of dmd gene deletion and duplication in the chinese patients compared to global populations. behav brain funct. 2008, 4:20. doi: 10.1186/1744-9081-4-20. 9. stuppia l, antonucci i, palka g, gatta v: use of the mlpa assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. int j mol sci. 2012, 13(3): 324576. 10. lai kks, lo ifm, tong tmf, cheng lyl, lam sts: detecting exon deletions and duplications of the dmd gene using multiplex ligationdependent probe amplification (mlpa). clinical biochemistry. 2006, 9 :367-72. 11. long f, sun w, ji x, li xh, liu xq, jiang wt, tao j: clinical application of multiplex ligationdependent probe amplification for the detection exonic copy number alterations in the dystrophin gene. zhonghua yi xue yi chuan xue za zhi. 201,28(6):699-704. 12. zamani gr, karami f, mehdizadeh m, movafagh a, nilipour y, zamani m. analysis of dystrophin gene in iranian duchenne and becker muscular dystrophies patients and identification of a novel mutation. neurol sci. 2015. doi: 10.1007/s10072015-2290-2. 13. zimowski jg, massalska d, holding m, jadczak s, fidziańska e, lusakowska a, et al: mlpa based detection of mutations in the dystrophin gene of 180 polish families with duchenne/becker muscular dystrophy. neurol neurochir pol. 2014, 48(6):416-22. 14. nouri n, fazel-najafabadi e, salehi m, hosseinzadeh m, behnam m, ghazavi mr, et al: evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an iranian population subset. adv biomed res. 2014, 3: 72. doi: 10.4103/2277-9175.125862. 15. cirak s, arechavala-gomeza v, guglieri m, feng l, torelli s, anthony k, et al: exon skipping and dystrophin restoration in patients with duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. lancet. 2011, 378(9791):595-605. 16. mitrpant c, fletcher s, wilton sd: personalised genetic intervention for duchenne muscular dystrophy: antisense oligomers and exon skipping. curr mol pharmacol. 2009,2(1):110-21 nepal j biotechnol. 2 0 2 0 d e c ; 8 (3): 102-110 doi: https://doi.org/10.3126/njb.v8i3.33664 research article ©njb, bsn 102 screening of actinomycetes from soil for antibacterial activity shailesh budhathoki1 and anima shrestha2 1department of microbiology, st. xavier’s college, maitighar, kathmandu, nepal. 2department of microbiology, tri-chandra multiple campus, ghantaghar, kathmandu, nepal. received: 30 oct 2020; revised: 21 dec 2020; accepted: 22 dec 2020; published online: 30 dec 2020 abstract actinomycetes are gram positive, free living saprophytes which are distributed in soil as one of the major populations and are primary source of antibiotics. this study was carried out with a quest to isolate actinomycetes from soil samples of different places and assess their antibacterial activity. isolation of actinomycetes was carried out by serial dilution of soil sample followed by spread plate method. the antimicrobial extract was extracted using ethyl acetate. assessment of antimicrobial activity was performed by using agar cup plate assay against test organisms (pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella typhi, salmonella paratyphi, bacillus subtilis, staphylococcus aureus). antibacterial activity was tested against methicillin sensitive staphylococcus aureus and methicillin resistant staphylococcus aureus in the isolates having effective inhibitory activity against staphylococcus aureus. from 15 soil samples of 12 different locations, 121 actinomycetes isolates were isolated. among them, 58 (47.9%) isolates were inhibitory against at least 1 test organism in primary screening, of which 22 isolates effective against more than 1 test organism was chosen for secondary screening. out of them, 8 were inhibitory against 2 test organisms while 14 were inhibitory against 3 test organisms. staphylococcus aureus was found to be the most susceptible test organism with its susceptibility against 12 actinomycetes isolates. among 12 isolates effective against staphylococcus aureus, 10 were found to have an inhibitory effect against methicillin susceptible staphylococcus aureus while 6 were found to have inhibitory effect against methicillin resistant staphylococcus aureus strain. the findings of this study highlight the inhibitory potential of actinomycetes and the need forfurther investigation for obtaining novel antimicrobial agents from actinomycetes from various unexplored areas. keywords: actinomycetes, inhibitory activity, isolates, antimicrobial, antibiotic corresponding author, email: animashrestha77@gmail.com introduction year by year microbial diseases are increasing, which have become the biggest threat to public health. there are about 200 known diseases transmitted by microorganisms such as bacteria, fungi, viruses, etc. to humans [1]. antibiotic refers to the chemical compound derived from microorganisms or living cells that inhibit and/or stop the growth of a microorganism. they are used in the treatment of external or internal infections. while some antibiotics are produced by a microorganism, most are now manufactured synthetically [2]. bacteria, fungi, actinomycetes, algae, lichens and green plants produce antibiotics. actinomycetes are one of the most important microorganisms that hold the prime position in the production of bioactive metabolites. they are responsible for the production of almost half of the discovered bioactive secondary metabolites notably antitumor agents, immunosuppressive agents, enzymes and especially antibiotics [3]. in industrial microbiology, actinomycetes make its position at the top as a potential source of antibiotics [4]. actinomycetes are prokaryotic organisms with filamentous nature, branching pattern, and conidia formation, which are like those of fungi. for this reason, they are also known as ray fungi. they are grampositive, free-living, saprophytic bacteria [5, 6]. actinomycetes populations are identified as one of the major groups of soil population, which may vary with soil type [7]. the number and types of actinomycetes present in soil would be greatly influenced by geographical location such as soil temperature, soil type, soil ph, organic matter content, cultivation, aeration and moisture content [8]. these are inhabitants of soil that also play important role in recycling of organic matter, production of novel pharmaceuticals, nutritional materials, cosmetics, enzyme inhibitors, immunemodifiers and vitamins [9, 10]. numbers of antibiotics have now been isolated from cultures of actinomycetes, such as actinomycetin, micromonosporin, mycetin, and actinomyces lysozyme etc. have been only partially purified, whereas others, including actinomycin, proactinomycin, streptothricin, and streptomycin, have been isolated and crystallized these substances differ greatly chemical structure and their antimicrobial properties, toxicity to animals, and in vivo activity [11]. compounds isolated from actinomycetes have numerous chemical structures such as macrolides, tetracyclines, aminoglycosides, glycopeptides and ansamicines which are used in nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:animashrestha77@gmail.com https://orcid.org/0000-0002-9919-476x mailto:animashrestha77@gmail.com nepal j biotechnol. 2020 dec; 8 (3): 102 -110 budhathoki & shrestha ©njb, bsn 103 antibacterial treatments whereas anthracyclines supplement anticancer chemotherapy. toxic polyethertype antibiotics are used as anti-coccicidal agents [12]. more than 70–80% of all known antibiotics have been isolated from actinomycetes, which are used in medicine and agriculture. the two major groups of soil actinomycetes that serve as important sources of antibiotics are streptomyces and micromonospora. streptomyces spp. are the biggest producer of antibiotics that account for about 80% of the total antibiotic products. micromonospora closely follows with less than one tenth as much as streptomyces [1]. members of streptomyces are a rich source of bioactive compounds, notably antibiotics, enzymes, enzyme inhibitors and pharmacologically active agents [13]. streptomyces are valuable resources for a novel products like antifungals, antivirals, antitumorals, anti-hypertensives, immunosuppressants and especially antibiotics [14]. the nature of the active agents or the antibiotics produced by actinomycetes depends upon the species; frequently upon the strain; the composition of the medium in which it is grown, and the conditions of cultivation. the antimicrobial properties of a given actinomycetes culture also depend upon the composition of the medium in which it is grown [11]. also, it is essential to maintain the optimum temperature, ph and salinity to produce bioactive secondary metabolites. the absence of optimum conditions can lead to failure in production or no growth could also be observed [15]. however, with the time of the discovery of antibiotics, the emergence antibiotic resistance bacteria have been a major problem. also, there is a rapid emergence of drug resistant strains of the pathogen than the rate of discovery of new drugs and antibiotics [16]. the development of resistance by the pathogens as well as the emergence of new pathogens has led to the necessity for the discovery of new antibiotics/antimicrobials for their infection. hence screening of antimicrobial activity of actinomycetes and study of their antimicrobial action against pathogens is an important process for the discovery of an antibiotic. in nepal, various infections are a major public health problems. antibiotics may be prescribed by the physician and other healthcare workers inappropriately or they may be purchased directly by consumers without prescription to the healthcare system. many patients selftreat with antibiotics, including prior to hospital admission, which can contribute to increased resistance rate [17]. drug resistance in microorganisms has been increasing and this has posed a serious threat for the mankind. discovery of the novel antimicrobials that can control the growth of these microorganisms is of global importance. as, actinomycetes are the most important source of antibiotics this study is intended to find out the antimicrobial activity of actinomycetes and their potential to inhibit the microbial growth. this study will serve as baseline data for future studies of the antimicrobial potential of actinomycetes isolates and the discovery of novel metabolites from actinomycetes in nepal. materials and methods collection of sample 15 soil samples from 12 different locations (nuwakot, lagankhel, katunje, chovar, lubhu, budanilkantha, chapagaun, godawari, sundarijal, sankhamul, gokarna and chandragiri) were collected for study (figure 1). laboratory processing of soil samples was carried out at research laboratory of kathmandu centre for genomics & research laboratory (kcgrl) and microbiology laboratory of st. xavier’s college. duplicate soil samples from a depth of 5-10 cm from the surface were collected in two separate sealed plastic containers [4, 14]. figure 1. map of sample collection sites soil treatment (before isolation) soil samples so collected were left to air dry at room temperature for (10-15) days, to reduce the number of contaminant bacteria (especially gram-negative bacteria) in soil samples as previously reported [15, 18]. isolation of actinomycetes the soil sample was serially diluted to 10-2, using distilled water as diluents. the dilutions were vigorously shaken in a vortex shaker to liberate actinomycetes spores from the soil into the supernatant liquid. aliquots of supernatant liquid from 10-1 and 10-2 dilutions of soil sample was plated on selective, solidified, starch casein agar (sca) incorporated with amoxicillin 20 mg/l as reported previously [9] and ketoconazole 30 mg/l [4] so nepal j biotechnol. 2020 dec; 8 (3): 102 -110 budhathoki & shrestha ©njb, bsn 104 as to eliminate contaminant gram positive soil dwelling bacteria and fungi respectively, using spread plate method. the inoculated sca plates were left at room temperature for 5 minutes to allow the agar to absorb the liquid and were incubated at 28°c for 7 days [19]. sub-culture of actinomycetes isolates after 7 days of incubation at 28°c, colonies exhibiting a dry chalky appearance were selected. starch casein agar (sca) plate was divided into a number of sections and each of characteristic actinomycetes colonies was subcultured in sections of sca plate. the plates were incubated at 28°c for 7 days. after colonies had developed, they were further sub-cultured in a test tube containing sca slants, incubated at 28°c for 7 days and then stored in the refrigerator as the stock [19]. the isolates were sub-cultured again at an interval of 2-3 weeks to prevent the cultures from dying out. primary screening of isolates the duplicate perpendicular streak method was employed for the primary screening of inhibitory action of isolates against bacterial species. actinomycetes isolates were streaked vertically down the center of the nutrient agar plate and then incubated at 28°c until visible growth of actinomycetes appear as a confluent vertical line of the colony (usually 7 days). after incubation, each of the test organisms (pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella typhi, salmonella paratyphi, bacillus subtilis, staphylococcus aureus), isolated from hospital admitted patients and confirmed with microscopic, cultural and biochemical characteristics [20], were streaked 1 cm apart and 2mm on the side of actinomycetes colony on nutrient agar, perpendicular to the colony and then incubated at 37°c for 24 hours. after incubation, plates were observed for any inhibition in the growth of test organisms [1, 6]. inhibition in growth was noted as a lack of growth of test organisms on the streak line. selection of isolates for fermentation: isolates inhibiting two or more test organisms (pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella typhi, salmonella paratyphi, bacillus subtilis, staphylococcus aureus) in primary screening were selected for further processing i.e. fermentation for production of antibiotic fractions. fermentation among isolated actinomycetes, 22 isolates exhibiting the greatest action against test organisms in primary screening were selected based on comparative inhibition against one or more isolates and the ability to inhibit two or more test organisms. yeast extractmalt extract broth (isp-2) was prepared in a conical flask. the colony of selected actinomycetes was cut from their pure culture and put into the broth. the conical flasks were placed into a rotary shaker incubator at 28°c for 8 days. [13, 21] extraction of antibiotic fraction: after incubation, the broth was collected and centrifuged at 4000 rpm for 30 minutes. the supernatant was collected and an equal amount of ethyl acetate was added to it. the two phases were vigorously shaken for 1 hr. then, the mixture was poured into a separating funnel and allowed to stand for 5 minutes during which aqueous and organic phases separated. the lower aqueous phase was discarded, while the upper organic phase was collected and heated in a water bath at 40°c to evaporate ethyl acetate [22]. the residue so left was weighed and dissolved in a small amount of phosphate buffer of ph 7 to solubilize crude antibiotic extract [13]. the mixture was transferred to an eppendrof tube and stored at refrigerator temperature for further use. secondary screening: agar well diffusion method was employed for secondary screening of inhibitory action against test organisms. test organisms (pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella typhi, salmonella paratyphi, bacillus subtilis, staphylococcus aureus) were inoculated into the nutrient broth and incubated at 37°c for 4 hrs. after incubation, the turbidity of the broth was compared with mcfarland standard 0.5. lawn/carpet culture of test organisms was performed on mueller hinton agar plates from mcfarland adjusted broth cultures. wells of 8 mm was bored in mueller hinton agar plates using sterile agar borer and 40 μl of ethyl acetate extracted antibiotic fractions were added into wells. plates were left at room temperature for 20-30 mins to diffuse antibiotic fractions, and then incubated at 37°c for 24 hrs. after 24 hrs, plates were examined and the diameter of the zone of inhibition around each well was measured [13]. results soil harvest large number of antibacterial actinomycetes from 15 soil samples, 121 isolates of actinomycetes were obtained. among them, 58 isolates were found to have effective inhibitory activity against at least 1 test organism while 63 were ineffective against test organisms (table 1). twenty-two isolates produced observable inhibitory effects against two or more test organisms during primary screening (table 2). among 58 isolates exhibiting inhibitory activity, 36 were active against one test organism and 22 against more than one test organism. of those 22, 8 and 14 isolates were active against 2 and 3 test organisms respectively (figure 2). nepal j biotechnol. 2020 dec; 8 (3): 102 -110 budhathoki & shrestha ©njb, bsn 105 figure 2. pie-chart showing primary screening of actinomycetes isolates against test organisms. 36 (62%) of isolates were found to be effective in inhibiting at least one test organism. 22 (38%) of isolates were effective on inhibiting 2 or more test organisms. of these 22 isolates 14 (24%) of them inhibited 2 test organisms while 8 (14%) effectively inhibited 3 test organisms. actinomycetes have broad spectrum antibacterial activity among test organisms, staphylococcus aureus was found to be the most susceptible with susceptibility toward 12 actinomycetes isolates followed by klebsiella pneumoniae, susceptible towards 10 isolates of actinomycetes. the least susceptible test organism was found to be salmonella paratyphi with susceptibility towards 5 actinomycetes isolates (figure 3). on secondary screening (testing of effect of antibiotic fraction obtained through fermentation followed by extraction) against test organisms, staphylococcus aureus was found to be the most effectively inhibited test organism followed by klebsiella pneumoniae. salmonella paratyphi was found to be the least inhibited test organism (table 3). table 1. number of actinomycetes isolated from soil samples with a screening of antibacterial activity s. n soil sample place of collection number of colonies number of colonies with inhibitory activity against 1 test organism against ≥2 test organism 1 a nuwakot (damp land) 5 1 1 2 b lagankhel (agri land) 14 5 3 3 c katunje (agri land) 10 3 2 4 d katunje (riverbank land) 8 3 1 5 e budhanilkantha (agri land) 13 4 3 6 f chapagaun (agri land) 19 5 4 7 g chapagaun (forest land) 8 2 2 8 h godawari (damp land) 3 0 1 9 i chovar (forest land) 3 1 0 10 j sundarijal (riverbank land) 5 4 1 11 k sundarijal (forest land) 9 1 2 12 l lubhu (damp land) 4 0 0 13 m sankhamul (riverbank land) 8 2 1 14 n gokarna (forest land) 7 2 0 15 o chandragiri (forest land) 5 3 1 total 121 36 22 table 2: primary screening of effective actinomycetes against test organisms s. n. actinomycetes colony p. aeruginosa e. coli k. pneumoniae s. typhi s. paratyphi b. subtilis s. aureus 1 a3 + + 2 b2 + + + 3 b4 + + + 4 b9 + + + 5 c5 + + + 6 c8 + + 7 d6 + + 8 e3 + + + 9 e7 + + 10 e12 + + + 11 f1 + + + 12 f2 + + + 13 f17 + + + 14 f19 + + 15 g1 + + + 16 g3 + + 17 h4 + + 18 j2 + + + 19 k1 + + + 20 k6 + + + 21 m3 + + 22 o4 + + + 36, 62% 8, 14% 14, 24% isolates effective against 1 test organism isolates effective against 2 test organism isolates effective against 3 test organisms nepal j biotechnol. 2020 dec; 8(3): 102 -110 budhathoki & shrestha ©njb, bsn 106 figure 3. bar diagram showing susceptibility of test organisms towards actinomycetes isolates. staphylococcus aureus was found to be most susceptible organism toward actinomycetes with 12 isolates inhibiting its growth, followed by klebsiella pneumoniae (10 isolates). salmonella paratyphi was least inhibited organism with only 5 actinomycetes isolates able to inhibit its growth. actinomycetes have inhibitory activity against drug-resistant strains the antibiotic fractions found to be effective against staphylococcus aureus from secondary screening were tested against methicillin susceptible staphylococcus aureus (mssa) and methicillin resistant staphylococcus aureus (mrsa) variants. among 12 isolates, 10 were found to have an inhibitory effect against mssa while only 6 were found to have an inhibitory effect against mrsa strain (table 4). table 4: screening results of effective antibiotic producers against mssa and mrsa s.n . actinomycetes colony primary screening secondary screening (zoi in mm) mssa mrsa mssa mrsa 1 a3 + + 16 10 2 b4 + _ 7 3 b9 _ 4 c5 + + 9 5 5 d6 + + 12 8 6 e3 + 6 7 7 e12 + + 8 6 8 f2 + 8 9 g3 + 6 10 j2 + 13 11 k1 + + 9 7 12 k6 + 5 out of 58 effective isolates in primary screening, ph range (7.4-7.8) showed higher number of isolates followed by ph range (7.9-8.4) with 18 and 17 effective isolates respectively (table 5). the least number of effective isolates were found in acidic ph range while neutral and basic ph range showed larger proportion of effective isolates. on distribution of effective isolates based on moisture of soil, moisture range of (11-20) % showed highest number of effective isolates (17). even though the moisture range (31-40) and (41-50) % showed high number of isolates, it has to be noted that the soil samples of river bank has high moisture due to the constant flow of water over them. this moisture range was observed only in river bank soil and the growth of actinomycetes on such soil is affected by humus and various other factors as well. table 3: secondary screening of antibiotic fractions against test organisms (zone of inhibition in mm) s.n. actinomycetes colony p. aeruginosa e. coli k. pneumoniae s. typhi s. paratyphi b. subtilis s. aureus 1 a3 8 20 2 b2 12 9 7 3 b4 10 7 11 4 b9 12 12 7 5 c5 8 15 9 6 c8 8 6 7 d6 9 11 8 e3 5 3 8 9 e7 7 6 10 e12 6 4 12 11 f1 11 12 4 12 f2 3 11 8 13 f17 13 5 12 14 f19 2 9 15 g1 7 8 5 16 g3 8 6 17 h4 5 4 18 j2 6 9 15 19 k1 9 8 13 20 k6 7 9 7 21 m3 9 6 22 o4 8 5 2 9 9 10 6 5 7 12 0 2 4 6 8 10 12 14 n o o f a ct in o m y ce te i so la te s test organisms nepal j biotechnol. 2020 dec; 8 (3): 102 -110 budhathoki & shrestha ©njb, bsn 107 discussion the soil samples undertaken for the study were of different environmental sites which include forest land, agricultural lands, river-bank land, and damp lands. on the isolation of the actinomycetes, it was observed that the highest number of actinomycetes isolates were isolated from 4 soil samples of agricultural land (56 out of 121 isolates i.e. 46.28%). three soil samples from damp land were found to have 12 isolates of actinomycetes which were lowest among the soil samples classified into different ecological conditions. the higher number of isolates from agricultural and forest land can be due to the positive impact of the vegetation in the colonization of the actinomycetes. this finding is in correspondence to the report by ghorbani et al which states that the abundance of actinomycetes isolates decreases from irrigated cultivated land to pastures i.e., damp lands [22]. klemmedson also reported that the actinomycetes may form the number of roots nodulated symbiotic relationship with various plants thus increasing the actinomycetes population in the lands with vegetation [23]. on the distribution of soil samples and actinomycetes isolates as per the ph value of soil, slightly alkaline ph (7.4-7.8) or moderately alkaline ph (7.9-8.4) was found to be most favorable for the proliferation of actinomycetes as 70 out of 121 isolates were obtained from these ph range (table 5). acidic ph has decreased number of actinomycetes and is not favorable for actinomycetes agricultural lands was neutral to slightly alkaline in ph (7.2-8.2) while the ph of the forest lands was in acidic range (5.2-6.4) except for soil sample from forest land of gokarna (ph value of 7.6) i.e. neutral ph (table 5). the results of the study agree with those of akond et al [15], ameerah et al [24], palanichamy et al which reported that optimum growth of actinomycetes occurs in neutral to alkaline ph [25]. however, the findings were in contrast to kontro et al which reported 10 species of streptomyces isolated from finland were found to have maximum growth at ph range of (4.0-5.5) [26]. this shows that the major factor limiting actinomycetes development in forest soils is believed to be the low soil ph, as the development of most actinomycetes is favored by a neutral or slightly alkaline ph as reported by golinska et al [27]. in this study, it was found that black colored soil harbors most of the antibiotic producing actinomycetes followed by black-brown soil in comparison to the red soil. this is in contradiction with the findings by guo et al who reported that red soil is ideal habitat for acidophilic actinomycetes and harbors a diverse group of actinomycetes which are a promising sources of novel secondary metabolites [28]. the distribution of effective isolates as per the ph value of soil of origin showed that ph of the soil has a significant effect on the effectiveness ofthe production of the antimicrobial compounds by actinomycetes species. also, the effective isolates were found to be from neutral to alkaline soil. these findings are in accordance with the ameerah et al [24], akond et al where the maximum growth of actinomycetes was reported in neutral to alkaline ph range [15]. the findings are in conformity with those of singh et al which reported that maximum growth as well as highest antimicrobial activity by s. sannanensis su118 was achieved at ph 7 while it does not exhibit any activity at ph 9 [29]. out of 121 actinomycetes isolates, 58 isolates were found to have inhibitory activity against at least 1 test organism i.e. 47.9% of isolates were active against at least 1 test organism. a similar result was noted in studies of kumar et al [30] and patel et al [12]. in contrast to the findings, table 5: physical characterization of soil samples s.n. soil sample place/location of collection color ph moisture 1 a nuwakot (damp land) red 5.3 9 % 2 b lagankhel, (agri land) blackbrown 7.2 32 % 3 c katunje (agri land) brown 8.2 20% 4 d katunje (river bank land) blackbrown 7.7 52% 5 e budhanilkantha (agri land) black 8.1 38% 6 f chapagaun (agri land) dark black 7.5 43% 7 g chapagaun (forest land) brown 6.2 12% 8 h godawari (damp land) brown 5.4 15% 9 i chovar (forest land) brown 5.2 18% 10 j sundarijal (river bank land) blackish 8.4 47% 11 k sundarijal (forest land) brown 6.4 29% 12 l lubhu (damp land) brown 5.8 22% 13 m sankhamul (river bank land) black 7.8 42% 14 n gokarna (forest land) blackbrown 7.6 15% 15 o chandragiri (forest land) reddish brown 5.7 12% nepal j biotechnol. 2020 dec; 8 (3): 102 -110 budhathoki & shrestha ©njb, bsn 108 gurung et al reported that 34.18% of 79 isolates were active against test organisms [18] and pandey et al found 33.96% actinomycetes isolated from the khumbu region showed inhibitory activity against at least 1 of 5 test organisms (bacillus subtilis, staphylococcus aureus, proteus, salmonella typhi) [31] . among 58 active isolates, only 22 (37.9 %) were active against 2 or more test organisms. this is in accordance with the reports of sharma et al [32] and salam and rana [33]. on the contrary devadass et al stated that only 21 out of 150 i.e. 14% of isolates from western ghats of tamil nadu were active against more than one test organism [34]. from the study staphylococcus aureus was found to be most susceptible organism towards actinomycetes isolates. also, on comparison of the zone size of inhibition against the gram-positive organism i.e. staphylococcus aureus against that of the gram-negative test organisms, it can be inferred that the actinomycetes isolates were more active against gram-positive bacterium. this may be due to the difference in cell morphology of gram-positive and gram-negative cells and furthermore, the antibiotic compound could have been more functional against gram positive cell components than that of gram-negative cell components. a study carried out by tyagi et al reported that actinomycetes isolates were highly active against staphylococcus aureus, streptococcus, bacillus and e. coli strains. it also suggests that actinomycetes have more potent inhibitory activity against gram-positive bacteria [35]. the secondary screening of the antibacterial activity showed that out of 22 antibiotic fractions, only 8 were ineffective against gram-positive bacteria while only one fraction from isolate (g3) was ineffective against gramnegative bacteria. 13 fractions were found to be effective against both gram positive and gram-negative bacteria. these findings showed that the 9 fractions were found to have a narrow spectrum of activity while the 13 fractions were found to have a wide spectrum of inhibitory activity against the test organism. however, they cannot be confirmed as broad spectrum and narrow spectrum until they are tested multiple times with a wider range of the test organisms. on testing of inhibitory activity of the 12 isolates active against staphylococcus aureus towards drug resistant strains mrsa and mssa, 11 were found to inhibit mssa while 6 of them showed inhibitory activity against mrsa. these findings showed that drug-resistant strains of staphylococcus aureus can be treated with the bioactive secondary metabolites from actinomycetes isolates. sharma et al reported 6 strains of actinomycetes isolates having highly inhibitory activity against mrsa strains (mrsa-97, mrsa-67, and mrsa p-169) [32]. in a similar study chaudhary et al reported the inhibitory activity of the 31 isolates towards staphylococcus aureus among which number of them being inhibitory towards mrsa [5]. on the distribution of effective isolates as per moisture content highest number of effective isolates was found to be from soil having a moisture content of (11-20) %. the findings are inconformity with those of mavordi et al which stated that pseudomonas spp. producing the antibiotics phenazine-1-carboxylic acid were abundant in the rhizosphere of native plant species growing in nonirrigated areas adjacent to the sampled dryland wheat fields i.e. the lands with less moisture [36]. conclusion soil harbor large number of actinomycetes isolates which exhibit inhibitory activity against number of bacterial strain. acidic ph favors the growth of actinomycetes and its ability to produce antimicrobial compounds. actinomycetes has been a constant source of antimicrobial agents and the further studies exploring the antimicrobial potential of these isolates from various unexplored areas need to be done. author’s contribution sb is the principal investigator, carried out all the laboratory works, also prepared the manuscript. as is the corresponding author, an academic supervisor of the research and prepared the manuscript. competing interests the authors declare no competing or financial interests. funding the materials, reagents and chemicals required for the laboratory work was funded by microbiology research society, nepal. acknowledgments we would like to thank kathmandu centre for genomics and research laboratory as well as st. xavier’s college for providing the research space. we would like to thank microbiology research society for providing the materials, reagents and chemicals required to accomplish the research work. we are thankful to ms. sunita shrestha and ms. naina byanjankar, research assistants of nepal academy of science and technology (nast) for their help in the preparation of map of sample collection sites. ethical approval and consent not applicable. nepal j biotechnol. 2020 dec; 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[retrieved from elsevier on 13/07/2018]. 31. pandey b, ghimire p and agrawal vp. studies on the antibacterial activity of the actinomycetes isolated from the khumbu region of nepal. journal biology science. 2004; 23:44-53. 32. sharma d, kaur t, chadha bs and manhas rk. antimicrobial activity of actinomycetes against multidrug resistant staphylococcus aureus, e. coli and various other pathogens. tropical journal of pharmaceutical research. 2011; 10(6): 801-8. https://doi.org/10.4314/tjpr. v10i6.14 33. salam md and rana s. antimicrobial potential of actinomycetes isolated from soil samples of punjab. india journal of microbiology & experimentation. 2014; 1(2): 3-10. 34. devadass bj, paulraj mg, ignacimuthu s, theoder pas and dhabi naa. antimicrobial activity of soil actinomycetes isolated from western ghats in tamil nadu, india. j bacteriolmycol. 2016; 3(2): 00059. https://doi.org/10.15406/jbmoa.2016.03.00059 35. tyagi j, bhatnagar t and pandey fk. isolation and characterization of actinomycetes from soil and screening their nepal j biotechnol. 2020 dec; 8 (3): 102 -110 budhathoki & shrestha ©njb, bsn 110 antibacterial activities against different microbial isolates. international journal of life sciences research. 2014; 2(4): 101-5. 36. mavrodidv, mavrodi ov, parejko ja, bonsall rf, kwak ys, paulitz tc et al. accumulation of the antibiotic phenazine-1carboxylic acid in the rhizosphere of dryland cereals. applied and environmental microbiology. 2012; 78(3): 80412. https://doi.org/10.1128/aem.06784-11 nepal j biotechnol. 2020 dec; 8 (3): 95-101 doi: https://doi.org/10.3126/njb.v8i3.33663 research article ©njb, bsn 95 comparative study of bioactive compounds in different varieties of pears in nepal barsha koirala , angela shrestha st. xavier’s college, kathmandu, nepal article history:received: 5 may 2020; revised: 7 dec 2020; accepted: 19 dec 2020; published online: 30 dec 2020 abstract this study was conducted to evaluate the physicochemical parameters, perform qualitative tests (for sugars and phytochemicals), and quantitative tests (phenolics, antioxidants, anthocyanins, tannins, vitamin c) of six different varieties of pears i.e. bartlette, chinese pears, chojuro, kosui, pharping local, and yakumo. the juices extracted from respective pears were used for the analysis. the phenols were determined by the folin-ciocalteu method, antioxidants by the dpph scavenging activity, and anthocyanins by a so₂ bleaching technique. the pharping local pears were found to have the highest anthocyanins (85.95±0.1 mg/l), total phenolic content (600±0.01 mg gae/l), antioxidants (ic50 value 250±0.00 mg of phenol/l) and vitamin c content (12.2±0.01 mg/100 ml) and tannins were observed to be highest in yakumo pears (0.93±0.01 g/l). likewise, the highest clarity i.e. 1.960±0.00 was observed in bartlette pears and the highest acidity (2.01±0.01%) in chojuro pears. various sugar/carbohydrate tests like molisch’s test, benedict’s test, barfoed test, bial’s test, seliwanoff test, fehling’s test and iodine test were performed for the pear varieties. all the pears gave positive results for all the sugar tests except iodine test. the positive results for sugar/carbohydrate signifies the presence of various sugars that help for the better taste, texture, and aroma of pear. the pear varieties showed the presence of phytochemicals like flavonoids, terpenoids, catechins, cyclic glycosides, and proteins. the phytochemicals are responsible for fruit preservation and act as anticarcinogenic components. among the varieties of pears, pharping local pears were observed to be most nutritional because of high antioxidants, phenols, anthocyanins, and vitamin c. keywords: pears, physicochemical parameters, antioxidants, phenolic content, pharping local corresponding author, email: varshakoirala@gmail.com introduction pear is the second most important deciduous fruit found in nepal [1]. it is cultivated in both mid and high hills ranging from 800 to 1200 m above sea level. the pear covers 4396.5 hectors of land, a productive area of 3386 hectors with productivity of 34151 mt which yields 10.1 mt/hectors [1]. every year, tons of pears decay during the harvesting season, because of their low consumption rate [1,2]. pears are rich in carbohydrates, vitamin b6, vitamin a, vitamin c, sugars, iron, calcium, sodium, potassium, thiamine, water, dietary fibers, phosphorus, etc. pears are also used as a medication to prevent the lungs’ function, bones deformation, coughs, and chills, ulcers, pulmonary disease, improving immunity, etc. [1,2]. usually, there are two types of pear grown in nepal, i.e. european and asian. asian pears, which are also called apple pears, salad pears, nashi, oriental, chinese or japanese pears, are a large group of pears that are crispy and ready to eat as soon as they are harvested. kosui, chojuro, yakumo, hosui, pharping local, etc are the varieties of asian pears. european pears are harvested when they are hard and green, and stored at room temperature for the ripening process; so, they are sweeter. the pears like bartlette, comice, d’ anjou are the varieties of european pears [3,4]. pears are rich in sugars like fructose, sucrose, glucose, sorbitols, etc. sucrose is the source of high energy and helps in cold tolerance capability of fruits; glucose and fructose act as antioxidants. pears are also responsible for maintaining the quality of fruits and their maturity [2]. the pear juice contains 9-15 % of soluble solids [5]. the various analyses (optical and chemical) are performed to maintain the quality of juices which are called physicochemical analysis. the acidity and ph of the fruits are responsible for color, brightness, and freshness and taste of the juices [6,7]. polyphenols are a group of compounds that use phenol as a building block. some phenolic compounds found are gallic acid, quercetin, flavonoids, anthocyanins, (+)-catechin, tannins, epigallactocatechins, resveratrol, rutin, myricetin [8,9]. various free radicals are generated by oxidative stress and their accumulation in the cells causes oxidative damage and degeneration leading to various complications like premature aging, cataract, heart disease, and neurodegenerative disorders [10]. the compounds such as phenolics, antioxidants, and vitamin nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0001-8281-8671 mailto:varshakoirala@gmail.com https://orcid.org/0000-0001-7355-2162 mailto:varshakoirala@gmail.com nepal j biotechnol. 2020 dec; 8 (3): 95-101 koirala & shrestha ©njb, bsn 96 c are responsible for the prevention as well as damage repair caused by the free radicals. fruits are rich in these compounds, therefore, their consumption can help with bone formation, collagen, anti-inflammatory, antitumors/cancerous functions, etc. [11]. this research is focused on the comparative analysis of different types of pear and can help people to understand the nutritional values of pears. the data generated from this research could be useful to compare different characteristics of pear available in different origins. this research provides information about the phytochemicals, nutritional values, and sugar concentration in pears grown in nepal. the nutritional profiling of pears could be helpful for its product commercialization in the international market. materials and methods collection of samples six varieties of fully ripen pears (bartlette, chinese, chojuro, kosui, pharping local, and yakumo) were collected from the warm temperate horticulture centre, kirtipur, nepal in august, 2019. physicochemical analysis ph, tss, acidity, clarity, moisture, and ash content were determined. ph was determined using a ph meter. tss was measured using a brix refractometer. clarity was determined by taking the absorbance of pear juice at 660 nm using a spectrophotometer [11,12,13]. moisture content for moisture content, the sample was ground and 10 gm of the sample was kept on the petri dish. the initial weight of the petri dish was noted. the plates were further placed in the oven at 105°f and the decrease in weight was noted every hour till it decreases to ±5 mg. before weighing, the plate was kept on a desiccator [12]. the moisture content was calculated as: moisture content = initial weight final weight ×100 % initial weight ash content silica crucible was washed and dried in a hot air oven at 150 degrees for 30 minutes. the samples were ground and 10 gram was weighed. the sample was burned over a low flame furnace and was transferred to the temperature-controlled muffle furnace using long tongs. the temperature of muffle furnace was kept at 500 degrees and was left for 3-4 hrs to cool. the crucible was left to cool and was weighed [12]. the ash content was calculated as: total ash = weight of ash × 100 % weight of sample where, the weight of ash = wt. of the crucible with ash wt. of the crucible vitamin c content the dye solution (mixture of sodium salt of 2, 6dichlorophenol-indophenol dye and sodium bicarbonate) was standardized by titrating ascorbic acid with the dye solution until the appearance of pink color persists for 10-15 seconds. the dye factor i.e. mg of ascorbic acid per ml of dye was calculated using the following formula: dye factor = 0.25×10/titre for titration, 10-20 ml sample was titrated against dye till the appearance of pink color persists for 15 seconds. the reading in the burette was noted. ascorbic acid (%)= titre×dye factor×volume made up ×100 weight of sample × volume of the sample taken metaphosphoric acid (hpo₃) acetic acid solution (3%) was prepared by dissolving 15 g metaphosphoric acid in 450 ml water, and 40 ml glacial acetic acid. the standard for ascorbic acid was prepared by dissolving 0.05 gm of l-ascorbic to 250 ml with the metaphosphoric acetic acid solution. for the preparation of dye solution, 0.05 g of the sodium salt of 2, 6-dichlorophenol-indophenol dye, and 0.04 g sodium bicarbonate were dissolved in 200 ml water. it was then filtered and stored in a dark-colored bottle at a refrigerated condition [13]. qualitative tests for sugars/carbohydrates in pear juice the various tests for sugars (molisch’s test, benedict’s test, barfoed’s test, seliwanoff test, fehling’s test, bial’s test, and iodine test), protein test and phytochemicals (catechins, flavonoids, cyclic glycosides, terpenoids) were performed for pear juices. molisch’s test initially, 2 ml of the sample was taken, and 2-3 drops of molisch’s reagent was added. after some time, purple ring formation was observed which gives a positive test indicating the presence of all types of sugars like monosaccharides, disaccharides, and polysaccharides [12]. benedict’s test one ml of the sample was taken initially, and 2-3 drops of benedict’s reagents were added and placed in a water bath and boiled. after some time, the color of the precipitate was observed; the presence of red precipitate nepal j biotechnol. 2020 dec; 8 (3): 95-101 koirala & shrestha ©njb, bsn 97 gives a positive test for benedict’s test. it indicates a positive test for reducing sugars like glucose [12]. barfoed’s test initially, 1 ml of the sample was taken, and 2 ml of barfoed’s reagent was added. it was left to boil in the water bath and the color change was observed. brick red color appearance indicates the positive test for reducing sugars [12]. seliwanoff test first of all, in 1 ml of the test solution, 2 ml of seliwanoff reagent was mixed and kept in a water bath for 1 minute. the appearance of deep color gives a positive test for keto sugars i.e. fructose and sucrose [14]. fehling’s test first of all, 1 ml of the sample was taken, and 1 ml of fehling’s reagent was added. it was left in a boiling water bath. the precipitation of red color is an indication of a positive test for sugars like glucose and fructose [14]. bial’s test bial’s reagent was added initially to the test sample. it was then kept in boiling water for some time. the appearance of blue-green color indicates positive for ribose sugars [12]. iodine test this test was carried out by taking 1 ml of the sample in which 4-5 drops of iodine were added. blue color indicates a positive test for complex sugars like starch in a sample [12]. phytochemicals in pear juice flavonoids one ml test sample was tested with mg metal and 5-6 drops of conc. hcl. the change in color was observed. red color denotes flavonoids, orange stands for flavones, and violet indicates flavonones [14]. terpenoids in the mixture of 2 ml chloroform and 3 ml conc. sulfuric acid, the sample was added and heated for 2 minutes. a grayish-reddish brown color was observed which indicates a positive test for terpenoids [14]. cyclic glycosides the sample was mixed with 2 ml of chloroform. sulfuric acid was added and shake gently. the brown ring on the interface indicates the presence of cyclic glycosides [14]. protein test the sample was boiled with 2 ml of 0.25 % w/v of ninhydrin solution. the presence of violet-blue color is the positive test for protein [14]. catechin test the sample was mixed with the fecl₃ solution. the olive green color gives the test positive for the catechins [14]. total phenolic content (tpc) the aliquots of 1 ml gallic acid of various concentrations in methanol were added. then, 5 ml of 10 % folin ciocalteu reagent and 4 ml of 7% na₂co₃ were mixed up making the final volume 10 ml. the absorbance was measured at 760 nm against a blank (fc reagent+ na₂co₃) and the graph of the standard was plotted using the data of absorbance versus concentration (µg/ml) [13]. for determining total phenolic content in sample, 20 µl juice sample was taken and the further procedure was carried out as for the calibration solutions. the absorbance was taken and the levels of phenolic content were determined using the standard graph as gallic acid equivalents (gae) [16]. antioxidant content (aoc) the stock solutions of the sample were prepared by diluting 5 ml sample(pear juice) and 10 ml of 13.5% ethanol. diluted samples of pear juice i.e. 50, 100, 150, 200 and 250 μl were mixed with dpph maintaining final volume 3 ml and left for 30 minutes in dark and absorbance was measured at 517 nm. the volume of wine in the diluted solutions needed to decrease the initial dpph concentration by 50% together with the amount of phenol in mg/l was calculated. the results were used to obtain the ic 50 values in mg of phenol/l. the % inhibition was calculated as: inhibition (%) = (ac as) x 100 ac where, ac = absorbance of the control (100 μl of meoh instead of the sample) as = absorbance of the sample the percent inhibition was plotted against volumes of wine using microsoft excel and the volume needed to decrease dpph concentration by 50% was calculated from the graph. the volume of sample (diluted) that is required for decrement in the initial dpph concentration by 50 % together with the amount of phenol in mg/l was used for attaining the value of ic 50 in mg of phenol/l [15]. total tannin content (ttc) 200 μl of pear juice, 300 μl conc. hcl and 100 μl of distilled water were mixed in two different test tubes. the first test tube was incubated at 100°c for 30 min, whereas in the second sample, 50 μl alcohol was added and the absorbance of both samples was taken at 470 nm. the absorbance of the two samples was differentiated and represented as δa520 [17]. koirala & shrestha ©njb, bsn 98 δa520 = 1.1 × δa470 and δa520 = 1.54 × δa470 the lowest δa520 value was chosen for the estimation of total tannin content and was represented as g/l of juice. it is calculated as [17]: ttc = 15.7 × lowest δa520 total anthocyanin content (tac) 50 μl pear juice, 50 μl hcl in ethanol (0.1%), and 100 μl aqueous hcl (20%) were mixed in two different test tubes. to the first test tube, 220 μl of distilled water was added and the same amount of sodium bisulfite (26%) was added to the second test-tube. then the absorbance was measured at 520 nm against a blank (50 μl hcl in ethanol (0.1%), 100 μl aqueous hcl (20%), and 270 μl distilled water). the difference was calculated and represented as δa520 [17]. the tac as mg/l of juice was calculated as: tac = 875 × δa520 data analysis the tests were performed on triplicates (n=3) and the results for quantitative tests were reported as mean± standard deviation (s.d.). the level of significance between various parameters were determined using one way anova in microsoft excel 2013 and the data presented were found to be statistically significant (p < 0.05). results physicochemical parameters of pears total acidity and ph were observed to be the highest in chojuro pears i.e. 2.01±0.01 % and 5.23±0.01, respectively. likewise, clarity was observed to be the highest in bartlette pears i.e. 1.960±0.00. tss (°bx) was observed to be highest in chinese pears i.e. 11±0.00°bx. likewise, moisture content were observed to be highest in chojuro pears i.e. 17.42±0.01 % and ash content in kosui and yakumo pears i.e. 1.5±0.04 and 1.5±0.02 % respectively (table 1). tests for sugars/ carbohydrates the qualitative tests were performed for the pear juices. all the varieties gave positive tests for all sugars except the iodine test which indicated absence of starch in pears (table 2). phytochemicals screening all the pears gave positive tests for the phytochemicals (table 3). the highest concentration of flavonoids, catechins, and cyclic glycosides were observed in bartlette and pharping local pears. likewise, terpenoids were found to be highest in pharping local pears only. nepal j biotechnol. 2020 dec; 8 (3): 9 5 -1 0 1 table 1: physicochemical parameters of pears s.n. name of sample physicochemical parameters acidity (%) ph clarity °bx moisture content (%) ash content (%) 1. bartlette 0.134±0.01 4.13±0.01 1.960±0.00 10±0.00 16.68±0.00 0.6±0.01 2. chinese 0.67±0.02 4.77±0.01 2.058±0.00 11±0.00 16.97±0.02 0.21±0.00 3. chojuro 2.01±0.01 5.23±0.01 2.159±0.00 8±0.00 17.42±0.01 0.25±0.01 4. kosui 0.73±0.01 5.06±0.03 2.303±0.01 8±0.00 16.32±0.02 1.5±0.04 5. pharping local 0.87±0.01 4.38±0.01 2.454±0.00 7±0.00 15.85±0.00 1.3±0.01 6. yakumo 0.67±0.01 5.05±0.00 2.301±0.02 9±0.00 14.84±0.015 1.5±0.02 all the values (n=3) were expressed as mean ± standard deviation and found to be statistically significant (p < 0.05) table 2: various sugar tests in pear varieties s.n. name of sample molisch’s test iodine test benedict’s test barfoed test bial’s test seliwanoff test fehling’s test 1. bartlette +++ + ++ + ++ + 2. chinese +++ + ++ + +++ + 3 chojuro +++ ++ +++ + +++ ++ 4. kosui +++ ++ +++ + +++ ++ 5. pharping local +++ ++ +++ + ++ ++ 6. yakumo +++ + ++ + ++ + note: (-) denote absence, (+) denote trace, (++) denote moderate and (+++) denote high amount of sugars . the comparison were done on the basis of colour intensities nepal j biotechnol. 2020 dec; 8 (3): 9 5 -1 0 1 koirala & shrestha ©njb, bsn 99 determination of tannins, anthocyanins, total phenolic content(tpc), antioxidant content(aoc) and vitamin c in pear juice tannins were observed to be highest in yakumo pears i.e. 0.93±0.01 g/l, and anthocyanins, total phenolic content, antioxidants, and vitamin c were observed to be highest in pharping local pears i.e. 85.95±0.1 mg/l, 600±0.01 mg gae/l, ic 50 value 250±0.00 mg of phenol/l and 12.2±0.01 mg/100 ml respectively (table 4). discussion table 1 focuses on the physicochemical parameters like ph, tss, clarity, moisture, and ash content. the highest clarity was found in bartlette pears i.e. 1.960±0.00, as it showed the lowest absorbance (clarity is inversely proportional to absorbance). on similar research conducted on two different pears i.e. shughri and physhun pears, shugri pears had tss 13.58°bx, 83.1% moisture, 3.94% ash, and 13.71°bx, and 54.51% moisture, 1.86% ash respectively [18]. chinese pears were found to have highest sucrose (i.e.11±0.00°bx) among the 6 varieties. likewise, the moisture was observed to be the highest in chojuro pears i.e. 17.42±0.01% and ash content in kosui and yakumo pears i.e. 1.5±0.04% and 1.5±0.02% respectively. shughri and physhun pears had higher moisture content, ash content, and tss than the pears in this research. bartlette pears in earlier research was found to have the acidity 3.50-4.60% while the bartlette pears in this research had the acidity of 0.134±0.01%. different acidity for the same variety of pear could be because of the different climatic conditions, storage temperature, and other environmental parameters [19]. table 2 shows the presence of sugars in different pear juice. all the pears gave positive tests for molisch’s test, benedict’s test, barfoed test, bial’s test, seliwanoff test, and fehling’s test but negative for iodine test. this indicates the presence of various sugars like glucose, fructose, and, sucrose and the absence of complex sugars like starch in the pears varieties. sugar acts as a flavor enhancer, making pear sweet increase the taste, texture, color, and aroma. they also act as food preservatives. all the pears gave positive test for molisch’s test which indicates the presence of various sugars in high amount. positive test for benedict’s test and barfoed test indicate presence of simple sugars like glucose, fructose, galactose, etc. [11]. those sugars were found high in chojuro, kosui and pharping local pears. positive bial’s test indicates presence of ribose sugars. the positive seliwanoff test indicates presence of sugars like sucrose. sucrose was found to be high in chinese, chojuro and kosui pears. positive fehling’s test indicates presence of reducing sugars like glucose, fructose, lactose, etc. [14]. they were present in high amount in chojuro, kosui and pharping pears. likewise, a study was done for the analysis of various sugars in two different pear cultivars (pyrus communis) [18,20]. fructose was found to be in highest concentration, followed by other sugars like glucose, sucrose, fructose, and sorbitol. table 3 highlights the presence and absence of different phytochemicals in pear juice. the pears gave positive tests for phytochemicals like flavonoids, terpenoids, catechins, cyclic glycosides, and proteins. another research conducted on pear [21] showed a high amount of catechins and flavonoids present in pears. likewise, in table 3: qualitative analysis of phytochemicals s.n. name of sample flavonoids terpenoids catechins cyclic glycosides proteins 1. bartlette ++ + ++ ++ + 2. chinese + + + + + 3 chojuro + + + + + 4. kosui + + + + + 5. pharping local ++ ++ ++ ++ + 6. yakumo + + + + + note: (+) denote trace, (++) denote moderate and (+++) denote high amount and (–) indicates the absence of phytochemicals table 4: tannins, anthocyanins, total phenolic content (tpc), antioxidant content (aoc), and vitamin c in pears s.n. name of pears tannins (g/l) anthocyanins (mg/l) tpc (mg gae/l) aoc (ic 50= mg of phenol/l) vit. c (mg/100 ml) 1. bartlette 0.05±0.01 45.55±0.01 501.1±0.05 299.40±0.05 6.57±0.05 2. chinese 0.91±0.00 55.65±0.05 273.5±0.2 549.45±0.01 6.94±0.00 3. chojuro 0.06±0.02 30.63±0.00 399.5±0.28 375.93±0.00 5.55±0.02 4. kosui 0.02±0.00 40.85±0.00 304.9±0.01 501.68±0.01 2.3±0.05 5. pharping local 0.04±0.00 85.95±0.1 600±0.01 250±0.00 12.2±0.01 6. yakumo 0.93±0.01 68.75±0.02 301.8±0.05 509.2±0.01 2.2±0.01 all al the values (n=3) were expressed as mean ± standard deviation and found to be statistically significant (p < 0.05). nepal j biotechnol. 2020 dec; 8 (3): 9 5 -1 0 1 koirala & shrestha ©njb, bsn 100 similar research [22], various phytochemicals like catechins were found. in table 4, the amount of tannins, anthocyanins, total phenolic content, antioxidants, and vitamin c in pear varieties are presented. tannins were observed to be the highest in yakumo pears i.e. 0.93±0.01 g/l; anthocyanins, total phenolic content, antioxidants, and vitamin c were observed to be the highest in pharping local pears i.e. 85.95±0.1 mg/l, 600±0.01 mg gae/l, ic 50 value 250±0.00 mg of phenol/l, and 12.2±0.01 mg/100 ml respectively. compared to a previous similar research [23], where the level of tannin was observed to be 1.6 g/l, this study, found the level of tannin in yakumo pear equal to 0.93±0.01 g/l [24]. anthocyanins are responsible for the red coloration in pear fruits and its development depends on heat and light. the anthocyanin level was found to be 89.5 mg/l in pears which is higher compared to pear in this research i.e. 85.95±0.1 mg/l. given that, the anthocyanin level is higher in high temperature, those pears might have grown in high temperatures as compared to the pears cultivated in nepal [25]. varieties of oriental pear and occidental pear had total phenols 78.5-83.9 mg gae/l and high antioxidant activities. jules d’airolles and abate fetal pears showed the lowest dpph scavenging capacity; and cheongbae, niitaka, and hanareum pears were found to have high total phenolic, flavonoid contents, and higher antioxidants than other varieties [26,27]. the highest phenolic content was observed in pharping local pears i.e. 600±0.01 mg gae/l which is higher compared to oriental and occidental pears. the amount of the phenolic compounds present is based on fruit source and environmental factors as well [26]. it also acts as a primary antioxidant or free radical terminators and are effective hydrogen donors [26]. the lower ic 50 value indicates greater antioxidant activity because the value indicates the level of antioxidants essential for the reduction of free radical i.e. dpph by 50% of initial concentration. the vitamin c content was observed to be 12.2±0.01 mg/100 ml in pharping local pears while in similar research conducted [27], it was found in the range 2.26.57 mg/100 ml which is less than that of this research. this could be because of the difference in the various factors like variety, seasonal variation, environment, climate, and the difference in protocols for the determination of vitamin c. conclusion pharping local pears are found to be the most nutritious when compared to the other five varieties. pears are the fruits that are rich in vitamin c, antioxidants, phenolic contents, anthocyanins etc. along with those components, various sugars, phytochemicals like catechins, flavonoids, terpenoids, glycosides, and little protein as well. given such richnesspears in general and pharping pears in particular are recommended as rich sources of vitamins, antioxidants, health-promoting factors. author’s contribution bk performed the experiment in the lab under the supervision of as. bk and as contributed for original draft preparation and during revision. bk and as contributed significantly in editing, revising and rendering the write-up. all authors have read and approved the final manuscript. competing interests the authors have no competing interests regarding the publication. funding no funds have been provided for 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18:536–546. doi: https://doi.org/10.1080/10942912.2013.835821 https://doi.org/10.1016/j.foodchem.2016.04.009 https://dx.doi.org/10.1155%2f2008%2f937651 https://doi.org/10.20546/ijcmas.2017.607.403 https://dpr.gov.np/wp-content/uploads/2019/08/journal-2019.pdf https://dpr.gov.np/wp-content/uploads/2019/08/journal-2019.pdf https://dx.doi.org/10.3390%2fantiox2030110 https://doi.org/10.1016/s0076-6879(99)99017-1 https://dx.doi.org/10.1002%2ffsn3.794 https://www.semanticscholar.org/%20paper/comparative-study-of-two-pear-(pyrus-communis-l.)-hussain/cf1fd30f331397758d764b%20bb7795%2028b11712094b https://www.semanticscholar.org/%20paper/comparative-study-of-two-pear-(pyrus-communis-l.)-hussain/cf1fd30f331397758d764b%20bb7795%2028b11712094b https://www.semanticscholar.org/%20paper/comparative-study-of-two-pear-(pyrus-communis-l.)-hussain/cf1fd30f331397758d764b%20bb7795%2028b11712094b https://doi.org/10.1007/978-3-030-11048-2 https://dx.doi.org/10.1097%2fnt.0000000000000112 https://dx.doi.org/10.1097%2fnt.0000000000000112 https://dx.doi.org/10.1097%2fnt.0000000000000112 https://doi.org/10.1021/ac011001s https://doi.org/10.1080/10942912.2013.835821 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: -62-68 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 62 nepjol.info/index.php/njb isolation of polyhydroxybutyrate (phb) producing bacteria, optimization of culture conditions for phb production, extraction and characterization of phb christina thapa¹, pallavi shakya¹, rabina shrestha¹, sushovita pal¹, prakash manandhar² department of biotechnology, sann international college, purbanchal university, nepal1 department of microbiology, st. xavier’s college, nepal2 abstract polyhydroxybutyrates (phbs) are energy reserves synthesized by different micro-organisms such as alcaligenes, pseudomonas, staphylococcus, algae, in excess of carbon and limitation of nutrients like nitrogen. these biopolymers are suitable alternate to synthetic carbon-based polymers. however, the high production cost limits their commercialization. the aim of this study was thus, focused on optimization of culture condition for maximum phb production in an attempt to reduce the production cost. the micro-organisms for this purpose were isolated from 4 different soil samples and screened for phb production. culture conditions for these organisms were optimized by changing the parameters, viz., incubation time, ph, carbon source and nacl concentration. thus, optimized culture condition was used to culture the isolates for extraction of phb and its analysis. the extracted compounds on ftir-analysis gave characteristic c=o peak of phb, thus, confirming the seven isolates to be phb producers. results for optimized parameters for the isolated phb positive species showed that synthesis of phb was maximum at 48 hours i.e. during the early stages of stationary phase. however, different isolates favored different culture conditions. highest phb accumulation and growth of isolates were seen at ph 7 and 9. similarly, it was observed that glucose was favored by 4 isolates and sucrose was favored by 3 isolates. interestingly, nacl concentration did not cause significant effect on neither the bacterial growth nor the phb production. during the extraction of phb from the optimized culture conditions, extraction of phb from broth gave significant yield than that from agar. a good phb yield from broth amounting to 36.41% and 34.59% was observed for bacillus pasteurii and micrococcus luteus respectively, showing a potential for their exploitation in industrial phb production. at optimized conditions, 7 isolates exhibited significant phb yields, thus showing a potential for further exploitation. keywords: bioplastics, biopolymer, polyhydroxybutyrates, phb, fourier transform infra-red spectroscopy (ftir) *corresponding author email: rabeenastha@gmail.com introduction traditional plastics are synthetic carbon-based polymers that are made from non-renewable source, mostly from petroleum. due to their relatively low cost, ease of manufacture and flexibility, the demand of plastics is evergrowing. however, plastics, being man-made, are not recognized by micro-organisms. [13] thus, they take very long to degrade i.e. 450 years on average for degradation of a plastic bottle. [7] plastic debris also poses considerable threat by choking and starving wildlife, distributing nonnative and potentially harmful organisms, absorbing toxic chemicals and degrading to micro-plastics that may subsequently be ingested. [2] also, due to high cost of recycling, plastics are rarely recycled leading to crammed up landfills. for eradication of these and various other problems such as carbon emission during incineration, biodegradation of plastic is a must. considerable amount of interest in the development and production of an alternative, biodegradable plastics or bioplastics is being done. among them polyhydroxy alkanoic acids (phas) are drawing much attention as they have nontoxic residual products and low environmental permanence. [9] depending on the types of carbon sources available and the biochemical pathways operating in the cell, microorganisms are capable of synthesizing various types of phas. poly [r3 hydroxybutyrate] (p[3hb]) is the first type of pha identified and is the most common pha found in nature. [1] nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 62-28 thapa et al. ©njb, biotechnology society of nepal 63 nepjol.info/index.php/njb phbs are carbon and energy reserve polymers produced in bacteria, archaea, and in few eukaryotes, such as yeasts and fungi when carbon source is in plentiful and other nutrients such as nitrogen, phosphorous, oxygen or sulphur are limited. the storage molecule is then metabolized under unfavorable conditions when other common energy sources are not available. some bacterial species which naturally produce phb are ralstonia eutrophes, alcaligenes, pseudomonas, bacillus, rhodococcus, staphylococcus and micrococcus. [1] phb is ecofriendly, biodegradable, biocompatible and is accumulated up to 90% of cell dry weight. [10] phb based plastics made by combining phb with other biocompatible polymers (like 3-hydroxyvalerate) [8] find many applications in agriculture, packaging, and medical field including drug delivery and tissue engineering. [10] in spite of these interesting properties, industrial production of phb is still not well established due to its high production cost. this has made it unable to compete with conventional plastics in the commercial market. the phb content and its composition are influenced mainly by the strain of the microorganism, the type of substrate employed and its concentration, and other growth conditions such as ph, time and temperature. [11] therefore, much research is needed to discover and identify novel species with vastly superior production capacity and optimization of conditions for maximal synthesis of phb. this research focuses on isolation and characterization of phb producing bacteria from easy and convenient sources i.e. sewage soil sample. the objective was to analyze the extracted phb by different isolated organisms in optimized physical and chemical conditions. biochemical and morphological tests are performed for the identification purpose. in an attempt to overcome the limitations associated with costly substrates, this research is designed to use and test the efficiency of readily available and relatively cheap carbon sources such as sucrose, glucose and fructose in phb production. moreover, it also focuses on optimization of various growth conditions such as incubation time, ph and nacl concentration for increase in the polymer production from respective phb positive organism. considering the optimum growth conditions and carbon source of each organism, phb extraction and its characterization using ftir analysis was done. the extracted phb was calculated as percentage yield of the cell dry weight obtained. materials and methods sample collection and isolation of pure cultures soil samples were collected aseptically from topsoil of four sites viz., teku dumping site, balaju industrial site, banks of dhobi khola and budhanilkantha animal waste manure. one gram of each sample was dispersed in 10ml of sterile distilled water and heated at 80°c for 10 minutes to isolate only endospore forming bacteria. serial dilution of these samples was done up to 10-3, followed by spread plating of 100µl diluted samples on nutrient agar plates. thereafter, the plates were incubated at 30°c for 48 hours. pure culture of morphologically distinct colonies was grown in modified agar plates. the constituents of modified agar plates are: beef extract (0.3%), peptone (0.5%), sodium chloride (0.8%), glucose (1%), and agar (1.5%). [12] primary screening of phb producing bacteria detection for phb production was employed by using lipophilic stain sudan black b. [3] stain was prepared by dissolution of 0.3 gm powdered stain in 100 ml of 70% ethanol. for microscopic studies, smears of colonies were heat-fixed on clean, grease-free glass slides, followed by staining with 0.3% solution of the sudan black b. after leaving the slides undisturbed for 15 minutes, immersion in xylene and counterstaining with safranin (5% w/v in sterile distilled water) was performed. cells appearing blue-black under microscope were accredited as phb positive strains. phb positive strains were preserved on two vials, viz., working and stock vials, containing agar slants with 2% glycerol for preservation. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 62-28 thapa et al. ©njb, biotechnology society of nepal 64 nepjol.info/index.php/njb morphological and biochemical characterization of phb positive isolates distinct morphological features of the isolates were recorded on the basis of shape, color and size. similarly, cellular morphology was studied under the microscope using gram staining and endospore staining. standard microbiological methods were employed for identification of isolated bacteria by biochemical tests. the tests performed were imvic test, nitrate test, sugar utilization test, catalase test, oxidase test, starch utilization test and oxidative-fermentative test. growth curve study of isolates phb producing medium was used to study the growth and production of phb. the components of the media are: glucose 1g, peptone 0.25g, yeast extract – 0.25g, nacl – 0.01g, kh2po4 – 0.05g, mgso4 – 0.02g and ph at 7. [6] one percent inoculums from activated phb positive isolates were inoculated in conical flasks containing phb producing media, followed by incubation of the culture for 48 hours at 37°c with occasional shaking. at an interval of every 4 hours, the samples were collected to perform sudan staining and the biomass reading was done using spectrophotometer at 640nm. optimization of phb production effect of ph every microorganism has a minimum, an optimum and a maximum ph for growth. to standardize the optimum ph for the production of phb, the phb positive bacterial cultures were inoculated in phb producing media at different ph (2, 4, 7, 9 and 11) and incubated at 37°c for 48 hours with occasional shaking. the ph values were taken in order to cover different acidic, neutral and basic ph ranges. after incubation, the samples were screened using sudan stain to confirm phb production and turbidity of the media due to bacterial growth was measured by spectrophotometer at 640nm. effect of nacl concentration microorganisms vary widely in their nacl tolerance. thus, phb producing media with different nacl concentrations (0.1%, 0.5%, 2%, 5%, and 10%) was prepared. after autoclaving, 1% of activated culture was added to each tube and incubated at 37°c for 48 hours. the samples were collected after 48 hours for sudan staining and for measurement of o.d. at 640nm. effect of carbon sources 2% glucose, sucrose, and fructose were added into phb producing media as carbon sources and the selected isolates were grown in it. after incubation and screening by sudan stain, the phb produced by the isolates was quantified spectrophotometrically for the selection of carbon source that showed highest phb production. extraction of phb the optimized ph and carbon source for each bacterium were used for the extraction of phb by solvent extraction method [4] with slight modifications. firstly, 1% of phb positive strain was inoculated in phb producing media of optimized ph and carbon source and it was incubated at 37°c. after each 4-hour, 1 ml of media was centrifuged at 11,800 rpm for 20 minutes and sudan staining was done to confirm phb production. when the phb production was confirmed, which mostly ensued after 48 hours, 50 ml of bacterial cell culture growth was taken and pelleted at 5000 rpm for 25 minutes. the dry weight of the pellet was taken and then it was washed with acetone and ethanol successively. for the recovery of phb, equal volume of 6% sodium hypochlorite was used to re-suspend the pellet and it was incubated at 37°c for 10 minutes. this was followed by centrifugation at 5000 rpm for 30 minutes to sediment the lipid granules. the pellet obtained was washed with acetone and ethanol followed by hot chloroform treatment. after the pellet dissolved in chloroform, whatman filter paper was used to filter out the cell residues so that only phb is present in the chloroform solution. finally, the filtrate was evaporated in hot air oven at 40°c and dry weight of extracted phb was measured. the percentage of phb accumulation was calculated using the formulae: nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 62-28 thapa et al. ©njb, biotechnology society of nepal 65 nepjol.info/index.php/njb 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑃𝐻𝐵 ( 𝑔 𝑚𝑙 ) × 100 𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 this procedure was repeated for all the phb positive strains and phb was extracted from the strains inoculated in broth media as well as in agar media. characterization of phb the presence of prominent functional groups such as ch3, ch2, c=o, c-o, ch, and oh is a critical decider of presence of phb. atr-ftir spectroscopy (spectrum65 ftir spectrometer) was used for qualitative identification and for checking the presence of such groups in the extracted compounds. result and disscussion isolation and screening altogether, 23 colonies, which were distinct, were chosen based on their shapes and colors. after 24-48 hours culture period, sudan black b staining was done to confirm the presence of phb granules. among 23 bacteria, 7 were found to be sudan positive, i.e. they were capable of producing lipid granules which could have the presence of phb. morphological characterization the result of morphological and biochemical characterization of the 7 sudan positive bacteria is shown in table 1 and table 2 respectively. it was found that five of the strains belonged to bacillus species and 2 strains, y202 and k302, belonged to arthrobacter species and micrococcus luteus respectively. figure 1: graph showing growth curve of isolates it was found that high amounts of black stained granules were obtained in the 48-hour period for all the bacteria. therefore, 48 hours was chosen as the optimized incubation time for phb production. growth curve analysis the inoculated cultures were incubated at 37°c for 48 hours and readings were taken at 640 nm using spectrophotometer. 640 nm was taken as the required wavelength because the wavelength of measurement (wm) of od depends on growth of the culture and here, we expect higher growth. the absorbance vs. incubation hour plot of bacterial strains plotted using r-programming has been shown in the figure 1. optimization of culture conditions the 7 strains of sudan positive bacteria were subjected to growth in the phb producing media prepared with different ph, nacl and table 1. morphological characteristics of strains morphology t101 b139 y202 k302 x102 d301 l402 gram’s test + + + + + + + cell size l: 1μm b:2 μm l: 3μm b: 1μm diameter: 2 μm diameter: 2 μm l: 5 μm b:1 μm l:5 μm b:1 μm l: 5 μm b: 1 μm shape rod rod cocci cocci rod rod rod spore + + + + + margin irregular smooth smooth smooth wooly smooth smooth color white light yellow yellow yellow pinkish white white white elevation flat drop-like convex drop-like flat flat convex opacity translucent opaque opaque opaque translucent opaque opaque table 2. identification of strains s.n. strain identification from bergey’s manual of determinative bacteriology 1. t101 bacillus pumilus 2. b139 bacillus megaterium 3. y202 arthrobacter sp. 4. k302 micrococcus luteus 5. x102 bacillus pasteurii 6. d301 bacillus cereus 7. l402 bacillus sphaericus nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: thapa et al. ©njb, biotechnology society of nepal 66 nepjol.info/index.php/njb carbon sources. the graphs of absorbance vs the optimized conditions of bacterial strains were plotted using r-programming (figure 4, 5 and 6 respectively). figure 2: sudan stained black phb granules of strain l402 figure 3: sudan stained black phb granules of strain d301 figure 4: graph showing absorbance vs. ph plot of bacterial strains. it was established that, for bacillus megaterium (b), arthrobacter sp.(y), bacillus cereus (d) and bacillus sphaericus (l) the optimized ph was 9, for micrococcus luteus (k) and bacillus pasteurii (x), the optimized ph was 7 and for bacillus pumilus (t), ph 7 and ph 9 were compatible. thus, we can deduce that acidic ph is not suitable for phb production. extraction of phb extraction was performed from both phb producing broth and agar. it was observed that extraction from broth gave much better results than that from agar from all of the species except arthrobacter spp. the highest percentage of phb accumulation from broth culture was shown by bacillus pasteurii and the lowest was shown by arthrobacter spp. similarly, from agar, figure 5: graph showing absorbance vs. nacl concentration plot of bacterial strains. it was established that bacillus pumilus(t), bacillus megaterium (b) and bacillus cereus (d) grew the most in the concentration of 0.5%, arthrobacter spp. (y) and bacillus sphaericus (l) grew the most in the concentration of 2% while micrococcus luteus (k) and bacillus pasteurii (x) grew uniformly in all the concentrations except in 10%. figure 6: graph showing effect of different carbon sources on bacterial growth. it was observed that bacillus species could grow and produce phb in both glucose and sucrose. bacillus pumilus (t), bacillus pasteurii(x) and bacillus sphaericus preferred sucrose while bacillus megaterium (b) preferred glucose and bacillus cereus preferred both glucose and sucrose. arthrobacter spp (y) and micrococcus luteus (x) preferred glucose. arthrobacter spp. showed highest accumulation of phb. bacillus pasteurii, bacillus pumilus, bacillus sphaericus did not show any growth in nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: thapa et al. ©njb, biotechnology society of nepal 67 nepjol.info/index.php/njb agar possibly due to limited availability of nutrients in their respective growth conditions. table 3. comparison between percentage phb accumulation from isolates grown in broth and agar ftir analysis the functional groups of extracted phb were identified using ftir analysis. the functional groups of phb extracted from bacillus pasteurii, arthrobacter spp., micrococcus luteus and bacillus cereus was confirmed as c=o groups. figure 7: ftir analysis of extracted product from x102 (bacillus pasteurii) figure 8: ftir analysis of standard phb conclusion the present study was designed for the isolation of effective poly-hydroxybutyrate producing strains from soil to yield maximum phb under optimized conditions. from our research, we found out that cosmopolitan “everything is everywhere” population such as bacillus, arthrobacter and micrococcus species were able to produce phb in considerably good quantity compared to other isolated species. consequently, the effect of various parameters like carbon source, incubation time, ph and nacl concentration on phb production were seen to be species specific. similarly, the production from broth and fermentation methods gave much better results than that from agar in all of the isolated species except arthrobacter. phb yield from broth amounting to 36.41% and 34.59% was observed in bacillus pasteurii and micrococcus luteus respectively, showing a potential for their exploitation in table 3. comparison between percentage phb accumulation from isolates grown in broth and agar s.n. bacterial code bacteria %phb accumulation from broth %phb accumulation from agar 1. x102 bacillus pasteurii 36.41 2. k302 micrococcus luteus 34.59 17.65 3. b139 bacillus megaterium 28.63 4.12 4. t101 bacillus pumilus 21.46 5. l402 bacillus sphaericus 18.45 6. d301 bacillus cereus 14.91 9.35 7. y202 arthrobacter spp. 8.56 20.65 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: thapa et al. ©njb, biotechnology society of nepal 68 nepjol.info/index.php/njb industrial phb production. further characterization of extracted products with the help of atr-ftir analysis showed prominent functional groups ch3 , ch2 , c=o, c-o, ch and oh, which when compared with the standard phb curve, confirms the extracts as phb. hence, this project focused on the isolation of microorganisms from soil samples of polluted sites and the optimization of conditions for the production of phb effectively and frugally. acknowledgement we sincerely thank the biotechnology department and management of sann international college for funding our research work. we are also grateful to mrs. manjushree hada for her constant support and supervision. references 1. ansari s, fatma, t: polyhydroxybutyrate a biodegradable plastic and its various formulations. international journal of innovative research in science, engineering and technology 2014, 3(2):9494–9499. 2. barnes dka, galgani f, thompson rc, barlaz m: accumulation and fragmentation of plastic debris in global environments. philosophical transactions of the royal society b: biological sciences 2009; 364(1526): 1985–1998. 3. burdon kl, stokes jc: studies of the common aerobic spore-forming bacilli staining for fat with sudan black b stain. journal of bacteriology 1942, 43: 717-724 4. chang y, hahn s, kim b, chang h: optimization of microbial poly (3 hydroxybutyrate) recovery using dispersions of sodium hypochlorite solution and chloroform. biotechnol. bioeng. 1994, 44(2): 256-261. 5. godale c, ambarshetti s: media optimization for phb production and its application as precursor for bioplastics. european journal of biotechnology and bioscience 2015, 3(12):49–51. 6. mikkili i, karlapudi ap, venkateswarulu, nath sb, kodali vp: isolation, screening and extraction of polyhydroxybutyrate (phb) producing bacteria from sewage sample. international journal of pharmtech research 2014, 6(2): 850-857 7. leblanc r: how long does it take garbage to decompose? the balance 2017. 8. nehra k, chhabra n, sidhu p, lathwal p, rana j s: molecular identification and characterization of poly-β-hydroxybutyrate (phb) producing bacteria isolated from contaminated soils. asian journal of microbiology, biotechnology and environmental sciences 2017, 17:281-290. 9. pachekoski wm, agnelli jam, belem lp: thermal, mechanical and morphological properties of poly (hydroxybutyrate) and polypropylene blends after processing. materials research 2009, 12(2):159-164 10. rydz j, wanda s, mariya k, and darinka c: polyester-based (bio) degradable polymers as environmentally friendly materials for sustainable development. international journal of molecular sciences 2015, 16(1): 564–596. 11. saharan bs, grewal a, kumar p: biotechnological production of polyhydroxyalkanoates : a review on trends and latest developments. chinese journal of biology 2014. 12. singh g, mittal a, kumari a, goel a, aggarwal nk, yadav a: optimization of poly-b-hydroxybutyrate production from bacillus species. european journal of biological sciences 2011, 3 (4): 112-116. 13. wolchover n: why doesn’t plastic biodegrade? livescience 2011. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 11-15 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 11 nepjol.info/index.php/njb comparative study of growth statistics of two species of paulownia and optimization of rooting methods hari krishna saiju*, abhishesh bajracharya, brishav rajbahak, stuti ghimire department of biotechnology, asian institute of technology and management (aitm) lalitpur, nepal abstract paulownia is a fast-growing woody tree, native to the forests of china. it belongs to the family scrophulariaceae and is mainly used as a source of wood for furniture and musical instruments. due to its fast-growing nature and high-quality of wood, there has been growing interest in cultivation and research of paulownia in nepal. growth comparison was performed by measuring shoot length in in vitro condition. among two species of paulownia paulownia tomentosa (thunb.) steud and paulownia fortuneii (seem.) hemsl., the growth rate of p. tomentosa was found to be 0.355 cm/week while that of p. fortuneii was found to be 0.637 cm/week in in-vitro conditions in ms medium supplemented with 0.1 mg/l naa and 1mg/l bap. optimization of rooting methods was also performed, in which, sand rooting was found to be easier and more effective than in-vitro rooting. dipping the plantlets in 1 mg/l of naa was found to produce longer and denser roots than lower or higher concentrations during sand rooting. keywords: paulownia tomentosa, paulownia fortuneii, growth comparison, in-vitro rooting, sandrooting, nodal culture *corresponding author email: hk.saiju@aitm.edu.np introduction paulownia is a fast-growing, woody tree native to the forests of china [1]. it is a deciduous tree but becomes evergreen in the tropics [2]. it belongs to the family scrophulariaceae and is majorly used as a source of wood for furniture and musical instruments [3]. it has a very low thermal conductivity making it ideal for construction of insulative structures [4]. each tree can produce 44 cubic feet of wood in average and can be harvested after 8-10 years of plantation. it is also tolerant to pollutants and can grow in many types of soils. its leaves and flowers show medicinal properties and can also be used as fodder and fertilizers due to their high nitrogen concentration [5]. its tolerance to drought and soil extremes makes it commercially important for use in the reclamation of surfacemined land [6]. it is also a suitable raw material for pyrolysis conversion into liquid and gaseous products [7]. the generic name, paulownia, honors anna pavlovna of russia [8]. paulownia tomentosa was first introduced in nepal in 1988 ad by international centre for integrated mountain development (icimod) in godavari. it was normally propagated through seeds but due to seed dormancy and slow seedling growth, tissue culture products have been used. among the several species of paulownia, paulownia tomentosa (thunberg) steudel and paulownia fortuneii (seemann) hemsley are the most widely used species in the context of nepal. the former is usually found below an altitude of 1800m while the latter is usually found below an altitude of 2000m [1, 9]. due to the booming market for paulownia plants in nepal, the plantlets have to be manufactured in huge amounts which can be performed by tissue culture. but there have been no publications of research about the in-vitro growth statistics and very few data regarding lab-to-land techniques like acclimatization and rooting methods. in this study, we aim to compare the in-vitro growth statistics of the two species to shed light on their growth patterns. furthermore, we have extrapolated the type and concentrations of hormones and the method of rooting required for optimal root initiation. materials and methods sample and material collection two different species of paulownia, namely, paulownia tomentosa and paulownia fortuneii were used in this experiment. paulownia fortuneii samples were brought in sterile culture jars from the department of plant resources (dpr), thapathali. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 11-15 saiju et al. ©njb, biotechnology society of nepal 12 nepjol.info/index.php/njb paulownia tomentosa samples available in the plant tissue culture laboratory, himalayan white house international college, khumaltar were used for the project. multiplication due to the requirement of a high number of plants, the samples were mass propagated in ms medium supplemented with hormone concentrations of 0.1 mg/l naphthalene acetic acid (naa manufactured by sigma chemical co.) and 1 mg/l benzyl amino purine (bap manufactured by s.d. fine-chem limited) in 150 ml culture vessels. the growth medium and culture equipment were autoclaved (manufactured by life steriware) at 121°c temperature and 15 lb./sq. inch pressure and nodal culture of the samples was performed in a laminar air flow hood (manufactured by amar chand & company (acco). as a result, we performed subcultures twice and prepared a total of 30 vessels for each sample, with an average of 5 explants per vessel, which were stored in an incubation room, under fluorescent tube-lights (2000 lux) at a constant temperature of 25°c [1]. shoot growth comparison after two subsequent subcultures, we met the required explant number which was estimated to be 250 explants. following this, nodal cultures were performed using ms medium with 0.1 mg/l naa and 1 mg/l bap, with only one explant per vessel, for ease of measurement. 20 vessels were produced for each plant species, which were also stored in the incubation room, under 2000 lux fluorescent tube lights at 25°c temperature. culture vessels were recorded alongside a scale every week for 7 consecutive weeks [1]. rooting optimization sand rooting in this experiment, a total of 30 culture vessels were removed from the incubation room and exposed to indirect sunlight at room temperature for 10 days for acclimatization process. nodal cuttings of the invitro plants, including at least one leaf, were prepared and placed into solutions of differing concentrations of naa (0.5 mg/l, 1 mg/l and 1.5 mg/l) for about 10 minutes. the cuttings were then transplanted into rooting trays packed with autoclave-sterilized wet sand and placed into a polythene chamber. after a week of incubation (with water spraying twice a day) at room temperature, liquid ms media was added to the sand twice a week. the plantlets were removed from the sand after 4 weeks of incubation, for the measurement of root density and length [10-12] in-vitro rooting in this experiment, a total of 30 plantlets from culture vessels were transferred into ms media with differing naa concentrations (0.5 mg/l, 1 mg/l, and 1.5 mg/l). these vessels were stored in the incubation room under 2000 lux fluorescent lights at 25°c temperatures for two months. plants were carefully extracted from the media using forceps for measurement of root density and length [13, 15]. results in-vitro growth statistics according to the data in table 1, p. fortuneii was found to have a higher average growth rate of 0.637 cm (sd=0.22) per week in comparison to 0.355 cm (sd=0.12) per week growth rate of p. tomentosa. there was a significant difference in the growth rates of p. tomentosa and p. fortuneii; t (6)=5.150, p = 0.002. both the line graphs represented peaks followed by a gradual decline in growth rate. in-vitro growth was found to peak during the 3rd week for p. tomentosa (0.543 cm) and during the 4th week for p. fortuneii (0.727 cm). in the following weeks, the growth receded slowly for the rest of the culture period. within the 7th week, plant height reached to an average of 5.188 cm (n=16; sd=0.98) for p. tomentosa and 7.197 cm (n=16; sd=1.68) for p. fortuneii. rooting optimization sand rooting: comparative study of root development between two species out of 64 explants, 35% survived after 4 weeks of incubation in the polythene chamber. among those, 55% were p. fortuneii samples and 45% were p. tomentosa samples. as shown in table 2, the average root length of p. tomentosa samples was estimated to be 3.72 cm (n=11; sd=1.32) and the average root number was calculated to be 10.22 (n=11; sd=5.64). for p. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 11-15 saiju et al. ©njb, biotechnology society of nepal 13 nepjol.info/index.php/njb fortuneii samples, the average root length was estimated to be 3.04 cm (n=12; sd=1.72) and the average root number was calculated to be 7.5 (n=12; sd= 7.38). table 2: rooting optimization with different concentrations of phytohormone naa, performed in sand and invitro medium type of rooting species hormone concentration (mg/l) sample size (n) avg. root length (cm) avg. root number sr pt 0.5 3 3.33±1.44 7.50±5.00 1 8 3.87±1.35 11.25±5.82 1.5 0 na na total 11 3.72±1.32 10.22±5.64 pf 0.5 3 4±1.32 5.83±2.88 1 5 3.4±2.16 11.50±10.24 1.5 4 1.87±0.75 3.75±2.50 total 12 3.04±1.72 7.50±7.38 sum total 23 3.36±1.55 8.80±6.60 ivr pt 0.5 1 2.60±0 3.30±0 1 2 1.61±0.43 1.37±0.53 1.5 2 2.13±0.38 2.35±1.20 total 5 1.96±0.44 2.15±1.04 pf 0.5 4 3.91±1.03 2.92±1.40 1 3 2.36±0.11 2.51±0.72 1.5 1 4.40±0 4.80±0 total 8 3.39±1.09 3.00±1.25 sum total 13 2.84±1.13 2.67±1.20 abbreviations: sr sand rooting, ivr in-vitro rooting, pt paulownia tomentosa, pf paulownia fortuneii, na – not applicable figure 1: line graph showing the growth trend of the two species in in-vitro conditions (n=16). plants grown in ms medium with supplementation of 0.1 mg/l naa and 1 mg/l bap. peaks in growth rate can be seen in week 3 for p. tomentosa (0.543 cm/week) and in week 4 for p. fortuneii (0.908 cm/week) figure 2: figure represents a sample of p. tomentosa undergoing in-vitro growth for 7 weeks. the gradual invitro growth in plant height as well as biomass can be clearly visualized from this figure. figure 3: figure represents a sample of p. fortuneii undergoing in-vitro growth for 7 weeks. the gradual invitro growth in plant height as well as biomass can be clearly visualized from this figure. 0 0.2 0.4 0.6 0.8 1 p la n t g ro w th ( in c m ) growth trend of two species of paulownia (in-vitro) p. fortuneii p. tomentosa table 1: average height growth rate (in cm/week) of both species of plants for 7 weeks (n=16). the average in-vitro growth rate of p. fortuneii was found to be higher than that of p. tomentosa. species\time week 1 week 2 week 3 week 4 week 5 week 6 week 7 avg. net growth rate p. fortuneii 0.186 0.547 0.789 0.908 0.72 0.65 0.662 0.637 p. tomentosa 0.142 0.331 0.543 0.397 0.359 0.301 0.411 0.355 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 11-15 saiju et al. ©njb, biotechnology society of nepal 14 nepjol.info/index.php/njb sand rooting: comparative study of effect of varying concentrations of auxin (naa) treatment in root development as shown in table 2, among the surviving samples, 26% had been treated with 0.5 mg/l naa, 56% had been treated with 1mg/l naa and 17% had been treated with 1.5 mg/l naa. samples treated with 1mg/l of naa showed highest root density in both p. tomentosa and p. fortuneii. none of the p. tomentosa plants treated with 1.5mg/l naa survived the sand rooting, but both the average root length and root number from p. fortuneii plants treated with 1.5 mg/l were found to be low. p. fortuneii plants dipped in 0.5 mg/l naa showed the highest average root length of 4cm (n=3; sd=1.32). in-vitro rooting: comparative study of root development between two species callus formation was observed in all the in-vitro rooting samples. root initiation was observed after 3 weeks of sub-culture. out of 13 viable samples, 38% were p. tomentosa samples and 61% were p. fortuneii samples. as shown in table 2, the average root length of p. tomentosa samples were observed to be 1.96 cm (n=5; sd=0.44) and the average root number was calculated to be 2.15 (n=5; sd=1.04). for p. fortuneii samples, the average root length was observed to be 3.39 cm (n=8; sd=1.09) and the average root number was calculated to be 3 (n=8; sd= 1.25). in-vitro rooting: comparative study of effect of varying concentrations of auxin (naa) treatment in root development as shown in table 2, among the viable samples, 38% had been treated with 0.5 mg/l naa, 38% had been treated with 1 mg/l naa and 23% had been treated with 1.5 mg/l naa. samples treated with 0.5mg/l naa showed the highest average root length and density for p. tomentosa, while samples treated with 1.5mg/l showed the highest average root length and density for p. fortuneii. comparison of root length and root number between in-vitro rooting samples and sand rooting samples as shown in table 2, while comparing average root length and root number between different rooting techniques, sand rooting was clearly better suited for root development than in-vitro rooting technique. in case of sand rooting samples, their average root length was estimated to be 3.36 cm (n=23; sd=1.55) and the average root number was calculated to be 8.80 (n=23; sd=6.60). for in-vitro rooting samples, the average root length was estimated to be 2.84 cm (n=13; sd=1.13) and the average root number was calculated to be 2.67 (n=15; sd= 1.20). discussion mass propagation of paulownia plants by tissue culture is gaining popularity in nepal. due to this, many research works are being carried out on different species of paulownia both in and outside nepal. p. fortuneii was found to have higher average growth rate among the two species. this result could be interpreted as p. fortuneii having a greater metabolic ability to utilize the energy and nutrients from ms medium than that of p. tomentosa. despite the lower average growth rate in in-vitro condition, p. tomentosa overcame its flaws by demonstrating superiority during sand rooting. p. tomentosa samples were found to have higher average root length as well as higher root density than that of p. fortuneii. optimization test of hormone pre-treatment showed that the samples treated with 0.5 and 1 mg/l naa had greater average root length than those treated with 1.5mg/l. out of 10 p. tomentosa samples dipped in 1.5mg/l naa, none of the samples survived the sand rooting process. p. fortuneii samples dipped in 1.5mg/l naa also resulted in shorter and sparser roots. it was also observed that samples treated with 1mg/l naa had higher average root density. these results suggest that concentrations of 0.5 and 1mg/l are optimum for treatment before transfer to sand. callus formation was observed in in-vitro rooting samples which are probably induced by mechanical damage to the nodal cutting and presence of auxin (naa) during the culture process. p. fortuneii demonstrated higher densities and lengths of roots in in-vitro rooting. this result also supports our interpretation that p. fortuneii may have a greater ability to utilize energy and nutrients from ms medium. conversely, root length and density were found to be higher in in-vitro media having nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 11-15 saiju et al. ©njb, biotechnology society of nepal 15 nepjol.info/index.php/njb hormone concentration of 0.5 mg/l and 1.5 mg/l. this inconsistency in the result may have stemmed from inadequate sample size, so further studies must be conducted. while comparing the results from sand and in-vitro rooting techniques, we found sand rooting technique resulted in longer and denser roots. due to the high availability of water and nutrients during in-vitro conditions, smaller and sparse roots may have been enough to sustain the plant whereas the scarcity of water and nutrients during sand rooting may have promoted higher root growth. a study performed by rodrigues et al. in 1995 demonstrated a significant increase in root density during water deficit, which is similar to our findings [14-16]. conclusion in the comparison of in-vitro growth statistics of p. tomentosa and p. fortuneii, the latter was found to have higher average growth rate. growth curves were found to peak at 3rd and 4th weeks respectively for the two species. in the study to optimize rooting, p. tomentosa was found to have higher root length as well as root density during sand rooting, but p. fortuneii showed better root development in in-vitro conditions. among the different concentrations of naa, 0.5 and 1 mg/l were found to bear the best results during sand rooting. in case of in-vitro rooting, 0.5 and 1.5 mg/l concentrations of naa were found to give better roots. comparison between the two rooting techniques showed that sand-rooting is the better method for root induction in case of paulownia plants as both p. tomentosa and p. fortuneii demonstrated better results during sand rooting. references 1. rajbahak s, et al: clonal propagation of paulownia tomentosa steud. for commercial production. bull. dept. pl. res. 2014, 36:56-60. 2. siebold and zuccarini fl: paulownia. flora of china 1998, 18:8-10. 3. melhuish jh, gentry ce and beckjord, p r: paulownia tomentosa seedling growth at differing levels of ph, nitrogen, and phosphorous. j environ hort. 1990, 8:205-207. 4. akyildiz mh and kol hs: some technological properties and uses of paulownia tomentosa steud. wood. j environmental biology 2010, 31:351-355. 5. zhu zh, chao cj, lu xy, xiong yg: paulownia in china: cultivation and utilization. asian network of biological sciences. edia.n. rao et al. 1986, 1-65. 6. tang, rc, et al: paulownia a crop tree for wood products and reclamation of surface-mined land. south j appl for. 1980, 4:19-24. 7. yorgun s and yildiz d: slow pyrolysis of paulownia wood: effects of pyrolysis parameters on product yields and bio-oil characterization. j. anal. appl. pyrolysis. 2015, 114:68-78. 8. coombes aj: the a to z of plant names. usa timber press 2012, 312. 9. zima a, hosek j, treml j, muselík j, suchý, p et al: antiradical and cytoprotective activities of several c-geranyl-substututed flavonones from paulownia tomentosa fruit. molecules. 2010 15:6035-6049. 10. rajkarnikar km and rajbahak s: in-vitro multiplication of paulownia fortuneii (seem.) hemsl. through seed culture. bull dept pl res. 2015 37:63-65. 11. kumar pp, rao cd, rajaseger g, rao an: seed surface architecture and random amplified polymorphic dna profiles of paulownia fortunei, paulownia tomentosa and their hybrid. annals of botany 1999, 83:103-107. 12. rajbhandary sb and bajaj yps: rooting of in vitro produced shoots in nonsterile sand an inexpensive and efficient technique for enmasse micropropagation. biotechnology in agriculture and forestry 1991, 17:262-268. 13. venkateswarlu b, mukhopadhyay j, sreenivasan e, kumar vm: micropropagation of paulownia fortuneii through in vitro axillary shoot proliferation. indian j exp biol 2001, 39:594-599. 14. magar lb, shrestha n, khadka s, joshi, j., acharya, j., gyanwali, g., marasini, b., rajbahak, s., & parajuli, n: challenges and opportunity of in vitro propagation of paulownia tomentosa steud for commercial production in nepal. int j appl sci biotechnol 2016, 4:155-160. 15. bergmann ba and whetten r: in vitro rooting and early greenhouse growth of micropropagated paulownia elongata shoots. new forests 1997, 15:127138. 16. rodrigues ml, pacheco cma and chaves mm: soilplant water relations, root distribution and biomass partitioning in lupinus albus l. under drought conditions. j exp bot 1995, 46:947-956 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 33-36 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 33 nepjol.info/index.php/njb hormonal effect on mandarin orange (citrus reticulata blanco) micro-propagation resham babu amgai1*, hari kumar prasai1, yama raj pandey1 1regional agriculture research station-lumle, nepal agricultural research council, kaski, nepal. abstract tissue culture is the best option to produce disease free seedling of the fruit crop rapidly. micro-propagation and use of the in-vitro grafting (micro-grafting) is very helpful for production of virus free planting materials in mandarin. different levels of the in-vitro hormone affect the success of callusing, shooting and plant regeneration in mandarin. shoot bud, flower bud and in-vitro seedling epicotyl was used as explants to study the hormonal effect on mandarin micro-propagation. similarly, 10 levels of bap and iaa combination on ms media for mandarin tissue culture were used. observation was done for 100 test tubes per treatment combination after 4, 8 and 12 weeks of culture. data was arc sine transformed for analysis. shooting from explants was significantly higher (71.72%) on medium level of the bap (0.5 mg/l) and iaa (0.2 mg/l) using in-vitro seedling stem as explant, however, it was 27.91% for stem bud as explant. stem bud showed higher level of callusing (6.15%, p<0.001) in mandarin orange. however, flower bud didn’t develop shoot in mandarin tissue culture. increment of the in-vitro regeneration of the shooting and callusing was observed by the increment of the in-vitro incubation duration in mandarin orange tissue culture. keywords: mandarin orange, citrus reticulata, in-vitro shooting, hormone, callusing. *corresponding author email: reshamamgain@yahoo.com introduction citrus is the major fruit in nepal that shares 26.84% of the total fruit growing area in the country. mandarin orange (citrus reticulata balnco) is predominant occupying 26495 ha i.e. 71.65% of total citrus growing area in nepal [1]. nepalese mandarin industry is facing a lot of citrus decline related problem that can be overcomed through use of disease free planting materials. seed propagation is 85-90% in mandarin; however, grafting is gaining popularity [2]. certain pathogens are difficult to be eliminated from mother plant like citrus exocortis and stubborn, which might be eliminated by a process of shoot tip grafting in-vitro. indian citrus ringspot virus (icrsv) can also be eliminated by using the shoot tip (0.7mm) of sprouts of cultured nodal segment of kinnow through in-vitro shoot tip grafting (micro-grafting) [3]. similarly, plant obtained by micro-grafting do not have the same problems as nucellar plants such as reversion to juvenile state, excessive thorniness, vigorous and upright habit of growth, slowness to fruiting, alternate bearing in early years and physical differences in fruit characteristics [4]. the scion taken from in-vitro cultured buds produced healthy plants than scions from whole trees [5]. similarly, it reduced the dependency for the seasonal flush of the mandarin for scion during micro-grafting. youtsey [6] reported that rate of successful grafts (as high as 50%) often influenced by the condition of the plant material and time of year. therefore, the continue supply of the in-vitro scion material will enhance the micro-grafting efficiency of citrus for all year around. similarly, in-vitro callusing, regeneration and conservation of mandarin is very important. martin et al. [7] also established a protocol by using nodal stem segments of sweet orange for in-vitro conservation for two years and its recovery that was successfully applied on mandarin. therefore, this study was conducted to identify the suitable in-vitro culture media for mandarin in-vitro shooting and callusing. materials and methods variety and ex-plant selection mandarin orange variety ‘manakamana local’ was used in this study. immature shoot with single bud, mature flower bud and in-vitro seedling epicotyl (2 cm long) was used as ex-plant. in-vitro seedling of 8 week was used to harvest epicotyl. explants were collected during citrus flowering season. immature shoot with single bud was used as explant to study the shooting from callus. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 33-36 amgai et al. ©njb, biotechnology society of nepal 34 nepjol.info/index.php/njb in-vitro culture media different hormonal composition was applied for the murasige and skoog (ms) basal media [8] (as in table 1) ex-plant culture and incubation the explant was sterilized with 4% sodium hypochlorite solution for 3 minutes. it was washed with distilled water for 3 times and cultured on test tube with 20 ml specified media (table 1). the test tubes were incubated at 22±2oc with 16:8 hours light: dark. 4. observation and data analysis observation was taken after 4 weeks, 8 weeks and 12 weeks after incubation. three replications were used for each treatment. observation for each treatment consists of 100 test tubes. each test tube is observed for callusing and shooting directly from ex-plant or from callus. data was angular transformed for variance analysis. results and discussion shooting flower bud don’t show shooting directly, however, it showed highest callusing rate (81.77%). similarly, highest shooting directly from explants (figure 1) (31.195%) was observed from in-vitro epicotyle (table 2). mendes et al. [9] also observed that epicotyl explants from 35-day-old seedlings produced significantly more shoots per explants in sweet orange variety ‘para’. no shooting from callus was observed on ms basal media+bap 4 mg/l with/without iaa. similarly, highest shooting from callus (24%) was observed on ms basal media+0.5 mg/l bap. highest callusing was observed on ms basal media+ bap 2mg/l with/without iaa 0.2mg/l (table 3). ms basal media+1mg/l bap gave highest shooting (49.81%) directly from explant. shooting directly from explant and from callus was found increased by increment of the incubation period (figure 2). similarly, significant interaction between explant and media, explant and incubation period was observed for callusing from explant and shooting directly from explant. no interaction was observed among explant, media and incubation callusing flower bud gave highest callusing for all media composition (figure 3). it was highest on ms basal media+0.5 mg/l bap with or without 0.2 mg/l iaa and ms basal media+2 mg/l bap from flower bud explant. no callusing was observed under hormonal level 1 mg/l bap in in-vitro shoot tip. highest callusing from immature shoot with single bud was observed on ms basal media+1 mg/l bap+0.2 mg/l iaa (table 5). table 1: different level of growth hormone used in the in-vitro culture of mandarin [t denotes media composition with ph 5.70] hormone concentration (mg/l) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 6-benzylaminopurine (bap) 0 0 0.5 0.5 1.0 1.0 2.0 2.0 4.0 4.0 indole-3-acetic acid (iaa) 0 0.2 0 0.2 0 0.2 0 0.2 0 0.2 table 2: mean percentage of the callusing and shooting directly from explants observed according to different plant parts used as explants. [value in parentheses is angular transformation; letters after mean is the comparisons using duncan's multiple range test (dmrt) for mean differences] explant callusing from explant shooting directly from explant immature shoot with single bud 21.043 (21.175)b 19.69 (19.002)b flower bud 81.77 (71.872)a no shooting in-vitro shoot tip (2 cm long) 21.216 (20.347)b 31.195 (33.431)a probability <0.001 <0.001 lsd at 5% 4.829 4.494 cv% 3.522 5.807 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 33-36 amgai et al. ©njb, biotechnology society of nepal 35 nepjol.info/index.php/njb figure 4. effect of different level of hormones on mandarin orange immature-shoot with bud explant shooting directly from ex-plant flower bud didn’t show any direct shooting nor does it produce any shoot after callusing. ms basal media+4mg/l bap+0.2mg/l iaa don’t produce any shooting directly from immature shoot with single bud (table 6). it didn’t produce shoot from callus too (table 3). highest shooting was observed on ms basal media+1 mg/l bap from in-vitro epicotyle (71.722%) and from immature shoot with single bud (27.906%) (figure 1 and figure 4). similarly, immature shoot with single bud produced highest shooting in ms basal media+0.20mg/l iaa (table 6). however, mendes et al. [9] observed that the percentage of shoots that produced roots in sweet orange variety ‘para’ was significantly higher in media with naa and iba than with naa alone. conclusion since the requirement of bap for shoot development was genotype specific [9], for mandarin orange cv. ‘manakamana local’ to produce in-vitro scion for micro-grafting the ms basal media+0.20mg/l iaa in immature shoot is table 3: mean percentage of the callusing, shooting directly from explants and shooting from callus observed according to different media composition. [value in parentheses is angular transformation; letters after mean is the comparisons using duncan's multiple range test (dmrt) for mean differences. ms-murashige and skoog [8], bap-benzayl amino phosphate, iaa-indole acetic acid] media composition callusing from explant shooting from callus shooting directly from explant media bap, mg/l iaa, mg/l ms 0.00 0.00 33.02(30.244)c 6.1(10.310)a 20.574(22.875)bc ms 0.00 0.20 33.844(30.398)c 12.4(17.720)ab 27.355(28.907)b ms 0.50 0.00 39.441(35.993)bc 24(25.360)b 21.726(24.37)bc ms 0.50 0.20 41.316(37.254)abc 8.4(12.260)a 31.18731.838b ms 1.00 0.00 40.879(36.210)bc 8.3(12.130)a 49.814(44.503)a ms 1.00 0.20 49.201(46.483)a 11.9(17.310)a 36.316(33.575)b ms 2.00 0.00 50.396(45.978)abc 8.3(12.000)a 23.46(24.727)bc ms 2.00 0.20 42.652(38.895)abc 8.3(12.200)a 20.392(22.105)bc ms 4.00 0.00 39.136(36.189)bc no shooting 11.666(14.584)c ms 4.00 0.20 43.545(40.336)abc no shooting 11.938(14.678)c probability 0.0031 0.018 <0.001 lsd at 5% 8.817 7.964 10.05 cv% 3.522 3.540 5.807 table 4: f-probability value for interaction between explants, media and incubation period. interaction callusing from explant shooting from callus shooting directly from explant explant x media <0.001 only immature shoot used as explant 0.0379 explant x incubation period <0.001 only immature shoot used as explant <0.001 media x incubation period 0.240 0.255 >0.650 explant x media x incubation period >0.650 only immature shoot used as explant >0.650 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 33-36 amgai et al. ©njb, biotechnology society of nepal 36 nepjol.info/index.php/njb best for high shooting. similarly, the ms basal media+0.5 mg/l bap is suitable for shooting from callus (24%) on this cultivar. references 1. moad: statistical information on nepalese agriculture 2012/13. government of nepal. ministry of agriculture development. agriculture business promotion and statistical division, statistics section, singhadurbar, kathmandu, nepal, 2013. 2. fao: training manual for combating citrus decline problem in nepal. directorate of agriculture, ministry of agriculture and cooperatives, government of nepal and food and agriculture organization of united nations, 2011, tcp/nep/3302:(d). 3. sharma s, singh b, rani g, zaidi aa, hallan v, nagpal a, virk gs: production of indian citrus ringspot virus free plants of kinnow employing chemotherapy coupled with shoot tip grafting. j cen eur agri. 2007, 8(1): 1-8. 4. nauer em, roistacher cn, carson tl, murashigue t: in-vitro shoot tip grafting to eliminate citrus viruses and virus-like pathogens produces uniform budliness. hortscience 1983, 18: 308-309. 5. navarro l: shoot-tip grafting in-vitro (stg) and its application: a review. proceeding of the international society of citriculture 1981, 1: 452-456. 6. youtsey co: a method for virus-free propagation of citrus: shoot tip grafting. citrus industry 1978, 59:39-47. 7. martin ml, duran-vila n: conservation of citrus germplasm in-vitro. j amer soc hort sci. 1991, 116 (4): 740-746. 8. murasige t, skoog f: a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiol. plant. 1962, 15: 473-497. 9. mendes afs, cidade lc, manzoli gn, otoni wc, filho wss, costa mgc: tissue culture parameters in sweet orange cultivars. pesq. agropec. bras, brasilia 2008, 43(8): 1093-1096. table 5: mean percentage of the callusing from explants observed with respect to different media composition and explants used (lsd = 15.27 at 5%). [value in parentheses is angular transformation; letters after mean is the comparisons using duncan's multiple range test (dmrt) for mean differences. ms-murashige and skoog [8], bap-benzayl amino phosphate, iaa-indole acetic acid] media composition immature shoot with single bud flower bud in-vitro shoot tip (2 cm long) media bap mg/l iaa mg/l ms 0.00 0.00 33.434(30.385)ghi 65.622(60.061)bcd 0.000(0.287)l ms 0.00 0.20 20.677(21.556)hijk 80.853(69.352)abc 0.000(0.287)l ms 0.50 0.00 25.618(26.992)ghij 92.704(80.700)a 0.000(0.287)l ms 0.50 0.20 30.737(30.366)ghi 93.208(81.108)a 0.000(0.287)l ms 1.00 0.00 31.998(31.180)ghi 90.635(77.164)ab 0.000(0.287)l ms 1.00 0.20 34.999(33.592)fgh 88.644(77.054)ab 23.958(28.802)ghij ms 2.00 0.00 13.196(15.258)ijkl 90.703(79.234)a 47.289(43.443)efg ms 2.00 0.20 8.097(10.009)kl 60.832(56.382)cde 59.028(50.294)def ms 4.00 0.00 0.000(0.287)l 77.197(68.928)abc 40.208(39.354)fg ms 4.00 0.20 11.668(12.125)jkl 77.300(68.737)abc 41.667(40.146)efg table 6: mean percentage of the shooting directly from explants observed with respect to different media composition and explants used (lsd = 14.21 at 5%). [value in parentheses is angular transformation; letters after mean is the comparisons using duncan's multiple range test (dmrt) for mean differences. ms-murashige and skoog [8], bap-benzayl amino phosphate, iaa-indole acetic acid] media composition immature shoot with single bud in-vitro shoot tip (2 cm long) media bap, mg/l iaa, mg/l ms 0.00 0.00 12.938(13.974)efg 28.209(31.775)bcd ms 0.00 0.20 27.117(26.317)bcdef 27.593(31.497)bcd ms 0.50 0.00 26.236(24.501)cdef 17.216(24.240)cdef ms 0.50 0.20 26.930(27.202)bcde 35.444(36.475)bc ms 1.00 0.00 27.906(28.415)bcde 71.722(60.590)a ms 1.00 0.20 26.314(24.306)cdef 46.319(42.844)b ms 2.00 0.00 17.356(16.732)def 29.565(32.721)bcd ms 2.00 0.20 22.224(18.764)def 18.561(25.446)cdef ms 4.00 0.00 9.878(9.519)fg 13.453(19.648)cdef ms 4.00 0.20 0.000(0.287)g 23.874(29.069)bcde nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 7-12 research article doi: https://doi.org/10.54796/njb.v10i1.225 ©njb, bsn 7 microbial quality assessment of raw freshwater fish sold in local markets of kathmandu valley shristi prasai, puja shrestha, sriniwas pandey, ishika adhikari, srijana gurung, kamil prajapati st. xavier’s college, maitighar, kathmandu, nepal received: 14 may 2022; revised: 8 jul 2022; accepted: 15 jul 2022; published online: 30 jul 2022 abstract microbial quality of labeo rohita, cyprinus carpio and clarias batrachus collected from the markets of kathmandu valley was evaluated. 9 freshwater fish (skin, gills, intestine) were sampled and were analyzed for total plate count (tpc), total coliform count (tcc) and total fecal coliform count (tfcc). the average tpc ranged from 4.1 x 107 to 1.02 x 108 cfu/gm, with the highest count in c. batrachus and the lowest in c. carpio, whereas the organ wise load was the highest in intestine with 1.3 x 108 cfu/gm and the lowest in skin with 1.02 x 107 cfu/gm. the highest tcc and tfcc was found in c. carpio and c. batrachus respectively, whereas organ wise distribution showed the highest count in intestine for both tcc and tfcc. the pathogens isolated from the samples were escherichia coli, staphylococcus aureus, coagulase negative staphylococcus (cons), vibrio cholerae, salmonella typhi and s. paratyphi. e. coli was isolated from 67% of l. rohita, 44.44% of c. carpio and 66.67% of c. batrachus. s. aureus was isolated from 44.44% of both l. rohita and c. batrachus whereas 55.55% of c. carpio. cons were isolated from 33.33% of l. rohita, 22.22% of c. carpio and 33.33% of c. batrachus. s. typhi was isolated from 11.11% of c. carpio and 22.22% of c. batrachus. s. paratyphi was isolated from 11.11% of both l. rohita and c. batrachus, v. cholerae was isolated from 11.11% of l. rohita, 33.33% of c. carpio and 22.22% of c. batrachus. the observation of this study showed higher bacterial load in all of the fishes above the acceptance level and presence of total coliform, fecal coliform and potential human pathogens suggests that the microbial quality of the fish available in the market is not satisfactory. hence, the fishes possess a threat to public health safety and there is an urgent need to improve the quality control and quality assurance systems for fish markets of kathmandu valley. keywords: raw fish, microbial quality, e. coli, s. aureus, vibrio cholerae. corresponding author, email: kalkam2013@gmail.com introduction fish is one of the chief sources of protein and has remained an important part of consumption for many centuries[1]. the poikilothermic nature of fresh fish allows a wide variety of bacteria such as pseudomonas, moraxella, acinetobacter, shewanella, flavobacterium, and vibrio among gram negative and gram-positive bacteria such as bacillus, micrococcus, clostridium, lactobacillus, and corynebacterium [2]. the non-indigenous ones that contaminate the fish or the habitat include escherichia coli, clostridium botulinum, aeromonas, shigella dysenteriae, staphylococcus aureus, listeria monocytogens and salmonella spp. the indigenous bacterial pathogens that are found naturally in the fish habitat are vibrio spp. and aeromonas spp. [3]. microbiological quality of raw fish results from microbiological load of aquatic habitat, methods of capture, transportation, chilling and storage conditions imposing a threat of food-borne infections as the pathogen can be conveyed to consumers at retail level through raw fish. coliform, especially escherichia coli are often used as criteria to assess the quality and safety of foods [1]. extraneous bacteria, escherichia coli is the fecal indicator capable of surviving in fish and found to be surviving and even multiplying in the digestive tract of rainbow trout (oncorhynchus mykiss) [4]. water being the habitat, fish is continually bathed in aqueous suspension of various microorganisms and their exterior surface, hence is in constant contact with these organisms. some of the microorganisms may colonize the external parts of fish becoming the resident microflora. the presence of the microflora adds to the defense system of fish, thereby inhibiting the accession and consequent colonization by other potential pathogens. obviously, the bacterial flora of fish depends on the fish’s recent intake diet and the extent of contamination in the food [5]. a study performed on fish skin sampled from the lake hawassa of southern ethiopia by [6], resulted in finding of the pathogenic strains of e. coli to be contaminating the fish with statistically significant results as defense to the fact that fish are contaminated enough to cause foodnepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: https://nepjb.com/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-9739-4848 mailto:kalkam2013@gmail.com nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 7-12 prasai et al. ©njb, bsn 8 borne illness. according to sichewo et al. (2014) [7], inspection of various organs of fish such as skin, intestine, gills and mouth collected from different fish ponds of zimbabwe showed presence of salmonella typhi from nhengo, imbayago and nyamakwe. according to kumari et al. (2001) [8], gill and intestine samples of labeo rohita when processed revealed to be contaminated with coagulase negative staphylococcus spp. and staphylococcus aureus. according to xu et al. (2019) [9], v. cholerae was detected from the intestines of ten freshwater fish species collected, including astatotilapia flaviijosephi, barbus longiceps, c. idella, cyprinus carpio, mugil cephalus, myripristis murdjan, oreochromis aureus, sarotherodon galilaeus, and tilapia spp. and tilapia zilli. improper handling or consumption of undercooked or raw fish may contribute to the intake of pathogens which can potentially cause diseases. diseases in human that can be caused by bacteria present in fish includes food poisoning and gastroenteritis, diarrhoea, superficial wound infections and ulcers, bacillary dysentery (shigellosis), clonorchiasis, dracunculiasis and paragonimiasis due to larvae and metacercariae ingested in fish and crustaceans, cholera, typhoid and paratyphoid, etc. [4]. salmonella, staphylococcus spp., escherichia spp., vibrio parahaemolyticus, clostridium perfringens, clostridium botulinum e and enteroviruses are held responsible for majority of fish concerned with food borne diseases [2]. the safety concerns instigated the need for the study i.e. to investigate the presence of any human pathogenic bacteria from live freshwater fishes that are popularly consumed in kathmandu valley. materials and methods collection of fish sample simple random sampling method was used for selecting samples. the freshly slaughtered 9 fish samples were collected on different day from different retail markets of kathmandu valley (sundhara, baneshwor, thapagaun, gairigaun, ghatthaghar) and brought to the laboratory in an icebox. fishes were killed by the retailers without causing any physical injury just before the sample collection. the three species of fish, l. rohita (rohu), c. carpio (common carp) and c. batrachus (mungri) were chosen based on popularity among the consumers and commercial availability. sample preparation and processing sample preparation was done according to sichewo et al. (2014) [7]. fish was cut ventrally to collect intestine and gills using sterile surgical blades and forceps in aseptic condition. one g of intestine and gills were taken and crushed into fine solution by adding 10ml of normal saline in sterile mortar, from where one ml of aliquot volume was taken and serially diluted up to 10-5. skin samples were taken by rolling sterile cotton swabs all over the skin surfaces of all 9 fish and then inoculated into 10ml of normal saline. it was then serially diluted up to a dilution of 10-5. bacteriological analysis of fish samples for total plate count (tpc), 0.1ml sample from 10-3 and 10-5 dilutions were taken and spread plating was done on pca agar. the plates were incubated for 24 hours at 37°c. for total coliform count (tcc) and total fecal coliform count (tfcc), spread-plating was done from 0.1 ml of every sample of 10-3 dilutions in vrba agar plates and incubated at 37°c and 44.5°c, respectively for 24 hours. for isolation of salmonella spp. one ml of sample was inoculated in 9 ml of enrichment media, selenite f broth, from where a loopful of sample was taken and cultured on ss agar. for vibrio cholerae, one ml of sample was inoculated in 9ml of enrichment media, alkaline peptone water, incubated at 37°c for 24 hours. a loopful of sample was taken from enrichment broth and cultured on tcbs agar. a loopful of original sample was streaked on macconkey agar (for e. coli) and on mannitol salt agar plate (for staphylococcus aureus) and the plates were incubated at 37°c for 24 hours. the colonies obtained from tcbs, ss, ma and msa were further sub-cultured on na [10] and identified by gram staining and biochemical tests (imvic, tsia, urease, catalase, oxidase, oxidative/fermentative test). for vibrio cholerae string test was also performed while staphylococcus aureus was confirmed by coagulase test. results table 1. average tpc of fish samples sample organs (cfu/gm in average) skin gills intestine average lr 6.5 x 106 8.2 x 107 8.05 x 107 5.6 x 107 cc 1.23 x 107 2.96 x 107 8.03 x 107 4.1 x 107 cb 1.2 x 107 6.6 x 107 2.3 x 108 1.02 x 108 average 1.02 x 107 5.9 x 107 1.3 x 108 note: lr = labeo rohita, cc = cyprinus carpio and cb = clarias batrachus in this study, 9 fish sample of 3 different varieties, l. rohita (lr), c. carpio (cc) and c. batrachus (cb) were analyzed for its microbial quality and antimicrobial susceptibility testing was done for the isolated strain. the average tpc of lr, cc and cb ranged from 4.1 x 107 to 1.02 x 108 cfu/gm (table 1). the highest bacterial load was found in c. batrachus whereas the lowest in c. carpio. among the nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 7-12 prasai et al. ©njb, bsn 9 body parts, intestine was found to be highly loaded with the bacteria in all the fishes. total coliforms were present in all parts of the three species of fishes. tcc ranged from 2.47 x 105 to 8.7 x 105 cfu/gm while tfcc was found to be 1.6 x 105 to 3.07 x 105 cfu/gm (table 2). fecal coliforms were present in all parts of all the analyzed fishes except for skin of l. rohita, and skin and gills of c. carpio. all parts of c. batrachus showed presence of fecal coliforms. six different organisms were isolated from gills, skin and intestine of the three fish samples (table 3). in case of l. rohita, gills were found to harbor the diverse species of bacterial pathogens compared to skin and intestines. gills were found to harbor s. aureus (66.67%), v. cholerae (33.33%), e. coli (100%) and cons (33.33%). e. coli, s. aureus and cons each were present in 66.67% of skin sample. e. coli was found in 100% and s. paratyphi in 33.33% of intestine. s. typhi was not isolated from any of the three body parts. as for c. carpio, s. aureus was present in 100% of gills, 66.67% of skin. v. cholerae was found in 66.67% of gills and 33.33% of intestine. e. coli was found in 66.67% of gills and 33.33% of both skin and intestine. cons were present in 33.33% of both skin and gills. s. typhi was present in only 33.33% of intestine whereas s. paratyphi was not isolated from any of the three body parts. in case of c. batrachus, gills showed the prevalence of most of the type of bacteria. e. coli was present in 100% of gills, 66.67% of intestines and 33.33% of skin. s. aurues was in 100% of skin and 33.33% of gills. cons was present in 66.67% of skin and 33.33% of gills. s. typhi was present in 33.33% of gills and intestines each. discussions this study was carried out in the quest to determine the microbial quality of raw and freshly killed freshwater fishes available in retail markets of kathmandu valley. the skin, gills, and intestine of the samples were analyzed for tpc, tcc and tfcc. the average total plate count of l. rohita, c. carpio and c. batrachus was found to be 5.6 x 106, 4.1 x 107, 1.02 x 108, respectively indicating that latter had higher microbial load than the other two (table 1). as for organ-wise distribution, skin of l. rohita contained least microbial load compared to gills and intestine where as in c. batrachus intestine had the highest microbial load. however, in c. carpio, all three organs had similar table 2. average tcc and tfcc of fish samples sample total coliform count (cfu/gm) total fecal coliform count (cfu/gm) skin gills intestine average skin gills intestine average lr 1.5 x 105 3.5 x 105 2.4 x 105 2.47 x 105 no growth 9.4 x 106 2.6 x 105 1.77 x 105 cc 1.2 x 106 2.1 x 105 1.2 x 106 8.7 x 105 no growth no growth 1.6 x 105 1.6 x 105 cb 7.6 x 105 2 x 105 tmtc 4.8 x 105 1.7 x 105 2.9 x 105 4.6 x 105 3.07 x 105 average 7.03 x 105 2.53 x 105 7.2 x 105 1.7 x 105 1.92 x 105 2.93 x 105 note: lr = labeo rohita, cc = cyprinus carpio, cb = clarias batrachus, tmtc = too many to count table 3. distribution of bacteria in skin, gills and intestine of fish samples. sample organ (n=3) isolates s. aureus s. paratyphi s. typhi v. cholerae e. coli cons lr skin 2(66.67%) nd nd nd nd 2 (66.67%) gills 2 (66.67%) nd nd 1 (33.33%) 3 (100%) 1 (33.33%) intestine nd 1 (33.33%) nd nd 3 (100%) nd cc skin 2(66.67%) nd nd nd 1 (33.33%) 1 (33.33%) gills 3 (100%) nd nd 2 (66.67%) 2 (66.67%) 1 (33.33%) intestine nd 1 (33.33%) nd 1 (33.33%) 1 (33.33%) nd cb skin 3 (100%) nd nd nd 1 (33.33%) 2 (66.67%) gills 1 (33.33%) 1 (33.33%) nd 2 (66.67%) 3 (100%) 1 (33.33%) intestine nd 1 (33.33%) 1 (33.33%) nd 2 (66.67%) nd % = calculated according to total number of fish samples (3 each) nd= not detected note: lr = labeo rohita, cc = cyprinus carpio, cb = clarias batrachus, tmtc = too many to count nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 7-12 prasai et al. ©njb, bsn 10 microbial load. high load of bacteria possibly resulted from keeping fishes in wells with contaminated water. total bacterial count of more than 105 cfu per gram elevates the concern of hygiene. according to icmsf (2011), for a newly caught fish, aerobic count may range from 104 to 107 cfu per cm2. much lower count is associated with properly skinned ones [11]. detectable spoilage is usually associated with spoilage bacteria exceeding 107 cfu per gram. goja (2013) did similar study in three freshwater fish and found that viable bacterial counts in intestine and skin ranged from 1.5 x 103 to 8.4 x 104 cfu per gram and 2.8 x 103 to 9.8 x 103 cfu per gram respectively which is much lower than in the three fish samples in this study[12]. comparable research conducted on 150 fish samples from nile bream by sichewo et al. (2014) revealed the tpc of intestine, skin, gills ranging from 4.6 x 103 to 8.03 x 103 cfu per gram [7]. every organ of the fish samples was found to contain total coliform (table 2). similarly, presence of fecal coliforms was also true for all samples analyzed except skin of l. rohita and skin and gills of c. carpio. all samples contained fecal coliform in at least one of the body part whereas the coliform count was the highest in the intestines. according to liu et al. (2016), gut microbiota and their diversity is influenced by many independent factors as different niches have variation in diet availability [13]. in addition, gut microbiota is also influenced by metabolic capacity and gut content enzyme activity. coliform or fecal coliforms are not considered to be normal flora of fish which reflects contamination of fish during transportation, handling or during rearing in water contaminated with human or animal waste [3]. four of the fish samples had a very high count of coliform in their intestines (tmtc). the feed and trophic level (carnivores, omnivores and herbivores) might have some association with the intestinal microbiota. the salinities of water in fish habitat also have some influence on the microbiota of fish intestine [14]. prevalence of microorganisms varied among the three fish samples analyzed with e. coli being most common isolate followed by s. aureus and cons (figure 1). prevalence of v. cholerae was higher among c. carpio, while s. typhi is absent in l. rohita and s. paratyphi was absent in c. carpio. e. coli was present in the gills of l. rohita (100%), c. carpio (66.67%) and c. batrachus (100%) respectively (table 3). similar study conducted by yogoub (2009) revealed that e. coli was the most dominant isolate from fishes [2]. enterobacteriaceae genera were isolated from gills, skin, intestine and muscles of 83 out of 150 randomly collected fishes which also included pathogenic salmonella and shigella spp. during rainy season due to rain surface runoff of organic matters into water bodies are increased which favors multiplication of bacteria. previous study performed by shabeeb et al. (2016) revealed that most isolates were gram negative rods [15]. figure 1: distribution of pathogenic bacteria in l. rohita, c. carpio and c. batrachus %= calculated according to the total number of samples (9 each) salmonella being enteric bacteria, their presence in freshwater fish undoubtedly attributes to fecal contamination of such source from where it is harvested. ponds with higher temperature or from hot regions harbors more salmonella with high prevalence rates. according to bibi et al. (2015) there has been occurrence of salmonella typhi in freshwater fish like labeo rohita (skin, gills, intestine) and cyprinus carpio (intestine)[16]. in our study, s. typhi was isolated from one sample each of gills and intestine of c. batrachus and a sample of intestine of c. carpio while none of the sample from l. rohita showed the presence of the bacterium (table 3). as for s. paratyphi, it was not isolated from any of the tested samples of c. carpio but isolated from one sample of intestine of both l. rohita and c. batrachus. it was noted that none of the skin samples of all the three fish tested showed the presence of salmonella spp. however, these isolates were not confirmed through serological tests. all 3 gills samples of the c. carpio contained staphylococcus aureus (table 3). intestines of l. rohita and c. batrachus were not found to contain the pathogen. staphylococcus is related with unhygienic handling as these are inhabitant of human skin [17]. presence of such opportunistic human pathogens advocates the possibility of cross contamination between handlers and fish. a study conducted in andhra pradesh, india by bujjamma and padmavathi (2015) in 192 fish samples disclosed that over 24.47% of fish were contaminated with s. aureus which also included similar samples [18]. similarly, sampled skins were mostly found to harbor these 0 10 20 30 40 50 60 70 s. aureus s. paratyphi s. typhi v. cholerae e. coli cons p er ce n ta g e (% ) labeo rohita cyprinus carpio clarias batrachus nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 7-12 prasai et al. ©njb, bsn 11 pathogens. however, all intestine samples were negative for cons. skin was observed as the most contaminated area with cons and staphylococcus aureus. composition and count of microflora in fish is a function of water quality, age, type of species and fish [19] along with rearing density and diet impact [20]. in relation to this, hazard escalates when livestock manure is fed to fish which is also the case in nepal. as an established fact, chicken has been remarkably associated with a high load of salmonella. in this study, v. cholerae was isolated from gills of all three types of fish and from intestines of c. carpio (table 3). samples were expected to yield positive results for v. cholerae as this bacterium is the indigenous member of fish associated aquatic habitats. however, their distribution was outnumbered by that of e coli and s. aureus. gills bear a high load of bacteria as a consequence of water and organic substance precipitation[19, 21]. a study conducted in bangladesh on hilsha, a freshwater fish by hossain et al. (2018) revealed that out of 48 fishes, 39 fishes tested positive for v. cholerae in specific ompw gene assay [22]. gills tested positive in highest number (79%) on market fish whereas the same happened for scale swab in case of fresh fishes which is similar to our findings as most gills tested positive for the pathogen. furthermore, pcr results in this study, revealed (66.7%) higher prevalence of vibrio cholerae in fish purchased from local markets than those collected from river banks. e. coli being highly prevalent in all intestinal samples, lower yield of v. cholerae can probably be attributed to glucose metabolism of e. coli yielding acid products which can potentially reduce survival chances of v. cholerae as evidenced by in-vitro studies [23]. halpern and izhaki (2017) emphasized the mutualistic relation between v. cholerae and fish with the evidence of the former helping fish to properly digest chitinous prey like zooplanktons [21]. however, fish serve as an important vehicle for v. cholerae and hence is the key to the dissemination process for epidemics. vibrio parahemolyticus has been sporadically associated with freshwater fish and is seasonal. none of the freshwater fish contained v. parahemolyticus [22]. these findings and observations seem alarming and demand assessing of the freshwater fish and other aquatic products that are kept for sale in the markets. high load of bacteria in the samples including the presence of total coliforms, fecal coliforms and pathogenic bacteria can be attributed to the type of water used in the shops for keeping the fishes. the bacterial load in the fish can probably be the function of frequency of change of water. besides, indigenous bacteria on surface as well as normal flora of other sites, rapidly multiply and invade the previously sterile part after death of fish and hence, spoilage is a consequence if proper keeping conditions are not maintained after harvesting or killing (after being bought). in addition to that, fish handlers at different stages of the supply chain are at greater risk apart from consumers eating the fish. bacteria can pave a way into wounds, cracks or lacerated regions of skin of fish handlers and might potentially cause various infections. moreover, the bad hygiene practice of the handler can potentially cause fish borne infection. conventional cooking systems might reduce the bacterial load to acceptable or possibly kill pathogens. however, the risks remain high when consumers prefer it raw or smoked. in addition, undercooking of such contaminated fish can pose dangers to public health. conclusion nine (3 l. rohita, 3 c. batrachus, 3 c. carpio) fish samples were taken from retailers of kathmandu which were processed for assessing their microbial quality and were assayed for presence of potential pathogenic bacteria. the samples were found to contain a high load of bacteria along with coliforms. the samples were also found to contain potential pathogens like s. aureus, s. typhi, s. paratyphi, v. cholerae, e. coli and cons with the most prevalent being e. coli. these findings suggested the poor microbial quality of freshwater raw fishes that are being sold in the market. c. batrachus was found to be the most contaminated fish. the research indicated that there is an immediate need for quality assessment of fish and such aquatic products on a large scale. to conclude, the fish whether alive, dead, dried or frozen should be consistently monitored for bacterial load and presence of pathogenic forms. author’s contribution project coordinator: kp conceptualization: sp, ps, sp, ia, sg and kp writing – original draft preparation: sp, ps writing – review & editing: kp all authors read and approved the final manuscript: yes competing interests no competing interests were disclosed funding no grants were involved in supporting this work acknowledgements we would like to express our deepest gratitude to the department of microbiology, st xavier’s college, nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 7-12 prasai et al. ©njb, bsn 12 maitighar. we are also thankful to staff members for their help, support and their kind cooperation during the research period. ethical approval and consent not applicable references 1. eizenberga i, terentjeva m, valciņa o, et al. microbiological quality of raw fish at retail market in latvia. in: food quality and safety. njf latvia, 2015, pp. 16–18. 2. yagoub so. isolation of enterobacteriaceae and pseudomonas spp. from raw fish sold in fish market in khartoum state. j bacteriol res 2009; 1: 85–88. 3. atwa ei. bacteriological study of fish samples collected from different markets in some egyptian governorates and antimicrobial sensitivity of isolates. int j curr microbiol appl sci 2017; 6: 2765– 2776. 4. megha p u and harikumar p s. isolation and identification of pathogenic bacteria in edible fish: a case study of mogral river, kasargod, kerala, india. bio sci 2016; 100: 43672–43677. 5. jalal, k.c.a., akbar john, 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izhaki i. fish as hosts of vibrio cholerae. front microbiol 2017; 8: 282. 22. hossain zz, farhana i, tulsiani sm, et al. transmission and toxigenic potential of vibrio cholerae in hilsha fish (tenualosa ilisha) for human consumption in bangladesh. front microbiol 2018; 9: 222. 23. nag d, breen p, raychaudhuri s, et al. glucose metabolism by escherichia coli inhibits vibrio cholerae intestinal colonization of zebrafish. infect immun 2018; 86: e00486-18. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 review article doi: https://doi.org/10.3126/njb.v9i1.38643 ©njb, bsn 85 advances in agricultural biotechnology mamata kc , anuj lamichhane agriculture and forestry university, rampur, chitwan; nepal received: 04 nov 2020; revised: 20 jul 2021; accepted: 24 jul 2021; published online: 31 jul 2021 abstract agricultural biotechnol.ogy is becoming the major sector in crop improvement through the use of scientific techniques for the modification of genes conferring resistance to biotic, abiotic stress and improving the quality of crops. with the evolvement from mendelian genetics to molecular biotechnology, there have been several developments in the field of crop improvement. recent biotechnological advances have aimed towards removing the physiological constraints of the crops and increasing crop yield potential. with the use of different tools of agricultural biotechnologies like genetic engineering, tissue culture, embryo rescue, somatic hybridization, molecular marker-assisted selection, genome doubling, and omics technologies, various transgenic crops have been developed over the decades and have been approved for commercialization. this development and adoption of transgenic technology have been shown to increase crop yields, reduce co2 emission, reduce pesticide and insecticide use and decrease the costs of crop production. even though the biotechnological approach and transgenic organisms have immense potential to contribute to the world’s food security, several concerns of genetically modified crops being a threat to the environment and human health have developed. this review will address applications and concerns of biotechnology in crop improvement considering health hazards and ecological risks. keywords: agricultural biotechnology, genetic engineering, transgenic organisms, benefits, concerns. corresponding author, email: kcmamata24@gmail.com introduction biotechnology refers to the implementation of comprehensive scientific techniques to alter and enhance the characteristics of different plants, animals, and microorganisms that are of economic importance [1]. biotechnology is a broad term that includes applications of microorganisms and different foreign genes (gene of interest) in the processing of food; agriculture and forestry; environmental protection, medical sector, etc. [2]. agricultural biotechnology is the branch of biotechnology that involves the exertion of scientific techniques for the modification and improvement of crops as well as livestock [3]. with the increasing population, traditional agriculture is not sufficient to meet the demands of food worldwide, thus the continuous increase in agricultural productivity depends on effective unification of biotechnology with classical breeding to create an "evergreen revolution" [4]. crop productivity has advanced largely during the 20th century based on applications of mendelian genetics, but if farmers are to address the demands that will be laid on them over the next half-century more effectively, research in biotechnology and molecular biology should be aimed towards removing the physiological constraints of the crops and increasing crop yield potential [5]. recent developments in plant molecular biology and genomes not only has provided us the knowledge and understanding of plant genomes but also the possibility of modifying them [6]. biotechnology provides series of techniques that give access to a wider gene pool and also permits the accurate progress to produce new and useful plant and animal genotypes working along with conventional breeding techniques side by side [7]. the use of traditional techniques, without any question, has profoundly improved important heritable characters such as yield, resistance to disease, etc. in crops, however, there are certain restrictions to these techniques like it may take a very long time to introduce, select and establish a trait into a cultivar or it may be impossible to incorporate certain traits with these techniques. genetic engineering overcomes these limitations by introducing the desired trait in short time without altering other characters of the plant [8]. in this technological era, agriculture faces a new stream of technological revolution associated with biotechnology which could offer considerable assurance for agricultural sustainability by quality enhancement of the product, disease and insect pest resistance, environmental protection, and improving agricultural productivity [9]. with the advances in the field of molecular biology, scientists can manipulate dna to produce transgenic organisms, the process is known as “genetic engineering” and offers a range of benefits along with possible risks [3]. there are controversial social and regulatory consequences with genetic engineering and food made from transgenic crops [10]. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-9302-6554 mailto:kcmamata24@gmail.com https://orcid.org/0000-0002-3593-604x nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 kc & lamichhane ©njb, bsn 86 so, all of transgenic crops developed are not released for commercial cultivation. this review tries to address the recent advances of biotechnology in agriculture and its major concerns. background and history of biotechnology in agriculture agriculture is the backbone of the human food supply. agriculture was practiced manually, in the beginning, using primitive technologies based on plow and harrow. the industrial revolution (1875-1885) enabled accelerated economic development which led to the movement of people from rural areas to industrialized cities. it was around this time the chemical fertilizers were introduced for protection against disease and attainment of higher yields [11]. the human population at present is 7.87 billion increasing at 1.1% average annual rate of population change in year 2015-2020 [12]. this continuous increase in population, estimated to reach 9 billion by 2050, poses a serious challenge to global food security. with the increasing world population, agricultural land has been utilized for settlement purpose. this has decreased the land under cultivation and ultimately the productivity. so, increasing food demands of the world can be met by increasing the global agriculture productivity. but lower land under agriculture cultivation demanded a drastic innovation in technology which not only increase the agriculture productivity but also sustain it for long time. this was provided by the breakthrough of biotechnology field [13]. gregor mendel’s paper “experiments on plant hybridization”, published in 1866; included how different traits were passed from generation to generation which marked the beginning of new technologies designed for improvement in crop species [1]. but, gene modification in crops is supposed to have begun around 10,000 years ago as a result of random or chance through the selection of novel crop types [14]. in 1960, green revolution helped in increasing productivity of three main cereal crops viz. rice, maize, and wheat. a particularly important finding was the discovery of the molecular structure of deoxyribonucleic acid (dna) and the fact that dna was involved in inheritance. the genetic code was cracked in the 1960s and made a way for the transfer of genetic material even easier. with the transfer of genes from one organisms to another, different novel organisms are created, often referred as ‘genetically modified organisms (gmos)’ [15]. with development of several gmos; modern biotechnology has focused on genetic manipulation for agriculture, horticulture, environment, medicine, forensic science, and many other fields [16]. the major events in history of biotechnological development is presented in table 1 [11]. table 1: summary of the main events in the development of biotechnology [11] classical biotechnology 1664 discovery of microorganisms. 1884 discovery of bacteria. 1857 microbiology of lactic fermentation. 1860 end of the spontaneous generation theory 1866 theory of inheritance (gregor john mendel) 1902 chromosomal theory of inheritance 1910 discovery of linkage 1928 transformation in bacteria 1941 one gene-one enzyme hypothesis 1946 bacterial conjugation. 1947 chargaff’s rule modern biotechnology 1953 dna structure. 1958 semi-conservative replication of dna 1959 gene regulation. 1960 green revolution 1966 genetic code decoding 1970 the high specificity of restriction enzymes. rise of phyto-genetics cimmyt foundation 1973 recombinant dna replication in e.coli 1978 human proinsulin gene isolation 1985 polymerase chain reaction. 1992 beginning of the golden rice project 1996 full-fledged commercialization of gm crops agricultural biotechnology in crop improvement agricultural biotechnology refers to the use of biological organisms or range of tools for the improvement of the plants, animals, microorganisms, or food derived from them. following are some biotechnology tools used in agriculture: transgenesis transgenesis also called genetic engineering or recombinant dna (rdna) technology; includes multiple techniques used for the desired manipulation of genetic material (cutting and joining together) particularly dna from various species, and subsequent introduction of the resulting hybrid dna into a new organism to form new combinations of heritable genetic material [17][18]. organisms resulting from transgenesis are called genetically modified organisms (gmos). around 530 different transgenic events in 32 crops have been approved for cultivation in different parts of the world [19]. among them, maize accounts for the maximum number of events (240), followed by cotton (67), potato (50), argentine canola (42), soybean (42), carnation (19), and so on. transgenesis has been applied to develop herbicide-tolerant (ht) transgenic crops, nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 kc & lamichhane ©njb, bsn 87 insect-resistant (ir) transgenic crops, abiotic stresstolerant (ast) transgenic crops, disease-resistant transgenic crops, and nutritionally improved transgenic crops. herbicide tolerant transgenic crops the first herbicide-tolerant transgenic crop to be commercialized was glyphosate-tolerant soybean (roundup ready soybean), which harbored epsps gene from cp4 strain of agrobacterium tumefaciens. most of the commercialized glyphosate-resistant crops harbor this gene [20]. two different genes from streptomyces spp., namely pat and bar, were utilized for developing glufosinate-resistant crops. similarly, other ht transgenic crops specific to other herbicides like 2,4-d, isoxafutole, oxynil, and sulfonylurea, have been commercialized recently [21]. a total of 351 herbicide tolerance events have been approved for cultivation [19]. of these, the maximum number of ht events (212) has been commercialized in maize, followed by cotton (45), argentine canola (34), and others. insect resistant transgenic crops most of the insect-resistant transgenic crops are developed from cry genes from bacillus thuringiensis (bt); which provides resistance against a wide variety of insect pests (lepidopterons, coleopterans, and dipterans) [22]. cry genes not merely provide resistance against insect pests but also is non-toxic to mammals. the first commercially successful crop was cotton in which cry gene was inserted that provided resistance against its lepidopteron insect pest. after the success of transgenic cotton, cry genes have been incorporated in many crops, viz., potato, rice, canola, soybean, maize, chickpea, alfalfa, and tomato [21]. similarly, vip genes isolated from bacillus species (b. thuringiensis and b. cereus) are incorporated in cotton and maize for insect resistance [23][19]. genes encoding protease inhibitor (pi) from different sources (plants, bacteria, and fungi) have been used to produce insect resistant plants. the cptii and potato protease inhibitor ii genes have been introduced in tobacco, and rice, and cotton, respectively to provide resistance against insects [21][19]. to date, 305 insect resistance events have been approved for cultivation [19]. of these, the maximum number of insect-resistant events (208) has been commercialized in maize, followed by cotton (50), potato (30), and others. abiotic stress tolerant transgenic crops the impact of abiotic stresses is increasing in crops with changing climatic conditions. certain plants adapt to these abiotic stresses at the molecular level by altering the expression of an array of genes. this helps to create nearoptimal conditions for plant growth and development [21]. due to the complexity of the abiotic stress adaptation trait (many genes are involved), a lesser number of abiotic stress tolerance events have been commercialized as compared to traits like disease, insect, and herbicide tolerance. a total of 12 abiotic stress tolerance events have been approved for cultivation in maize(7), sugarcane(3), and soybean (2) [19]. the use of bacterial cold shock proteins (csp) to mitigate the effects of abiotic stresses, like cold in arabidopsis, cold, heat, and water deficit in rice, and water deficit in maize, has been demonstrated by castiglioni et al. in 2008 [24]. two genes: the cspa gene from e. coli and the cspb gene from soil bacterium b. subtilis were incorporated in maize, which not only showed better adaptation during waterscarce conditions but also did not lead to pleiotropic effects in maize. recently, hahb-4 gene from helianthus annus (sunflower) is introduced in verdeca’s drought tolerant transgenic soybean commercialized as verdeca hb4 soybean. the gene produces isolated nucleic acid molecule encoding the transcription factor hahb-4 which binds to a dehydration transcription regulating region of plant [19]. similarly, using beta gene from e. coli and rhizobium meliloti drought-tolerant transgenic sugarcane has been made. these transgenic sugarcane crops withstand drought conditions up to 36 days and produce 10-30% higher sugar as compared the non-transgenic plants under drought conditions in field trial [25,26]. disease resistant transgenic crops diseases are caused by pathogens (fungi, bacteria, viruses, and other micro-organisms), and cause huge loss in crop yield. despite the environmental hazards caused by the use of agrochemicals, management of diseases in plants is usually done using agrochemicals, which pose the challenge of the development of chemical-resistant pests [21]. scientists have been able to breed plants with disease resistance traits using transgenesis. so far, 29 disease resistance events have been approved for cultivation [19]. of these, the maximum number of disease-resistant events (19) has been commercialized in potato, followed by papaya (4), squash (2), and others. most of the disease-resistant crops commercialized confer resistance against viruses [21]. using gene encoding the viral coat protein of tobacco mosaic virus (tmv), the first disease-resistant plant was found, which was resistant to tmv infection [27]. similarly, transgenic papaya conferring resistance to papaya ringspot virus (prsv) has been developed through a “pathogenderived resistance mechanism”, where the ‘prsv cp’ gene is introduced by microparticle bombardment into papaya [28]. in bean (phaseolus vulgaris l.), rnai-mediated nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 kc & lamichhane ©njb, bsn 88 resistance against bean golden mosaic virus (bgmv) was developed by silencing the sequence region of the ac1 viral gene which inhibited the synthesis of the viral replication protein of the bgmv [29]. in potato (solanum tuberosum l.), the rpi-vnt1.1 gene from solanum venturii is introduced using agrobacterium-mediated gene transfer, which produces late blight resistance protein and confers resistance to potato late blight [30]. the major constituents of the fungal cell wall (chitin and α-1, 3 glucan) are degraded by the chitinase enzyme thus when the chitinase gene was introduced in tobacco and rice, it has been reported to enhance fungal resistance in the plant [31]. nutritionally improved transgenic crops many successful efforts have been made to improve nutritional qualities in crops using transgenesis. the most recent example includes biofortified rice line gr2e (golden rice), developed by the introduction of gene ‘crt1’ from pantoea ananatis and gene ‘psy1’ from zea mays. golden rice is capable of synthesizing carotenoids in the endosperm. gr2e was approved for use as food in the philippines, australia, new zealand, canada, and the united states [19]. similarly, to improve the nutritive value of potato, the transgenic potato tubers were developed by expressing amaranthus seed albumin gene ‘ama1’, which is plentiful of all essential amino acids for human diet specification according to the who standard [32]. an effort was made to enhance the pro-vitamin a content in tomato by producing transgenic tomato and converting phytoene to lycopene with the transference of bacterial gene for phytoene-desaturase enzyme. and also three times more β carotene content was produced by these transgenic plants than normal plants [33]. antisense fae1 gene transferred to brassica napus and brassica juncea has resulted in low erucic acid content [34]. in maize, the introduction of the ‘cordapa’ gene from corynebacterium glutamicum has increased the production of amino acid lysine [19]. tissue culture tissue culture is the culture of cells, tissues, organs, or their components in a nutrient medium under sterile conditions [35]. it usually involves the use of small pieces of plant tissue (explants) which are cultured in aseptic conditions [36]. tissue culture manipulates and extends the period of cells, anthers, pollen grains, or other tissues and develops a whole, living growing organisms. using tissue culture, genetically engineered cells can be transformed into genetically engineered organisms [37]. tissue culture has been used extensively to create genetic variability through the in-vitro culture of protoplasts, anthers, microspores, ovules, and embryos, to improve crop plants and to increase the number of desirable germplasm available to the plant breeder. it is one of the pivotal tools of biotechnology [38]. tissue culture is used in the germination of seeds that are difficult to germinate like banana. grand naine (g9) variety of banana is prepared using tissue culture, which results in mass propagation of disease-free high yielding clones, and true to type plants [39]. similarly, the meristem tip culture of banana plants produces plants devoid of banana bunchy top virus (bbtv) and brome mosaic virus (bmv) [40]. in vitro cell and organ, culture can be used for the conservation of endangered germplasms. the plants that do not produce seeds (sterile) or produce seeds that cannot be stored for a long period (recalcitrant seeds), can be preserved using tissue culture techniques for the maintenance of gene bank [36]. embryo rescue for wide hybridization embryo resulting from inter-specific or inter-generic crosses may fail to produce a hybrid because of pre or post-fertilization incompatibility barriers. these barriers can be overcome by rescuing such embryos and culturing them for producing a whole plant, which facilitates the transfer of desirable genes from wild relatives into cultivated species [38][18][41]. this technique is known as embryo rescue or wide hybridization. wide hybridization and embryo rescue were done in capsicum to transfer fruit rot-resistant traits by debbarama et al. in 2013 [42]. somatic hybridization somatic hybridization is a technique that integrates somatic cells from two different cultivars, species, or genera of plants for the manipulation of cellular genomes [43]. somatic hybridization by protoplast fusion helps in the regeneration of novel germplasm and into whole organisms through tissue culture [44][45]. similarly, incompatibility barriers at inter-specific or intergeneric levels can be overcome by somatic hybridization. fusion between protoplasts of potato (solanum tuberosum) and tomato (lycopersicum esculentum) has created pomato (solanopersicon, a new genus). it not only overcomes barriers of sexual incompatibility but also creates novel genotypes [46] a salt-tolerant hybrid callus culture was developed by somatic hybridization between rice (oryza sativa) and mangrove grass (myriostachya wightiana), which is useful in the development of salt-tolerant rice varieties [47]. disease resistance genes are also transferred using somatic hybridization like asymmetric somatic hybridization was used to transfer bacterial blight resistance trait from wild oryza meyeriana l. to oryza sativa l. ssp. japonica [48]. similarly, those genetic traits nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 kc & lamichhane ©njb, bsn 89 that are cytoplasmically controlled like male sterility, resistance to certain antibiotics and herbicides, can be easily transferred using protoplast transformation followed by somatic hybridization [43]. cybridization has been used to transfer cytoplasm male sterility (cms) in rice [49]. molecular marker aided genetic analysis and selection molecular marker aided genetic analysis helps in gene identification i.e. it studies dna sequences particularly to identify the genes, qtl (quantitative trait loci), and molecular markers; as well as associate them with the organism. molecular marker aided selection helps to identify and trace the inheritance of previously identified dna fragments through a series of generations [37]. molecular marker-assisted breeding uses molecular markers along with linkage maps and genomics to alter and improve plants or animal traits based on genotypic assays [50]. rice genotypes having resistance to bacterial blight(bb) and basmati quality and desirable agronomic traits were identified using phenotypic and molecular marker-assisted selection, which can be either directly used in the development of commercial varieties or used as a donor of bb resistance in basmati breeding programs [51]. similarly, marker-assisted selection allowed identification of sources of coffee berry disease and coffee rust resistance for use in preventive breeding for resistance to these diseases. several genes from other coffea species were important sources for gene pyramiding in breeding programs aimed at multiple and durable resistance [52]. genetic analysis of fusarium head blight resistance in cimmyt bread wheat line c615 was done using traditional and conditional qtl mapping by yi et al. in 2018 [53]. this study showed genetic relationships between fhb response and related traits at the qtl level providing useful information for marker-assisted selection for the improvement of fhb resistance while breeding. doubled haploid/ genome doubling a doubled haploid (dh) is a genotype formed when haploid cells undergo chromosome/genome doubling. haploid cells like pollen, egg cells, or other cells of gametophyte are subjected to spontaneous chromosome doubling, giving a doubled haploid cells, which is then grown into a doubled haploid plant [54]. it allows the development of pure line varieties or inbred parental lines quicker compared to traditional breeding [55]. double haploid technology in wheat accelerated time to market and faster genetic gains in yield and resistance gain, which helped in reducing varietal development time [56][57]. similarly, anther-culture followed by dh offers a great opportunity to accelerate breeding progress and improve grain quality. dh plants through antherculture provide an efficient method for rapid production of homozygous lines of rice which are found to be more viable than other lines [58]. similarly, in another study by bakhshi, bozorgipour, and shahriari-ahmadi in 2017, chromosome elimination method was used to develop double haploid wheat lines via crosses with maize as the male parent [59]. further 3 wheat lines were selected to develop and adapt under heat stress conditions. ‘omics’ technologies ‘omics’ technologies are subcategories of bioinformatics which include genomics, proteomics, transcriptomics, genome sequencing, and metabolomics [60]. genomics is used to understand the structure, function, and evolution of genes; and identify dna that confers to traits in the organisms. proteomics helps to analyze the protein in tissue for identifying gene expression in that tissue as well as decipher the specific function of proteins encoded by particular genes[61][37]. omics based approach helps to decipher the entire genome for gaining insights into plant molecular responses, which provides specific strategies for crop improvement. using the omics approach, we can identify dna (gene) encoding for a certain trait (genomics), rna coded by it (transcriptomics), proteins formed (proteomics), metabolites produced (metabolomics), and phenotype expressed (phenomics). omics technology provides valuable information on the structure and behavior of crop genomics. any gene responsible for a particular trait can be used to enhance breeding in different ways [62]. a herbicide-tolerant maize line was developed by precise insertion of a target gene using site direct mutagenesis [63]. concerns of agriculture biotechnology biotech crops were grown in 29 countries in 2019, contributing significantly to food security, sustainability, climate change mitigation, and upliftment in the lives of farmers and families worldwide [64]. however, some concerns regarding gene manipulation in crops being ecologically harmful and unsafe for human consumptions. major concerns of agriculture biotechnology are briefly discussed below: adverse effects on non-target organisms the use of transgenic crops for a specific cause (disease/pest resistance) has caused unintended effects on non-target organisms. reduction in monarch butterfly population has been reported on the adoption of glyphosate-resistant transgenic crops in the usa and mexico [65]; and higher mortality was reported when its nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 kc & lamichhane ©njb, bsn 90 larva fed on milkweed leaves dusted with the genetically modified bt maize as compared to laboratory conditions [66]. similarly, wide-scale adoption of bt cotton in china increased the population of minor pest (mirid bug), which acquired the status of major pest later [67]. biosafety issues there have been concerns about the safety of transgenic food being a threat to human health and the environment. risks associated with human health include allergenicity, toxicity, horizontal gene transfer, and feed safety [68]. when introducing a gene into an organism, the level of allergens might increase in the modified organism above the natural range or new allergen might be introduced. so, bean crops modified to increase the level of cysteine and methionine content were discarded after the discovery of the expressed protein of transgene being highly allergenic [69]. so testing of transgenic food may be required to avoid harm to the consumers. similarly, who has claimed genetic material can be transferred from transgenic food to cells of the human body or bacteria in the intestinal tract or to soil microbes mainly because the dna ingested from transgenic food is not completely degraded by digestion [68]. the possibility of horizontal transfer of antibioticresistant marker genes from transgenic food to animal and human gut microbes may result in antibiotic resistance in the gut microflora, though its possibility is extremely low [21]. similarly, the cultivation of genetically modified crops could cause “genetic erosion” as farmers restrict themselves to few popularly grown varieties. gm crops are not part of the natural process, so they could cause unpredictable changes in ecology and evolutionary response; the resurgence of pests and emergence of superweed are the results of these. resistance breakdown extensive cultivation of insect-resistant and herbicidetolerant crops increases the chances of the development of resistance in the targeted insect population through high selection pressure. new insect biotypes may evolve with resistance against transgenic technology. similarly, superweed having resistance against herbicides may emerge. the field evolved pest resistance to bt maize has been reported in spodoptera frugiperda (fall armyworm) in brazil to cry1f expressing corn and cry1ac expressing soybean [67]. in china, field evolved resistance to bt cotton in cotton bollworm (helicoverpa armigera) to cry1ac expressing cotton has been reported [70]. economic, social and political concerns there are economic concerns about gm crops, as the price of seeds will be so high that small farmers and farmers in developing countries are unable to afford seeds for gm crops [71]. concern about negative socioeconomic impacts of rapid technological change on-farm or rural structure is also present. in muslim communities, the use of gmos is considered halal or haram [72]. the labeling of genetically modified foods is one of the major political concerns. usa does not label gm foods, but there must be a common consensus on labeling genetically modified foods and their products in all countries. similarly, differences in biotechnology regulations differ in the us and eu, due to minor differences in consumers' preferences [68]. conclusion agriculture has come a long way from the green revolution to the gene revolution. it is being applied and updated more and more daily. with the ability to know and modify the genetic makeup of organisms using biotechnological tools, we can cope with the increasing demand for food through the development of novel varieties of crops with a higher yield, better resistance against biotic and abiotic factors, and ensure environmental sustainability. the use of biotechnology in agriculture has not only helped to increase the productivity of crops but also reduced the cost of production by decreasing needs for inputs (pesticides) and improved the livelihood of the farmers. similarly, new varieties of plants with higher yields in fewer inputs have wider environment adaptability; give better rotation to conserve natural resources has been developed through biotechnology applications. despite these rapid developments, concerns regarding the safety issues of gm crops on human health, food/feed safety, on the environment, social, economic, and political are raised continuously. complete and transparent assessment of gm crops application and their effects should be done, with strong regulatory implementation mechanism for use of gm crops. alternatively, new methods such as cisagenesis, intragenesis, and genome editing can be utilized for developing improved crops. competing interests the authors declare that they have no competing interests. author’s contribution all authors contributed equally. acknowledgments not applicable. references 1. persley gj, siedow jn. applications of biotechnology to crops: benefits and risks. 1999. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8 5 9 2 kc & lamichhane ©njb, bsn 91 2. gavrilescu m, chisti y. biotechnology a sustainable alternative for chemical industry. vol. 23, biotechnology advances. elsevier inc.; 2005. p. 471–99. 3. wieczorek a. use of biotechnology in 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foods. sci relig glob perspect philadelphia, pa, usa. 2005;4–8. nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 review article doi: ttps://www.doi.org/10.54796/njb.v9i2.41914 ©njb, bsn 39 efficacy and toxicity of different forms of asparaginases against acute lymphoblastic leukemia: a review adesh baral, ritesh gorkhali, amit basnet, shubham koirala, hitesh k. bhattarai department of biotechnology, kathmandu university, dhulikhel, nepal. received: 15th jun 2021; revised: 22nd dec 2021; accepted: 25th dec 2021; published online: 31st dec 2021 abstract acute lymphoblastic leukemia (all) is a form of blood cancer that affects white blood cells and is among the most common forms of leukemia with children and adolescents showing the highest number of cases. most treatment protocols include chemotherapy using asparaginase. asparaginase converts asparagine to aspartic acid and ammonia. unlike normal, healthy cells, cancerous cells depend on asparagine for their growth. when these cells are deprived of asparagine by the action of the enzyme, the cancer cells selectively die. as of date, several forms of asparaginases are commercially available and are administered in all therapy. but due to limited study, it will be early and inaccurate to predict which forms of the enzymes are better. in this review, we aim to compare the efficacy and toxicity of four different asparaginases—native escherichia coli asparaginase, peg escherichia coli asparaginase, erwinia chrysanthemi asparaginase and a recombinant escherichia coli asparaginase—used in all therapy in children and adolescents using available clinical trial data. pubmed and clinical trial.org databases were used to select studies. asparaginase activity, toxicity, anti-asparaginase antibody level and event-free, overall survival was compared for different asparaginases. seventeen randomized and non-randomized controlled trials were included. evidence was insufficient to ascertain which asparaginase is the best. peg escherichia coli asparaginase seems to be better with a high activity among the treated patients but there remains high toxicity for all available asparaginases. this study highlights a need to discover alternative sources of asparaginase from the organisms, which are evolutionarily distant from escherichia coli and erwinia chrysanthemi with high higher enzyme activity and reduced toxicity. keywords: efficacy, acute lymphoblastic leukemia, asparaginase, clinical trials. corresponding author, email: hitesh321@gmail.com introduction acute lymphoblastic leukemia or all is among the most prevalent forms of leukemia and most commonly affects children. all is caused by unchecked and malignant proliferation of lymphoid progenitor cells in blood, bone marrow, and extramedullary sites [1]. all presents itself via three main pathological processes: first is the failure of bone marrow, second is the infiltration of other tissues by blasts via malignancy and third is the systemic effects arising from cytokines released by the cancerous cells. all often shows common signs and symptoms among children which include: anemia, thrombocytopenia, and pronounced hepatosplenomegaly or lymphadenopathy [1]. american cancer society estimates that 6590 cases were diagnosed in 2016 alone with 1400 death [2]. it is suspected that a combination of prenatal mutations and environmental factors cause all. l-asparaginase is an enzyme that catalyzes the formation aspartic acid and ammonia from asparagine. it is commonly produced in bacteria, but not in humans. organisms produce it in the course of their normal life cycle and it can be extracted and purified for industrial and medical purposes. l-asparaginase was introduced in the early 70s as part of the treatment protocols for all in children. now, l-asparaginase has become a routine part of treatment protocols of all [3]. asparaginase works via depletion of asparagine in the blood. l-asparagine is an important amino acid used by cells in protein synthesis. most normal cells produce lasparagine for their growth using l-asparagine synthetase enzyme, synthesizing l-asparagine from aspartic acid and glutamic acid. neoplastic cells like all cells are incapable of producing their own lasparagine because they lack l-asparaginase synthetase enzyme. thus, they are dependent on the extracellular supply of asparagine for their existence and reproduction. exogenous source can be the serum where asparagine from diet and normal cells are pooled. consequently, l-asparaginase is used as a therapeutic agent against all. l-asparaginase catalyzes the degradation of asparagine into ammonia and aspartate and depletes the asparagine in the blood serum, which leads to starvation of cancer cells causing cell death [4]. l-asparaginase is commercially extracted mostly from erwinia chrysanthemi and escherichia coli. the enzyme can be used in its purified native state or can be nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-7147-1411 mailto:hitesh321@gmail.com mailto:hitesh321@gmail.com nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 40 conjugated to increase its half-life. peg escherichia coli asparaginase is a variant made by conjugating the escherichia coli l-asparaginase with polyethylene glycol. there are many commercially available examples of peg escherichia coli asparaginase as well. usually, pegasparaginase can be used in lower doses compared to native state asparaginases due to its greater half-life [5]. some people can have allergic reactions to escherichia coli asparaginase. erwinia chrysanthemi asparaginase is used as an alternative for these cases [3]. therapeutic results of combining l asparaginase with chemotherapy protocols have been generally very successful. many variations have been made to improve the results of these therapies, usually centered around reducing enzyme related side effects that are common in these therapies[6]. though all three forms of l asparaginase have been extensively studied for their effectiveness and safety, unintended enzyme-related side-effects like hypersensitivity and allergic reactions abound [7]. till date, there has been limited study to address the efficacy and toxicity of different forms of asparaginases available for therapy because of which individuals who are under therapy are at high risk. this creates an urgent need to address and minimize the risk by providing necessary and relevant information on safety and fill in the research gap. in this study, we have attempted to compile and examine a list of studies to understand efficacy, safety and toxicity of peg escherichia coli asparaginase, erwinia chrysanthemi asparaginase, and native escherichia coli asparaginase to determine if there is a need for an alternative source of asparaginase to reduce these unwanted side effects. asparagine concentration: ogawa et al. reported that the average plasma asparagine concentration of patients treated with e. chrysanthemi asparaginase was 0.218 𝝁m [8]. concentration of < 0.5 𝝁m is considered as complete depletion of asparagine from the blood [9]. van der sluis et al reported that complete depletion of asparagine was seen in 97.8% of patients treated with recombinant asparaginase and 97.9% of patients treated with native e. coli asparaginase [10]. likewise, pieters et al. claimed that the mean asparagine concentration dropped to 0.5 µm under both recombinant e. coli asparaginase and native e. coli asparaginase in 99% of patients [11]. in another study by van der sluis et al. the mean asparagine concentration dropped to <0.5 𝝁m in all patients at all time points measured with recombinant e. coli asparaginase[12]. in a comparison done between intermittent and continuous routes of peg e. coli asparaginase among 625 children of europe, it was found that 95% of patients were asparagine depleted during treatment [13]. in 2011, a study conducted to demonstrate peg e. coli asparaginase as a viable alternative in patients that have shown allergic reactions to treatments using native e. coli asparaginase, it was found that asparagine level depleted to 40% and 20% at day 7 and day 14 respectively for hypersensitive patients using peg asparaginase. similarly for non-hypersensitive patients, it was depleted to 50% at day 14 but while using native e. coli asparaginase it was depleted to only 86% on day 14 [14]. 26% of patients receiving e. chrysanthemi asparaginase showed asparagine depletion during reinduction. with e. coli asparaginase receiving patients, asparagine depletion was seen in 60% to 90% during the re-induction phase. serum asparagine levels recovered after 4 days for patients administered with e. chrysanthemi asparaginase compared to 11 days for e. coli asparaginase [15] (table 1). table 1. average asparagine concentration in treated patients from individual trials clinical trial type of asparaginase average concentration(𝝁m) [8] erwinia chrysanthemi 0.218 [16] native escherichia coli 0.13 [10] native escherichia coli (asparaginase medac) <0.5 [10] recombinant escherichia coli <0.5 [17] native escherichia coli (asparaginase medac) <0.5 [17] recombinant escherichia coli <0.5 [12] recombinant escherichia coli <0.5 [13] peg escherichia coli <0.5 [14] native escherichia coli <0.5 asparaginase activity: measurement of asparaginase concentration during the therapy has lots of technical limitations, which is why asparaginase-enzyme activity is generally used to monitor asparaginase. as per u.s fda, effective plasma level of asparaginase was defined as ≥ 0.1 iu/ml and used for determination of efficacy in the approval process for asparaginase [18]. ogawa et al. reported that the average activity of e. chrysanthemi asparaginase in treated patients throughout the study was 0.36 iu/ml, which was much higher than the therapeutic level of asparaginase [8]. all the treated patients in their study achieved a therapeutic level of asparaginase. likewise, vyas et al. noted in patients treated with native e. coli nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 41 asparaginase and peg e. coli asparaginase, the activity of the enzymes were 0.13 iu/ml for native e. coli asparaginase and 0.30 iu/ml for peg e. coli asparaginase with 86% in the native e. coli group and 94% generic peg e. coli group achieving a therapeutic level of asparaginase [16]. in case of recombinant escherichia coli asparaginase, van der sluis et al. reported that the average asparaginase activity was 0.17 iu/ml with 62.2 % of patients achieving the therapeutic level of asparaginase and in native e. coli asparaginase average asparaginase activity was 0.16 iu/ml with 65.9% patients achieving the therapeutic level of asparaginase [10]. similarly, place et al. claimed that peg e. coli asparaginase activity was around 0.7 iu/ml is treated patients and native e. coli asparaginase activity was around 0.1-0.2 iu/ml[19]. in a phase ii study conducted by dinndorf et al. for the fda, the asparaginase activity and the depletion of asparagine were measured in days after the first dose. they found that between the 2nd and 7th day after the first dose both native e. coli asparaginase and the peg escherichia coli asparaginase had activities above 0.03 iu/ml in 50 patients. this number decreased to below 10 patients for native e. coli asparaginase group while it reached 20 for the peg group in the remission induction phases [20]. in a study, the therapeutic peg e. coli asparaginase activity was observed to be 0.234 iu/ml among 86% of surviving patients [17]. in another study by pieters et al. median asparaginase activity for native e. coli asparaginase was found to be 0.19 iu/ml while recombinant asparaginase showed an activity of 0.14 iu/ml (pieters et al. 2008). moreover, van der sluis et al. reported the serum asparaginase activities of recombinant e. coli asparaginase to be >0.10 iu/ml in 74% patients and was 0.13 iu/ml of all measured samples respectively [12]. in rau et al. none of the study population completed the trail and only one patient had tolerated the peg e. chrysanthemi asparaginase with activity >0.1 iu/ml [21]. significantly shorter serum half-life of 0.65 days was observed for e. chrysanthemi asparaginase enzyme compared to 1.24 days for e coli asparaginase in a study by duval et al. [15] (table 2). as stated earlier, asparaginase activity in-vivo was standardized to be less than 0.1 iu/ml during the therapy, but it happens to be between 0.13-0.70 iu/ml (table 3). this showcases that most of the individuals, who were under therapy achieved the threshold. toxicity: in a therapy involving asparaginase, toxicity is directly table 3. average range of asparaginase activity in treated patients from all the studied trials. type of asparaginase average activity range (iu/ml) from all trials native escherichia coli 0.13-0.19 peg escherichia coli 0.23-0.70 erwinia chrysanthemi 0.13-0.58 recombinant escherichia coli 0.13-0.17 related to the dose administered. this is why controlled administration of quantity is a must. a trial reported that e. chrysanthemi asparaginase hypersensitivity reaction (urticaria) of grade 1-2 were seen in 2(8%) of patients, pancreatitis of grade 1-3 in 3(12%) of the patients, and hyperglycemia of grade 1-2 in 5 (20%) of the patients [8]. similarly, hypersensitivity reaction of grade 3-4 was seen in 7(12%) patients in native e. coli asparaginase treated group, 3 (6%) in peg e. coli asparaginase treated group, hyperglycemia of grade 3-4 was seen in 2(4%) patients in native e. coli asparaginase treated group and in 1(2%) patient in peg e. coli asparaginase group [16]. later, van der sluis et al reported that hypersensitivity reaction was seen in 2(2.1%) of the patients in recombinant e. coli asparaginase group and 5(5%) in native e. coli asparaginase group, pancreatitis of grade ≥ 2 was seen in 1 (1%) of patient in native e. coli asparaginase group [16]. also the hypersensitivity reaction of grade 1-4 was seen in 28(12%) of patients receiving peg e. coli asparaginase and in 21(9%) in native e. coli asparaginase receiving group. pancreatitis of grade ≥ 2 was seen in 27 (12%) of the patients receiving peg asparaginase and 22(10%) receiving native e. coli asparaginase group [19]. in 1999, in a study by liang et al. 10,000 iu/m2 dose escherichia coli asparaginase was used during the remission induction therapy on 93 children with table 2. average asparaginase activity from individual trials in treated patients clinical trial type of asparaginase average activity (iu/ml) [8] erwinia chrysanthemi 0.36 [16] native escherichia coli 0.13 [16] peg escherichia coli 0.30 [10] native escherichia coli (asparaginase medac) 0.16 [10] recombinant escherichia coli 0.17 [19] native escherichia coli 0.1-<0.2 [19] peg escherichia coli 0.70 [17] native escherichia coli (asparaginase medac) 0.19 [17] recombinant escherichia coli 0.14 [12] recombinant escherichia coli 0.13 nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 42 standard-risk or sr (spontaneous remission) all. they found that 26.8% or 25 of the participants showed signs of toxicity. of them, 15 or 16% showed signs of sepsis, 2 or 2% had pneumonia, 6 or 6% showed signs of hyperglycemia, and 6 or 6% had hemorrhage. during remission induction, 19 of 93 or 20.4% of patients developed a severe infection. death during induction occurred in 6 patients [22]. a phase ii clinical trial was conducted by dinndorf et al. for the peg escherichia coli asparaginase oncaspa® by enzon pharmaceuticals in 118 children aged 1 to 9 years. it was a comparative study between a native e. coli asparaginase and the peg e. coli asparaginase. they concluded, 14 of the 58 patients in the peg asparaginase group compared to 18 of the 59 patients in the native escherichia coli group suffered from toxic effects. hyperglycemia was much more common in the peg escherichia coli asparaginase group with 5% or 3 patients suffering from it and abnormal liver conditions were much more common in the native escherichia coli asparaginase group, 10 patients with abnormal liver tests compared to 6 in the peg escherichia coli asparaginase group [20]. another study conducted on 144 patients aged below 22 years to study the effect of weekly vs. bi-weekly dosing regimens with peg e. coli asparaginase and native e. coli asparaginase in patients with first relapses of all in reinduction therapy, found that out of the 143 patients whose data was evaluated 72 or 50% of them suffered from severe infections. 29 of them had hypoalbuminemia, 32 of them had low fibrinogen and 9 of them showed weight loss. there were also specific toxicities seen in the group given peg e. coli asparaginase. only 6 of the 144 tested showed peg-asparaginase hypersensitivity, 4 of whom only showed grade i allergic reaction. the other 2 had grade iii hypersensitivity and were given alternative e. chrysanthemi asparaginase instead [23]. additionally, in the e. coli asparaginase 5000 arm 9 (2.7%) deaths were observed, whereas in e. coli asparaginase 10000 arm 23 (6.5%) deaths were observed. pneumonia was seen in about 50% of patients and hypersensitivity reaction was reported in 4.5% (n=31) patients in e. coli asparaginase 10000 arm. in e. coli 5000 arm 1.8% (n=13) patients showed hypersensitive reaction [24]. later, 15 out of 16 deaths were for patients over 40 years. sepsis together with hepatotoxicity occurred in 50% of the dead patients. among surviving people treated with peg e. coli asparaginase pancreatitis and hypoalbuminaemia of grade 3+ were recorded on 2 patients (3%) [17]. pieters et al. reported no death during the trail but recorded deep venous thrombosis and severe hyperglycemia in two separate patients given with recombinant asparaginase and similarly, deep venous thrombosis and severe neutropenia in two different patients treated with native e. coli asparaginase [11]. a study conducted by van der sluis et al. found that 12 patients under treatment showed hemorrhage, nose bleeding, thrombosis of the superior vena cava and increased alanine aminotransferase ctc grade iii [12]. rau et al. reported complications of chest tightness and facial erythema with mild swelling and anaphylaxis in three patients [21]. albertsen et al. in 2019, demonstrated that 60 (9.6%) patients experienced toxicity during peg e. coli asparaginase treatment and 23 (3.7%) after the last dose. among those showing symptoms, hypersensitivity was seen in 13 (2%), osteonecrosis in 29 (4.6%), pancreatitis was seen in 24 (3.84%) and thromboembolisms in 17 (2.72%). in a 3-year period of observation, incidence of any form of toxicity associated with first asparaginase treatment after randomization was found to be much higher in children of age 10 years or older compared to children younger than 10 years, but did not differ between boys and girls or between patients at intermediate risk or standard risk [13]. additionally in another study comparing native e. coli asparaginase with e. chrysanthemi asparaginase, e. coli asparaginase arm had more instances of coagulation abnormalities (30.2%) compared to e. chrysanthemi asparaginase arm (11.8%) [15]. another trial conducted in spain by ribera et al concluded that during induction therapy, percentage of patients with detectable infection, hypersensitivity, thrombosis, hepatic toxicity, pancreatitis and coagulopathy (all of them within grade 3-4) for native e. coli asparaginase was 56%, 1%, 6%, 21%, 1%, 11% respectively while for peg e. coli asparaginase was 45, 0, 9, 38, 3, 18 respectively. similarly during consolidation therapy, percentage of patients with detectable infection, hypersensitivity, thrombosis, hepatic toxicity, pancreatitis and coagulopathy (all within grade 3-4) for native e. coli asparaginase was 16%, 1%, 0.4%, 3%, 0%, 0.4% respectively while for peg e. coli asparaginase was 13%, 1%, 0%, 11%, 0%, 5% respectively [25]. toxicities caused by different forms are asparaginases are relatively similar in every patient group (table 4). nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 43 event-free survival (efs): following initial treatment for cancer, patients generally remain free of any complications or symptoms that were intended to delay or prevent their treatment. this is known as an event-free survival. vyas et al. reported that 2-years event-free survival of patients treated with peg e. coli was 84% and treated with native e. coli asparaginase was 80.7% [16]. place et al evaluated 5years of event-free survival of patients to be 90% in peg e. coli asparaginase treated group and 89% native e. coli asparaginase treated group[19]. pession et al. study found that 5 year and 10 year eventfree survival (efs) of native e. coli asparaginase group was 84.6% and 82.5% for the 494 patients enrolled in the trial. 58 cases failed to achieve event-free status with 1 case of second malignancy and 22 relapses. the study by liang et al. was a comparison between the use of native e. coli asparaginase and epidoxorubicin in the treatment of sr all in the remission induction therapy. they also saw 5 relapses in their asparaginase arm of the study group. they estimated an efs at 3 years to be 72%[22]. the fda phase ii trial study by dinndorf et al. did not attempt to find long-term efs. they estimated an 80% efs for both their erwinia asparaginase group and peg asparaginase group. karachunskiy et al. conducted a study on event-free survival rates and found that at 10 years the probabilities of event-free survival rates for escherichia coli 5000 arm (79 ± 2%) were not significantly different from escherichia coli 10000 arm (75 ± 2%) [24]. while comparing native e. coli asparaginase with e. chrysanthemi asparaginase, event-free survival predicted was 6 years and percentage patient survival was 73.4% versus 59.8% [15] (table 5). by observing years of event free survival ranges, it can be discerned that peg e. coli asparaginase gives low degree of disease reoccurrence (table 6). follow up period for event free survival patients were reported for 10 years for native e. coli (10 years), 6 years for peg e. coli and 6 years for e. chrysanthemi asparaginases. overall survival: a clinical trial reported 2-year event-free survival of patients at 93% treated with peg e. coli asparaginase table 5: time period of event-free survival in treated patients from individual trials. clinical trial type of asparaginase event free survival year % of patients survived follow up period (years) [16] native escherichia coli 2 80.7 2 peg escherichia coli 2 84 [19] native escherichia coli 5 89 6 peg escherichia coli 5 90 [26] native escherichia coli 5 84.6 10 native escherichia coli 10 82.5 [24] native escherichia coli 10 73-81 10 [15] native escherichia coli 6 73.4 6 erwinia chrysanthemi 6 60 [22] native escherichia coli 3 72 3 table 6: average year range of event-free survival in treated patients from all the studied trials. type of asparaginase average year range of eventfree survival year (% range) native escherichia coli 5(84%-89%)-10 (75%-82%) peg escherichia coli 5(90%) erwinia chrysanthemi 6 (59%) and 84% treated with native e. coli asparaginase [16] and another trial evaluated 5-years of overall survival of patients. the statistics was 96% treated with pegasparaginase and 94% treated with escherichia coli asparaginase [19]. likewise, karachunskiy et al. reported that patients of e. coli asparaginase 5000 arm (86 ± 2% ) had slightly superior probability of overall survival at 10 years compared to e. coli asparaginase 10000 arm (82 ± 2%) [24]. overall survival estimated at 5 and 10 years was 94.4% and 93.7% in the group with asparaginase versus 89.8% and 88.6% in the group without asparaginase , respectively [26] (table 7). moreover, study conducted in 2002 demonstrated that 6 year survival rate using native e. coli asparaginase was greater than using e. chrysanthemi asparaginase (83.9% vs 75.1%) [15]. table 4. average range of occurring toxicity in treated patients from all the studies trials. asparaginase types toxicity escherichia coli asparaginase (%) pegescherichia coli asparaginase (%) erwinia chrysanthemi asparaginase (%) recombinant escherichia coli asparaginase (%) hypersensitivity 1.8 – 12 2– 12 1.3 – 12 2.1 pancreatitis 1 – 10 3-12 na na hyperglycemia 4 – 6 2 5 – 20 na the range of toxicities are of 1-4 grade from all the trials. nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 44 in terms of overall survival peg asparaginase from escherichia coli showed better result than other forms of asparaginases (table 8) table 8. average year range of overall survival in treated patients from all the studied trials. type of asparaginase average year range of overall survival year (% range) native escherichia coli 5 (94%) 10 (93%) peg escherichia coli 5 (96%) follow up period of overall survival of patients were reported for 10 years for native e. coli asparaginase, 6 years for peg e. coli asparaginase and 6 years for e. chrysanthemi asparaginase. anti-asparaginase antibody (aaa) the most common adverse reactions of asparaginase in children are produced by anti-asparaginase antibodies. these adverse reactions can manifest as mild or severe allergic reactions. it was reported that 10% (9 in native e. coli asparaginase and 10 in recombinant escherichia coli asparaginase group) patients were detected positive for aaa [10]. the dinnodorf et al. fda study found that 16 out of 57 or 28% of their patients treated with native e. coli asparaginase had anti-aparaginase antibodies at any given time in of the treatment. three subjects were known to have pre-existing antiaparaginase antibodies. in the patients treated with the peg e. coli asparaginase 11% of the 55 or 6 of them had asparaginase antibodies. rau et al. 2018 reported very low anti-peg igm among three patients. anti-peg igg was observed in three patients except one after 5.5-years of exposure to peg e. coli asparaginase [21] (table 9). table 9. individual patients positive with anti-asparaginase antibody (aaa) from trials. clinical trial type of asparaginase % of patients positive for anti-asparaginase antibody (aaa) [10] recombinant escherichia coli 10 native escherichia coli (asparaginase medac) [20] native escherichia coli 28 peg escherichia coli 11 peg asparaginase from escherichia coli has shown promising advantages over other forms of asparaginases. anti-asparaginase antibodies are found in lower numbers in peg asparaginase . (table 10). table 10. average % range of patients positive for antiasparaginase antibody (aaa) from all the studies trials. type of asparaginase average % range of patients positive for anti-asparaginase antibody (aaa) native escherichia coli 10%-28% peg escherichia coli 11% discussion and conclusion data was obtained from various clinical trials (table 11) on asparaginase activity, concentration, and toxicity of the three major types of asparaginases used for all therapies: peg-asparaginase, erwinia chrysanthemi asparaginase and native escherichia coli asparaginase. peg e. coli asparaginase activity was seen to be between 0.3-0.7 iu/ml, which is the highest in comparison to others (table 3). in terms of toxicity, all three forms of asparaginases showed similar results (table 4). toxicities like hyperglycemia and pancreatitis were seen in a significant number of cases leading to a decrease in the effectiveness of the enzyme in the treatment of the patients. in several studies, we can see that incidence of toxicity increases with the dose of the enzyme. another minor conclusion that we can derive from clinical trials by place et al and vyas et al is that overall survival was slightly higher for peg e. coli asparaginase than native e. coli asparaginase (table 8). this difference is very slight and the results stay the same for event free survival making conclusions about the higher efficacy of one type of asparaginase over the other difficult (table 6). there were number of other studies that did not directly evaluate the safety and efficacy of asparaginases in clinical trials for children. since these studies were also included in this review, we will discuss the findings of these studies. in the study by rau et al. pegcrisantaspase (erwinia chrysanthemi pegylated asparaginase) treatment also reported hypersensitivity reaction to the patients table 7. time period of overall survival in treated patients from individual trials. clinical trial type of asparaginase overall surviva l year % of patients survived follow up period (years) [16] native escherichia coli 2 84 2 peg escherichia coli 2 93 [19] native escherichia coli 5 94 6 peg escherichia coli 5 96 [26] native escherichia coli 5 94.4 10 native escherichia coli 10 93.7 [24] native escherichia coli 10 80-88 10 [15] native escherichia coli 6 84 6 erwinia chrysanthemi 6 75 nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 45 with previously showing hypersensitivity reaction to peg asparaginase treatment. table 11. general data of selected studies. author, year country type of enzyme (asparaginase) route of administration period of treatment of enzyme total number of participant study group(age) ogawa et al. 2017 japan erwinia chrysanthemi intramuscular 2weeks 24 2-16 years vyas et al. 2018 india native escherichia coli) intramuscular 10 weeks 106 less than 18 years generic peg escherichia coli) intravenous van der sluis al, 2018 netherlands recombinant escherichia coli intravenous 5 weeks 199 children native escherichia coli place et al. 2015 usa and canada peg escherichia coli) intravenous 30 weeks 463 1-18 years escherichia coli intramuscular pession et al. 2005 italy 90% of the patients received erwinia chrysanthemi 10% escherichia coli intramuscular 24 months 490 1-15 years liang et al. 1999 taiwan escherichia coli intramuscular 110 weeks 201 1-15 years dinndorf et al. 2007 usa escherichia coli[elspar® from merck] peg escherichia colioncaspa® by enzon pharmaceuticals, inc both intramuscular 12 weeks 118 1-9 years abshire et al. 2000 usa peg escherichia coli erwinia chrysanthemi intramuscular 4 weeks 144 below 22 years of age karachunskiy et al moscow– berlin native escherichia coli intramuscular 200 days 774 1-19 years patel et al. 2017 uk peg escherichia coli 8 weeks 91 25-65 years pieters et al. netherlands recombinant escherichia coli asparaginase asparaginase medac intravenous 39 days 32 1-14 years van der sluis et al. 2013 netherlands and germany recombinant escherichia coli -asparaginase 39 days 12 below 1 years rau et al. 2018 usa peg erwinia chrysanthemi intravenous 29 days 4 1-20 years albertsen et al. 2019 denmark, finland, iceland, norway, or sweden pegescherichia coli intramuscular 30-33 weeks 625 children duval et al. 2002 belgium, france and portugal escherichia coli intravenously 6 weeks 700 less than 18 years erwinia chrysanthemi kurtzberg et al. 2011 usa, canada peg escherichia coli asparaginase intramuscularly 4 weeks 76 less than 21 years native escherichia coli ribera et al. 2017 spain native escherichia coli intravenously 4 weeks 126 18-60 years it is possible that peg (poly ethylene glycol) can be immunogenic and anti-peg igg antibodies are formed during peg-asparaginase treatment. the remaining immunological memory may mediate hypersensitivity reaction during pegcrisantaspase treatment. one of the patients involved in this study had not been exposed to pegaspargase for 5.5 years. he did not experience a pegcrisantaspase hypersensitivity reaction. a lack of a durable immunologic memory from anti-peg-mediated immune reactions may be the case for this patient. it is suggested that patients who have been recently exposed to peg in the formulation of other medicine in any disease should not use any peg asparaginase in any form. instead, native escherichia coli or erwinia chrysanthemi asparaginase should be used. in another study by patel et al. it was shown that asparaginase toxicity can be substantial in older patients, making it difficult to deliver safely to those aged above 40 years [17]. similarly, when ribera et al used native or peg asparaginase in adult patients there no significant difference observed in complete remission, diseases free survival, and overall survival with no influence in patient response and outcome [25]. based on numerous previous studies teenagers and younger adults typically have better outcome from induction and consolidation treatment compared to adults (aged above 40 years). careful timing of administration and avoidance of overlapping toxicities are recommended for the older patients. nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 39-47 baral et al. ©njb, bsn 46 in the liang et al. study authors state that the increased mortality was due to immunosuppression via depletion of blood asparagine by the enzyme. they had increased the doses of the leunase brand asparaginase to match that of the dose preparation by nesbit et al. of escherichia coli preparation called crasnitin. authors attribute the severe infection and unexpected mortality to this dose change [22]. the product sold as medac, is also known to cause excessive toxicity when given at a high dose, leading to a 50% reduction in the dosage [27]. karachunskiy et al. have reported that patients treated with e. coli asparaginase of dose 10000 u/m2 have reported the severe hypersensitivity reaction more frequently than patients provided with 5000 u/m2 of dose. also, death in complete remission occurred significantly more in 10000 u/m2 provided patients [24]. an advantage of higher doses was not found in the group. thus, it will be advantageous to discover and use a higher activity asparaginase, thereby allowing use of lower dose of enzyme to reduce the incidences of toxicity. enzymes with low km value will have higher activity against l-asparagine. besides the l-asparagine activity of l-asparaginase, there is the secondary activity of mentioned asparaginase enzymes against lglutamine, that has been linked to the different toxic side effects[28,29]. also, the role of l-glutamine activity has not been seen in anti-cancer activity of the enzyme[30]. one method of finding new asparaginase is to extract it from an organism other than escherichia coli or erwinia chrysanthemi. we can expect an organism that is evolutionarily distant from escherichia coli or erwinia chrysanthemi to have different enzyme activity. thus, we can expect to find better alternatives to commercially available asparaginases with higher activity than escherichia coli and erwinia chrysanthemi [31, 32]. according to this study, peg asparaginase provides better enzyme concentration than e. coli or erwinia chrysanthemi asparaginase in various clinical trials. similarly, two studies show that peg asparaginase has higher 2-year overall survival than native e. coli asparaginase. the difference is very minor to conclusively say peg asparaginase is superior. using erwinia chrysanthemi asparaginase when e. coli and peg asparaginase fail, as is currently done, is recommended from this study as well. furthermore, alternative source of asparaginase from the organisms, which are evolutionarily distant from escherichia coli and erwinia chrysanthemi and with a lower km value i.e., higher enzyme activity toward l-asparagine, and low activity towards the l-glutamine need to be discovered. such novel asparaginases can be used in lower dose thereby by reducing toxicity. acknowledgement the 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approach. jmirx med. 2021 sep;2(3):e29844. https://doi.org/10.2196/29844 32. thomas j. kindt, barbara a. osborne rag. kuby immunology, sixth edition. 6th ed. w. h. freeman & company; 2006. 9–11 p. https://doi.org/10.1182/blood-2008-04-149443 https://doi.org/10.1200/jco.18.01877 https://doi.org/10.1097/mph.0b013e31822d4d4e https://doi.org/10.1182/blood.v99.8.2734 https://doi.org/10.1080/08880018.2018.1538277 https://doi.org/10.1038/leu.2016.219 https://doi.org/10.1182/blood-2013-01-480822 https://doi.org/10.1016/s1470-2045(15)00363-0 https://doi.org/10.1016/s1470-2045(15)00363-0 https://doi.org/10.1002/pbc.26873 https://doi.org/10.1007/s00432-019-02854-x https://doi.org/10.1080/10428194.2017.1397661 https://doi.org/10.1200/jco.2005.11.411 https://doi.org/10.1016/j.leukres.2015.04.008 https://doi.org/10.1182/blood-2013-10-535112 https://doi.org/10.1182/blood-2013-10-535112 https://doi.org/10.2196/29844 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 8-17 research article doi: https://doi.org/10.3126/njb.v9i1.38645 ©njb, bsn 8 antibiotic susceptibility pattern of staphylococcus aureus isolated from pus/wound swab from children attending international friendship children's hospital bidhya maharjan1 , shovana thapa karki2, roshani maharjan3 ¹department of microbiology, st. xavier's college, tribhuvan university, maitighar, nepal ²department of pathology, international friendship children's hospital, maharajgunj, kathmandu, nepal 3department of microbiology, tri-chandra multiple college, tribhuvan university, ghantaghar, kathmandu, nepal received: 04 nov 2020; revised: 05 jul 2021; accepted: 19 jul 2021; published online: 31 jul 2021 abstract a wound gets infected when the organism gets invaded through the breached skin, proliferated and production of various enzymes, toxins, etc. in order to treat the wound infection, antibiotic susceptibility pattern of organism should be determined before the prescription of the medicine. the present study was conducted from september 2017 to march 2018 with an aim to determine antibiotic susceptibility pattern of staphylococcus aureus identified from the pus/wound swab among the patients visiting the international friendship children's hospital, kathmandu, nepal. total 270 sample were processed, isolated and identified using standard microbiological procedure and biochemical test. antibiotic susceptibility test was carried out by using modified kirby bauer's disc diffusion method. out of total sample, 51.48% (139) showed growth. the growth distribution was found to be high in out-patient department 84.9% (118) than in-patient department 15.1% (21). among 139 positive growth, 83.5% were gram positive and 16.5% were gram negative. all together 12 different organisms were identified, among which s. aureus was found to be predominant organism 105 (75.5%). s. aureus was found to be sensitive towards linezolid followed by doxycycline whereas it was found resistant towards ciprofloxacin. among s. aureus identified, 50% were multidrug resistant (mdr) s. aureus and 55% were methicillin resistance s. aureus (mrsa). mrsa was found to be sensitive towards linezolid followed by doxycycline and resistant towards ciprofloxacin. the association between mdr and mrsa was found positively significant (i.e. p-value = 0.000). all strains of s. aureus were found to be sensitive towards vancomycin. 22.86% were double disk diffusion test (d-test) positive. the prevalence of d-test was found to be high in mrsa (75%). the relationship between d-test and mrsa was found to be significantly correlated with each other (r = 0.39). linezolid, chloramphenicol, vancomycin and doxycycline is a drug of a choice for both s. aureus and mrsa infection. keywords: pus/wound swab, staphylococcus aureus, antibiotic susceptibility test (ast), multidrug resistance (mdr), methicillin resistance staphylococcus aureus (mrsa), d-test corresponding author, email: 23bidhya@gmail.com introduction human skin acts as an excellent barrier to infection, protect underlying tissues, bones, organs, etc. and prevents the entry of microbes (i.e. potential pathogens) into our body unless the mechanism is breached due to injury, trauma or surgical intervention [1, 2, 3]. a break in the integrity of the skin or tissues which may be associated with disruption of the structure and compromises its protective function is called a wound [4]. a wound gets infected when proliferating microorganisms invade to a level that invokes a local or systemic response in the host [5]. during wound infection, the bacteria multiplies, healing is disrupted and wound tissues get damage and also spread to nearby tissues. the consequences of any tissue damage, wound infection or any internal tissue injury is pus [6]. pus is defined as the accumulation of dead cells and microorganisms, together with accumulated fluid and various proteins [7]. wound infection is a common problem during injury, mainly in the case of children [4, 8]. injuries in the children may be due to falls followed by burns, cuts and animal bites which causes both financial and psychological strain on the family because it drags the patient to the health care facilities [9, 10]. wound infection account for 70-80% mortality and also an important cause of morbidity among surgical patients and 75% of mortality following burn injuries [11, 12, 13]. the common organism responsible for pus formation or wound infection are: coagulase negative s. aureus (cons). s. aureus, bacillus spp., clostridium spp., peptostreptococcus spp., actinomyces spp., e. coli, proteus spp., neisseria spp., vibrio vulnificus, candida spp., etc. [14]. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-9769-1201 mailto:23bidhya@gmail.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 9 s. aureus is a versatile pathogen capable of causing a wide range of human diseases [15]. it is a significant human pathogen that causes wound infection, soft tissue infection and produces the pus [16, 17]. it belongs to the family micrococcaceae, gram positive cocci having grape like cluster arrangement of 0.5-1.5 µm diameter, aerobic, facultatively anaerobic, ꞵ-hemolytic, fermentative, oxidase negative, non-sporing, non-motile, noncapsulated, yellow zone formation around the colonies on msa and oil paint appearance on na slopes [18, 19]. there has been a huge problem all over the world in the treatment of infectious disease due to increase in antibiotic resistant cases [20]. multi-drug resistant (mdr) is defined as the non-susceptibility of organism to at least one agent in 3 or more antimicrobial categories, extremely drug resistance (xdr) is non-susceptibility to at least 1 agent in all but 2 or fewer antimicrobial categories and pan drug resistance (pdr) is nonsusceptibility to all agents in all antimicrobial categories [21]. methicillin resistant s. aureus (mrsa) has been identified as one of the major risk pathogens associated with the development of antimicrobial resistant [22]. mrsa is defined as a strain of s. aureus that is resistant to a large group of antibiotics called ꞵ-lactams, which include penicillin and cephalosporin [23]. in nepal, various laboratories have reported the emergence of mrsa mainly community-associated mrsa (camrsa) which have been detected in the lumbini medical college and teaching hospital while doing cross-sectional studies of prevalence of mrsa [24]. in another study, study carried out to assess the extent of mrsa in the kathmandu model hospital kathmandu, mrsa were more frequently isolated from pus samples and that too from hospitalized patients [23]. vancomycin is a glycopeptide antibiotic that inhibits cell wall biosynthesis, remains a drug of choice for treatment of severe mrsa infections. s. aureus isolates with complete resistance to vancomycin (mic≥16µg/ml) are termed as vancomycin resistant s. aureus (vrsa). vrsa was first reported in the u.s in 2002 [25]. in one of the studies conducted in the manmohan memorial college and teaching hospital, kathmandu, nepal, there all the mrsa identified was found to be susceptible towards the vancomycin [26]. d-test is a simple disc diffusion test to study the macrolide lincosamide streptogramin b resistance (mlsb), both constitutive and inducible as well as macrolide streptogramin b resistance (msb) in s. aureus. macrolide group (erythromycin, azithromycin, rokitamycin) is a drug used to treat of s. aureus infection and also used for those allergic to the penicillin [24]. after a few years of drug's introduction in therapy, staphylococci developed resistance to erythromycin in 1956. these resistant strains were found in france, u.k and in the u.s.a [27]. lincosamide (clindamycin) is used for the treatment of mrsa infection [28]. since these both antibiotics have the same site of drug target, there is a high chance of cross resistant among these antibiotics due to modification of drug target [29]. this study helps to perceive the current status in prevalence of s. aureus in pus/wound swab, the antibiotic susceptibility pattern of the isolated s. aureus and also any presence of multidrug resistant strain among the isolates. it also helps to know the resistant towards commonly used antibiotic and aware the practitioner from misusing the antibiotic. hence, the aim of the study was to assess the prevalence of s. aureus and the antibiotic susceptibility pattern of s. aureus isolated from the pus/wound swab from children attending international friendship children's hospital (ifch), maharajgunj, kathmandu. materials and methods sample collection and identification of isolates the research was conducted at the microbiology laboratory of international friendship children's hospital, maharajgunj, kathmandu from september 2017 to march 2018. a hospital based cross-sectional study was carried out among the patients visiting to the hospital having wound infection below 16 years, requesting for culture and susceptibility testing. in total, 270 pus/wound swab samples were collected using aseptic technique. out of total sample, 228 samples were collected from out-patient department (opd) and 42 samples were collected from in-patient department (ipd). within ipd also, 12 samples were collected from general ward (gw), 4 samples from special ward (sp. ward), 12 samples from surgical/burn ward (s/b ward), 6 samples from infant icu (iicu), 3 samples from pediatric icu (picu), 4 samples surgical icu (sicu) and 1 sample from neonates icu (nicu). here, 130 samples were of male and 140 samples were of female. 17 samples were of age group 0-1 month, 54 of 1 month-1year, 75 of 1-3 years, 54 of 4-6 years, 57 of 6-12 years and 13 of 12-15 years. the specimens were well labelled and then transported to the laboratory and processed immediately. after macroscopic and microscopic observation, it was cultured on blood agar (ba) and mac-conkey agar (ma) and incubated at 37°c for 24 hrs. the isolates were identified by colony morphology, gram staining and nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 10 various biochemical tests [30]. the gram-positive cocci in cluster observed under microscope was considered as staphylococcus species and was subjected under different biochemical test for the confirmation of s. aureus. the staphylococcus species showing catalase positive, oxidase negative, fermentative, yellow colony on mannitol salt agar (msa), coagulase positive and dnase positive were confirmed as s. aureus [30]. for gram negative organism, different biochemical tests such as: catalase test, oxidase test, sulphur indole motility (sim) test, methyl red (mr) test, voges-proskauer (vp) test, citrate test, oxidative/fermentative (o/f) test, urease test and triple sugar iron (tsia) test were performed for the identification of the organism. antibiotic susceptibility test the antibiotic susceptibility testing was done by using modified kirby-bauer disc diffusion method [31] on mueller hinton agar using antibiotic discs of hi-media laboratories pvt. ltd. the antibiotic used was selected by following clinical and laboratory standards institute (clsi) 2017 guideline [31] for s. aureus. the antibiotics used were cotrimoxazole (1.25/23 mcg), chloramphenicol (30 mcg), ciprofloxacin (5 mcg), gentamycin (10 mcg), doxycycline (30 mcg), linezolid (30 mcg), vancomycin (30 mcg), azithromycin (15 mcg), meropenem (10 mcg) and piperacillin (100/10 mcg). novobiocin (30 mcg) was used to identify s. epidermidis and s. saprophyticus. if the identified s. aureus was found to be resistant to at least one agent in three or more antimicrobial categories, then the organism was considered as multidrug resistant (mdr) and if the identified s. aureus was found to be resistant to at least 1 agent in all but 2 or fewer antimicrobial categories, then it was considered as extremely drug resistant (xdr) [21]. after screening mdr, the identified s. aureus was then screened for methicillin resistant s. aureus (mrsa) using cefoxitin disc (30 mcg).the organisms resistant i.e. ≤21 mm zone of inhibition (zoi) towards the cefoxitin were confirmed as mrsa and those sensitive were confirmed as methicillin sensitive s. aureus (mssa) [25]. if the organism was found to be vancomycin resistant in disc diffusion method, it was further processed for the confirmation of vancomycin resistant s. aureus (vrsa) by using minimum inhibitory concentration (mic) method [31]. s. aureus atcc 25923 was used as control strain. d-test d-test was performed by using erythromycin disc (15 mcg) and clindamycin disc (2 mcg). the antibiotic discs were placed on a lawn cultured mha plate at 15 mm apart and was incubated at 37° c at 18-24 hrs [24]. the organisms that showed flattening zone of clindamycin adjacent to the erythromycin disc were considered as dtest positive (mlsbi resistant, i.e. inducible macrolidelincosamide-streptogramin b resistance). if the organism was found to be resistant towards both discs then, it was taken as constitutive mlsb (mlsbc) and if organism showed sensitive towards clindamycin but resistant towards erythromycin, then it was taken as d-test negative [24]. data analysis all the data was entered in statistical package for the social science (spss) version 16. most of the data was analysed by using spss version 16 (spss for windows, chicago, spss inc). the association between mdr and mrsa was determined by performing chi-square test analysed by spss version 16 whereas the correlation coefficient between d-test and mrsa was calculated by using statistical method, i.e. karl pearson's correlation coefficient. in the study, we used pearson's chi-square test to test whether mrsa influences the increase in mdr cases or not whereas karl pearson's correlation coefficient test was used to test whether there is significant correlation between mrsa and d-test. results growth pattern of culture and distribution of culture positive within the departments out of 270 pus/wound swab samples, 139 (51.48%) were found to be culture positive while remaining 131 (48.52%) showed no growth. opd showed highest positive culture 118 (85%) compared to that of the ipd 21 (15%). within the hospital department, highest growth was seen in the department of neonates icu (nicu) 100% (1/1) followed by surgical icu (sicu)75% (3/4), infant icu (iicu) 66.67% (4/6), opd 51.75% (118/228), surgical/burn ward (s/b ward) 50% (6/12), general ward (gw) 41.67% (5/12), pediatric icu (picu) 33.33% (1/3) and lowest in special ward (sp. ward) 25% (1/4) (figure 1). bacteriological profile of pus/wound swab in the study, 116 (83.5%) out of 139 were gram positive and 23 (16.5%) were gram negative. out of total 139 culture positive cases, s. aureus 105 (75.5%) was found to be common isolates followed by escherichia coli 7 (5.04%) and staphylococcus epidermidis 7 (5.04%); pseudomonas aeruginosa 6 (4.3%); unidentified organism 4 (2.9%); enterococcus 2 (1.45%), proteus mirabilis 2 (1.45%) and staphylococcus saprophyticus 2 (1.45%); salmonella typhi 1 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 11 (0.72%), klebsiella oxytoca 1 (0.72%), klebsiella pneumoniae 1 (0.72%) and citrobacter species 1 (0.72%) (table 1). figure 1. distribution of culture positive cases within the departments. table 1. bacteriological profile of pus/wound swab microorganism identified number percentage s. aureus 105 75.5% e. coli 7 5.04% citrobacter spp. 1 0.72% p. aeruginosa 6 4.3% enterococcus 2 1.45% s. saprophyticus 2 1.45% s. epidermidis 7 5.04% s. typhi 1 0.72% p. mirabilis 2 1.45% k. oxytoca 1 0.72% k. pneumoniae 1 0.72% unidentified 4 2.8% total 139 100.00% distribution of s. aureus according to the gender, age and within the hospital departments among 105 positive sample showing s. aureus, 51% (54) were found to be female patient and 49% (51/105) were male patient. the highest prevalence of s. aureus was found among age group 12-15 yrs. 87.5% (7/8) followed by the age group 1-3 yrs. 84.21% (32/38), age group 1 month-1 yrs. 76.92% (20/26), age group 6-12 yrs. 75% (24/32), 0-1 month 71.42% (10/14) and age group 4-6 yrs. 57.14% (12/21) (figure 2). most of the s. aureus was highly isolated from iicu department 100% (4/4), picu 100% (1/1), nicu 100% (1/1) followed by gw 80% (4/5), opd 78.81% (93/118), s/b ward 33.33% (2/6) and no s. aureus were isolated from sp. ward) 0% (0/1) and sicu 0% (0/3) (table 2). figure 2. distribution of s. aureus within the age group of the patient table 2. distribution of s. aureus within hospital departments departments s. aureus total number percentage opd 118 93 78.81% general ward 5 4 80% special ward 1 0 0% surgical / burn ward 6 2 33.33% infant icu 4 4 100% pediatric icu 1 1 100% surgical icu 3 0 0% neonates icu 1 1 100% total 139 105 75.5% table 3. antibiotic susceptible pattern of s. aureus (n= 105) antibiotic used antibiotic susceptible pattern resistant intermediate sensitive gentamycin 11(10%) 9(9%) 85(81%) ciprofloxacin 71(68%) 10(10%) 24(23%) chloramphenicol 4(4%) 4(4%) 97(92%) cotrimoxazole 26(25%) 8(8%) 71(68%) cefoxitin 58(55%) 0(0%) 47(45%) erythromycin 58(55%) 18(17%) 29(28%) clindamycin 28(27%) 2(2%) 75(71%) piperacillin 4(4%) 5(5%) 96(91%) meropenem 1(1%) 2(2%) 102(97%) azithromycin 54(51%) 7(7%) 44(42%) doxycycline 0(0%) 2(2%) 103(98%) linezolid 1(1%) 0(0%) 104(99%) vancomycin 9(9%) 0(0%) 96(91%) antibiotic susceptibility pattern of s. aureus and multidrug resistant (mdr) s. aureus while performing antibiotic susceptibility test (figure 3), out of 105 s. aureus, 104 (99%) were found to be sensitive towards linezolid followed by doxycycline 103 (98%), meropenem 102 (97%), chloramphenicol 97 (92%) and vancomycin 96 (91%). the organism was found to be 14 26 38 21 32 8 139 10 20 32 12 24 7 105 71.42% 76.92% 84.21% 57.14% 75% 87.50% 100.00% 0 50 100 150 200 250 300 total s. aureus number s. aureus percentage 228 12 4 12 6 3 4 1 118 5 1 6 4 1 3 1 51.75% 41.67% 25% 50% 66.67% 33.33% 75% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% growth percentage growth number departments total nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 12 resistant towards ciprofloxacin 71 (68%) followed by cefoxitin 58 (55%) and erythromycin 58 (55%) respectively (table 3). in our study, 50% (52) were found to be multidrug resistant. among multidrug resistant also, one strain was found to be resistant to all the antimicrobial agent used to be tested, i.e. extremely drug resistant (xdr). figure 3. antibiotic susceptibility test of staphylococcus aureus on mha. (va = vancomycin, azm = azithromycin, ptz = piperacillin, dox = doxycycline and lz = linezolid). distribution of mrsa among s. aureus positive sample and its antibiotic susceptibility pattern in the study, 55% (58) were found to be cefoxitin resistant showing methicillin resistant strains (mrsa) whereas 45% (47) were found to be cefoxitin sensitive showing methicillin sensitive strains (mssa). all the resistant strains were further tested for vancomycin susceptible test. table 4. antibiotic susceptibility pattern of mrsa (n=58) antibiotics resistant intermediate sensitive cotrimoxazole 18(31.04%) 3(5.17%) 37(63.79%) chloramphenicol 3(5.17%) 3(5.17%) 52(89.66%) gentamycin 6(10.35%) 7(12.07%) 45(77.58%) ciprofloxacin 51(87.93%) 6(10.35%) 1(1.72%) clindamycin 21(36.21%) 0(0%) 37(63.79%) erythromycin 38(65.53%) 8(13.79%) 12(20.68%) piperacillin 4(6.9%) 4(6.9%) 50(86.20%) meropenem 1(1.72%) 1(1.72%) 56(96.56%) azithromycin 43(74.13%) 5(8.62%) 10(17.25%) linezolid 1(1.72%) 0(0%) 57(98.28%) doxycycline 0(0%) 2(3.44%) 56(96.56%) vancomycin 9(15.52%) 49(84.48%) here, mrsa was found sensitive towards linezolid 98.28% (57) followed by doxycycline 96.56% (56), meropenem 96.56% (56), chloramphenicol 89.66% (52), piperacillin 86.20% (50), vancomycin 84.48% (49), gentamycin 77.58% (45), cotrimoxazole 63.79% (37) and clindamycin 63.79% (37). mrsa was found to be resistant towards ciprofloxacin 87.93% (51) followed by azithromycin 74.13% (43), erythromycin 65.53% (38) and was found to be zero resistant towards doxycycline. (table 4). vrsa and mic among isolated s. aureus, 9 were found to be resistant towards the vancomycin disc. while performing minimum inhibitory concentration test, all positive strains were found to be sensitive towards vancomycin in a very low concentration, i.e. 0.25 µg/ml and minimum bactericidal concentration was found to be 0.25 µg/ml. association between mdr and mrsa in the study, 44 (84.61%) mrsa were found to be mdr and 14 (26.42%) mrsa were found to be mdr negative. by analyzing the data of mdr and mrsa using chisquare test, the value was found to be chi-square (1, n=105) =35.958, p˂.01. therefore, mdr was found to be statistically significant associated with mrsa. d-test of s. aureus and correlation between dtest positive and mrsa in d-test (figure 4), out of total 105 s. aureus identified, 24 (22.86%) were found to be d-test positive, 21 (20.0%) were d-test negative, 31 (29.52%) were sensitive to both erythromycin and clindamycin and 29 (27.62%) were constitutive resistant (table 5). figure 4. double disk diffusion test (d-test) on mha medium showing sensitive (k) and positive result (l). cd = clindamycin and e = erythromycin) table 5. correlation between mrsa and d-test d-test positive d-test negative constitutive resistant sensitive total r value mrsa 18 (75%) 8(38.1%) 20 (69%) 12 (38.71%) 58 0.39 mssa 6 (25%) 13 (61.9%) 9 (31%) 19 (61.29%) 47 total 24 21 29 31 105 (100%) nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 13 here, 18 (75%) mrsa were d-test positive, 8 (38.1%) mrsa were d-test negative, 20 (69%) mrsa were constitutive resistant and 12 (38.71%) mrsa were sensitive as shown in table 5. the correlation coefficient (r) between d-test and mrsa was found to be 0.39 (r = .313, p˂.01), i.e. d-test and mrsa was found to be positive but lowly correlated with each other. discussion out of total sample, 139 (51.48%) showed growth and 131 (48.52%) showed no growth. the growth result was found nearly similar to the study conducted by hanumanthappa et al, where they found 56% growth rate [32]. the result was lower to the study conducted by rai et al [10] 58.6%; khan et al [33] 65.2% and patil et al [34] 86%. the lower growth might be due to difficult-togrow fastidious organisms, inappropriate methods of collection and transportation of specimens or the administration of antibiotics prior to specimen collection. among 139 positive growth result, 118 (85.65%) were found to be positive from out-patient department and 21 (14.4 %) from in-patient department. high prevalence of growth in opd might be due to increase in community acquired infection. higher positive growth in opd was also found in the study carried by kc et al [35] and found contrary to the study carried out by pant et al, where they found 63.1% from ipd and 56.2% from opd [36]. out of total growth 139, 116 (83.5%) were found to be gram-positive and 23 (16.5%) were found to be gram negative. the high prevalence of gram-positive organism might be due to the presence of gram-positive bacteria as a normal flora of the human body. the result was found to be similar to the research conducted by kc et al [35]; devi et al [37] and pant et al [36]. the result was found contrast to the study conducted by patil et al (2019)78% gram negative and 22% gram-positive bacteria [34]. the predominance of s. aureus (75.5%) in the study might be due to s. aureus being normal flora of skin, glands, nails, etc. and having various virulence factors. the result was seems to be related to the study conducted by sultana et al (2015) 40.45% s. aureus followed by e. coli 28.18% [38]; barakoti et al (2017) 41.45% s. aureus followed by e. coli 22.79% [39]; bankar et al (2018) 34.21% s. aureus followed by e. coli 23.02%[40] and shahi et al (2018)70.6% s. aureus [41]. however mahat et al [42] and patil et al [34] pseudomonas spp. as predominant organism. the infection in the age group 12-15 yrs. might be due to the various activities performed in the school, environment, involved in fight, their playmates and contact with various object. the children under 5 years are also prone to get pus/wound infection because of unintentional falls, burns, etc. and not only that these group also has low immune power to overcome any kind of infection, therefore it is likely to get infected. the outcomes were found to be contrast with the study conducted in 2017 by pokhrel et al, where they got higher prevalence of s. aureus in age group 1-3yrs [43]; rai et al in age less than 1 year [10] and pant et al in age group 15yrs [36]. the distribution of s. aureus among gender was found to be high in female patient 54 (51%) than the male patient 51 (49%). the finding resembled with the research conducted by muluye et al [20] and bhatt et al [44]. the result obtained from our study was contrast to the research conducted by shrestha et al [28] and garoy et al [45]. the high prevalence of s. aureus in icu departments might be due to the colonization of s. aureus from patient's own flora, transmission through staff hands, air, procedure of surgery, inanimate object, longer period of hospital stays, etc. similar study was carried out by bhatta et al. (2014) who had reported higher prevalence of s. aureus in hospital setting accounting [44]. s. aureus was found to be highly sensitive towards linezolid (99%) followed by doxycycline (98%). the outcome was found similar to the study conducted by nirmala et al [2] of 100% sensitive towards linezolid and vancomycin and by khan et al [33]. it was found to be a bit different from the research carried out in 2018 by tadesse et al [46] in which they found 100% sensitive towards ampicillin. from the study, out of 105 isolates, 52 (50%) were found to be mdr. among mdr also one strain was found to be resistant to all the antimicrobial agents to be tested (extremely drug-resistant). mdr cases may be due to accumulation of multiple genes, expression of genes that code for multidrug efflux pumps, extruding a wide range of drugs, mutational alteration of the target protein, enzymatic inactivation of drugs, etc. [47]. here, 44 (84.61%) mdr were found to be mrsa and 8 (15.39%) mdr were found be mssa. the increase in mdr in mrsa may be due to a distinctive feature of mrsa, i.e. their resistance to β-lactam antibiotics. therefore, once the s. aureus is resistant to methicillin, it may also show resistance towards other antibiotic classes like: aminoglycosides, macrolides, tetracycline, chloramphenicol and lincosamide. our result was lower in comparison to upreti et al [48] with 68.2% mdr, pahadi et al [49] with 86.41% were mdr; tadesse et al nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 14 [46] 82.3% mdr; whereas higher than he study conducted by kadariya et al [50] with 44.2% were mdr and mama et al with 27.8% were mdr [51]. the strong association between the mdr and mrsa was found (p˂.01) while performing pearson chi-square test. hence, we can say that the prevalence of mdr increases as the prevalence of mrsa increased. the data obtained from the research was found to be similar to the study conducted by joachim et al in which 21.3% were mdr, out of which 72.7% of mrsa strains were mdr showing statistically significant association between mrsa and mdr among s. aureus isolates (p=0.001) [52]. the prevalence of mrsa was found to be 58 (55%) and methicillin sensitive s. aureus (mssa) was found to be 47 (45%). the study resembles to the study carried out by devi et al (2017), where 50.79% were mrsa and 49.21% were mssa [37]. however, the study was in contradiction to the study carried by kayastha et al (2010) 8.92% mrsa [23]; ansari et al (2014) 43.1% mrsa [53]; jaiswal et al (2016) 72% mrsa [54] and adhikari et al (2017) 35.50% mrsa [55]. since our research was conducted from september 2017 to march 2018, the prevalence of mrsa seems to be increasing in nepal as well [56, 53, 55, 57, 48, 45, 41]. the development of resistance of s. aureus towards methicillin may be due to the acquisition of staphylococcal chromosome cassette mec (scc mec) in its chromosome, which carries a mec a gene facilitating resistance to methicillin via penicillin binding protein (pbp-2a). although the acquisition of the meca gene, the organism cannot exhibit resistant towards methicillin unless the gene is activated. mrsa was found to be sensitive towards linezolid 98.28% (57) followed by doxycycline 96.56% (56) and resistant towards ciprofloxacin 87.93% (51) followed by azithromycin 74.13%. similar sensitive pattern in mrsa was found in the study carried out by choudhury et al (2016) in which organism was found sensitive towards linezolid (99.3%), vancomycin (99.3%) and resistant towards cefuroxime (59.50%) [58]. in our study, vancomycin resistant was found to be 9% (9/105) from disc diffusion method but while performing the mic, s. aureus was found to be 100% sensitive towards vancomycin, i.e. 0.25 μg/ml and mbc was found to be 0.25 μg/ml. hence, isolated s. aureus was found to be 100% susceptibility towards vancomycin. therefore, we need to perform mic for the confirmation of vancomycin resistant strain. the cause of vancomycin resistance may be due to the activation of van a and van b gene. the finding was found to be similar to the research conducted by kshetry et al, where organism was found sensitive towards vancomycin while performing mic test [59] and study by bamigboye et al showed 1.4% vrsa but found to be van a and van b gene negative [25]. from the study, only 22.86% (24) were found to be d-test positive, 20% (21) were found to be d-test negative, 29.52% (31) were found to be susceptible to both erythromycin and clindamycin and 27.62% (29) were found to be constitutive resistant. the resistance of the erythromycin and clindamycin may be due to the resistance encoded in erythromycin methylase (erm) genes. the constitutive expression may be due to the organism being resistant to all macrolides, lincosamides and type b streptogramin antibiotics. the study resembled to the study carried out by mama et al [51] with 24.1% d-test positive, 1% d-test negative, 2% constitutive d-test and 60.85% sensitive towards erythromycin and clindamycin. in this study, d-test positive was also seen high in mrsa 75% (18/24) compare to the mssa 25% (6/24). similar result was obtained in research conducted by pal et al [60]. the correlation (r) between d-test and mrsa was found to be 0.39 which means d-test and mrsa are positively but lowly correlated, i.e. d-test cases may increase as increase in mrsa cases. the result obtained was contrast with the study carried out by gosh et al [61]. the increase in reported inducible clindamycin resistant shows the increase in prevalence of inducible clindamycin resistance along with constitutive resistant among the clinical isolates of s. aureus. hence, the screening of inducible clindamycin resistant should be done in every clinical laboratory. conclusion prevalence of wound infection was found to be high (51.48%) in our study. the growth rate was found high in opd patient than ipd. s. aureus was predominant organism followed by e. coli and s. epidermidis. the prevalence of s. aureus was seen high in the age group of 12-15 years. the cases were also seen high in the department of iicu, picu, and nicu. high prevalence of mrsa was observed in this study. the isolates were sensitive mainly towards linezolid, doxycycline, meropenem, and chloramphenicol, vancomycin. the organism was found highly resistant towards ciprofloxacin. 50% of isolates were found to be mdr. among mdr, one strain was found to be xdr. mdr was mainly found in mrsa than mssa strain. hence, all mrsa are considered as mdr. d-test positive cases was found higher in mrsa cases. since, inducible d-test has nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 15 been reported, it is necessary to screen the inducible clindamycin resistance before the prescription of the medication for the effective treatment of infection. vancomycin, linezolid, doxycycline, meropenem, and chloramphenicol were effective drug for s. aureus and mrsa. author's contribution bm conducted laboratory experiments, data analysis, interpretation and manuscript writing; stk designed the research conception, reviewed the manuscript; rm designed the research, contributed in data analysis, manuscript writing, reviewing and editing. all authors read and approved the final manuscript. competing interests we have read nepal journal of biotechnology policy on declaration of competing interest and declare that we have no competing interests. funding the author(s) declared that no grants were involved in supporting this work. acknowledgements we are very beholden for the support provided by international friendship children's hospital and the st. xavier's college, kathmandu, nepal. ethical approval and consent this research was approved by nepal health research council (nhrc), kathmandu, nepal (ref. no.-2610), international friendship children's hospital, maharajgunj, nepal and the st. xavier's college, kathmandu, nepal. informed consent was obtained from parents of 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prevalence of methicillin resistant staphylococcus aureus, multidrug resistant and extended spectrum beta-lactamase producing gram negative bacilli causing wound infections at a tertiary care hospital of nepal. antimicrob resist infect control. 2018 oct 8;7:121. doi: 10.1186/s13756-018-0408-z. 49. pahadi p, shrestha u, adhikari n, shah p, amatya r. growing resistance to vancomycin among methicillin resistant staphylococcus aureus isolates from different clinical samples. journal of nepal medical association. 2014 dec 31;52(196):977981.https://doi.org/10.31729/jnma.2797 50. kadariya j, thapaliya d, bhatta s, mahatara rl, bempah s, dhakal n, et al. multidrug-resistant staphylococcus aureus colonization in healthy adults is more common in bhutanese refugees in nepal than those resettled in ohio. biomed res int. 2019 jul 1;2019:5739247. https://doi: 10.1155/2019/5739247 51. mama m, aklilu a, misgna k, tadesse m, alemayehu e. methicillinand inducible clindamycin-resistant staphylococcus 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https://doi.org/10.3126/jpn.v4i7.10297 https://doi.org/10.31729/jnma.2797 https://doi.org/10.1155/2019/2965490 https://doi.org/10.1186/s13104-017-2668-8 https://doi.org/10.1186/1471-2334-14-157 https://www.researchgate.net/publication/310331234 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):8 1 7 maharjan et al. ©njb, bsn 17 vancomycin for staphylococcus aureus isolated from pus/wound swab samples of the patients attending a tertiary care hospital in kathmandu, nepal. can j infect dis med microbiol. 2017 jan 5;2017:2191532. https://doi.org/10.1155/2017/2191532 56. khanal lk, jha bk. prevalence of methicillin resistant staphylococcus aureus (mrsa) among skin infection cases at a hospital in chitwan, nepal. nepal medical college journal: nmcj. 2010 dec;12(4):224-228. pmid: 21744763 57. neopane p, nepal hp, shrestha r, uehara o, abiko y. in vitro biofilm formation by staphylococcus aureus isolated from wounds of hospital-admitted patients and their association with antimicrobial resistance. int j gen med. 2018 jan 18;11:25-32. doi: 10.2147/ijgm.s153268. 58. choudhury d, chakravarty p. prevalence and antimicrobial susceptibility pattern of methicillin resistant staphylococcus aureusin silchar medical college and hospital, assam, india. int j basic clinpharmacol. 2016 oct;5(5): 2174-2177. https://dx.doi.org/10.18203/2319-2003.ijbcp20163257 59. kshetry ao, pant nd, bhandari r, khatri s, shrestha kl, upadhaya sk, et al. minimum inhibitory concentration of vancomycin to methicillin resistant staphylococcus aureus isolated from different clinical samples at a tertiary care hospital in nepal. antimicrobial resistance and infection control. 2016;5:27. https://doi.org/10.1186/s13756-016-0126-3 60. pal n, sharma b, sharma r, vyas l. detection of inducible clindamycin resistance among staphylococcal isolates from different clinical specimens in western india. j postgrad med. 2010;56(3):182-185. http://www.jpgmonline.com/text.asp?2010/ 56/3/182/68637 61. ghosh s, banerjee m. methicillin resistance & inducible clindamycin resistance in staphylococcus aureus. the indian j med research. 2016 mar;143(3):362-364. doi: 10.4103/0971-5916.182628. https://doi.org/10.1155/2017/2191532 https://dx.doi.org/10.18203/2319-2003.ijbcp20163257 https://doi.org/10.1186/s13756-016-0126-3 nepal j biotechnol. 2 0 2 1 d e c ; 9 (2): 7-13 research article doi: https://www.doi.org/10.54796/njb.v9i2.41908 ©njb, bsn 7 microbial and physico-chemical quality assessment of rivers of kathmandu valley santosh poudel1 , akash paudyal1, bishnu prasad sharma1, kamana sharma1, yubaraj baral1, shailaja adhikari2, manju shree shakya hada1 1department of microbiology, tri-chandra multiple campus, tribhuvan university, kathmandu, nepal 2kathmandu upatyaka khanepani ltd. (kukl), kathmandu, nepal received: 05th jun 2021; revised: 13th dec 2021; accepted: 16th dec 2021; published online: 31st dec 2021 abstract water quality refers to the chemical, physical, biological characteristics of water. it is a measure of condition of water relative to requirement of one or more biotic species and or to any human need or purpose. the main objective of the study is to detect the physio-chemical and microbiological parameters of water sample from the bagmati river and its tributaries of kathmandu valley along with antibiotic susceptibility. in physico-chemical parameters, turbidity, temperature, ph, electrical conductivity, dissolved oxygen, biological oxygen demand, ammonia, alkalinity, hardness, chloride, phosphate, iron, nitrate, total dissolved solids, and color were analyzed. iron and turbidity was found to be above the world health organization and nepal standard guideline in all the samples (100%), while ammonia was found to be above the who guideline in 10(90%) samples. among 11 samples, 10(90%) showed a low dissolved oxygen level. most probable number method was followed for counting total load of coliform and fecal coliform. escherichia coli was isolated from the sample and subjected to antibiotic susceptibility. coliform was detected in all the samples and e. coli was identified as highly resistant towards gentamicin (81.8%) and sensitive towards chloramphenicol (81.8%). high value of ammonia, turbidity and low value of dissolved oxygen in the lower belts of river was due to large inputs of wastewater and organic loads caused by anthropogenic activities. high value of coliform in all the samples indicates bacterial contamination in river water. the comparative study for the water quality variables in the urban areas showed that the main rivers and its tributaries were equally polluted. keywords: e. coli, bagmati, physico-chemical parameters, antibiotic susceptibility, most probable number method corresponding author, email: poudelsantosh550@gmail.com introduction water is the main constituent of earth's hydrosphere and the fluids of all known living organisms. it is vital for all known forms of life [1]. among the type of surface water, river is the most accessible and widely available source of water to human beings. river water has various uses like irrigation, drinking, and water hydropower, directly related to people. river water has an economic backbone in the upliftment of the country [2]. bagmati is one, with religion and geographical importance. various important religious shrines like gokarneshwor, pashupatinath, guhyeshwari, teku dovan, and others lie on the river's bank [3]. the bagmati river is considered the source of nepalese civilization and urbanization. in contrast to the various large snowfed rivers of the himalayas, it is a river originating from baghdwar and shivapuri hill at an altitude of 2650m. bagmati river is the principal river of the kathmandu valley river system. it drains the entire valley with its seven tributariesbishnumati river, manohara river, dhobi river, nakkhu river, balkhu river, hanumante river, and tukucha river [4]. the quality of the bagmati river and its tributaries have been rapidly degrading. it contains large amounts of untreated sewage, and high levels of pollution of the river exist due primarily to the region's large population. many residents of the kathmandu valley empty personal garbage and waste into the river [5]. in kathmandu, urbanization and industrialization contribute to deterioration in water quality with regional consequences for the aquatic system and health of downstream sub-basin user groups. sewer lines for domestic and industrial wastewater have been connected to the river [6]. these activities are responsible for serious pollution and the production of foul odors near the riverside. river system water entering the core urban area is visibly black with filth stinks badly. in particular, the hanumante river, dhobi river, tukucha river, and bishnumati river are the most polluted [7]. a few kilometers of the uppermost section (high mountain with a catchment area of 17 km2) is only suitable for drinking water supply [8]. the remaining sections are not nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0001-9327-8850 mailto:poudelsantosh550@gmail.com nepal j biotechnol. 2021dec;9 (2): 7-13 poudel et al. ©njb, bsn 8 used for potable purposes due to greater water quality deterioration [9]. although many wastewater treatments plants have been constructed in the kathmandu valley, only one of the guheshwori wastewater treatment plant is currently functional. various restoration project has been ongoing within kathmandu valley focused on improving water quality and establishing minimum flow requirements [10]. assessment of the physico-chemical and microbiological parameters of the river water indicates the level of pollution and provides progression to understand its effect on the aquatic life as well as on the human population [11]. water is the principal vehicle for the transmission of a wide range of communicable diseases. from the microbiological view, the presence of total coliform and fecal coliform shows then water quality. coliform bacteria e. coli, which is a medically important bacteria causing many significant illnesses like bacteremia, cholangitis, uti, travelers' diarrhea, neonatal meningitis, pneumonia [12,13]. in addition, other bacteria like salmonella, shigella, pseudomonas, proteus, and vibrio in drinking water are major causes of water-borne diseases [14]. various antibiotics are used for the treatment of the diseases caused by e. coli. in recent years the number of antibiotic-resistant strains of e. coli is increasing by the haphazard use of antibiotics [15]. antimicrobial resistance is a global public health concern contributing to increased morbidity and mortality, particularly in lowincome countries. studies on commensal bacteria are essential as they reflect the state of antimicrobial susceptibility patterns in populations. overpopulation of kathmandu valley and inappropriate use of antimicrobials signal significant rates of resistance among flora circulating within the community. increased use of antibiotics in agriculture, domestic, livestock, and the hospital are likely to develop resistance by e. coli strains. this study was carried out to analyze different physicochemical parameters and microbiological parameters of the bagmati river and its tributaries. materials and methods sampling site and sample a total of 11 samples were tested in the study. water samples were collected from four different bagmati river sites (sundarijal, before guheshwori treatment plantgaurighat, after guheshwori treatment plant-gaurighat and chovar) and seven sites of its tributaries in kathmandu valley (bishnumati river-teku dhovan, balkhu river-balkhu, dhobi river-baneshwor, nakkhu river-nakkhu, tukuchu river-tripureshwor, mahohara river-jadibuti, hanumante river-jadibuti). the sample was collected in a sterile bottle of 500 ml capacity for microbiological analysis and a sample bottle of 500 ml for physico-chemical analysis. the bottles were labeled with time, place, and date of sample collection and transported to the laboratory as soon as possible on the icebox. for dissolved oxygen (do) and biological oxygen demand (bod), two bod bottles each of 300 ml were taken at the site, processed at the lab. the study was conducted from november 2019 to january 2020 in kathmandu upatyaka khanepani limited (kukl) laboratory, mahankal chaur, kathmandu. sample analysis analysis of physico-chemical parameters the standard method for examining water and wastewater [16] was followed to analyze the physicochemical parameters of water as temperature (by mercury thermometer), ph (by digital ph meter), e.c. (by e.c. meter), turbidity (by turbid-meter), color and ammonia (by colorimeter), iron, phosphate and nitrate (by uv-visible spectrophotometer), chloride, alkalinity, and hardness (by titration) and do and bod (by winkler’s method). analysis of microbiological parameters microbiological parameters analysis was carried out following standard methods [16]. enumeration of total coliform count and fecal coliform were done by most probable number (mpn) method. isolation of e.coli was done by enrichment in buffered peptone water and cultured in emb (eosin methylene blue)[17]. the colony was identified and confirmed by following respective biochemical characteristics[18] and subjected to antibiotic susceptibility using kirby bauer disk diffusion method following clsi guidelines[19]. the antibiotics used were ampicillin (10 µg), gentamicin (10 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), ceftriaxone (5 µg) and (chloramphenicol (30 µg). results physical parameters assessment temperature of sample ranges from 90c of sundarijal (sl), chobar (chob), and 16.50c of hanumante (han) and guheshwori before treatment (gbt). color was observed highest in tukucha (tuk) i.e., 40hu and lowest in sundarijal (sl) i.e., 2.5hu. only the water sample from sundarijal was clear while all other samples appeared turbid and hazy. sl sample was found to have lowest turbidity i.e., 2.7 ntu whereas mahohara (man) sample nepal j biotechnol. 2021dec;9 (2): 7-13 poudel et al. ©njb, bsn 9 had the highest i.e., 759.32. only sl sample follows who and ndwqs guidelines (≤5 ntu). electrical conductivity of samples varied from sl 49.2 µs/cm being lowest and bal 1109µs/cm being highest. out of 11 samples hanu, tuk, bal donot follow who guidelines with value (1039, 1090, 1109) µs/cm respectively. value of tds was highest in tuk i.e. (371 mg/l) and lowest in sl (9 mg/l). all tds value of samples lies within the guideline of who (1000 mg/l). chemical parameter assessment ph of the sample manohara was found to be lowest i.e., 7.22 and sample dhobi(dhobi) was highest i.e., 8.65 which doesn’t follow who guidelines. water in most of the river samples were slightly alkaline. dissolved oxygen in the sample sl has highest i.e., 12.4 mg/l while bishnumati has lowest i.e.,0.77 mg/l. in the study performed bod of the sample bishnumati has highest i.e., 354 mg/l and sl has lowest 8.9mg/l. ammonia of the sample sundarjal being lowest i.e., 0.2mg/l lies within the who guidelines while other sample don’t follow who guidelines. sample balkhu has highest value i.e., 90 mg/l. highest value of total hardness was obtained from the sample balkhu i.e., 344 mg/l and lowest value from sundarijal i.e,.12 mg/l. all values were within the ndwqs guidelines (≤500 mg/l). chloride value was highest in tukucha i.e., 149.76mg/l and lowest in sundarjal i.e., 1.92 mg/l. in the study performed phosphate value was highest in tukucha i.e., 3.5 mg/l and lowest in sundarijal i.e., 0.0 mg/l. all the values of iron don’t follow the guidelines of who and ndwqs (≤0.3). iron value was highest in sample table 1. physical parameters of water samples. (* temperature during measurement) p h y s ic a l p a ra m e te r s u n d a ri j a l (s l ) g u h e s w o ri b e fo re t re a tm e n t (g b t ) g u h e s w o ri a ft e r t re a tm e n t (g a t ) m a n o h a ra r iv e r (m a n ) h a n u m a n te r iv e r (h a n u ) t u k u c h a r iv e r (t u k ) d h o b i r iv e r (d h o b i) n a k k h u r iv e r (n a k ) b a lk h u r iv e r (b a l ) b is h n u m a ti (b m ) c h o b a r (c h o b ) a p p e a ra n c e c le a r t u rb id t u rb id t u rb id t u rb id t u rb id t u rb id t u rb id t u rb id t u rb id t u rb id temperature (°c) 9° 19° 16.5° 12° 16.5° 16° 13° 11° 13° 15° 9° color (hu) 2.5 25 30 35 25 40 20 35 25 15 25 turbidity (ntu) 2.7 52.59 295.12 759.32 122.66 179.78 186.71 261.33 112.03 305.80 15.57 e.c.* (µs/cm) 49.2 (9.1°) 258.7 (13°) 167.0 (18.6°) 567 (13.0°) 1039 (16°) 1090 (14°) 820 (13.9°) 701 (14.5°) 1109 (12.5°) 986 (14°) 983 (11.6°) tds 9 89 57 143 367 371 300 162 354 349 268 table 2. chemical parameters of water samples (* temperature during measurement) p h y s io c h e m ic a l p a ra m e te r s u n d a ri ja l (s l ) g u h e s w o ri b e fo re t re a tm e n t (g b t ) g u h e s w o ri a ft e r t re a tm e n t (g a t ) m a n o h a ra r iv e r (m a n ) h a n u m a n te r iv e r (h a n u ) t u k u c h a r iv e r (t u k ) d h o b i r iv e r (d h o b i) n a k k h u r iv e r (n a k ) b a lk h u r iv e r (b a l ) b is h n u m a ti (b m ) c h o b a r (c h o b ) ph* 7.7 (9.1°) 7.41 (13°) 7.72 (18.6°) 7.22 (13°) 7.45 (16°) 7.3 (14°) 8.65 (13.9°) 8.29 (14.6°) 7.61 (13°) 7.27 (14°) 7.56 (11°) do (mg/l) 12.4 3.02 4.87 2.24 1.05 1.10 1.26 2.8 1.4 0.77 1.59 bod (mg/l) 8.9 66 48 160 279 261 242 187 291 354 213 ammonia (mg/l) 0.2 15 12 24 35 80 40 24 90 40 32.5 alkalinity (mg/l) 24 108 88 88 264 288 248 160 404 296 294 hardness (mg/l) 12 128 56 80 264 152 312 148 344 176 178 chloride (mg/l) 1.92 26.88 19.2 65.28 103.68 149.76 69.12 46.08 111.36 80.64 88.46 phosphate (mg/l) 0.0 0.4 0.2 1.0 2.0 3.5 2.5 0.5 2.0 2.5 2.5 iron (mg/l) 0.4 1.6 6 23.2 8.8 4.4 6.4 3.2 6.4 15.2 4.8 nitrate (mg/l) 0.2 0.9 2.1 3.7 5.7 5.7 4.8 1.0 4.6 5.3 2.1 nepal j biotechnol. 2021dec;9 (2): 7-13 poudel et al. ©njb, bsn 10 manohara i.e., 23.2 and lowest in sample sl i.e., 0.4 mg/l. hanumante and tukucha sample have highest nitrate value i.e., 5.7 mg/l while sundarijal sample has lowest i.e., 0.2 mg/l. table 3. total coliform count by mpn method sample number of tubes giving positive reactions mpn index (cfu/100ml) 1 of 50ml each 5 of 10ml each 5 of 1ml each sundarijal 1 5 5 >180 guheshwori before treatment 1 5 5 >180 guheshwori before treatment 1 5 5 >180 manohara 1 5 5 >180 hanumante 1 5 5 >180 tukucha 1 5 5 >180 dhobi 1 5 5 >180 nakkhu 1 5 5 >180 balkhu 1 5 5 >180 bishnumati 1 5 5 >180 chobar 1 5 5 >180 microbiological analysis all 11 sample (100%) contain coliform above 180 per 100 ml. antibiotic susceptibility table 4. susceptibility patterns of e. coli isolates. r= resistant, i= intermediate, s= sensitive a m p ic il li n 1 0 µ g g e n ta m ic in 1 0 µ g c o tr im o x a z o le 2 5 µ g c ip ro fl o x a ci n 5 µ g c e ft ri a x o n e 3 0 µ g c h lo ra m p h e n ic o l 3 0 µ g sundarijal r r r s s s gbt s i s s s s gat r i s s s s man r r s r i s hanu r r s i r s tuk r r r r i i dhobi r r s s s s nak s r s s s s bal s r s s i s bm r r r r i s chob r r r i i r table 4 showed that e.coli isolated from sites tuk, bm and chob were resistant against more than 50 percent of the antibiotics testes while isolates from sites gbt, gat, dhobi, nak and bal were susceptible to more than 50 percent of the antibiotics tested. figure 1. green metallic sheen colony growth on m-endo agar (e. coli). from 11 isolates, 6 (54.4%) were found multi drug resistant (mdr) i.e., resistant to 3 or more class of antibiotics. most of the mdr isolates (45.5%) were resistant to the gentamicin in this study. figure 2. antibiotic susceptibility by disk diffusion method discussion turbidity in this study was recorded in range of 2.7ntu in sundarijal to 759.32ntu in manohara. the values of turbidity except sundarijal were much higher compared table 5. antibiotic susceptibility of e. coli (n=11) sensitive intermediate resistance ampicillin (10 µg) 27.27% (3) 0 72.72% (8) gentamicin (10 µg) 0 18.18% (2) 81.82% (9) cotrimoxazole (25 µg) 63.6% (7) 0 36.4% (4) ciprofloxacin (5 µg) 54.55% (6) 18.2% (2) 27.27% (3) ceftriaxone (30 µg) 45.5% (5) 45.5% (5) 9.1% (1) chloramphenicol (30 µg) 81.9% (9) 9.1% (1) 9.1% (1) nepal j biotechnol. 2021dec;9 (2): 7-13 poudel et al. ©njb, bsn 11 to the previous study conducted i.e. 14.2ntu before mixing of tributaries of kathmandu valley[20]. discharge of industrial effluents such as alums and chemicals might contribute to the high value of turbidity. tributaries of bagmati are major contributors to turbidness in bagmati river. the ph value was found within the range of who standards and ndwqs guidelines value except the sample of dhobi river, which is found to be 8.65. it is higher than the value reported in the previous study be 8.20 [21]. high value of ph may be due to the increasing waste discharge and industrial effluent along with microbial decomposition of organic matters. temperature ranged from 90c to190c, while previous study conducted [4] during summer season recorded a maximum of 200 c. since the study was conducted in winter explains the low temperature recorded. electrical conductivity of the samples hanumante, tukucha, and balkhu were 1039 µs/cm, 1090µs/cm, and 1109µs/cm respectively that do not lie within the guidelines of who standard 0-1000 µs/cm. in the previous study [21], highest value was 889.59 µs/cm. higher value indicates the presence of a higher amount of dissolved ions as well as plant nutrients in the water which might be due to the wash off of the fertilizer from agricultural lands. alkalinity refers to the capability of water to neutralize acid. in natural water, there are many salts of weak acids such as silicates, borate-causing alkalinity. in this study, highest alkalinity was of balkhu 404mg/l. the alkalinity observed in this study is higher than the result of previous study 360 mg/l [20]. samples were collected during winter season, the flow and level of water were low. higher value of alkalinity might be due to increase concentration of natural soil and minerals. the ammonia concentration for sundarijal was only 0.2 mg/l which was limit for surface water recommended by who [22]. but the concentration of ammonia for other samples was a lot higher than the recommended value which might be due to more amount of municipal waste dumping in the river of kathmandu valley. highest ammonia concentration was from balkhu sample, 90 mg/l, and tukucha sample, 80 mg/l. similar results reported by [20], also had a higher value of ammonia in balkhu river indicating that it was more polluted than other rivers. the highest value of chloride recorded was 149.76mg/l in tukucha followed by balkhu 111.36mg/l, indicating most contaminated about chloride content. previous study [23] , presented a similar result being teku and sundarighat as most contaminated sites[23] considering tukucha river and balkhu river joins on bagmati in teku and sundarighat respectively. high chloride concentration in river is toxic to aquatic life and also increases the potential corrosivity of water[24]. nitrate was recorded highest in tukucha and hanumante i.e.5.7mg/l which is higher than the data previously recorded [23], 3.95mg/l. both studies showed similarity in the increment of nitrate content from upstream to downstream[23]. excess levels of nitrates can be considered to be a contaminant of river waters. most sources of excess nitrates come from human activity. the source of excess nitrates can usually be traced to agricultural activities, human wastes, or industrial pollution. rainwater can wash nitrates in the fertilizer into streams and rivers[25]. highest phosphate value was 3.5 mg/l in tukucha. sundarijal sample phosphate value was 0.0 mg/l and that of chobar 2.5 mg/l which are low compared to that of the data of similar research [23], 0.24 mg/l at sundarijal and 12.3 mg/l at chobar [26]. significant increase was seen in the level of phosphate as the river enters an urban core area, which is from manmade sources such as septic systems, fertilizer runoff, and improperly treated wastewater. at sundarijal, bod level was found to be within the guideline by bbwmsip. however, the level of bod increases with the increase in the organic waste in the river. bod indicated the pollution of organic waste resulting low level of dissolved oxygen. highest bod was observed in bishnumati. previous study [26] showed a low level of bod in sundarijal but an increment in the level of bod towards downstream. dissolved oxygen concentrations in the core urban areas were significantly lower than that of sundarijal showing that the water is anoxic. as the river flows downstream, the dissolved oxygen gets more reduced. lowest concentration was observed in bishnumati of 0.77 mg/l. bacterial decomposition of incorporated organic matter was most likely for low level of dissolved oxygen similar research [23,26] also observed low level of do in bagmati river. in this study, coliform was present in the entire river water sample. the presence of a high amount of coliform might be due to the fact; samples were taken from the river before it was subjected to a treatment of disinfection. previous studies on the river water from kathmandu valley showed the presence of various viruses [27], human enteric viruses, protozoa, and indicators of pathogens[28] except in the sample of nepal j biotechnol. 2021dec;9 (2): 7-13 poudel et al. ©njb, bsn 12 sundarijal. the water is contaminated by various anthropogenic activities as it flows downstream. all 11 samples were positive for e. coli. identification of e. coli during the study indicates the fecal contamination of human origin in bagmati river and its tributaries. although there was a low detection rate of pathogen in sundarijal during a previous study[28] but the detection of e. coli from the sundarijal suggest the contamination of water even upstream of the bagmati river, which cannot be neglected. multiple drug-resistant (mdr) was 54.4% in this study. other researchers have reported an increasing pattern of e. coli isolates against common antibiotics in nepal[29], [30]. it is observed from the study that gentamicin and ampicillin were most resisted by 81.82% and 72.72% respectively by the e. coli isolates, similar results of high resistance against β-lactamase(ampicillin)[31] and aminoglycoside (gentamicin) [30] were reported. among the antibiotics, chloramphenicol was more sensitive to the isolates. a high rate of effectiveness by the chloramphenicol against mdr isolates of e. coli was reported in previous studies[31, 32]. conclusion the study showed the variation in water quality of the bagmati river and its tributaries. the quality of water is worse in the urban core areas compared to that of upstream. the presence of fecal coliform in river water is the prime indication of a possible source of an outbreak for waterborne diseases and water is not suitable for drinking purposes without proper treatment. high resistance towards some antibiotics shows the threat to the exposure of antibiotics resistance bacterial strains by the population of kathmandu valley. immediate action is needed to prevent further deterioration of the river and amplify efforts to slow the emergence and spread of resistance. author’s contribution the development of concept, preliminary work and laboratory analysis, was done by sp, ap, bps, ks and yb under the guidance of mssh and sa. all authors read and approved the final manuscript. competing interests the authors declare that they have no conflicts of interest. funding this study was not funded by any agency or institution. acknowledgments all authors are grateful to the faculty and laboratory staff of microbiology department of tri-chandra multiple campus for their continuous support in this research work, and special mention to the kathmandu upatyaka khanepani limited(kukl) and all the staffs for providing laboratory and support. ethical approval and consent this study was carried out with the approval from the concerned authorities. data availability the data can be made available upon request. reference 1. 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[kim ks. current concepts on the pathogenesis of escherichia coli meningitis: implications for therapy and prevention. current opinion in infectious diseases. 2012 jun 1;25(3):2738.https://doi.org/10.1097/qco.0b013e3283521eb0 13. kaper jb, nataro jp, mobley hl. pathogenic escherichia coli. nature reviews microbiology. 2004 feb;2(2):12340.https://doi.org/10.1038/nrmicro818 14. lee sh, kang hj, park hd. influence of influent wastewater communities on temporal variation of activated sludge communities. water research. 2015 apr 15;73:132-44.. https://doi.org/10.1016/j.watres.2015.01.014 nepal j biotechnol. 2021dec;9 (2): 7-13 poudel et al. ©njb, bsn 13 15. fair rj, tor y. antibiotics and bacterial resistance in the 21st century. perspectives in medicinal chemistry. 2014 jan;6:pmcs14459. https://doi.org/10.4137/pmc.s14459 16. american public health association, american water works association, water pollution control federation, water environment federation. standard methods for the examination of water and wastewater. american public health association.; 1912. 17. singh p, prakash a. isolation of escherichia coli, staphylococcus aureus and listeria monocytogenes from milk products sold under market conditions at agra region. acta agriculturae slovenica. 2008 nov;92(1):83-8. 18. cheesbrough m. district laboratory practice in tropical countries, second edition, dist. lab. pract. trop. countries, second ed., pp. 1– 434, jan. 2006. https://doi.org/10.1017 /cbo9780511543470 19. clsi c. performance standards for antimicrobial susceptibility testing. clinical lab standards institute. 2016;35(3):16-38. 20. adhikari mp, neupane mr, kafle m. physico-chemical parameterization and determination of effect of tributaries on enhancement of pollutants in bagmati river. journal of nepal chemical society. 2019 dec 31;40:3643.https://doi.org/10.3126/jncs.v40i0.27276 21. kannel pr, lee s, lee ys, kanel sr, pelletier gj. application of automated qual2kw for water quality modeling and management in the bagmati river, nepal. ecological modelling. 2007 apr 10;202(3-4):503-17.https://doi.org/10.1016 /j.ecolmodel.2006.12.033 22. edition f. guidelines for drinking-water quality. who chronicle. 2011;38(4):104-8. 23. gautam r, shrestha jk, shrestha gk. assessment of river water intrusion at the periphery of bagmati river in kathmandu valley. nepal journal of science and technology. 2013 oct 14;14(1):13746.https://doi.org/10.3126/njst.v14i1.8934 24. water resources. chloride, salinity, and dissolved solids | u.s. geological survey. u.s. geological survey [internet] mar 1 2019 available from: https://www.usgs.gov/mission-areas/waterresources/science/ chloride-salinity-and-dissolved-solids (accessed dec. 11, 2020). 25. us epa. the sources and solutions: agriculture [internet] available from: https://www.epa.gov/nutrientpollution /sources-and-solutions-agriculture (accessed dec. 11, 2020). 26. shrestha n, lamsal a, regmi rk, mishra bk. current status of water environment in kathmandu valley, nepal. 27. haramoto e, yamada k, nishida k. prevalence of protozoa, viruses, coliphages and indicator bacteria in groundwater and river water in the kathmandu valley, nepal. transactions of the royal society of tropical medicine and hygiene. 2011 dec 1;105(12):711-6.https://doi.org/10.1016/j.trstmh.2011.08.004 28. tandukar s, sherchand jb, bhandari d, sherchan sp, malla b, ghaju shrestha r, haramoto e. presence of human enteric viruses, protozoa, and indicators of pathogens in the bagmati river, nepal. pathogens. 2018 jun;7(2):38. https://doi.org/10.3390 /pathogens7020038 29. singh sd, madhup sk. clinical profile and antibiotics sensitivity in childhood urinary tract infection at dhulikhel hospital. kathmandu university medical journal. 2013;11(4):31924.https://doi.org/10.3126/kumj.v11i4.12541 30. parajuli np, maharjan p, parajuli h, joshi g, paudel d, sayami s, khanal pr. high rates of multidrug resistance among uropathogenic escherichia coli in children and analyses of esbl producers from nepal. antimicrobial resistance & infection control. 2017 dec;6(1):1-7.https://doi.org/10.1186/s13756-0160168-6 31. ansari s, nepal hp, gautam r, shrestha s, neopane p, gurung g, chapagain ml. community acquired multi-drug resistant clinical isolates of escherichia coli in a tertiary care center of nepal. antimicrobial resistance and infection control. 2015 dec;4(1):18.https://doi.org/10.1186/s13756-015-0059-2 32. ast as. physicochemical and bacteriological analysis of bagmati river in kathmandu valley. annals of applied bio-sciences. 2018;5(3). https://doi.org/10.21276/aabs.2213 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 96-102 doi: https://doi.org/10.3126/njb.v7i1.26949 ©njb, biotechnology society of nepal 96 nepjol.info/index.php/njb. review article infectious sources of histoplasmosis and molecular techniques for its identification sudip bhandari1, binod rayamajhee2, laxmi dhungel1, sami poudel1, bhagwati gaire1, sunil shrestha1, niranjan parajuli3* 1department of biotechnology, national college, tribhuvan university, kathmandu, nepal 2department of infectious diseases and immunology, kathmandu research institute for biological sciences (kribs), lalitpur, nepal 3central department of chemistry, tribhuvan university, kathmandu, nepal abstract histoplasmosis, a fungal infection caused by histoplasma capsulatum (h. capsulatum), acquired from contaminated soil with droppings of chicken or birds and found to be distributed in many parts of the world. the prevalence of histoplasmosis has not well studied in nepal. the common symptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia and weight loss. most of the infections associated with histoplasmosis are asymptomatic. people with compromised immune systems such as hiv/aids (plwha), cancer, and organ transplant recipients are at risk of developing this disease. in this review, we have summarised the current status of histoplasmosis in nepal and molecular techniques available for its identification. to date, the significant outbreak is not reported in nepal, but the risk of infection for the vulnerable population cannot be undermined. appropriate preventive measures and treatment on time can reduce the burden of this fungal disease. further, this review is also focused on molecular identification of h. capsulatum. hence, careful considerations by concerned stakeholders for national surveillance programs and the treatment of patients on time after proper diagnosis is highly recommended. keywords: histoplasmosis, asymptomatic, vulnerable, treatment *corresponding author: email: nparajuli@cdctu.edu.np introduction histoplasmosis is acquired by inhalation of spores of h. capsulatum, which is usually found in the soil contaminated by bird droppings, which reveals the higher risk of disease in farmers, gardeners, poultry keepers, construction workers, pest control workers and in some instances travelers visiting caves and tunnels[1]. histoplasma is a dimorphic fungus, which produces mycelial form in the soil environment and converts to the yeast form at host body temperature (37 °c), and it usually does not develop the symptomatic infection. so people need not concern about the infection [2]. however, for immune-compromised people, the fungus can result in severe infection. very young children and elderly people who have a weak immune system; are more likely to get histoplasmosis disease [3]. the development of infection and the dissemination of h. capsulatum are initially dependent on the condition of the host body[4]. the majority of infected persons could have either no symptoms or a very less mild sickness, which is hard to recognize as the cause of histoplasmosis [5]. the clinical symptoms generally occur only in a small number (around 1%) of the population when exposed with h. capsulatum spores [6]. persons who are usually immuno-compromised and are unable to develop effective cell-mediated response are likely to develop symptomatic disease during the period of acute dissemination in body [5], which includes infant child, patients with hiv/aids, organ transplant recipients, and those with hematologic deficiencies, and also those patients, who are on corticosteroids drugs treatment [7]. an individual is under the risk of developing symptoms even after years leaving the endemic vicinity of histoplasmosis if the man or woman was in an immuno-suppressive situation at that time [8]. histoplasmosis is of four major types: pulmonary histoplasmosis, progressive disseminated histoplasmosis, nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 96-102 bhandari et al. 2019 ©njb, biotechnology society of nepal 97 nepjol.info/index.php/njb. cutaneous histoplasmosis, and african histoplasmosis [9, 10]. pathogenesis of histoplasmosis the fungal infection starts after the inhalation of spores produced by the mycelial form of h. capsulatum and the spores deposited inside the alveoli of the lungs [11]. after some time, the spores start to germinate inside the alveoli at normal body temperature to become dimorphic form. then pulmonary macrophages engulf that yeast form of dimorphic fungus [12]. after the engulfment, the yeasts become a parasite and multiply within the alveolar cells, then travel to hilar and mediastinal lymph nodes [13]. when yeast form fungus gets access to the blood circulation system, then disseminates more rapidly and swiftly across various organs of the body. about after two weeks of exposure, the macrophages become fully fungicidal, then the cellular immunity starts a defense against the fungal particles [14]. it leads to necrosis at the site of infection like in the lungs, liver, spleen, lymph nodes, and on bone marrow, adrenal glands, and mucocutaneous membranes, which results in progressive disseminated histoplasmosis [4] as shown in figure 1. the progressive type of histoplasmosis is much more lethal and severe as compared to other kinds of histoplasmosis [15]. epidemiology histoplasmosis is distributed worldwide, but incidence cases are often reported around the periphery of river valleys [16]. the majority of infections in humans are reported from the central united states, whereas the small number of cases are also reported from brazil, argentina, india, and south africa [17]. currently, due to an increase in the occurrence of histoplasmosis worldwide, scientists have been developing several diagnostic strategies. histoplasmosis endemicity can be evaluated by population-based use of a histoplasmosis skin test [18]. the skin test was found to be useful when a major endemic area of histoplasmosis identified in the north-eastern and midwestern united states [19]. increased number of infectious diseases like histoplasmosis is being reported in areas where it was previously not thought to be prevalent, and the changing distribution of infectious diseases can become an important step to guide diagnostic workup and direct public health awareness [17]. notably, in tuberculosisendemic regions, disseminated histoplasmosis can easily be misdiagnosed as tuberculosis because of its similar clinical symptoms and must be considered as histoplasmosis infection in patients who do not respond to empiric antitubercular therapy [20]. an estimated discern indicates that nearly 3,000 people develop kidney failure yearly in nepal [21]. similarly, there are additionally a high number of the population in nepal with a diabetic condition; the 2016 diabetes profile has shown that 9.1 %of the nepali people are dwellings with diabetes [22]. likewise, the cases of hiv/aids is also in excessive number inside the country [23]. according to national centre for aids and std control (nepal), there are around 31,020 hiv/aids sufferers in the country, and it is estimated to upward thrust at a rate of 2 patients per day. those persons having conditions like a diabetic, organ transplant, and the person with hiv/aids have a weak immune system, which means that they are at high risk of developing other secondary infections like histoplasmosis during their lifetime. according to the published reports, the geographical distribution of histoplasmosis has not been clearly understood [4]. the occurrence of histoplasmosis in asia has not fully appreciated until recently. malaysia is the first country in asia, where h. capsulatum was isolated from soil samples [24]. india, a close neighboring of nepal, has also reported a high number of histoplasmosis cases in west bengal and assam till 2018[24]. from, 1995 to 2018, about 388 cases of h. capsulatum has been reported, and the number is in increasing order in india [24]. in recent years, an increasing number of histoplasmosis cases have been recognized among hiv-positive and/or diabetic patients in india [1]. in case of nepal, histoplasmosis has only been described four times, amatya et al., (2014) reported that the nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 96-102 bhandari et al. 2019 ©njb, biotechnology society of nepal 98 nepjol.info/index.php/njb. cases of histoplasmosis in an indian national who traveled to nepal for medical care and once in a nepali citizen, making this the second case described in the literature by subedi et al., (2016) [25]. recently, another case has been published by sharma and adhikari (2019). according to them, a 52 years old man with diabetes shows adrenal involvement in histoplasmosis [26]. additionally, one case has reported in a nepalese migrant to the usa with evidence of infection being acquired in nepal by gandi et al., in 2015 [27]. nepal and india have an open border, and a large number of nepalese migrants is directly dependent on indian economic and cultural aspects such as employment, education, social relationship, and trade among other elements. such socioeconomic relationship directly has an impact on the dissemination of the diseases, including histoplasmosis. identification of h. capsulatum and diagnosis of histoplasmosis traditionally, identification of causative agent of histoplasmosis is carried out through tissue culture or body fluids at 25°c on sabouraud’s dextrose agar to allow the growth of mycelial phase of h. capsulatum. for the proper growth of mold, it takes about six weeks. after the incubation period, two different types of conidia are formed. the tuberculate or macroconidia are of about 8 – 15 µm in diameter and have a thick wall. similarly, the microconidia are tiny about 2 -4 µm in diameter and are more infectious when compared to the more abundant conidia. however, the procedure of culturing is timeconsuming [28]. likewise, histopathological examination is another technique for detection of the histoplasma. it is generally done for severely ill patients [29]. the method includes tissue biopsy. during the histopathological observation, other organisms also could mimic the appearance like that of h. capsulatum. so, this could be eliminated by using another stain like methenamine silver or periodic acidschiff stains, which visualize h. capsulatum [30]. detection of circulating histoplasma associated antigen in urine and serum is another diagnostic option to detect the presence of the fungi using an immunological technique like sandwich enzyme immunoassay (eia), and it was first described in 1986 [31]. this technique generally uses h. capsulatum antigen, which is a polysaccharide, a polyclonal rabbit antihistoplasma immunoglobulin g linked to biotin, and horseradish peroxidase [4, 18]. the assay also shows the highest sensitivity against aids patients who had disseminated histoplasmosis [9]. antibody tests are also performed to detect several forms of histoplasma cases. there are two standard assays for antibody detection they are, (i) complement fixation (cf) test, which is based on the use of two separate antigensyeast and mycelial (or histoplasmosis)and (ii) immunodiffusion (id) assay [32]. the id assay tests generally detect the presence of m and h precipitin bands. an m band often existing in persistent types of histoplasmosis and lasts for many months to years after the infection. an h band is also indicative of the severe form of histoplasmosis [17]. the id assay is approximately only 80% sensitive but is more specific than cf assay because in cf assay, cross-reaction may occur with other fungal infections like sarcoidosis [33]. molecular identification of histoplasmosis in recent days, the molecular approach is widely used as a reliable and rapid technique for the detection of diseases with higher sensitivity and specificity. at present, the use of chemoluminescence labeled dna probe for the detection of specific sequences of ribosomal rna (rrna) has been able to detect and confirm all the h. capsulatum isolates from the culture [32]. accuprobe is one of them, which is based on the principle of nucleic acid hybridization. a single-stranded chemiluminescent labeled dna probe is complementary to the sequence of ribosomal rna of fungus. after the rrna released from fungi, the steady dna-rna hybrid formed and that hybrids are detected and measured by using hologicluminometer. a positive result is given by reading equal to or higher than the predetermined cut-off values, and an adverse nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 96-102 bhandari et al. 2019 ©njb, biotechnology society of nepal 99 nepjol.info/index.php/njb. effect is the cost less than that of cut-off values [39]. polymerase chain reaction (pcr) assay pcr is based on the amplification of the fungal gene for identifying mycoses. in2003, guedes et al. designed a nested pcr based technique for the rapid detection of fungi. the oligonucleotides used in this method is based on the m antigens of h. capsulatum var. capsulatum. the oligonucleotidesmsp1f msp1r (pair 1) and msp2f msp2r (pair 2) results amplicon of size 111 bp and 279 bp, respectively[34]; (msp1f: 5′ aca aga gac gac ggt agc ttc acg 3′ andmsp1r: 5′ gcg ttg ggg atc aag cga tga gcc 3′), (msp2f: 5′ cgg gcc gcg ttt aac agc gcc 3′ and msp2r: 5′ acc agc ggc cat aag gac gtc 3′). the pcr reaction was performed with 100ng dna amplified in a 25 µl reaction using 20 pmol of oligonucleotides(35 cycles of denaturation, annealing, and chain extension) [34]. by using this protocol,100% specificity and accuracy were mentioned in the 31 examined strains from the animal, soil, and human [34]. the results also confirmed the absence of other fungal strains such as h. capsulatum var. farciminosum, paracoccidioides brasiliensis, candida spp., sporothrix schenckii, cryptococcus neoformans,etc in the amplified products[34]. real-time pcr (rtpcr) the real-time rt-pcr or qpcr shows promising results in the diagnosis of histoplasmosis infection, which currently identified the h. capsulatum among several other fungi [35]. simon et al., (2010) carried out research for the detection of h. capsulatum based on real-time pcr using the taqman probe [36]. for this study, specific primers and probe (taqmanlabeled with fluorescent dye 6carboxyfluorescein at 5′ end and a nonfluorescent quencher at the 3′ end) were used for the analysis of the internal transcribed spacer (its) region of the rrna. the oligonucleotides hcits-167f (5′ aacgattggcgtctgagcat-3′) and hcits229r (5′-gagatccgttgttgaaagttttga3′) were used. the following probehcits-188p 5′-6famagagcgataataatccmgb -3′) was used. the specificity of rtpcr was calculated to be 95.4% in comparison to the culture method. during the process, there is no pcr inhibitor detected. among the 275 samples which were previously reported to be negative in culture method, 11 samples were reported as positive by rt-pcr with a specificity of 96.0%. similarly, among the 341 samples tested, zero nonspecific signals were recorded [36]. despite this promising research result, for the detection of h. capsulatum in clinical samples, there are no currently fda-approved molecular assays available. moreover, to date, the molecular approach for the detection of histoplasmosis is in a developing stage. similarly, an improved and reliable technique has to be designed as a dependable approach for the detection of the disease. regarding the future accessibility of molecular techniques for the detection of histoplasmosis is not unclouded in resource-limited countries like nepal [37]. treatment of histoplasmosis based on the current status, most patients with mild acute histoplasmosis limited to lungs will be resolved after a month without specific treatment. however, the disseminated or the severe form of histoplasmosis should be treated with antifungal agents[17]. the treatment of chronic and severe form of pulmonary histoplasmosis is generally done by prescribing amphotericin b (3.0–5.0 mg/kg daily for 1–2 weeks, intravenously), followed by itraconazole (200 mg 3 times per day for 3 days and after that 200 mg for two times a day, for a total of 3 months) is required and recommended[38]. similarly, for progressive disseminated histoplasmosis amphotericin b (3.0 mg/kg daily) is recommended for about 715days, which is followed by oral drug itraconazole having 200 mg concentration for daily three times for three days and after that 200 mg daily for two times for a total of at least a year). likewise, for the patients with the immunosuppressed condition, itraconazole daily (200 mg) is recommended as prophylaxis[19,38]. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 96-102 bhandari et al. 2019 ©njb, biotechnology society of nepal 100 nepjol.info/index.php/njb. research status of histoplasmosis in nepal in nepal, there is poor hygienic practices and sanitation in major parts of the country and is offering the home for the continuous emergence and re-emergence of several life-threatening infectious diseases. so, histoplasmosis could be a serious issue of public health in days to come. in nepal, the majority of the population lives in rural areas with minimal health care facilities. the burden of the disease is much higher in rural settings compared to the urban areas. although the pattern of diseases might change regularly, infectious diseases remain the leading cause of mortality and morbidity in nepal. in many rural settings, cases of some diseases are often identified only through their clinical signs and symptoms. lack of proper public health awareness and treatment of infectious diseases made people prone to several fungal infections, including histoplasmosis, which may be a severe issue [40]. as the pervasiveness of hiv/aids and tuberculosis are budding swiftly, the infection from the histoplasma may lead to a significant effect [41]. thus, careful diagnosis and treatment on time are paramount to control the future outbreaks of histoplasmosis. moreover, the probability of undiagnosed histoplasmosis dissemination also urges for the need of proper diagnostic strategies of histoplasma spp. infection in nepal. as per institutional information, there is no routine examination and treatment of histoplasmosis infection in tribhuvan universityteaching hospital and national public health laboratory (nphl-nepal), while both of them are considered as reference health service centres of the country. challenges in nepal as nepal is under the way of development, the budgeting for the health and its related sector is very less. lack of proper investment in the health sector for the diagnosis and treatment of diseases is a significant problem in nepal. death of hundreds of people by unknown disease is also prevalent. lack of proper government strategies and operative ventures in the health sector has been a cardinal cause of those strange deaths. allocation of the budget on the health sector is only attentive to the treatment of some severe disease, which shows that the country has less interest in disease diagnosis. it is one of the significant consequences of the death of people from unknown conditions. therefore, early diagnosis of histoplasmosis is mandatory to reduce the burden of illness. conclusion the clear and accurate picture of the dissemination of histoplasmosis is complicated to understand in the resource-limited clinical sector of nepal. many factors could contribute to this, like lack of proper lab facilities, lack of expertise on fungal infections, etc. additionally, not much reliable data are available from the government and academia due to incompetence in the diagnosis strategies. the published data are also in the form of case reports based on a hospital visit. it is already emphasized that the pockets of endemicity do exist in nepal, which could lead to a significant impact on public health. hence, careful considerations and the molecular approach of disease identification may be a suitable alternative. to address this problem, further research should be done immediately in nepal conflict of interest the authors declare no conflict of interest. references 1. 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antibiotic susceptibility profile of respiratory pathogens obtained at tertiary care hospital from western nepal deepak subedi1, surya prasad devkota1,2,3*, dharm raj bhatta4, binita koirala sharma1, ashmita paudel2, krishna gurung1,5, damodar gautam1 1pokhara bigyan tatha prabidhi campus, nayabazar, pokhara 2regional college of health science and technology, nayabazar, pokhara 3 school of health and allied sciences, pokhara university, pokhara 4manipal college of medical sciences, pokhara 5prithvi narayan campus, pokhara abstract the prevalence and drug resistance of the respiratory pathogens is increasing gradually in nepal. however, their detail study is rare in the western region of nepal. hence, this study was carried out to know the incidence and antibiotic susceptibility profile of the respiratory pathogens obtained at a tertiary care center located at pokhara. 139 pathogens were isolated from 460 clinical samples included. significant pathogens were gram-negative bacteria 94 (67.62%), followed by 28 (20.15%) candida, and gram-positive isolates 17 (12.23%). the growth rate was significantly higher for sputum samples in comparison to throat swabs. pseudomonas aeruginosa, klebsiella pneumoniae, and acinetobacter spp were significant gram-negative isolates while staphylococcus aureus, streptococcus pneumoniae, and streptococcus pyogenes were grampositive pathogens. sensitivity rate was higher for colistin and imipenem among gram-negative isolates while lower sensitivity was for cefepime. vancomycin was effective against all tested gram-positive isolates while erythromycin and ciprofloxacin were less effective. keywords: respiratory pathogens, western nepal, pseudomonas aeruginosa, acinetobacter spp, klebsiella pneumonia, s. aureus. *corresponding author email: devkotasp1@gmail.com introduction infection of the respiratory tract is a significant cause of mortality as well as morbidity among the elderly and young people of nepal [1]. management of these infections is more complex in developing countries due to the lack of the proper identification of pathogens and their suitable treatment [2]. the causative agents of the respiratory infections are not detected in many cases hence physicians depend on clinical manifestations for the diagnosis [3]. klebsiella pneumaniae, streptococcus pneumoniae, and haemophilus influenzae are the significant pathogens causing respiratory infections [4, 5]. in addition to this, pathogens like staphylococcus aureus, acinetobacter, and pseudomonas aeruginosa are also frequently isolated from respiratory specimens [6, 7]. polymicrobial respiratory infections by two or more bacteria, two or more viral pathogens, and mixed viral and bacterial pathogens also have been reported [4, 5]. pneumonia and infection of lower respiratory tract are the causes of more than 4 million deaths per year and this problem is more frequent in middle-and low-income nations [8]. drug resistance among these pathogens has been increasing as there are multiple reports of multidrug resistance among various respiratory pathogens and many of them are not susceptible to several routine antibiotics [4, 9]. though most of the bacterial respiratory infections are treatable, the huge death is due to a lack of proper preventive measures and unavailability of healthcare facilities [8]. several reports are indicating a gradual increase in antibiotic resistance among many bacterial pathogens responsible for respiratory diseases [10, 11]. the imprudent use of antibiotics for treating these infections has resulted in a very rapid increase in drug resistance of the respiratory pathogens [12]. though various pathogens [13] and risk factors [14] are associated with nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 1-7 subedi et al. 2019 ©njb, biotechnology society of nepal 2 nepjol.info/index.php/njb. respiratory infections, their detail study is very limited in this region. surveillance study on respiratory pathogens was imminent in this part of the nation. hence this study was done to access the prevalence, distribution and drug resistance profile of the respiratory pathogens isolated at a tertiary care center of western nepal. materials and methods study site and duration the study site was manipal teaching hospital, a 750-bed multi-specialty tertiary care hospital located at pokhara, nepal. the study was conducted for a period of six months (july 2016 to january 2017) at the microbiology laboratory of the hospital. sample collection samples were collected from patients with clinical symptoms of respiratory tract infection as indicated by physicians. for the collection of sputum samples, patients were given various instructions on how to collect a sputum sample correctly. clean, well-labeled, wide-necked, dry and leak-proof screw-cap container was provided to the patients for sputum collection. the sample was accepted only if it was sputum but samples containing saliva, nasal secretions, mucus, etc. were not analyzed. in the case of unacceptable samples, repeated samples were requested. throat sample was collected by trained personals. the patient was first allowed to sit comfortably in a good light and using a tongue depressor the throat was observed for any swelling, redness, pus, ulcerations, exudates, and presence of the membrane. a sterile cotton swab was used to collect a sample of the infected throat. special care was taken not to contaminate the swab with saliva and placed it into a sterile container. macroscopic and microscopic examination of the sample appearance, as well as presence or absence of blood in the given sputum, was observed and noted. similarly, gram staining was performed from both sputum and throat swabs to observe pus cells and bacteria. isolation and identification respiratory tract samples obtained from both admitted and outpatients visiting the hospital were included in this study. soon after collection, all the specimens were cultured on chocolate agar and 5% sheep blood agar. the inoculated plates were then incubated on candle jar for 18-24 hours. optochin and bacitracin discs were placed on the primary inoculation for the presumptive screening of s. pneumoniae and h. influenzae as well as s. pyogens respectively. identification of the pure culture was carried out by observing colony characteristics and gram staining followed by oxidase test, catalase test, coagulase test, urease test, tsi test, and imvic tests. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 1-7 subedi et al. 2019 ©njb, biotechnology society of nepal 3 nepjol.info/index.php/njb. antibiotic susceptibility test kirby-bauer disc diffusion method was used using muller hinton agar (hi-media, india). clinical and laboratory standards institute (clsi) guidelines were used for the interpretation of the results [15]. 0.5 mac farland suspension of the isolates were used for inoculation. amikacin (30µg), azithromycin (15µg), cefepime (30µg), ceftriaxone (30µg), ciprofloxacin (5µg), colistin, (10µg) cotrimoxazole (25µg), imipenem (10µg), piperacillin/ tazobactam (100µg/10), vancomycin (30µg), levofloxacin (5µg), teicoplanin (30µg), and erythromycin (15µg) were used for the test. e. coli atcc 25922 was used as a control organism for gram-negative isolates while staphylococcus aureus atcc 25923 was used as a control organism for gram-positive isolates during this test. results among 460 samples, 139 (30.2%) showed the growth of respiratory pathogens. the incidence of the pathogens was higher from the sputum sample in relation to the throat sample (table 1). pseudomonas aeruginosa, acinetobacter spp., klebsiella pneumoniae, staphylococcus aureus, escherichia coli, and candida spp. were the common pathogens isolated followed by haemophilus influenzae, streptococcus pneumoniae, streptococcus pyogenes, and enterobacter spp. streptococcus pyogenes were isolated only from throat swab while all gram-negative isolates were only from the sputum sample. only candida was isolated from both of the samples (table 2). table 1: total cases and types of samples samples growth positive (%) growth negative (%) total (%) sputum 134 (29.13%) 299 (65%) 433 (94.13%) throat swab 5 (1.07%) 22 (4.8%) 27 (5.87%) total 139 (30.2%) 321 (69.8%) 460 (100%) vancomycin was the most effective drug for all three gram-positive pathogens as all of these isolates were sensitive to vancomycin. teicoplanin and clindamycin were also highly table 2: distribution of gram-positive cocci, gramnegative bacilli and candida spp. in throat swab and sputum samples microorganisms samples total (%) throat swab (%) sputum (%) pseudomonas aeruginosa 0 31(22.3) 31 (22.3) acinetobacter spp 0 26 (18.7) 26 (18.7) klebsiella pneumoniae 0 23 (16.55) 23 (16.55) staphylococcus aureus 0 9 (6.47) 9 (6.47) escherichia coli 0 8 (5.76) 8 (5.76) haemophilus influenza 0 4 (2.88) 4 (2.88) streptococcus pneumoniae 0 4 (2.88) 4 (2.88) streptococcus pyogenes 4 (2.88) 0 4 (2.88) enterobacter spp 0 2 (1.44) 2 (1.44) candida spp. 1 (0.72) 27 (19.42) 28 (20.14) total 5 (3.6) 134 (96.4) 139 (100) active against s. aureus and streptococcus pyogenes respectively while other drugs were less effective as there was resistance ranging from 22 to 100% for antibiotics other than vancomycin, teicoplanin, and clindamycin (table 3). colistin was the most effective drug for treating pseudomonas aeruginosa and acinetobacter. least drug resistance was noted on enterobacter while the highest resistance was observed on acinetobacter. resistance towards all common antibiotics was observed in all gram-negative pathogens excluding enterobacter with varying percentages (table 4). discussion nearly one-third of the samples were positive for the respiratory pathogens. findings of the shrestha et al (2005) also reported that 31% of the sputum samples were positive for pathogens [16]. among the isolated pathogens gramnegative isolates were more prevalent than gram-positive pathogens. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 1-7 subedi et al. 2019 ©njb, biotechnology society of nepal 4 nepjol.info/index.php/njb. table 3: antimicrobial susceptibility profiles of gram-positive isolates antibiotics used pathogens staphylococcus aureus (n=9) streptococcus pyogenes (n=4) streptococcus pneumoniae (n=4) sensitive no. (%) resistant no. (%) sensitive no. (%) resistant no. (%) sensitive no. (%) resistant no. (%) amikacin 7 (77.8) 2 (22.2) 3 (75) 1 (25) ciprofloxacin 3 (33.3) 6 (66.7) clindamycin 2 (22.2) 7 (77.8) 4 (100) 0 2 (50) 2 (50) co-trimoxazole 1 (25) 3 (75) erythromycin 2 (22.2) 7 (77.8) 0 4 (100) 2 (50) 2 (50) levofloxacin 0 4 (100) 3 (75) 1 (25) teicoplanin 9 (100) 0 vancomycin 9 (100) 0 4 (100) 0 4 (100) 0 a very high incidence of gram-negative pathogens among respiratory samples are also reported earlier [2, 12, 16]. gram-negative isolates like pseudomonas aeruginosa, acinetobacter spp., klebsiella pneumoniae were predominant. these findings were in accordance with the previous studies [13, 17]. similarly, staphylococcus aureus was the major gram-positive pathogens. pathogens like streptococcus pneumoniae, streptococcus pyogenes were also isolated from the respiratory samples. the predominance of staphylococcus aureus among gram-positive pathogens followed by streptococcus spp. is common from respiratory samples [1, 3, 18]. candida isolates were also the major cause of table 4: antimicrobial susceptibility profiles of gram-negative bacilli antibiotics used pathogens pseudomonas aeruginosa (n=31) acinetobacter spp (n=26) klebsiella pneumoniae (n=23) e. coli (n=8) haemophilus influenzae (n=4) enteroba cter spp (n=2) s (%) s (%) s (%) s (%) s (%) s (%) amikacin 26 (83.9) 8 (30.8) 15 (65.2) 8(100) 2 (100) azithromycin 2 (50) cefepime 7 (22.6) 6 (23.1) 6 (26.1) 0 2 (100) ceftriaxone 3 (75) ciprofloxacin 22 (71) 8 (30.8) 10 (43.5) 2 (25) 4 (100) 2 (100) colistin 31 (100) 26 (100) co-trimoxazole 8 (34.8) 5 (62.5) 2 (50) 2 (100) imipenem 23 (74.2) 9 (34.6) 21 (91.3) 7(87.5) 2 (100) piperacillin/ tazobactam 13 (42) 5 (19.2) 7 (30.4) 3 (37.5) 2 (100) nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 1-7 subedi et al. 2019 ©njb, biotechnology society of nepal 5 nepjol.info/index.php/njb. respiratory infections as indicated by previous authors [3, 19]. vancomycin was the most reliable treatment option for all of the gram-positive pathogens isolated. the high sensitivity of vancomycin against s. aureus and streptococcus spp isolated from respiratory tract infection is found elsewhere [18, 19, 20]. more than 66% of s. aureus were resistant to ciprofloxacin, clindamycin, and erythromycin. increasing resistance of s. aureus towards these drugs is common globally [18, 20]. all streptococcus pyogenes isolated in this study were sensitive to clindamycin and vancomycin while they were absolutely resistant to levofloxacin and erythromycin. very similar antibiotic sensitivity patterns of streptococcus pyogenes are reported earlier [21, 22]. the majority of streptococcus pneumoniae were resistant against co-trimoxazole, clindamycin, and erythromycin. an elevated level of resistance among streptococcus pneumoniae obtained from sputum and other samples are reported from different countries [23]. colistin resistance was not observed in pseudomonas aeruginosa and acinetobacter spp. indicating that this drug is still effective as a last resort. there are many reports of absolute colistin sensitivity among gram-negative respiratory pathogens [24, 25]. however, there is a report of colistin resistance among multidrug-resistant acinetobacter spp isolates obtained from icu patients suffering from respiratory tract infection in nepal [26]. imipenem and amikacin resistance was less in gram-negative isolates in comparison to other antibiotics like cefepime, ciprofloxacin, piperacillin/ tazobactam. less resistance is detected in common respiratory gram-negative pathogens against imipenem and amikacin [24, 27]. drug resistance was higher in acinetobacter spp among gram-negative pathogens. highly elevated antibiotic resistance among acinetobacter isolated from respiratory tract infection is reported previously in nepal [17, 26]. we also noticed drug resistance among haemophilus influenzae as some isolates were not sensitive to azithromycin, ceftriaxone, and co-trimoxazole. antibiotic-resistant haemophilus influenzae causing respiratory tract 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[http://snims.org/files/microbiology/2.pdf] nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 47 nepjol.info/index.php/njb study of phytochemical, antioxidant and antimicrobial activity of artocarpus heterophyllus nita thapa, pratiksha thapa, jay bhandari, prasodhan niraula, nikita shrestha, bhupal g. shrestha* department of biotechnology, kathmandu university, dhulikhel, nepal. abstract in today’s world, search for natural medicines is increasing as a result of drug resistance of pathogens and also due to negative consequences of antibiotic. presence of phytochemicals, antioxidant potential and antimicrobial activity of artocarpus heterophyllus was carried out in this study. leaf of this plant was subjected to warm extraction with three different solvents namely methanol, aqueous methanol and ethyl acetate. leaf extract showed the presence of coumarin, alkaloid, terpenoid in methanol solvent; tannin, coumarin, saponin in aqueous methanol extract and coumarin, terpenoids in ethyl acetate solvent. further, antimicrobial activity was assessed through disc diffusion method with six pathological bacteria and two fungi strains in four different concentrations of plant extract. largest zoi of 16mm was obtained against b. subtilis in 200mg/ml concentration for ethyl acetate extract. antioxidant potential was measured by dpph (diphenyl-2-picrylhydrazyl) assay. dpph free radical scavenging activity was expressed in % inhibition with l ascorbic acid as standard and leaf extract in methanol showed the best activity. keywords: phytochemical, antioxidant, antimicrobial, ic50, zoi (zone of inhibition). *corresponding author email: bgs@ku.edu.np introduction in the written record, the study of herbs dates back over 5,000 years to the sumerians, who created clay tablets with lists of hundreds of medicinal plants such as myrrh and opium [1]. the traditional use of herbs to prevent, treat and even cure various illnesses and diseases has largely been replaced by modern medicine. however, it has been estimated by world health organization (who) that 80 percent of the population of some asian and african countries presently depend on traditional herbal medicine for their most basic health care needs. also who notes that between 25-40% of pharmaceuticals drugs are derived from plants. it is also noted that 40-50% of medicines are direct or synthetic copies of plant ingredients. the use of medicinal plants offers poorer populations the ability to fight diseases at low costs. the use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed [2]. herbal medicine is still the mainstay of about 75 80% of the whole population, and the major part of traditional therapy involves the use of plant extract and their active constituents [3]. in recent years, antimicrobials derived from the plants have been receiving increasing attention as synthetic antibiotics have shown ineffectiveness against several pathogenic organisms due to increasing drug resistance [4]. nepal is botanically rich in all the three levels of biodiversity which are species diversity, genetic diversity and habitat diversity. in nepal, there are various plants with potent medicinal properties and have been used since ancient ages. among the 7,000 species of medicinal plants recognized all over the world, more than 900 types of precious medicinal plants are said to be found in nepal [5]. a. heterophyllus bark and fruit are medicinally used to treat sprains, fractures, diabetes and are also used for laxative effect of abdomen and to increase the breast milk production in nursing mothers [6]. in addition to the antimicrobial activity of a. heterophyllus, anti-inflammatory, antioxidant, anticholinergic, anti-diabetic, immune modulatory effect, inhibition of protease, oestrogen regulation and inhibition of melanin biosynthesis have also been reported through several pharmacological research investigations of the plant parts [7]. a. heterophyllus is a plant belonging to moraceae family and is commonly referred to as ‘jackfruit’ in english and ‘katahar’ in nepali. it is found to originate from nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 thapa et al. ©njb, biotechnology society of nepal 48 nepjol.info/index.php/njb southeast asia and is widely cultivated in tropical lowlands and are found to have distinct aroma. the bark, roots, leaves and fruit are found to contain medicinal properties and are widely used in various traditional and folk systems of medicine in order to treat a range of ailments. materials and methods sample preparation a. heterophyllus leaves were collected from pachkhal, nepal. the leaves were washed thoroughly under tap water and shade dried at room temperature (24– 26°c) and then pulverized by a mechanical grinder. the powder was then passed through a 40-mesh sieve and stored in a well closed container before its use. solvent extraction warm extraction was done using soxhlet apparatus using methanol and ethyl acetate as solvent. 6gm of the powdered sample was packed into the filter paper and were placed into the thimble of the soxhlet apparatus. 250ml of methanol /ethyl acetate/aqueous methanol was added to the thimble. the apparatus was operated continuously for 3 days monitoring the circulation of water into the condenser. the soxhlet extract was then treated with hexane in order to remove chlorophyll pigment by the help of the separating funnel. the process was repeated until the chlorophyll pigment was completely extracted in hexane. then the final extract was collected in eppendorf tube after the extract was dried of solvent on water bath at 50˚c [8]. various concentrations were then made by dissolving in dimethyl sulfoxide (dmso). phytochemical screening presence of phytochemicals were analyzed by following standard procedure which are as follows: a. test for basic alkaloids (mayer’s test) 5ml of extract was concentrated to yield a residue. residue was dissolved in 3ml of 2% (v/v) hcl. 3 drops of mayer’s reagent were added. appearance of the dull white precipitate indicated the presence of basic alkaloids. b. test for coumarin 4ml extract solution was taken. 1-2 drops of water (hot) was added. volume was made half (for uv fluorescence). 10% nh4oh was added to another half volume (for strong fluorescence). presence of green fluorescence indicated the presence of coumarin. c. test for saponins 2ml extract was shaken vigorously for 30 seconds in a test tube. persistence of thick forth even after 30 minutes indicated the presence of saponins. d. test for glycosides 2ml of extract was dried till 1ml.1-2ml nh4oh was added and shaken. appearance of cherish red color indicated the presence of glycosides. e. test for reducing sugar (fehling’s test) 1ml of extract was taken.1ml distill water was added.5-8 drops of fehling’s solution (hot) was added. presence of brick red precipitation indicated the presence of reducing sugar. f. test for steroids 1ml extract was dissolved in 10 ml chloroform. equal volume of conc. h2so4 was added by the side of test tube. upper layer turned red and sulphuric acid layer turned yellow with green fluorescence. this indicated the presence of steroids. g. test for quinone ml of extract was taken.1ml of conc. h2so4 was added. formation of red color indicated the presence of quinone. h. test for terpenoids 1 ml of extract was mixed with 2 ml of chloroform. 3ml of conc. h2so4 was added to form a layer. reddish brown precipitate coloration at the interface formed indicated the presence of terpenoid. antioxidant assay diphenyl-2-picrylhydrazyl (dpph) radical scavenging activity was done for determining antioxidant activity. preparation of dpph solution dpph solution of 100µm was prepared by dissolving 3.94mg of dpph in 100ml of methanol. it was nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 thapa et al. ©njb, biotechnology society of nepal 49 nepjol.info/index.php/njb protected from light by covering the bottle with aluminum foil. preparation of standard solution 10mg/ml stock solution of ascorbic acid was prepared and test solution of 1, 2, 3, 4, 5 and 10 µg/ml of ascorbic acid was prepared from stock solution by dilution. preparation of test sample 10mg of plant extract was dissolved in 1ml of methanol to prepare stock solution of 10mg/ml. test solution 1, 2, 3, 4, 5 and 10 µg/ml was prepared from stock solution by dilution. experiments were done in triplicate. estimation of dpph scavenging activity for this, 1 ml methanol and 1 ml dpph solution were mixed as control. 2ml methanol was taken in eppendorf tube as blank. 1ml ascorbic acid was mixed with 1 ml dpph as standard and 1ml plant extract was mixed with 1ml dpph as sample. all these mixtures were immediately kept in dark to prevent from light. after 30 minutes, absorbance was taken in 517nm. antimicrobial screening agar disc diffusion assay was performed to test antibacterial and antifungal activities [9]. fungal strains rhizopus, aspergillus flavus and bacterial strains pseudomonas aeruginosa, bacillus subtilis, bacillus thuringiensis, escherichia coli, proteus mirabilis were subjected to sensitivity test. whatman filter 1 disc (6mm) was autoclaved and dipped in extract of various concentrations and introduced on the upper layer of agar plate earlier swabbed with microbial concentration with the help of inoculating loop. standard antibiotic discs chloramphenicol 30 µg(c 30) , gentamicin 10 µg (gen 10), ciprofloxacin 30 µg (cf 30), cefotaxime 30 µg (ctx 30) and tetracycline 30 µg (te 30) were used. the plates were incubated overnight at 37oc. microbial growth inhibition was determined by measuring the diameter of zone of inhibition (zoi). results of antimicrobial activity are expressed as average of triplicates. results in our study, when six gram of dried sample was used for extraction, aqueous methanol exhibited highest yield percentage i.e. 10.98% and least yield was given by methanol solvent i.e. 0.22%. ethyl acetate solvent gave yield of 4.68% when used as shown in table 1. table 1: yield value of artocarpus heterophyllus s.n. solvent sample loaded (gm) yield amount (gm) yield percentage (%) 1 ethyl acetate 6 0.28 4.68 2 aqueous methanol 6 0.66 10.98 3 methanol 6 0.013 0.22 extraction and characterization of several active phytocompounds from these green factories have given birth to some high activity profile drugs [10]. secondary metabolites of plants serve as defense mechanisms against predation by many microorganisms, insects and herbivores [11]. methanol extract showed presence of coumarin, steroids; ethyl acetate showed presence of coumarin, terpenoid whereas aqueous methanol showed presence of tannin, coumarin and saponin as shown in table 2. table 2: phytochemical screening phytochemicals methanol ethyl acetate aqueous methanol alkaloids + tannins + reducing sugar coumarin + + + glycosides quinone steroids terpenoids + + saponin + nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 thapa et al. ©njb, biotechnology society of nepal 50 nepjol.info/index.php/njb this finding is similar to the findings of rawat et al [12]. coumarin was present in all three extracts tested and it can be said that this plant’s leaf exhibit blood-thinning, anti-fungicidal and anti-tumor activities. presence of terpenoid is responsible for its aromatic properties whereas it also possesses antibacterial and anti-cancer properties. this plant can also reduce problem of cholesterol as presence of steroid is noted. saponin is only present in aqueous methanol extract and it can also contribute to lowering of blood cholesterol and inhibition of cancer cell growth. saponin containing plants are used as traditional medicines, especially in asia and are intensively used in food, veterinary and medicinal industries [13]. tannin is found to present in aqueous methanol extract which is naturally occurring plant polyphenols that have a characteristic binding and precipitating proteins. phytochemical screening from other research confirmed the presence of phytosterols, anthraquinone, terpenoids, phenols, glycosides, flavonoids and diterpenes in both of the trees i.e. artocarpus heterophyllus and artocarpus altilis [14]. the antioxidant activity of tannins results from their free radical and reactive oxygen species-scavenging properties, as well as the chelation of transition metal ions that modify the oxidation process [15]. table 3: antimicrobial assay solvent microorganism extract concentration (in mg/ml) and zone of inhibition (in mm) 200mg/ml 100mg/ml 50mg/ml 25mg/ml methanol b. cereus 11 9 7 7 b. thuringiensis 11 10 7 6.5 e. coli 6.5 6 6 6 p. aeruginosa 7 7 6.5 6.5 p. mirabilis 7.5 7 6..5 6.5 rhizopus 9 8 7 6.5 a. flavus 9 9 7.5 7 b. subtilis 9 7.5 7 6.5 ethyl acetate b. cereus 11 7 6.5 6.5 b. thuringiensis 7 6.5 6.5 6.5 e. coli 8 7.5 7.5 6.5 p. aeruginosa 12 11 8 7 p. mirabilis 9 7.5 7 6.5 rhizopus 8 7 6.5 6.5 a. flavus 9 8 7.5 7 b. subtilis 16 13 11 10 aqueous methanol b. cereus 10 7.5 7.5 7.5 b. thuringiensis 6.5 6 6 6 e. coli 7.5 6.5 6.5 6.5 p. aeruginosa 6.5 6.5 6.5 6 p. mirabilis 7.5 6.5 6 6 rhizopus 9 8 7 6.5 a. flavus 8 7 7 5.5 b. subtilis 9 7.5 6.5 6.5 table 4: zone size interpretative standards for selected antimicrobial discs (ctx30, gen10, cf30, c30 te30) and their observed zoi during experiment antibiotics zone of inhibition (mm) b. cereus e. coli b. thuringiensis p. mirabilis rhizopus a. flavus p. aeruginosa b. sub tilis ctx30 18 20 11 20 23 26 34 34 gen10 24 25 20 14 26 23 34 22 cf30 30 25 23 20 32 30 47 32 c30 23 29 24 12 33 36 30 24 te30 31 24 25 11 25 26 34 31 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 thapa et al. ©njb, biotechnology society of nepal 51 nepjol.info/index.php/njb 0 2 4 6 8 10 12 z o i (m m ) antimicrobial activity of aqueous methanol solvent 200mg/ml 100mg/ml 50mg/ml 25mg/ml figure 1: a, b and c are antimicrobial activity of extract prepared in methanol, aqueous methanol and ethyl acetate. figure d represent antimicrobial activity of the standard antibiotics with 8 antimicrobial assay was performed by agar disc diffusion method by using six bacterial strains and two fungal strains. this activity was assessed by measuring the diameter of zoi of four concentrations used as shown in table 3. the obtained zoi of standards for selected antimicrobial discs is shown in table 4. largest zoi of 16mm was obtained against b. subtilis in ethyl acetate extract, whereas 11mm zoi was largest in methanol extract obtained against b. cereus. antimicrobial activity was comparatively better in ethyl acetate extract in comparison to methanol and aqueous methanol extract. besides this, antibacterial activity was higher than antifungal activity as largest zoi of 9mm is obtained against fungi rhizopus and a. flavus. also zoi was largest in 200mg/ml and least in 25mg/ml concentration, showing a dose dependent response. clinical microbiologists are showing interest in the table 5: antioxidant activity concentration (µg/ml) % scavenging activity methanol ethyl acetate aqueous methanol ascorbic acid leaf extract ascorbic acid leaf extract ascorbic acid leaf extract 1 8.07 6.63 14.37 3.26 11.38 10.41 2 18.45 11.93 17.69 6.52 15.78 14.13 3 25.81 15.25 22.54 8.01 31.67 18.15 4 35.46 20.68 28.72 10.95 41.42 22.76 5 44.25 28.03 34.66 14.43 44.19 26.63 10 89.25 61.84 38.45 17.86 94.04 47.17 0 5 10 15 20 25 30 35 40 45 50 z o i (m m ) antimicrobial activity of standard antibiotics ctx30 gen10 cf30 c30 te30 c 0 2 4 6 8 10 12 z o i (m m ) antimicrobial activity of methanol solvent 200mg/ml 100mg/ml 50mg/ml 25mg/ml 0 2 4 6 8 10 12 z o i (m m ) antimicrobial activity of ethyl acetate solvent 200mg/ml 100mg/ml 50mg/ml 25mg/ml c nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 thapa et al. ©njb, biotechnology society of nepal 52 nepjol.info/index.php/njb ic50 value of ascorbic acid = 5.62µg/ml ic50 value of artocarpus heterophyllus = 8.33µg/ml ic50 value of ascorbic acid = 12.96µg/ml ic50 value of artocarpus heterophyllus = 29.29µg/ml ic50 value of ascorbic acid=5.27µg/ml ic50 value of artocarpus heterophyllus=10.69µg/ml figure 2: a, b and c represents antioxidant activity of methanol, ethyl acetate, aqueous methanol respectively. topic of antimicrobial plant extracts as they believe that these phytochemicals will find their way as alternate to antimicrobial drugs as those prescribed by physicians. also several of these plant extracts are already being tested in humans and this is probably because of increased public awareness of problems with the over prescription and misuse of traditional antibiotics. also nowadays multiple drug resistance has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious disease [16]. dpph stable free radical method is an easy, rapid and sensitive way to survey the antioxidant activity of a specific compound or plant extracts [17]. in our study, effective antioxidant activity was shown by methanol extract where ic50 value for ascorbic acid was 5.62µg/ml and leaf extract showed value of 8.33 µg/ml as shown in table 5. similarly, ic50 value for ascorbic acid was 12.96µg/ml and leaf extract showed value of 29.29 µg/ml when ethyl acetate was used as solvent. ic50 value for ascorbic acid was 5.27µg/ml and leaf extract showed value of 10.69µg/ml when aqueous methanol was used as solvent. among three solvents used for the study, methanol extract showed best the antioxidant activity. so, the leaf extract of the plant could have some anti-cancer activity as has been earlier reported for ashwagandha [18, 19]. conclusion we have been able to show that the leaf of a. heterophyllus contains presence of different phytochemicals that have potential health benefits. extraction efficiency of leaf extract was higher in aqueous methanol solvent. preliminary study indicated the presence of phytochemicals which can further be studied to explore medicinal properties. the plant extract also showed more antibacterial activity than antifungal activity against major pathogens. methanol extract showed effective antioxidant activity in comparison to other solvent used in this study. further, these can be subjected to isolation of the therapeutic antimicrobials which can be beneficial to mankind. acknowledgement we would like to express our deep gratitude to kathmandu university biotechnology department for providing us work space and necessary guidance for the successful completion of this research work. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 47-53 thapa et al. ©njb, biotechnology society of nepal 53 nepjol.info/index.php/njb references 1. sumner j: the natural history of medicinal plants. timber press. 2000. 2. culture and health, orientation texts – world decade for cultural development 1988 – 1997, document clt/dec/pro – 1996, paris, france, pg. 129. 3. akerele o: summary of who guidelines for the assessment of herbal medicines herbal gram. 1993, 22: 13-28. 4. akgul c, saglikoglu g: antibacterial activity of crude methanolic extract and its fractions of aerial parts of anthemis tinctoria. ind j biochem biophy. 2005, 42: 395-397. 5. manandhar np: plants and people of nepal. timber press. usa. 2000: 50p. 6. fernando s: herbal food and medicines in sri lanka. new delhi. (original work published in 1982).2003. 7. jitendra r, kalpana s, shweta s, kumar m s, manish b: artocarpus heterophyllus (jackfruit) potential unexplored in dentistryan overview. ujp. 2014, 3(1): 50-55. 8. shrestha p et al: phytochemical screening, antimicrobial activity and cytotoxicity of nepalese medicinal plants swertia chirayita and dendrobium amoenum. njb 3.1. 2015 : 48-57. 9. lamichhane b, adhikari s, shrestha p, shrestha b g: study of phytochemical, antioxidant, antimicrobial and anticancer activity of berberis aristata. j trop life sci. 2014, 4(1): 01-07. 10. mandal v, mohan y, hemalatha s: microwave assisted extraction—an innovative and promising extraction tool for medicinal plant research. pharmacognosy reviews. 2007, 1(1):7-18. 11. lutterodt g d, ismail a, basheer r h, baharudin h m: antimicrobial effects of psidium guajava extracts as one mechanism of its antidiarrhoeal action. malaysian j med sci. 1999, 6(2): 17-20. 12. rawat a, mahajan s, gupta a, agnihotri r k, wahi n, sharma r: detection of toxigenic fungi and mycotoxins in some stored medicinal plant samples. ijasbt. 2014, 2(2): 211-216. 13. hostettmann k, marston a: chemistry and pharmacology of natural products, saponin. 1995. 14. suman r, arti t: evaluation of phytochemical and antimicrobial effect of artocarpus heterophyllus leaves extracts. j pharm sci. 2014. 15. serrano j, puupponen-pimia r, dauer a, aura a, saura-calixto f: tannins: current knowledge of food sources, intake, bioavailability and biological effects. mol nutr food res. 2009, 53:310–329. 16. joshi b, lekhak s, sharma a: antibacterial property of different medicinal plants: ocimum sanctum, cinnamomum zeylanicum, xanthoxylum armatum and origanum majorana. kathmandu university journal of science, engineering and technology. 2009, 5(1): 143-150. 17. koleva i i, van beek t a, linssen j p, groot a d, evstatieva l n: screening of plant extracts for antioxidant activity: a comparative study on three testing methods. phytochem anal. 2002, 13(1): 8-17. 18. widodo n , kaur k, shrestha b g, takagi y, ishii t, wadhwa r , kaul s c: selective killing of cancer cells by leaf extract of ashwagandha: identification of a tumor-inhibitory factor and the first molecular insights to its effect. clin cancer res. 2007, 13(7): 2298-2306. 19. widodo n, takagi y, shrestha b g, ishii i, kaul s c, wadhwa r: selective killing of cancer cells by leaf extract of ashwagandha: components, activity and pathway analyses. cancer letters. 2008, 262: 37–47. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 66-67 issn 2091-1130 (print) / issn 2467-9319 (online) case study ©njb, biotechnology society of nepal 66 nepjol.info/index.php/njb fasciolopsis buski vomited out by a child; the first case reported from nepal narayan dutt pant1*, manisha sharma2, saroj khatiwada3 1department of microbiology grande international hospital, dhapasi, kathmandu, nepal 2department of microbiology, kathmandu medical college, kathmandu, nepal 3cist college, kathmandu, nepal abstract live adult worms of fasciolopsis buski are rarely seen in humans except in autopsy. only a few such cases have been reported in the world literature. we reported a case of fasciolopsiasis in a child of age 14 months who coughed out the live adult fasciolopsis buski after administration of antihelminthic drug. the patient was a resident of terai (far western) region of nepal and had history of travelling to india. this is the first case of fasciolopsis reported from nepal. key words: fasciolopsiasis, nepal, antihelminthic drug, diarrhea. *corresponding author email: ndpant1987@gmail.com introduction fasciolopsiasis is a gastrointestinal infestation by a trematode; fasciolopsis buski mainly involving duodenum and jejunum. the fluke is the largest intestinal fluke parasitizing humans and was first noted by busk in 1943 from the duodenum of a deceased indian sailor. fasciolopsiasis is prevalent in various parts of south east asia including the neighboring countries china and india. the infections by fasciolopsis buski are common in impoverished countries where proper sanitation systems are lacking [1]. the disease occurs due to ingestion of encysted metacercariae on aquatic vegetation or direct water [2]. mostly the infection is asymptomatic but in severe infection the common symptoms are abdominal pain, diarrhea, lowgrade fever, toxemia, allergy, anemia, ascites, generalized edema, obstruction of intestine sometimes leading to death [1, 3]. diagnosis is made by detection of eggs in stool but the differentiation between fasciolopsis buski and fasciola hepatica is very difficult in routine examination of stool [1]. here we report a case of fasciolopsiasis in a child of age 14 months who coughed out the live adult fasciolopsis buski after administration of antihelminthic drug. this is the first case of fasciolopsis reported from nepal. case report: a 14 months male child attended outpatient department with the chief complain of diarrhea, vomiting, refusal to eat, fever, irritability, and weakness. the child was a resident of terai (far western) region of nepal and had travel history to lucknow, india for treatment of urinary tract infection and follow up for the treatment of epilepsy. the patient was under medication (ofloxacin and carbamazepine). he had significant leucocytosis (14000 cells/mm3) with eosinophillia (12%). stool routine examination and culture didn’t reveal any significant findings. since the patient was already in broad spectrum antibiotic for treatment of urinary tract infection the possibility of bacteria being the cause of the illness was quite low. so the patient was given metronidazole and mebendazole to cover all other possible causes of the diarrhea. after around 12 hours of administration of the medication the patient vomited out a moving worm of size 30×19 mm2, leaf shaped with anterior end narrower and the posterior end broadly rounded, dorsoventrally flattened, unsegmented and flesh colored (figure 1). the worm was identified as fasciolopsis buski on the basis of the morphological characteristics such as lack of cephalic cone, poorly-developed suckers (oral and ventral) and the unbranched ceca. all symptoms subsided after the full course of treatment and total leucocyte counts and eosinophil counts became normal. figure 1. fasciolopsis buski vomited out by the child. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:66-67 pant et al. ©njb, biotechnology society of nepal 67 nepjol.info/index.php/njb discussion fasciolopsiasis is endemic in india, cases being reported mainly from areas including bihar and uttar pradesh but no cases have been yet reported from nepal which is surprising as those areas are connected with the terai region of nepal with open borders and shares cultural and geographical similarities [4]. the cases may have been underdiagnosed due to poor health facility and hence unreported. in the country like nepal where the open defecation around the water bodies is common and the pigs are kept in close contact with humans the prevalence of the disease may be alarming as the habit of eating aquatic vegetation and drinking untreated water is common in nepal. so a study is necessary to determine the prevalence of the fasciolopsis buski infection at least in the areas of nepal which are connected with the high prevalence area of india. the child had history of drinking water from a pond during his stay in india. so it is high chance that the child may have got infection from the water he drank from the pond as no other history which might involve the risk of getting infection by fasciolopsis buski could be elucidated. live adult worms of this parasite are very rarely seen in humans except in autopsy [5]. there are two more reports of live adult worms being vomited out [1, 5]. other cases of live adult worms causing different clinical conditions are reported by cao et al. [2], mahajan et al. [4] and lee et al. [6]. the present case raises the possibility of unidentified cases of fasciolopsiasis in nepal. conclusion with reporting of this case it can be concluded that the cases of fasciolopsiasis are possible in nepal and it should also be considered as differential diagnosis in case of the suspected patients with gastrointestinal symptoms. competing interests the authors declare that they have no competing interests. statement regarding the patient’s consent patient’s guardian’s consent was taken for the publication of this case report. references 1. mohanty i, narasimham mv, sahu s, panda p, parida b: live fasciolopsis buski vomited out by a boy. ann trop med public health 2012,5:403-405. 2. cao yh, ma ym, qiu f, zhang xq: rare cause of appendicitis: mechanical obstruction due to fasciolopsis buski infestation. world j gastroenterol 2015,21(10):31463149. 3. sen–hai y, mott ke: epidemiology and morbidity of food borne intestinal trematodes infections. trop dis bull 1994, 91:r126-r150. 4. mahajan rk, duggal s, biswas nk, duggal n, hans c: a finding of live fasciolopsis buski in an ileostomy opening. j infect dev ctries 2010,4(6):401-403. 5. le th, nguyen vd, phan bu, blair d, mcmanus dp: case report: unusual presentation of fasciolopsis buski in a vietnamese child. trans r soc trop med hyg 2004, 98(3):193-194. 6. lee th, huang ct, chung cs: gastrointestinal: fasciolopsis buski infestation diagnosed by upper gastrointestinal endoscopy. journal of gastroenterology and hepatology 2011, 26(9):1464. http://www.ncbi.nlm.nih.gov/pubmed/?term=le%20th%5bauthor%5d&cauthor=true&cauthor_uid=15024930 http://www.ncbi.nlm.nih.gov/pubmed/?term=nguyen%20vd%5bauthor%5d&cauthor=true&cauthor_uid=15024930 http://www.ncbi.nlm.nih.gov/pubmed/?term=phan%20bu%5bauthor%5d&cauthor=true&cauthor_uid=15024930 http://www.ncbi.nlm.nih.gov/pubmed/?term=blair%20d%5bauthor%5d&cauthor=true&cauthor_uid=15024930 http://www.ncbi.nlm.nih.gov/pubmed/?term=mcmanus%20dp%5bauthor%5d&cauthor=true&cauthor_uid=15024930 http://www.ncbi.nlm.nih.gov/pubmed/15024930 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 25-31 research article doi: https://doi.org/10.54796/njb.v10i1.227 ©njb, bsn 25 phytochemical and antimicrobial screening of bark extract of shorea robusta (sal) bijay kumar shrestha , bidhya dhungana , jenish shakya , romika shrestha , sujata chauhan department of microbiology, central campus of technology, tribhuvan university, hattisar, dharan, nepal received: 10 oct 2020; revised: 29 dec 2021; accepted: 21 jul 2022; published online: 30 jul 2022 abstract different parts of shorea robusta (sal) are being used in ancestral and ayurvedic medicines and are known to cure health ailments. the different phytochemicals present in s. robusta is known to possess antimicrobial property. the different botanical parts of this plant have been used in ayurvedic medicines to cure certain infectious diseases. the main aim of this study was to screen phytochemicals and antimicrobial activity of bark extract of s. robusta. literatures were collected through books, journals and further additional information were collected from residents and traditional ayurvedic practitioners. the ethanolic bark extract of s. robusta was obtained through 70% ethanol in rotatory shaker for 72 hours at 37 ℃ and then the crude extract was dried, preserved and analyzed for phytochemical analysis and antimicrobial activity. the phytochemical screening of ethanolic extract of bark of s. robusta indicated presence of phytochemicals like, alkaloids, flavonoids, tannins, steroids, anthraquinone and absence of phlobatannins, terpenoids, starch and proteins. the extract of s. robusta on staphylococcus aureus exhibited clear zone of inhibition of 21mm at minimum inhibitory concentration (mic) of 2 mg/ml while on escherichia coli exhibited clear zone of inhibition of 9 mm at mic of 4 mg/ml. the antimicrobial activity may be conferred due to the presence of plant phytochemicals. s. robusta bark extract exhibiting significant minimum inhibitory concentration and antimicrobial activity indicates the efficacy of this plant to be considered for discovering and extracting new antimicrobial products against the pathogens. these findings need further support for appropriate formulation of the drug and its therapeutic use in clinical settings. keywords: antimicrobial screening, bark extract, minimum inhibitory concentration, pathogens, phytochemical profile, shorea robusta corresponding author, e-mail: interfacebj@gmail.com introduction shorea robusta is a deciduous large tree, exceptionally reaching a height of 50 m. [1]. s. robusta has always been a tree of medical, cultural and economic importance and is known by common name ‘sal’. s. robusta belongs to family dipterocarpaceae and is reported to possess antimicrobial properties [2]. the entire tree of s. robusta is used for different purposes such as timber in house construction, firewood, leaves for making leaf-plates and cups [3]. the different chemical composition of s. robusta plant such as asiatic acid, triterpenic acid, tannic acid and phenol is known to possess antimicrobial property [4]. the different parts of s. robusta like bark, flower, resins are known to possess pharmacological action to treat diarrheal diseases, diabetes mellitus and bacterial infections etc. [5]. staphylococcus aureus is a pathogenic superbug that causes bloodstream infection, tissue infections and often associated with infective endocarditis [6]. whereas, escherichia coli is prominent cause of gastroenteritis, urine infection and bloodstream infections [7]. these two are the most prevalent and common cause of human infections that induce clinical mastitis and need to be correctly cured by appropriate antimicrobials for preventing and controlling emerging drug resistance and nosocomial infections [8]. since the pathogens are emerging with drug resistance, the need for research in antimicrobial products from medicinal plants can address the issue brought by drug resistant strains in clinical settings [9, 10]. the number of infections caused by emerging drug resistant pathogens grow daily and the hospitalized patients with immunocompromised condition are more prone to severe infections [11]. there is a line of antibiotics on market, but bacterial species shows resistance to most antimicrobials used in the clinical treatment [12]. it has been documented that the bark of s. robusta is traditionally used as astringent, acrid, cooling, anthelminthic, anodyne, constipating, and urinary astringent, union promoter depurative and tonic [13]. they are useful in vitiated conditions of cough, ulcers, wounds, bacterial infections diarrhea, dysentery, nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: https://nepjb.com/index.php/njb issn (print): 2091-1130 mailto:interfacebj@gmail.com https://orcid.org/0000-0002-6542-829x mailto:interfacebj@gmail.com https://orcid.org/0000-0002-5390-9889 https://orcid.org/0000-0002-9397-4511 https://orcid.org/0000-0002-4925-9297 https://orcid.org/0000-0002-7528-3792 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):2 5 3 1 shrestha et al. ©njb, bsn 26 gonorrhea, leucorrhea, pruritus, leprosy, cough, and anemia [14]. therefore, present study was aimed to screen and evaluate phytochemicals and antibacterial activity of crude ethanolic extracts of s. robusta bark extract against s. aureus and e. coli. material and methodology research design this study was conducted from february 2017 to april 2017 at microbiology laboratory of central campus of technology, dharan. bark of s. robusta was collected from bijaypur hill forest of dharan-14 which extends at an altitude of 390 meters (latitude 26° 49′ 12″ n and longitude: 87° 18′ 0″ e). the selected plants were firstly identified from herbarium collection of postgraduate campus biratnagar, nepal. the different botanical information was collected by field study, research articles and books. medicinal, cultural and economic information about the plants was obtained from experienced traditional ayurvedic practitioners and local individuals. all the information about plants, medicinal values and uses were documented. about 500 grams bark samples were collected from the forest and were brought in microbiology lab of central campus of technology, dharan. microorganisms used the bacterial strains used in this research were s. aureus (25923) and e. coli (cft073) strain. these microbial cultures were requested and received from microbiology department of central campus of technology, dharan. the growth media used were mannitol salt agar (msa) (himedia, india) for s. aureus and macconkey agar (mac) (himedia, india) for e. coli. these selective growth medias were used for recovering the bacterial strains from preserved culture for further study. the isolated bacterial strains from selective media were further subcultured in brainheart infusion broth media (himedia, india). extract preparation the extraction methodology was carried out according to agrawal and paridhavi (2012) [15]. the bark of s. robusta was chopped and dried under shade at room temperature of 25 ℃ for two weeks. the dried bark of s. robusta was powdered using mortar and pestle at room temperature. about 20 grams of the powdered plants was extracted with 400 ml of 70% ethanol in rotatory shaker for 72 hours at 37 ℃. the obtained extract was concentrated and dried by evaporation in hot air oven at 60 ℃. stock solutions of 32 mg/ ml was prepared in 10% sterile dimethyl sulfoxide (dmso). the stock solution was stored at 4 °c until use. crude powder extract or aqueous suspension of ethanolic dried extract was used in phytochemical screening. phytochemical assays screening of the phytochemical constituents was carried out for detection of documented chemical constituents of bark extract as described by thilagavathi et al., (2015) [17] and harborne (1998) [18]. test for alkaloids in a test tube, 2 ml ethanolic bark extract was inoculated with 2-3 drops of hcl (dilute hydrochloric acid). to this suspension about 1 ml of dragendorff’s reagent was added. the presence of alkaloids is indicated by the appearance of orange to red precipitate. test for flavonoids in a test tube, 4 ml ethanolic bark extract was suspended with 1.5 ml methanol. the solution was gently heated to warm with addition of magnesium with 4 drops of conc. hcl (concentrated hydrochloric acid). development of color change is indicative for presence of flavonoids. test for phlobatannins bark extract sample was boiled with 1% aqueous hydrochloric acid. suspension of red precipitate is evidence for the phlobatannins. test for tannins about 2 ml of ethanolic bark extract was treated with 23 drops of 10% lead acetate. tannins are indicated by the development of white precipitate. test for steroids about 10 ml of chloroform was taken in a test tube and 2 ml of ethanolic bark extract was added to it. this suspension was treated with 1 ml of acetic anhydride and then 2 ml of concentrated sulphuric acid. the presence of steroids is indicated by the development of blue green color at the junction. test for anthraquinone in a test tube, dilute sulphuric acid and 1 ml of diluted ammonia were added to 5 ml of the ethanolic bark extract. development of pink color indicates the existence of anthraquinone. test for terpenoids about 10 ml of chloroform was added to 2 ml of ethanolic bark extract. the resulting suspension was inoculated with 1 ml of acetic anhydride and 2 ml of concentrated sulphuric acid. the existence of terpenoids is indicated by development of red, pink or violet color at the junction. nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):2 5 3 1 shrestha et al. ©njb, bsn 27 test for starch benedict’s test: one litre of benedict’s solution was be prepared from 100 gm of anhydrous sodium carbonate, 173 gm of sodium citrate and 17.3 gm of copper (ii) sulfate pentahydrate. in a test tube, 0.5 ml of benedict’s reagent was added to 0.5 ml of the ethanolic bark extract. the suspension was heated on water bath at 100 ºc for 2 minutes. the existence of starch is indicated by the development of red color precipitate. test for proteins ninhydrin test: about 1 ml of the ethanolic bark extract was treated with 2-3 drops of ninhydrin agent and heated in a boiling water bath. the presence of proteins is indicated by the appearance of purple blue color. antimicrobial assay antibacterial tests were carried out by well diffusion method as described by aneja (2009) [16]. for antibacterial bioassay, the fresh bacterial inoculum with standard turbidity of 0.5 mcfarland standard for microorganism was arranged in mueller hinton broth (himedia, india) and about 100 µl culture was seeded over the mueller hinton agar (himedia, india). mueller hinton agar (mha) with 2% nacl was seeded by s. aureus culture and mha without 2% nacl was seeded by e. coli culture. with the cork borer no. 6, the wells of about 6mm were created in the media plates. the different test concentrations ranging from, 0.0625-16 mg/ml of bark extract was developed in 10% dmso solution. about 50 µl aliquot of extracts with different concentrations were inoculated into the wells of mha plates seeded by the s. aureus strains. similarly, about 50 µl aliquot of extract with different concentrations were inoculated into the wells of mha plates seeded by the e. coli strains. the inoculated culture medias were allowed to incubate at 37 ℃ for 24 hours. about 50 µl of 10% sterile dmso solution was used for the negative control and penicillin (10 µg) and gentamicin (10 µg) was used as the positive control. after the incubation, the plates were observed for the halo zone around the well. the halo zone around the well represented zone of inhibition which was measured and documented. the experiments were performed for three times and the mean zone of inhibition was computed. minimum inhibitory concentration the minimum inhibitory concentration (mic) of plant extracts was studied using 96-well microtitre plates as explained by clsi (2012) [19]. the 96-well plates were prepared from 95 µl aliquot of mueller hinton broth (mhb) (himedia, india) suspended in wells. in each well, 5 µl bacterial culture of 0.5 mcfarland standard, prepared in mhb medium was inoculated. about 100 µl of stock extract was suspended in first well and so was the same volume of suspension serially diluted to achieve two-fold dilution ranging from 16-0.0625 mg/ml. for negative control dmso solution was used. the microtitre plates were covered with sterile lid and incubated at 37 °c exactly for 24 hours. the minimum concentration of the extract sample, that inhibited growth of tested organism after overnight incubation, was determined as mic. quality control complete aseptic condition was maintained during media preparation, sample collection, sample processing. reagents and culture media were regularly monitored for their manufacture and expiry date and proper storage. laboratory equipment like incubator, refrigerator, autoclave and hot air oven were regularly monitored for their efficiency. the temperature of the incubator and refrigerator was monitored every day. contamination of biological samples was prevented by performing the work in laminar flow cabinet. data analysis the information collected was documented and the data would be analyzed by microsoft excel 2010. results phytochemical screening the phytochemical screening of ethanolic extract of bark of shorea robusta indicated presence of phytochemicals like, alkaloids, flavonoids, tannins, steroids, anthraquinone and absence of phlobatannins terpenoids, starch and proteins (table 1). antimicrobial assay and mic the ethanolic bark extract was tested for antimicrobial activity against both bacteria s. aureus (25923) and e. coli (cft073). the antimicrobial assay with zone of inhibition with positive controls are shown in table 2. table 1. phytochemicals screening of bark of shorea robusta phytochemicals results intensity of color alkaloids present +++ flavonoids present ++ phlobatannins absent tannins present ++ steroids present + anthraquinone present ++ terpenoids absent starch absent proteins absent nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):2 5 3 1 shrestha et al. ©njb, bsn 28 the ethanolic bark extract of s. robusta on s. aureus exhibited clear zone of inhibition of 21 mm at mic of 2 mg/ml. the ethanolic bark extract of s. robusta on e. coli exhibited clear zone of inhibition of 9 mm at mic of 4 mg/ml (figure 1 and 2). the ethanolic bark extract of s. robusta expressed suppressive activity on both s. aureus (25923) and e. coli (cft073). the findings of this study provide significant in vitro antimicrobial activity of ethanolic extract of bark of s. robusta against s. aureus and e. coli. figure 1. antimicrobial screening of bark extract of s. robusta against s. aureus figure 2. antimicrobial screening of bark extract of s. robusta against e. coli figure 3: antimicrobial susceptibility test by s. robusta extract. medicinal information the medicinal, cultural, and economic information was collected from ayurvedic practitioners and locals are included in table 3. table 3. the medicinal, cultural, and economic information of s. robusta plant parts medicinal use other cultural and economic use s. robusta bark enhance immunity power, treat typhoid, diarrhea, ulcer. used in incense stick leaves reduce obesity, inhibit pain. biodegradable leaf plates and cups resins lower fever, skin disorder, diarrhea. used in incense stick discussion nepal has always stood a nation of natural biodiversity rich in natural vegetation that includes herbs, shrubs at different climate and altitude [20]. many of such herbs have been used as traditional medicines by local people including some even been market as ayurvedic medicine [21]. nowadays, pharmacology industries are also seemed interested in incorporating natural drugs since because of modern drugs imposing many health side effects with growing incidence of antibiotic resistance. the s. robusta which is one of the species of plant found in nepal has been known to be best timber producing tree [22]. its many parts have been used by local people for medicinal and cultural purposes [23]. in this study the phytochemical screening of ethanolic extract of s. robusta bark showed the presence of alkaloids, flavonoids, anthraquinone tannins and steroids whereas phytochemicals like terpenoids, starch, phlobatannins and proteins were absent. this result table 2. antimicrobial assay of positive controls microorganisms type of bacteria antibiotic zone of inhibition in mm s. aureus gram + penicillin g (10µg)30 mm e. coli gram gentamicin (10 µg) – 17 mm 0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 7 8 9 16 8 4 2 1 0.5 0.25 0.125 0.0625 14 11 9 0 0 0 0 0 0 mic (mg/ml ) zone of inhibition (mm) 0 5 10 15 20 25 30 1 2 3 4 5 6 7 8 9 16 8 4 2 1 0.5 0.25 0.125 0.0625 26 24 23 21 0 mic (mg/ml ) zone of inhibition (mm) nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):2 5 3 1 shrestha et al. ©njb, bsn 29 coincides with the study performed by [2, 24] which showed the presence of common phytochemicals. similarly, the study performed by [3] showed the absence of anthraquinone which has shown similarity with this present study. there are other many factors that affect phytochemical composition such as geographical condition, climatic condition, collection procedure, storage condition, etc. [25, 26, 27]. the effectiveness of the plant extract against selected bacterial strains may be due to the collective antimicrobial action of different phytochemical constituents [28]. the botanical biomolecules like flavonoids, alkaloids and variety of other phenolic constituents have been identified with antimicrobial properties [29]. the phytochemical screening of ethanolic extract of bark of s. robusta showed the presence of flavonoids, which is an antioxidant compound having antimicrobial property to suppress both certain species of gram-positive and gram-negative bacteria [30]. studies have forwarded different antimicrobial mechanism of phytochemicals. some phytochemicals are supposed to suppress growth and development of microorganism, bacterial cell membrane disarrangement, halting microbial metabolism and modifying microbial genetic expression [31]. investigators have proved antibacterial activity of s. robusta extracts against bacterial pathogens [32]. in one study, s. robusta extract exhibited antibacterial activity against different clinical pathogens [2]. in agreement with this study even in the present study the ethanolic extract of bark of s. robusta exhibited significant antimicrobial activity with significant level of minimum inhibitory concentration against both bacterial pathogens. these results clearly suggest its antimicrobial efficacy in treating the infection caused by those pathogens. plant extract for natural antibiotic could be a strong potent drug against many pathogenic species and could be drug of choice against emerging drug resistant strains [33]. the extended study for purification, activation and therapeutic uses of plant extracts should be conducted to examine its effective antimicrobial role against pathogenic microorganisms. if being effective the nature’s best metabolite could be effective in treating infections caused by antibiotic drug resistance pathogens that have become one of the major therapeutic challenges in clinical settings. the indigenous community of developing nations are still using the plant based traditional drugs for treating many diseases. resins, leaves, flower and bark extracts of s. robusta has been identified with rich medicinal importance [34]. potent antimicrobial drugs extracted from s. robusta may be safe for treating many infections and inflammations. in some asian nations the research on antimicrobial and immunomodulatory effect of the plant has been carried out and even they have come up with excellent results on its antimicrobial and immunological property [35]. these findings will support for scientific research on this plant for pharmacological importance against nosocomial and community acquired pathogens. with new emerging infectious diseases people have been depending upon allopathic, homeopathic and ayurvedic medicines. if proper research could be carried out, then this botanical species could be used for excellent antimicrobial and immunomodulatory medicine. in nepal, the study has been limited to taxonomic study and conservation. the bark of s. robusta is not available like that of other plant products. it is the tree which requires long tenure for its proper growth and development [36]. therefore, scientific tissue culture, conservation and promotion can be achieved through community forest and commercial forestry. where in context of nepal the scientific screening and study of s. robusta based drugs are not adequately performed and analyzed, thus this study will help to provide lead to explore more about the pharmacological importance of s. robusta. conclusion ethanolic extract of s. robusta bark extract displayed acceptable inhibitory activity against both s. aureus and e. coli. the antimicrobial activity may be conferred due to the presence of plant phytochemicals like alkaloids, tannins, anthraquinone etc. further development of the plant-based drugs could bring sustained drug release and would help to reduce side effects of synthetic drugs. in-vivo studies of these plant-based extracts are required for therapeutic application. authors contribution bks and bd designed the study, participated in sample collection, extraction and processing. bks and js participated in literature review, sample extraction, sample processing quality control, data analysis and result interpretation. rs and sc participated in drafting and proofreading the manuscript. all five authors participated in drafting the manuscript and approved for publication. competing interests there is not any competing interest. funding self-financed nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):2 5 3 1 shrestha et al. ©njb, bsn 30 acknowledgements authors want to thank the microbiology department of central campus of technology, hattisar, dharan for laboratory support. authors extend sincere thanks to the residents, traditional ayurvedic medicine practitioners and herbarium collection of postgraduate campus biratnagar, nepal for immense support and response. ethical approval and consent not applicable. abbreviations msa: mannitol salt agar mac: macconkey agar mha: mueller 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2(2):77-85. 32. 32. vashisht s, singh mp, chawla v. in-vitro antioxidant and antibacterial activity of methanolic extract of shorea robusta gaertn. f. resin. international journal of pharmaceutical and phytopharmacological research. 2016;6(4):68-71. doi: 10.24896/eijppr.2016641. 33. 33. subramani r, narayanasamy m, feussner k. plantderived antimicrobials to fight against multi-drug-resistant human pathogens. 3 biotech. 2017; 7(172). doi: https://doi.org/10.1007/s13205-017-0848-9. 34. 34. luitel dr, rokaya mb, timsina b, münzbergová z. medicinal plants used by the tamang community in the makawanpur district of central nepal. j ethnobiol ethnomed. 2014;10(5):1-11. doi: 10.1186/1746-4269-10-5. 35. 35. dhama k, tiwari r, chakraborty s, saminathan m, kumar a, karthik k, wani my, amarpal ss, rahal a. evidence based antibacterial potentials of medicinal plants and herbs countering bacterial pathogens especially in the era of emerging drug resistance: an integrated update. international journal of pharmacology. 2014;10(1):1-43. doi: 10.3923/ijp.2014.1.43. 36. 36. sapkota p, meilby h. modelling the growth of shorea robusta using growth ring measurements. bankojanakari. 2009;19(2):25-32. doi: https://doi.org/10.3126/banko.v19i2.2982. nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 3 2 3 9 research article doi: https://doi.org/10.54796/njb.v10i1.228 32 ©njb, bsn 32 quality evaluation of apis laboriosa and apis mellifera honey collected from bagmati province, nepal abhishek bajgain, bashu dev neupane, diwakar sarraf, jwalant karmacharya, saksham ranjitkar, rajan shrestha, rajendra gyawali department of pharmacy, school of science, kathmandu university, dhulikhel, kavre, nepal received: 9 jun 2022; revised: 22 jun 2022; accepted: 4 jul 2022; published online: 30 jul 2022 abstract honey is a natural sweet substance produced by apis sp. from floral nectar or other plant parts which are gathered, modified and stored in the honeycombs by honeybees. the current research was aimed to analyze the quality parameters of locally available honey. honey samples of apis laboriosa and apis mellifera were collected during spring of 2019 & 2021 and autumn 2021 from the bagmati province, nepal. samples were analyzed their physicochemical and phytochemical properties. the result shows that, the ph was ranged between [4.467±0.0306 5.05±0.02], rheological studies showed newtonian flow and pseudoplastic type of non-newtonian flow, specific optical rotation was ranged between [(+) 5.75±0.4684 (-) 12.71±0.234], specific gravity was ranged between [1.35±0.00017 1.409±0.00022], moisture content was ranged between [19.2% 25%]. secondary metabolite screening showed the honey samples possesses flavonoids, saponins, glycosides, tannins, amino acids, protein and reducing sugar. total phenolic content was ranged between [1.0427 6.86288] gm gae/kg honey while total flavonoid content ranged between [0.016755 0.353132] gm qe/kg honey. ic50 obtained from dpph assay ranged between [649.6465 9867.1617] ppm. properties and qualities of honey are affected by seasonal factors and various floral sources. the samples were in positive correlation between flavanoid content, phenolic content and their respective anti-oxidant potency. keywords: honey, quality, physicochemical properties, phytochemicals, tpc, tfc, antioxidant. corresponding author, email: ragyawali@gmail.com introduction honey is a natural sweet substance produced by honey bee, which can be classified based on type of honey source, floral and extra floral honey[1]. apis laboriosa is world’s largest honeybee species with measurement of up to 3.0 cm, which are found at an altitude range from 2,500 m to 4,000 m above the sea level, building their nests commonly in higher altitude, 1,200 m. above the sea level. honey is harvested twice a year in spring season and in autumn season, with spring one showing strong medicinal property than autumn one [2].the medicinal property of honey is that, the floral distribution of the region, where apis laboriosa lives; is the plants that belong to ericaceae (rhododendron) family, which have a psychoactive and hallucinogenic group of phytochemistry known as grayanotoxins[3]. in nepal, it is estimated that over 10,000 mt of honey is produced and every year growing the production. honey is reported to contain at least 181 substances, high nutritional value, high refractive index, high viscosity and specific gravity [4][5][6]. to the best our knowledge, this is the first comparative study on nepalese honey to investigate wide range of quality parameters. the present study was aimed to carry out the quality assessment and characterization of honey of apis laboriosa harvested from bagmati province, nepal. material and methods honey collection in this study, honey samples of apis laboriosa and apis mellifera were harvested on spring of 2019 & 2021 and autumn 2021 from bagmati province, nepal. the information about samples profile is as given below in the figure 1, figure 2 and table 1. physical properties in a view to identifying the purity of honey based on physical properties following preliminary tests were performed. sand sinking test in this test, 50 g sand was taken from nearby and was sieved through mesh size 30. the sand was then allowed to dry completely in oven at a temperature of 95℃ for 15 minutes. the particle distribution profile of the sand was examined using electromagnetic sieve shaker (model: ems-8, instrument manufacturer: electropharma). after that, four petri plates were kept in a row. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: https://nepjb.com/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-5745-0702 mailto:ragyawali@gmail.com nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 33 ©njb, bsn 33 figure 1. google earth snapshot showing honey harvested location figure 2.f different honey samples (from left to right; s1, s2, s3, and s4). with the help of a tripod stand, the funnel was set and the sand was allowed to flow through the funnel, which created a conical shape of sand over the petriplates. to the sandhill, three drops of each honey sample were placed in each petriplates, and the time required to sink through the sand surface was recorded. delayed sinking honey samples are considered to be pure as those honey samples contain less amount of moisture and are not adulterated [7]. water sinking test in this test, a 5 g of each sample was taken and it was poured into a beaker containing deionized water. after that, the nature by which it interacts with water was observed. directly sinking honey samples to the bottom of the vessel without mixing with water unless stirred are considered to be pure [7]. air-flow test in this test, a clean glass stirrer was taken and was dipped into the vessel containing the honey sample. the stirrer was then rotated through the honey sample table 1. honey collection site of bagmati province, nepal sample no. harvested time types of honey honeybee species harvested location year season s1 2019 spring honeydew honey apis laboriosa uttargaya r.m. – 1, karyangmaryang, rasuwa [39.1 km perimeter, 71.9 sq. km area] s2 2021 spring general nectar honey apis laboriosa uttargaya r.m. – 2, thulogaun, rasuwa [28° 1'28.36"n, 85° 9'50.73"e] s3 2021 autumn general nectar honey apis laboriosa uttargaya r.m. – 2, thulogaun, rasuwa [28° 1'28.36"n, 85° 9'50.73"e] s4 2021 autumn diploknema butyraceae nectar honey apis mellifera rakshirang r.m., silinge, makwanpur [42.7 km perimeter, 48.8 sq. km area] nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 34 ©njb, bsn 34 and it was raised to a certain height above the honey surface to allow for free-flow of the honey samples. the flowing nature of honey was then observed. honey samples, which flow in a continuous thread-like pattern, are considered to be pure [7]. physicochemical properties colour colour tone of honey was noted, which also revealed the identity and nature of various floral sources and helped in the differentiation of types of honey. ph firstly, the ph meter (model: ph 211 microprocessor, instrument manufacturer: hanna instruments) was calibrated using qualigen (fisher’s scientific) buffer tablets of ph 4.0, 7.0, and 9.2. as specified in the label of the buffer tablets, each buffer tablet was dissolved in distilled water to the volume mark of 100 ml volumetric flask. after the device has been calibrated, each honey sample was checked for its ph readings. the ph range of unadulterated honey should lie between the range of 3.5 to 5.5 [8]. rheological properties to make uniform treatment on viscosity, overall samples were maintained at 41±1℃ by using an electric air-heater. viscosity was analyzed by viscometer (model: dv-iii ultra programmable rheometer, instrument manufacturer: brookfield) attaching spindle size of 63, the rheological properties of honey were studied [1]. optical rotation for this experiment, 1.0% (w/v) of the honey sample in distilled water was prepared at first. to the prepared sample, using blank correction as distilled water; the prepared sample was filled in the tube of polarimeter (model: bk-p2s, instrument manufacturer: biobase biodustry [shandong] co. ltd.) and its triplicate reading was taken. honeydew honey should exhibit positive optical rotation while nectar honey exhibit negative optical rotation to the incident plane-polarized light [6]. by using the following equation, specific optical rotation was computed: [𝛼]𝜆 𝑇 = 𝛼𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑏 × 𝑐 where, [𝛼]𝜆 𝑇 = specific rotation in degree at t℃ and light wave-length (𝜆) 𝛼𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 = observed rotation in degree b = path-length in the decimeter c = concentration in gm/ml relative density using the pycnometer (glassware manufacturer: jain scientific glass works [jsgw]), digital analytical balance (instrument manufacturer: bel engineering), and laboratory thermometer, the density of our four (4) honey samples were determined. the room temperature of the lab during the time of the experiment was also measured with the help of a laboratory thermometer. after that, an empty pycnometer was taken and its mass was noted using digital analytical balance. now, it was filled with the honey sample and again its mass was weighed. the density of uncontaminated honey typically ranges between 1.38 and 1.45 gm/ml [9]. 𝐷𝑒𝑛𝑠𝑖𝑡𝑦(𝜌) = 𝑊𝑡𝐹𝑖𝑙𝑙𝑒𝑑 − 𝑊𝑡𝐸𝑚𝑝𝑡𝑦 25 where, wtfilled =the mass of pycnometer after sample filling wtempty =the mass of the empty pycnometer before filling refractive index the refractive index of the honey samples was measured by using abbe refractometer (model: sn 4040, instrument manufacturer: guru nanak instruments, new delhi). sample holding prism of the instrument was cleaned well by rinsing it, with the help of ethanol and soft tissue paper. after that, a drop of honey sample was then loaded into the lower sample prism. now, the upper sample prism was interlocked with the lower sample prism which facilitated contact between the two sample prisms and ultimately formed a film layer of honey sample. by viewing through the eye-piece, coarse scale adjustment and fine scale adjustment knobs were rotated and readings were noted. refractive index of unspoiled honey typically ranges from 1.474 to 1.504 indicating the presence of water content in honey from 25% to 13% respectively [10]. pollen contents honey sample was prepared in a conical flask and it was left to shake in incubator shaker (manufacturer: biobase biodustry [shandong] co. ltd.) at 100 revolution per minute (rpm) for 15 minutes at 45℃. the stock solution was then poured into the centrifugation tube. after that, it was allowed to centrifuge at 5000 rpm for 10 minutes in centrifugation apparatus (model: nf 200, manufacturer: nüve laboratories). the settled precipitate was scraped out and finally, it was observed in the microscope for determination of its shape and size. nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 35 ©njb, bsn 35 qualitative screening test of phytochemical classes plant metabolites were screened according to previously established methods [11-13] total phenolic content total phenolic content was determined by folinciocalteu (thermo fisher’s scientific india pvt. ltd.) method, according to previously published method [12].the honey stock solution was prepared at 10% in water. a portion of 1 ml of the honey and 0.8 ml of 10 % aqueous reagent and followed by 2 ml of 15% sodium carbonate was added. final volume was made by adding water. mixture was incubated for 2 hrs, and absorbance was measured at 765 nm against the blank. a standard curve of gallic acid was prepare for quantification, using a concentration range of 250, 500, 750, 1000 and 1250 ppm and result were expressed as mg gallic acid/kg honey. calculation of tpc was done using the following formula expressed in mg gallic acid equivalent (gae) per kg of honey sample, 𝑇𝑃𝐶 = 𝐺𝐴𝐸(𝑚𝑔/𝑙) × 𝑉(𝑚𝑙) × 10−3 (𝑙/𝑚𝑙) × 𝐷𝑓 𝑊𝑡𝑆𝑎𝑚𝑝𝑙𝑒 (𝑔𝑚) × 10 −3(𝑘𝑔/𝑔𝑚) where tpc =total phenolic content (in mg gae/kg honey) gae = gallic acid equivalent (in mg/l) v = total volume of methanol extract (in ml) df = dilution factor total flavonoid content total flavonoids content in honey was determined by a calorimetric method according to previous method. briefly, 1.0 gm of honey sample was taken, which was dissolved in 10 ml 80% ethanol to make sample stock solution. the sample stock solution was then allowed to incubate in incubator shaker (biobase biodustry [shandong] co. ltd.) at 100 rpm for 15 minutes at 45℃. total 1.0 ml of supernatant sample stock solution was pipette out to which 0.2 ml of 10% (w/v) aqueous aluminum chloride was then added and subsequently, 0.2 ml of 1 m. aqueous potassium acetate and 3 ml of 80% ethanol was added. finally, volume make-up was done to the mark adding sufficient distilled water and absorbance reading was measured at the wavelength of 415 nm against blank solution from the standard stock solution.[12].tfc was done using the following formula expressed in mg quercetin equivalent per kg of honey; 𝑇𝐹𝐶 = 𝑄𝐸(𝑚𝑔/𝑙) × 𝑉(𝑚𝑙) × 10−3 (𝑙/𝑚𝑙) × 𝐷𝑓 𝑊𝑡𝑆𝑎𝑚𝑝𝑙𝑒 (𝑔𝑚) × 10 −3(𝑘𝑔/𝑔𝑚) where, tfc is total phenolic content (in mg quercetin/kg honey) qe is quercetin equivalent (in mg/l) v is total volume of ethanol extract (in ml) df is dilution factor anti-oxidant dpph assay dpph assay was estimated using the 2,2-diphenyl-1picrylhydrazyl hydrate radical (dpph) (glentham life science ltd, uk)) according to previous method of kačániová[14 15]. the honey samples were diluted in methanol at concentrations of 400, 800, 1200, 1600 and 2000 ppm solutions and from each dilution 0.3 ml was mixed with dpph. the mixtures were vortexed, left in dark room temperature for 60 min and the absorbance was measured at 517 nm under uv-visible spectrophotometer (uv 1800, manufacturer: shimadzu scientific instruments) correcting baseline blank correction by methanol. the inhibition % is given by the relation 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑆𝑎𝑚𝑝𝑙𝑒 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100% where acontrol = mean absorbance reading of 0 ppm solution against methanol as blank asample =e mean absorbance reading of 400, 800, 1200, 1600, and 2000 ppm solutions results and discussions preliminary purity test sand sinking test s1 took shortest period of time among all the samples while s4 took longer period of time to sink through sand-hills. various time (in seconds) taken by samples to sink through the sand surface is as shown in table 2. table 2. time of sand sinking test s.n. sample time (in seconds) 01. s1 62.33±2.08 02. s2 197.33±2.52 03. s3 262.33±2.517 04. s4 900±0 water sinking test all of our honey samples went down to the bottom of the cup without mixing up with the water except when stirred, which showed that all our honey samples complied the water sinking test as mentioned in literatures mentioned above. air flow test all the honey samples went down like a thread without breaking ranging 0.5 to 3 seconds as a continuous thread, which also showed that all our honey samples nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 36 ©njb, bsn 36 complied for the air flow test as mentioned in literatures mentioned above. confirmatory purity test colour from colour shade analysis of honey samples with that of reference shade of yellow colour, our different honey samples demonstrated varieties of colour as listed in table 3. table 3. different shades of honey as per base yellow colour s.n. sample shade of yellow colour 1. s1 amber 2. s2 light amber 3. s3 white 4. s4 extra-light white ph presence of carbohydrate in dominant amount is the reason to which honey shows slightly acidic nature. due to the reason, honey has a good anti-microbial potency. among four samples, s3 showed highly acidic with ph of (4.467±0.0306) where s1 showed lowest ph with the value of (5.05 ± 0.02). various values of ph shown by different honey samples are tabulated down in table 4. table 4. various ph readings of different honey samples s.n. sample ph 1. s1 5.05±0.02 2. s2 4.833±0.0379 3. s3 4.467±0.0306 4. s4 4.767±0.0416 rheological properties among the samples, only s1 showed newtonian type of flow property, while rest of the samples showed pseudo-plastic flow property upon checking rheological properties of honey samples using brookfield viscometer. plot of rotation per minute (rpm) versus torque% and rpm versus viscosity (in centi-poise) of different honey samples as shown in figure 3-6. figure 3. rpm versus torque% and rpm versus viscosity (cp) (i.e. rheological properties of s1) figure 4. rpm versus torque% and rpm versus viscosity (cp) (i.e. rheological properties of s2) 320 345 370 395 0 100 200 300 v is co si ty ( cp ) rpm s2 0 20 40 60 80 100 0 50 100 150 t o rq u e % rpm s3 0 20 40 60 80 100 0 50 100 150 t o rq u e % rpm s1 0 20 40 60 80 100 0 100 200 300 t o rq u e % rpm s2 0 50 100 150 200 250 300 0 50 100 150 v is co si ty ( cp ) rpm s1 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 37 ©njb, bsn 37 figure 5. rpm versus torque% and rpm versus viscosity (cp) (i.e. rheological properties of s3) figure 6. rpm versus torque% and rpm versus viscosity (cp) (i.e. rheological properties of s4) specific optical rotation from the literatures, it is established fact that honeydew honey and adulterated honey only shows positive specific optical rotation, while nectar honey shows negative specific optical rotation to the plane polarized light. s2 showed highest angle of rotation among the four samples while s1 showed positive angle of rotation. various values of degree of rotation are shown in table 5. specific gravity honey is denser than water with 1.3 to 1.4 folds. s3 and s1 are the respectively highest and lowest dense honey samples among the samples involved in this study. table 6. shows different values of specific gravity which was obtained during analysis. table 5. specific optical rotations of different samples at concentration of 1.0% (w/v) in water. s.n. sample specific optical rotation 1. s1 (+)5.75±0.4684 2. s2 (-)12.71±0.234 3. s3 (-)8.79±0.3098 4. s4 (-)2.299±0.3098 table 6. specific gravity of samples observed at room temperature of 14℃. s.n. sample specific gravity 1. s1 1.35±0.00017 2. s2 1.404±0.0016 3. s3 1.409±0.00022 4. s4 1.376±0.00497 refractive index and moisture content moisture content in honey samples is indicated by the refractive index values of honey samples. s1 has highest refractive index and thus moisture content and s4 has lowest refractive index and moisture content upon analysis of four different samples. table 7 is listed with different observations of refractive index and moisture content of all four honey samples involved in the study. table 7. refractive index and moisture content (in %) of samples s.n. sample refractive index moisture content (in %) 01. s1 1.473 25 02. s2 1.476 24.2 03. s3 1.476 24.2 04. s4 1.489 19 figure 7. microscopic view of pollen content at 400x magnification of s1(1), ,s2(2), s3(3) ands4(4). microscopic studies of pollen contents upon viewing microscopic slides of different honey samples, we found out the spring season harvested honey containing the same kind of pollen and it was different from autumn harvested sample. for the case of s4, as it is nectar honey of diploknema butyraceae; the honey sample exhibit different kind of pollen contents in it, which is totally different from all of the honey samples. figure 7 contains the pictures of microscopic slides of different honey samples. 100 150 200 250 300 0 50 100 150 v is co si ty ( cp ) rpm s3 0 20 40 60 80 0 50 100 150 t o rq u e % rpm s4 640 660 680 700 720 740 0 50 100 150 v is co si ty ( cp ) rpm s4 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 38 ©njb, bsn 38 secondary metabolites upon performing qualitative screening of secondary metabolites in honey samples; there was positive test for flavanoids, glycosides, saponins, tannins, reducing sugar, terpenoids, amino acids and proteins. there was negative test for alkaloids and phlobatanins. table 8 shows the summary of results for all four honey samples as obtained from qualitative screening of secondary metabolites. table 8. qualitative screening of secondary metabolites in different honey samples s.n. name of test s1 s2 s3 s4 01. mayer’s test for alkaloid 02. flavanoid test +++ +++ +++ +++ 03. browntoger’s test for glycosides + + + + 04. foam test of saponins ++ ++ ++ ++ 05. ferric chloride test for tannins + + + + 06. benedict’s test for reducing sugar ++ +++ ++ +++ 07. xanthoproteic test for amino acids and protein +++ +++ +++ +++ 08. terpenoids test +++ +++ +++ +++ 09. phlobatannins test negative, + weak positive, ++ moderate positive, +++ strong positive quantitative determination of secondary metabolites tpc and tfc upon plotting of standard calibration plot of gallic acid for tpc and quercetin for tfc, we obtained following graphs as shown in figures with r2 of 0.9658 and 0.981 respectively for the linear regression keeping concentration (in ppm) along x-axis and mean absorbance along y-axis. mean absorbance is the average of absorbance reading taken from triplicate data. figure 8. standard calibration curve of gallic acid for tpc figure 9. standard calibration curve of quercetin for tfc using above standard calibration curve and linear regression equation of line, tpc and tfc was found to be highest in s1 and lowest in s3. table 9 shows the results of estimated tpc and tfc of different honey samples. table 9. quantitative determination of secondary metabolites s.n. sample tpc (gm gae/ kg honey) tfc (gm qe/ kg honey) 01. s1 6.862884943 0.3531322456 02. s2 1.626269056 0.1607248345 03. s3 1.042708154 0.01675598334 04. s4 2.615476423 0.1883074849 tpc = total phenolic content and tfc = total flavanoid content figure 10. concentration versus inhibition% graph from dpph assay (anti-oxidant of different samples at varying concentration) table 10. results of anti-oxidant assays (ic50 values and times potency with the respect to standard) s.n. samples ic50 (in ppm) times potency 01. quercetin 346.9112586 1.000 02. s1 649.6465517 0.534 03. s2 2353.956344 0.1475 04. s3 9867.161765 0.0353 05. s4 1390.792215 0.2497 y = 0.00338x + 0.09117 r² = 0.96580 0 0.2 0.4 0.6 0.8 1 1.2 0 100 200 300 m e a n a b so rb a n ce concentration (ppm) tpc y = 0.0426x + 0.0519 r² = 0.9811 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 a b so rb a n ce concentration (ppm) tfc y = 0.00348x + 47.73923 r² = 0.93397 y = 0.00733x + 32.74550 r² = 0.96365 y = 0.00272x + 23.16132 r² = 0.91245 y = 0.01824x + 24.63195 r² = 0.93571 y = 0.02076x + 42.78935 r² = 0.97686 20 30 40 50 60 70 80 90 300 1300 in h ib it io n % inhibition concentration [ppm] dpph assay s1 s2 s3 s4 quercitin extract from allium cepa nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):3 2 3 9 bajgain et al. 39 ©njb, bsn 39 anti-oxidant dpph assay upon plotting, concentration (in ppm) versus % inhibition of different honey samples and similarly, standard of quercetin extract extracted from allium cepa, we obtained various ic50value from the plot, through which we became able to compare between the times potency value of anti-oxidant ability of different samples to that with standard quercetin extract. from the observation, we found out that s1 had maximum potency with 0.534 times and s3 has minimum potency with 0.0353 times of standard quercetin extract from allium cepa. figure 10 is the plot of inhibition concentration (in ppm) versus % inhibition of different samples and standards involved in the study. table 10 is the result of ic50 value and times potency of different honey samples obtained from the calculation. conclusions different tests performed during this research complied with the various standard research articles published in different journals. potency of honey is affected by the types of honey and seasons at which it has been harvested. honey with lower anti-oxidant potency can be used in formulation of topical cosmetological products or as daily dietary supplement. honey with higher values of anti-oxidant potency can be used in combination with other different active ingredients for the formulation of different therapeutic products. conflict of interest the author declares no conflict of interest. authors contribution all authors have equal contribution references 1. bambang n, ikhsan m, tensiska, sukri n, mahani, rheological properties of honey and its application on honey flow simulation through vertical tube. iop conf ser earth environ sci. 2019 oct 1;334(1):012041. 2. kitnya n, prabhudev mv, bhatta cp, pham th, nidup t, megu k, et al, geographical distribution of the giant honey bee. 2020;15. zookeys 951: 67 81. doi: 10.3897/zookeys.951.49855 3. jansen sa, kleerekooper i, hofman zl, kappen if, staryweinzinger a, van der heyden ma. grayanotoxin poisoning: 'mad honey disease' and beyond. cardiovasc toxicol. 2012 sep;12(3):208-215 4. khalil mi, sulaiman sa, boukraa l, antioxidant properties of honey and its role in preventing health disorder. :11. the open nutraceuticals journal 2010, 3, 6 -16. 5. nwankwo, cm, ezekoye, cc, igbokwe so, phytochemical screening and antimicrobial activity of apiary honey produced by honey bee (apis mellifera) on clinical strains of staphylococcus aureus, escherichia coli and candida albicans. afr j biotechnol, 2014 jun 4;13(23):2367–72. 6. dinkov d, a scientific note on the specific optical rotation of three honey types from bulgaria. :2. page 319 320 7. asokan, s. and jayanthi, 2017, “phytochemical analysis of various honey samples obtained from theni district, south india", international journal of current research, 9, (01), 45387-45390. 8. kivima e, tanilas k, martverk k, rosenvald s, timberg l, laos k. the composition, physicochemical properties, antioxidant activity, and sensory properties of estonian honeys. foods. 2021 march 1;10(3):511. 9. tomasik p, chemical and functional properties of food saccharides, chapter 6, page no 74, isbn 978-0-203-49572-8. international standard book no-13:978-0-203-49572-8 10. stefan bogdanov, honey as nutrient and functional food, in book of honey, chapter 8, bee product science, 2016, 1-47. 11. amabye tg, phytochemical and biochemical compostion of wild honey a case study in estern zone areas in tigray ethiopia. moj food process technol. 2017;4(3):88‒94. doi: 10.15406/mojfpt.2017.04.00094 12. özkök a, d’arcy b, sorkun k, total phenolic acid and total flavonoid content of turkish pine honeydew honey. j apiproduct apimedical sci. 2010 apr 1;2(2):65–71. 13. adalina y, kusmiati e, pudjiani m, phytochemical test and physical chemical properties of rubber honey from three types of bees ( apis mellifera, apis dorsata and trigona itama ). iop conf ser mater sci eng. 2020 sep 23;935:012007. 14. subroto e, lembong e, filianty f, indiarto r, primalia g, theodora hc, et al, the analysis techniques of amino acid and protein in food and agricultural products. 2020;9,29-36. 15. kačániová et al, antimicrobial and antiradical activity of slovakian honeydew honey samples. journal of microbiology, biotechnology and food sciences 2011/12 1 (3) 354 368. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):29-35 doi: https://doi.org/10.3126/njb.v8i1.30207 research article ©njb, bsn 29 phytochemicals levels and antioxidant capacities of figs flowers fruits mohammed messaoudi , maroua merah university dr moulay tahar, po box 138 al-nasr district 20000, saida –algeria article history:received: 7 dec 2019; revised: 5 apr 2020; accepted: 16 jun 2020; published online: 31 jul 2020 abstract since antiquity, phenolic compounds produced by plants were known as free radical scavengers and as powerful antioxidants. huge interest has been made by researchers to the traditional uses of medicinal plants against illnesses related to oxidative stress. this study measures the correlation that can be existed between the antioxidant capacity and phytochemicals levels of four varieties of ficus carica fruits, figs flowers or "bakor" as called locally in algeria. therefore, extracts were assessed for determining their antioxidative potentials using both test of total antioxidant capacity and dpph (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test followed by quantitative phytochemical analysis to estimate the total flavonoid level (tfl), the total phenolic level (tpl), the total anthocyanin level (tal) and the condensed tannins level of plants methanolic extracts. a positive correlation was observed between phenolics content and the antioxidant capacity of figs flowers methanol extracts. the methanolic extract of bechar (meoh var.2) chelated 87. 9± 1.23 % of the dpph free radical with ic50 value equal to 0.185 mg/g dw. a high antioxidant ability of almost all extracts is, probably, related to the appreciable rates of flavonoids, phenolics and tannins showed by those fig extracts. the highest value of phenolics level was detected among the variety 1 methanolic extract of bechar (meoh var.1) of 10.4 mg gae/g dw. keywords: antioxidant capacity; bakor; figs flowers; ficus carica; methanol extracts; dpph. corresponding author, email: microbiologistemed@yahoo.fr introduction phytochemicals, including phenolics, flavonoids, flavonols, ascorbic acid, lignin, xanthones, stilbenes, etc., are plant-based secondary metabolites, which are associated with the protection of human health against chronic diseases [1, 2, 3, 4]. the relative importance of medicinal and food plant species can be assessed by their use-value. plant species with more traditional uses exhibit high use value compared to those which have fewer ones [5]. nowadays, medicinal plants considered as an important source of drugs as about 25% of the drugs prescribed worldwide derive from plants [6]. fig tree ficus carica linn. originated in the middle east areas such as syria, asia minor, and iran, then, it was spread to the mediterranean basin countries by old humans [7, 8]. it belongs to the family of moraceae. f. carica l. is one of the unique widely spread ficus species that has edible fruits with high commercial value. the production of commercial fig is situated in regions that possess a mediterranean climate [9]. f. carica l. has three figs yields, early fig stays on the tree; late fig of autumn or figs flowers carries from august to winter and is locally known as bakor and the green or winter figs [10, 11]. oxidative stress is an inequality between prooxidants and antioxidants in favor of the first contributing to the appearance of several pathologies. the uncontrolled oxygen species resulted will have serious and severe consequences for the human organism [12]. several studies focus on natural antioxidant sources to find new effective, safe and cheap antioxidants as there is a strong relationship between the decrease of certain chronic diseases and plants-produced antioxidants [13]. fruits are essential functional foods that maintain the human vital functions as they providing a well-balanced diet [14]. viewing the biological properties of f. carica fruits, our study focuses on the correlation between phytochemicals contents and antioxidant capacity of dried fruits methanolic extracts of figs flowers or "bakor" originated of four different varieties of f. carica. two varieties of bechar and the two others from mascara. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:microbiologistemed@yahoo.fr http://orcid.org/0000-0001-9893-5987 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):29-35 messaoudi and merah ©njb, bsn 30 materials and methods collection of plant samples fruits from four different varieties of f. carica figs flowers or "bakor" as called locally were collected between may and august 2018. two varieties from bechar located at the southwest of algeria: var.1 (lahmar), var.2 (ouakda). the other two ones from mascara situated at the north of algeria: var.3 (el bordj), var.4 (ghriss). plants specimens were identified by the laboratory of biotoxicology, pharmacognosy and biological valorisation of plants (university of saida). figure 1. fruit of ficus carica l. (left: whole fruit; right: cross-section) [15, 16]. preparation of methanolic extracts four samples of fig flowers were air-dried and crushed. after, 1 g of f. carica fruits was soaked, under ultrasound, using pure methanol (20 ml) for 24 h. plants extracts were filtered, concentrated and stored at 4°c until used (meoh var.1, meoh var.2, meoh var.3, meoh var.4) [17]. phytochemical screening chemical products and reagents purchased from merck company, darmstadt, germany and phytochemical screening tests were repeated for three times. determination of percentage yield the percentage yield was calculated for each extract using the formula: percentage yield (%) = a/b × 100 where: (a)=the dry weight of extract, (b)=soaked samples material [18]. determination of total flavonoids level (tpl) to measure the total flavonoids level, volumes of 2 ml from both plant extracts and 2% ethanolic solution of aluminum trichloride (alcl3) were mixed and incubated 10 min at room temperature. after measurement of absorbance at 430 nm, tfl resulted in mg quercetin per g dry weight [19]. determination of total phenolic level (tpl) total phenolics level was estimated via folin– ciocalteu's reagent. a mixture of sodium carbonate solution (2 ml, 2%) and 0.1 ml of plant extract was incubated for 5 min. then, a volume of 100 μl folin– ciocalteu's reagent was added. the mixture was incubated for 30 min and absorbance was read at 700 nm to determine the tpl as mg gallic acid per g dry weight (mg gae/g) [20]. determination of total anthocyanins level (tal) the ph differential method was used to deduce total anthocyanins level, by which, the absorbance of the reaction solution was measured at both 510 nm and 700 nm at two ph 1.0 then ph 4.5 using buffer systems: hydrochloric acid (0.2 m) and sodium acetate (1 m). a= (a510-a700)ph1,0 (a510-a700)ph4,5 tal=[(a x mw x df) / ma] x 100 a: absorbance; mw: molar mass; df: dilution factor; ma: molar absorption. tal was expressed as mg cyanidin-3-glucoside per g of dry weight (mg c3g/g) [2, 21]. determination of condensed tannins level (ctl) the vanillin test was used to estimate the condensed tannins level (ctl). the mixture contained methanolic solution of vanillin (4%, w/v), methanol (37%, v/v), hcl (8%, v/v), at equal volume was kept at 30 ° c until used. then, a solution of 1500 μl of vanillin methanolic solution was added to 50 μl of plant extracts and 750 μl of concentrated hcl was incubated for 20 min. the absorbance was read at 550 nm and compared to a blank of equal volumes of both 37% methanol and 8% hcl. results were expressed in mg of catechin equivalent per gram of the dry weight (mg ce/g) [22, 23, 24]. tests of antioxidant capacity the antioxidant capacity of the four varieties were evaluated using free radical scavenging test (dpph) and total antioxidant capacity (tac). nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):29-35 messaoudi and merah ©njb, bsn 31 dpph free radical scavenging test dpph scavenging test was used to estimate the capacity of each extract to scavenge hydrogen atom generated of 2,2-diphenyl-1-picrylhydrazil radical. a mixture of 1 ml of 100 μm methanol solution of the free radical dpph and different concentrations of each extract was incubated 20 min. the absorbance of the solution was read at 517 nm and compared to the blank that contained both 1 ml of dpph methanolic solution and 0,1 ml of methanol solvent [25, 26]. the inhibition percentage (% ip) of dpph solution was estimated by the following formula: % ip = [(at0at20) / at0× 100)] where, at0= absorbance of the blank solution after 20 min. at20= absorbance of each sample after 20 min. then, the concentration of an extract that allowing to 50% inhibition of dpph solution (ic50) was obtained graphically. the less is the ic50, the higher is the antioxidant capacity. a positive control with various concentrations was prepared by the methanolic solution of ascorbic acid [27]. total antioxidant capacity (tac) the test of phosphomolybdenum reagent allows the determination of total antioxidant capacity (tac) of plant samples. a mixture that contains a 3 ml volume of a solution of ammonium molybdate reagent (4 mm), sulphuric acid (0.6 m) and sodium phosphate (28 mm) added to a volume 0.3 ml of each extract was incubated at 95°c. after, 90 min, the absorbance was measured at 695 nm against a control solution prepared under the same conditions, constituted of 0.3 ml of methanol and 3 ml of all the reagents used before. total antioxidant capacity expressed as mg ascorbic acid per g dry weight (mg aae / g). a calibration curve with various concentrations was prepared using ascorbic acid [28]. statistical analysis the experimental data obtained from the tfl, tpl, tal, ctl and antioxidant capacity tests were expressed as means ± standard deviation. statistical analysis of data was performed using microsoft excel. statistical differences were estimated, oneway anova and student’s t-test were used. the correlation coefficient of antioxidant capacities as determined by the pearson test. values are to be statistically significant at p < 0.05. results and discussion total flavonoids level, total anthocyanins level, total phenolics level, condensed tannins level of fig flowers fruits (bakor) methanolic extracts are presented in figure 2. the total phenolics level of the four extracts varied from 4.7 to 10.4 mg gae/g dw. the highest level was significantly (p<0.05) conferring to the variety 1 methanolic extract of bechar (meoh var.1) and the lowest in the extract meoh var.3 of mascara (see figure 2). figure 2. levels of various phytochemicals of fig flowers fruits methanolic extracts. meoh var.1; meoh var.2: variety 1, 2 bakor (fig flowers) of bechar. meoh var.2; meoh var.3: variety 2 bakor of mascara. tfl: total flavonoids level, tal: total anthocyanins level, tpl: total phenolics level. ctl: condensed tannins level. mg qe/g : quercetin equivalents; mg c3g/g : cyanidin-3-glucoside equivalents; mg gae /g : acid gallic equivalents; mg ce/g: catechin equivalent. the extract of bechar meoh var.2 has the highest total flavonoids value 7.1 mg qe/g dw while meoh var.4 has the lowest one of 3.2 mg qe/g dw. an important total anthocyanin levels were detected in meoh var.1 of 2.6 mg c3g/g dw compared to the methanolic extract of mascara meoh var.3 which has a low value (1.7 mg c3g/g dw). a statistical significant differences existed on phytochemicals levels between plant samples (p < 0.05). total phenolics amounts resulted in f. carica skin extracts ranged between 28.6 and 211.9 mg gae / 100 g fw and between 24 and 237 mg gae / 100 g fw [21, 29]. the considerable phytochemicals levels obtained can be explained by the sonication extraction using pure methanol as solvent and as such, is not an ignored proposition as the extraction of flavones, polyphenols, anthocyanins, tannins can nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):29-35 messaoudi and merah ©njb, bsn 32 be very good using alcohol such as methanol solvent [30]. furthermore, the special climate conditions such as the low monthly rainfall and the high temperature characterizing the harvest year of plant samples can also affect widely their phytochemicals amounts. according to vallejo et al. 2012, the skin of the early fig fruit (first crop) is richer in phenolic components than late fruit (second crop) possibly as a consequence of climatic factors [31]. figures 3, 4 and 5 illustrate the total antioxidant capacity and the % inhibition of dpph. figure 3. total antioxidant capacity of fig flowers fruits methanolic extracts. meoh var.1; meoh var.2: variety 1, 2 bakor (fig flowers) of bechar. meoh var.3; meoh var.4: variety 3, 4 bakor of mascara. figure 4. dpph radical scavenging activity of fig flowers fruits methanolic extracts. meoh var.1; meoh var.2: variety 1, 2 bakor (fig flowers) of bechar. meoh var.3; meoh var.4: variety 3, 4 bakor of mascara the inhibition concentration value that exhibits 50% of radical scavenging activity for different varieties of extracts (table 1). table 2 summarizes the correlation coefficients among antioxidant tests, total flavonoid level, total anthocyanins level and total phenolic level. figure 5. % inhibition of dpph. meoh var.1; meoh var.2: variety 1, 2 bakor (fig flowers) of bechar. meoh var.3; meoh var.4: variety 3, 4 bakor of mascara compared to the positive control ic50 value 0.004 mg/g dw, the lowest ic50 equal to 0.185 mg/g dw was deduced for methanolic extract of bechar (meoh var.2), which chelated 87.9± 1.23 % of the dpph free radical. however, the extract meoh var.4 has presented the lowest %ip of 63.5 ± 0.87% with an ic50 value of 0.448 mg/g dw. our results showed a statistically significant difference between studied extracts and positive controls (p < 0.05). table 1. ic50 value of fig flowers fruits methanolic extracts. varieties of fig flowers (bakor) ic50 (mg/ml) meoh var.1 0.260a meoh var.2 0.185b meoh var.3 0.345c meoh var.4 0.448d a.a 0.004e meoh var.1; meoh var.2: variety 1, 2 bakor (fig flowers) of bechar. meoh var.3; meoh var.4: variety 3, 4 bakor of mascara. a.a: ascorbic acid. capital letters (a–b) and lowercase letters (a–b) indicate significant differences at p < 0.05. the high tpl and tfl levels measured can explain widely such a considerable percent of inhibition. phenolics are the most effective antioxidants, which function as free radical scavengers, and absorb oxygen radicals [32, 33], because of their acidity, ability to transfer electrons and characteristic benzene rings [34]. solomon et al. 2006 have proven that, compared to the fruit pulp, fruit skins have the most contribution to phytochemicals amounts and antioxidant capacity and both darkand browncolored fig varieties are the most effective ones by their important amounts of flavonoids, polyphenols, and anthocyanins. besides, anthocyanins compounds of the darkand brown-colored fig skin varieties contribute to 36 and 28% of their total antioxidant capacity [35]. 0 20 40 60 80 100 0.0625 0.125 0.25 0.5 0.75 1 % i n h ib it io n o f d p p h concentration mg/ml meoh var.1 meoh var.2 meoh var.3 meoh var.4 0 20 40 60 80 100 120 meoh var.1 meoh var.2 meoh var.3 meoh var.4 a.a% i n h ib it io n o f d p p h 0 20 40 60 80 100 120 meoh var.1 meoh var.2 meoh var.3 meoh var.4 a.a t o ta l a n ti o x id a n t a ct iv it y (m g a a e / g ) http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=total+antioxidant+capacity nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):29-35 messaoudi and merah ©njb, bsn 33 the total antioxidant capacity ranged from 63.5 to 92.1 mg aae/g dw for meoh var.2 and meoh var.1, respectively in comparison with ascorbic acid 98.2 ± 1.43 mg aae/g dw. the total antioxidant capacity estimated for methanolic extracts of fig varieties fruits from the same region (mascara) as our studied fig-flowers varieties 3, 4 (el bordj, ghriss) was ranged from 68.8 to 88.6 mg aac/g dw and the highest values were observed for the ethanolic extracts. also, the max of the total tannin content of methanolic extracts was max of 122.35 mg aae/g dw [36]. according to the statistical analysis, separately, there are non-significant differences between the two fig varieties of bechar: var.1 (lahmar), var.2 (ouakda) and also no significant differences between fig varieties originated of mascara var.3 (el bordj), var.4 (ghriss) (p > 0,05). a positive and strong correlation was registered between the antioxidant capacity of plant extract and its total phenolic composition [37, 38, 39, 40, 41]. in vitro tests have shown that dried fig fruits possess a significant antioxidant capacity subsequently to their human consumption. as mentioned previously, the total antioxidant capacity correlated well with anthocyanins and phenolic compositions (r = 0.989, r = 0.515), but the correlation is low with the flavonoid amount r = 0.248 [15, 42]. however, the existence of other unidentified molecules with antioxidant properties in those extracts can not be overlooked. peel extracts of fig fruits had a higher ability to scavenger free radicals, at all concentrations than pulp extracts with ic50 values of 80.04 and 28.85 mg/ml. for the pulp, ic50 values were 105.85 and 176.88mg/ml [43]. results showed a positive and strong correlation between antioxidant capacity and different phytochemical compounds amounts. phenolics and tannins components of meoh var.2 have an appreciable correlation with dpph radical test r2 =0.948 and r2 =0.972, respectively. total phenolics had a strong correlation with the dpph radical test (r2 =0.948). furthermore, an important relation between both total flavonoids and total phenolics and dpph radical presented by the extract meoh var.3 with r2 =0.954 and r2 =0.943, respectively. a high correlation coefficient was deduced between antioxidant capacity and the levels of polyphenols and anthocyanins with r2 = 0.985 and r2 =0.992, respectively [34]. the differences observed in the antioxidant capacity of plant extracts can be related to certain parameters like those that the solvent used for extraction, its polarity, and the tests used [44]. moreover, as the mechanisms of antioxidant effect are dissimilar and the most natural antioxidants are multifunctional, it is important to use various antioxidant capacity tests to take a global observation of it. conclusion the important antioxidant capacity of methanolic extracts of fig flowers fruits is impressive and probably it is the result of their exceptional richness in phenolic compounds. such bioactive molecules reacting as natural antioxidants and then, they are well known to display a positive impact on human health and can be considered for future uses as antioxidant components in agro-food industries. the algerian flora known for its high richness and biodiversity as well as algerian folk medicine is also considered as an appreciable source of both new drugs and bioactive molecules since ancient times. future studies will be needed to elucidate more and more medicinal plants and traditional preparations used for therapeutic purposes. conflict of interest the authors declare that they have no competing interests. table 2. the correlation coefficient among antioxidant capacity tests, total flavonoid level, total anthocyanins level, total phenolic level, and tannins level. meoh var.1 dpph tac meoh var.3 dpph tac tfl 0.812a 0.531a tfl 0.954a 0.208a tpl 0.873a 0.715b tpl 0.943b 0.681b tal 0.601a 0.224c tal 0.176c 0.566c ctl 0.730a 0.478d ctl 0.658d 0.619b meoh var.2 dpph tac meoh var.4 dpph tac tfl 0.914a 0.574a tfl 0.507a 0.288a tpl 0.948b 0.724b tpl 0.426a 0.397b tal 0.632c 0.587c tal 0.311a 0.141c ctl 0.972d 0.598d ctl 0.529a 0.336b tfl: total flavonoids level, tal: total anthocyanins level, tpl: total phenolics level, ctl: condensed tannins level, tac: total antioxidant capacity. meoh var.1; 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1 0 (1): 1-6 research article doi: https://doi.org/10.54796/njb.v10i1.224 ©njb, bsn 1 development of effective protocol for four varieties of large cardamom anna balzer1, sujit shah1,2 , yam kumar ale1, dipti adhikari1, sanjit niroula1, jawaharlal mandal1, amar bahadur pun1, krishna poudel1, parashuram bhantana1, govinda timsina1, ravindra karn1, sujwal karki1 1agricultural research station, pakhribas, dhankuta, nepal 2daffodil agro biological research center, lalitpur, nepal received: 24 mar 2021; revised: 9 jan 2022; accepted: 16 jan 2022; published online: 30 jul 2022 abstract large cardamom is one of the most important spices that can significantly contribute to the economical farming in the country of nepal. it is grown in nepal and north-eastern states of india which provide suitable agroclimatic growing conditions of high humidity, ambient temperature and high rainfall. meeting the demand for high quality plants and yield of cardamom is challenging with traditional methods of propagation. the present study has used the plant tissue culture technique to produce high quality plants. in this regard, ms media with three different hormonal combinations were used for the development protocol for 8 weeks. shoot length, root length, shoot number and root number were assessed at intervals. the best protocol for growth was ms media with 1 mg/l bap + 0.5 mg/l iba for the ramsey variety, with no significant difference for golsai, dambarsai, or sikkimae varieties. similarly, the acclimatization and field transfer study was done. the use of any substrate composition in ratio of coco peat: soil 1:2; moss: coco peat 1:2 and sawdust: coco peat 1:2 enables transfer of healthy plants to the field. the results indicate that the varieties respond differently to the micropropagation process and to hormone concentrations indicated by differing root and shoot production. the protocol of 1mg/l bap and 0.5mg/l iba could be used for the ramsai while optimal shoot production for golsai and sikkimae should be at 0.5mg/l and 5mg/l for shoot production. all varieties showed optimal root production at 0mg/l bap and 0.5mg/l iba. this study sheds light on the different responsiveness of varieties to tissue culture and hormone concentrations for both root and shoot development in micropropagation. keywords: cardamom, 6-benzylaminopurine, micropropagation, amomum subulatum, variety, tissue culture corresponding author, email: sujitaug16shah@gmail.com introduction large cardamom, amomum subulatum roxb. belongs to the family zingiberaceae and its cultivation is confined to the sub-himalayan range of eastern nepal, northern india (sikkim and west bengal) and bhutan [1]. nepal is the world's largest producer of large cardamom supplying close to 50% of the world's market demand with 14200 ha cultivated [1], these are concentrated in taplejung, sankhuwasabha, panchthar, and ilam comprosing 80% of nepal’s production [2]. the annual production is 6026 mt from productive area of 11665 hectares and supports the livelihood of more than 70,000 families directly and indirectly [3]. it has been regarded as one of the important spices upon which the livelihood of most farmers depends. however, the quality and quantity of the cardamom have declined over the years to disease associated with it [4]. common cardamom diseases include foorkey (nanovirus) [5], and chirke diseases (macluravirus) [6], and phoma leaf spot (phoma hedericola) [7]. these have directly or indirectly affected the stakeholders and the economy of the country. the demand and the price have been declining over the years because of low quality and for this reason many farmers have stopped cultivating large cardamom. large cardamom has a high price volatility which poses a risk to long term farming, and reduced production is caused by aging orchards, disease, poor management, and lack of new planting material [8, 9]. these problems pose potential risks to the large cardamom industry in nepal. a plant tissue culture technique (shoot tip culture) provides an alternate way to meet the quality and quantity of the cardamom, producing vigorous diseasefree plantlets in large numbers. the techniques will help to restore the high quality of plantlets and fulfill the demand of disease-free plants in the cardamom pocket zone of nepal [10]. this will in turn help to reestablish the ability to supply the regional and international cardamom markets. in this regard, present investigation has developed an effective protocol for producing highly demanded large nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: https://nepjb.com/index.php/njb issn (print): 2091-1130 mailto:sujitaug16shah@gmail.com https://orcid.org/0000-0002-6175-5242 mailto:sujitaug16shah@gmail.com mailto:sujitaug16shah@gmail.com nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 6 balzer et al. ©njb, bsn 2 cardamom varieties such as ramsai, dambarsai, golsey, and sikkimme. material and methods explants were collected from the nursery of narc substation pakhribas. the freshly collected sucker (explants) were washed under running tap water for at least 30 minutes to remove soil and other external particles attached on their surface. then, the explants were dipped in water containing tween 20 (0.1%) for 15-20 minutes, shaken well and again washed in running tap water until all the detergents washed off clearly and rinsed with distilled water. thereafter, dipped into 1% sodium hypochlorite solution for 15 minutes and followed subsequently by with 70% ethyl alcohol for 2 minutes [11]. finally, explants were rinsed thoroughly with sterile water for 5 times and ready for cut after drying in filter paper. multiplications were made from sterile mother stock which had been established through several rounds of subculturing with the aim to minimize the need for rhizome collection and allow year-round multiplication. plants were assessed daily to monitor growth and contamination, and were sub-cultured every 7-21 days. plant growth assay the protocorms produce from the explants were used for the plant growth assay. ms (murashige & skoog) media was prepared [12]. the different hormonal compositions (ms + 0mg/l bap + 0.5mg/l iba; ms+0.5 mg/l bap + 0.5 mg/l iba; ms + 1mg/l bap + 0.5 mg/l iba; ms + 5mg/l bap + 0.5mg/l iba) were used for the plant growth assay for all the varieties ramsey, golsey, dambarsai, and sikkimme. the plantlets were grown in aseptic conditions under a 16 h photoperiod, at 25± 2ºc. growth pattern of the plantlets were recorded after 60±5 days. shoots were then excised and placed on fresh media to grow until field transfer. acclimatization and field transfer the in-vitro plantlets were acclimatized using different substrate compositions. the substrates used were sterilized cocopeat, moss, sawdust and soil. the ratio of substrate cocopeat:soil 1:2; moss: cocopeat 1:2 and sawdust:cocopeat 1:2. the trial was kept under observation for 4 weeks in a greenhouse. the plant height was measured at weekly intervals. statistical analysis & growth ranking plant growth assays were performed independently for a minimum of eight replicates to measure the shoot and root length and number. both were tested by anova with the alpha error level set at p≥0.05 (graphpad prism). normal distribution was tested on each data set (d’agostino & pearson), with 22% of data sets being considered normally distributed. non-gaussian distribution was therefore assumed for anova. the control was considered to be 0mg/l bap media. a growth ranking system was implemented to identify optimal hormone concentrations within the study due to high variability and low sample sizes. growth rankings were determined by firstly normalizing each parameter to the highest average value. shoot or root growth ranking was then a multiplicative product (unitless) of the two parameters (length and number). results plant growth assay figure 1. the plant growth assay perfomerd for 6o days in-vitro condition for all four varieties of cardamom dambarsai (a), golsai (b), sikkimae (c), and ramsai (d) with ms+1mg/l bap +0.5mg/l iba the in-vitro plant growth assay performed for four varieties of cardamom to investigate the optimum concentration of hormone required of the plant growth (figure 1). length and numbers of shoots and roots are indicated in table 1. shoot number was significantly higher in the sikkimae variety on 5mg/l bap producing 8.4±0.9 shoots per cutting. root number was significantly lower in the ramsai variety at 5mg/l bap producing 1.5±0.4 roots per cutting. a b c d nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 6 balzer et al. ©njb, bsn 3 table 1: growth patterns of dambarsai, golsai, sikkimae, and ramsai cardamom varieties in response to differing cytokinin concentration in micropropagation. table 2: growth ranking for optimal shooting and rooting of dambarsai, golsai, sikkimae, and ramsai cardamom varieties in response to differing cytokinin concentration in micropropagation. optimal raking for the trail for shoot and root responses are shown in bold . in ramsai this concentration also produced a lower root number and length than all other treatments with a length of 27.5±6.8 mm length. shoot number was highest in the 0.5mg/l bap media for golsai at 7.8±1.0 shoots per cutting. no other measurements were significantly different (table 1). the number of shoots and roots produced differed widely between varieties. the maximum number of shoots produced in a media protocol per variety from 2.6 for ramsey to 8.4 for the sikkimae variety. root number varied from 3.1 (golasi) to 5.4 (sikkimae). table 2 indicates growth ranking for best shoot and root development. ranking based on growth of shoots and roots indicated that shoot and root optimization differed within each variety. the best ranking media to produce root for all varieties was the 0.5mg/l iba media with no exogenous bap (table 2). highest ranking media for shoot production was 1mg/l bap for dambarsai, 0.5mg/l bap for golsai, 5mg/l bap for sikkimae and 1mg/l bap for ramsai (table 2). values are indicated as mean ± sem. * indicates significant difference at the end of the trial period from the control bap concentration of 0mg/l. acclimatization and field transfer the plants that were grown for 65 days were taken for acclimatization and field transfer (figure 2). the plant height in all three different substrate compositions (cocopeat: soil 1:2; moss: cocopeat 1:2 and sawdust: cocopeat 1:2) was recorded at 15 and 30 days. the total growth accumulated from day 0 (transfer date) is shown in figure 2. the growth of plants in each substrate composition did not differ between substrates. survival and health of plants did not differ in the 30 day trial (figure 2 a-c). discussion the previous study has shown the use of 0.5mg bap + 1.0mg iba in one liter ms media as a standard protocol for the single ramsey variety [12]. similarly, use of ms+ sucrose 40 g + bap 3mg/l + 0.5 naa + 2mg/l iba was dambarsai shoot number 2.5 ± 0.5 2.9 ± 0.5 4.3 ± 0.8 3.4 ± 0.7 shoot length (mm) 36.3 ± 15.7 41.3 ± 13.6 42.7 ± 10.3 37.9 ± 10.0 root number 3.0 ± 1.0 2.6 ± 1.2 3.7 ± 1.0 0.9 ± 0.7 root length (mm) 100.0 ± 52.9 68.8 ± 44.8 71.7 ± 23.1 12.9 ± 12.0 golsai shoot number 3.8 ± 1.1 7.8 ± 1.0* 4.8 ± 1.0 6.0 ± 1.3 shoot length (mm) 53.0 ± 17.2 38.5 ± 6.4 33.3 ± 5.8 32.1 ± 7.6 root number 3.1 ± 1.1 0.7 ± 0.4 0.9 ± 0.5 0.1 ± 0.1 root length (mm) 51.0 ± 20.9 6.0 ± 4.1 19.6 ± 14.6 0.5 ± 0.5 sikkimae shoot number 4.4 ± 0.6 3.2 ± 1.5 3.7 ± 1.1 8.4 ± 0.9* shoot length (mm) 48.1 ± 20.8 35.0 ± 19.7 42.2 ± 7.1 82.3 ± 13.1 root number 4.9 ± 1.8 5.4 ± 2.0 3.3 ± 1.0 2.3 ± 0.8 root length (mm) 101.3 ± 47.0 63.8 ± 40.4 93.3 ± 32.6 20.5 ± 10.5 ramsai shoot number 2.2 ± 0.5 2.3 ± 0.4 2.6 ± 0.4 1.5 ± 0.2 shoot length (mm) 61.7 ± 16.0 47.1 ± 12.2 57.5 ± 10.8 35.0 ± 3.6 root number 5.2 ± 1.1 2.3 ± 0.8 2.7 ± 0.7 1.5 ± 0.4* root length (mm) 112.8 ± 29.3 96.3 ± 31.9 111.5 ± 38.4 27.5 ± 6.8* 0mg/l bap + 0.5mg/l iba 0.5mg/l bap + 0.5mg/l iba 1mg/l bap + 0.5mg/l iba 5mg/l bap + 0.5 mg/l iba 0mg/l bap + 0.5mg/l iba 0.5mg/l bap + 0.5mg/l iba 1mg/l bap + 0.5mg/l iba 5mg/l bap + 0.5 mg/l iba dambarsai shoot 0.49 0.64 1.00 0.70 root 0.82 0.49 0.72 0.03 golsai shoot 0.48 0.73 0.39 0.47 root 1.00 0.03 0.11 0.00 sikkimae shoot 0.31 0.16 0.22 1.00 root 0.90 0.63 0.57 0.09 ramsai shoot 0.86 0.68 0.93 0.32 root 1.00 0.37 0.51 0.07 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 6 balzer et al. ©njb, bsn 4 demonstrated [13]. revisiting the investigation, we have demonstrated the use of 1mg/l bap + 0.5mg/l iba is far better for the overall in-vitro plant growth in the ramsai variety. we also show that the optimal concentration of bap with 0.5mg/l iba, based on this study, for shoot number production in the golsai and sikkimae varieties are 0.5mg/l and 5mg/l respectively. while it is useful to have high shoot numbers, the shoots themselves must also show sufficient development in size. therefore, we implemented a ranking system to look at both number and length of shoots and roots. the optimal media for shoot growth differed by variety, however for root growth the optimal media was that which contained no exogenous bap. figure 2. the plants after 65 days of growth were transfer to three different sterile substrate composition; (sawdust: coco peat 1:2 fig. (a); coco peat: soil 1:2 fig (b); moss: coco peat 1:2 fig(c)) for 30 days. growth measured intern of plant height for 4 weeks showing no significant difference in height during the trail at level of p>0.05. most significantly, root initiation is higher in the presence of a lower cytokinin concentration. the role of cytokinin’s in root formation is complex and depends on many interacting factors, and affects both the type and amount of root growth [14-16]. in all varieties, the optimal root production as a rank was best in a media containing no exogenous cytokinin with 0.5mg/l iba. previous studies have shown variability across cytokinin concentrations with respect to root number formation in zingeriberaceae. no clear trend between bap concentration and root growth was shown in kaempferia parviflora tissue culture studies with 0, 1.5 and 2.5mg/l producing the same number of roots [17]. similarly micropropagation experiments using bap and naa showed no consistent correlation with cytokinin concentration and root number in amomum subulatum, whereas increasing cytokinin resulted in increasing shoot number up to 4µm bap concentration [13]. it may be beneficial to implement 2 stage in vitro propagation systems for cardamom whereby shoot multiplication is carried out on 0.5mg/l bap + 0.5mg/l iba for golsai, 1mg/l bap + 0.5mg/l iba for dambarsai and ramsai, and 5mg/l bap + 0.5mg/l iba sikkimae. for root development all should be transferred to media containing nil exogenous cytokinin. although not the primary aim of this study, it has become apparent that there are different responses of varieties to tissue culture. when looking at the number of shoots and roots produced per variety, these varied widely per variety. this outcome suggests that the sikkimae variety is more responsive to the micropropagation process than others. this is not unexpected and has been observed in other species including oil palm (elaeis guineensis) [18], potato (solanum tuberosum) [19], gerbera (gerbera jamesonii) [20], and grapes (vitis vinifera) [21], among a b c nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 6 balzer et al. ©njb, bsn 5 many others. further studies may establish this so we suggest additional research is undertaken. this outcome presents an opportunity to focus production on varieties who respond better to tissue culture, provided they also have desirable field characteristics. varieties with lesser responses may need to be the focus of additional research to optimize and meet demands. the acclimatization and field transfer are important steps for the mass propagation. in this regard, the all threesubstrate combination showed uniform growth and survival rate for plants growth. any of these substrate combination (cocopeat: soil 1:2; moss: cocopeat 1:2 and sawdust: cocopeat 1:2) can be used for acclimatization based on the availability which allows for greater production flexibility. other studies have shown optimal hardening of zingiberaceae species cow dung, coir dust,soil:sand and cow dung mixes all with high survival rates of 90-100% [22]. studies using peatmoss, sand, vermiculite and perlite mixtures found optimal survival of 100% on peatmoss alone [23]. further work may be carried out to determine whether golsai, sikkimae, dambarsai, and ramsai varieties respond differently to other auxins and cytokinins to optimize the protocol further. establishments of in vitro mother stock healthy plantlets is the major outcome of the present study. this will allow for ongoing propagation of clones year-round and will no longer be dependent on rhizome formation. this will also reduce the risk of disease contamination being introduced by continued field collection and allow clonal propagation every 60 days which will increase productivity to meet grower demand. conclusion the present investigation revisited the protocol that was already established to optimize the hormonal concentrations for maximum plant growth. in this regard, the present study has developed the protocol with the use of a lower concentration of hormone for the four varieties of plant. the ms media composition with 1mg/l bap + 0.5mg/l iba can be recommended as standard protocol for cardamom at this stage. similarly, substrate (cocopeat: soil 1:2; moss: cocopeat 1:2 and sawdust: cocopeat 1:2) depending upon the availability can used for acclimatization process. further research may elucidate better protocols including the use of different plant growth regulators, carbon sources, and nutrient sources. similarly, a two-stage process should be studied; where shoot production is carried out on media containing optimal bap concentrations per variety, and transferred to cytokinin free media for root development. author contributions author 1 and 2 designed the research experiment and performed the entire research. author 3 established the in-vitro plantlet. author 2 wrote the manuscript. authors 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12 revised the manuscript. author 6 supervised the entire research work. competing interests the authors declare no competing interests. funding this study was funded by the nepal agricultural research council. acknowledgements we would like to acknowledge the support and commitment of all staff involved in the cardamom production program at the pakhribas agricultural research station. data availability please contact the corresponding author for access to the data. ethical approval no ethical approval was required, sought or granted for this study. references 1. gautam n, et al., technology, chemistry and bioactive properties of large cardamom (amomum subulatum roxb.): an overview. int. j appl. sci. biotech.2016. 4(2): p. 139-149. http://dx.doi.org/ 10.3126/ijasbt.v4i2.15104 2. khatiwada a, subedi a, dangol r. a review on status of production of large cardamom in nepal and its marketing in national and global scenario. malay. j halal res. 2019. 2(1): p. 1621.http://dx.doi.org/10.2478/mjhr-2019-0004 3. rao, y., et al., large cardamom (amomum subulatum roxb.)-a review. 1993 4. deka t, sudharshan m, saju k. new record of bumble bee, bombus breviceps smith as a pollinator of large cardamom. current sci. 2011. 100(6): p. 926-928 5. mandal b, shilpi s, barman ar, mandal s, varma a. nine novel dna components associated with the foorkey disease of large cardamom: evidence of a distinct babuvirus species in nanoviridae. virus res. 2013;178(2):297-305. doi:10.1016/ j.virusres.2013.09.027 6. mandal b, vijayanandraj s, shilpi s, pun kb, singh v, pant rp, jain rk, varandarasan s, varma a. disease distribution and characterisation of a new macluravirus associated with chirke disease of large cardamom. ann. appl. biol. 2012; 160(3): p. 225236.https://doi.org/10.1111/j.1744-7348.2012.00537.x 7. saju k a, deka t n, sudharshan m r, gupta u, biswas a k. incidence of phoma leaf spot disease of large cardamom (amomum subulatum roxb.) and in vitro evaluation of fungicides against the pathogen. j spice 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51(4): p. 175-182 22. lincy a, sasikumar b. enhanced adventitious shoot regeneration from aerial stem explants of ginger using tdz and its histological studies. turk. j bot. 2010; 34(1): p. 2129.https://doi.org/10.3906/bot-0805-6 23. abbas ms, taha hs, aly ui, el-shabrawi hm, gaber es. in vitro propagation of ginger (zingiber officinale rosco). j genet eng biotechnol. 2011; 9(2): p. 165-172.https://doi.org/10.1016/ j.jgeb.2011.11.002 http://dx.doi.org/10.15835/nsb10210234 https://doi.org/10.3906/bot-0805-6 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 issn 2091-1130 (print)/issn 2467-9319 (online) review article ©njb, biotechnology society of nepal 74 nepjol.info/index.php/njb technologies for the extraction, separation and purification of polyphenols – a review shyam suwal1* and alice marciniak2 1 department of food science, faculty of science, university of copenhagen, rolighedsvej denmark 2institute of nutrition and functional foods (inaf), department of food sciences, université laval, québec, canada abstract polyphenols are high molecular weight, organic molecules mainly found in plant kingdom. they are mostly known for their positive impact on health, specifically for their antioxidant activity. indeed, they are widely studied for the prevention of multiple diseases such as cancer, inflammatory, cardiovascular and neurodegenerative diseases. nevertheless, extractions of these growing interest molecules remain challenging using conventional methods such as solvent extraction. that is why recent researches have focused on improving the extraction of polyphenol by using different technologies such as ultrasound, microwave, pressurized liquid, pulsed electric field, supercritical fluid and high hydrostatic pressure. in the current context, the assistedextraction should demonstrate their potential to improve the extraction efficiency while being cost-effective and with a low environmental impact. to this end, technologies ought to, for instance, increase the solubility of polyphenol and the permeability of the cell wall. consequently, this review is focused on the use and potential of these technologies to improve polyphenol extractions from plants as well as their purification using various methods. it discusses of the advantages and disadvantages with some examples of all these technologies assisted-extraction in comparison with conventional extraction method as well as purification technology. keywords: polyphenols, conventional extraction process, emerging technologies *corresponding author email: shyam@food.ku.dk introduction the prevention and treatment of chronic diseases mainly cardiovascular diseases (cvd) and cancer are the major challenge in modern day health care system. about 60% of deaths were caused by the chronic diseases in 2001 and according to the study of world health organization (who) the heart disease will overcome the infectious disease by 2020 [1]. therefore, the burden (economical as well as social) of chronic diseases is huge and tends to increase day by day alarming an urgent need of preventive measures. undoubtedly, the primary cause of these diseases is associated with unhealthy diets. there is an increasing interest in diets and nutrition which has resulted in an increased importance and beliefs in functional foods and nutraceuticals [2]. several bioactive molecules (peptides, vitamins, polyunsaturated fatty acids and polyphenols) have been isolated from food sources of plant as well as animal origin and are claimed to possess health benefits against cvd and cancer [3-5]. very diverse nature of polyphenol in their chemical composition and their wide spread prevalence in edible plant make them very interesting as an ingredient of functional foods or nutraceuticals [3]. different beneficial roles on human health such as antihypertensive anticancer, neuroprotector etc. have been confirmed [6]. therefore, an immense interest of researchers on polyphenols mainly in their physiological roles (both in vitro and in vivo), their extraction processes and product development in an industrial scale can be noticed. however, extraction of such molecules from the complex plant matrices is the rate limiting factor. several methods of polyphenol extraction from variety of food and food by-products exist such as solvent (soxhlet), ultrasound assisted, microwave assisted, pressurized liquid, pulse electric field, supercritical fluid and high hydrostatic pressure extractions. the extraction methods not only affect the input cost, final productivity and purity but also their biological activity in laboratory assay [7, 8]. a cost effective and environment friendly method are thus desired for the isolation of polyphenols. a separation and purification process is another vital step in product development with polyphenols. therefore, the nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 75 nepjol.info/index.php/njb present study aims to review different techniques used for the isolation of figure 1: chemical structure of principal polyphenols (adapted from [3]). . polyphenols published in the literature. in addition, methods of fractionation and purification of extracted polyphenol or free polyphenols present in liquid beverages are also discussed polyphenols and health claims polyphenols are readily available in the plantderived food sources consumed by human in daily basis such as fruits, vegetables, cereals, tea, wine etc. [3]. they are responsible for the major organoleptic properties such as color and taste of plant-derived food and beverages [9]. plant polyphenols comprise of diverse groups of compounds that make them difficult to be characterized except for some well-known ones whose structure, chemical composition and functionality are well defined. ample of polyphenol structures (having several hydroxyl groups in aromatic ring) are found in plants are therefore classified according to the number of phenol rings to one another. they can be mainly classified into the phenolic acids, flavonoids (flavones, anthocyanidins), stilbenes, and lignans (cinamic acid) as shown in figure 1. each of these phenolic compounds can be subclassified according to the type of heterocycle involved. the chemical nature of phenolic compound can vary from a simple to polymerized structure and may be found in a complex form with some carbohydrate, protein or plant substances making them highly insoluble [10]. furthermore, a single may contain several polyphenols whose concentration (several mg to few g per kg or l), stability and activity vary from one to another. more than 8000 phenolic compounds are known to exist and about 4000 flavonoids have been identified [11]. further details about different type of phenolic compounds in food sources, their characteristics and relevance can be found in the literature [3, 12]. polyphenols are very well known as the most abundant antioxidant in the diet and they have been claimed to possess potential benefits against cardiovascular diseases, cancer and neurodegenerative diseases in vitro as well as in vivo [6, 13, 14]. due to the vast structural diversity of polyphenols many of them have not yet been identified and characterized consequently the functional mechanism are not well established. amongst them, the tea polyphenol: catechin also known as epigallocatechin-3-gallate (egcg) is well characterized and a large number of researches are focused on its mode of action. lorenz [14] discussed the different mechanisms of http://en.wikipedia.org/wiki/anthocyanidin nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 76 nepjol.info/index.php/njb anticancer (apoptosis, inhibition of tumor growth and inhibition of telomerase), antioxidant (free radical scavenging, chelation of metal ions, inhibition of ros producing enzyme and reduction of inflammatory cytokines), antihypertensive (vasodilation) and neuroprotective (antineuro inflammatory effect, activity of egcg in the cellular and molecular levels. in addition, a detail pharmacokinetic study (absorption, distribution metabolism and elimination) of tea polyphenols is recently reviewed by clifford et al. [15]. extraction process due to positive effects on human health, phenolic compounds (isolated form or in extract) could be used as food supplements (functional food) or nutraceuticals. to do so, a cost-effective methods for the extraction and separation of polyphenols is necessary. a lot of research has focused on the analysis of biological activities and characterization of phenolic compound and the extraction process are often ignored. the extraction and isolation of polyphenols remain challenging especially due to structural complexity and instability (degradation and reaction during processing) [9, 11]. therefore, the efficiency of the extraction of phenolic compounds from plant material are influenced by several parameters: the chemical nature and location within the plant matrix of the phenolics, the extraction method used, the size of sample particle, storage conditions and presence of interfering substance and consequential biochemical and chemical reactions [10, 16]. phenolic compound are essentially found in cell walls, vacuoles, associated with nuclei, leaves, gymnosperm and rhizomes [17]. in addition, most of the phenolic compounds in plant sources are bound to plant material by covalent bond making it a real challenge to liberate them into the extractable form [18]. therefore, different extraction methods have been developed in this domain. the rate limiting step for phenolic compound extraction from plant material is the (a) solubility, (b) diffusibility through the cell walls and (c) washing out (rinsing) of the cell wall [16, 19]. 1. conventional solvent extraction solvent extraction (se) is one of the conventional methods of extraction of bioactive molecules from plant sources but is still widely used for the extraction of various natural bioactive compounds specially the phenolic compounds from various sources [20]. pretreated plant material (washing, drying, etc.) are exposed to varieties of solvents such as, water, hexane, ether, chloroform, acetonitrile, benzene, ethanol and methanol which take up the molecules of interest (polyphenols). however, the efficiency of solvent extraction methods is affected by the solvent used (solubility of phenolic compounds depend on the solvent type or its polarity) and the nature of phenolics (degree of polymerization) and their interaction with other plant or food constituents [10]. the choice of extraction techniques have to take into consideration of the location of phenolic compounds in the plant, most of which are stored in the vacuoles only extractable by alcoholic or organic solvents except that are bound to insoluble carbohydrate or protein [21]. soxhlet extraction also known as solid-liquid extraction is one of the first techniques known to be used for the extraction of polyphenols compounds [22]. this technique is more often considered as a leaching or lixiviation. the working principal of soxhlet apparatus is simple as shown in figure 2. the sample is placed in a thimble-holder and solvent (extractant) and is filled with condensed solvent from a distillation flask. a siphon aspirator is used to unload the excess of solvents when it reaches to overflow level that carries the extracted molecules to the bulk liquid. this process is repeated until a complete extraction is achieved. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 77 nepjol.info/index.php/njb figure. 2: conventional soxhlet extractor (adapted from [23]) the soxhlet extraction process is easy to operate not requiring any professional expertise. in addition, extraction productivity could be increased by simultaneous extraction in parallel and further treatment such as filtration is not required as no plant residues are present. however this method of extraction is often criticized as a large volume of solvent used which eventually cause environmental problems in addition to higher cost involved, long treatment period and thermal degradation (due to excessive exposure to heat during extraction and evaporation post extraction) of extracted molecules as the extraction is carried out at boiling point of solvent [22]. in addition, excessive exposure to light and oxygen lead to the degradation of other sensitive compounds [24]. therefore, various other improved methods of solvent extraction are researched and rapidly being developed which mainly focus on decreasing the quantity of organic solvent, treatment time and energy and increase the yield. these methods are discussed in the following sections in details. 2 novel and emerging methods of extraction 2.1 ultrasound assisted extraction the use of ultrasound (wave frequencies of more than 20 khz to 10mhz) in the extraction of bioactive molecules known as ultrasound-assisted extraction (uae) are believed to reduce the organic solvent, find other alternative solvents which are economically, environmentally sound and safe to human health [25]. the irradiation of plant tissues with ultrasound increase the number of extraction solvent exposed to the molecules by breaking down the cell wall, increasing swelling and hydration degree thus improving the diffusion and mass transfer [19]. the use of uae have been found to improve the extraction efficiency up to 35 % during the extraction of phenolic compounds from various plant sources [25]. chen et al. [26] found an optimized condition of uae treatment (solvent to solute ratio of 4:1 (ml/g), extraction time 200 s and ultrasonic power was 400 w) and found 3.45 mg of anthocyanins per gram of fresh raspberries at moderate temperature (less than 40oc. another study was carried out for the retention (stability) of anthocyanins in grape juice as function of ultrasound amplitude during ultrasound treatment [27]. at higher amplitude and treatment time degradation of certain anthocyanins were observed due to the mechanism called sonolysis of water molecules due to cavitation inducing the formation of hydroxyl radicals which eventually lead to chemical degradation. a comparative study of different method of extraction namely uae, high hydrostatic pressurized extraction (hhpe) and pulse electric field (pef), which will be discussed later, showed higher total phenolic content as compared to that of control (water as solvent at 70oc) [28]. but least yield and antioxidant activity was observed in case of uae as compared to hhpe and pef. however, no reason was discussed for the least antioxidant activity observed. this could be large quantity of plant residues (impurities) present in the extracts. uae is a non-thermal process which could be easily integrated to other existing methods without major modification to improve the extraction efficiency and yield. therefore, the main advantage of uae over other technique could be mild temperature thus minimum thermal degradation [29]. however, the drawback of this technique is the requirement of post treatment like filtration to get rid of resulting plant residues during the process and inability to reuse the extraction solvent [22]. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 78 nepjol.info/index.php/njb 2.2 microwave assisted extraction microwave assisted extraction (mae), developed in 1980s, is a process which involves the heating of solvent by in contact with sample with microwave energy to partition the molecules of interest from plant matrix to the solvent used [30]. in comparison to other traditional extraction method, mae possesses advantages like shorter time, controlled temperature and other extraction environment, less solvent, higher extraction rate, energy saving and better quality product (higher antioxidant activity) with lower cost [31]. in addition, mae method could adapt easily the already existing extraction process for example that of soxhlet. the extraction method is different from other conventional method because the extraction occurs due to the changes in cell structure caused by electromagnetic waves [32]. unlike the conventional method where the heat is transferred from the solvent to the interior of sample, in mae the heat is dissipated volumetrically inside the irradiated medium therefore the distribution of temperature is equal all over the sample the increase in free phenolic acid content with increase in microwave power and treatment time was observed in citrus mandarin pomace and peels consequently increasing their antioxidant capacity [33, 34]. this study also demonstrated that at 250w of microwave power, the total flavonoid compounds decreased with increase in the duration of treatment suggesting a possible degradation due to longer exposure to heat. therefore, microwave treatment could be economically viable, rapid and used in an industrial scale production but careful choice of heating temperature and duration is needed. 2.3 pressurized liquid extraction a pressurized liquid extraction (ple) method which is comparatively a new method, is operated at elevated temperature (50 to 200°c) and pressure (3.5 to 20 mpa) and is supposed to enhance the extraction efficiency as compared to conventional methods carried out at room temperature and atmospheric pressure [16]. this technique uses a solid samples packed in an extraction cell and uses organic solvent or water at high pressure and temperature above the boiling point [35]. a typical instrumental set up of a pressurized liquid or fluid extraction (ple) is shown in figure 3. the system consists of a pumping system for the supply of solvent from the solvent vessel which enters to a heating system to increase the temperature of the solvent, an extraction cell containing sample, a back pressure controller and a collector at the end [35]. the extraction efficiency using ple are improved by increasing the solubility of phenolic compounds and mass transfer to the extracting solvent. [36]. in addition, increasing the pressure at elevated temperature, the diffusivity of a solvent could be increased which will better penetrate through the plant matrix and disrupt the solute-matrix interaction due to der waals forces, hydrogen bonding and dipole attractions between solute molecules and active sites on the matrix [37]. in the study of denery et al. [24] similar extraction efficiency of caretenoids were observed using ple and conventional (which used only sonication and centrifugation) extraction method reducing the extraction solvent by 50% and extraction time by about 77 %. in addition, ple was advantageous for the oxygen and light liable carotenoids. fig. 3: schematic diagram of pressurized liquid extraction system adapted from [35] the thermal degradation which even favors the oxidation of phenolic compound such as anthocyanins could be main issue related to ple. however, palma et al. [38] have demonstrated that among 9 phenolic compounds tested most of nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 79 nepjol.info/index.php/njb them are stable except for catechin and epicatechin at 150 °c and maintain the recovery rate above 85 % with methanol as extracting solvent. similar result was observed by piñeirol et al. [39] during the extraction of catechin and epicatechin from tea leaves and grape seeds with recovery rate below 95% at 130oc. however, this study demonstrated the best recovery of catechin and epicatechin from grape seeds using ple using methanol as solvent as compared to simple stirring assisted extraction and uae. water is an extremely polar solvent, therefore not usually used as solvent for the extraction of organic compounds. however, water is nonflammable, non-toxic, widely distributed ecological solvent [36]. at high temperature and pressure water can act as good solvent as the polarity approaches (decreases) to that of alcohols. to maintain the water in liquid state a pressure ranging from 15 (at 200oc) to 85 (at 300oc) bars has to be applied [37]. therefore, pressurized hot water extraction (phwe) method is getting interest in the extraction of bioactive molecules from plant and food material. it could also be possible to combine the ple with phwe in order to increase the efficiency of extraction process. the temperature of solvent, pressure applied and extraction time vary widely depending strongly upon the type of phenolics, the solvent used and extraction instrument. therefore, at this stage nothing could be concluded about the optimum ple parameters (time, solvent, pressure and temperature). 2.4 pulsed electric field pulsed electric field (pef) is one of the newest technologies developed as a non-thermal process which have been widely studied in the food processing sectors. pretreatment of plant material with pef enhance the extraction of bioactive molecules by electro-permeabilization (also known as electroporation) of plant cell increasing the mass transfer even at lower electric field [40]. biological membrane plays an important role to amply the electrical signal as it is more conductive as compared to its surrounding environment (cytoplasm and extracellular medium). the mechanism of electroporation is shown in the figure 4. when an external electric field (e) is applied to a cell with certain thickness, the electrical potential across the membrane (called transmembrane electric field, which is far greater than applied electric field) increases and when the e exceeds the critical value a reversible or irreversible pores are formed. effectively, the number pore formation depends directly on the electric field applied as well as the number of pulse. a significant improvement by 21.5 % and 28.6 % in anthocyanins content in wine with the grape skin pretreated with pef (50 pulses and 1 hz) using 5 and 10 kv/cm electric fields as compared to that of untreated one was shown by lópez et al. [41]. in addition, application of pef (50 pulses at 122 hz) at 5 kv/cm electric field have shown to maintain higher anthocyanins and polyphenols concentration during the aging process of red wine [42]. furthermore, treatment with pef of merlot grapes was found to improve the sensorial and visual quality of wine in addition to increase in the polyphenols and anthocyanins [43, 44]. the four fold increase in antioxidant activity was observed for pef (30 pulses, 2 hz and 3 kv/cm) treated grape as compared to untreated grape byproducts [28]. the increase in antioxidant activity was directly related to significant increase in phenolics as compared to the untreated samples. therefore, pef can be regarded as a promising tool for valorization of low cost industrial waste to nutraceuticals or functional food. the pef treatment to enhance the extraction and increase the phenolic compound concentration during maceration and fermentation process of red wine making is extensively researched. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 80 nepjol.info/index.php/njb fig. 4: mechanism of electroporation of biological membrane in an external electric field (e). ec represent the critical value of electric field applied (adapted from [40]). 2.5 supercritical fluid extraction supercritical fluid extraction (sfe) has been widely used in large scale in order to extract natural solid materials especially for food ingredients, supplementation and phytopharmaceuticals. sfe is getting its popularity due to a very low or no organic solvent, innovative, high valued and quality final product, reduced extraction time and higher selectivity but often criticized for high investment cost [45, 46]. at the critical values of pressure and temperature a fluid behaves like a supercritical fluid having an intermediate properties (diffusivity, solubility and density) of gas and liquid [46]. carbon dioxide and water are most widely used as supercritical fluid. in comparison to water co2 is comparatively easier to operate and commonly used as it has a moderate critical temperature (31.2oc as compared to 101.1oc for water) and pressure (72.9 atm vs. 217.6 atm for water). in addition, co2 is generally regarded as safe (gras) and does not leave the residue after extraction simply by depressurization at room temperature. however, co2 is not very suitable for extraction of highly polar compounds (such as flavonoids) as polarity of co2 is lower thus, other solvent (e.g. methanol and ethanol) at low concentration could be used as co-solvent, known as modifiers, which subsequently increase the extraction efficiency. le floch et al. [47] used supercritical co2 extraction using methanol as modifiers to extract polyphenols from olive leaves at different temperature, pressure, concentration of modifiers and extraction time. the phenolic content increased with temperature, pressure and concentration of modifiers but a plateau was observed at 140 minutes. at the same time, a comparison was made with a uae with different solvents. the sfe with 10 % modifier (methanol) gave rise higher phenolic concentration as compared to uae with low polarity solvent (nhexane, diethyl ether or ethyl acetate) but significantly lower as compared to uae with high polarity solvent (methanol). only 45 % extraction efficiency was observed using sfe. another comparative study for the extraction of isoflavones from soybean flour were conducted between sfe (co2) with methanol as modifiers (10 %), soxhlet (80 % ethanol) and uae (70 % methanol) [56]. as before, a strong interaction between pressure and temperature was noticed. similarly, least amount of isoflavones (86.28 µg/g of dry weight) were extracted using sfe than uae (311.55 µg/g) and soxhlet (212.86 µg/g). despite of lower recovery of isoflavones using sfe techniques, it was the most selective process as very less co-extracts were present and require fewer steps for the extraction. moreover, the selectivity of the sfe process was further proven by the results found by kitzberger et al. [57] who demonstrated that only the shiitake mushroom extracts (which contained flavonoids) obtained by sfe (at 40 °c, 20 mpa and 15% ethanol) with ethanol as a co-solvent had an antibacterial activity against micrococcus luteus and bacillus cereus as compared to the conventional extraction with n-hexane, ethyl acetate and dichloromethane. however, the antibacterial activity was supposed to be due to selective extraction of flavonoids with sfeethanol however, the authors did not compare the results with conventional extraction with only ethanol as solvent. thus, it was not clear if the selective extraction of flavonoids and thus bioactivity was dependent on extraction technique or also with the type of solvent (more polar solvent like ethanol. therefore, sfe technique undoubtedly carries a huge application nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 81 nepjol.info/index.php/njb on polyphenols extraction but considerable innovations are needed for their industrial and economic feasibility. 2.6 high hydrostatic pressure high hydrostatic pressure is a non-thermal and green process governed by le chatelier’s principle (mozhaev 1994). while first application of hhp dates from few decades, the technology is considered as an emerging process since the first industrial-scale systems were available in 1990 in japan. high hydrostatic pressure processing consists to apply a uniform pressure (100 1000 mpa) which is instantaneously and uniformly transmitted, independent of the size and geometry of food, on flexible packaging materials filled with liquid or solid food products. the pressure transmitter fluid is generally water and the process can be used with or without utilization of heat. its main application concerns the pasteurization of food product in order to improve their shelf-life and preserve components of a wide range of food products. nowadays, a large number of studied focused on innovative applications of hhp such as the improvement of polyphenol’s extraction. jun et al., (2009) compared the assisted extraction of polyphenol from green tea leaves by hhp (hhpe) with conventional methods, using different solvents, pressure parameters and ratio liquid:solid. they stated an increase of polyphenol extraction yields is governed by an increase in pressure level (from 15 to 30% from 100 to 600 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 82 nepjol.info/index.php/njb table 1: methods of polyphenol extraction from different food sources and byproducts. source extraction method total phenolic content (mg/g) or mg/ml reference citrus mandarin peels mae-methanol 6.37 ± 0.200 [34] citrus mandarin pomace mae-methonol 6.18± 0.12 [33] medicinal plant-lien tze hsin il-mae-methanol 15.76±0.329 [48] peanut skin mae-ethanol 144 [49] grape marcs se-ethanol 28.06 [50] blueberry se-ethanol (50%) 1.48 [51] lemon peel enzyme –assisted 1.13 ± 0.0076 [52] black tea solvent-acetone -n,n-dimethylformamide -ethonol -methanol 98.19±1.10 109.36±0.71 86.31±3.50 68.69±1.34 [8] algae and cynobacteria ple-spe [53] green algae ple (1500-2000 psi) 11.4 [24] herbal medicine (epimedium sp.) ple70% ethanol (1500psi) 48.51 [54] grape seeds tea leaves ple-methanol 1.82 0.65 0.62 3.31 [39] raspberries uae (200w), 22khz 3.45 [26] grape seeds uae (200w), 24khz 0.23 0.07 [39] grape skin pef (50 pulses, 122 hz) 5kv/cm -10 kv/cm 9.65 10.6 [41] red grapes pef (50 pulses, 122 hz) -5 kv/cm -7 kv/cm 6.02 5.20 [55] grape byproduct pef (30 pulses, 2 hz, 3kv/cm) 61.2 [28] olive leaves sfe-10 % methanol, 330 atm and 100 oc 7.6 ± 0.5 [47] soybean flour sfe-10 % methanol, 355 atm and 50 oc 0.086 [56] shiitake mushroom sfe-15 % ethanol, 296 atm and 50 oc 10.2 [57] grape skin hhp-50% ethanol (200 mpa) 8.91 ± 0.13 [58] chilean papaya seeds hhp-500 mpa 15 min 50% ethanol [59] green tea leaves hhp-500 mpa 1 min 50% ethanol [60] watercress hhp-600 mpa 3.1 min 100% ethanol 64.68 ± 2.97 [61] mpa). globally, they achieve in 1 min 500 mpa the same extraction yield obtained by 90 min ultrasonic extraction, 45 min heat reflux extraction or 20h room temperature extraction. more recently, a research on chilean papaya seeds have reach the same conclusion concerning the efficiency of hhp to improve the extraction of polyphenol. indeed, briones-labarca et al., (2014) could extract more efficiently polyphenol by hhpe (5, 10 and 15 min) in comparison with nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 83 nepjol.info/index.php/njb conventional extraction (30 min) or even ultrasound assisted-extraction (5, 10 and 15 min). lastly, pinela et al., (2018) determined by response surface methodology, the optimal hhpe parameters to achieve the higher extraction yields of polyphenol from watercress. once again, there is a significant positive correlation between the level of pressure and the extraction yield. broadly speaking, hhp enable the solvent to enter cells and lead to an increase of the cell permeability. during the instantaneous release of pressure, the cell wall is disrupted leading to a spill of cytoplasm and target molecules such as polyphenols (jun et al., 2009). the different methods of extraction process used for polyphenol extraction from various food sources and their by-products are summarized in table 1. to this point, it is not very clear which is the best extraction method due to the complexity in plant source, wide nature of polyphenols (size and physicochemical) and varieties of solvent used. various studies made the comparison among different methods. however, the same method showed different yield which largely depended on type of source and solvent used. table 2 shows the comparison between different extractions methods discussed above. therefore, more comprehensive studies are needed that can clearly distinguish the mechanism of extraction and its relation to different type source, molecules of interest, time and cost. 4. separation and purification processes 4.1 ion exchange chromatography ion exchange chromatography (iec) is widely used technique in food processing which separates the molecules according to their charge [62]. the later can be modified by changing the ph or ionic strength of the solution. ottens et al. [62] emphasized that for a preparative purification of bio-molecules an iec process in indispensable that need to take into consideration of the choice of resins, the adsorbent (cost, resin capacity, regeneration cycle, life time and safety). adsorption and desorption of molecules to be separated (target molecules) on resins packed in a column are the main steps in the iec process. equilibration of resins (ph, ionic strength and solvent concentration), loading of samples with impurities (that will adsorb onto the resin), washing (to desorb the impurities) and elution (of target molecule to elution buffer) that can be carried out in isocratic or gradient mode, are the main steps of a typical iec process. a high recovery efficiency of egcg was found by using cyclic iec techniques that uses less volume of solvents (30% ethanol) as compared to conventional maceration extraction using ultrasound [63]. anthocyanins from strawberry were purified by iec using amberlite xad-7 as ion exchange resin before analytic separation [64]. similarly, cation exchange chromatography technique was used to purify polyphenols from red wine after solvent extraction [65]. as this technique uses solvents as an eluent to desorb the adsorbed polyphenols from the ion exchange resin further purification is needed to ensure the purity of the final product. in addition, the use of non-solvent iec process could be a better option. polyphenols such as epigallocatechin-gallate (egcg), epicatechin-gallate (ecg) and epigallocatechin (egc) were separated by using weakly acidic cation exchange gels (dextran based) from crude tea extracts without using any solvent [66] 4.2 membrane based process a membrane based separation process has been widely used in an industrial scale. in addition, all the above mentioned extraction processes contained more or less organic solvent which are proven to be toxic to human health, thus has an important role in consumer acceptance of these product. in addition, the separation and purification efficiency of membrane process depends upon the molecular weight polyphenols to be concentrated which ranges from 290 to 1200 g/mol, membrane molecular weight cut off (mwco) sizes and the transmembrane pressure (the driving force) applied in the system. generally, ultrafiltration (uf), nanofiltration (nf) and reverse osmosis (ro) process are used. the membrane process are particularly interesting to separate and concentrate polyphenols from or in nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 84 nepjol.info/index.php/njb table 2: comparison between different extraction process adapted and modified from [29] solvent extraction microwave assisted extraction supercritical fluid extraction ultrasoundassisted extraction pulsed electric field pressurized solvent extraction high hydrostatic pressure extraction brief description solvent is heated by a conventional oven and passed by the sample immersion of the sample in solvent and microwave energy is submitted a high pressure vessel is filled with sample and crossed continuously by the supercritical fluid immersion of the sample in solvent and submission to ultrasound using a us probe or us bath pulses of high electric voltages are applied to the sample placed in between two electrodes heat of the sample by a conventional oven and crossed by the extraction solvent under pressure sample is pressurized (100 – 1000 mpa) through a pressure transmitter liquid extraction time 6-8 hours 3–30 min 10–60 min 10–60 min 10–20 min 1 – 30 min sample size 1–10 g 1–5 g 1–30 g 1–30 g solvent volume 10–40 ml 2–5 ml (solid trap) 30–60 ml (liquid trap) 50–200 ml 15–60 ml cost moderate high low high high high advantages rapid and easy to handle rapid easy to handle moderate solvent consumption rapid low solvent consumption concentration of the extract no filtration necessary possible high selectivity easy to use rapid and non-thermal process rapid no filtration necessary low solvent consumption rapid green technology high selectivity high extraction yield no degradation of target molecules disadvantages high solvent consumption, long treatment time and thermal degradation extraction solvent must absorb microwave energy filtration step required many parameters to optimize large amount of solvent consumption filtration step required mechanism not well known and process intensification is difficult possible degradation of thermolabile analytes high cost equipment nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 85 nepjol.info/index.php/njb nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 86 nepjol.info/index.php/njb aqueous solutions (fruit juices) or from the complex solute-solvent after the extraction process using conventional methods can significantly minimize the amount of solvent being used [67, 68]. the efficiency of concentration of polyphenols from grape seeds by ultrafiltration (dead end filtration) with 50 kda membrane was found to depend on the method of extraction used [68]. in fact, the different extraction processes result to in different quality final product in terms of solute (polyphenols) content. significantly higher concentration (4050%) of total polyphenols was recovered in retentate after the ultrafiltration. the studies with different mwco membranes have shown that low molecular weight phenolic compounds are mostly present in permeate solution however in the concentrate of reverse osmosis process [69]. another possibility of concentration of polyphenols with higher selectivity is to use the sequential filtration process. for the low molecular free polyphenols found in olive mill wastewater (omw) were extracted by sequential filtration process [70]. the first step, microfiltration was followed by nanofiltration and osmotic distillation (also known as isothermal membrane distillation) and vacuum membrane distillation. the preliminary step of microfiltration reduced significantly total organic carbon and 0.5 g/l of low molecular weight polyphenols. the osmotic distillation process is relatively a new method which can potentially be used in the concentration of beverages and other liquid food stuffs. in this process, a micro-porous, non-liquid-wettable membrane (hydrophobic) separates a mixture of liquid containing a volatile in one side and a second liquid phase capable of absorbing the volatile component. membrane distillation process could be advantageous to selectively remove the solvent (ethanol) used during the extraction process. the details about the membrane distillation process are discussed in the literature [71]. this process was used to concentrate polyphenols from olive mill wastewater using micro-porous membranes with up to 99 % separation coefficient [72]. however, the membrane processes are frequently criticized due to membrane fouling and reduced selectivity for polyphenols separation. fouling by polyphenols and therefore significant reduction in water flux was observed by susanto et al. [73]. they proposed different mechanisms of interaction between polyphenols molecule (or aggregate of polyphenols) with membrane materials. the different interaction could be hydrophobic, hydrogen bonding and benzene ring interaction via π–π stacking when the membrane contains benzene rings (polyethersulfone, pes). the polyphenols can reduce the size of pore or block entirely. 4.3 electromembrane process electrodialysis with ultrafiltration membrane electromembrane process is widely used to separate charged molecules from a complex mixture of solution. one of the recent electromembrane processes is the combination of the conventional electrodialysis process with membrane filtration known as electrodialysis with filtration membrane (edfm), with a diverse application in food processing, biotechnology and biopharmaceutical [74]. in this method, the electric field applied acts as the driving force for the migration of charged molecules (ionic species) across a porous membrane (microfiltration) from one solution to another. as there is no pressure applied in the system in contrast to the pressure driven filtration process, there are no or very less accumulation of solutes on the membrane surface. bazinet et al. [75] reported the first application of electrodialysis (ed) process in the separation of polyphenols from tobacco extract. the authors successfully separated chlorogenic acid with migration rate up to 28 % using in 3 hours of treatment. a significant improvement in migration rate of 90.8 % for chlorogenic acid and 86.5 % and 81.3 % for scopoletin and rutin respectively by increasing the membrane stacks by 3 folds in the same system treated for 4 hours as shown in figure 5 [76]. later on the edfm process was used to separate catechins from green tea. labbé et al. [77] successfully and selectively separated egcg and egc from green tea infusion by in an ed system with the migration rate as high as 50 % within 1 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 87 nepjol.info/index.php/njb fig. 5: schematic diagram of electrodialysis system used for polyphenol separation from tobacco extract (adapted from [74]) hour of treatment. this study, in contrast to the study of bazinet et al. [75], a better migration rate was observed using ultrafiltration membrane (1000 da) in comparison to anion selective membranes. therefore, it was concluded that separation and purification process using ed depend on the type (physicochemical and molecular cut off values) of the membrane used. catechins are negatively charged at their initial ph (5.6 to 5.8) and thus migrate towards anode. in addition to isolating the polyphenols, another interesting application of edfm is to concentrate polyphenols with antioxidant activity in fruit juices. the anthocyanins and proanthocyanins were concentrated to 52.9 and 34.8 % respectively from natural cranberry juice by [78]. the authors also proposed a feasibility of direct integration of edfm system in the cranberry juice processing plant in order to produce anthocyanins enriched cranberry juice. however, attenuation polyphenol concentration in the feed cranberry juice leads to the degradation of juice quality and membrane fouling during the treatment are major issues that should be solved for its successful application. another study was carried out with larger volume of raw cranberry juice in the diluate compartment and smaller volume in the concentrate compartment [79]. this study demonstrated no change in physicochemical properties (sugar, organic acid, vitamin c, color index ph and conductivity) of raw cranberry juice in the diluate solution. another study were carried out by increasing the number of membrane stacking and using different volume ratio (diluate to concentrate side) of 30 which increased the anthocyanins content by 24 % [80]. a continuous but single passage of cranberry juice in diluate side of edfm system was found to increase the polyphenol content of another cranberry juice continuously circulated in the system. moreover, titrable acidity was found to significantly decrease in the enriched juice thus increase the organoleptic characteristic. however, above mentioned studies clearly demonstrated a problem related to the membrane fouling. therefore, optimisation of process for a proper membrane type (both mwco as well as membrane material), ph, other electrodialytic parameters are needed. in addition, this process seems very promising for the separation and purification of polyphenols present in industrial byproducts such as grape seeds and marcs, olive oil wastewater, etc after an appropriate extraction process. conclusion significant improvements in polyphenol extraction have been made as compared to tradition method of solvent extraction. however, although the quantity solvent used is significantly reduced, the use of solvent at less concentration is vital for better productivity and yield. the studies clearly show that the efficiency of an extraction nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 74-91 surname of autheor et al. ©njb, biotechnology society of nepal 88 nepjol.info/index.php/njb process is largely fig. 6: configuration of the edfm cell and of the global system used to enrich cranberry juice polyphenols (anthocyanins). aem: anionexchange membrane, fm: filtration membrane, cem: cation-exchange membrane (adapted from [77]). dependent on the type of polyphenol to be extracted, its location in plant as well as type of plant material, selectivity and quality desired. the extraction process design therefore has to take into account of the source, the total yield, the productivity and selectivity. moreover, more and more researches are inclined in the valorization of industrial by products such as from wine making process, olive oil mill and other fruit juices. this unquestionably shows both economical as well as environmental benefits. once the polyphenol has been extracted and for the polyphenols that are already present in an aqueous solution such as juices and wines, different separation and purification could be employed. ion exchange resins and pressure driven membrane process are widely used. however, electromembrane process 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y: evolution of cranberry juice physicochemical parameters during phenolic antioxidant enrichment by electrodialysis with filtration membrane. sep purif technol 2012, 87(0):31-39. 80. husson e, araya-farias m, desjardins y, bazinet l: selective anthocyanins enrichment of cranberry juice by electrodialysis with ultrafiltration membranes stacked. innov food sci emerg technol 2013, 17(0):153-162 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 4 0 4 4 research article doi: https://doi.org/10.54796/njb.v10i1.229 ©njb, bsn 40 screening of carbapenem resistance klebsiella pneumoniae and its mic against imipenem sarada saud1, ashwani agrawal1, soniya pokhrel1, sushma subedi1, sanjit shrestha2, and niroj man amatya 1 1department of medical microbiology, nobel college, sinamangal, kathmandu, nepal affiliated to pokhara university. 2pathology department, b & b hospital, lalitpur, nepal. received: 26 feb 2022; revised: 20 jul 2022; accepted: 26 jul 2022; published online: 30 jul 2022 abstract klebsiella pneumoniae is a common opportunistic pathogen causing a wide range of infections; pneumonia, urinary tract infection, bacteremia, and liver abscesses. it infects primarily immunocompromised and immunocompetent individuals. it presents itself as an antibiotic-resistant bacterium, especially for third-generation cephalosporins and carbapenems, creating serious global challenges. therefore, this cross-sectional study was conducted in b & b hospital, lalitpur to screen the distribution of carbapenem resistance k. pneumoniae through ertapenem and to assess the minimum inhibitory concentration of imipenem for screened carbapenem resistance k. pneumoniae. from 3447 different clinical samples collected according to standard guidelines, k. pneumoniae was identified using standard microbiological techniques; staining and a panel of biochemical tests. the antibiotic susceptibility test of the isolates was performed by the kirby-bauer disc diffusion method as per clsi 2018 guidelines. the screening of carbapenem resistance was assessed by using ertapenem disc and the mic of imipenem for carbapenem resistance and intermediate was performed using an epsilometer. a total of 85 k. pneumoniae were identified and their antibiotic susceptibility test revealed that ceftriaxone was the least effective antibiotic. the number of mdr, carbapenem-resistant and intermediate isolates was 51, 46, and 3, respectively. the mic of imipenem through an epsilometer from ertapenem resistant and intermediate revealed that 31, 5, and 13 isolates were resistant, intermediate, and sensitive respectively. these findings showed the inconsistency in the detection of carbapenem-resistant isolates in routine microbiology laboratories and further support the other tests for the detection of carbapenem resistance as suggested by clsi. keywords: carbapenem-resistant klebsiella pneumoniae, ertapenem, mic corresponding author, e-mail: mahaju@gmail.com introduction klebsiella pneumoniae, an important member of the enterobacteriaceae family; residing in the gastrointestinal tract of us, is not only the most clinically isolated opportunistic pathogen from immunecompromised individuals, neonates, critically ill patients, or patients with other risk factors in healthcare settings [1] but also from immunocompetent individuals causing a wide range of infections mostly urinary tract infection, pyogenic liver abscess, necrotizing pneumonia or other life-threatening infections [2]. management of its infection becomes complicated after it is found to be not susceptible to the third-generation cephalosporin antibiotics, including monobactams [3]. this is further aggravated by the non-response of carbapenem antibiotics through either the expression of carbapenemase enzymes that make bacteria almost resistant to a β-lactam group antibiotic [4, 5] or alteration of permeability due to loss of porin or overexpression of the efflux pump [5, 6]. therefore, who prioritizes extended-spectrum β-lactamase [esbl] and carbapenemresistant klebsiella pneumoniae [crkp] as a critical public health threat [7]. the epidemiological distribution of crkp fluctuates in all countries [8] with significantly higher morbidity and mortality rate than those of carbapenem susceptible k. pneumoniae, which initiates devastating public health conditions [9]. this bacterium notoriety gained its name among antibiotic-resistant bacteria. the european antimicrobial resistance surveillance network (ears-net) showed that from 2005 the non-susceptibility rate of this bacterium had increased significantly against thirdgeneration cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems that have greater variation in different countries of the european union [10] and other parts of the world, increasing global public health concerns [11]. hence, who recognized this bacterium with acinetobacter baumannii, pseudomonas aeruginosa as a who priority pathogen list for “research and development” of new antibiotics [7]. therefore, this study was carried out to find out the frequency of crkp along with their antibiotic susceptibility profile and mic nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: https://nepjb.com/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0003-3398-7422 mailto:mahaju@gmail.com nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):4 0 4 4 saud et al. ©njb, bsn 41 of imipenem for screened carbapenem-resistant and intermediate isolates. materials and methods it is a hospital-based prospective cross-sectional study; carried out in the microbiology department of b and b hospital, nepal, from 15 july 2018 to 15 october 2018. the target group of this study was irrespective of sex, all age groups of patients attending the hospital for medical treatment. all collected data were entered and analyzed using spss v17.0. ethical consent was obtained from the nobel institutional review committee (irc). bacterial isolation and identification samples (blood, pus, urine, respiratory specimen, catheter tips, and joint fluid) were collected aseptically according to standard microbiological guidelines [12]. good quality specimens were accepted, while unlabeled or mislabeled specimens, dry swabs, specimens that leak from a container, delay in specimen transport, and inappropriately stored samples were excluded from this study. all samples, except blood, were cultured on blood agar and macconkey agar; incubated at 370c overnight, and identified as k. pneumoniae using gram staining and conventional biochemical tests (catalase, oxidase, indole test, motility, citrate utilization, triple sugar iron utilization and urease test) [13]. the reagents and culture media were purchased from himedia laboratories, india. the bd™ bactec™ fx40 automated blood culture system was used for blood culture, and a positive culture bottle was further subcultured on blood agar and macconkey as previously for identification [13]. antibiotic susceptibility test the antibiotic susceptibility test was performed following clsi guidelines 2018 through the disc diffusion method. eleven different antibiotics (himedia laboratories, india); amikacin (30 µg), cefepime (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), cotrimoxazole (25 µg), gentamicin (10 µg), nitrofurantoin (300 µg), piperacillintazobactam (100/10 µg), and ofloxacin (5µg) were tested in isolated klebsiella pneumoniae. the bacterium showing resistance to at least one antibiotic from three or more than three different classes was categorized as multidrug resistance [14]. the resistance to carbapenem was screened using ertapenem (10μg) disc. according to zone size diameter, isolates were differentiated as sensitive, intermediate, and resistant with zone size inhibition ≥22 mm, 19-21mm, and ≤18 mm, respectively [15]. the minimum inhibitory concentration (mic) of the imipenem was tested for isolates showing intermediate or resistance to carbapenem using the imipenem ezy mictm strip following the manufacture instructions (himedia laboratories, india). the mic of imipenem was interpreted and the isolates were differentiated as sensitive (≤1 µg/ml), intermediate (2 µg/ml), and resistant (≥4µg/ml) [15]. for quality control of the mic test strip, carbapenem susceptible escherichia coli atcc 25922 was used. results patient characterization a total of 3447 specimens were received in the trimester period in which 771 samples showed culture-positive from which 815 bacteria were isolated. among 815 bacterial isolates, 85 isolates were confirmed as klebsiella pneumoniae. the rest data are shown in table 1. table 1. clinical and social demography of patients s.n. status of patients number (%) 1 opd 15 (17.65%) 2 inpatients 70 (82.35%) s.n. sex number 1 male 50 (58.82%) 2 female 35 (41.18%) s.n. age group number 1 below 20 12 (14.11%) 2 20-30 14 (16.47%) 3 30-40 12 (14.11%) 4 40-50 9 (10.59%) 5 50-60 14 (16.47%) 6 60 and above 24 (28.24%) antibiotic susceptibility test the antibiotic susceptibility test showed that ceftriaxone (59, 69.42%) was the most non-susceptible antibiotic, followed by ciprofloxacin (49, 57.65%) and gentamicin (48, 56.47%). furthermore, 60% (51 of 85) isolates were multidrug-resistant. the remaining data are shown in table 2. carbapenem-resistant the screening of carbapenem resistance showed that in 85 k. pneumoniae isolates, the carbapenem resistance and intermediate isolates were 46 (54.13%) and three (3.52%) respectively. the mic test of both intermediate and resistant isolates was done as suggested by the clsi guideline 2018. among the 49 isolates, the mic test of imipenem showed that 31 isolates were resistant, five intermediate, and 13 sensitives. furthermore, in 85 isolates, 51 isolates were mdr k. pneumoniae isolates, in which 45 isolates were resistant to carbapenem and six isolates were susceptible to carbapenem. nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):4 0 4 4 saud et al. ©njb, bsn 42 figure 1. mic test discussion klebsiella pneumoniae is an opportunistic pathogen responsible for causing various community-acquired and healthcare-associated infections. furthermore, the infection caused by this bacterium cannot be neglected, as it is included in eskape pathogens and the increasing incidence of crkp strains attracts attention to clinicians and other stakeholders. in our study, the highest percentage of k. pneumoniae was obtained in the inpatient department than others. this higher incidence of k. pneumoniae in long-term hospitalized patients may be related to the immune status of the patients, as the bacterium was isolated from the surgery unit with the use of invasive devices and administration of immunosuppressive drugs. in hospital settings, the transmission of the pathogen increases drastically because the colonization rate increases with an extended stay in the hospital and prolonged antibiotic therapy. a similar study carried out in the united states also claimed a higher incidence of k. pneumoniae infections in long-term acute care hospitals than in the short-term hospital stay [16]. antibiotic resistance is a common problem in k. pneumoniae. it is naturally resistant to the penicillin group of antibiotics [17] or acquires antibiotic resistance genes from mobile genetic cassettes called integrons, often carried out by transposons and transferable plasmids that transmit horizontally to receptor cells, integrated on plasmids or chromosomes through homologous recombination, expressing its fitness in the presence of antibiotics [18]. in this study, we evaluated 11 different antibiotics, in which amikacin, gentamicin, ciprofloxacin, ofloxacin, ceftriaxone, ertapenem were tested in all isolates, and the rest antibiotics were tested either as second-line antibiotics or depending on a source of clinical samples. our results showed a mixed antibiotic resistance profile compared to others in terms of antibiotic use, time period, bacterium source and country. approximately 48.24% and 56.47% of the isolates were not susceptible to aminoglycosides, amikacin, and gentamicin, respectively. a range of studies shows a wide variation in resistance pictures that range from 1% to 86% for gentamicin. a study in the eu/eea region showed that its resistance per cent ranges from below 1% to greater than 50 [19]. a comparative study from france and algeria revealed that its resistance was 28% and 86%, respectively, [20] while in a study from iran and india, the resistance rate was found to be 24% and 37.5%, respectively [21, 22]. a slightly lower percentage, 41%, was observed in nepal [23] which is nearly equal to our study. cephalosporins, such as ceftriaxone, are frequently used antibiotics for k. pneumoniae until they become esbl producers. our study showed that nearly 70% of the isolates were resistant to ceftriaxone. a similar finding was described from ethiopia [24] and a four-year consecutive study from greece [19]. ciprofloxacin and table 2. antibiotic susceptibility pattern of k. pneumoniae s.n. antibiotic susceptibility pattern total isolates antibiotics used sensitive (%) intermediate (%) resistant (%) 1 amikacin 24 (28.23) 20 (23.53) 41 (48.24) 85 2 gentamicin 34 (40) 3 (3.53) 48 (56.47) 85 3 ciprofloxacin 28 (32.94) 8 (9.41) 49 (57.65) 85 4 ofloxacin 33 (38.82) 5 (5.88) 47 (55.30) 85 5 ceftriaxone 24 (28.23) 2 (2.35) 59 (69.42) 85 6 ertapenem 36 (42.35) 3 (3.52) 46 (54.13) 85 7 cefepime 11 (16.18) 8 (11.76) 49 (72.06) 68 8 piperacillin-tazobactam 9 (14.06) 12 (18.75) 43 (67.19) 64 9 chloramphenicol 38 (61.30) 11(17.74) 13 (20.96) 62 10 cotrimoxazole 28 (50) 1 (1.78) 27 (48.22) 56 11 nitrofurantoin 6 (13) 6 (13) 34 (74) 46 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):4 0 4 4 saud et al. ©njb, bsn 43 ofloxacin are alternative antibiotics of choice if the isolates are esbl producers. the non-susceptibility rates for ciprofloxacin and ofloxacin were 57.65 % and 55.3 % in our study. similar to aminoglycosides, a wider range of variations in the non-susceptibility of quinolones was observed, ranging from less than 1% to more than 90%. the study of bulgaria, italy, and romania was in line with our study, while from germany, denmark, iran, and india, the non-susceptibility rate was lower than in our study [19, 21, 22]. a few studies from nepal showed that the resistance rate is more than 85 %, which is quite much higher than ours [23, 25]. in general, different studies conducted at different times revealed significant variability in antibiotic resistance patterns. such a type of variation was also observed in other antibiotics mentioned in table 2, which might be due to the low number of sample studies or how meticulously antibiotics were used in that country to mitigate antibiotic resistance problems. hence, resistance to these first-line agents represents an unprecedented challenge to clinicians, scientists, and healthcare systems. carbapenems (imipenem, meropenem, and ertapenem) are the antibiotics of choice for those k. pneumoniae that are mdr and esbl producers [26]. the phenotypic lab detection of carbapenem resistance is quite confusing and for confirmation of carbapenemase producer, different additional tests should be performed [27]. therefore, clsi recommends that ertapenem non-susceptibility is the most sensitive indicator for carbapenemase producers, and additional tests should not be performed for purposes other than epidemiological or infection control after breaking point evaluation of ertapenem, meropenem, and imipenem [15]. in our study, 49 (both intermediate and resistant) isolates showed positive screening through non-susceptibility to ertapenem. this suggests that resistance to carbapenem arises due to the formation of carbapenemase enzymes of classes a, b and d of the amber class of β-lactamase, restricting the treatment option [27]. the crkp was subjected to the etest to determine the mic of the imipenem. the test results showed that susceptible, intermediate and resistant were 13 [mic ≤1µg/ml), 5 [4µg/ml ≤mic≥1µg/ml) and 31 [mic ≥4 μg/ml), respectively. this implies that results from phenotypic methods can vary, as suggested by clsi. conclusions the fast-growing antibiotic-resistant klebsiella pneumoniae is a global problem, including in nepal, such that the choice of an effective antibiotic is now chaotic. the continued emergence of crkp has wreaked havoc on its routine diagnosis, as well as on its therapeutic treatment options. susceptibility testing [disc diffusion and mic) provides valuable information for therapeutic inference but does not adequately address carbapenem resistance, which is significant for infection control and epidemiological evidence necessary to curb the spread of carbapenem-resistant enterobacteriaceae. authors' contributions the laboratory works were performed by sarada saud, ashwani agrawal, soniya pokhrel and sushma subedi. research conceptualization, protocol selection and preliminary manuscript preparation were done by sarada saud, ashwani agrawal, soniya pokhrel, sushma subedi, sanjit shrestha and niroj man amatya. final manuscript preparation was done by niroj man amatya. the study was supervised under the guidance of sanjit shrestha and niroj man amatya. all authors read and approved the final manuscript. competing interests on behalf of all authors, the corresponding author states that there is no competing interest. financial disclosure it is a self-funded pilot study and no funding is granted. acknowledgements we would like to thank all the participants attained in this study and the pathology department of the b and b hospital, nepal. ethics approval the ethical consent was procured from nobel institutional review committee [irc) with reference number bch irc 161/2018. additional disclosure this manuscript has been already published in preprint form with title “screening ofklebsiella pneumoniaefor carbapenem resistance and 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thomas r, ramyashree a. isolation and antimicrobial sensitivity pattern of klebsiella pneumoniae from sputum samples in a tertiary care hospital. int j biomed adv res. 2016;7(2):53-7. 23. subedi s, maharjan j, shrestha b. antibiotic susceptibility test of klebsiella pneumoniae and k. oxytoca isolated from different clinical samples and perform random amplified polymorphic dna among k. pneumoniae. microbiology research journal international. 2016:1-11. 24. gashe f, mulisa e, mekonnen m, zeleke g. antimicrobial resistance profile of different clinical isolates against thirdgeneration cephalosporins. journal of pharmaceutics. 2018;2018 25. parajuli np, acharya sp, mishra sk, parajuli k, rijal bp, pokhrel bm. high burden of antimicrobial resistance among gram negative bacteria causing healthcare associated infections in a critical care unit of nepal. antimicrobial resistance & infection control. 2017;6(1):67 26. paterson dl. recommendation for treatment of severe infections caused by enterobacteriaceae producing extended-spectrum βlactamases (esbls). clinical microbiology and infection. 2000;6(9):460-3. 27. miller s, humphries rm. clinical laboratory detection of carbapenem-resistant and carbapenemase-producing enterobacteriaceae. expert review of anti-infective therapy. 2016;14(8):705-17 http://atlas.ecdc.europa.eu/public/index.aspx?instance nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 50-62 research article doi: https://doi.org/10.3126/njb.v9i1.38667 ©njb, bsn 50 evaluation of phytochemical, antioxidant and antibacterial activities of selected medicinal plants shrimita shrestha1 , sudip bhandari1 , babita aryal2 , bishnu p marasini1 , santosh khanal1 , pramod poudel3 , binod rayamajhee4 , bikash adhikari2 , bibek raj bhattarai2 and niranjan parajuli2 1department of biotechnology, national college, naya bazar, kathmandu, nepal 2central department of chemistry, tribhuvan university, kirtipur, kathmandu, nepal 3central department of biotechnology, tribhuvan university, kirtipur, kathmandu, nepal 4department of infectious diseases and immunology, kathmandu research institute for biological sciences (kribs), lalitpur, nepal received: 08 mar 2021; revised: 10 jul 2021; accepted: 15 jul 2021; published online: 31 jul 2021 abstract medicinal plants are important reservoirs of bioactive compounds that need to be explored systematically. because of their chemical diversity, natural products provide limitless possibilities for new drug discovery. this study aimed to investigate the biochemical properties of crude extracts from fifteen nepalese medicinal plants. the total phenolic contents (tpc), total flavonoid contents (tfc), and antioxidant activity were evaluated through a colorimetric approach while the antibacterial activities were studied through the measurement of the zone of inhibition (zoi) by agar well diffusion method along with minimum inhibitory concentrations (mic) by broth dilution method. the methanolic extracts of acacia catechu and eupoterium adenophorum showed the highest tpc (55.21 ± 11.09 mg gae/gm) and tfc (10.23 ± 1.07 mg qe/gm) among the studied plant extracts. acacia catechu showed effective antioxidant properties with an ic50 value of 1.3 μg/ml, followed by extracts of myrica esculenta, syzygium cumini, and mangifera indica. morus australis exhibited antibacterial activity against klebsiella pneumoniae (zoi: 25mm, mic: 0.012 mg/ml), staphylococcus aureus atcc 25923 (zoi: 22 mm, mic: 0.012 mg/ml), pseudomonas aeruginosa (zoi; 20 mm, mic: 0.05 mg/ml), and methicillin-resistant staphylococcus aureus (mrsa) (zoi: 19 mm, mic: 0.19 mg/ml). morus australis extract showed a broad-spectrum antibacterial activity, followed by eclipta prostrata, and hypericum cordifolium. future study is recommended to explore secondary metabolites of those medicinal plants to uncover further clinical efficacy. keywords: antibacterial activity; medicinal plants; secondary metabolites; minimum inhibitory concentration corresponding author, email: niranjan.parajuli@cdc.tu.edu.np introduction the separation and identification of physiologically active chemicals and molecules from medicinal plants has resulted in innovative treatments and pharmaceutical advances. secondary metabolites extracted from medicinal plants have played a significant role in upholding human health against various infectious diseases since ancient times. plant extracts or their active phytoconstituents have been used as folk medicine by 80 % of the world's population in conventional therapies [1]. it is believed that over 50% of all modern clinical drugs are of natural product origin [2]. multidrug resistance (mdr) is characterized as an acquired non-susceptibility to at least one antimicrobial agent from three or more categories [3]. mobile genetic elements such as interferons, plasmids, and transposons are the most common carriers of antibiotic resistance among bacteria [4]. the rapid emergence of resistance to newly introduced antimicrobial agents, suggests that even a new antimicrobial agent would not be a complete solution to the problem [5]. mdr pathogens have raised a significant problem in public health by undermining the existing antibiotic-based treatment era, resulting in an increased mortality rate in patients [6]. mdr pathogens worsen the disease severity and put the value of antibiotics at risk, affecting the global economy [7]. it is anticipated that if the race of antimicrobial resistance (amr) keep rising, it would take the lives of nearly ten million peoples annually by 2050 [8]. thus, a new antibacterial agent is urgently needed to treat mdrinduced infections caused by pathogens such as enterobacteriaceae, staphylococcus aureus, extendednepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0003-2896-9799 https://orcid.org/0000-0002-7899-2885 https://orcid.org/0000-0001-9731-8485 https://orcid.org/0000-0001-6153-5234 https://orcid.org/0000-0002-0186-6119 https://orcid.org/0000-0001-5304-7812 https://orcid.org/0000-0003-3007-8901 https://orcid.org/0000-0002-5532-6644 https://orcid.org/0000-0003-0259-5004 https://orcid.org/0000-0002-9233-6489 mailto:niranjan.parajuli@cdc.tu.edu.np nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 51 spectrum β-lactamase (esbl) producing bacteria, among others [9]. table 1: description of medicinal plants used in this study. medicinal plants voucher specimen local name parts used eclipta prostrata ncdb203 bhringaraj whole plants shorea robusta ncdb212 saal leaves smallanthus sonchifolius ncdb214 yacon leaves hypericum cordifolium ncdb201 arelu leaves mangifera indica ncdb211 mango leaves morus australis ncdb210 kimbu barks psidium guajava ncdb206 guava leaves chrysanthemum indicium ncdb205 godawari leaves myrica esculenta ncdb208 kafal leaves urtica ardens ncdb213 sisnoo buds pterocarpus marsupium ncdb204 bijayasal barks eupoterium adenophoium ncdb202 banmara leaves zingiber officinale ncdb200 aaduwa leaves acacia catechu ncdb209 khair barks syzygium cumini ncdb207 jamun leaves acinetobacter baumannii, pseudomonas aeruginosa, medicinal plants produce secondary metabolites that can tackle mdr pathogens. furthermore, medicinal plants have immunomodulatory and antioxidant activity, which result in antibacterial properties. they have a wide range of immunomodulatory effects stimulating both non-specific and specific immunity [10]. antimicrobial and antioxidant activity is found in phytochemicals such as vitamins (a, c, e, and k), tannins, carotenoids, polyphenols, flavonoids, alkaloids, saponins, pigments, enzymes, terpenoids, and minerals [11]. nonetheless, analgesic, antibacterial, deodorizing, febrifuge, fungicidal, antiseptic, astringent, galactagogue, diuretic, antidepressant, insecticidal, antipyretic, and sedative properties have been recorded for volatile oils from plants (blanco et al., 2009; bekoe et al., 2018; iscan et al. 2002). however, microorganisms have continuously evolved with a wide range of metabolic mechanisms to overcome drug effects [6]. plant-derived drugs are a superior choice over synthetic drugs because of fewer side effects and adverse effects (bindu jacob & narendhirakannan r.t., 2019; verma et al., 2018). nepal is rich in biodiversity and geographical condition with diverse flora, and numerous species are believed to possess curative properties. however, most of these claims lack scientific validation. the plants selected for this study are being used routinely by the indigenous people as remedies against various human diseases since ancient times. therefore, the selected plants may contain certain important bioactive compounds that could have some medicinal and antimicrobial properties and some therapeutic value based on phytochemical constituents and their secondary metabolites. hence, the antibacterial activity of plant extracts reported here would be beneficial to identify some potent secondary metabolites as future drug candidates for the therapeutic measures of mdr-strains-induced infections in nepal and beyond. materials and methods bacterial isolates eight mdr bacterial strains: acinetobacter spp. (628), citrobacter freundii (377), methicillin-resistant staphylococcus aureus (mrsa) (338), klebsiella pneumoniae (386), pseudomonas aeruginosa (484), escherichia coli (2a), morganella morganii (4331), and xanthomonas spp. (767) were collected from the national public health laboratory (nphl), kathmandu, and transferred aseptically to the laboratory of the department of biotechnology, national college for further study. all isolates were obtained from clinical specimens. besides, atcc strains such as e. coli 25922, s. aureus 25923, salmonella typhimurium 14028, and k. pneumoniae 700603 were also collected from the nphl stored at -20°c for further studies. collection of plant materials different parts (leaves, bark, fruit, roots, and stem) were collected based on the ethnomedicinal and traditional medicinal practices from different geographical regions of nepal as depicted in table 1 (collection period: january to june 2017). the plant samples were identified by national herbarium and plant laboratories, godawari, lalitpur, nepal, and herbarium collections were deposited in the department of botany, national college, khusibu, kathmandu. preparation of plant extracts the plant parts (mentioned in table 1) were dried in the shade at room temperature, pulverized into the powders with the help of a grinding mill, and then soaked in methanol for 24 hours. then, they were filtered, and the process was repeated three times with fresh methanol. to obtain plant extracts, the filtrates were concentrated in a rotary evaporator at 50 °c. determination of tpc and tfc using folin-ciocalteu reagent and a 96-well plate-based colorimetric process, the tpc was calculated (ainsworth & gillespie, 2007; bhandari et al., 2021). initially, 20 µl of plant extract was mixed with 100 µl of nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 52 folin-ciocalteu's reagent (1:10 v/v) and 80 µl of sodium carbonate (7.5%, w/v) in each well-containing standard and sample before incubation. then, the sample was incubated at room temperature, and absorbance was measured at 765 nm[15]. by comparing tpc to standard gallic acid, milligrams of gallic acid equivalents per gram of extract (mg gae/gm) were determined. likewise, for tfc, 20 µl of plant extract was mixed with 60 µl of methanol, 5 µl of potassium acetate (1 m), 5 µl of 10% aluminum chloride, and 110 µl of distilled water, then incubated at room temperature for 30 minutes, and the absorbance was measured at 415 nm[17]. likewise, tfc was expressed as milligrams of quercetin equivalents per gram of extract (mg qe/gm extract) by comparing to standard quercetin [17]. determination of antioxidant activity the antioxidant property was determined by discoloration assay based on the scavenging of 2, 2 diphenyl-1-picrylhydrazyl (dpph) free radical (0.1 mm ) (brand-williams et al., 1995; aryal et al., 2021) at 517 nm using a multi-plate reader (epoch 2, biotek, instruments, inc., usa), maintaining 1 mg/ml of quercetin as a control. crude extracts were allowed to react with dpph free radicals for 30 minutes at room temperature. the scavenging of dpph radical was calculated by using the following expression: (where optical density (od) is the absorbance). % scavenging = 100 − (od of extract) (od of control) × 100 antibacterial activity using sterile cotton swabs moistened with the bacterial suspension, an inoculum suspension containing 1.5 x108 cfu/ml of bacteria was spread on firm muller-hinton agar (mha) plates (balouiri et al., 2016; marasini et al., 2015; valgas et al., 2007). using a sterile cork borer, wells were punched in plates (6 mm diameter) and micropipettes were used to fill the wells with a functioning suspension (50µl) of plant extracts (50 mg/ml), as well as neomycin (20 µg /ml), amikacin (30 mcg), and nitrofurantoin (30 mcg) as positive controls and 50 % dmso as negative controls [23]. the mha plates were incubated for 24 hours at 37°c and finally, the zoi was determined after overnight incubation. determination of mic the broth dilution method was followed to determine mic values of plant extracts as recommended by the clinical and laboratory standards institute [24]. extracts of e. adenophorum, m. australis, e. prostrata, a. catechu, z. officinale, p. marsupium, s. robusta, m. indica, s. sonchifolius, m. esculenta, u. ardens, h. cordifolium, s. cumini, p. guajava, and c. indicium showed significant antibacterial activity with larger zoi, so they were selected for the determination of mic value. the plant extracts were two-fold diluted to get a series of concentrations ranging from 25 mg/ml to 0.012 mg/ml in freshly prepared sterile nutrient broth. then 20 µl of bacterial culture adjusted to 0.5 mcfarland standard was inoculated in each dilution tube and incubated at 37˚c for 24 hours. the set-up included bacterial growth controls containing test tubes with media inoculated with 20 µl of bacterial inoculum only and negative controls with media and plant extract without bacterial inoculum. the mic value was measured by choosing the lowest concentration of plant extract that inhibited the organism's growth in the test tubes, as determined by unaided observation. the bacterial growth in the tubes containing the plant extracts was compared to the control sample without the plant extracts to establish the growth endpoints. each assay was carried out in triplicate to confirm the results. results the researches on medicinal plants have been carried throughout the world to explore the bioactive compounds which could be used to make a preventive or treatment approach against various health complications. the ethnopharmacological applications of plants under study were depicted in table 2. yields, tpc and tfc of plant extracts the percentage yield of plant extracts varied from 5.94% to 28.47% (table 3). extracts of h. cordifolium had the highest percentage yield (28.47%), followed by a. catechu (23.0%), p. guajava (21.82%), and m. esculenta (19.02%). noticeably all plant extracts were found to be in semi-solid inconsistency. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 53 table 2: medicinal plants selected understudy with their ethnopharmacological applications medicinal plants family ethnopharmacological applications eclipta prostrata asteraceae used as an anti-inflammatory, antivenom [25], anti-aging, hepatoprotective, anti-viral, antimicrobial agents. bithiophenes and 5-(but-3-yne-1,2-diol)-5′-hydroxy-methyl2,2′bithiophene isolated from this plant used as antibacterial and antihyperglycemic [26], [27]. shorea robusta dipterocarpaceae used in the treatment of ulcer, cough, itching, leprosy, anthelmintic [28]. antibacterial wound healing and anti-inflammatory activity due to the presence of polyphenols, flavonoids, and triterpenoids, etc. ursolic acid extracted from this plant is responsible for showing antibacterial activity [29]. smallanthus sonchifolius asteraceae used as a functional food, antioxidant, antimicrobial, prebiotic, growth promoter [30]. leaves extract contains the compounds fluctuanin and enhydrin show antibacterial activity [31]. hypericum cordifolium hypericaceae treatment of back pain and broken bones, an antidepressant [32]. dermatological, neurological, and traumatological problems, antibacterial activity [33]. mangifera indica anacardiaceae used for gastric disorders, mouth sores, tooth pain, and dermatological disorders. [34] treatment for diabetes, infertility, ethanolic extract of m. indica showed significant antibacterial activity. methanolic extract displayed cytotoxicity against the pancreatic cancer cell line. magniferin (5) from plant extract showed antimicrobial effect [35], [36]. morus australis moraceae treatment for fever, protect the liver, improve eyesight, strengthen joints, lower blood pressure [37]. leaves contain 1-deoxynojirimycin known to have potential α glucosidase inhibition activity. the piperidine alkaloid and glycoproteins from the extract of m. austrralis have been used for antidiabetic agents [38]. psidium guajava myrtaceae used for ulcers, wounds, toothache, anti-allergic effects, anti-cancer effects, and antihyperglycemia [39]. used effectively in diabetes, diarrhea, dysentery, pain relief, cough, gastroenteritis, hypertension, caries. the hypoglycemic components in psidium guajava might be due to oleanolic acid, arjunolic acid, ursolic acid, and glucuronic acid [40]. chrysanthemum indicium asteraceae used for hypertension, pneumonia, colitis, stomatitis, fever, neurological problems, headache [41], antipyretic purpose, treatment of cephalgia, vertigo, and eye inflammations [42]. myrica esculenta myricaceae used for cough, anemia, asthma, chronic dysentery, fever, sores, tumors, nasal catarrh, piles, throat complaints, ulcers, and urinary discharges[43]. used against different disease conditions such as; antidiabetic, antiallergic, antimicrobial, anti-ulcer, antihypertensive, antioxidant, and higher phenolic and flavonoid compounds including myricetin, myricanol, and myricanone have anti-inflammatory properties. [44]. urtica ardens urticaceae used for diabetes, diarrhea, excessive menstrual bleeding, urinary disorders, respiratory problems, ulcers, asthma, rheumatism, high blood pressure [45]. treatment for sprains, kidney stones, hemorrhoids, flu, fever, hepatoprotective, nephroprotective effect, etc. [46]. pterocarpus marsupium fabaceae stomachache, cholera, dysentery, urinary complaints, tongue disease, toothache, and cough are all treated. [47]. treatment of diabetes, jaundice, and an ulcer [48]. eupoterium adenophoium asteraceae used for treatment of emetic, diaphoretic, stimulant, tonic, fever, cuts and wounds, analgesic [49]. used as an anti-inflammatory, blood coagulant, antimicrobial, antiseptic, and analgesic, antipyretic. isomers of mono-caffeoylquinic acid present in e. adenophoium exhibit potent anti-inflammatory, anti-bacterium, and anti-obesity properties [50]. zingiber officinale zingiberaceae treatment of diabetes, high blood pressure, cancer, stomachache, nausea, asthma, respiratory disorders [51]. treatment for diabetes, blood pressure, stomach ache, weight loss, diarrhea, and nausea. geraniol present in z. officinale shows potential antiinflammatory and antioxidant effects [52]. acacia catechu fabaceae it can be used to treat colds, coughs, ulcers, boils, and skin eruptions, bleeding masses, antipyretics, and acute and chronic wound healing. [53]. the key constituents of a. catechu are catechin and taxifolin, which have antifungal, antiviral, antibacterial, antiinflammatory, and antioxidant properties. [53]. syzygium cumini myrtaceae used for diabetes mellitus, constipation, stomachache, hiv, inflammation leucorrhoea, fever, strangury, and dermopathy [54], [55]. ferulic acid and catechins possess antioxidant properties [56]. gallocatechins are used to treat diabetes. quercetin isolated from s. cumini is used to treat diabetes and treat cytotoxicity. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 54 table 3: physical characteristics and percentage yield of the crude extracts. medicinal plants local name dry weight of plant (gm) percentage yield (%) hypericum cordifolium arelu 40 28.46 acacia catechu khayr 50 23.0 psidium guajava guava 50 21.82 myrica esculenta kafal 50 19.02 syzygium cumini jamun 50 17.0 mangifera indica mango 50 14.9 chrysanthemum indicium godawari 50 13.44 zingiber officinale ginger 50 12.5 smallanthus sonchifolius ground apple 50 11.16 pterocarpus marsupium bijayasal 50 11.02 eupoterium adenophorum banmara 50 10.42 shorea robusta sal 50 9.1 eclipta prostrata bhringraj 70 6.54 morus australis kimbu 34.8 6.03 urtica ardens sisnoo 50 5.94 table 4: tpc of medicinal plants. medicinal plants tpc (mg gae/gm) acacia catechu 55.21 ± 11.09 urtica ardens 50.01 ± 5.0 mangifera indica 49.88 ± 19.2 psidium guajava 45.21 ± 2.73 shorea robusta 45.21 ± 4.15 eupoterium adenophorum 37.61 ± 4.14 hypericum cordifolium 36.28 ± 2.37 chrysanthemum indicium 32.95 ± 4.43 syzygium cumini 28.28 ± 1.85 myrica esculenta 23.21 ± 4.42 pterocarpus marsupium 22.68 ± 1.35 morus australis 19.75 ± 2.94 zingiber officinale 19.21 ± 2.0 eclipta prostrata 18.95 ± 1.24 smallanthus sonchifolius 9.08 ± 1.01 table 5: tfc of medicinal plants. medicinal plants tfc (mg qe/gm) eupoterium adenophorum 10.23 ± 1.07 morus australis 9.10 ± 0.98 eclipta prostrata 8.67 ± 0.57 acacia catechu 8.34 ± 0.77 zingiber officinale 7.78 ± 0.71 pterocarpus marsupium 7.70 ± 0.85 shorea robusta 7.68 ± 0.71 mangifera indica 7.52 ± 1.12 smallanthus sonchifolius 7.40 ± 0.83 myrica esculenta 6.84 ± 1.30 urtica ardens 5.89 ± 0.35 hypericum cordifolium 5.89 ± 1.68 syzygium cumini 5.72 ± 0.52 psidium guajava 5.26 ± 1.15 chrysanthemum indicium 4.93 ± 0.66 tpc of plant extracts was expressed in terms of gallic acid equivalent (mg gae/gm dry weight of extract) and placed in the order from higher to lower using a calibration curve of gallic acid (y =0.0025x + 0.0413, r² = 0.981). tpc of plant extracts ranged from 55.21 ± 11.09 to 9.08 ± 1.0 mg gae/gm. extract of a. catechu exhibited the highest tpc, followed by u. ardens, m. indica, p. guajava, and s. robusta respectively (table 4). similarly, tfc of plant extracts was expressed in terms of quercetin equivalent (mg qe/gm) and placed in the order from higher to lower using a calibration curve of quercetin (y = 0.0202x – 0.972, r² = 0.972). the extract of e. adenophorum showed the highest tfc (10.23 ± 1.07 mg qe/gm), followed by m. australis and e. prostrata respectively (table 5). antioxidant activity free radical scavenging activity was used to assess the antioxidant activity of plant extracts, and the resulting degree of decolorization is stoichiometric in terms of the number of electrons captured from plant extracts. the results of antioxidant abilities of plant extracts were compared with standard quercetin (ic50 2.28 µg/ml). among them, methanolic extract of a. catechu, m. esculenta, s. cumini, and m. indica showed promising antioxidant properties with ic50 ranging 1.3-1.80 µg/ml (table 6). evaluation of antibacterial activity plant extracts were examined for antibacterial activity against eight mdr bacteria and four atcc bacterial species adopting the agar well diffusion technique. the extracts of m. australis, s. robusta, and m. indica showed the largest zoi i.e. 21 mm at 50 mg/ml towards e. coli atcc 25922 in agar plates. meanwhile, only e. prostrata extract showed 7 mm of the zoi against k. pneumoniae atcc 700603. the m. australis extract showed 22 mm of the zoi against s. aureus atcc 25923, which was the highest among the zoi shown by plant extract. similarly, m. australis extract showed the highest zoi against three mdr bacterial strains, k. pneumoniae, mrsa, and p. aeruginosa with 25 mm, 19 mm, and 20 mm, respectively (table 8). nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 55 table 6: ic50 values of plant extracts for antioxidant assay. medicinal plants ic50 (µg/ml) smallanthus sonchifolius 329.0 ± 0.01 morus australis 208.60 ± 0.02 pterocarpus marsupium 38.50 ± 0.04 shorea robusta 2.50 ± 0.01 mangifera indica 1.80 ± 0.06 syzygium cumini 1.60 ± 0.04 myrica esculenta 1.50 ± 0.03 acacia catechu 1.30 ± 0.05 quercetin (standard) 2.28 note: only significant results were shown and placed in order from higher to lower ic50 value. the extract of s. robusta and m. indica showed 17 mm of the zoi against mdr a. baumannii. figure 1, presents zoi of plant extracts against atcc strains e. coli and s. aureus while figure 2, presents zoi of plant extracts against the mdr k. pneumoniae and xanthomonas species. figure 1. antibacterial activity of plant extracts against atcc organism e. coli and s. aureus: a) neomycin; b) 50% dmso; c) e. prostrata; d) p. marsupium; e) a. catechu: f) m. indica; g) s. robusta; h) c. indicium. although some plant extracts exhibited potent antimicrobial activity towards some bacterial species, a higher number of plant extracts had a minimum antibacterial effect. the mic of plant extracts against atcc strains was between 0.012 mg/ml to 25 mg/ml (table 9). extracts of m. australis and h. cordifolium showed a broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria such as k. pneumoniae, e. coli, and s. aureus. the most potent antibacterial activity (mic = 0.012 mg/ml) was shown by extracts of m. australis, h. cordifolium, and p. guajava, and the least antibacterial activity (mic = 25 mg/ml) was observed in extracts of e. prostrata and s. cumini against atcc strain of s.aureus. regarding mdr strains, the most potent antibacterial activity (mic = 0.012 mg/ml) was shown by the extracts of m. australis and h. cordifolium against k. pneumoniae (386), followed by m. australis against xanthomonas species (4331) and p. aeruginosa (484) (table 10). figure 2. antibacterial activity of plant extracts against mdr k.pneumoniae and xanthomonas species; a) neomycin; b) 50% dmso; c) h. cordifolium; d) s. cumini; e) m. australis: f) a. catechu; g) m. indica; h) p. marsupium; i) m. esculenta. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 56 table 7: antibacterial activity of plant extracts against atcc bacterial strains. medicinal plants bacterial strains e. coli atcc 25922 k. pneumoniae atcc700603 s. typhimurium atcc 14028 s. aureus atcc 25923 eupoterium adenophorum 12 11 morus australis 21 22 eclipta prostrata 9.0 7.0 11 acacia catechu 18 15 zingiber officinale pterocarpus marsupium 12 14 shorea robusta 21 17 mangifera indica 21 14 smallanthus sonchifolius myrica esculenta 16 19 urtica ardens hypericum cordifolium 10 18 syzygium cumini 17 13 psidium guajava 16 15 chrysanthemum indicium 8.0 12 neomycin 22 10 15 20 50% dmso diameter of zone of inhibition in mm, well diameter = 6 mm, (-) = no antibacterial activity discussion in developing health care, the search for new medicines with better or enhanced therapeutic actions derived from medicinal plants with ethnobotanical significance has become increasingly valuable [57,58]. extraction is the most important step in obtaining the plant's bioactive compounds, and the yield is determined by the solvent and extraction method used [59]. in this study, methanol was used as a solvent with a table 8: antibacterial activity of plant extracts against mdr bacterial strains. medicinal plants bacterial strains 2a 386 338 628 377 767 4331 484 eupoterium adenophorum 13 15 11 morus australis 25 19 14 15 20 eclipta prostrata 10 16 10 9.0 acacia catechu 14 12 14 zingiber officinale pterocarpus marsupium 13 12 17 11 shorea robusta 17 mangifera indica 17 12 smallanthus sonchifolius 9.0 myrica esculenta 13 15 16 urtica ardens 9 hypericum cordifolium 20 12 16 20 syzygium cumini 16 16 14 17 psidium guajava 12 14 17 chrysanthemum indicium 15 15 8.0 neomycin 15 23 15 11 10 50% dmso amikacin 23 20 15 23 nitrofurantoin 22 18 16 17 16 15 (-) no antibacterial activity, 2a = e. coli, 338 = methicillin-resistant s. aureus (mrsa), 386 = k. pnemoniae, 628 = a. baumannii, 377 = c. freundii, 767 = xanthomonas species, 4331 = m. morganii, 484 = p. aeruginosa nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 57 percentage yield of h. cordifolium being the highest (28.46 %) followed by a. catechu (23 %) (table 3). the methanolic extract of a. catechu showed the highest tpc, while the extract of e. adenophorum showed the highest table 9: mic of plant extracts against atcc reference strains. medicinal plants bacterial strains e. coli atcc 25922 k. pneumoniae atcc 700603 s. typhimurium atcc 14028 s. aureus atcc 25923 eupoterium adenophorum morus australis 3.125 6.25 0.012 eclipta prostrata 6.25 25.0 acacia catechu 0.39 6.25 zingiber officinale pterocarpus marsupium 12.5 1.56 shorea robusta 3.125 12.5 mangifera indica 0.39 12.5 smallanthus sonchifolius myrica esculenta 0.097 1.56 urtica ardens hypericum cordifolium 6.25 6.25 0.012 syzygium cumini 0.39 25.0 psidium guajava 0.39 0.012 chrysanthemum indicium 6.25 neomycin 0.39 3.12 0.78 0.39 50% dmso diameter of zone of inhibition in mm, well diameter = 6 mm, (-) = no antibacterial activity reported. neomycin serves as positive control while 50% dmso serves as a negative control for the test. the concentration of plant extracts expressed in mg/ml. table 10: mic of plant extracts against mdr bacterial strains. medicinal plants bacterial strains 386 338 628 767 4331 484 eupoterium adenophorum 1.56 morus australis 0.012 0.19 3.12 0.05 0.05 eclipta prostrata 1.56 6.25 3.12 12.5 acacia catechu 0.78 6.25 1.56 zingiber officinale pterocarpus marsupium 0.39 1.56 3.12 0.39 12.5 shorea robusta 6.25 mangifera indica 3.12 0.78 smallanthus sonchifolius 6.25 myrica esculenta 0.39 12.5 3.12 1.56 6.25 urtica ardens 12.5 hypericum cordifolium 0.012 0.19 6.25 0.78 syzygium cumini 0.19 6.25 0.78 psidium guajava 3.12 3.12 1.56 chrysanthemum indicium 1.56 6.25 1.56 50% dmso neomycin 0.78 6.25 12.5 0.012 amikacin 3.12 3.12 6.25 0.78 nitrofurantoin 3.12 3.12 0.78 (-) no antibacterial activity, 2a = e. coli, 338 = methicillin-resistant s. aureus (mrsa), 386 = k. pneumoniae, 628 = a. baumannii, 377 = c. freundii, 767 = xanthomonas species, 4331 = m. morganii, 484 = p. aeruginosa. neomycin, amikacin and nitrofurantoin were used as positive control and 50% dmso as negative control for test. the concentration of plant extracts expressed in mg/ml. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 58 tfc values of 55.21 ± 11.09 mg gae/gm and 10.23 ± 1.07 mg qe/gm respectively (table 4 and table 5). a. catechu had the highest free radical scavenging activity in the dpph assay, followed by m. esculenta, s. cumini, and s. robusta. flavonoid and phenolic compounds from plants have been shown to have free radical scavenging activity and antioxidant properties, according to previous research [60]. the methanolic extract of a. catechu shows the ic50 of about 84.9 ± 1.9 µg/ml while 1.30 ± 0.05  µg/ml in our study [19]. the difference might be due to environmental variation, temperature, harvesting time, and temperature. these antioxidant mechanisms defend humans from infections and degenerative diseases by inhibiting and scavenging free radicals [61]. the present study showed selected plant extracts possessed antibacterial activity; e. prostrata showed potential antibacterial activity against the atcc strain of e. coli, s. aureus, and k. pneumoniae with zoi ranging from 7 mm to 11 mm. meanwhile, against mdr strains, the extract of e. prostrata showed zoi against acinetobacter spp. (628), k. pneumoniae (386), morganella morganii (4331), and xanthomonas spp. (767). previous studies also support the antibacterial and antifungal activity of e. prostata (chung et al., 2017; khanna & kannabiran, 2008). cherdtrakulki at et al. (2015) reported that bioactive compounds isolated from the aerial parts of e. prostrata such as triterpenoids, 3acetylaleuritolic acid, stigmasterol, a mixture of triterpenoids, fatty esters, and aromatic components, had effective antimicrobial activity against corynebacterium diphtheria nctc 10356, morexella catarrhalis, streptococcus pyogenes and saccharomyces cerevisiae atcc 2601. another study suggests the presence of alkaloids, cardiglycosides, phytosterol, beta-amyrin, polyacetylene, caffeic acid, stigmasterol, daucosterol on e. prostrata extracts and are found to be effective against k. pneumoniae, s. dysenteriae, e. coli, s. typhi, b. subtilis, p. aeruginosa, and s. aureus [26]. recently, ecliprostins a, b, and c isolated from this plant showed mic of 25.0, 6.25 and 25.0 m, respectively towards the growth of s. aureus [64]. m. australis extract showed a wide range of antibacterial activity against the mdr strains of acinetobacter spp. (628), methicillin-resistant s. aureus (mrsa) (338), k. pneumoniae (386), p. aeruginosa (484), and xanthomonas spp. (767) with mic value of 3.12 mg/ml, 0.19 mg/ml, 0.012 mg/ml, 0.05 mg/ml and 0.05 mg/ml respectively. a similar kind of result was observed by wei et al. (2016) against a wide range of pathogens such as s. aureus, fusarium roseum, s. faecalis, b. cereus, e. coli, k. pneumoniae, p. aeruginosa, salmonella enterica serovar typhi, c. freundii, candida albicans, microsporum audouinii, b. subtilis, micrococus flavus, and salmonella abony due to presence of phytoconstituents such as that mulberrofuran, moracins, oxyresveratrol, morusin, and kuwanon c isolated from methanolic extract of morus plant’s root bark. other plant extracts such as p. marsupium, m. esculenta, h. cordifolium also exhibited antibacterial activity against mdr strains with varying mic values (table 9 and table 10). the plant extracts might have a wide variety of phytochemicals that have different mechanisms of action for their antimicrobial activity [66]. by inhibiting enzymes and highly oxidizing compounds, phenol or hydroxylated phenol inhibits bacterial development, likely through reaction with sulfhydryl groups or nonspecific interactions with proteins [67]. antimicrobial effects are possibly due to flavonoid’s ability to bind to extracellular and soluble proteins, as well as bacterial cell walls, inactivate enzymes, and disrupt microbial membranes [68]. tannins function as antimicrobials by binding to adhesins, inhibiting enzymes, depriving bacteria of their food, forming a complex with the cell wall, disrupting membranes, and complexing metal ions [69]. terpenoids and essential oils show antimicrobial activity by membrane disruption by the lipophilic compounds. alkaloid acts as an antimicrobial agent by intercalating into the cell wall and dna of parasites [10]. these results indicate that nepalese medicinal plants contain different phytochemicals that need to be explored further to acquire a future drug candidate against mdr pathogens. conclusion medicinal plants have long been used as traditional healers for a range of infections, and they are also useful in the formulation of drugs to treat a variety of conditions. the leaves extract of e. andenophorum showed the highest tfc (10.23 ± 1.07 mg qe/gm) while bark extract of a. catechu showed a high tpc (55.21 ± 11.09 mg gae/gm). morus australis showed a broadspectrum antibacterial activity that might be a potential source of the future drug to treat mdr-associated infections. similarly, other plant extracts such as e. prostrata, m. esculenta, p. marsupium, and h. cordifolium also showed potential antibacterial activity against clinical isolates of mdr bacteria. future studies are anticipated to examine the possibility of these plants in nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 5 0 6 2 shrestha et al. ©njb, bsn 59 ethnomedicine and drug discovery to treat infections caused by drug-resistant pathogens. availability of data and materials plant specimen herbaria are kept in the national college, kathmandu, and can be retrieved as needed. data supporting this manuscript are accessible upon appropriate request to the corresponding author. conflict of interests we announce that none of the writers have a conflict of interest in reporting these results. funding statement not applicable list of abbreviations american type culture collection (atcc); minimum inhibitory concentrations (mic); multidrug-resistant (mdr); optical density (od); the inhibitory concentration of drug/extract that gives half-maximal response (ic50); total phenol contents (tpc); total flavonoid contents (tfc); zone of inhibition (zoi); 2,2diphenyl-1-picrylhydrazyl (dpph) references 1. who. 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(2017). antibacterial activity of tannins isolated from sapium baccatum extract and use for control of tomato bacterial wilt. plos one, 12(7), e0181499. https://doi.org/10.1371/journal.pone.0181499 ©njb, biotechnology society of nepal 39 nepjol.info/index.php/njb. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 doi: https://doi.org/10.3126/njb.v7i1.26950 original research article isolation, identification and production of encapsulated bradyrhizobium japonicum and study on their viability til kumari chhetri1, bijay raj subedee2, bijaya pant1* 1central department of botany, tribhuvan university, kathmandu, nepal 2research centre for applied science and technology, tribhuvan university, kathmandu, nepal abstract rhizobium, a nitrogen-fixing bacteria is the essential feature of leguminous plants which is essential for the regeneration of nutrient-deficient soil. this study was aimed to isolate, identify, mass culture and immobilize bradyrhizoium japonicum in encapsulated form and test their viability. root nodules were sterilized, grinded and cultured aseptically in yema media containing congo red. the obtained colon was sub-cultured to get a pure culture and different biochemical tests were conducted which proved bradyrhizobium japonicum as the slow-growing species. the test shows a positive result of catalase production and nodulation test whereas the ph tolerance test shows more tolerance to the acidic ph. similarly, bradyrhizaobium japonicum can tolerate 1% and 2% nacl concentration and it doesn’t show resistance to the penicillin disc of 10mg. the mass culture and encapsulation with sodium alginate adding sucrose as nutrient proved the simplicity for handling. altogether 548 beads were prepared from the 100ml of the cultured broths which were viable for more than 190 days at 1%, 2% and 3% sucrose concentration but less viable at 5% and 10% sucrose concentration under room temperature. keywords: bradyrhizobium, encapsulation, immobilization, viability, legumes, symbiotic bacteria *corresponding author email: bijayapant@gmail.com introduction a distinctive characteristic of the majority of legumes is their ability to enter into a nitrogenfixing symbiosis with a distinct group of soil bacteria collectively called root nodules bacteria or the rhizobia [1,2]. the rhizobia reduce the atmospheric nitrogen into ammonium which is termed as the biological nitrogen fixation and is more advantageous in the perspective of soil quality. the productivity and sustainability of agriculture throughout the globe are being significantly enhanced through nitrogen fixation from effectively nodulated legumes [3]. however, only certain combinations of legumes and rhizobia result in the formation of effective nitrogen-fixing nodules even though many moderately effective and ineffective combinations may and do arise. thus the bradirhizobium japonicum is host specific and nodulate only the species of soybean. apart from direct benefit from effective nitrogen fixation [4] legumes and rhizobium provides added value in weed, pathogen and insect control when rotated with crop in farming system [5] together with improving soil structure and increasing soil organic matter content [6]. the importance of legume crops to world production and compelling needs to exploit the nitrogen fixing potential of those crops have focused attention on technologies for the production of more effective legume inoculants. most legume inoculants have been prepared by adsorbing broth culture of selected rhizobia on a suitable carrier such as peat, clays, charcoal, lignite, cellulose powder, various powdered crop residues or soil compost mixtures. in 1979, dommergues et al. [7] proposed to entrap rather than adsorb rhizobium cells by incorporating the bacteria in a polymeric gel. the encapsulation of the inoculants with polyacrylamide maintained the suitable moisture content. these formulations of immobilized cells protect the microorganism against the environmental stresses and release them to the soil gradually when the polymers are degraded [8]. increasing the efficiency of the use of available soil nitrogen can meet the additional nitrogen demand by making cereal plants capable of fixing its own nitrogen through close nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 40 nepjol.info/index.php/njb. association with diazotrophic bacteria will pay off in term of increasing cereal production and helping resource poor farmers as well as saving the environment [9]. symbiotic nitrogen fixation is an important source of nitrogen and the various legumes crops and pasture species often fix as much as 200-300 kg nitrogen per hectare [10]. globally, symbiotic nitrogen fixation has been estimated to amount to at least 70 million metric tons of nitrogen per year [11]. in 1990, world consumption of fertilizer nitrogen is 88 million tones and apart from the consumption of nonrenewable energy sources, environmental pollution from fertilizer nitrogen escaping the root zones is high because in many cases nitrogen fertilizers are not used efficiently by crops [10]. therefore biological nitrogen fixation is an important and integral component of sustainable agricultural system. furthermore, biological nitrogen fixation from legumes offers more flexible management than fertilizer nitrogen because the pool of the organic nitrogen becomes slowly available to non-legumes species [10]. concomitant with nitrogen fixation, the legumes in rotation offers the control of crop disease and pests [3,12]. the bellagio conference on n2 fixation [13] acknowledged that with the decline in the price of manufactured fertilizer in 1990s, biological nitrogen fixation with legumes and rhizobia, was most likely to remain in extensive rather than intensive agricultural systems. thus the present study is emphasized for the mass production and immobilization of rhizobial inoculants in the most effective and cost effective ways of encapsulation. rhizobia are the gram negative, rod shaped, aerobic and heterotrophic soil bacteria, which includes genera rhizobium, bradyrhizobium, sinorhizobium, mesorhizobium, allorhizobium, and azorhizobium, which are able to form symbiosis with leguminous plants. they are facultative symbionts that have adapted to persist for long period in soil in a free living state if the suitable legume host is absent [2]. they could form the specialized organs, called nodules, on roots or stems of their hosts. rhizobia inside nodule could reduce atmospheric nitrogen and make it available to the plant. symbiotic rhizobia are common colonizers of the rhizosphere of both legume and non-legume plants and in addition to legumes they are also endophytes of several nonlegumes like rice and maize [14]. however, nonsymbiotic rhizobia can also be present in soil [15]. in the old system of classification the rhizobium fall into two groups based on their growth characteristics i.e. fast growing and the slow growing rhizobium. fast growing rhizobium are acid producers which develop pronounced turbidity in liquid media within 2-3 days and have the mean doubling time of 2-4 hours. the cells are rod shaped to pleomorphic, 0.5-0.9 microns in diameter and 1.2 to 3.0 micron long, and are motile by 2-6 peritrichous flagella. whereas slow growing rhizobium are alkali producing rhizobia and require 3-5 days to produce moderate turbidity in liquid media and have the mean doubling time of 6-7 hours. the cells are predominantly rod shaped and motile by a single polar or sub-polar flagellum [16]. since 1886, when it was discovered that bacteria caused the formation of the nitrogen-fixing nodules; then, the isolation of rhizobia from the nodules as pure cultures opened the way for artificial inoculation to replace the ‘soil transfer’ method, in which dry soil, from a location where the legume had been grown previously, was coated onto the seed just before sowing [17]. this dust method was modified to the “soil-paste or muddy water process”, in which the soil was mixed with water before pouring over the seed [18]. the first commercial pure (agar) culture inoculants have been patented by nobbe and hiltner in 1896 [44]. their patented culture was placed on the market under the name nitragin, which consist of a pure culture of desired strain of rhizobia grown in flat glass bottle containing only a small amount of solid gelatin medium. this material was either to be applied to seed or mixed with soil and spread over the field [19]. then, solid carrier such as soil or peat was first suggested in 1914 [17]. present day inoculants production techniques have been changed from those of the early 1900s. even many types of inoculant have been investigated; peat is the best carrier and is widely accepted in the inoculant industry. however, the challenge today is to nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 41 nepjol.info/index.php/njb. develop further improved inoculant formulations and methods of application. in nepal, rhizobial inoculants has been used from few years. rhizobial inoculants had been produced in soil science department of nepal agricultural research council. this was studied and conducted by sanu kesheri bajracharya. powder inoculums were made in soil and goal mixture in 3:1 ratio. but for the research proposes liquid inoculums is being used. the work was performed under the supervision of soil science department and farmer centered agricultural resource management (farm), asian biotechnology and biodiversity sub–program nepal (annual reports of soil science department of nepal agricultural research council). some research has been done on the effect of the peat based inoculums of the bradyrhizobium japonicum on the glycine max in the university researches. although rhizobia seem to be widely distributed in the soil, however soil in different places contains different strain of rhizobia and these rhizobia may not be effective for nitrogen fixation, and may not be appropriate for all legume. some soil may have effective rhizobial strain, but the number of rhizobia is low or containing higher number of ineffective strain [20]. inoculation of legume seed is a simple and practical means of ensuring effective nitrogen fixation. however, to answer the question “is inoculation of seed necessary?” is critical, even the use of rhizobial inoculant is not necessary in that area. therefore, allen [21] has listed four indicators that, if positive, the inoculation would be beneficial i.e. the absence of the same or symbiotically-related legume in the immediate past history of the land ; poor nodulation when the same crop was grown on the land previously; when the legume followed a non-legume in the rotation and when the land was undergoing reclamation. rhizobial inoculants can be immobilized in different materials. the material for peat based carrier is obtained from a naturally occurring organic material. the supply of peat based organic material is limited. even other solid materials such as lignite, charcoal, coir dust and compost of various agricultural wastes have been used instead of peat but their performance characteristics are not equivalent to peat based inoculants product [22]. therefore it is important to immobilize the rhizobium in any other suitable form as sodium alginate encapsulation. in solid and liquid inoculants three basic contaminant types were observed, such as bacteria, actinomycetes, and fungi. these include the possibilities of pathogenicity to human, animal, plant or rhizobia, which reduce the effectiveness of inoculant [23]. thus it is necessary to immobilize the bacterial cells in the form of encapsulated beads made in aseptic condition which prevents the contaminants and well as preserved the cells for several months without losing their viability. also the encapsulated beads are easy to handle, to use and to do packaging and distribute to the farmers. unbalanced use of chemical fertilizers had led to a reduction in soil fertility and to environmental degradation [24] and the cost of chemical fertilizers has increased so that it is unaffordable for farmer of developing country such as nepal. as a consequent, there has recently been a growing level of interest in environmentally friendly sustainable agricultural practices including organic farming systems [25]. for example, rhizobium and phosphate solubilizing microorganisms would reduce the need for n2 and p chemical fertilizers and decrease adverse environmental effects. therefore, in the development and implementation of sustainable agriculture techniques, bio-fertilization is of great importance in alleviating environmental pollution and the deterioration of nature [26]. a tightening of the agricultural n2 cycling to reduce n losses and an increase of n2 inputs through bnf to replace artificial fertilizer n2 use can help achieve this goal while at the same time maintaining agricultural production and reducing greenhouse gas emissions and energy consumption for the production of artificial n fertilizers [27-29]. materials and methods the materials used for the present study were root nodules of glycine max (white seeded species) grown at the earthen pot. the seed of nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 42 nepjol.info/index.php/njb. glycine max were taken from the market for the test. the necessary equipment and the chemicals required for the completion of the research were provided from the biotechnology and biochemistry unit of central department of botany. preparation of yema media [30]: all the ingredients required for the preparation of the yema media except agar and congo red were dissolved in the 950 ml of the sterilized water. congo red was dissolved separately in the next conical flask in 50 ml of water and sterilized them separately. then ph was maintained to 6.8-7.0. agar was added in the mixture of the ingredients and sterilized in autoclave at 121 degree celsius and 15 lb. pressure for fifteen minutes. from the autoclave, media was directly taken to the laminar air flow chamber and congo red was mixed with the ingredients mixture and poured in the sterilized petri plates and allowed it to cool down. finally the media was ready for the inoculation of the rhizobia. isolation of bradyrhizobium japonicum collection of root nodules: the roots of the soybean (white seeded species) were collected from putalisadak, kathmandu which were cultivated in the pots at the rooftops. the soil from the root was removed by washing with tap water. then only the fresh, turbid, matured and pinkish colored nodules were selected and collected on the beaker. only 0.2 gm. of nodules were taken for the present study. surface sterilization of the root nodules: root nodules were rinsed with tap water to remove the soil particle followed by rinsing with detergent and few drops of tween-20 for 1 hour in the running tap water. roots nodules were dipped in 95% ethanol for 5-10 seconds under laminar air flow chamber and transferred to 2.5% sodium hypochlorite for 2-4 minutes. then rinsed with sterile water for five times. preparation of the inoculants: the root nodules were crushed in 1ml of sterile water in the test tube with the sterile glass rod. then the solution was made 10 ml by adding sterile water. with the help of the pipette, 1 ml of the solution was taken in the next test tube and the final volume was made 10 ml by adding 9 ml of the sterile water to make 10-1 dilution of the solution. similarly, the solution was serial diluted upto 10-6 by transferring 1ml solution from the former test tube to the next one. from each of the serial dilution, 0.5 ml solution was taken and inoculated in the yema media by spreading with the help of l-shaped glass rod. finally, the plates were incubated at 300c in dark in inverted position for 4 days. to isolate the pure culture of rhizobia, only red colony from 4th day cultures were taken with the inoculating loop and streaked in the yema media with congo red and incubated at same condition as before. identification of rhizobium: the species of rhizobium were identified on the basis of its host as well as some biochemical tests as mentioned below: catalase production test [31]: the dark red portion of 18 to 24 hours pure colony was picked with the help of an inoculating loop and placed in the clean glass watch. then few drops of the 3% h2o2 were added over the organism on the watch glass with the help of the pasteur pipette. the immediate emergence of bubbles shows the production of catalase. ph tolerance test: yem broth was prepared without adding the agar in the yema media and adjusted to different ph as 4.5, 7, 9 and 9.5 by adding hcl and naoh. then the media was sterilized and rhizobium strain was inoculated and incubated for 14 days at 30 degree celsius and observed the growth of the rhizobia. nacl tolerance test: yema plates with different concentration of nacl (1%, 2%, and 4%) was prepared, sterilized and inoculated with rhizobium and incubated for 14 days at 30 degree celsius and observed the specific growth of the rhizobium. penicillin resistance test (kirby-bauer method) [32]: yema plates were prepared and placed right side up in an incubator at 37 0c for 10 to 20 minutes with the cover adjusted so that the slides are slightly opened. each plates were labeled with the name of test organism to be inoculated. a sterile cotton swab was dipped into a test culture and removes excess inoculums by pressing the saturated swab against the inner wall of the beaker containing the test organism. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 43 nepjol.info/index.php/njb. using the swab, the entire agar surface was streaked horizontally and vertically to ensure a heavy growth over the entire surface. the culture plates were allowed to dry for about 5 minutes. using the aseptic technique the penicillin disc was applied on the agar surface by using sterile forceps. each disc were kept at least 15 mm from the edge of plate. each disc were gently pressed down with the sterile forceps to endure that the disc adhere to the surface of the media. the plate cultures were then incubated in an inverted position for 24 to 48 hours at 300c. finally all the plates were examined for the presence or absence of a zone of inhibition surrounding each disc. nodulation test [33]: the seeds of soya bean were taken and surface sterilized in running tap water followed by dipping in 95% ethanol for 1 minutes. seeds were then washed with 6 consecutive washing with sterilized water. then the earthen pots along with 1:1 ratio of sand and soil were sterilized in hot air oven at 1600c for three hours. the sticking solution was made by adding 10% sucrose in distilled water which was first heated and then cooled to make sticky. the rhizobial inoculants of 4 days culture were added in the sticker solution to make slurry. the seeds of soybean were mixed in that slurry and stirred completely to make the inoculants attached on the seeds. they were then taken out and rolled on the caco3 to maintain the alkalinity, the process is called pelleting. the seeds were then dried in the air and sown in the sterilized earthen pots at the depth of one inch. similarly the seeds without inoculating the rhizobia are also sown in the next pot. finally the pots were watered and covered with transparent polyethylene sheet and tied around the pots. the polyethylene were made to have lots of holes for watering as well as for providing ventilation and kept in the green house. they were watered regularly and observed for the nodulation when the plant becomes 10-15 cm high. the presence of nodules in the inoculated plants and absence in un-inoculated plants shows the positive result of the respective rhizobial strain. color change of btb: yema plates containing btb were prepared similarly as the yema plates with bromothymol blue and inoculated with test organism and incubated at 300 c and observed the color change after 4-5 days. the appearance of blue color shows that the rhizobial strain is slow growing and the appearance of the yellow color shows that the rhizobial strain is of fast growing type. mass production of rhizobium starter culture of rhizobium: yem broth medium (100 ml) was prepared and autoclaved by transferring in a flask. thereafter, pure rhizobium colony was transferred into sterilized yem broth. inoculated yem broth was incubated at the water bath at 300c for four days. this was the starter culture of the rhizobium. mass culture of rhizobium: for the mass culture of rhizobium, yem broth was prepared in the large quantity in the conical flask and sterilized as mentioned before. the ph was maintained 6.5 to 7.0. then the sterilized yem broth was inoculated with the broth of starter culture prepared in advance. this was incubated for 3-4 days on the water bath at 300c. the culture was tested for the purity by inoculating in the yema plates staining with congo red. the broth culture was then transferred to the large flask and incubated for 4-9 days for the bacterial growth. encapsulation of rhizobium [34]: the rhizobium were immobilized by encapsulating with sodium alginate along with different concentration of sucrose as their nutrient. beads were prepared aseptically in laminar air flow chamber by using dropper and the micropipette. from the mass culture of rhizobium of 4-9 days, 25ml of broth was taken in four different beaker. then the 2% sodium alginate was weighted and mixed in the broth in each beaker. the sucrose concentration of 1%, 2%, 5% and 10% was added in different beaker and leveled them. the sterilized magnet was kept in the beaker and covered with the aluminum foil. then the beaker was kept on magnetic stirrer at 250 rpm for 8 minutes in order to dissolve the sodium alginate and the sucrose. on the other hand the solution of the 0.2 m cacl2 was prepared in the 1 liter beaker. the solution of the inoculums, sucrose and the sodium alginate was allowed to settle down for few minutes so that the air bubbles get disappeared. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 44 nepjol.info/index.php/njb. then the mixture was dropped from about 30 cm height by using the blunt ended pipette and collected in beaker containing 0.2 m cacl2 solution. the rounded beads being formed in the beaker were stirred regularly to prevent them from being attached with each other. after 30 minutes beads were formed which were taken out from the cacl2 solution by filtering with a muslin cloth and kept in the filter paper to be air-dried and left overnight. finally, the air-dried beads were kept in the lead closed culture tubes for further use to test their viability. viability tests of the encapsulated beads of bradyrhizobium japonicum (modified from [35]): the sodium alginate encapsulated beads hence prepared were stored in the airtight culture tube at room temperature for the further viability test. for the viability test yema media was prepared and sterilized as mentioned before. with the sterilized forceps the beads were inoculated in the surface of the media. the beads with different sucrose concentration were inoculated in different plates for testing the viability. then they were incubated at 300c for 48 to 72 hours in the incubator. the plates were observed for the formation of the rhizobial colony in the surface of the media. the same process was repeated at the interval of 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, and so on up to 7 months. results isolation and enumeration of the colonies the bradyrhizobium were isolated in the yema media and the number of colonies formed in the plates were enumerated and the average number of the cell forming unit were calculated by using following formula: dilution factor = volume of the sample used 𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑛𝑑 𝑡ℎ𝑒 𝑑𝑖𝑙𝑢𝑒𝑛𝑡𝑠 number of organism = dilution × number of colonies number of cfu per ml = number of organisms formed in average inoculums size × dilution here one colony is considered as one colony forming unit (cfu). the enumeration by spread plate method shows random result where 1st dilution have more number of colonies and the 6th dilution have the lowest number of colonies but the other have the ascending number of the colonies except the 4th serial dilution which have the less number of the colonies than the 5th dilution which alter from the principle of the serial dilution. the calculation shows that altogether 1156×10-2colony forming units are present in the 1 ml of the original sample obtained from the root nodules. shape, size, color and texture of organism on the plate: in the first plate many colonies were formed by the inoculation of the bradyrhizobium inoculums which were irregular shaped and some were concentric and spreading. some colonies were large enough and some were too small. the colony formed after re-streaked had shown smooth, the raised and convex shape at the place of the streak. the size of the colonies in average was 4-5 mm and the maximum colony was achieved in 6-8 days of culturing as it was noticed under visual estimation. the colonies were watery translucent with dark red rib like marking in the center of the streak. their color was noticed pinkish red on the plate of re–streak. biochemical tests: many biochemical tests have performed which confirmed the isolated bacterial strains as the bradyrhizobium japonicum. table 1: enumeration of organism by spread plate technique. s.n dilution factor inoculums size(ml) no. of colonies per plate no. of organisms average no. of organisms number of c.f.u. per ml 1. 10-1 0.5 339 33.9 1156× 10-2 2. 10-2 0.5 76 0.76 3. 10-3 0.5 17 0.017 578×10-2 4. 10-4 0.5 2 0.0002 5. 10-5 0.5 5 0.00005 6. 10-6 0.5 1 0.000001 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 45 nepjol.info/index.php/njb. table 4: viability test of the encapsulated beads of rhizobium japonicum s.n periods of viability test (in days) concentration of the sucrose 1% 2% 3% 5% 10% 1 7 + + + + + 2 20 + + + + + 3 50 + + + + + 4 75 + + + + + 5 100 + + + + + 6 120 + + + + + 7 145 + + + + + 8 170 + + + 9 190 + + + *(+)means viable and (–) means not viable immobilization of rhizobial strain: as the rhizobial strain was immobilized by encapsulating in the beaded form with sodium alginate hardened by cacl2 and mixing the sucrose as the additives, the number of beads formed from every 25 ml of broth were enumerated and the beads formed per liter of the broth was calculated which is mentioned in the table 3. the number of beads formed from every 25 ml of the cultured broth was different. an average of 137 beads were formed from 25 ml of the solution. viability tests of the encapsulated beads: the encapsulated beads of the bradyrhizobium japonicum were kept in the sealed bottle and they were tested periodically for the viability of the bacterial cells. the result of the viability test done up to 190 days is shown in the table 4: the result of the viability tests shows the diversified results. the beads prepared at 1%, 2% and 3% sucrose concentration had shown the viability up to six months. on the experiment done on 170th day and 190th day, the rhizobial strain was absent and the zone clearance rings were observed around the beads having 5% and 10% sucrose concentration on the yema plates. discussion present study was carried on bradyrhizobium japonicum and it was based on the rhizobium present on the root nodules of the soybean species found in nepal. different methods and the materials were used for the isolation, identification, mass culture, immobilization and viability tests. the data obtained have been table 2: biochemical tests on rhizobium spp s.n biochemical tests result remarks 1. catalase production test + ve 2. penicillin resistance test -ve 3. ph tolerance test ph 4.5 +ve ph 7 +ve slow growing rhizobia ph 9 -ve ph 9.5 -ve 4. nacl tolerance test 1% nacl extreme 2% nacl more 4% nacl less 5. color change of btb yellow 6. nodulation test +ve +ve = positive, -ve= negative table 3: number of beads formed from the 25ml of cultured solution in different concentration of sucrose. s.n % of soidum. alginate % of sucrose calcium carbonate cacl2(m) beads per 25ml average beads 1. 2% 10% 0.2 169 2. 2% 5% 0.2 127 3. 2% 3% 0.2 126 137 4. 2% 2% 0.2 146 5. 2% 1% 0.2 117 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 46 nepjol.info/index.php/njb. discussed with the relevant information and the similar works carried out by the different investigators. very few works have been done in nepal but several works have been done by the foreign researcher. from the present study performed on the bradyhzobium japonicum, varied responses were obtained. for the identification of the bacterial species present in the root nodules of the soybean, different tests have been performed. different biochemical tests performed for present study reveals that the strain of the rhizobium under study was the slow growing species. the catalase production test of the bradyrhizibium japonicum shows the positive result which is adjacent to the rhizobial isolates of the alfalfa as in the biochemical characterization performed by shahzed et al.[36]. it means that the rhizobial isolates of the present study contain the catalase enzyme which decomposes the hydrogen peroxide to release oxygen. this conforms that the bradyrhizobium japonicum is the cytochrome containing aerobic bacteria as described by buchanan and gibbons [37] that rhizobia are aerobic bacteria utilizing oxygen as the terminal electron acceptor. similarly the rhizobial isolates of the present study shows the less resistance to the penicillin disc 10µg which indicate that penicillin is effective to the bradyrhizobium japonicum which reduced the growth of the rhizobium showing the antimicrobial activity to rhizobia. the ph tolerance test performed for the present study shows that the rhizobial isolates can tolerate the low ph but cannot tolerate the high ph. it means that bradyrhizobium japonicum is the acid tolerant species of the rhizobium. as thornton & davey [38]; richardson & simpson [39] mentioned that slight change in ph alone can significantly affect the growth of root nodule bacteria ,the bradyrhizobium shows the high growth in ph 4.5 and 7 whereas it can’t grow in ph 9 and and 9.5. the concentration of the sodium chloride also effects the growth and the survival of the rhzobium species. as mentioned by singleton et al [22] that increasing salt concentration may have detrimental effects on rhizobial population, the bradyrhizobium japonicum grow well in the 1% and 2% nacl but do not grow well in 4% nacl concentration and it has also vital role in the cell viability for 7 weeks in the yema plates. also the nodulation test shows the positive result of the present study confirmed the isolates as the bradyrhizobium japonicum since it is host specific. the color change of btb to yellow showed that it is the fast growing species but the all other results biochemical tests points it as the slow growing bacteria. the mass culture of the rhizobial isolates of the present study shows the dense growth of the bacteria at 7-9 days of the inoculation forming the dense mass at the surface of the yema broth. it also indicate that it is the slow growing species of rhizobium since the fast growing species grow densely at 4-6 days of inoculation at the temperature of 300c. the cultured mass of the rhizobium was immobilized in the form of the encapsulated beads by using the sodium alginate extracted from algae as studied by neely & pettitt (1973) [40]. the preparation of encapsulated beads of rhizobium was not easier and it has many limitations in its procedure. the missing of one step hampers severely the formation of beads. the height of dropping, time and rotation of magnetic stirrer are the most important factors. an average of 137 beads was prepared from 25ml of broth. thus 548 beads per 100 ml can be prepared within the limitation of time and rotation of magnetic stirrer. the less rotation and the over rotation results in the deformation of beads. the large volume of the inoculant in the small beaker with small magnet could not dissolve the sodium alginate and hence the beads formation is effected which could not remain in the beaded form for the longer period at room temperature and dissolves itself. as saiprasad (2001) [14] reported that sodium alginate was the most accepted hydro-gel and frequently used as a matrix for the synthetic seeds because of its low toxicity, low cost, quick gelation and biocompatibility characteristics, it was used as the gelling agent along with the sucrose as the additives for their survival on the basis of study performed by vincent [41] and found that 24-44% of cells suspended in a 10% sucrose solution nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 39-49 chhetri et al. 2019 ©njb, biotechnology society of nepal 47 nepjol.info/index.php/njb. survived primarily drying whereas only 0.1 % survived when suspended in water. 2% of sodium alginate was found to be the best for the encapsulation which are hardened by 0.1 m cacl2 as noticed by kierstan & bucke (1977) [42]. when the beads encapsulated with sodium alginate were stored at the room temperature and tested for their viability, they showed the viable cells for six months. the air dried beads kept sealed in the culture tube have maintain their beaded structure for several months. the different sucrose concentration mixed as the additives for their survival have played the important role. in 1%, 2% and 3% sucrose concentration the cells were viable for 190 days of inoculation whereas in 5% and 10% sucrose concentration the cells were survived only for 145 days. mcleod (1961) [43] had found that the incorporation of 10% sucrose in yeast mannitol broth improved the survival on glass beads compared with un-amended broth cited by vincent [41] but the survival of the bradyrhizobium japonicum is less at higher sucrose concentration and vice versa. it shows that the sucrose at low concentration maintains the moisture content and support for the viability of the rhizobium whereas the higher concentration of the sucrose effects their survival after few months. thus it can be said that beads of bradyrhizobium japonicum prefers the lower concentration of the sucrose. conclusion findings of the present study carried on bradyrhizobium japonicum concluded that it can be isolated, identified and encapsulated in the forms of beads which looks like chemical fertilizers found in the market. it also shows that bradyrhizobium japonicum is slow-growing bacteria. besides soil, peat, charcoal as the solid inoculants and the broth as a liquid inoculant, the rhizobial inoculants can be immobilized in the form of encapsulated beads by using 2% sodium alginate, 1-3% sucrose as additives and 0.1m cacl2 as the hardening substances. this maintains the moisture content of the beads as well as prevents the contaminants and preserved the cells for several months. this study concludes that the encapsulated beads with sucrose (1-3%) as the additives can be viable for more than 190 days whereas with the 5% and 10% sucrose cells survive only for five months. also, they are easy for handling as well as can be viable for more than six months in the room temperature. thus the rhizobial strain can be easily immobilized by using sodium alginate and sucrose as additive. references 1. fred eb, baldwin ll, mccoy e: root nodule bacteria and leguminous plants. 1932 university of wisconsin press, madison. 2. graham ph:. ecology of root nodule bacteria of legumes. in m. j. dil-worth et al. 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(ed) 1973 2nd edition. academic press , new york, 49-88 41. vincent jm: survival of root-nodule bacteria. in e.g. hallsworth (ed.). nutrition of the legumes (p108–123). soil biol biochem. 1958 36: 1275-1288 quoted in deaker r, roughley rj, kennedy ir. (2004). legume seed inoculation technology-a review. 42. kierstan m, bucke: the immobilization of microbial cells, subcellular organelles and enzymes in calcium alginate gels. biotech bioengin. 1977 14: 387-397 43. mcleod rw, roughley rj: freeze-dried cultures as commercial legume inoculants. j exp agric annimal husband.19611: 29-33 44. nobbe f, hiltner l: inoculation of the soil for cultivating leguminous plants. 1896 usa. us patent no. 570813 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 13-18 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 13 nepjol.info/index.php/njb characterization of nepalese barley gene pool for leaf rust resistance resham babu amgai1*, sumitra pantha2, madan raj bhatta3 1biotechnology division, nepal agriculture research council, khumaltar, lalitpur, nepal. 2agriculture botany division, nepal agriculture research council, khumaltar, lalitpur, nepal. 3national plant genetic resource centre, nepal agriculture research council, khumaltar, lalitpur, nepal. abstract barley (hordeum vulagare l) is the major crop for the people living in the high hills and mountainous region of nepal. leaf rust (caused by puccinia hordei) is one of the major production threats for barley cultivation. a lot of variation can be observed on nepalese barley accessions with respect to leaf rust resistance characteristics. two hundred and forty one barley accessions were screened for leaf rust resistance characteristics on heading stage at khumaltar, lalitpur, nepal. among them, one hundred and nine nepalese barley accessions showing promising for disease resistance were screened using six ssr markers linked to leaf rust resistance genes. bonus and local jau was used as the resistant and susceptible check respectively. leaf rust resistance genes rph1, rph2, rph3, rph7, qblr-p and qtl on chromosome 5hs were detected on nepalese barley accessions using respective ssr markers. eight nepalese barley accessions showed presence of three and more leaf rust resistant genes. the poor relationship between the field disease resistance and molecular markers linked with specific leaf rust resistance gene proved that nepalese barley gene pool contains other leaf resistance genes. keywords: leaf rust, puccinia hordei, resistant gene, nepalese barley, simple sequence repeats (ssr) *corresponding author email: reshamamgain@yahoo.com introduction barley (hordeum vulgare l.) occupies total area of 29598 hectare and total production 33782 metric ton with average productivity of 1.141 metric ton/ha in nepal [1]. the maximum area of the crop lies in the mid-western development region. out of the total barley area, more than 50 % is in the hill region, while 40% is in the mountain region [2]. however, its production and productivity is declining due to diseases and unpredictable climatic condition of the mountain area [3]. leaf rust (caused by puccinia hordei) is one of the major problematic diseases for barley production in nepal [4]. therefore, rust resistant barley varieties are another need of mountain farmers. nepal harbours hundreds of the barley landraces. high level of genetic variation can be observed in nepalese barley [5]. similarly, a lot of variation was observed among the jumla collection of nepalese barley for many yield attributing characters [6]. many of these landraces possess one or more characteristics for abiotic and biotic stress tolerance [2,3]. variation on rust resistance characteristics is very important for rust resistance breeding program. therefore, use of these germplasm for rust resistance gene pyramiding is highly beneficial to nepalese farmers. identification of particular rust resistance gene and its incorporation is the only option for the development of barley varieties for leaf rust rust resistance. use of the molecular marker for this purpose is highly sought for this. material and methods germplasm collection one hundred and fifty five nepalese barley accessions (npgr no.s) collected from different parts of nepal were obtained from national plant genetic resources centre (npgrc); and forty seven jumla collection (jc# series), two local collection (acc# series) and thirty six barley breeding lines (nb, b, gr and xveola series) were collected from hill crops research program (hcrp), dolakha of nepal agricultural research council (narc) (table 1). similarly, one hundred and nine nepalese barley accessions were selected based on their disease resistance data (table 2) for nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:13-18 amgai et al. ©njb, biotechnology society of nepal 14 nepjol.info/index.php/njb table 1: nepalese barley accessions showing variation on leaf rust resistance characteristics. genotype leaf rust genotype leaf rust genotype leaf rust genotype leaf rust genotype leaf rust 1522 2060 2496 7436 jc#14 1535 2062 2505 7441 jc#15 1537 2064 2506 8252 jc#16 1538 2065 2507 9436 jc#17 1540 2066 2508 9963 jc#18 1543 2069 2511 12069 jc#19 80s 1544 2072 2512 12538 jc#21 40s 1545 2073 2513 40s 20774 jc#22 1546 2074 2514 22463 jc#23 1547 2075 2515 112-14 jc#24 1550 2078 2518 acc#1545 jc#25 1574 2079 2520 acc#1574 jc#26 60s 1575 2080 2523 arupos/oyb-oy jc#27 1576 2081 2525 b86019-1-0 jc#28 1589 2082 2527 b86019-1k2 jc#29 1664 2083 2530 b86023-1k ts jc#31 1999 2084 2532 b86065-1-4 jc#32 80s 2000 2086 2533 b86099-1k jc#33 80s 2001 2087 2534 tms b86099-2-1 jc#34 20s 2002 2088 2539 b86099-2k jc#35 2003 2089 2542 b86115 jc#36 60s 2004 2090 5177 b86122-1-5 jc#37 90s 2005 2181 5617 b86122-1-50k3 jc#38 ts 2008 2244 6035 b86122-1k jc#39 2009 2447 6036 b86152-2 jc#41 2010 2453 6038 b86152-2-2-00k jc#42 60s 2011 2454 6041 b86157-1-1-50-0k3 jc#43 20s 2013 2456 6044 b86615-1-4 jc#44 40ms 2014 2457 6045 b90k-007-1 jc#45 40s 2016 2458 6055 b90k-01-2k jc#48 40s 2018 2459 6063 80s b90k-014-1 ts jc#49 2023 2461 6235 b90k-024-1 jc#50 2024 2464 6289 b90k-038 jc#51 60s 2027 2465 6292 b90k-090 jumla coll 2029 2467 6293 bonus lg51/xve 2033 2468 6304 coq/ki/pes cii local jau 2035 2469 6309 gr-25-85 maticos 2037 2470 6310 jc#01 nb-100337 2040 2471 6311 jc#02 nb-100337/1034 2042 2472 6315 jc#03 nb-100337/1038 2043 90ms 2473 6316 jc#04 nb-100337/1214 2046 2482 6319 jc#05 nb1207/ci 2048 2483 6320 jc#06 20s xveola-12 2049 2485 6326 jc#07 60s xveola-13 2050 2486 6327 jc#08 10s xveola-28 2051 2487 6334 jc#09 30s xveola-38 2056 2488 6342 jc#11 20s xveola-43 2057 2491 6447 jc#12 2058 2494 40s 6557 jc#13 note: r-resistance, mr-moderately resistance, tms-trace moderately susceptible, tmr-trace moderately resistance, ts-trace susceptible, ssusceptible; blank=no disease i.e. 0 score nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:13-18 amgai et al. ©njb, biotechnology society of nepal 15 nepjol.info/index.php/njb table 2: nepalese barley accessions used for screening leaf rust resistance using simple sequence repeats marker 1535 2002 2046 2081 2458 2483 6036 6327 8242 jc#18 1537 2003 2051 2082 2459 2489 6293 6340 8244 jc#46 1538 2004 2062 2084 2461 2494 6304 6342 8245 jc#49 1540 2008 2064 2086 2464 2496 6309 6343 8246 local jau 1544 2009 2069 2087 2467 2505 6310 6344 8252 maticos 1545 2010 2072 2088 2468 2506 6314 6350 9436 nb-1003-37 1572 2014 2073 2089 2470 2508 6315 6557 9963 nb-1003-37/1038 1574 2023 2074 2090 2471 2515 6316 7436 arupos/oyb-oy solu uwa 1575 2026 2075 2441 2478 2537 6319 7441 b86122-1-50k3 xveola-12 1594 2033 2078 2456 2480 2541 6320 8239 bonus xveola-13 1601 2043 2080 2457 2482 2542 6326 8240 gr-25-85 xveola-43 2000 note: name as numbers without any alphabet denote the nepalese plant genetic resource (npgr) number. table 3: ssr markers used to identify presence of leaf rust resistance gene in nepalese barley gene pool marker name sequence-f [5'… … 3'] sequence-r [5'… … 3'] annealing temperature stripe rust resistance gene pcr product size chromosome no. ref ay642926ca11 ccaaaaac aattgagaa aacccta cctccc tgagag acctcctat t 58 rph7 183 3h [12] bmac096 gctatggcg tactatgta tggttg tcacgatga ggtatgatca aaga 58 rph2 173 5hs [13] bmag0225 aacaca ca aaatattac at ca cgagtagttc cc atg tga c 58 rph7 162 3h [14] bmag337 acaaagag ggagtagta cgc gacccatgat atatgaaga tca 55 qtl 129-150 5hs [13] ebmac075 5 agccttgtg tatcaggac a ctgctggtgt tctctaaaag t 55 rhp3 rhp19 144-155 7hl [15] ris44 acggatcta ctttagcta gca aaacaaccc cacacaatc 58 qblr-p 105-110* 7hl [16] note: *=product size determined based on field data for disease resistance nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:13-18 amgai et al. ©njb, biotechnology society of nepal 16 nepjol.info/index.php/njb table 4: nepalese barley germplasm with different leaf rust resistant gene identified using different molecular markers rph7 (ay642926ca11) rph2 (bmac096) rph3 & rph1 (ebmac0755) rph7 (bmag0225) qtl on 5hs (bmag0337) qblr-p (ris44) 2480 gr-25-85 1538 gr-25-85 1540 gr-25-85 2062 2470 2008 1545 1601 1544 2483 2471 2033 1574 2010 1545 2541 2515 2062 2010 2023 1574 solu uwa 6036 2478 2080 2033 1575 1537 2480 2505 2043 1601 2074 2483 2508 2051 2002 2082 2489 6304 2478 2003 2506 2515 6310 2480 2004 nb-1003-37 2541 6315 2482 2072 nb-100337/1038 6342 6343 2489 2073 xveola-43 8252 6350 2494 2075 9963 2000 2505 2078 1594 2026 2515 2080 2506 6309 2537 2081 2537 arupos/oy-b-oy 2541 2084 6557 bonus 6036 2086 jc#46 jc#49 6340 2088 jc#49 local jau 6342 2089 nb-1003-37/1038 maticos 6344 2090 solu uwa nb-1003-37 8246 2456 xveola-43 xveola-13 8252 2457 9963 2459 1537 2461 2000 2464 2014 2467 2046 2468 2074 2470 2082 6304 2506 6310 2542 6314 6557 6315 7436 6316 8239 6319 8245 6326 b86122-1-5-0k3 6327 jc#49 1535 local jau 2009 nb-1003-37 2064 nb-1003-37/1038 2069 solu uwa 2458 xveola-43 2471 6293 6309 6320 bonus nb-1003-37 note: combination on parenthesis is respective ssr markers used to detect particular gene. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:13-18 amgai et al. ©njb, biotechnology society of nepal 17 nepjol.info/index.php/njb table 5: nepalese barley germplasm having three and more leaf rust resistance gene detected by molecular markers genotype rph3 & rph1 qtl on 5hs rph2 qblr-p rph7 gr-25-85 0 0 1 1 1 nb-1003-37 0 1 1 1 1 npgr no. 2506 1 1 1 0 0 xveola-43 1 1 1 0 0 nb-1003-37/1038 1 1 1 0 0 npgr no. 2515 1 1 1 0 0 jc#49 1 1 0 0 1 solu uwa 1 1 0 0 1 note: 1=present, 0=absent molecular marker screening. bonus and local jau were used as resistant and susceptible check respectively. leaf rust evaluation at field barley lines were screened for leaf rust at heading stage at khumaltar, lalitpur, nepal during normal barley growing season. bonus (origin sweden) and local jau (nepalese landrace) was used as resistance and susceptible check respectively. two rows (spacing 20cm) of 1.5m long per accession were sown. the resistance and susceptible checks were repeated after every 15 test lines. two spreader rows of local jau were sown around the disease screening plots. disease scoring was conducted according to the modified cobb scale [7]. molecular marker six ssr markers were selected for screening leaf rust resistance gene (table 3). molecular markers are selected based on their linkage with particular leaf rust resistance gene. dna extraction, pcr reaction and data analysis genomic dna of barley accessions was prepared using modified ctab method as described by sul and korban [8]. each pcr reaction was conducted with100ng of genomic dna, 1 µm of each primer and 7.5 µl of 2x gotaqgreen pcr master mix (promega corporation, madison, wi, usa). pcr mixture was amplified in mj research ptc-100tm programmable thermal controller (mj research, inc, watertown, ma, usa) with the following temperature regimes: initial denaturation for 2 min at 95oc followed by 30 cycles of 95oc for 30 sec, annealing as per primer for 1 min, extension at 72oc for 2 min and final extension at 72oc for 7 min followed by holding at 4oc as described on table 3 and scottish crop research institute [9]. amplified pcr products were separated in 2% analytical grade agarose gel at 100v for 1h. gels were stained with 0.1 µg/ml ethidium bromide (promega corporation, madison, wi, usa) and then visualized under uv trans illuminator gel documentation system (wilber lourmat, marnela-valleen, france) using 1 µg guide size dna ladder (genetix, biotech asia pvt. ltd.). the presence and absence of particular band size was scored for screening disease resistance genes. results and discussion a lot of variation was observed in nepalese barley germplasm for leaf rust resistance characteristics (table 1). leaf rust resistance gene rph1, rph2, rph3, rph7, qblr-p and qtl on 5hs was detected on nepalese barley accessions using respective ssr markers. twelve landraces showed presence of rph2, 22 accessions showed presence of rph1 and rph3, 27 accessions showed presence of rph7, forty two accessions showed presence of qtl on chromosome 5hs and 47 accessions possessed leaf rust resistant qtl qblr-p (table 4). similarly, eight nepalese barley accessions showed presence of three and more leaf rust resistant gene (table 5). nepalese barley germplasm showed good resistance with leaf rust which may be due to the presence of leaf rust resistance major genes and quantitative trait loci (qtls) as detected by different ssr markers. tyryshkin [10] and henderson [11] also concluded that nepalese barley has good resistance against the leaf rust. the released hulless barley variety “solu uwa” show good resistance with leaf rust and have nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1:13-18 amgai et al. ©njb, biotechnology society of nepal 18 nepjol.info/index.php/njb qtls (table 4) and major genes including rph7 (figure 1). similarly, adult plant resistance for leaf rust was also observed by tyryshkin [10] for nepalese barley nb-3002 while screening world collection of barley for leaf rust and proved to have one dominant gene. this gene may be rph7. similarly, the poor relationship between the field disease resistance and molecular markers linked with specific leaf rust resistance gene proved that nepalese barley gene pool contains new leaf resistance genes that cannot be defined by the tested molecular markers. conclusion many nepalese barley landraces showed field resistance with leaf rust, however, some promising lines still lacks any major resistant genes as defined by molecular markers and need to be incorporated from other lines to address future unwanted leaf rust spread. barley genotypes with more than three resistant genes could be the choice of donor parents for leaf rust resistant breeding through molecular marker assisted selection in nepal. acknowledgement this work was conducted under global biodiversity trust grant no. gs10027. references 1. gon: statistical information on nepalese agriculture 2012/13. ministry of agriculture development. agri-business promotion and statistics division, kathmandu, nepal, 2013. 2. upreti rp: status of food barley in nepal. in-s. grando and h. gormez macpherson (eds.), food barley: importance, uses and local knowledge. proceedings of the international workshop on food barley improvement, 14-17 january 2002, hammamet, tunisia. icarda, aleppo, syria, 2005: 99-114. 3. hcrp: barley research reports 2007/08. hill crops research program, dolakha, nepal, 2008. 4. prasad rc, karki cb, sharma s: pathological report on barley. in-hill crops proceedings. national hill crops research program, kabre dolakha, nepal, 1993: 52-78. 5. bajracharya j, tiwari pr, shakya dm, baniya bk, sthapit br: genetic variation in barley landraces (hordeum vulgare l.) of jumla ecosite revealed by isozyme analysis. in br sthapit, a subedi, mp upadhaya and bk baniya, (eds.), proceedings of a national workshop on strengthening the scientific basis of in situ conservation of agricultural biodiversity during 24-26 april 2001at lumle, kaski, nepal. narc/libird/ipgri, 2001. 6. gupta sr, upadhyay mp, shah us: agromorphological variability study of barley (hordeum vulgare l.) landraces in jumla, nepal. nepal agric. res. j. 2009, 9:1-11. 7. peterson rf, campbell ab, hannah ae: a diagrammatic scale for estimating rust intensity of leaves and stem of cereals. can j res sci 1948, 26:496-500. 8. sul iw, korban ss: a highly efficient method for isolating genomic dna from plant tissues. plant tiss. cult. biotech. 1996, 2: 113-116. 9. scottish crop research institute: barley ssrs 1.0. http://bioinf.scri.ac.uk/ssr/barley_s.html (accessed 24 april 2016). 2005. 10. tyryshkin, lg: genetic control of effective leaf rust resistance in collection accessions of barley hordeum vulgare l. russ j genet. 2009, 45 (3): 376-378. 11. henderson mt: studies of sources of resistance and inheritance of reaction to leaf rust puccinia anomala rostr. in barley. ph.d. thesis, university of minnesota, minneapolis. 1945. 12. mammadov ja, brooks ws, griffey ca, saghai maroof ma: validating molecular markers for barley leaf rust resistance genes rph5 and rph7. plant breeding 2007, 126: 458-463. 13. karakousis a, barr ar, chalmers kj, ablett ga, holton ta, henry rj, lim p, langridge p: potential of ssr markers for plant breeding and variety identification in australian barley germplasm. aust j. agri. res. 2003, 54: 1197-1210. 14. czembor pc, pietrusinska a, czembor hj: mapping new resistance gene to puccinia hordei otth. in barley. abstract in: j.l. molinacano, p. christou, a. graner, k. hammer, n. jouve, b. keller, j.m. lasa, w. powell, c. royo, p shewry and a.m. stanca (eds.), cereal science and technology for feeding ten billion people: genomics era and beyond. options mediterraneennes, series a, 2008, no.81:151p. 15. park rf, poulsen d, barr ar, cakir m, moody db, raman h, read bj: mapping genes for resistance to puccinia hordei in barley. aust j agri. res. 2003, 54: 1323-1333. 16. rossi c, cuesta-marcos a, vales i, gomezpando l, orjeda g, wise r, sato k, hori k, capettini f, vivar h, chen x, hayes p: mapping multiple disease resistance genes using a barley mapping population evaluated in peru, mexico, and the usa. mol breed. 2006, 18: 355 – 366. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:68-70 issn 2091-1130 (print) / issn 2467-9319 (online) review article ©njb, biotechnology society of nepal 68 nepjol.info/index.php/njb microbial diversity in freshwater and marine environment sagar aryal1*, gaurab karki1, sunil pandey2 1department of microbiology, st. xavier’s college, kathmandu, nepal 2 department of microbiology, nobel college, kathmandu, nepal abstract water covers seven tenths of the earth's surface and occupies an estimated total volume of 1,386,000,000 cubic kilometers (km3). of all the water found on earth, 97% is marine. maximum of this water is at a temperature of 2 to 3°c and devoid of light; 62% is under high pressure (>100 atm). microscopic phytoplankton and associated bacteria generate a complex food web that can extend over long distances and extreme depths. the marine environment looks so vast that it will not be able to be exaggerated by pollution; however, in coastal areas human activities are increasingly disrupting microbial processes and damaging water quality. keywords: ecology, microorganisms, nutrition, water, phototrophs. *corresponding author email: broneps1@gmail.com introduction microbial diversity that we see today is the result of nearly 4 billion years of evolutionary change. microbial diversity can be seen in many forms, including cell size and cell morphology, physiology, motility, pathogenicity, developmental biology, adaptation to environmental extremes, phylogeny and mechanism of cell division [1]. microorganisms are present everywhere on earth that will support life. these include habitats we are all familiar withsoil, water, animal, and plants-as well as virtually any structures made by humans. some microbial habitats are ones in which humans could not survive, being too hot or too cold, too acidic or too caustic, or too salty. although such environments would pose challenges to any life forms, they are often teeming with microorganisms. organisms inhabiting such extreme environments are called extremophiles, a remarkable group of microorganisms that collectively define the physiochemical limits to life [2]. approximately 6000 species of prokaryotes and 100,000 species of protists have been formally described [3]. in the case of the diversity of microorganisms, even the right order of magnitude is unknown and the issue is highly controversial [4–6]. the total number of prokaryotic cells in the oceans has been estimated to be 1029 [7]. all prokaryotic organisms are classified as bacteria, whereas eukaryotic organisms include fungi, protozoa, and helminths as well as humans. prokaryotic organisms are divided into two major groups: the eubacteria, which include all bacteria of medical importance, and the archaebacteria, a collection of evolutionarily distinct organisms [1]. freshwater microbial diversity freshwater and marine environments differ in many ways including salinity, average temperature, depth, and nutrient content, but both provide many excellent habitats for microorganisms. large numbers of microorganisms in a body of water generally indicate high nutrient levels in the water. water contaminated by inflows from sewage systems or from biodegradable industrial organic wastes is relatively high in bacterial numbers. similarly, ocean estuaries (fed by rivers) have higher nutrient levels and therefore larger microbial populations than other shoreline waters [8]. in water, particularly water with low nutrient concentrations, microorganisms tend to grow on stationary surfaces and on particulate matter. in this way, a microorganism has contact with more nutrients than if it were randomly suspended and floating freely with the current. many bacteria whose main habitat is water often have appendages and holdfasts that attach to various surfaces. freshwater environments are highly variable in the resources and conditions available for microbial growth. both oxygen producing and oxygenconsuming organisms are present in aquatic environments, and the balance between nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:68-70 aryal et al. ©njb, biotechnology society of nepal 69 nepjol.info/index.php/njb photosynthesis and respiration controls the natural cycles of oxygen, carbon, and other nutrients (nitrogen, phosphorus, metals). among microorganisms, oxygenic phototrophs include the algae and cyanobacteria. these can either be planktonic (floating) and distributed throughout the water columns of lakes, sometimes accumulating in large numbers at a particular depth, or benthic, meaning they are attached to the bottom or sides of a lake or stream. because oxygenic phototrophs obtain their energy from light and use water as an electron donor to reduce co to organic matter, they are called primary producers [9]. a typical lake or pond serves as an example to represent the various zones and the kinds of microbiota found in a body of fresh water. the littoral zone along the shore has considerable rooted vegetation, and light penetrates throughout it. the limnetic zone consists of the surface of the open water area away from the shore. the profundal zone is the deeper water under the limnetic zone. the benthic zone contains the sediment at the bottom [10]. areas of the limnetic zone with sufficient oxygen contain pseudomonads and species of cytophaga, caulobacter, and hyphomicrobium. photosynthetic algae are located in the limnetic zone [11]. deeper waters of the profundal and benthic zones have low oxygen concentrations and less light. algal growth near the surface often filters the light, and it is not unusual for photosynthetic microbes in deeper zones to use different wavelengths of light from those used by surface-layer photosynthesizers. purple and green sulfur bacteria are found in the profundal zone. these bacteria are anaerobic photosynthetic organisms that metabolize h2s to sulfur and sulfate in the bottom sediments of the benthic zone. clostridium species are common in bottom sediments and may include botulism organisms, particularly those causing outbreaks of botulism in waterfowl [8]. marine microbial diversity the marine environment represents a major portion of the biosphere and contains 97% of the earth’s water. much of this is in the deep sea at a depth greater than 1,000 meters, representing 75% of the ocean’s volume. the ocean has been called a “high-pressure refrigerator,” with most of the volume below 100 meters at a constant 3°c temperature. the ocean, at its greatest depth, is slightly more than 11,000 meters deep. the pressure in the marine environment increases approximately 1 atm/10 meters in depth, and pressures are in the vicinity of 1,000 atm at the greatest ocean depths [2]. much of the marine environment is covered by sea ice that may comprise up to 7% of the world’s surface. microorganisms actually grow and reproduce at the interface between the ice and the seawater. the microbes that have been recovered from these ice cores have been given intriguing generic names: these include polaromonas, marinobacter, psychroflexus, iceobacter, polibacter, and psychromonas antarcticus [2]. the world’s oceans are teeming with microscopic life forms. nominal cell counts of >105 cells per ml in surface sea water [12, 13] predict that the oceans harbor 3.6 x 1029 microbial cells with a total cellular carbon content of 3 x 1017g [7]. communities of bacteria, archaea, protists, and unicellular fungi account for most of the oceanic biomass. these microscopic factories are responsible for 98% of primary production [7, 14] and mediate all biogeochemical cycles in the oceans [12]. coastal and shelf sediments play a significant role in the remineralization of organic matter. in shelf areas, an estimated 32 to 46% of the primary production settles to the sea [15]. much of the primary productivity in the open oceans, even at significant depths, comes from photosynthesis by prochlorophytes, tiny prokaryotic phototrophs that are phylogenetically related to cyanobacteria. prochlorophytes contain chlorophylls a and b or chlorophylls a and d. the organism prochlorococcus is a particularly important primary producer in the marine environment. aerobic anoxygenic phototrophs include bacteria such as erythrobacter, roseobacter, and citromicrobium, all genera of alphaproteobacteria. deepwater archaea are almost exclusively species of crenarchaeota, and many or perhaps even most are ammonia-oxidizing chemolithotrophs, these organisms play an important role in coupling the marine carbon and nitrogen cycles. very small planktonic heterotrophic bacteria inhabit pelagic marine waters in numbers of 105 – 106 cells/ml. the most abundant of these is pelagibacter, a genus of class alphaproteobacteria. cells of pelagibacter are small nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:68-70 aryal et al. ©njb, biotechnology society of nepal 70 nepjol.info/index.php/njb rods that measure only 0.2– 0.5 µm, near the limits of resolution of the light microscope. bacteroidetes and actinobacteria, and has also been found in nonhalophilic species of archaea, such as the thermoplasma group. in the oceans, viruses are more abundant than cellular microorganisms, often numbering over 107 virions/ml in typical seawater [1]. conclusion major bacterial groups now recognized as abundant in the open ocean include alphaand gammaproteobacteria, cyanobacteria, bacteroidetes, and to a lesser extent, betaproteobacteria and actinobacteria; firmicutes are only minor components. with the exception of the cyanobacteria, most marine bacteria are thought to be heterotrophs adapted to extremely low nutrient availability, some augmenting energy conservation through proteorhodopsin or aerobic anoxygenic phototrophy. recent anthropogenic interventions in marine environment have threatened all lives, including microorganisms. study of marine microbial biodiversity is of dynamic importance to the understanding of the different processes of the ocean, which may present effective novel microorganisms for screening of bioactive compounds. references 1. madigan mt, martinko jm, stahl da, clark dp: brock biology of microorganisms. 13th ed. 2012. san francisco: pearson benjamin cummings. 2. prescott lm, harley jp and klein da. microbiology. 5th ed. 2002. the mcgraw−hill companies. 3. corliss jo, margulis l, melkonian m: handbook of protoctista 1st ed. 1990. jones & bartlett publishers. 4. finlay bj and esteban gf: ubiquitous dispersal of freeliving microorganisms. microbial diversity and bioprospecting 2004. asm press. pp. 216–224. 5. hedlund bp and staley jt: microbial endemisms and biogeography. microbial diversity and bioprospecting 2004 asm press. pp. 225–231. 6. whitfield j: biogeography: is everything everywhere? science 2005. 310:960–961. 7. whitman wb, coleman dc and wiebe wj: prokaryotes: the unseen majority. proc. natl. acad. sci 1998. 95:6578– 6583. 8. tortoraand gj, funke br: microbiology: an introduction. 9th ed. 2008. san francisco: pearson benjamin cummings. 9. tortora gj, funke br, case cl: microbiology: an introduction. 10th ed. 2010. san francisco: pearson benjamin cummings. 10. rathi j: microbial physiology genetics and ecology. 1st ed. 2009. manglam publishers & distributors. 11. britannica.com: limnetic zone. ecology. retrieved 201508-14. from http://www.britannica.com/science/limnetic-zone 12. porter kg and feig ys: the use of dapi for identifying and counting aquatic microflora limnol. oceanogr.1980, 25:943–948. 13. hobbie je, daley rj and jasper s: use of nuclepore filters for counting bacteria by fluorescence microscopy. appl. environ. microbiol. 1977, 33:1225–1228. 14. atlas rm and bartha r: microbial ecology: fundamentals and applications. 1993. (benjamin/cummings, redwood city, ca). 15. wollast, r: the coastal organic carbon cycle: uxes, sources, and sinks. ocean margin processes in global change 1991. pp. 365-381. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 1 nepjol.info/index.php/njb comparative antioxidant, antimicrobial and phytochemical assesments of leaves of desmostachya bipinnata l. stapf, hordeum vulgare l. and drepanostachyum falcatum (nees) keng f. pragya nepal, minu singh, amina baniya, sushma singh, hari krishna sainju, rajani shrestha department of biotechnology, asian institute of technology and management (aitm), khumaltar, lalitpur, nepal abstract nepal is rich in varieties of religious plants. the locally used religious plants also carry medicinal importance. desmostachya bipinnata l. stapf, hordeum vulgare l. and drepanostachyum falcatum (nees) keng f. are three plants belonging to the family poaceae having religious significance in different practices of hinduism. they were also used as traditional medicines by our ancestors but nowadays they are underutilized. in this research, our core objective was to validate the traditional assumption of use of these plants in medicinal purposes by carrying out the assessments like antimicrobial assessment, antioxidative assessment and phytochemical assessment. methanolic extracts produced from leaves of all three plants were examined for antimicrobial activities through agar well diffusion method. the same extracts were also assessed for determining their antioxidative potentials with the use of dpph (1, 1-diphenyl-2-picryl hydrazyl) free radical scavenging assay followed by qualitative phytochemical analysis and gcms (gas chromatography mass spectroscopy). most promising antimicrobial activity was shown by desmostachya bipinnata l. against salmonella typhimurium and staphylococcus aureus, drepanostachyum falcatum (nees) keng f. against salmonella typhimurium and klebsiella pneumoniae and hordeum vulgare l. against salmonella typhmurium and staphylococcus aureus. the antioxidant activity of the plant extracts were observed in descending order of hordeum vulgare l.>desmotachya bipinnata l. > drepanostachyum falcatum (nees) keng f. and phytochemical assessment of the extracts indicated the presence of alkaloids, glycosides, sterols, triterpenes, saponins, flavonoids, coumarins, phlobatanin and reducing sugars. through this project, we can clarify that the above mentioned plants have bioactive compounds which contributed for the presence of antimicrobial and antioxidative property in the plants. key words: antimicrobial, antioxidant, desmostachya bipinnata l. stapf, drepanostachyum falcatum (nees) keng f., phytochemical screening, hordeum vulgare. *corresponding author email: pragya.nepal07@gmail.com introduction religions have created major linkage between human and nature by bounding human beings to several aspects of natural systems. all hindu families in nepal and india perform pujas (religious rituals) on certain occasions. there is no religious ritual, which does not require plants and their products [1]. most of these plant species also have important medicinal value. for example; ocimum tenuiflorum locally known as tulasi is a holy plant having many religious significances and on the other hand, it has been used as a traditional medicine to cure common cold and flu. these kind of religious plants were involved by our ancestors in rituals to create a remark upon their importance as medicine or as a valuable product [2]. now, we are unaware about the main reasons of use of these plants but we are still using them in religious purposes. since, their uses have only been limited to religious practices; we tried to disclose the hidden medicinal importance of these plants by conducting different assessments on them. it is also reported that edible wild plants carrying religious importance such as, ocimum tenuiflorum and desmostachya bipinnata are the important source of fibre, vitamins, minerals and other nutrients which are used for the therapy of various diseases [3]. desmostachya bipinnata l., belonging to family poaceae is a perennial grass abundantly distributed in india. this plant is an important nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 2 nepjol.info/index.php/njb ingredient while performing ‘hom’, which is a sacred practice in hindu rituals. its root stock and leaves are used in indian traditional system of medicine. many medicinal properties and use of desmostachya bipinnata l. have been reported in reference literatures [4]. hordeum vulgare (barley) is an annual grass from the poaceae family and it is stated to be one of the earliest known crops to human kind. barley seedling’s leaves are locally called as jamara and it is one of the most important plants in our greatest festival, vijaya dashami meanwhile, it is also traditionally used extensively to cure jaundice and gall bladder’s stones. barley is consumed for curing digestive disorders such as; diarrhoea, stomach pain and inflammatory bowel conditions. drepanostachyum falcatum (arundinaria falcata nees) is a common species of hill bamboo and is locally called as nigalo. it is found in the homesteads in an abundant amount. this plant is used as an effective soil stabilizer for farming in nepal and is used in religious practices like; hindu marriage, house warming rituals etc. materials and methods plant materials leaves of desmostachya bipinnata (kush), hordeum vulgare l.(jamara) and drepanostachyum falcatum (nees) keng f. (nigalo) were collected from birgunj (located at parsa district, narayani zone), kupandole (located at lalitpur district, bagmati zone) and balaju (located at kathmandu district, bagmati zone) respectively during october of 2016. extraction the collected leaves were first washed then dried in hot air oven at 40ºc-70ºc followed by grinding. sample were dissolved in parts of 80% methanol in 1:3 ratios and placed in hot water bath shaker for 16 to 18 hours followed by filtration using muslin cloth [5]. the filtrate was stored and the residue was re-extracted under the same. the extracts obtained were pooled and filtered. the combined methanol specimen was evaporated to dryness using a vacuum rotary evaporator at 65ºc. gc-ms analysis of compounds present in plant extracts gas chromatography-mass spectrometry (gcms) is a method, which combines the features of gas liquid chromatography and mass spectrometry to identify different substances present in a test sample [6].the process of gcms was operated on gc-ms equipment i.e. thermo gc-trace ultra ver: 5.0 and thermo ms dsq ii. features of the gc-ms system were; tr 5-ms capillary non-polar column having 35 mts dimension and thickness of film used in the process was 0.35μm.using helium as the carrier gas, flow rate of the mobile phase was set at 1.0 ml/min. in the gas chromatography section, prior temperature programme of 45°c was elevated to 255°c at 5°c/min and1 μl sample was injected. samples were first dissolved in chloroform to form a solution and were finally run at a range of 50655 m/z. the final results were compared by the use of wiley spectral library search programme. phytochemical screenings qualitative phytochemical screening was carried out by using following standard protocols [7]. test for basic alkaloids (dragondroff's test): 2 mg of extract was dissolved in 3 ml of 2 % (v/v) hcl .to the solution, dragondroff's reagent was added thoroughly. appearance of orange brown precipitate indicated the presence of basic alkaloids. test for glycosides: 100µl of glacial acetic acid was added to about 2 mg of extract, followed by addition of few drops of 5% ferric chloride (fecl3). to the solution, few drop of h2so4 was added until the formation of blue colour, which then, confirmed presence of glycosides. test of sterols and triterpenes (salkwoski's test): 2 mg of extract was dissolved in 1ml of chloroform. later, 1 ml conc. h2so4 was added to the solution. eventually, appearance of brown ring between junctions of two liquids http://www.tipdisease.com/2013/11/diarrhea-causes-symptoms-treatment.html nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 3 nepjol.info/index.php/njb indicated the presence of sterols and the brown ring with upper layer green indicated the presence of triterpenes. test for tannins and polyphenol: 2 mg extract was dissolved thoroughly in 1 ml of water. to the solution, 3 drops of 1% (w/v) ferric chloride (fecl3) was added. appearance of blue black/ violet colour indicated the presence of tannins and polyphenols. test for saponins: 2 mg extract was dissolved in 2 ml of water. the solution was shaken vigorously for 30 minutes. persistence of thick froth (about 1 cm) height even after 30 minutes indicated the presence of saponins. test for flavonoids: to 2 mg of extract, 5ml of 10% (v/v) ammonium hydroxide (nh4oh) was added and to the solution, 1 ml of conc. h2so4 was added. disappearance of yellow coloration indicated the presence of flavonoids. test for coumarins: 2 mg extract was thoroughly dissolved in hot water of 2ml. after cooling, the solutions were divided into two test tubes. to one of the test tubes, 10% (v/v) ammonium hydroxide (nh4oh) was added until the solution became basic. the other test tube was used as control. the test tubes were kept under uv light. fluorescence indicated the presence of coumarins. test for phlobatanin: 1mg extract solution was boiled with 2ml of 1% hcl until the appearance of red precipitate was visible, which then, indicated the presence of phlobatanin. test for reducing sugars (fehling's test): about 1 mg of extract was mixed with 1 ml of water. to this solution, 1 ml of fehling's reagent (1:1 mixture of fehling's reagent a and b) was added. appearance of red precipitate indicated the presence of phlobatatin. antimicrobial screening antimicrobial screening was conducted by agar well diffusion method. sample was prepared by adding 25 mg extract to 500µl of 100% dimethyl sulfoxide (dmso) followed by 10-15 minutes of vortexing. 200µl of aforementioned dmso solution was added to 200µl of double distilled water and was termed as 'pure solution'. again, pure solution was added to dmso in 1:1 and 2:1 ratios. for positive control, antibiotics; chloramphenicol, tetracycline and cefoxitin were used whereas, imidazole prepared in dmso (0.1g/ml) was used as antifungal agent. similarly, for negative control 100% dmso was used. bacterial inoculums were prepared in nutrient broth by taking a loop full of pure culture of microorganism and incubating them at 37ºc for 3 hours. turbidity of the broth was compared to mcfarland turbidity standard number 0.5. microorganisms used a total of seven reference microbial strains (three gram negative bacteria; escherichia coli atcc 25922, klebsiella pneunomiae atcc 700603, salmonella typhimurium atcc 14028,one gram positive bacteria; staphylococcus aureus atcc 25923 and four fungi; fusarium spp., trichoderma spp., aspergillus flavus , aspergillus niger) were used as the test organisms for the anti-microbial screening. antibacterial screening mha (mueller hinton agar) plates were swabbed with 3 hours broth culture using cotton swab. wells of 6mm were bored using sterile borer and 50µl of extract solution was poured using micropipette. the plates were kept for half an hour for diffusion in room temperature and incubated at 37ºc for 24 hours. anti-bacterial activity of each extract was expressed in terms of diameter of zone of inhibition (mm) produced by respective extract against microorganisms after incubation. the procedure was done in replicate manner. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 4 nepjol.info/index.php/njb antifungal screening fungal inoculums were prepared in pdb (potato dextrose broth) by taking a loop full of pure culture of microorganisms and incubating them at 28ºc for 24 hours. petri plates containing pda (potato dextrose agar) were swabbed with 24 hours broth culture using cotton swab. wells of 6mm were bored using sterile borer and 50µl of extract solution was poured in them using micropipette. the plates were kept for half an hour at room temperature for diffusion and incubated at 28ºc for incubation. the antifungal activity of each extract was expressed in terms of diameter of zone of inhibition (mm) produced by respective extract against fungus after incubation. antioxidant assay the antioxidant potentials of different extracts were determined using dpph radical scavenging assay. dry extract was dissolved in 1ml methanol at different concentrations (100µg/ml, 200µg/ml, 300µg/ml, 400µg/ml and 500µg/ml) and was added to 4ml of 0.004% methanol solution of dpph. after 30 minutes incubation at room temperature, the absorbance was read against a blank at 517nm in spectrophotometer (cary 60 uv-vis) from agilent technologies at nast. inhibition of free radical by dpph in percent (%) was calculated by using equation as: i (%) = [(a blank-a sample)/a blank] ×100, where a blank is the absorbance of control reaction (containing all reagents except the test compound and a sample is the absorbance of the test sample. extract concentration providing 50% inhibition (ic50) was calculated from the graph plotted for inhibition percentage against extract concentration. preparation of ascorbic acid ascorbic acid solution of 100 µg/ml, 200 µg/ml, 300 µg/ml, 400 µg/ml and 500 µg/ml were prepared in methanol. screening of anti-oxidant activity 1000 µl of extracts solutions in methanol (100 µg/ml, 200 µg/ml, 300 µg/ml, 400 µg/ml and 500 µg/ml) was added to 4ml of 0.004% methanol solution of dpph. after 30 minutes incubation at room temperature, the absorbance was read against a blank at 517nm in spectrophotometer (cary 60 uv-vis) from agilent technologies. inhibition of free radical by dpph in percent (%) was calculated by using equation as: i (%) = [(ablank asample)/a blank] ×100, where ablank is the absorbance of control reaction (containing all reagents except the test compound and asample is the absorbance of the test sample. extract concentration providing 50% inhibition (ic50) was calculated from the graph plotted for inhibition percentage against extract concentration. statistical analysis data are expressed as means and statistical analysis was performed with microsoft excel 2010 and ibm spss statistics 23. difference on statistical analysis of data was considered significant at p < 0.05. results analysis of gc-ms of plant extracts the graph generated by gas chromatography showed composition of the extracts and the graph showed by mass spectrophotometer gave the percentage of each component (figures 1, 2 and 3). the horizontal axis represented percentage area whereas the vertical axis represented the retention time of several components found in each extract. there were variations in retention time and percentage area of the three different extracts. total of 10, 32 and 28 compounds were identified through gc-ms from desmostachya bipinnata l., hordeum vulgare l. and drepanostachyum falcatum (nees) keng f. respectively (figures 1, 2 and 3). the major compounds found in desmostachya bipinnata l. were 2,3 benzofuran dihydro and octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15,hexacamethyl (table 1) and the major compounds found in drepanostachyum falcatum (nees) keng f. were phenol 2, 6-dimethoxy, benzoic acid and n-hexadecanoic acid (table 1). similarly, compounds like quinoline, pthalic anhydride, 1, 2-benzenedicarboxylic acid and indole (table 1) were identified in hordeum vulgare. l.. the compounds were nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 5 nepjol.info/index.php/njb figure 1: gc-ms graph for drepanostachyum falcatum (nees) keng f figure 2: gc-ms graph for hordeum vulgare l. figure 3: gc-ms graph for desmostachya bipinnata l. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 6 nepjol.info/index.php/njb table 1: major compounds present in three plants extracts observed through gc-ms. s.n extract name of compound retention factor peak area 1 drepanostachyum falcatum (nees) keng f. phenol, 2, 6-dimethoxy 10.823 95 benzoic acid, 4-ethoxy, ethyl ester 13.649 93 n-hexadecanoic acid. 19.478 98 2 hordeum vulgare l. phthalic anhydride 10.372 95 1h-indole, 4-methyl 11.325 94 quinoline, 2-ethyl 14.624 81 3 drepanostachyum falcatum (nees) keng f benzofuran 2,3dihydro 8.934 56 octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15,hexacamethyl 19.731 72 identified with the database of national institute standard and technology. majority of the compounds extracted from all three extracts belonged to hydrocarbon class. gc-ms results of drepanostachyum falcatum (nees) keng f. showed presence of wide range of compounds like saturated fatty acids like n-hexadecanoic acid, unsaturated cyclic chemical compound like pyran-4-one, aromatic organic compound like syringol and indene, ranges of aldehydes like vanillin and benzaldehydes. similarly, the gc-ms result of hordeum vulgare l. also showed the presence of aromatic heterocyclic organic compounds like indole and indolizine, anhydrides of thalic acid, heterocyclic aromatic organic compounds like; quinoline and pyrrole and other organic compounds like; acetamide, guanidine etc. phytochemical screening phytochemical screening (table 2) of drepanostachyum falcatum (nees) keng f. showed presence of alkaloids, glycosides, sterols, saponin, coumarins and reducing sugar in good amount and triterpenes in fair amount. whereas, hordeum vulgare l. extract contained phytochemicals like alkaloid, sterols, saponins, flavonoids, coumarins, phlobatanin and reducing sugar in fair amount (table 2). similarly, desmostachya bipinnata l. extract showed presence of glycosides and flavonoids in fair amount. from table 2, presence of various phytochemicals in various amounts in the three plant extracts could be observed. drepanostachyum falcatum (nees) keng f. showed presence of most of the phytochemicals in comparatively good amount than hordeum vulgare l. and drepanostachyum falcatum (nees) keng f. antibacterial assay the antibacterial test showed a very promising result as all the extracts showed zone of inhibition against both gram positive and gram negative bacteria which was significantly comparable to the action of antibiotics against table 2: phytochemical assessment of all three plant extracts s.n tests for phytochemicals plant extracts desmostachya bipinnata l. hordeum vulgare l. drepanostachyum falcatum (nees) keng f 1. basic alkaloids + ++ +++ 2. glycosides ++ +++ 3. sterols + ++ +++ 4. triterpenes + ++ 5. tannis and polyphenols + 6. saponins + ++ +++ 7. flavonoids ++ ++ 8. coumarins + ++ +++ 9. phlobatanin ++ 10. reducing sugar + ++ +++ index: (+++) strongly positive, (++) medium positive, (+) weak positive and (-) negative https://en.wikipedia.org/wiki/saturation_(chemistry) https://en.wikipedia.org/wiki/heterocyclic https://en.wikipedia.org/wiki/aromaticity https://en.wikipedia.org/wiki/organic_compound nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 7 nepjol.info/index.php/njb figure 4: zoi shown by various concentrations of the three extracts against salmonella typhimurium figure 5: zoi shown by various concentrations of the three extracts against staphylococcus aureus. figure 6: zoi shown by various concentrations of the three extracts against klebsiella pneumoniae. bacteria. most promising antimicrobial activity was shown by desmostachya bipinnata l with zone of inhibition of 21.9mm against salmonella typhimurium and 19.65 mm against s. aureus (figure 4 and 5). hordeum vulgare showed zone of inhibition of 20.5 mm l. against salmonella typhi and of 24.45 mm against s. aureus (figure 4 and 5). drepanostachyum falcatum (nees) keng figure 7: zoi shown by various concentrations of the three extracts against e. coli. figure 8: zoi shown by antibiotics against bacteria. f. showed zone of inhibition of 22.85 mm against salmonella typhi and 22.5 mm against klebsiella pneumonia (figure 4 and 6). hordeum vulgare showed zone of inhibition of 22.5 mm against e. coli (figure 7). the zone of inhibitions shown by the extracts solution was compared to the zone inhibition showed by the antibiotics (figure 8) although, all three plants belonged to the same family poaceae, the presence of biomolecules in each plant varied from another yet, all three plants showed a appreciable result against both gram-positive bacteria and gram negative bacteria. antifungal assay in the case of antifungal assessment, widest zone of inhibition of 15mm was shown by sample 2:1 of hordeum vulgare against trichoderma spp. (figure 9) and pure sample of 0 5 10 15 20 25 30 pure 2:01 1:01 z o n e o f in h ib it io n (i n m m ) plant extracts used in different … h.vulgare d.bipinnata d.falcatum 0 5 10 15 20 25 pure 2:01 1:01z o n e o f in h ib it io n ( in m m ) plant extracts used in different concentrations h.vulgare d.bipinnata d.falcatum 0 5 10 15 20 25 pure 2:01 1:01 z o n e o f in h ib it io n (i n m m ) plant extracts used in different concentrations h.vulgare d.bipinnata d.falcatum 0 5 10 15 20 25 30 35 40 45 z o n e o f in h ib it io n ( in m m ) bacteria used chloraphenicol cefoxitin tetracycline b c d a e b a d c 0 5 10 15 20 25 pure 2:01 1:01 z o n e o f in h ib it io n ( in m m ) plant extracts used in various … h.vulgare d.bipinnata d.falcatum nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 8 nepjol.info/index.php/njb drepanostachyum falcatum against fusarium spp. (figure 10). figure 9: zoi shown by hordeum vulgare against fungus figure 10: zoi shown by drepanostachyum falcatum against fungus dpph radical scavenging assay table 3: ic50 values of desmostachya bipinnata l., hordeum vulgare l. and drepanostachyum falcatum (nees) keng f. s.n plant extracts ic50 1. desmostachya bipinnata l. 143.36 2. hordeum vulgare l. 135.625 3. drepanostachyum falcatum (nees) keng f 590 4. abscorbic acid 86.20 in the dpph method, the antiradical activity was evaluated by the capacity of antioxidant compound to reduce the dpph radical as indicated by the decrease in its absorbance at 517 nm until the reaction reached a plateau. the antiradical activity was defined as the amount of antioxidant necessary to decrease the initial dpph concentration by 50% [8]. the ic50 values of all the plant extracts and ascorbic acid (table 3) illustrates that the antioxidant activity of the three plants extracts are in the descending order of hordeum vulgare l.>desmotachya bipinnata l. > drepanostachyum falcatum (nees) keng f. dpph radical scavenging assay of the three plants are graphically represented from figure 11-14. hordeum vulgare l. and desmotachya bipinnata l. were able to inhibit 50% concentration of dpph radicals at 135.625µg/ml and 143.36µg/ml (table 3) against, ic50 of the ascorbic acid's which was at 86.20µg/ml (table 3). figure 11: dpph radical scavenging assay of desmostachya bipinnata l. figure 12: dpph radical scavenging assay of hordeum vulgare l. figure 13: dpph radical scavenging assay of drepanostachyum falcatum (nees) keng f. 0 5 10 15 20 pure 2:01 c(+) c(-) 0 5 10 15 20 25 pure 2:01 c(+) c(-) 0 20 40 60 80 100 120 100 200 300 400 500 % i n h ib it io n concentration of extract(µg/ml) 0 10 20 30 40 50 100 200 300 400 500 % i n h ib it io n concentration of extract(µg/ml) 0 20 40 60 80 100 100 200 300 400 500 % i n h ib it io n concentration of abscorbic acid(µg/ml) nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 9 nepjol.info/index.php/njb figure 14: dpph radical scavenging assay of ascorbic acid. discussion the benzoic acid present in drepanostachyum falcatum (nees) keng f.is a key component in prevention of infection caused by several bacteria. compounds present in desmostachya bipinnata l. such as, 4-hydroxynorephedrine is an active sympathomimetic metabolite of amphetamine in humans [9]. amphetamine is a potent central nervous system (cns) stimulant which have been used for treatment of attention deficit hyperactivity disorder (adhd) [10]. unlike our test sample, the result on essential oil of gc-ms of desmostachya bipinnata l., conducted by kumar & patel [10] showed the presence of 16 compounds with the similarity values ranging from 97% to 100% whereas, in our project only 10 compounds with much lesser similarity values (approx. 60%) were observed. the reasons for these differences might be due to several variations in samples .as, our solution was in crude form whereas the other one was the essential oil. there is also a difference in extraction procedures and it may also be due to the differences in the geographical inheritance of the plants. as a major class of natural products, alkaloids have been used in various parts of the world as a source of remedies to treat a wide variety of illnesses yet they are underrepresented in the context of newly introduced medicines [11]. whereas, another group of glycosides i.e. saponins are used widely for their effects on ammonia emissions in animal feeding and saponins have also been used as adjuvants in vaccines e.g. quil a; component qs-21, isolated from the bark of quillaja saponaria molina, to stimulate both the th1 immune response and the production of cytotoxic t-lymphocytes (ctls) against exogenous antigens [12].phytochemical analysis showed presence of coumarin in all three plant extracts where, drepanostachyum falcatum (nees) keng f. showed strongly positive result. in a research done by kumar [13] for antimicrobial assay in essential oil of desmostachya bipinnata ,the diameter of the inhibition zone of oil of desmostachya bipinnata varied from 15.2 to 56.2 mm where the largest zone of inhibition was obtained for s. epidermidis (56.2mm) and lowest for s. aureus (33.9mm) which is opposite to ours and the reason behind this could be dissimilarities in presence of phytochemicals due to condition from where the plant had originated including the difference in the temperature, humidity and other environmental stresses. madineni [14] found that thaumatin like proteins was extracted from barley have antimicrobial activity against candida albicans, bacillus subtilis, e. coli, saccharomyces cerevisiae. those proteins could be present in our samples too as we had good result upon e.coli. antimicrobial assay verified that the plants rich in phytochemicals like saponins, coumarins, sterols, alkaloids and reducing sugars showed more promising results. from this research, we figured out antifungal properties of these plant extracts aren’t as promising as antibacterial properties. .desmostachya bipinnata l. didn't show zone of inhibition against aspergillus flavus and aspergillus niger at all , although different concentration of plant extracts showed zone of inhibition against different fungus (table: 2). our research has also focused on the use of antioxidants, particularly, on the importance of naturally derived antioxidants, which may reduce free radicals, ros production and may exhibit defensive property. the antioxidant activity of the three extracts were in the order of hordeum vulgare l.>desmotachya bipinnata l. 0 10 20 30 40 50 60 70 80 90 100 200 300 400 500 % i n h ib it io n concentration of extract(µg/ml) https://en.wikipedia.org/wiki/potency_(pharmacology) https://en.wikipedia.org/wiki/central_nervous_system https://en.wikipedia.org/wiki/central_nervous_system https://en.wikipedia.org/wiki/stimulant https://en.wikipedia.org/wiki/attention_deficit_hyperactivity_disorder https://en.wikipedia.org/wiki/attention_deficit_hyperactivity_disorder https://en.wikipedia.org/wiki/adjuvant https://en.wikipedia.org/wiki/vaccine https://en.wikipedia.org/wiki/quil_a https://en.wikipedia.org/wiki/qs-21 https://en.wikipedia.org/wiki/quillaja_saponaria https://en.wikipedia.org/wiki/cytotoxic_t-lymphocytes nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 1-10 nepal et al. ©njb, biotechnology society of nepal 10 nepjol.info/index.php/njb > drepanostachyum falcatum (nees) keng f. the antioxidative effect was mainly due to phenolic components, such as phenolic acids and phenolic diterpenes [15]. phenolic compounds have been proved to be responsible for the antioxidant activity in many medicinal plants. conclusion the importance of religious plants is still prevailed in our society although scientific validation on use of these plants has not been done much. there was an urge to identify the bio-active compounds present in those plants for which we planned to conduct this project on religious plants. different bioactive compounds such as alkaloids, saponins, steroids, reducing sugars and glycosides were present in the plants. plant which showed most promising result in phytochemical screening is drepanostachyum falcatum (nees) keng f. similarly, these plants also showed significant antioxidant property through dpph radical scavenging assay. since, the plants were rich in various phytochemicals; they had a moderate to significant antibacterial property but they showed quite weaker antifungal property which may be due to absence of some other bioactive compounds which were required to inhibit growth or kill the fungus. it can be concluded that the religious plants that we use in our daily life requires scientific validation for being use which can only done with research. these plants shouldn't only be limited to its religious practices, instead it could be used in multidisciplinary approaches. as we have shown in this project, the plants of religious value have important phytochemicals, antioxidative capacity and antimicrobial property, these plants could be also used in medicinal and therapeutic fields references 1. niroula g: religion and conservation: a review of use and protection of sacred. journal of institute of science and technology, 2015, 20(2): 61-66, iost, tribhuvan university. 2. sapkota pp: religious culture and medicinal plants: an anthropological study. dhaulagiri journal of sociology and anthropology. 2013 7:197. 3. dandapat s, kumar m, kumar a, sinha mp: therapeutic efficacy and nutritional potentiality of indian bay leaf (cinnamomum tamala buch. hem.). intnl j pharm. (2013), 6. 4. santhanam r, okoro ck, rong x, huang y, bull at, andrews ba, asenjo ja, weon hy, goodfellow m: streptomyces deserti sp. nov., isolated from hyper-arid atacama desert soil. antonie van leeuwenhowk, 2012 8 5. chang-geun k, dae-sik h, young-hwan k, euikyung k, jong-shu k: evaluation of antimicrobial activity of the methanol extracts from 8 traditional medicinal plants. toxicol res. 2011 27(1)31-32. 6. kumar j, kamajaj m, nandagopalan v, anburaja, v., & thiruvengadam, m. a study of phytochemical constituents in caralluma umbellata. international journal of pharmaceutical science invention 2013 37. 7. chhetri h, yogol ns, sherchan j, kc a, mansoor s, & thapa p: phytochemical and antimicrobial evaluations of some medicinal plants of nepal. kathmandu university journal of science, engineering and technology. 2008 4(1):49-54. https://doi.org/10.3126/kuset.v4i1.2883. 8. lahouar l. phytochemical content and antioxidant properties of diverse varieties. food chem. 2014 581. 9. santagati na, marrazzo a, ronsisvalle g: simultaneous determination of amphetamine and one of its metabolites by hplc with electrochemical detection. j pharm biomed anal. 2002 30(2):247-55. 10. kumar a and patel j: chemical composition and antimicrobial activity of the essential oil of coriandrum sativum. international journal of phytomedicine. 2010 436-439. 11. vafa amirkia mh: alkaloids as drug leads a predictive structural and biodiversity-based analysis. elsevier phytochemistry letters, 2014 56. 12. patrick h demana, c. f. effect of incorporation of the adjuvant quil a on structure and immune stimulatory capacity of liposomes. immuno. cell biol., 2004 5. 13. kumar ak, sharvanee s, patel j, choudhary rk: chemical composition and antimicrobial activity of the essential oil of desmostachya bipinnata linn. int j phytomed. 2010 4. 14. jebor ma: characterization and antimicrobial activity of barley grain. int j curr microbiol appl sci 2013. 47. 15. fereidoon sjp: phenolic antioxidants crc critical rev. food sci nutr. 1992. 2:67-103. nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1): 27-34 research article doi: https://doi.org/10.54796/njb.v11i1.244 ©njb, bsn 27 effect of coconut water and ga3 concentrations on in vitro clonal propagation of potato cultivars from nepal durga prasad kafle1 , sanam parajuli1, aastha upreti1, dhurva prasad gauchan1 1department of biotechnology, school of science, kathmandu university, dhulikhel, nepal received: 22 jun 2023; revised: 22 jul 2023; accepted: 24 jul 2023; published online: 31 jul 2023 abstract nodal propagation plays a crucial role in mass multiplication of potato plants. growth regulators and media selection have an impact on the efficacy and quality of propagation. both coconut water, a naturally occurring organic source of growthpromoting compounds, and the synthetic growth regulator ga3 (gibberellic acid-3), have the ability to accelerate plant growth. the purpose of this study was to evaluate the effects of growth regulators and media, specifically ga3 and coconut water (cw) in agar-based media (abm) and clarigel-based media (cbm) which is a gellan gum-based media, on potato nodal propagation. in contrast to the control group, ga3 in abm did not produce definitive results, however ga3 in cbm showed a considerable level of efficacy. for the janak dev variety, cbm surpassed abm in terms of root length, root hairs, leaf size, and dry biomass, whereas abm demonstrated superior root length for the cardinal variety. in comparison to the ga3 alone at concentration ranges from 0 to 2 mg/l, adding cw at 200 ml/l to cbm or combining ga3 (0.25 mg/l) and cw (10 ml/l) substantially enhanced features including shoot length, leaf size, and root growth for both kinds. acclimatized plantlets had a survival efficiency of 85% to 95%, with cbm supplemented with ga3 showing the highest survival rate and cbm supplemented with cw coming in second. these results highlight the significance of growth regulator and media choice in enhancing potato nodal propagation for improved plant quality and multiplication. keywords: potato, pbs, ga3, clarigel, agar, coconut water, micropropagation corresponding author, email: gauchan@ku.edu.np introduction micropropagation has revolutionized plant propagation and is a key factor in the mass production of disease-free plants. this method entails growing plant cells, tissues, or organs in an aseptic environment in a controlled laboratory setting [1]. the potato (solanum tuberosum l.), among the plant species extensively researched utilizing tissue culture, stands out for its nutritional value and tolerance to varied agro-climatic situations [2]. the major goal of potato tissue culture techniques is the disease-free propagation and maintenance of plants through meristem culture [3]. this method requires isolating and cultivating the apical meristem, a tiny area near the plant's growing tip that contains cells that are actively dividing. since meristems frequently have no viral infections, meristem culture is especially useful for getting rid of viruses. but the likelihood of getting virusfree seedlings increases when chemo and thermotherapy are applied to the explants before meristem culture [4]. in nepal, potatoes have emerged as one of the most important cash crops and vegetables, covering a total of 193,997 hectares of land and producing 3,112,947 metric tons annually as of 2019 [5]. potatoes like janakdev, khumal seto, cardinal, kufri jyoti, and khumal rato-2, desiree, are among the most popular kinds in nepal. in addition to these, there are several domestic and foreign variations. janak dev and cardinal are the most popular in the nepalese hills, where there are many others. this work focuses on improving the tissue culture conditions of these two types in that environment based on growth regulators and solidifying agents. the success of commercial potato tissue culture depends on the selection of the right culture media and technology. plant performance in terms of growth can vary depending on the composition of the medium and the container system. since the beginning of tissue culture, the most widely used method for micropropagating plants, including the potato itself, has been solid media-based tissue culture utilizing glass jars. the choice of solidifying agent for an example has also been a factor in driving the results [6]. agar is utilized to build a strong support system in the medium that aids in both the support of the plants and the media's diffusibility. interest has been generated by gellan gum (clarigel), an efficient replacement for the commonly employed agar-based media. clarigel has shown nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0009-0000-5369-9829 https://orcid.org/0000-0002-3728-5076 mailto:gauchan@ku.edu.np mailto:gauchan@ku.edu.np nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 28 encouraging results in terms of plants’ shoot multiplication and root growth [7]. in plant tissue culture, the basal media compositions almost remained unchanged since the development of murashige and skoog (ms) media in 1962 that supplies with the macro-nutrients, micro-nutrients, iron, and vitamins [8]. the basic elements like nitrogen, phosphate, potassium, iron, and manganese are supplied by the basal media. the basal media has to be enriched with carbon sources (dextrose, sucrose etc.), inositol and hormones depending upon the plants’ requirements [9]. apart from that, plant hormones including auxins, cytokinins and gibberellic acid (ga3) are commonly employed as plant growth regulators, that play a vital role in regulating physiological processes during tissue culture. in potato tissue culture, ga3 is the most frequently used hormone that promotes shoot elongation and multiplication of the in vitro plantlets [10]. however, the high cost and limited availability of such hormones have prompted researchers to explore alternative growth-promoting substances, such as coconut water (cw), which has shown potential as a natural substitute [11,12]. figure 1: selected potato varieties (a); dissection of sprouts to expose meristem (b); meristem at the tip of surgical blade (c); meristem with leaf primordia (d); meristem regenerated plantlets in jam jar (e); nodal sub culture to obtain enough plants for the experiment (f); clonal varieties from nodal subculture if the meristem cultured potato plants (g); acclimatized plants in a screenhouse (h); well survived plantlet in the screenhouse transplanted from the seedling tray (i); janak dev variety (left) with agar without growth regulators (a), with agar+ga3 (g+a) and with clarigel and cw (c+c); cardinal variety (right) with agar without growth regulators (a), with agar+ga3 (g+a) and with clarigel and cw (c+c); comparison of plant morphology of janak dev and cardinal (l and m respectively); comparison of leaf area using graph paper (n and o). in light of these key components, this study was conducted with an aim to explore the advantages of the cw over commercially available plant growth regulators, virus elimination, and growth performance in different solidifying agents (agar vs. clarigel) were examined in two varieties of potato; janak dev and cardinal as these varieties are the most common ones for the hilly regions of nepal. furthermore, the study was set to evaluate the effectiveness of ga3 versus cw as growth-promoting substances. additionally, the use of the ms basal medium will be investigated for its effectiveness in supporting potato tissue culture. it was discovered that ga3 (gibberellic acid) in cbm (coconut broth medium) had notable efficacy while ga3 in abm failed to produce definitive results. when compared to abm, the janak dev variety outperformed cbm in terms of root length, root hairs, leaf size, and dry biomass. the cardinal variant, however, showed longer roots in abm. additionally, both types' shoot length, leaf size, and root growth were greatly enhanced by the combination of ga3 and cw at particular concentrations. the cbm supplemented with ga3 showed the best survival rate for acclimatized plantlets, followed by the cbm supplemented with cw. these results highlight the significance of using the proper growth regulator and medium to improve potato nodal propagation for improved plant quality and multiplication. through this study, we aim to contribute to the optimization and advancement of potato tissue culture techniques, ultimately enhancing potato production and global food security. materials and methods selection of potato tubers and sprouting janak dev and cardinal varieties of s. tuberosum that were suspected to be virus infected were chosen for the study as shown in figure 1. average sized tubers were labelled separately and stored in warm dark chamber for 8 to 10 days until they sprouted. healthy sprouts measuring 1.0 to 2.0 cm in length were excised and surface sterilized as suggested by [13]. to begin, potato sprouts were carefully excised from the tubers. these sprouts underwent a thorough cleansing process using distilled water, followed by a brief immersion in 70% alcohol for 30 seconds. subsequently, they were rinsed again with distilled water and subjected to sterilization within a laminar air flow cabinet using a 0.1% aqueous solution of hgcl2 for a duration of 4 to 7 minutes. after sterilization, the sprouts were further washed with sterilized distilled water, repeating this procedure 4 to 5bntimes to ensure their surface cleanliness and complete removal of sterilant. apical meristem culture meristem culture procedure was followed as done by [14]. primarily, surface sterilized sprouts’ tip was nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 29 carefully removed, and the outer leaves and leaf primordia were dissected under a dissecting microscope in a controlled laminar airflow cabinet. the resulting meristem tip was then put onto test-tube with hormone free potato culture media that was composed of murashige and skoog [8] media (ms media) supplemented by 2 mg/l calcium d-pantothenate (himedia, india), 30 g/l plant tissue culture grade sucrose (thermo fisher, india) and 0.8% agar (himedia). this meristem tip was made up of the apical dome and the first pair of leaf primordia, which were roughly 0.1 to 0.15 mm in length depending on the particular cultivar. in order for the meristem tips to multiply and generate adventitious shoots measuring roughly 2 cm in length, these culture tubes were put in a growth environment under precise circumstances (22 ± 1°c, 16/8 hours of light/dark frequency per day with an intensity of 18 w/m2). the plants developed from meristem were again explanted for another cycle of meristem culture and this process was repeated for two more times to make sure of viral sterility. plant micropropagation after the development of plants from cultured meristem, they were micropropagated by single nodal sub-culture technique as suggested by [10,15] in jam jars using previously used potato tissue culture agar media supplemented by 0.25 mg/l of ga3 provided by himedia, india and the media ph was maintained at 5.8 using ph thermo orion-420 ph meter. the process of sub-culture was repeated until the enough number of plants for the further experiment were achieved. media composition for optimization of plant’s growth media composition that remained unchanged: ms basal media [8], sucrose (30g/l), 2 mg/l calcium dpantothenate and myo-inositol provided by himedia (0.1g/l). two experimental sets were prepared as described below: i) unchanged media composition + varying concentrations of ga3 + agar (himedia) as gelling agent the media composition mentioned earlier that remained unchanged was prepared using an already prepared concentrated stock. in order to generate ga3 concentrations of 0, 0.1, 0.25, 0.5, 1, and 2 mg/l, each present in triplicate media jam jars with 50 ml media for each potato variety, a stock solution of ga3 at a concentration of 50 mg/l was first made and added to the media. agar, at a concentration of 8 g/l, was utilized as the solidifying agent. single node with identical sized were cultured in the prepared media under laminar air flow (laf) condition and the cultured jam jars were incubated at 25 ± 2°c, 16/8 hours of light/dark frequency per day with an intensity of 18 w/m2 [16] for 30 days. results for the plants’ shoot length, root length, number of roots, number of leaves, number of nodes, internode length, leaf size and dry biomass were observed at the 30th day of incubation. ii) unchanged media composition + varying concentrations of coconut water (cw), 0.25 mg/l ga3 and combination of 0.25 mg/l ga3 and 10 mg/l coconut water 2.5 g/l gellan gum as gelling agent the unchanged media composition was supplemented in triplicates for each variety by 0, 10, 20, 50, 100 and 200 ml/l filtered cw in jam jar with net 50 ml media. for comparison, two other experimental sets of media, one with 0.25 mg/l ga3 and the other with the combination of 0.25 mg/l ga3 and 10 mg/l cw were prepared in similar manner. gellan gum (clarigeltm, plant tissue culture tested from himedia) at a concentration of 2.5 g/l, was utilized as the solidifying agent. nodal culture and result observation was done same as in previous section. analysis of plants growth performance as plants growth indicators, following parameters were analyzed as suggested by [17]: shoot length, root length, number of root hairs, number of nodes, dry biomass, leaf size and internode length. plant hardening: in vitro plantlets that were 30 days old in jam jars were brought out of the incubation chamber and placed in normal screen house temperature for 5 to 7 days. then the plants were transplanted into a plastic seedling tray with a sterile sand and soil substrate in a 2:1:1 ratio. the transplant procedure was carried out as suggested by [18] in an enclosed space (an aphid-proof screen house) with specified soil composition and fertilizer combination. after properly cleaning and removing all media residue from the plant roots, bavistin 200 mg/ml was applied for 30 seconds to the roots. prior to transplantation, soil was improved with 2 g/kg of urea, 2 g/kg of dap, and 1.2 g/kg of potassium fertilizer, taking into account our soil for 0.2 m2 area. the plants were irrigated with 5 ml sterile water on daily basis. the growth was observed for 15 days and survival rate of the plants was evaluated. nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 30 data analysis data framing, table construction and bar graph design was done using microsoft excel version 2013 and the analysis of variance (anova) followed by post-hoc analysis by tukey hsd for the mean comparison at 95% confidence interval was done using r studio v.2022.10.1. results and discussion the variation in growth regulators used for propagating potato nodal propagation resulted in different responses in plant growth. however, the application of ga3 at various concentrations in agar media did not yield conclusive results compared to the control group without any growth regulators. none of the observed attributes showed any improvement compared to the negative control when ga3 was applied in concentrations ranging from 0 to 2 mg/ml in agar-based media (abm), with a 95% confidence interval. nonetheless, the application of ga3 was found to be significantly effective when using clarigel-based media (cbm). for the janak dev variety, the cbm was found to exhibit greater root length (8.67 ± 1.15 cm), quantity of root hairs (4.33 ± 0.58), leaf size (33.67 ± 5.86 mm2), and dry biomass content of the plants (12.48 ± 0.88 mg) than the negative control of abm. however, there were only minor differences in the two varieties' node counts and internode lengths. with a value of 10.03 ± 0.31 cm, root length for the cardinal variety was determined to be superior in abm to cbm. the number of nodes and the number of nodes did not differ noticeably between the two circumstances. all other characteristics, including shoot length, number of root hairs, leaf size, and dry biomass, respectively being 6 ± 1 cm, 5.33 ± 2.31, 23.33 ± 8.02 mm2, and 11.84 ± 2.69 mg, were significantly better expressed in cbm than in abm, both of which lacked growth regulator additions. when supplemented with 0.25 mg/ml ga3 to both of these two basal media, cbm was better than abm for both cardinal and janak dev varieties in terms of all the attributes under investigation. the results are displayed in table 1 and table 2. further, comparing the performance of different concentrations of cw (varying from 10 to 200 mg/l) with that of 0.25 mg/l ga3 in cbm, addition of cw up to 200 ml/l expressed significantly higher values for root length, number of root hairs, plant dry biomass and number of nodes for both varieties. combination of 0.25 mg/ml ga3 and 10 ml/l cw was statistically found to be superior for the enhancement of shoot length and leaf sizes for both varieties. ga3 resulted in longer internode length than cw treatment in cbm for both varieties. the survival efficiency of the screen house acclimatized plantlets ranged in between 85 to 95% for both cultivars from all media systems used. plants from clarigel media supplemented with ga3 had survival rate of 95% (19 survivors out of 20 transplanted plants) followed by the plants from cw supplied clarigel media with the survival rate of 90%. plants grown in agm with ga3 had the least survival rate of all (85%). the study investigated the effects of growth regulators and media on potato nodal propagation. ga3 in abm showed no conclusive results, but was effective in cbm. cbm outperformed abm in root length, root hairs, leaf size, and dry biomass for janak dev variety. abm had better root length for cardinal variety. cbm with ga3 showed superior performance for both varieties. the ability of clarigel, a bacterial polysaccharide to gel more consistently than agar, a polysaccharide from seaweeds and its physical smoothness and softness when compared to agar aid in the uniform growth of in vitro plants [19]. study of clarigel’s advantages over agar in the tissue culture of potato remains limited, it’s better water retention qualities than agar, can provide a more constant and suitable moisture level for the tissue culture. water availability has a significant impact on plant metabolism and nutrient absorption. however, in some, gelrite have been reported to show hyperhydration which in case of some plants like those with hard woods was proven to be a drawback of this very nature of gelrite [20]. the inertness and purity of clarigel provide more accurate results, especially considering that the ms basal media is already defined based on salts. in contrast, agar contains various impurities, including salts of metals and sulfur. [21]. the results presented here represent the mean values ± standard deviation of three separate observations. the letters superscripted with the numerical values denote the groupings obtained from the tukey hsd test with an alpha of 0.05 conducted after anova. values sharing the same letter are not significantly different at the 95% confidence level. the results presented here represent the mean values ± standard deviation of three separate observations. the letters superscripted with the numerical values denote the groupings obtained from the tukey hsd test with an alpha of 0.05 conducted after anova. values sharing the same letter are not significantly different at the 95% confidence level. nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 31 table 1: variation of ga3 concentration using agar based media (abm) variety ga3 concentration (mg/ml) shoot length (cm) root length (cm) number of root hairs number of nodes internode length (cm) dry biomass (mg) biomass (%) janak dev 0 8.07a ± 0.42 4.47a ± 0.93 3.67a ± 1.15 8.33ab ± 1.53 0.99b ± 0.19 9.06a ± 1.76 8.58ab ± 0.85 0.1 8.57a ± 1.38 5.33a ± 0.15 2ab ± 0 10a ± 1 0.86b ± 0.16 10.05a ± 0.82 9.77ab ± 1.92 0.25 5.8a ± 0.87 4.37a ± 1.89 1b ± 0 6.67ab ± 2.08 0.92b ± 0.28 10.02a ± 0.61 11.31a ± 0.78 0.5 7.73a ± 1.42 6.73a ± 0.06 1.67ab ± 1.15 7.67ab ± 0.58 1.01b ± 0.12 9.34a ± 1.22 8.96ab ± 0.42 1 6.37a ± 1.59 4.4a ± 1.39 1.67ab ± 0.58 6ab ± 1 1.06ab ± 0.15 8.46a ± 2.44 8.35b ± 1.10 2 8.53a ± 3.06 3.8a ± 0.98 2ab ± 1 5.33b ± 2.08 1.67a ± 0.41 9.37a ± 1.58 9.42b ± 0.33 cardinal 0 5.5b ± 0.46 10.03a ± 0.31 2.67a ± 0.58 7.67a ±1.53 0.73b ± 0.10 10.46a ± 1.57 9.84a ± 0.68 0.1 7.93a ± 0.86 9.57a ± 0.50 2.33a ± 0.58 6.67ab ± 1.15 1.31ab ± 0.38 11.56a ± 3.08 10.48a ± 2.01 0.25 7.47a ± 0.67 8.47a ± 1.01 2.33a ± 0.58 4.33bc ± 1.15 1.18ab ± 0.03 12.33a ± 3.06 10.39a ± 2.03 0.5 8.27a ±1.17 8.83a ± 0.75 2a ± 0 7.33a ± 1.15 1.14ab ± 0.14 14.99a ± 3.25 12.44a ± 2.87 1 6.57ab ± .32 4.6b ± 1.11 2.33a ± 0.58 3.67c ± 0.58 1.62a ± 0.57 9.69a ± 1.43 9.04a ± 0.70 2 6.8ab ± 0.1 8.07a ± 1.34 2.66a ± 0.58 6.33abc ± 1.58 1.89a ± 0.33 13.59a ± 2.10 11.20a ± 0.77 cw in cbm enhanced root length, root hairs, biomass, and nodes compared to the most widely used concentration of ga3 in potato tissue culture (0.25 mg/l). ga3 and cw combination improved shoot length and leaf size as shown in figure 2. these findings indicate that the choice of growth regulator and media type significantly influenced the growth and development of potato plants. cw is a natural, organic source of growth-promoting elements such vitamins, minerals, amino acids, and phytohormone [22]. table 2: coconut water (cw) versus ga3 in clarigel-based media (cbm) variety treatments shoot length (cm) root length (cm) number of root hairs number of nodes leaf size (mm2) internode length (cm) dry biomass (mg) janak dev 0 7e ± 1 8.67a ± 1.15 4.33c ± 0.58 7.67b ± 0.58 33.67e ± 5.86 0.92c ± 0.2 12.48e ± 0.88 cw 10 ml/l 7.83e ± 0.29 8.33a ± 2.31 5.67c ±1.15 8.67b ± 0.53 52.33d ± 4.51 0.93c ± 0.19 15.25de ± 1.27 cw 20 ml/l 9.5de ± 0.5 5.67a ± 1.15 7c ± 2 9.33ab ± 0.58 54.33d ± 2.52 1.02bc ± 0.11 26.38c ± 0.67 cw 50 ml/l 12cd ± 1 6.83a ± 1.26 7.33bc ± 2.08 9.67ab ± 0.58 55.67cd ± 5.03 1.25bc ± 0.18 29.52bc ± 2.82 cw 100 ml/l 13.33abc ± 1.15 5.5a ± 0.5 11.67ab ± 2.52 9.67ab ± 0.58 76.67b ± 3.79 1.39bc ± 0.18 32.63ab ± 1.72 cw 200 ml/l 15ab ± 1 8.33a ± 2.31 13.67a ± 1.53 11a ± 1 82.67ab ± 7.64 1.37bc ± 0.18 36.42a ±1.83 ga3 0.25 mg/l 15.33a ± 1.15 5.67a ± 2.08 4.33c ± 0.58 7.67b ± 0.58 71.33bc ± 2.52 2.01a ±0.27 14.13de ± 0.83 cw 10 ml/l + ga3 0.25 mg/l 12.33bcd ± 1.53 6.67a ± 0.58 7.67bc ± 1.53 8b ± 0 97a ± 10.54 1.54ab ± 0.19 18.44d ± 2.40 cardinal 0 6e ± 1 7.67a ± 1.15 5.33cd ± 2.31 7.67 b ± 0.58 23.33d ± 8.02 0.78b ± 0.08 11.84e ± 2.69 cw 10 ml/l 7.83de ± 0.29 7.67a ± 2.08 6.33bcd ± 1.53 8.67ab ± 1.53 50 c± 7.21 1.08b±0.45 14.69de ± 1.16 cw 20 ml/l 9.5cd ± 0.5 5.67a ± 1.15 7bc ± 1 9.33ab ± 0.58 55c ± 6.24 1.02b ± 0.11 22.78bc ± 1.16 cw 50 ml/l 12bc ± 1 6.83a ± 1.26 8.67abc ± 2.52 9.67ab ± 0.58 65.33bc ± 12.1 1.25b ± 0.18 27.77b ± 2.25 cw 100 ml/l 13.33ab ± 1.15 5.5a ± 0.5 10 ab ± 1 9.33ab ± 0.58 86 ab ± 4 1.43ab ± 0.21 33.46a ± 1.26 cw 200 ml/l 15a ± 1 8a ± 1.73 11.67a ± 0.58 10.67a± 1.53 97.67 a ± 6.51 1.42 ab ± 0.25 38.46a ± 1.57 ga3 0.25 mg/l 15a ± 1.73 5.67a ± 2.08 2.33d ± 0.58 7.33b ± 0.58 56.33c ± 10.21 1.97a±0.27 15.04de ± 1.69 cw 10 ml/l + ga3 0.25 mg/l 11bc ± 1 6.67a ± 0.58 9abc ± 1 8ab ± 0 107.67a ± 5.86 1.37ab ± 0.13 19.7cd ± 3.19 nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 32 a ab ab a a abc ab c 0 2 4 6 8 10 12 14 gg 200c gg g gg (g+c) ag g r o o t le n g th ( c m ) condoitionsb cardinal janak dev a c bc c a bc c d 0 5 10 15 20 25 30 35 40 45 gg 200c gg g gg (g+c) ag g d ry b io m a ss ( m g ) condoitions d cardinal janak dev a a b c a a ab c 0 2 4 6 8 10 12 14 16 18 gg 200c gg g gg (g+c) ag g s h o o t le n g th ( c m ) conditions a cardinal janak dev a c bc c a cd bc d 0 2 4 6 8 10 12 14 16 gg 200c gg g gg (g+c) ag g n u m b e r o f ro o t h a ir s condoitionsc cardinal janak dev a c bc c a b b c 0 2 4 6 8 10 12 14 gg 200c gg g gg (g+c) ag g n u m b e r o f n o d e s condoitionse cardinal janak dev a b a c ab b a c 0 20 40 60 80 100 120 gg 200c gg g gg (g+c) ag g l e a f si z e ( m m 2 ) condoitionsf cardinal janak dev nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 33 figure 2: comparison of shoot length (a), root length (b), number of root hairs (c), dry biomass (d), number of nodes per plant (e), leaf size (f) and average internode length (g) of cardinal and janak dev varieties of potato plantlets in tissue culture condition grown in: gellan gum/clarigel based media with 200 ml/l coconut water [gg 200c], gellan gum/clarigel based media with 0.25 mg/l ga3 [gg g], gellan gum/clarigel based media with combination of 0.25 mg/l ga3 and 10 ml/l coconut water [gg(g+c)] and agarbased media with 0.25 mg/l ga3 (ag g). compared to synthetic phytohormones like ga3, it comprises a complex blend of substances that can offer a more complete and balanced nutrient profile for plant growth. auxins, cytokinins, and gibberellins, among other growth-promoting compounds found in cw, can jointly affect different aspects of plant growth and development [23]. this encompassing effect boost a variety of tissue culture performance factors, including root length, root hairs, biomass, and node development [24]. on root initiation and development, cw has reportedly been shown to have remarkable effects. it includes auxins, especially indole-3-acetic acid (iaa), which is essential for promoting root growth and expanding the quantity of root hairs [24]. in this very study, the addition of cw at certain concentrations showed positive effects on various attributes in cbm. these results provide valuable insights for optimizing potato nodal propagation techniques and selecting appropriate growth regulators and media combinations to enhance plant growth and biomass production. based on the findings of this study, it is recommended that adding cw supplements to clarigel or gelrite-based media in the range of 50 to 200 mg/ml can be helpful for improving a variety of traits in plant tissue culture. additionally, 10 ml/l of cw and 0.25 mg/l of ga3 in clarigel or gelrite-based media have demonstrated encouraging effects when combined. conclusion in comparison to ga3 in agar-based media, the use of coconut water (cw) in clarigel based medium greatly improves the health and development of in vitro potato plants. cw has the potential to be a safe and efficient growth-enhancing agent for potato nodal propagation due to its superior efficacy in promoting root length, root hairs, leaf size, dry biomass, shoot length, and root growth. these results underline how crucial it is to choose growth regulators and media carefully in order to achieve the best plant quality and multiplication in potato plantlet in in vitro conditions. exploring the underlying principles and possible uses of cw in enhancing potato propagation methods call for more investigation. acknowledgement our sincere gratitude to the kathmandu universityintegrated rural development program/nepal technology innovation center (ku-irdp/ntic) for grant support and khadyanna tatha biu aaalu alaichi tatha falful nursery udyog, panauti-3, kavre, for providing us with the potato materials and screen house as workspace. reference 1. gamborg ol. plant tissue culture. biotechnology. milestones. in vitro cellular & developmental biology-plant. 2002 mar;38:84-92. https://doi.org/10.1079/ivp2001281 2. hussain t. potatoes: ensuring food for the future. adv plants agric res. 2016;3(6):178-82. 3. gong h, igiraneza c, dusengemungu l. major in vitro techniques for potato virus elimination and post eradication detection methods. a review. american journal of potato research. 2019 aug 15;96:379-89. https://doi.org/10.1007/s12230-019-09720-z 4. shoala t, eid ke, el-fiki ia. impact of chemotherapy and thermotherapy treatments on the presence of potato viruses pvy, pvx and plrv in tissue-cultured shoot tip meristem. journal of plant protection and pathology. 2019 dec 1;10(12):581-5. 5. moal-2018/2019. statistical information on nepalese agriculture (2018/2019). 2019. 6. kaur a, kumar a. the effect of gelling agent, medium ph and silver nitrate on adventitious shoot regeneration in solanum tuberosum. biorxiv. 2020 jan 3:2020-01. 7. repalli sk, geda ck, pradhan ns, rao gj. influence of additional nutrients and gelling agents on in vitro response of selected indica rice varieties. international journal of biology. 2021;11(4):126. 8. murashige t, skoog f. a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiologia plantarum. 1962 jul;15(3):473-97. https://doi.org/10.1111/j.13993054.1962.tb08052.x 9. phillips gc, garda m. plant tissue culture media and practices: an overview. in vitro cellular & developmental biology-plant. 2019 jun 15;55:242-57. https://doi.org/10.1007/s11627-019-09983-5 10. mehmood a, shah ah, sajid m, ahmad h. investigation of ga3 effect on in vitro micropropagation of potato varieties. international journal of agronomical and agricultural research. 2016;9(5):21-30. 11. sembiring r, hayati m, kesumawti e. potato tuber (solanum tuberosum l.) formation due to the application of different concentrations of coconut water in in-vitro. iniop conference ab a b b c a ab bc 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 gg 200c gg g gg (g+c) ag g in te rn o d e l e n g th ( c m ) condoitions g cardinal janak dev nepal j biotechnol. 2 0 2 3 j u l y ; 1 1 (1):27-34 kafle et al. ©njb, bsn 34 series: earth and environmental science 2021 mar 1 (vol. 711, no. 1, p. 012021). 12. hafsan h, mustami mk, masriany m, aziz ir, mustakim m. the utilization of coconut water waste as a growth media of the invitro potato cutting. scientiae educatia: jurnal pendidikan sains. 2018 dec 31;7(2):108-16. 13. bhuiyan fr. in vitro meristem culture and regeneration of three potato varieties of bangladesh. research in biotechnology. 2013 jun 24;4(3). 14. cassells ac, long rd. the elimination of potato viruses x, y, s and m in meristem and explant cultures of potato in the presence of virazole. potato research. 1982 jun;25:165-73. https://doi.org/10.1007/bf02359803 15. sarkar d, naik ps. phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro. plant cell tissue organ cult. 2000;60(2):139–49. 16. da rocha ps, de oliveira rp, scivittaro wb. new light sources for in-vitro potato micropropagation. orignal article biosci j. 2015;(5):1312–8. 17. murshed r, najla s, albiski f, kassem i, jbour m, al-said h. using growth parameters for in-vitro screening of potato varieties tolerant to salt stress. 2018 feb 20. 18. sakha bm, rai gp, dhital sp, nepal rb. disease-free pre-basic seed potato production through tissue culture in nepal. nepal agric res j. 2014;8(november 2014):7–13. 19. mohamed gm, amer am, osman nh, sedikc mz, hussein mh. effects of different gelling agents on the different stages of rice regeneration in two rice cultivars. saudi journal of biological sciences. 2021 oct 1;28(10):5738-44. 20. zimmerman tw, cobb bg. vitrification and soluble carbohydrate levels in petunia leaves as influenced by media gelrite and sucrose concentrations. plant cell reports. 1989 jun;8:358-60. https://doi.org/10.1007/bf00716673 21. scherer pa, müller e, lippert h, wolff g. multielement analysis of agar and gelrite impurities investigated by inductively coupled plasma emission spectrometry as well as physical properties of tissue culture media prepared with agar or the gellan gum gelrite. ininternational symposium on propagation of ornamental plants 226 1987 aug 23 (pp. 655-658). https://doi/10.17660/actahortic.1988.226.91 22. yong jw, ge l, ng yf, tan sn. the chemical composition and biological properties of coconut (cocos nucifera l.) water. molecules. 2009 dec 9;14(12):5144-64. https://doi.org/10.3390/molecules14125144 23. hafsan h, mustami mk, masriany m, aziz ir, mustakim m. the utilization of coconut water waste as a growth media of the in vitro potato cutting. sci educ. 2019 jan 2;7(2):108. 24. aishwarya pp, seenivasan n, naik ds. coconut water as a root hormone: biological and chemical composition and applications. magnesium (mg/100ml). 2022;22(20.87):31-65. https://doi.org/10.17660/actahortic.1988.226.91 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 18 nepjol.info/index.php/njb detection of latent hiv-1 infection and drug resistant mutation testing in nepal: hiv-1 env v3 dna sequence and rt gene (m184v) mutation rupendra shrestha1,2*, sundar khadka1,2, susbin raj wagle1,2, alisha sapkota1,2 1department of laboratory medicine, nobel college, sinamangal, kathmandu, nepal. 2center for molecular dynamics nepal (cmdn), thapathali-11, kathmandu, nepal. abstract hiv-1 resistance to antiretroviral therapy (art) is a crucial issue, despite various effective drugs are available for the treatment. although the viral rna is suppressed below the detection limit (<50 copies/ml) with the use of potent antiviral drugs, the mutation can be archived in the cellular reservoir as proviral dna. the detection of proviral dna and mutation screening in hiv 1 rna for genotypic resistance is the sole basis for monitoring the effectiveness of art. our study aim to access the extent of latent hiv infections by detecting env v3 dna and also testing of m184v (meth184val; atg gtg substitution at 184th codon) specific mutations in hiv-1 rt gene to monitor the effectiveness of art. the hiv-1 env v3 dna sequence was amplified using multiple upstream and downstream primes to show the latent hiv infections, whereas polymerase chain reaction restriction fragment digestion assay (pcr-rfda) was used for testing m184v mutation in hiv-1 rt gene. in the study, out of 15 hiv infected patient blood samples, 12 shows amplification of env v3 dna, confirming the latent hiv infections while 3 were negative for env v3 dna. hiv-1 rt gene tested for m184v mutation in all 15 samples showed wild type after analysis using pcr-rfda. after digestion with cviaii, three bands were observed in wild type whereas in mutant only two bands. although the study shows negative for the m184v resistance mutation, screening of various panels of drug resistance mutations should be performed in recently infected hiv1 patients for planning the effective art strategy. the data is not enough to compare the overall scenario of the nepal thus warrant urgency for large scale study with standard genotypic tools. keywords: hiv-1, antiretroviral therapy, resistance mutation, env v3 dna, rt gene. *corresponding author e-mail: dph.rupendra@gmail.com introduction hiv/aids is the gradually increasing global epidemic threat with an estimated 36.7 million hiv positive people worldwide. among them 5.1 million people are in asia including 3,00,000 newly infected and 1,80,000 aids-related deaths occurred in asia per year [1]. till date 39,249 people are estimated to be living with hiv in nepal [2]. the hiv epidemic has changed due to use of antiretroviral therapies (arts) that suppress the hiv-1 rna load in plasma below the detection limit (<50 copies/ml). however, lifelong art is necessary for patients with latent hiv infections due to decrease in level of cd4+ lymphocytes [3, 4]. the decrease in level of cd4+ t cells caused by hiv infection leads into aids and cause opportunistic infections, like pneumocystis carinii pneumonia and kaposi’s sarcoma [5]. more than 50% of the worldwide epidemic are due to hiv-1 subtype c and are prone to genotypic resistance with treatment failure. hiv-1 has a higher rate of replication with lack of 3’ to 5’ proof reading mechanism and highly error-prone to reverse transcriptase [6-8]. the selection of drug specific resistance mutation in viral genome is due to treatment failure and those mutations may be archived into the cellular reservoir (proviral dna). thus, mutations can be detected in proviral dna even if hiv rna is below detection limit [9]. hiv1 resistance to art is a major problem which is due to suboptimum treatment and transmission of resistant variant at the time of infection [10-12]. resistance mutation is associated with high replicative activities of the viral genome during therapy. several drugs have been approved for the treatment of hiv-1 infection such as nucleotide and nucleoside reverse transcriptase inhibitors (nrtis), protease inhibitors (pis), nonnucleoside rt inhibitors (nnrtis), and fusion inhibitors. however, drug resistance mutations in hiv-1 remain crucial, even though effective (art) is available in the global market. one of the commonly associated drug resistance mutations in hiv-1 rt gene is m184v. the appearance of lamivudine nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 shrestha et al. ©njb, biotechnology society of nepal 19 nepjol.info/index.php/njb (3tc) resistance mutation, m184i, was found to be transiently proceeded to the outgrowth of m184v substitution [13-15]. the study of the nucleotide sequences of both 3tc-resistant variants shows that m184v (gtg) originates from wild-type met (atg) but not from the m184i variant (ata) [16]. both variants are generated from the wildtype atg sequence by transitional substitutions (g to a for 184i and a to g for 184v). the g to a substitution is the type of mutation that most commonly occur during replication of hiv-1 thus m184i appears before m184v [17, 18]. in addition, there have been no systematic studies of hiv-1 resistance mutation in nepalese population. here, we performed the preliminary study of common drug resistance mutation, m184v using a basic molecular tool called pcr-rfda and assessed the detection of proviral dna in hiv-1 infected patients with prolonged art. materials and methods patients counselling and collection of samples individual patient counselling was performed on the background of hiv/aids, diagnosis, monitoring, treatment, drug resistant and genotype testing. a questionnaire was performed with regards to drug use, sexual behavior and travel history. 15 samples were collected from hiv-1 infected patients undergoing long term art commonly with 2-3 nrtis and 1-2 pis or an nnrti for at least 3 4 years recruited in sparsha nepal, sanepa, kathmandu was included. out of 15 cases, only two were clinically approved as art resistance and viral loads of each patients are shown in table 1. data were collected as a part of a continuing study of antiretroviral treatment at the time of primary hiv-1 infection, which was approved by the department of laboratory medicine, research committee of nobel college and chief of sparsha nepal. extraction of nucleic acid the nucleic acid extraction procedure was performed in biosafety cabinet (bsl-2). hiv-1 rna was extracted using precision qiaamp viral rna mini kit (qiagen, hilden, germany) while dna was extracted using shine gene whole blood dna extraction kit (wuhe road, minhang district, shanghai, china) as per manufacturer's instructions. proviral dna detection: nested pcr nested pcr for hiv-1 env gene the nested pcr was performed in the env v3 dna sequences of hiv-1 for the amplification using highfidelity pfu dna polymerase. the amplification was performed as described in previous study [19]. the total reaction volume of 20μl containing 1x pcr buffer, 1.5 mm mgcl2, 200 μm dntps, and 0.25 u of pfu enzyme and rnase free water. the first round pcr was performed with 0.5 μm of upstream primers ja9ae (5’cacagtacaatgcacacatg-3’), ja9b (5’cacagtacaatgtacacatg-3’), and downstream primers ja12a (5’gcaatagaaaaattctcctc-3’), ja12b (5’acagtagaaaaattcccctc-3’). the reaction mixture was incubated at 95°c for 15 minutes, 94°c for 30 sec, 58°c for 30 sec, 72°c for 30 sec and 72°c for 10 minutes, for 35 pcr cycles. 1 μl of the firstround pcr product was used as template for second-round pcr. inner upstream primers used were a mixture of 0.33 μm each of ja10ub (5’ctgttaaatggcagtctagc-3’), ja10uc (5’ctgttaaatggtagtctagc-3’), and ja10ug (5’-ctgttaaatggcagtttagc-3’). inner downstream primers used were different for each reaction, namely, 0.33 μm each of ja11lae (5’aatttctagatcccctcctg-3’), ja11lb (5’aatttctgggtcccctcctg-3’), and ja11lc (5’aatttctaggtcccctcctg-3’). the reaction mixture was incubated at 95°c for 15 minutes, 94°c for 30 sec, 50°c for 30 sec, 72°c for 30 sec and 72°c for 10 minutes, for 35 pcr cycles. the final pcr product was resolved in 1.7% agarose gel electrophoresis and image was captured using gel documentation. hiv-1 rna mutational (m184v) analysis: pcr-rfda rt gene amplification: nested pcr the amplification of hiv-1 rt gene was performed using nested primer pairs which were reported in previous study [20]. the isolated hiv-1 rna was subjected to onestep rt-pcr (qiagen, hilden, germany). the rt-pcr product was then used as templates for second round pcr. the qiagen nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 shrestha et al. ©njb, biotechnology society of nepal 20 nepjol.info/index.php/njb onestep rt-pcr was performed as recommended by the manufacturer. the total volume of 50μl reaction was carried out containing rnase free water, 1x onestep rt-pcr buffer, 200 μm dntp mix, 0.6 μm of each primers a1 (5’aattttcccattagccctatt-3’) and ne1 (5’tatgtcattgacagtccagct-3’), 2μl of onestep rt-pcr enzyme mix, 5 units rnase inhibitor and 2 μg viral rna for reversetranscriptase gene. the reaction was incubated at 48°c for 20 minutes (reverse transcription) followed by 94°c for 4 minutes, 94°c for 30 sec, 52°c for 30 sec, 72°c for 30 sec, and 72°c for 10 minutes, for 35 pcr cycles. the second round pcr was done by using a qiagen master mix with 5 µl of the first rt-pcr product and primers nna (5’aagccaggaatggatggccca-3,) and e (5’ccatttatcaggatggagttc-3’). the reaction mixture was incubated at 95°c for 15 minutes, 94°c for 30 sec, 50°c for 30 sec, 72°c for 45 sec and 72°c for 10 minutes, for 30 pcr cycles. m184v mutation testing: restriction fragment digestion assay (rfda) the amplified rt gene was tested for m184v mutation by digestion approach called rfda. the rfda was performed using restriction enzymes cviaii. the total volume of 50μl reaction was performed containing 1x nebuffer, 10 units’ cviaii restriction enzyme, and 1μg rt-pcr product at 25oc for 1 hour. the digested product was resolved in 1.7% agarose gel electrophoresis and image were captured using gel documentation. results in the present study, detection of proviral dna by amplification of env v3 dna sequences was done by nested pcr while testing of resistance mutation in the hiv-1 rt (met 184 val) gene was performed by pcr and rfda. individual band, resulting from final pcr product and enzyme digested product was compared with art resistance strain and molecular size marker (100bp). the results are tabulated and is shown in table 1. table 1: patient’s information, art and genotypic testing in the study population n-15 code gender/age art used viral load copies/ml env v3 dna m184v mutation patient status hrm1 male/36 yrs azt, 3tc, nfv 1500 positive negative critical hrm2 male/33 yrs azt, 3tc, efz 1500 positive negative critical hrm3 male/26yrs azt, 3tc, nfv 1500 positive negative critical hrm4 male/35yrs azt, 3tc, nfv 1000 positive negative expired hrm5 male/23yrs azt, 3tc, nfv 1000 positive negative art hrm6 male/32yrs azt, 3tc, nfv 300 negative negative art hrm7 male/34yrs azt, 3tc, nfv 800 positive negative art hrm8 male/25yrs azt, 3tc, nfv 800 positive negative art hrm9 male/28yrs azt, 3tc, efz 500 positive negative art hrm10 male/30yrs azt, 3tc, nfv 250 negative negative art hrm11 male/36yrs azt, 3tc, nfv 300 negative negative art hrm12 male/25yrs azt, 3tc, nfv 750 positive negative art hrm13 male/27yrs azt, 3tc, nfv 400 positive negative art hrm14 female/28yrs azt, 3tc, efz 650 positive negative art hrm15 female/33yrs azt, 3tc, nfv 550 positive negative art key: hrm-hiv-1 resistance mutation, art-anti retroviral therapy, aztzidovudine, 3tclamivudine, efv-efavirenz, nfv nelfinavir table 2: most common mutations in rt gene and specific drugs resistance. resistance genes resistance level mutation gene high intermediate low m184v/i rt 3tc, ftc abc, ddi k65r rt abc, ddi, tnf, d4t 3tc, ftc m41l rt azt, d4t ddi, abc tnf k103n rt efv, nvp key: 3tclamivudine, ftc-emtricitabine, aztzidovudine, d4tstavudine, efv-efavirenz, nvpnevirapine, abcabacavir, ddi didanosine, tnftenofovir nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 shrestha et al. ©njb, biotechnology society of nepal 21 nepjol.info/index.php/njb identification of env v3 dna the env v3 dna sequence was analyzed to rule out latent hiv-1 infections. out of 15 samples amplified for env gene, only 12 of samples show positive for env v3 dna sequence by nested pcr. the size of the product was 335bp which was a band of our figure 1: nested pcr for env v3 dna sequences. marker (m) of 100bp was used to compare the molecular weight of amplified product resolved in gel of 1.7%. a) amplified product with band of interest (335bp) was visualized in all lane 1-6. b) amplified product was observed only in lane 7, 8, 10, 12 but not in lane 9 and 11. gel image of 3 positive samples is not shown. interest and is illustrated in figure 1 (a & b). the remaining 3 samples negative for the same gene was reamplified twice showed the similar results. the reason for env gene negative in those three samples might be due to complete elimination of proviral dna or due to its reduction below the detection limit after prolonged art. m184v mutation in hiv-1 rt gene hiv-1 rna was tested for the most common mutation; m184v which emerges in patients using lamivudine (3tc) monotherapy or can be a phenotypic reversion of zidovudine (zdv). pcrrfda molecular tool was used for testing the mutation in which gene was amplified by nested pcr and further final product was digested with cviaii restriction endonucleases. in the present study, we tested m184v mutation in 15 samples and all showed wildtype strain. the wildtype strains show three fragment after digestion with band size of 453bp, 60bp and 168bp are susceptible to 3tc while mutant type have 2 bands with size of 453bp and 228bp are considered as 3tc resistance which means the m184v resistance mutation was emerged by modification of the 184 restriction site substituting cviaii enzymes. the virtual schematic illustration of nested pcr and rfda of both wild type and mutant type is shown in figure 2 (a & b). discussion early detection of drug resistance mutation in hiv-1 infected patients in developing countries is a big challenge however, can aid clinicians in making proper art strategy plans. various molecular tools like real time pcr and sequencing facilities are still lacking. also, such tools are expensive and less sensitive to minor resistance variants. the development of cheap and sensitive tools to screen resistance mutation can be a breakthrough in the field of molecular biology for low economic countries like nepal. in the study, we used nested pcr and pcr-rfda to detect hiv-1 infections and screen the prevalence of m184v mutation in hiv-1 patients underwent prolong art. the cheap and sensitive methods for detecting primary infection in infants and latent hiv-1 infection is to identify proviral dna. the hiv proviral dna is positive after 28 days while most healthy individuals are accustomed to waiting 3 months for a conclusive result. we performed nested pcr to identify the env v3 dna sequences in 15 samples, out of which 12 showed positive for proviral dna. the pcr amplification on 3 negative samples was repeated twice with the same primers for the env v3 dna but consistently the results were negative. the reason might be a low copy number which is below the detection limit or can be complete elimination of proviral dna due to prolonged art. the standard methods for the detection of hiv-1 proviral dna are currently lacking and several discordant results are still present in different studies [21]. in the present study, we didn’t study for the nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 shrestha et al. ©njb, biotechnology society of nepal 22 nepjol.info/index.php/njb figure 2: nested pcr and rfda for detection of m184v resistance mutation. a) wildtype stain with three bands of size 453bp, 60bp, 168bp. b) mutant strain with two bands of size 453bp and 228bp resistance mutation in hiv-1 proviral dna. however, the presence of drug resistance mutations in the reservoir of latent hiv-1 provirus and their stability may impact future management and/or treatment possibilities. the presence of latent proviral dna doesn’t indicate the resistance to art but the specific mutation like m184v, k65r, m41l and k103n etc. can be detected if the similar mutation is observed in hiv-1 rna. this is due to retention of mutant from hiv-1 rna into cdna due to long term suppressive art that integrates into host genome. the evolution of drug resistance mutation in globally epidemic hiv-1 is due to suboptimal nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 shrestha et al. ©njb, biotechnology society of nepal 23 nepjol.info/index.php/njb treatment, a point mutation in viral genome and resistance variants leads to incomplete suppression of the viral genome. the development of drug resistance mutation threatens the success of the future therapy regimens. the challenge in treatment of hiv-1 is associated with the development of drug resistance mutation against antiretroviral drugs which can significantly reduce the viral rna to undetectable levels in plasma [22]. the various resistance mutation pattern has been reported with majorities in both nucleoside reverse transcriptase inhibitors (nrtis) and nonnucleoside reverse transcriptase inhibitors (nnrtis) while only a few with nnrtis and also in minor ratio with protease inhibitors (pi) and 3class resistance (nrtis, nnrtis and pis). the genotyping of the reverse transcriptase gene sequence revealed that almost 90% resistance mutation were m184v [23] and such mutation emerges in patients receiving lamivudine (3tc) therapy, but also it could be associated with phenotypic reversion of zidovudine (zdv) resistance [24]. in the present study, we screened for m184v resistance mutation in hiv-1 infected patients receiving 3tc in combination by pcrrfda methods. the only wild type strain was found in all the sample analyzed which means the m184v mutation was negative. our results completely contradict the other reports on m184v. in the study though phenotypic resistance and high viral load were included, m184v mutation was not detected which might be due to numerous reasons like 1) duration of 3tc art, 2) possibilities of other mutation like m184i, k65r, etc 3) change in drugs 4) less likely to be technical error . the commonly used drugs by the hiv-1 infected patients in nepal are azt, 3tc, efv and nfv and is shown in table 1. the high level resistance to 3tc associated with m184v mutation is also resistance to other drugs like ftc, abc and ddi. in addition to this, resistance to 3tc is also associated with k65r mutation and its variant forms but the level of resistance may vary from high-intermediate-low. the most common rt gene mutation and specific drug resistance is shown in table 2. there are several mutations in genes of hiv-1 associated with resistance to specific drugs [25]. the m184v mutation was detected in lesser than 10% by allele specific pcr but was consistently negative by standard genotyping [26] while the ratio of mutant and wild type is equal to 50:50 reported by almost half of the laboratories [27]. the prevalence of the m184v mutation is notably higher than that of other mutation [28-30] and is significantly associated with 3tc [31]. the early detection of resistance mutation in hiv-1 infected patients would help to change the treatment regimens susceptible to both proviral dna and viral rna and control measures can be taken to minimize the transmission of resistant variants. an understanding of the molecular mechanism of drug resistance will enable us to develop improved tools for resistance screening. conclusion in the present study, we set out to identify the hiv1 latent infections by detecting env v3 dna sequences using nested pcr. in addition to this, the m184v resistance mutation was not observed in the samples analyzed thus indicating as wild type strain. also, we optimized the pcr-rfda protocol using cviaii restriction enzymes for the hiv-1 rt strains for mutation testing. this strategy was meant to enhance assay discrimination between the specific drug resistance mutation (mutant allele) and its wild type (wild type allele) using nested primers and cviaii restriction enzymes. declaration of interests the authors declare that they have no competing interests. authors’ contributions rupendra came up with the study, frame experimental work and prepared manuscript. all authors have equally involved in completing the research experiment and data compiling. all authors read and approved the final manuscript for publication. acknowledgement the authors would like to thank the nobel college for funding the projects and center for molecular dynamics nepal (cmdn) for providing all the facilities to perform the research experiment. we also like to thank dr. sameer m. dixit, bhavesh mishra, raunak shrestha, sulochana manandhar, and prajwal rajbhandari for their valuable discussion and extensive support to make this work done. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 18-25 shrestha et al. ©njb, biotechnology society of nepal 24 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136-146. 27. frater aj, dunn dt, beardall aj, ariyoshi k, clarke jr, mcclure mo, weber jn: comparative response of african hiv-1-infected individuals to highly active antiretroviral therapy. aids 2002, 16(8): 1139-1146. 28. delaunay c, brun-vézinet f, landman r, collin g, peytavin g, trylesinski a, flandre p, miller m, descamps d: comparative selection of the k65r and m184v/i mutations in human immunodeficiency virus type 1-infected patients enrolled in a trial of first-line triplenucleoside analog therapy (tonus imea 021). journal of virology 2005, 79:9572-9578. 29. hira sk, panchal k, parmar pa, bhatia vp: high resistance to antiretroviral drugs: the indian experience. international journal of std & aids 2004, 15(3): 173-177. 30. sen s, tripathy sp, patil aa, chimanpure vm, paranjape rs: high prevalence of human immunodeficiency virus type 1 drug resistance mutations in antiretroviral treatmentexperienced patients from pune, india. aids research and human retroviruses 2007, 23(10): 13031308. 31. zaccarelli m, perno cf, forbici f, cingolani a, liuzzi g, bertoli a, trotta mp, bellocchi mc, di giambenedetto s, tozzi v, gori c, d'arrigo r, de longis p, noto p, girardi e, de luca a, antinori a: using a database of hiv patients undergoing genotypic resistance test after haart failure to understand the dynamics of m184v mutation. antiviral therapy 2003, 8(1): 51-56. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 93-108 review article doi: https://doi.org/10.3126/njb.v9i1.38671 special issue: icbsn 2021 ©njb, bsn 93 intellectual property right on basmati rice: current scenario and evidences of origin, diversity, cultivation and use values of basmati rice in nepal bal krishna joshi1 , krishna hari ghimire1, prakash raj bista2, ram baran yadaw3, ram krishna shrestha4, gaurish krishna kharel5, prakash paneru6, ram bahadur kc6 and deepak bhandari6 1national agriculture genetic resources center, narc; khumaltar, kathmandu, nepal; 2ministry of agriculture and livestock development, kathmandu; 3directorate of agricultural research, narc, province-2, parwanipur, bara; 4crop development and agro-biodiversity conservation center, department of agriculture, lalitpur; 5kto inc, kathmandu 6nepal agricultural research council, kathmandu, received: 0 mar 2021; revised: 14 may 2021; accepted: 22 may 2021; published online: 31 jul 2021 abstract basmati rice, also called the king/prince of rice landraces has very special values in nepalese society as well as in other countries of indian subcontinent. with the objectives of collecting, analyzing and documenting basmati related information in nepal, we visited different sites; carried out key informant surveys; organized focus group discussions, online interaction and discussion meetings; requested all relevant offices/ persons/ stakeholders through phone, website, and letter to share information; organized high level official meeting, and basmati rice expert meeting; documented video documentary and did online as well as library search. because of its high market value at global level, many countries and organizations have been attempting to get intellectual property rights (mainly patent and geographical indication tag) on basmati rice. india applied for gi tag to basmati rice in the european union (eu) in july 2018, and nepal submitted opposition letter along with proofs and evidences of origin, diversity, cultivation and use values of basmati rice on 9 december 2020. a total 133 basmati type rice landraces are grown in 60 districts of nepal. basmati rice is traditionally grown, sold, and consumed in geographically localized areas of nepal since ancient time. international and national scientists have defined lower altitude of nepal as one of the centers of origin of basmati rice. many nepalese basmati rice landraces have been characterized and evaluated using morphological traits, isozymes and dna markers. four basmati type of rice landraces have been registered in national seed board. many community seed banks have maintained different types of basmati rice landraces. national agriculture genetic resources center and international genebanks have collected more than 80 and conserved 68 basmati landraces. basmati rice landraces have geo-linked traits. the historical culture of production, consumption and marketing of native basmati rice in nepal should always be favored by both national and international rules and regulations. nepal has ample and valid evidences to get geographical indication (gi) right on basmati rice. keywords: geographical indication, basmati rice, origin, diversity, historical literature corresponding author, email: joshibalak@yahoo.com introduction rice diversities consisting of 2500 native landraces and 153 improved varieties are being grown in 75 out of 77 districts and within an altitude range from 60 to 3050 m in nepal [1–7]. before 1980, nepal was exporting rice including aromatic rice to india, china, singapore and bangladesh [1,8–12[. in 1977, a total of 105,000-t rice was exported [1]. the word ‘basmati’ is used as an adjective describing the things having aroma or fragrance. basmati, the prince/king of rice is a valued and expensive cereal. many landraces are very localized and possess specific traits, for instance, basmati with aroma [2,13,14]. in general, basmati type landraces include all aromatic rice landraces. aromatic rice is grown in 10% of total rice area (i.e. 150,000 ha out of total 1491,744 ha rice area) with total production of 375,000t in nepal [15]. average productivity of such landraces is about 2.5 t/ha. basmati rice emits aroma which could be a geographical indication (gi). geographical indication is a sign used on products that has a specific geographical origin and possess qualities or reputation that are due to that origin. it is a very common practice to provide gi tag to the agricultural products in the world to monopolize the marketing. germany has the highest number of gi tagged products with 9,499, but, nepal does not have any one [7]. a total of 361 gi products have been registered in india as of september 2019. darjeeling tea was the first gi tagged product in india, registered in 2004. in 2010, basmati rice also got registered as gi product in india. india has also submitted nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 http://orcid.org/0000-0002-7848-5824 mailto:joshibalak@yahoo.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 94 application to eu for geographical indication tag to basmati rice in 2018 [16]. in addition to eu, india has also sought registration of ‘basmati’ in different countries. basmati rice is grown in indian subcontinent and many countries have their own native basmati rice [16–18]. many diverse basmati types of rice landraces are being grown in different parts of nepal since ancient times [1,6,14,19,20]. therefore, the nepalese farming communities have rights on using basmati rice. nepal applied opposition letter to eu with regards to gi tag to indian basmati rice on 9 december 2020. to be eligible to get gi tag to basmati rice, nepal needs to develop and generate relevant proofs and evidences. the objectives of this paper, therefore, were to compile basmati rice related proofs and evidences of origin, diversity, cultivation and use values in nepal; to analyze historical cases of basmati and aromatic rice landraces and to aware and generate information about gi tag to basmati rice. methodologies basmati rice type (which include all aromatic rice landraces) have been grown in different parts of nepal since ancient time. there are key farmers and researchers who are well familiar with basmati type rice landraces. both primary and secondary data were collected, analyzed and discussed. information related to geographical indication and basmati rice were telecasted, published in different media and shared widely to make aware and improve understanding of geographical indication. nine different methods to generate and compile basmati rice related proofs and evidences were adopted. we visited 6 different sites; surveyed 15 key informants; organized 5 times focus group discussion; organized 3 times online interaction and discussion meetings; requested call through phone, email, website and different media to relevant organization across the country; organized two times high-level-official meetings; organized a basmati rice expert consultation meeting; developed three video documentaries, and surveyed literatures. proofs and evidences were grouped and analyzed under 10 different areas as shown in figure 1. database of genesys (https://www.genesys-pgr.org/), national genebank of nepal and community seed banks were analyzed. districts growing aromatic rice landraces were mapped in the country map. total number of aromatic rice landraces was estimated based on their name given by farmers. aromatic rice diversities were grouped under two: basmati group (any landraces that contain at least the word basmati in their name) and nonbasmati aromatic group (any landraces that emits aroma but do not have the word basmati in their name). figure 1. groups of potential proof and evidences of origin, diversity, cultivation and use values of basmati rice findings all relevant stakeholders, farming communities, experts and high officials attended in various official meetings agreed that basmati rice originated in nepal and different types of basmati rice are being grown. basmati rice has multidimensional values associated with farming communities, wealthy people and special occasions. farmers and researchers are also well familiar with the historical importance, diversity, market value and use of basmati rice in nepal. geographical indication in nepal because of climatic variation, nepal is rich in agrobiodiversity and some of them are produced in very specific areas e.g. jumli marshi, jethobudo, basmati, juju dhau, pharping pear, etc. more than 100 agricultural products are potential for geographical indication (gi) tag in nepal [7,21]. it is well known that if jethobudo grows other than pokhara valley, its quality decreases. none of the products are registered as gi in nepal; however, there are many products including basmati, marketed informally as gi and getting higher price for assured better quality in different parts of the country. three traits (famous, special trait and origin) are very important on gi system. basmati is very famous, has a very special trait and originated in nepal, and therefore, hold capacity to get gi tag. legally registration system as gi has not been existed in nepal, but there is a policy provision for gi [7]. basmati rice in nepal and india basmati rice emits a specific aroma in the field, at harvesting, in storage, during milling, cooking and eating. some landraces may emit aroma in only few stages e.g. at 12 34 5 6 8 7 9 10 deep rooted evidences for gi nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 95 harvesting, during cooking, etc. in nepal basmati type rice covers all aromatic rice landraces (short grain, medium and long grain types) and varieties [22]. it is grown in tarai and mid hill agricultural ecozones. this is highly reputed rice and cost very high. therefore, normal family cannot offer such rice all the day. basmati rice are sold in many locations by the name of production areas. many short and medium grain aromatic landraces are grown and consumed locally but they are not known much in the international market. in india, basmati type rice include only long grain rice that emits aroma in most of the time [16–18,23]. along with basmati, many short and medium grain aromatic rice varieties are grown in different parts of india. some of them are superior for taste and aroma as compared to long grain aromatic varieties [18]. though basmati rice includes all aromatic rice in nepal, we have to standardize the basmati rice for international trade as per the international standard including in india and pakisan. relatively traditional grown basmati rice are better in quality and aroma than basmati varieties developed by breeder and grown in high input conditions. tools and techniques now are available to check the adulteration of basmati that help protect the interest of consumers and farmers [18]. ready-to-use kit along with dna markers could be used for basmati authentication. an international code of practice has been developed for maintaining the reputation of basmati rice [18]. figure 2. historical events on intellectual property right (ipr) over basmati rice and its geographical indication (gi) cases in nepal historical events on ipr over basmati rice because of very highly recognized and preferred trait of basmati rice, different types of intellectual property rights are being tried on basmati rice around the world. for example, us-based ricetec company patented basmati rice in 1997 [24]. india registered basmati rice under the regime of geographical indication in 2010. in nepal ipr over agricultural products and technologies are very negligible. in case of basmati rice, nepal applied detail proofs and evidences of origin, diversity, cultivation and use values of basmati rice in eu in dec 2020. historical details of ipr over the basmati rice and gi cases are given in figure 2. evidences of origin, diversity, cultivation and use values of basmati rice basmati rice possess geo-linked trait i.e. quality and aroma. same genotype of basmati rice if grown in other than its original home localities, their quality differs and could not get same quality products as produced in their native localities. this property of basmati rice is then provided as gi tag. many different types of information and methodologies are needed to get gi tag [21]. evidences on aromatic rice in nepal are described below. 1997 detail evidences submitted to eu 2 sept 1997 nepal, member of wipo ricetech, us patented basmati rice june 2000 india opposed us patent on basmati rice 23 april 2004 2002 ricetech withdrew basmati rice patent nepal, member of trips/wto may 2010 july 2018 11 sept 2020 5 oct 2020 28 nov 2020 2 dec 2020 7 dec 2020 8 dec 2020 9 dec 2020 15 dec 2020 16 dec 2020 20 dec 2020 6 dec 2020 5 dec 2020 4 feb 2021 gi tag to basmati rice in india (7 states) by apeda india applied gi tag to basmati rice in eu notice on gi tag to basmati rice published in oj of the eu pakistan decided to oppose india claim over gi tag in eu gk kharel got informed notice of oj of the eu on gi tag to basmati rice notice of oj of the eu on gi tag to basmati rice published in dekhapadhi notice of oj of the eu on gi tag to basmati rice published in setopati basmati gi tag issue entered in di and moics after application notice by apaa nepal basmati gi tag issue entered in narc and moald proofs and evidences collected 1st high level meeting and decided to oppose oppose letter prepared and submitted and received acknowledgement from eu disseminated widely through media meeting with napa 2nd high level meeting 29 dec 2020panel discussion on basmati case, napa 1 jan 2021committee formed nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 96 linguistic and ancient literature evidences in literal meaning, basmati means aroma or scented [3,7,9,25]. it is made up of two sanskrit words, ‘vas’ means aroma and ‘mati’ means ingrained from the origin. in nepali language, the equivalent of vas is bas (aroma) and therefore, aroma related rice landraces and varieties are called basmati. almost all nepali understand the meaning and importance of basmati and basmati rice and therefore, this word basmati has become the very common nepali word. ancient literatures have mentioned basmati rice as an important food, nutritional and medicinal items [1,26–30]. chandranighantu (250 years old literature) has described sali dhan [29,31]. ayurved has grouped rice diversity in three categories and among them shukdhanya category includes 15 sali dhanya jaat including basmati type [29]. nepali literature of 1960 bs (1903 ad) reported 61 landraces including basmati type [27]. in lumbini, which is birth place of lord gautam buddha, there is aromatic rice landrace called kalanamak, which was used as holy grain during baudha period (900 bc). because of its religious important, community has started conservation works for kalanamak rice landrace (called bhaudhakalin kalanamak dhan) and reviving the culture [32,33]. the father of lord budhha is the king suddhodhan which mean pure rice with aroma. nepal has also registered aromatic rice by this name, suddhodhan kalanamak. kalanamak rice also contain high iron and zinc [34]. many researchers have documented and published about nepalese basmati rice landraces and many of these are available online [1,6,9,19,30,35–48]. folklore basmati is common word in nepal and has been used for giving name to ladies. other plant species having aroma are also named with this word e.g. basmati sponge gourd, basmati rayo, basmati banana, etc. we can see some stories, poems, songs associated with basmati in nepal. for example, there is deuda song in western nepal, “basmati ko dhan pakya garai basai gaya” which mean during maturity, basmati rice emits aroma and any one can feel its aroma around the field. similarly, fair dance song called hathhorha is held in baisakhi festival. there is a long song this fair dance and its part (“basmati ropanya syara o bamja jhuprai bamja”) also relate basmati rice [49]. traditions, specialty and reputation basmati rice is traditionally grown, sold, and consumed in nepal since ancient time [1,10–12,25,33–36,44,48,50–58]. historically, public mind always considers basmati rice as a special grain aromatic rice grown and produced in a particular geographical area. basmati rice landraces have geo-linked traits and they are grown in different parts of nepal [4–7]. it is highly valued and most important rice landraces fetching premium price in the market (8,9,19,21,25,36,38,38,41,50,57,59–62). basmati is a group of rice landraces used on special occasion [10,51,55]. farmers’ experiences indicated that organic basmati is far better than non-organic in quality and aroma. the aroma decreases when an aromatic rice is grown with chemical fertilizers. the application of home made compost (made up from native and local materials and livestock dungs) is a must if the real aromatic rice is to be produced. aroma is higher in recently harvested rice over old stock. rice dehulled with local dhiki is with higher aroma as compared to grain milled in a rice huller [19]. its straw is very soft and long therefore farmers prefer to make different home items, gundri, chakati, chataai, etc. from its straw. basmati rice straw is also used to make marriage-temple or house and this is very old tradition in tarai region. sociocultural, economical and market value basmati rice has social and cultural values in nepalese communities [10–12,14,33,52,55,57,58]. social status is very high for those family who consume and grow aromatic rice. common culture from ancient time is to offer basmati rice based food items to guest, relatives, vip, in festival, special function, marriage ceremony, etc. [57,60]. nepali community consider basmati rice grain as holy, pure, chokho, virgin and therefore used during fasting, offer to gods and goddess, and used in different religious ceremony (chhat, shraddha, etc.). it is also a component of axeta and vikxa. there are a lot of socio cultural evidences particularly in tarai area. it is used during ram janaki bibaha, in general marriage ceremony, kul deuta pooja, etc. some farmers use basmati rice grain in death ceremony and ritual shraddha. for this, they allocate separate land for continued growing of basmati rice and harvest from this land is used during ritual program. on the day of shraddha, their home use to be full of delicious rice smell. in many religious events, there is a function called homhalne where aromatic rice grains are used and one can feel smell of rice around during this function. basmati rice has a very high economic value [19,44,59,60]. the aromatic rice is very popular in both domestic and international markets and fetches premium price. gin and shahi [28] reported that nepal used to export about 200 metric tons of fine quality aromatic rice per annum earning about 4l million rupees in 1977. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 97 table 1. list of basmati landraces conserved in national genebank, nepal sn accession landrace collected site 1 ngrc01669 jhinuwa masino gorakhkali, gorkha 2 ngrc01698 basmati dhan kalsil, bajura 3 ngrc 01811 basmati dhan mundi, humla 4 ngrc 01815 basmati dhan tukche, mustang 5 ngrc 01825 basmati dhan makai, humla 6 ngrc 01835 sunaulo dhan badhu, bajura 7 ngrc 01867 masino basmati dhan dhading 8 ngrc 01945 basmati dhan lalitpur 9 ngrc 01967 kalanamak dhan khungai, rupandehi 10 ngrc 02022 hanse dhan dandagaon, salyan 11 ngrc 02030 basmati dhan dipayal, doti 12 ngrc 02036 sunaulo dhan martadi, bajura 13 ngrc 02066 hansaraj dhan madigaon, bajhang 14 ngrc 02093 hansaraj dhan manara, dadeldhura 15 ngrc 02094 basmati dhan manara, dadeldhura 16 ngrc 02103 sunaulo dhan bhandara, dadeldhura 17 ngrc02821 jhinuwa masino gorkha bazaar, gorkha 18 ngrc03016 kanakjira udayapur 19 ngrc03023 sunaulo ghaiya sanagaun -7, doti 20 ngrc03038 sunaulo ghaiya silgadhi-9, doti 21 ngrc03050 sunaulo ghaiya sallaghari-11, dadeldhura 22 ngrc03051 danda basmati dasharat chand-9, baitadi 23 ngrc03052 danda basmati dasharat chand-9, baitadi 24 ngrc03096 kalo masino taranagar (dado), gorkha 25 ngrc03249 jhinuwa basmati dhan raluka, nuwakot 26 ngrc03268 basmati dhan thumpakhar, sindhupalchok 27 ngrc03289 rato basmati dhan parsa 28 ngrc03291 rato basmati dhan bara 29 ngrc03293 basmati nokhi dhan bara 30 ngrc03326 kanakjira dhan sunsari 31 ngrc03364 basmati dhan chhinnamasta, saptari 32 ngrc03369 kalanamak dhan kapilvastu 33 ngrc03375 basmati dhan dhanusha 34 ngrc03389 basmati dhan beldari, kanchanpur 35 ngrc03415 basmati dhan phulkaha katti, siraha 36 ngrc04999 kalo masino gaikhur, gorkha 37 ngrc05007 hansaraj basmati dhan chaudhari-6, mauri bagar, bajhang 38 ngrc05017 hansaraj dhan banjh-8, bajhang 39 ngrc05018 shyamjiro banjh-8, bajhang 40 ngrc05691 shyam jira gadariya-1, kailali 41 ngrc07862 jarneli dhan barpak, gorkha 42 ngrc07869 begani ghaiya saurpani, gorkha 43 ngrc07889 masino basmati bichaur-4, lamjung 44 ngrc07900 mohanbhog patharaiya-9, kailali 45 ngrc07915 kalo jhinuwa ghanpokhara, lamjung 46 ngrc07923 lekali basmati ghanpokhara, lamjung 47 ngrc08267 seto basmati dhan shivasatasi municipality, jhapa 48 ngrc08273 kalo tuned basmati dhan shivasatasi municipality, jhapa 49 ngrc08276 chhoti basmati dhan shivasatasi municipality, jhapa 50 ngrc08277 rato basmati dhan shivasatasi municipality, jhapa 51 ngrc08278 hansaraj dhan shivasatasi municipality, jhapa 52 ngrc08289 kanakjira basmati dhan shivasatasi municipality, jhapa 53 ngrc08300 jorayal basmati dhan shivasatasi municipality, jhapa 54 ngrc08303 hansaraj dhan shivasatasi municipality, jhapa 55 ngrc08308 kalo basmati dhan shivasatasi municipality, jhapa 56 ngrc08372 kalo basmati dhan kawasoti n.pa.14, nawalpur 57 ngrc08442 hansaraj dhan satyawti gaupa, gulmi 58 ngrc08586 basmati dhan betali – 4, ramechhap 59 co 10271 hansaraj dhan satyawti gaupa, gulmi 60 co 10406 basnadaar lamda dhan sayaal -5, doti 61 co 10512 basmati dhan betali 4, ramechhap 62 co 10669 kalo tuned basmati shiwa satasi n. pa. 3, jhapa 63 co 10681 shyanmjira khjura ga. pa. 4, banke 64 co 10691 basmati dhan bhadrapur n. pa. 1, jhapa 65 co 10692 chulthe basmati bhadrapur n. pa. 1, jhapa nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 98 66 co 10751 basmati dhan bagmati 6, lalitpur 67 co 10764 aglo basmati dhan bagmati 7, lalitpur 68 co 10765 hocho basmati dhan bagmati 7, lalitpur 69 co 10915 basmati dhan gokulganga ga. pa. 4, ramechhap 70 co 11257 kalo basmati ganeshaman n. pa. 7, dhanusha 71 co 11258 sunaulo sugandha bhartapur mahangarpalika, chitwan 72 co 11381 gajiyabad basmati bharatpur sub mc 19, chitwan 73 co 11382 puspa basmati bharatpur sub mc 19, chitwan 74 co 11416 basnadar kalo bharatpur sub mc 19, chitwan 75 co 11427 thaniya basmati bharatpur sub mc 19, chitwan 76 co 11454 kalanamak bharatpur sub mc 19, chitwan 77 co 11462 basphool bharatpur sub mc 19, chitwan 78 co 11464 baspare bharatpur sub mc 19, chitwan 79 co 11468 basmati paschimko bharatpur sub mc 19, chitwan 80 co 11470 jhinuwa basmati bharatpur sub mc 19, chitwan note: there are other basmati (aromatic) type rice accessions in national genebank of nepal that need to further study and verification. many local rice millers (around 60 rice factories) are marketing basmati rice by different brand names at local and national levels. some of native aromatic landraces are as competitive as modern varieties [8,9]. many households prefer to grow economically valued traits ie aroma [63]. relatively quality of nepalese basmati rice is better than other countries. three landraces (basmati, rato basmati, and kalo nuniya) are very popular aromatic landraces in nepal and have a high market value in comparison with other varieties [64]. basmati comes under the group of five-qualities (pancha gudiya) product in nepal. these five qualities are purity, quality, tasty, healthy and nutritious. some of basmati landraces are medicinally important [29]. basmati rice landraces milled in local mill (dhiki) content low glycemic index and therefore are useful for diabetes patients. databases four basmati type of rice landraces have been improved and registered in national seed board of government of nepal. they are pokhreli jetho budho rice registered in 2006, lalka basmati registered in 2010, suddhodhan kalanamak and kalonuniya registered in 2020. these registrations have also been published in nepal gazette (nepal rajpatra) on different dates [3,65–67]. many community seed banks have maintained different types of basmati rice landraces in their localities [68–70]. national agriculture genetic resources center (national genebank) under nepal agricultural research council (narc, www.narc.gov.np) has collected and conserved more than 80 basmati type rice accessions from different areas of nepal (table 1). there are other landraces which are basmati type but recognized as different names. their examples include hansraj, jethobudho, jhinuwa, kalo masino, tilki, ghiu puri, begani, jarneli, gauriya, kalo nuniya, kala namak, kanak jira, kariya kamod, krishnabhog, sali dhan, shyamjira, etc [4–6,45,71]. international genebanks (https://www.genesyspgr.org/) has conserved more than 68 basmati rice accessions collected from different parts of nepal (table 2) and some of them were collected in early 1970s. some of basmati type accessions conserved in the international rice research institute (www.irri.org) genebank include asamiya basmati, basmati, basmati anpjhutte, basmati dhan, basmati gola, basmati lamo, basmati masino, basmati nokhi, basmati pahade, basmati red, basmati uzarka, basmati white, danda basmati, kalo basmati, masino basmati, rato basmati, sete basmati, seto basmati, etc. many of these accessions conserved in irri has already been shared with other countries for research and utilization [67]. table 2. nepali rice accessions named basmati available from genesys sn accession acquisition date local name 1 pi 549247 1984/12/13 basmati mutant 2 irgc 16213 1972/06/30 masino basmati 3 irgc 23861 1972/04/05 dhera dun basmati 4 irgc 23787 1972/04/05 basmati dhan 5 irgc 58881 1981/08/31 basmati lamo 6 irgc 58882 1981/08/31 basmati masino (purple tip) 7 irgc 58886 1981/08/31 basmati red 8 irgc 58884 1981/08/31 basmati nokhi 9 irgc 58883 1981/08/31 basmati masino 10 irgc 59054 1981/08/31 kalo basmati 11 irgc 58880 1981/08/31 basmati gola 12 irgc 58885 1981/08/31 basmati pahade 13 irgc 16136 1972/06/30 asamiya basmati 14 irgc 83309 1994/09/22 basmati 15 irgc 83679 1994/09/22 seto basmati 16 irgc 83317 1994/09/22 basmati mutant 17 irgc 83316 1994/09/22 basmati mixed 2 18 irgc 83650 1994/09/22 rato basmati 19 irgc 83310 1994/09/22 basmati dhan 20 irgc 83314 1994/09/22 basmati dhan http://www.narc.gov.np/ https://www.genesys-pgr.org/ https://www.genesys-pgr.org/ http://www.irri.org/ nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 99 21 irgc 83784 1994/09/22 basmati 22 irgc 86925 1996/10/24 basmati dhan 23 irgc 83313 1994/09/22 basmati dhan 24 irgc 83312 1994/09/22 basmati dhan 25 irgc 83661 1994/09/22 red basmati 26 irgc 83308 1994/09/22 basmati 27 irgc 83315 1994/09/22 basmati dhan 28 irgc 88761 1995/06/02 basmati mixed 1 29 irgc 83678 1994/09/22 seto basmati 30 irgc 83676 1994/09/22 sete basmati 31 irgc 83311 1994/09/22 basmati dhan 32 irgc 16130 1972/06/30 basmati 33 irgc 58887 1981/08/31 basmati white 34 irgc 58879 1981/08/31 basmati anpjhutte 35 irgc 59205 1981/08/31 rato basmati 36 irgc 58888 1981/08/31 basmati uzarka 37 irgc 110313 1996/10/24 danda basmati 38 irgc 62000 1982/04/20 masino basmati 39 irgc 117438 2008/12/15 basmati lamo::irgc 58881-1 40 irgc 127766 2011/05/01 rato basmati::irgc 59205-1 41 irgc 132324 2013/11/01 basmati red::irgc 588862 42 irgc 133780 2011/11/01 basmati dhan::irgc 83313-1 43 irgc 133781 2011/11/01 basmati dhan::irgc 83315-1 44 irgc 133779 2011/11/01 basmati dhan::irgc 23814-1 45 irgc 133785 2011/11/01 basmati gola::irgc 58880-1 46 irgc 133805 2011/11/01 basmati masino::irgc 58883-1 47 irgc 133810 2011/11/01 basmati mutant::irgc 83317-1 48 irgc 133824 2011/11/01 basmati white::irgc 58887-1 49 irgc 133809 2011/11/01 basmati mixed 1::irgc 88761-1 50 irgc 133806 2011/11/01 basmati masino (purple tip)::irgc 58882-1 51 irgc 133815 2011/11/01 basmati nokhi::irgc 58884-1 52 irgc 133962 2011/11/01 danda basmati::irgc 110313-1 53 irgc 134149 2011/11/01 kalo basmati::irgc 59054-1 54 irgc 134335 2011/11/01 rato basmati::irgc 83650-1 55 irgc 134357 2011/11/01 red basmati::irgc 83661-1 56 irgc 134410 2011/11/01 sete basmati::irgc 83676-1 57 irgc 134411 2011/11/01 seto basmati::irgc 83679-1 58 irgc 134645 2011/11/01 dhera dun basmati::irgc 23861-1 59 irgc 134791 2012/11/01 basmati dhan::irgc 83310-2 60 irgc 134792 2012/11/01 basmati dhan::irgc 83312-2 61 irgc 134836 2012/11/01 masino basmati::irgc 62000-2 62 irgc 134880 2011/11/01 basmati dhan::irgc 86925-1 63 irgc 134948 2013/11/01 basmati dhan::irgc 83311-2 64 irgc 135656 2011/11/01 basmati mixed 2::irgc 83316-1 65 irgc 135657 2011/11/01 basmati pahade::irgc 58885-1 66 irgc 135712 2014/05/01 basmati dhan::irgc 83314-2 67 irgc 136202 2015/05/01 basmati uzarka::irgc 58888-2 68 irgc 140367 2018/01/05 seto basmati::irgc 83678-1 note: there are many other basmati (aromatic) type rice accessions (with other than basmati name) collected from nepal in this genesys database. source: [67], https://www.genesyspgr.org/ center of diversity basmati rice has been originated in indian subcontinent [16–18,72–74]. the center of diversity of aromatic rice are the foothills of himalayas in the indian states of uttar pradesh (up) and bihar, and tarai region of nepal [17,38,54,75] and produced in geographically localized areas of nepal. the center of diversity and dispersal route are indicated in figure 3. international and national nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 100 scientists have defined lower altitude of nepal as one of the centers of origin of basmati rice [38,56,72,76,77]. the tarai belt of nepal was once considered as the bowel of aromatic rice landraces [38]. choi et al [76] reported three major geographically structured genetic groups of aromatic rice and they are bhutan and nepal which is admixture of cluster 2 and 3; bangladesh, india and myanmar which made distinct cluster, and iran and pakistan which also made distinct cluster. figure 3. center of diversity and dispersal route of aromatic rice in asia.source: [17] historical evidences and research ancient documents (ayurved, chandraniganthu), old scientific literatures [1,26,27,29,32,33] and culture of lord gautam bauddha [33] have mentioned different features and uses of aromatic rice. cultures and values associated with basmati rice have been passed from generation to generation. basmati rice is the most preferred by all nepali people. basmati rice was used by king family, very rich people, in special occasion, festival, very special function, etc. it is common culture in nepal to offer basmati rice to vip and guest. family having basmati rice also got respected by the communities. it has been used as indicator of rich people and neighbors easily know cooking variety of rice by smelling aroma. research on basmati rice started in 1951 with the collection of 930 rice germplasm (including aromatic landraces) from across 54 districts and their evaluation at parwanipur and khumaltar [1,26] in nepal. narc and other organizations have been working for developing aromatic rice varieties using local basmati rice landraces since early 1960s [8,9,40,67,78–82]. nepali students (bsc ag) in india have also experiences of taking basmati rice including wild rice from nepal to india with expecting good amount of money. they remembered; teacher taught about the importance of basmati rice and possibility of patenting this rice (prabeen dahal, 2020 personal communication). basmati rice diversity and production areas there are many different forms of basmati rice landraces in nepal, grown in different districts [1,4– 9,19,27,30,35,38,41,50,60,61,71,83–85]. we found total 133 aromatic rice landraces by name. among them, 43 landraces contain the words in association with basmati in their name (table 1 and 2) and 90 landraces were named by the word other than basmati (box 1). farmers in particular area may give their own name to aromatic rice landraces introduced from other areas. four aromatic landraces have been improved and registered in national seed board of government of nepal and two exotic aromatic varieties (sunaulo sugandha and sugandhit dhan-1) have been released for general cultivation. in irri genebank, there are about 86 landraces described by the name basmati irrespective of grain dimensions and intensity of aroma in irri [23]. maximum variation was observed in nepal, followed by india and bangladesh in aromatic germplasm [23]. very high diversity at both phenotypic and genotypic levels in basmati rice have been reported in nepal [6,7,38,45,71,78,86]. intra landrace diversity was also found commonly in many aromatic landraces [79,80]. these aromatic landraces possess very different traits and based on 12 bases, types of aromatic rice landraces and varieties along with meaning and examples are given in table 3. some are with awn and some are awnless with red, white and black grain. based on grain size, there are three types of basmati rice, namely short, medium and long grain basmati rice [30,70,72,87]. aromatic rice does not exist for deep water condition in nepal. rahmani and box 1. list of basmati (aromatic) type landraces (not named by the word basmati) in nepal (total= 90) aachame masino, anadi basnadar, anjana, bagane, bagari, bahani, baharni, barambhusi, basnadaar lamda dhan, basnadar kalo, baspare, basphool, batisara, bayarni, begani, belguthi, biramphool, chengul, chiniyapuri, chirankhe, dudhe marsi, gauria (gaure), ghaiya rato, ghyu puri, ghyu kumari, gude kalo, gude seto, gudgudo, gudura, gurdi kalo, gurdi seto, hansaraj, hanse dhan, hapsa, hapsa rato, indrabeli, jaran dhan (kalo), jarneli, jaswa, jethobudho, jhinuwa, jhinuwa ghaiya, jirasari, jogini, kalanamak, kalo bayarni, kalo jhinuwa, kalo masino, kalo nuniya, kalo nuniya thulo, kalo jhinuwa, kanak jira, kariya kamod, kasturi, khairo anadi, khalte kholo, koili, krishna bhog, krishna charcha, lajee, lalbachchhi, madhukar, mahabhog, mahajogani, malbhog, masino jhinuwa, motisor, pahenle, pakhe jhinuwa, pakhe tunde, pokhreli masino, pran peuri, rahumanuwa, rajbhog, ram tulsi, ramjoin, sali dhan, samundraphinj, seto bayarni, seto jhinuwa, shyamjira, sisuwapanheli, sunaulo dhan, sunaulo ghaiya, suwawat, thapachini, tilki, tulsi prasad, tulsiphool, tunde. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 101 harinkher can also grow in shade area. similarly koili is shade loving aromatic landrace. pranpyuri is very soft basmati rice landrace. table 3. grouping of nepalese rice landraces based on different criteria sn basis type meaning example 1 planting season chaite aromatic dhan spring rice, transplanting in chaitra tauli bhadayia aromatic dhan early type bhadaiya basmati barkhe or agahani aromatic dhan normal rice basmati, kasturi, jhinuwa, jaswa, chananchur , ujrka basmati, lalka basmati, tulsi prasad, gopalbhog hiunde aromatic dhan winter or boro rice pakhe masino 2 maturity early aromatic rice early maturity bhadaiya basmati, gyu puri medium aromatic rice medium maturity thapachini, anadi basnadar late aromatic rice late maturity gurdi, koili, basmati, kasturi, jhinuwa, jaswa, chananchur , ujrka basmati, lalka basmati, tulsi prasad, gopalbhog, tilki 3 grain size short grain aromatic rice small size grain with aroma jethobudho, panhele, motisar medium grain aromatic rice medium size grain with aroma mahajogani long grain aromatic rice long size grain with aroma anadi basnadhar 4 ecosystem (production environment) rainfed upland aromatic rice unbunded condition suwawat, begani ghaiya rainfed lowland aromatic rice bunded condition hansaraj, sali dhan irrigated aromatic rice bunded condition tilki, kalo masino 6 cultivation introduce d aromatic from abroad sunaulo sugandha, sugandhit dhan-1 improved aromatic developed by breeder lalka basmati, sudodhan kalanamak landrace aromatic maintained, developed by farmers kariya kamod, krishna bhog 7 morphotype tall aromatic rice tall height kalo masino, anadi medium aromatic rice medium height hamsaraj, thapachini ya dwarf aromatic rice short height tulsi kathey 8 grain color white aromatic rice grain with white husk tauli, hansaraj black aromatic rice grain with black husk shyam jira, kalanamak, kalo nuniya red aromatic rice grain with red husk begani ghaiya, sali dhan 9 photoperiod response aus (saro, gaddar or ghaiya) aromatic rice mature within a certain period, photoperiod non sensitive ghiu puri, begani ghaiya nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 102 aman (agahani or sarihan) aromatic rice mature at particular time, photo period sensitive kalanamak, tilki, gauriya, ujaraka basmati, tulsi prasad, kanakjira 10 awn awnless aromatic rice grain without awn pokhreli masino short awn aromatic rice grain with very short awn rato basmati, lalka basmati long awn aromatic rice grain with long awn seto basmati, hansaraj 11 aroma complete (universal ) aromatic rice emits aroma in all stages (field, harvesting, storage, milling, cooking and eating) hansaraj, sali dhan, kalo masino, jhinuwa, jethobudho, kalanamak, kalo nuniya partial aromatic rice emits aroma in only few stages (cooking and eating) lalka basmati, jorayal basmati, anadi, chananchur , jaswa, gauriya, ujarka basmati 12 name basmati named rice name has at least basmati word basmati, ujarka basmati non basmati aromatic rice name has other than basmati word kasturi, jhinuwa source: [1,7,19,22,36,50,52,57,61,70,86,88,89] source: [1,2,6,7,14,19,20,30,35,36,41,47,48, 50–53, 57,61,65,68,70,78, 81–83, 85–91] aromatic rice is produced in many asian countries [18]. aromatic rice landraces mainly basmati, kalanamak, kariyakamod, kalonuniya, hansraj, jethobudho, jhinuwa, syamjira, tilki, etc. are being grown in more than 30,000 hectares of 41 districts in nepal [4–6,57,65]. aromatic rice is cultivated from east mechi to west mahakali. there are many ghaiya aromatic landraces as well in doti and achham districts. aromatic rice gauri, paranpyuri, kalo gude and jhinuwa are still cultivated in small areas in surkhet valley. survey and literatures have shown that aromatic rices are grown in 60 districts out of 77 in nepal (figure 4). the tarai belt was once considered as the bowel of aromatic rice landraces [38]. altitudinal distribution of aromatic rice landraces is given in table 4. aromatic rices are grown from 60 to 1800 m altitude in nepal. figure 4. districts (marked by star sign) showing cultivation of native basmati (aromatic) type rice landraces in nepal source: [1,6,7,14,19,20,51,52,65,69,70,78] agro-morphological and nutritional based evidences collection, characterization and evaluation of native aromatic rice landraces have been started since 1951 in nepal [1,26]. a large number of landraces and introduced aromatic rice varieties were characterized and evaluated both at on-farm and on-station by many nepalese researchers using agro-morphological traits [1,7,9,21,28,30,39,43–45,51–53,58,70,71,78–80,82,84,86– 88,92]. cooking and eating qualities are the main important features in basmati rice. these qualities are associated with different factors. nutritional analyses have been done in some of native and improved basmati type rice varieties and landraces [21,22,48]. the protein content was maximum in red basmati (7.74%) and minimum in black basmati (6.51%) among the four basmati varieties [22]. these nutritional profiles indicates that there is variation within and among different types of aromatic rice landraces grown in nepal. isozymes and dna based evidences many nepalese aromatic rice landraces have been explored through isozymes and dna markers [18,38,45,76–78,92–94]. dna profile and fingerprints of some aromatic rice landraces using ssr markers are given in figure 5. national and foreign scientists have used nepalese aromatic rice genotypes for genetics study, molecular breeding and diversity study, and reported variation within and among landraces collected from different parts of nepal. bardiya bajhang darchula mustang dolpa mugu humla achham kailali doti baitadi bajura jumla jajarkot dailekh dang salyan surkhet banke myagdi rolpa rukum kalikot tanahu lamjung kaski manang gulmi palpa siraha m a h o tt a ri bara parsa chitwan gorkha ltp nuwakot rasuwa dolakha dhankuta taplejung ilam jhapa m o ra n g saptari b ktm sindhuli kavre k h o ta n g china india n syangja aromatic rice growing district, total: 52 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 103 table 4. distribution of basmati type rice landraces in nepal sn rice cultivar districts altitude, m achhame masino chitwan, jhapa, makawanpur, morang 200-800 asamiya basmati morang <600 badiya basmati bara, rautahat, parsa <600 bagari chitawan, siraha 60-300 baharni bara, parsa, saptari, siraha <500 basmati bara, bajura, dadeldhura, darchula, dhanusha, doti, humla, jhapa, kapilvastu, kanchanpur, kathmandu, lalitpur, mahottari, morang, parsa, pyuthan, ramechhap rautahat, rupandehi, sarlahi, siraha, sindhupalchok, taplejung, udayapur 200-1000 basmati anadi bara <300 basmati anpjhutte dolakha <800 basmati nokhi bara <300 belguthi jhapa, morang, sunsari, sankhuwasabha, panchthar, ilam, jhapa, terhathum <800 biramphool dhading, jhapa, kathmandu, kaski, lamjung, morang, parbat, siraha, sunsari, udayapur 400-800 charan basmati bajura 1000 chengul bara, parsa, sunsari <500 chhoti basmati jhapa, morang, sunsari <300 chirankhe bhojpur, dhankuta, illam, okhaldhunga, panchthar, terhathum <1800 chulthe jhapa, sunsari 60-300 danda basmati dadeldhura 1530 deradun basmati bake 300 gauria arghakhanchi, baglung, kapilvastu, lamjung, myagdi, nawalparasi, ramechhap, rupandehi, sankhuwasabha, sunsari, terhathum 300-1400 ghyu kumari bara, parsa, sarlahi, sindhuli <500 gola basmati sunsari <500 gude (seto, kalo) dailekh <1100 gudgudo gulmi <1100 hansraj bajhang, baitadi, darchula, dadeldhura, jhapa, kanchanpur, morang, palpa, pyuthan, salyan, sunsari, surkhet, syangja 60-1100 hapsa jhapa <300 hapsa rato jhapa 60-300 indrabeli dhading, dhankuta, gorkha, lamjung 800-1400 jaran dhan (kalo) arghakhanchi, bajhang, dang, gulmi, jajarkot, kaski, parbat, rukum, salyan and surkhet 800-1400 jaswa dhanusha, mahottari, morang, rautahat, saptari, siraha, sunsari 60-300 jethobudho kaski, myagdi, parbat, sunsari, syagnja, tanahun 600-1250 jhinuwa baglung, doti, gorkha, kailali, kanchanpur, kaski, kathmandu, lamjung, myagdi, nuwakot, parbat, shankhuwasaba, sindhupalchok, sunsari, syangja, tanahun 300-1300 jirasari jhapa, morang, panchthar, ramechhap, sunsari <600 jogini chitwan, ramechhap 500 jorayal basmati doti <800 jorpal basmati jhapa, morang, sunsari <1200 kalo basmati dhankuta, jhapa, kathmandu, morang, sunsari <1200 kalo jhuse basmati jhapa, morang, sunsari <300 kalo nuniya jhapa, morang, sunsari 60-300 kalo nuniya thulo jhapa, morang, sunsari 60-300 kalo/kala nimak bardiya, chitwan, nawalparasi, rupandehi 100-400 kalotunde basmati jhapa, morang, sunsari <300 kanak jira bara, bardiya, chitawan, jhapa, kailali, kanchanpur, kapilbastu, morang, salyan, sunsari, syanja <600 kariya kamod dhanusa, morang, saptari, siraha 200-400 kasturi bara, kailali, parsa 500-1400 krishna bhog achham, dhankuta, kanchanpur, ramechhap <1400 krishna charcha bajura <1400 lalbachchhi jhapa, morang, sunsari 60-300 lalka basmati bara, dhanusha, parsa, rautahat 60-300 lanjhi bara, parsa <500 lekali basmati lamjung 1500 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 104 mahabhog kailali, dhading, rasuwa, bara, parsa 200-600 mahajogini bara, parsa <300 masino basmati dhading, khotang <900 motisar bara, parsa <300 pahade basmati ilam <1000 pahenle bajhang, bardiya, gorkha, ilam, kaski, lamjung, myagdi, palpa, parbat, sinduplanchok, syanja 600-800 pokhreli masino solukhumbu, sankhuwasabha 600-800 pran peuri sallyan, surkhet 1200-1400 rajbhog dhading, kailali, kanchanpur <600 ram tulsi panchthar, terhathum 800-1100 ramjawain bara, parsa <600 rato basmati jhapa, morang, sunsari 60-300 rato basmati sano bara, jhapa, mahottari, morang, parsa, siraha, sunsari <300 ratotunde basmati jhapa, morang, sunsari <300 sali dhan baitadi, dadeldhura, gorkha <1200 samundrabakhi phim dhading, nuwakot <600 samundraphinj dhading, kaski, makawanpur, nuwakot 200-600 seto basmati bara, jhapa, morang, parsa, sunsari 60-300 shyamjira banke, doti, jhapa, kailali, kanchanpur, morang, sunsari 60-300 thapachini achham, bajhang, bajura, dadeldhura, kailali, lamjung, terhathum 2001400 tulsi prasad nawalparasi, parsa, dhanusa 200-1400 tulsiphool dhanusha, jhapa, mahottari, morang, saptari, sindhuli, siraha, sunsari, udayapur 60-300 ujarka basmati bara, parsa, rautahat 60-300 source: [2,4–6,14,19,63,68,90,91] figure 5. dna profile of aromatic and non aromatic rice landraces (upper figure) and dna fingerprint (down figure) of basmati type rice landraces using ssr markers. source: [78] gaps and policy implication research and mechanism for implication of gi is urgently needed in nepal as this sector is neglected and underutilized. research, development and education system should focus on native crop diversity and traditional knowledge along with traditional products and process. documentation of different kinds of information e.g. traditional, folklore, scientific information etc. should be done for all types of native nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):93-108 joshi et al. ©njb, bsn 105 agricultural genetic resources. accelerated research is necessary to identify the geo-linked traits and products, and geographical indicator of genetic resources. simple mechanism should be in place to register native agricultural products as gi at provincial and national levels. conclusion there are strong proofs and evidences of the origin, diversity, cultivation and use values of basmati rice in nepal based on survey, on-farm and on-station trials, lab research, and information from local, regional, national and global levels. basmati type rice possesses quality trait for getting geographical indication tag and it includes all types of aromatic landraces i.e. short, medium and long grain basmati rice in nepal. nepalese farming communities in many districts are maintaining, growing, using, marketing and sharing basmati type rice landraces since unknown time period. the rights of nepalese communities, therefore, should not be prohibited for production, consumption and marketing of their basmati rice landraces. legal system for gi tag should be immediately established in nepal and agricultural products must be registered. harmonized system (hs) on trade should also be established separately for aromatic rice in nepal. competing interests no competing interests. funding nepal agricultural research council funded this research. ethical approval and consent not applicable references 1. mallick rn. rice in nepal. kathmandu: kala 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p=0.121). the positive predictive value (ppv) and negative predictive value (npv) of mtb/rif were 96.85% (93.61, 98.72) and 94.95 % (93.52, 96.14), respectively. hiv co-infection did not impact sensitivity, specificity or liquid culture time to positivity (ttp). when mtb/rif accuracy was evaluated using composite reference standard culture positivity from sputum, the sensitivity and specificity were similar to those obtained in the primary analysis using either definite tb versus possible and non-tb combined; definite and possible tb combined versus non-tb. keywords: mycobacterium tuberculosis, prevalence, genexpert, nepal *corresponding authors e-mail: dr_roshankurmi@hotmail.com introduction tuberculosis (tb) is a leading cause of death worldwide. in the early 1990s, the government of nepal brought up a program to control tb at national scale called dots (directly observed treatment short course) [1]. dots program was initiated in april 2001 with a goal to diagnose and cure tb patients which has treated more than 20 thousand patients till date [2]. until december of 2014 there are 554 microscopy centres and 22 genxpert location in the country which specializes in diagnosis of tb [3]. the expansion of dots program has proven its efficacy in reducing the mortality and morbidity in nepal, however, despite of available diagnosis and treatment regimes, approximately 3000-5000 people are still dying per year of tb [4]. in a dots centre in kathmandu, prevalence of multi drug resistant (mdr) case of tuberculosis was found to be 3.6% [5], which could have been mitigated by early diagnosis and rapid identification of anti-tb agents resistance. recent data from east nepal suggests that 3.3% of all patients, who were smear microscopy negative, have rmp resistant tb [6]. despite of dots and available diagnosis, complications due to mdr are emerging and also there hasn’t been overall increment in number of people on anti tuberculosis treatment [6]. sputum smear light microscopy, patients symptoms combined with cxr results is the preferred algorithm for tb diagnosis in nepal. smear test is neither relevant nor sensitive enough for the diagnosis of tb alone and needs additional diagnosis as well clinical suspecting to decide either patients should be enrolled on antibiotic or not [7]. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 26-32 kurmi et al.. ©njb, biotechnology society of nepal 27 nepjol.info/index.php/njb this study aims to clarify if current algorithm of tb diagnosis is flexible enough to trace the tb rates at genexpert centre in parsa district of public health office, nepal. our aims were to determine epidemiological characteristics, hiv status of patients with tuberculosis and comparative study of different test routinely used for diagnosis of tb at a large outpatient clinic in parsa of nepal. materials and methods study sites and population study was conducted in parsa district in the narayani zone of southern nepal which lies 283 km (176 mi) south of the capital kathmandu, 3 km north of the border of the indian state of bihar with population 315,011 in 2011. the district is divided into 86 village development committees and one municipality which is one of the relatively well developed districts in the country located on the flat terai lowland. the public health sector has 49 facilities including 1 zonal hospital, 1 public health office, 3 primary health centres, 9 health posts and 35 sub-health posts, additionally there are 6 private sector facilities thus resulting in a total of 55 facilities providing dots services in parsa district. the reported notification rate of new pulmonary positive tuberculosis in parsa district was 69 per 100,000 populations in 2014 [3]. patient classification and diagnosis algorithm patients with suspected tb (≥18 ye of age) were enrolled between april 2014 and may 2015. who algorithm was basis for the classification of suspects of tb. based on who algorithm, two subgroups were formed to classify tb suspects; 1) patients with cough for ≥ 2 weeks and 2) patients without cough for ≥ 2 weeks but having symptoms like weight loss accompanied with night fever t>37.5°c, pleural effusion or pericarditis induced breathlessness, chronic headache, swelled armpit (>2cm) or abnormal chest radiogram, thus referred as, “with cough or without cough’’. patients with who defined danger sign with respiratory rate >30/minute, fever >39°c, pulse rate >120/minute and unambulatory patients were excluded from the study. smear positive were not enrolled for genxpert thus were directly enrolled for anti tb regime. remaining patients with smear negative were enrolled in genxpert and further confirmation was done by liquid culture for mtb positive and rif resistant samples. those cultures were further screened for mdr against first line and second line drugs. patients with sputum negative for tb were classified as possible tb (if they are on anti-tb treatment program) or as non-tb (if they are not on anti-tb treatment program). prospective patients (≥20 years of age) with suspected pulmonary tb (n=2091) were enrolled in the study who were attending the dots centre in parsa district of public health office, nepal from jul 2013 to jan 2015. patients had at least one symptom of tb including chest radiograph with infiltrates and smear microscopy negative results in duplicate. specimen collection and laboratory procedures sputum specimens were processed using standardized protocols. the length of time between sample collection and results being issued to the clinic was also recorded. smears stained with zn stain methods were examined microscopically following the standard protocol. at least 200 fields were evaluated before reporting negative. bacillary density was graded as scanty, 1+, 2+, and 3+, and all such smears were defined as ‘‘smear-positive’. following decontamination with n-acetyl-lcysteine and sodium hydroxide, centrifuged sputum deposits underwent microscopy, and following re-suspension in phosphate buffer, equal volumes were tested by the genexpert mtb/rif assay. the results of all tests were read by technologists blinded to the outcomes of the other assays. sputum pellets were also tested by trained technologists using the genexpert mtb/rif assay. genexpert mtb/rif and patient management the collected sputum sample was aliquoted in two equal volumes; first half for immediate primary clinical works and other for reference for future works at -20°c. from the first aliquot, the specimen was used for smear tests followed by genexpert mtb/rif assay. the diagnostic assays were followed by a single specialized clinical research associate blind to the patient's diagnosis in http://en.wikipedia.org/wiki/narayani_zone http://en.wikipedia.org/wiki/nepal http://en.wikipedia.org/wiki/kathmandu http://en.wikipedia.org/wiki/india http://en.wikipedia.org/wiki/states_and_territories_of_india http://en.wikipedia.org/wiki/bihar nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 26-32 kurmi et al.. ©njb, biotechnology society of nepal 28 nepjol.info/index.php/njb reference standard and index test, throughout the study period. statistical analysis collected data were analyzed and interpreted statistically using graphpad prism version 6.0 and spss 17.0. all the values are expressed as mean±sd and are analyzed using student’s t test which is parametric as well mann-whitney test wherever applicable. p value (p<0.05), was considered significant unless stated otherwise. results study population and tb diagnoses figure 1: district wise number of tuberculosis patients visited at genexpert centre in parsa the suspected pulmonary tb patients (n= 2091) were enrolled for sputum microscopy and genexpert mtb/rif for the confirmation of tb. among them, 1301(62.21%) were male and 790 (37.78%) were female. all the patients were referred to this centre by local doctors and physicians after having tuberculosis symptoms and abnormal chest radiograph for sputum microscopy and genexpert mtb/rif for the confirmation of makwanpur, mahottari, panchthar, saptari, siraha, morang, sindhuli, rasuwa and chitwan (figure 1). since the study conducted at parsa district, the actual number of case presented here does not highlight the prevalence of tb in the respective district. as bara is the adjacent district to parsa, the number of patients visited is more than other district. the prevalence of tb in parsa district is 3 times higher than the prevalence in the bara district. tb. the clinical symptoms showed most of the patients have fever, cough, and weight loss and night sweat (table 4). the maximum tb cases were from parsa district (555, 26.5%) followed by bara, rautahat, sarlahi, diagnostic accuracy of different method and mtb/rif performed on uncentrifuged sputum direct sputum microscopy is popular worldwide. in nepal, the tuberculosis diagnosis and monitoring rely in sputum microscopy because of its low cost and easier to perform. at present there are 533 microscopy centres catering the sputum microscopy examination throughout the country. the region wise distribution is given in table 1. most of the microscopy centres are established in government setting and few are established in nongovernmental organization and private sectors. ntc-national figure 2: mdr pattern in tb patients figure 3: gene xpert mtb rif pattern in tb patient 0 200 400 600 800 1000 1200 1400 1357 476 11890 17 12 6 4 2 1 1 1 1 5 0 500 1000 1500 2000 2500 s-ve s+ve not done total table 4: clinical feature of tuberculosis clinical feature present absent fever 1885 206 cough 1741 350 weight loss 1471 620 night sweat 1655 436 loss of appetite 1376 715 malaise 1117 974 chest pain 1744 347 0 500 1000 1500 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 26-32 kurmi et al.. ©njb, biotechnology society of nepal 29 nepjol.info/index.php/njb reference laboratory and genetup are providing culture and in the beginning of 2012, a new molecular diagnostic tool; gene xpert mtb/rif machines for rapid diagnosis of dr tb had been introduced to nine health facilities (7 in eastern region and 2 in central region) in collaboration with ntp nepal and international organization for migration (iom) through tb reach funding. gene xpert mtb rif pattern in tb patient and mdr pattern in tb patients are shown in figure 2 and figure 3 respectively. table 1: number of dots and microscopy centres by region development region/ centres edr cdr wd r mwd r fw dr total no. of treatment centres 237 366 239 199 143 1184 no. of subcentres 709 1008 667 406 284 3074 dr tb treatment centres 2 4 3 1 3 13 dr tb treatment sub-centres 11 37 15 5 3 71 microscopy centres 122 198 82 73 79 554 * showed the number of dots and microscopy centres in which number of sub-centres are more followed by number of treatment centres and microscopy centres. there is few dr tb treatment centres. (edr: eastern development region. cdr: central development region; wdr: western development region; mwdr: mid western development region; fwdr: far western development region) table 3: comparison of different test for diagnosis of pulmonary tuberculosis test result smear positive 585 smear negative 1506 gene xpert positive 788 gene xpert negative 1303 the comparative study of different diagnostic tools is given in table 3. of the 2091 patients enrolled in this study, the sensitivity of mtb/rif was 95.50% (91.87, 97.82) and significantly higher than smear microscopy performed on the same fluid, which had a sensitivity of 61.97% (55.41, 68.21, table 5). five of 127 smear-negative cases had mtb/rif-positive un-centrifuged sputum, resulting in a specificity of 81.23% (75.95, 85.78), which was similar to smear microscopy 98.29 % (97.34, 98.97; p=0.121). the positive predictive value (ppv) and negative predictive value (npv) of mtb/rif were 96.85% (93.61, 98.72) and 94.95 % (93.52, 96.14), respectively. hiv co-infection did not impact sensitivity, specificity (table 6) or ttp. when mtb/rif accuracy was evaluated using composite reference standard culture positivity from sputum, the sensitivity and specificity were similar to those obtained in the primary analysis using either definite tb versus possible and non-tb combined; definite and possible tb combined versus non-tb. discussion in nepal, the estimated incidence of tb is 163 per 100,000 with a prevalence rate of 241 per 100,000 populations. in 2014-15, the nepal tuberculosis programme (ntp) registered 17,788 sputum smearpositive cases and 8,367 sputum smear negative cases [6, 8]. a total of 2091 individuals from thirteen districts of nepal were included in the present study and the prevalence of smear positive pulmonary tuberculosis was found to be 788 out of 2091(37.06 %). we found the most of the patients were passively participated in the study and almost all patients are visited government hospital for the treatment. approximately 66.76% of the patient had the cough duration for last 2 days. although, only 37.09 % of patients had afb positive on smear, out of which 97.8 % were new table 5: pattern of sputum culture with active tb parameters cohort (n=2091) sputum smear positive with active tb (n=672) sputum smear negative compatible with active tb (n=1419) median age in years 37.3 (26.148.3) 33.8 (28.142.8) 37.4 (29.9-47.1) male 1301 464 725 female 790 208 694 hiv infected 65 22 43 median cd4 counts (if hiv positive) 98 78 20 previous tb treatment 89 9 80 smokers (past or current) 201 118 83 nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 26-32 kurmi et al.. ©njb, biotechnology society of nepal 30 nepjol.info/index.php/njb cases and only 2.2. % is repeated cases; the chest x ray reveals 99.43% had abnormal finding in chest. the genexpert result shows large number of patients diagnosed with tuberculosis. this might be that the afb is not present in initial few days because most of the patient has visited less than 5 days of appearance of cough. the similar study carried by shagufta iram et al[9], shows the genexpert mtb/rif is a sensitive method for rapid diagnosis of tuberculosis, especially in smear negative cases and in ptb as compared to the conventional zn staining. for countries endemic for tb genexpert can serve as a sensitive and time saving diagnostic modality for pulmonary tb. our study is consistent with other studies which have suggested the benefit of genexpert in smearnegative patients in developing countries [10, 11].the majority of these studies have been carried out in africa where there is a substantially higher hiv burden than in south asia. three studies have been reported from low-hiv prevalence regions, including peru and two hospitals (hinduja hospital and christian medical college hospital) in india [11, 12]. it is not clear how many of the patients testing positive for mtb by smear, culture and gene xpert in this study would have been started on tb treatment based on the judgement of the treating clinician if genexpert testing was not available. in the previous year (2013-2014) at parsa district, 2245 patients were treated for tb, approximately onethird of those detected by genexpert during the study period. it is possible that the availability of genexpert increased the recognition of tb symptoms, although we can claim that in this endemic setting tb awareness is consistently high among clinicians. conversely, it is possible that a negative smear test may have discouraged table 6: accuracy of xpert mtb/rif for the detection of smear positive tb in sputum all patients n=2091 hiv uninfected n=2026 hiv infected n=65 sensitivity, % (95% ci) specificity, % (95% ci) sensitivity, % (95% ci) specificity, % (95% ci) sensitivity, % (95% ci) specificity, % (95% ci) smear microscopy 61.97% (55.41,68.21) 96.48 % (95.24, 97.48) 78.38% (71.74, 84.08) 92.50 % (90.85,93.93) 62.36% (51.93,64.82) 95.23 % (93.84, 97.41) mtb/rif performed on uncentrifuged sputum 95.50% (91.87, 97.82) 94.69 % (93.23, 95.92) 77.66% (72.24, 82.46) 99.09 % (98.33, 99.56) 94.13% (90.16, 95.28) 92.47 % (90.67, 94.54) mtb/rif performed on uncentrifuged sputum in patients who were smear negative 81.23% (75.95, 85.78) 98.29 % (97.34, 98.97) 91.77% (87.45, 94.98) 95.70 % (94.36, 96.80) 83.82% (76.72, 86.26) 94.78 % (91.23, 97.76) mtb/rif performed on a resuspended pellet of centrifuged sputum 51.89% (44.94,58.78) 93.79 % (92.24, 95.11) 60.44% (52.94, 67.60) 91.42 % (89.68, 92.95) 52.62% (46.73, 56.28) 91.72% (89.91, 93.63) ppv† (95% ci) npv† (95% ci) ppv† (95% ci) npv† (95% ci) ppv† (95% ci) npv† (95% ci) smear microscopy 47.44% (40.61,54.34) 92.73 % (91.08, 94.16) 54.84 % (47.39, 62.13) 90.46 % (88.65, 92.08) 85.95% (80.92, 90.07) 98.76 % (97.93, 99.32) mtb/rif performed on uncentrifuged sputum 96.85% (93.61,98.72) 94.95 % (93.52, 96.14) 78.75% (73.42, 83.45) 99.36 % (98.69,99.74) 93.69% (89.65, 96.51) 97.04 % (95.89,97.94) mtb/rif performed on uncentrifuged sputum in patients who were smear negative 62.61% (56.12,68.77) 97.09 % (95.93, 97.99) 81.87% (75.49, 87.18) 92.51 % (90.87, 93.95) 51.89% (44.94, 58.78) 94.48 % (93.00, 95.72) mtb/rif performed on a resuspended pellet of centrifuged sputum 47.44% (40.61,54.34) 92.73% (91.08, 94.16) 54.84% (47.39, 62.13) 90.46 % (88.65, 92.08) 90.13% (87.25,92.47) 52.43% (49.10, 53.91) nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 26-32 kurmi et al.. ©njb, biotechnology society of nepal 31 nepjol.info/index.php/njb treatment in some cases which would otherwise have been treated, although clinicians were informed that a negative smear test does not exclude a tb diagnosis. patients with a strong possibility of pulmonary tb despite a negative smear test were referred for further investigations including culture at a tertiary centre. the rate of smear positivity at parsa is 15.0 % which is consistent with the results in other areas of similar endemicity; it is therefore unlikely that the genexpert diagnosis was inflated due to low smear microscopy confirmation [13-15]. if we look on test selection, smear microscopy, due to its simplicity, speed, and low cost, is used widely in low-resource settings; the low sensitivity precludes it from being an ideal test. the requirement for a rapid, simple tb diagnostic is evidenced by the widespread application of commercial serological tests which are inaccurate. these tests are widely provided at a cost to the patient and used to determine medical treatment. although the genexpert has several limitations, including requirement for stable electricity supply, limited temperature range, availability of maintenance, and bulky consumables, wider availability of the accurate genexpert assay may counter the use of these serological tests by providing a viable alternative to the patient and healthcare provider. it is highly probable that a small number of cases of tb were missed in this study as genexpert does not have as high sensitivity as culture. there is a danger that clinicians will “exclude” a tb diagnosis on the basis of a negative genexpert test and it is important that education is carried out to ensure clinicians are aware of the test limitations prior to the test being implemented. however, it is not sustainable to implement tb culture facilities at general hospitals in south asia and the long turnaround of results means loss to follow-up in the diagnostic pathway is high. this study was not an assessment of genexpert sensitivity and specificity, as this has been comprehensively evaluated in comparison with culture by others. conclusion the prevalence of tb is high in parsa dots centre in comparable to other districts of nepal. however, the relatively higher rate of rif resistance observed in our study signals the danger of increasing mdrtb in the study areas in the future in nepal. on the other hand, mtb rif positivity among sputum negative, rif resistant (dr tb) among sputum negative and rif resistant among retreatment cases is large in this study. the higher level of mdr-tb among previously treated patients suggests the need to strengthen the dots strategy and the capacity of laboratories to undertake effective treatment especially among previously treated patients in nepal. the genexpet is more specific and sensitive that smear afb detection. the government of nepal ministry of health and population should implement genexpert in each dots centre in nepal for better diagnosis. abbreviations cxr: chest x-ray; dots: directly observed treatment short course; dst: drug susceptibility testing; hiv: human immunodeficiency virus; mdr: multidrug-resistance; ntp: national tuberculosis program; ppv: positive predictive value; npv: negative predictive value. authors’ contributions rk, bpg was involved in the study conception and design, data analysis and drafting of the manuscript. aa, rr and kdm were involved in the design and reviewing of the manuscript. bpg was involved in reviewing of the manuscript. all authors have read and approved the final version of the manuscript. conflict of interests the authors declare no competing interests regarding the publication of this paper. acknowledgment authors acknowledge the role of the staff of dots centre, parsa during the conduct of this study for their invaluable help during the identification of cases and collection of data. references 1. his majesty’s government of nepal ministry of health and the world health organization. tuberculosis control in nepal 1995–1999. a development plan for the national tuberculosis programme. kathmandu, nepal, 1995. 2. national tuberculosis programme. tuberculosis control in nepal. status report. thimi, nepal: national tuberculosis centre. 1997. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 26-32 kurmi et al.. ©njb, biotechnology society of nepal 32 nepjol.info/index.php/njb 3. goverment of nepal. national tuberculosis center 2014. http://www.nepalntp.gov.np/ index.php?view=publication 4. . 5. bhatt cp, bhatt ab, shrestha b: drug resistant cases of tuberculosis in directly observed treatment short course. journal of nepal health research council 2010, 8(1):44-47. 6. creswell j, rai b, wali r, sudrungrot s, adhikari lm, pant r, pyakurel s, uranw d, codlin aj: introducing new tuberculosis diagnostics: the impact of xpert((r)) mtb/rif testing on case notifications in nepal. the international journal of tuberculosis and lung disease : the official journal of the international union against tuberculosis and lung disease 2015, 19(5):545-551. 7. desikan p: sputum smear microscopy in tuberculosis: is it still relevant? the indian journal of medical research 2013, 137(3):442-444. 8. banjara mr, kroeger a, huda mm, kumar v, gurung ck, das ml, rijal s, das p, mondal d: feasibility of a combined camp approach for vector control together with active case detection of visceral leishmaniasis, post kala-azar dermal leishmaniasis, tuberculosis, leprosy and malaria in bangladesh, india and nepal: an exploratory study. transactions of the royal society of tropical medicine and hygiene 2015, 109(6):408-415. 9. iram s, zeenat a, hussain s, wasim yusuf n, aslam m: rapid diagnosis of tuberculosis using xpert mtb/rif assay report from a developing country. pakistan journal of medical sciences 2015, 31(1):105-110. 10. rachow a, zumla a, heinrich n, rojas-ponce g, mtafya b, reither k, ntinginya en, o'grady j, huggett j, dheda k et al: rapid and accurate detection of mycobacterium tuberculosis in sputum samples by cepheid xpert mtb/rif assay--a clinical validation study. plos one 2011, 6(6):e20458. 11. boehme cc, nicol mp, nabeta p, michael js, gotuzzo e, tahirli r, gler mt, blakemore r, worodria w, gray c et al: feasibility, diagnostic accuracy, and effectiveness of decentralised use of the xpert mtb/rif test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study. lancet 2011, 377(9776):1495-1505. 12. vassall a, van kampen s, sohn h, michael js, john kr, den boon s, davis jl, whitelaw a, nicol mp, gler mt et al: rapid diagnosis of tuberculosis with the xpert mtb/rif assay in high burden countries: a cost-effectiveness analysis. plos medicine 2011, 8(11):e1001120. 13. hamid s, hussain sa, ali i: comparative analysis of case screening with varying cough duration and sputum samples for diagnosis of tuberculosis in patients attending the opd at a tertiary care hospital at srinagar, india. nigerian journal of clinical practice 2012, 15(4):430-435. 14. malhotra s, zodpey sp, chandra s, vashist rp, satyanaryana s, zachariah r, harries ad: should sputum smear examination be carried out at the end of the intensive phase and end of treatment in sputum smear negative pulmonary tb patients? plos one 2012, 7(11):e49238. 15. hooja s, pal n, malhotra b, goyal s, kumar v, vyas l: comparison of ziehl neelsen & auramine o staining methods on direct and concentrated smears in clinical specimens. the indian journal of tuberculosis 2011, 58(2):72-76. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:35-39 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 35 nepjol.info/index.php/njb marker assisted screening of nepalese rice for bacterial leaf blight (blb) resistance resham babu amgai1*, raj kumar niroula1, sumitra pantha2, shreya singh hamal1, bishal gole tamang4, bindeshwar prasad sah1, madan raj bhatta3 1biotechnology division, nepal agriculture research council, khumaltar, lalitpur, nepal 2agriculture botany division, nepal agriculture research council, khumaltar, lalitpur, nepal 3national plant genetic resource centre, nepal agriculture research council, khumaltar, lalitpur, nepal 4institute of agriculture and animal science, tribhuwan university, rampur, chitwan, nepal abstract bacterial leaf blight (blb) is the most important yield limiting factor in nepalese rice. blb resistance rice varieties are highly demanding in the country. breeding efforts for developing disease resistant depends on availability and use of resistant gene donors. nepalese rice landraces could be the source of resistant gene. therefore, ninety six nepalese rice accessions were screened using eight simple sequence repeats (ssr) markers and one sequence tagged sites (sts) marker for presence and absence of blb resistance gene. we have detected blb resistance gene xa-10 on five accessions, xa-13 on six accessions, xa-7 on 23 accessions, xa-3 and xa-4 on 52 accessions, xa-5 on 25 accessions, xa-8 on 30 rice accessions. no any rice accessions tested have xa-21. similarly, 17 rice accessions showed three and more than three blb resistance genes. presence of xa-13 on susceptible check variety cntrl-85033 confirmed that this resistant gene is not working in nepalese rice field. therefore, nepal need to pyramide the blb resistant genes for durable resistance. keywords: bacterial leaf blight, simple sequence repeats, resistant gene, nepalese rice, marker assisted screening *correspondence author email: reshamamgain@yahoo.com introduction bacterial leaf blight (blb) is one of the productions limiting biotic stresses in rice. it is caused by xanthomonas oryzae pv. oryzae (xoo). it can reduce the yield up to 50% [1] and in nepal; it reduced the yield from 5-60 % in terai and mid-hills during hot and humid periods [2]. twenty four genes conferring resistance to blb have been identified through classical genetic analysis [3] and 10 out of 24 genes have been mapped using restriction fragment length polymorphism (rflp), rapid amplified polymorphic dna (rapd) and microsatellite markers [4-7]. among them, 6 genes are recessive in nature [1]. similarly, two new genes xa-22 in rice variety zha-chang-long [8] and xa-23 in oryza rufipogon [9] were identified and mapped on different chromosomes. most common blb resistant gene used in rice breeding and blb screening worldwide are xa1, xa-2, xa-3, xa-4, xa-5, xa-7, xa-8, xa-10, xa-11, xa13, xa-14 and xa-21 [10]. blb resistance rice varieties are highly demanded worldwide. however, the continuous evolution of pathogenic races leading to the breakdown of resistance in many improved varieties [9]. thus, success of resistance breeding program depends on the availability of the resistant donors. similarly, pyramiding different resistant genes in a single rice variety will increase the resistance. however, two or more resistance gene pyramiding in a single variety is easy through molecular marker assisted selection (mas), and identification for the presence and absence of particular gene in a variety for mas as donor and recipient parent through molecular marker assisted screening is very fast, reliable and cheaper. therefore, this study was carried out to identify the accessions within nepalese gene pool with the potential of blb resistance genes. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:35-39 amgai et al. ©njb, biotechnology society of nepal 36 nepjol.info/index.php/njb methods germplasm collection seventy nepalese rice accessions (npgr no.s) collected from terai region of nepal were obtained from national plant genetic resources centre (npgrc); and five breeding lines (nr series) and released rice varieties (chandannath-1, chandannath3, chhomrong, macchapuchre-3, manjushree-2, palung-2 and taichung-176) from agriculture botany division of nepal agricultural research council (narc). similarly, rice varieties ir-64, sabitri and masuli, and breeding line cntrl-85033; terai rice landraces bagari, bhatti, karma, lal tenger and parewa pankha; and hill rice landraces belkuti, gerneli, jumli marshi and seto anadi were collected from different parts of nepal (table 1). molecular marker and check variety eight ssr markers and one sts marker (pta248) for the presence and absence of blb resistance gene. molecular markers are selected based on their linkage with particular blb resistance gene (table 2). ir-64 and cntrl-85033 were used as resistant and susceptible check respectively. dna extraction, pcr reaction and data analysis genomic dna of rice accessions was prepared using modified ctab method as described by sul and korban [11]. each pcr reaction was conducted with100ng of genomic dna, 1 µm of each primer and 7.5 µl of 2x gotaqgreen pcr master mix (promega corporation, madison, wi, usa). pcr mixture was amplified in mj research ptc-100tm programmable thermal controller (mj research, inc, watertown, ma, usa) with the following temperature regimes: initial denaturation for 2 min at 95oc followed by 33 cycles of 95oc for 30 sec, annealing as per primer for 1 min, extension at 72oc for 2 min and final extension at 72oc for 7 min followed by holding at 4oc as described on table 2 and gramene [12]. amplified pcr products were separated in 2% analytical grade agarose gel at 100v for 1h. gels were stained with 0.1 µg/ml ethidium bromide (promega corporation, madison, wi, usa) and then visualized under uv transilluminator gel documentation system (wilber lourmat, marne-la-valleen, france) using 1 µg guide size dna ladder (genetix, biotech asia pvt. ltd.). the presence and absence of particular band size was scored for screening disease resistance genes. results and discussion different resistance genes were identified in nepalese rice germplasm as defined by different molecular markers (table 3). we identified blb resistance gene xa-10 on two accessions, xa-13 on six accessions, xa-7 on 23 accessions, xa-10 on five accessions, xa-3 and xa-4 on 52 rice accessions (figure 2), xa-5 on 25 accessions, xa-8 on 30 rice accessions. no any rice accessions have xa-21 (figure 1). similarly, 17 rice accessions showed three and more than three blb resistance genes (table 4). nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:35-39 amgai et al. ©njb, biotechnology society of nepal 37 nepjol.info/index.php/njb nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:35-39 amgai et al. ©njb, biotechnology society of nepal 38 nepjol.info/index.php/njb nepalese rice accessions lack the xa-21 genes which is more important for controlling blb epidemics throughout the world. kameshwara rao [16] reviewed and noted that xa-21 gene is transferred from o. longistaminata and integrated into some irri developed rice varieties. lacks of such varieties in our study may be the result for this. similarly, our susceptible check ‘cntrl-85033’ showed the presence of xa-13 but amgai [17] reported that it was heavily infected by blb at nepalese rice field. this may be due to the difference on blb isolated found in nepal which may break the resistance reaction developed by xa-13 gene. resistant check ir-64 showed the presence of xa-5 indicating that it is effective for nepalese blb pathogen. conclusion nepalese rice landraces contains many marker alleles for different blb resistant genes. the rice landraces with effective resistant gene can be used as donor parent for mas. however, for the enhancement of resistance in nepalese rice with absence and/or ineffective resistance gene can be done by transferring broad spectrum resistance gene like xa-21. blb resistance is also affected by multiple resistance genes and their interaction [17]. therefore, pyramiding blb resistance gene on nepalese rice variety is most important for durable resistance with blb. acknowledgement this work was conducted under global biodiversity trust grant no. gs10027. references 1. rao kk, lakshminarasu m, jena kk: dna markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice. biotech. adv. 2002, 20: 33– 47. 2. burlakoti rr, khatri-chhetri gb: bacterial diseases of crop plants in nepal-a review. j. inst. agric. anim. sci., 2005 25:1-10. 3. lee ks, angeles er, khush gs: inheritance of resistance to race 6 of bacterial blight in rice. rice genet news letter 2001, 17:73–4. 4. mccouch, sr, tanksley sd: genetic and physical analysis of the rice bacterial blight disease resistance locus xa 21. mol. gene. genet. 1992, 236: 113–120. 5. ronald pc, albano b, tabien r, abenes l, wu k, mccouch sr, tanksley sd: genetic and physical analysis of the bacterial blight disease resistance locus, xa-21. mol. gen. genet. 1992, 236:113–120. 6. borines lm, veracruz cm, redona ed, hernandez jf, natural mp, raymundo ad and leung h: marker-aided pyramiding of bacterial blight resistance genes in maintainer lines for hybrid rice production. irri conference abstract 2000, 4(2):162. 7. lang nt, buu bc: molecular genetic analysis and marker assisted selection for restorer line and bacterial blight resistance in hybrid rice. sabrao 2004, 36 (2): 83-93. 8. gao hp, lin xh, yu gx, zhang dp, xie yf: inheritance of resistance to bacterial blight of four yunan rice varieties. rice genet newsl. 1999, 12: 233–234. 9. phuc, nv, lang nt, buu bc: sts and microsatellite marker-assisted selection for bacterial blight resistance in rice oryza sativa l. omonrice 2005 13: 18-25. 10. irri, 2012. disease resistance bb blast ver 5.0. host plant resistance to diseases group (hpr-d). plant breeding, genetics and biotechnology division (pbgb). international rice research institute (irri), the philippines. 2012 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:35-39 amgai et al. ©njb, biotechnology society of nepal 39 nepjol.info/index.php/njb 11. sul iw, korban ss: a highly efficient method for isolating genomic dna from plant tissues. plant tiss. cult. biotech. 1996, 2: 113-116. 12. gramene. 2015. http://www.gramene.org/ (accessed 30 april 2015). 13. davierwala ap, reddy apk, lagu md, ranjekar pk, gupta vs: marker assisted selection of bacterial blight resistance genes in rice. biochemical genetics 2001, 39: 261278. 14. pha, nt, lang nt: marker assisted selection in rice breeding for bacterial leaf blight. omonrice 2004 12: 19-26. 15. blair mw, mccouch sr: microsatellite and sequencetagged site markers diagnostic for the rice bacterial leaf blight resistance gene xa-5. theoretical applied genetics 1997, 95:174-184. 16. kameshwara rao, k, lakshminarasu m, jena kk: dna markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice. biotechnology advances 2002, 20: 33-47. 17. amgai rb, parajuli gp, khan ah, poudel b, gm pb, gc cb, bhattarai em, pandey yr: eco-friendly seed treatment procedures in rice identification and dissemination for western hill of nepal. in identification and dissemination of eco-friendly seed treatment procedures in rice. final technical report for nardf pp 408/2006/07. kaski: nepal agriculture research council regional agricultural research station lumle 2009: 2-11. 18. zhou y, cao y, huang y, xie w, xu c, li x, wang s: multiple gene loci affecting genetic backgroundcontrolled disease resistance conferred by r gene xa3/xa26 in rice. theoretical applied genetics 2009 < doi 10.1007/s00122-009-1164-5> . nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:48-54 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 48 nepjol.info/index.php/njb phytochemical screening, antimicrobial activity and cytotoxicity of nepalese medicinal plants swertia chirayita and dendrobium amoenum pritish shrestha, manisha bista, prativa sharma, shristi shrestha, basanta lamichhane, sandeep adhikari, binayak raj pandey, bhupal govinda shrestha* department of biotechnology, school of science kathmandu university, dhulikhel, nepal abstract research on medicinal plants are important to nepal because most of its rural population relies on it as mode of medicine. medicinal plants namely swertia chirayita and dendrobium amoenum were collected from mid hills of nepal. the present study was undertaken to find the antimicrobial activity, phytochemical presence and their cytotoxicity in different extraction medium. the percentage yield from the plants were highest in warm methanol extraction with 12.6%, followed by ethyl acetate and lowest was for cold methanol. plant extract showed the presence of antioxidants like alkaloid, terpenoids, flavonoids, tannin, glycosides. the brine shrimp bioassay of methanol and ethyl acetate extract showed cytotoxicity. chiraito extract showed lc50 of 199 ppm for dhunche sample, 128.82 ppm for daman sample and 131.82 ppm of illam sample. the antibacterial activity of methanol extract of chiraito and dendrobium amoenum showed significant bioactivity by inhibiting growth of microbial species selected for the test. the zone of inhibition shown by the extracts was comparable to the standard antibiotics. similarly, methanol extract of chiraito also showed significant antifungal activity with the zone of inhibition comparable to amphotericin. keywords: antioxidant, lc50 (lethal concentration-50), nauplii, zoi (zone of inhibition) *corresponding author email: bgs@ku.edu.np introduction for centuries, medicinal plants are being used as ayurveda or traditional medicine in nepal by local tribes and also in most of the rural areas of the indian subcontinent. medicinal plants and ayurveda practice in the sub continent are intertwined. different parts of plant are used in different herbal medicine as one of the constituents of final composition. some phytochemicals present in these medicinal plants, have medicinal values and are expected to yield positive biological activities. nepal, in the middle of the himalayan belt which extends from myanmar in the east to karakorum in the west, possess vast diversity of plants because of its geographical distribution [1,2,3,4]. some of these medicinal plants are used in indigenous rural remedies, homoeopathic medicines, and allopathic pharmacopeia [5,6,7,8]. the exploitation of locally available medicinal plants in health care and economic advancement is a necessity of nepal. among all those plants swertia chirayita and dendrobium amoenum is the focus of this research. among 100 species of swertia genus, 27 species are found in nepal. among them around nine species are reported to be traded for medicinal purpose. swertia chirayita is biennial erect herb which is approximately 50 to 125 cm tall. the plant is a native of temperate himalayas, found at an altitude of 1200–3000 m. its stem is robust, branched and cylindrical below, four angled upward and containing large pith, broadly lanceolate leaves with 5-nerve and sub-sessile. it has lurid greenish yellow flowers tinged with purple in large panicles, with egg-shaped capsules and minute seed which are smooth and many angled [13]. chiraito grows mainly in temperate himalayas and is reported from 40 districts of nepal. chiraito grows between open forest and margin of cultivated land, it is predominant in dolakha and spreads mainly in altitude of 1500 m to 3000 m of eastern and central region of nepal [2,9]. dendrobium contains about 1,200 species and is also known as orchid [10]. this genus also occurs in diverse habitats throughout much of south and east asia, stretching to oceania and some of pacific islands. this plant is commonly distributed between 660 m to 2000 m temperate forest of himalayan region and grow in little light exposure [11,12]. the orchids are mostly found in terrestrial, epiphytic and nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 48-54 shrestha et al. ©njb, biotechnology society of nepal 49 nepjol.info/index.php/njb saprophytic habitat. dendrobium amoenum grows in clustered pendulous five to six slender stems. morphologically, this species is highly evolved, tall, straight with elongated pseudobulbs covered by modest sized leaves. it has unique floral pattern which is fairly constant varying in size from very small to large, the shape and form of the stems and leaves are divergent. this traditional medicinal species is also ornamental plant and have been cultivated for decorative purpose. the flowers flourish on an older stem in cluster of two to three per node and the flower is amusingly perfumed [13,14,15]. materials and methods sample preparation swertia chirayita plants were collected from or near from dhunche, daman and illam and dendrobium amoenum from pokhara, during their flowering season. the plants were air dried under room temperature. the dried plant samples were cut and grinded to make it in powder form and kept for storage at room temperature. extraction three types of extraction method were carried out, viz. warm methanol extraction, cold methanol extraction and ethyl acetate extraction. for warm extraction, soxhlet apparatus was used. 10 g of the crushed sample along with 200 ml of methanol was put into the soxhlet apparatus. the soxhlet was run for 28 hours at 65⁰c. the methanol extract was taken out from the soxhlet apparatus. the pigment was removed using hexane in the separating funnel. the methanol fraction was then dried using water bath. for cold extraction methanol at room temperature was used. 100 g of powdered form of sample was taken and 450 ml methanol, that was just enough to cover the upper layer of sample, was poured on it and was shaken regularly. after 48 hrs in room temperature, filtration was done and the filtrate was stored at room temperature. for ethyl acetate extraction, 10 g of powdered material was dissolved in 25 ml ammonium hydroxide. 300 ml of ethyl acetate was then added and left for 72 hrs at room temperature. extract was then filtered. ethyl acetate was then left to dry in water bath [16,17,18]. phytochemical screening was done to check the presence of alkaloid, sterols, triterpenes, tannins and polyphenols, reducing sugar, saponins, flavonoids, glycosides and coumarin according to protocol described in [19,20,21]. brine shrimp bioassay preparation of test sample stock solution was prepared by dissolving 100 mg of the extract in little amount of dimethyl sulfoxide (dmso) for initial solublization and then addition of water to final volume of 25 ml to make a stock of concentration of 4000 ppm. the stock solution was further diluted to 1000, 100, 10 ppm concentration. for hatching of brine shrimp, 50 mg of brine shrimp eggs were sprinkled in a beaker with 300 ml of sea water. the transferred sample as then allowed incubating at 32 – 35oc for 24 hrs. bioassay cleaned test tubes were divided into four groups each group consisting of five test tubes. after 24 hrs of incubation, the nauplii were recovered with a pipette and 10 nauplii were transferred in each test tube. the groups were then treated with different dilutions of sample. the test tubes were then incubated at 32– 35oc overnight. the incubated tubes were observed for the number of survived nauplii and graph was plotted for death percentage versus log of concentration of the extract. this gives linear equation in the form of y = mx + c. calculation of lc50 the death of nauplii was calculated as death percentage as, % death = deaths/initial x 100. the % death was corrected for any control deaths by subtracting the %death control from % death test. the lethal concentration 50 (ld50) was derived from the graph from the equation of the straight line [19]. antimicrobial screening micro-organisms used six clinical samples of bacteria were collected from kathmandu university teaching hospital (kuth), dhulikhel namely staphylococcus aureus, escherichia coli, kleibsella pneumonia, salmonella paratyphi, salmonella typhi, pseudomonas aerugenosa. the yeast saccharomyces cerevisiae (ycs2) were taken from microbiology lab of department of biotechnology, kathmandu university. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 48-54 shrestha et al. ©njb, biotechnology society of nepal 50 nepjol.info/index.php/njb preparation of sample the different extraction of plant was dissolved in dmso and water, which was made into different concentrations. inoculation sterilized petri dishes were filled to a uniform depth, with respective sterilized medium (nutrient agar for bacteria and muller hinton agar for fungus respectively). the solidified media was then inoculated with respective organisms. holes were cut into the medium using a sterile borer. solution of various concentrations of the extracts under test was poured into the bored holes with the help of a micropipette and the standard antibiotic discs or solutions were gently placed at the different sites of the petri-dish containing the inoculated medium in the case of cup plate method. in case of filter disc method, filter paper discs prepared from watman’s filter paper were soaked with the test solution and the filter discs are then gently placed into the inoculated medium with the help of a sterile forceps. the plates were then maintained at room temperature for few minutes to allow the antibiotic and the extract solution to diffuse into the medium. in the uniform media, diffusion of the extract solution and the antibiotic discs will occur uniformly around the cup and the concentration gradient will be established around it and at a certain distance from the cup. the plates were then incubated at a suitable temperature (usually 37-39⁰c) for 24-48 hours. after 48 hours, the zones of inhibition were measured with the help of a measuring ruler or antibiotic zone reader [22]. result the percentage yield for a given plant is calculated as: figure 1: yield percentage of different plant from various solvents. [a] representsyield percentage from cold extract (methanol), [b] represents yield percentage from cold extract (ethyl acetate) and [c] represents yield percentage from warm extract (methanol). the various phytochemicals tested for chiraito and dendrobium amoenum extracts with their corresponding results can be tabulated in table 1 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 48-54 shrestha et al. ©njb, biotechnology society of nepal 51 nepjol.info/index.php/njb figure 2: zone of inhibition (zoi) of different plant samples from cold methanol extract of 2.5 % and 7.5 % against different bacteria. a, b, c, d, e and f represents zoi of different plant extract against s. paratyphi, e. coli, k. pneumoniae, s. typhi, p. aerugenosa and s. aureus respectively. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 48-54 shrestha et al. ©njb, biotechnology society of nepal 52 nepjol.info/index.php/njb figure 3: zone of inhibition (zoi) of different chiraito samples. a represents zoi of chiraito (dhunche) prepared from ethyl acetate against 5 microbes and b represents zoi of chiraito (daman) against saccharomyces cerevisiae. zoi (in mm) of plant extract against following fungus and comparison with fungicide solution figure 3 brine shrimp bioassay figure 4: brine shrimp assay graph for chirato sample of different places graph a= dunche, graph b= illam and graph c=daman figure 5: brine shrimp assay for different chirato samples in different concentration calculation: graph was plotted as death percentage versus log of extract concentration in ppm. this shows the linear equation in the form of y=mx+c. by substituting the value of y=50, the corresponding value of x gives the log value of lc50 value and antilog of that value gives the lc50 value in ppm. from fig 4, graph 1, equation: y=22x -1.33 for lc50, y=50, then x=2.3, antilog of x is antilog(2.3) = 199ppm therefore, lc50 of chirato (dhunche) methanol extract for brine shrimp is 199ppm. from fig 4, graph 2, from equation: y= 26.5x-6 for lc50, y=50, then x=2.11, antilog of x is antilog(2.11) = 128.82ppm therefore, lc500 of chirato (daman) methanol extract for brine shrimp is 128.82 ppm. from fig 4, graph 3, from equation: y= 20x + 7.6 for lc50, y=50, then x=2.12, antilog of x is antilog (2.12) = 131.82ppm therefore, lc50 of chiraito (illam) methanol extract for brine shrimp is 131.82ppm. 5 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 48-54 shrestha et al. ©njb, biotechnology society of nepal 53 nepjol.info/index.php/njb discussion the % yield from the chiraito extract was highest in warm methanol extraction with 12.6% as compared to cold methanol extraction and that for ethyl acetate was 9% as shown in figure 1. however in the case of dendrobium amoneum the % yield was higher for cold extraction at 4.4 % as compared to warm extraction at 3.4 %.this indicates that there are many compounds present in the plants soluble in methanol and ethyl acetate and that there is higher amount of bioactive component in chiraito compared to dendrobium amoneum. generally all alkaloids are highly soluble in methanol. high extract yield for methanol also suggests that there are high amount of alkaloids in the plant. the phytochemical screening of the plant extract showed the presence of alkaloid, terpenoids, tannins, coumarins, flavanoids and sterols, as shown in table 1, indicating that there are high value natural compounds in the plants. since there are presence of alkaloids, terpenoids, coumarins, flavanoids and sterols these plants could have anti cancer activity. the antibacterial activity of methanol and ethyl acetate extract of chiraito and dendrobium amoenum showed bioactivity by inhibiting growth of microbial species selected for the test as shown in figure 2 and 3. the zone of inhibition shown by the extracts was comparable to the standard antibiotics. cold methanol extract of chiraito didn’t show any activity against e coli. cold methanol extract of chiraito from dhunche had activity higher than that of the control antibiotic tetracycline. bioactivity of ethyl acetate extract was not significant as that of methanol extract and showed activity against only pseudomonas auregenosa. this may be due to absence of alkaloids. cold methanol extract of chiraito (daman) showed significant activity against saccharomyces cerevisiae. the zone of inhibition was significantly higher than that of control, amphotericin at 25µg/ml. the brine shrimp bioassay of methanol extract showed cytotoxic nature of plant extracts. if lc50 value of test sample is less than 1000 ppm the extract is considered to be biologically active. chirato extract showed lc50 of 199 ppm for dhunche sample, 128.82 ppm for daman sample and 131.82 ppm for illam sample. since cytotoxicity value given by brine shrimp assay directly correlates with its cytotoxicity ability, toxicity at low concentration of chiraito extracts can have toxicity against cancer cell lines and has the potential to be developed as drugs for cancer. conclusion: researchers have identified compounds used in mainstream medicine derived from plant resources. similarly, this research has assessed the phytochemical, biological screening of chiraito and dendrobium amoenum. this natural product can bring new and effective antimicrobial agents and serve as alternate source of combating infections in human beings. hence this research can have a promising potential in various traditional, complementary and alternate systems of treatment of human diseases. further work on isolation and characterization of these plants and their pharmacodynamics study can be a great contribution. the research also proved to be beneficial in exploiting medicinal plants having biological activities namely cancer. references 1. radcliffe-smith a, hara h, steam wt, williams lhj: an enumeration of the flowering plants of nepal. kew bulletin 1979, 34(1):198. 2. joshi k: swertia l. (gentianaceae) in nepal: ethnobotany and agenda for sustainable management. ethnobotanical leaflets 2008, 12:1-6. 3. cseke jl, kirakosyan a, kaufman pb, warber sl, duke ja, brielmann hl: natural products from plants. in 2nd edition. crc press taylor and francis group, 2006. 4. siddiqui s, verma a, rather aa, jabeen f, meghvansi mk: preliminary phytochemicals analysis of some important medicinal and aromatic plants. advances in biological research 2009, 3(5-6):188-195. 5. joshi b, lekhak s, sharma a: antibacterial property of different medicinal plants: ocimum sanctum, cinnamomum zelanicum, xanthoxylum armatum and origanum majorana. kathmandu university journal of science, engineering and technology 2009, 5:1-6. 6. bhattarai kr, ghimire m: commercially important medicinal and aromatic plants of nepal and their distribution pattern and conservation measure along the elevation gradient of the himalayas. banko jankari 2007, 16(1):1-6. 7. okamura t, ayajiki k, fujioka h, toda m, fujimiya m, nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 48-54 shrestha et al. ©njb, biotechnology society of nepal 54 nepjol.info/index.php/njb toda n: effects of endothelial impairment by saponin on the responses to vasodilators and nitrergic nerve stimulation in isolated canine corpus cavernosum. british journal of pharmacology 1999, 127(3):802-808. 8. sharma m, chapagain n, sah s: phytochemicals as emerging antimicrobials. plant resources (a scientific publication) 2010, 32:96-101. 9. joshi p, dhawan v: swertia chirayita – an overview, current science, 2005, 89(04):635-640 10. venkateswarlu s, mudunuri ssr, gottumukkala vs: synthesis and biological activity of isoamoenylin, a metabolite of dendrobium amoenum. bioscience, biotechnology and biochemistry 2002, 66(10):2236-2238. 11. rajkarnikar k; in vitro propagation of dendrobium amoenum wall. ex lindi. from seed culture. plant resources (a scientific publication) 2010, 32:90-92. 12. shrestha p: antibacterial activity of medicinal plant extracts. plant resources (a scientific publication) 2010, 32:51-54. 13. martha r, gutierrez p: orchids: a review of uses in traditional medicine, its phytochemistry and pharmacology. journal of medicinal plants research 2015, 4(8):592-638. 14. zhang cf, wang m, wang l, iinuma m, zhang m, xu ls, wang zt: chemical constituents of dendrobium gratiosissimum and their cytotoxic activities. indian journal of chemistry 2008, 47b:952-956. 15. buckingham j: dictionary of natural products. cdrom 13:1. boca raton, florida: chapman & hall/crc press, 2005. 16. bhandari j, naqvi a, niraula p, thapa p, thapa n, shrestha n and shrestha bg: phytochemical screening, antioxidant assay of junipers recurva and study of its invitro cytotoxicity against breast cancer cell lines. international journal of pharma and bio sciences. 2015, 6(3b):1134-1145. 17. lamichhane b, adhikari s, shrestha p, shrestha bg: study of phytochemical, antioxidant, antimicrobial and anticancer activity of berberis aristata. journal of tropical life science. 2014, 4(1): 1-7. 18. shrestha p, adhikari s, lamichhane b, shrestha bg: phytochemical screening of the medicinal plants of nepal. iosr journal of environmental science, toxicology and food technology. special issue 2015, 1(6):11-17. 19. chhetri hp, yogol ns, sherchan j, anupa kc, mansoor s, thapa p: phytochemical and antimicrobial evaluations of some medicinal plants of nepal. kathmandu university journal of science engineering and technology. 2008, 1:49-54. 20. setzer wn, flair mn, byler kg, huang j, thompson ma, setzer af, moriarity dm, lawton ro and windham-carswell.: antimicrobial and cytotoxic activity of crude extracts of araliaceae from monteverde. costa rica. brenesia 1992, 38:123-130. 21. zabri h, kodjo c, anoubilé b, békro m, bekro y a: phytochemical screening and determination of flavonoids in secamone afzelii (asclepiadaceae) extracts. african journal of pure and applied chemistry 2008, 2:80-82. 22. reuben kd, abdulrahman fi, akan jc and egwu go: phytochemical screening and in vitro antimicrobial investigation of the methanolic extract of croton zambesicus muell arg. stem bark. european journal of scientific research 2008, 23:134-140. nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 33-38 research article doi: https://www.doi.org/10.54796/njb.v9i2.41913 ©njb, bsn 33 simple method devised for rapid isolation and identification of vibrio cholerae from water resources of sunsari district, nepal bijay kumar shrestha and jenish shakya department of microbiology, central campus of technology, tribhuvan university, hattisar, dharan, nepal. received: 16th nov 2020; revised: 24th jun 2021; accepted: 26th dec 2021; published online: 31st dec 2021 abstract cholera is a gastrointestinal disease caused by pathogenic strain of vibrio cholerae, the disease clinically manifested by ricewater diarrhea, nausea and vomiting. this study aimed to study the incidence of vibrio species and employ simple method for rapid detection of vibrio cholerae from water samples of sunsari, nepal. identification of v. cholerae through biochemical tests requires extensive labor and costs. in resource limited laboratories, isolation and identification of v. cholerae often becomes difficult. therefore, this study also aimed for selecting scope of this methodology as a scientific outcome for rapid isolation and identification of vibrio cholerae. a total of 100 water samples were collected from sunsari district in which 25 samples were collected from sewage, 25 from pond, 25 from tap and 25 from tube well. the samples of collected water were sent to the microbiology laboratory of central campus of technology maintained in ice cold box and were enriched in alkaline peptone water and selectively isolated from tcbs agar and na agar without nacl. pathogens were isolated and identified by conventional microbiological techniques. out of 100 water samples collected, sucrose fermenting vibrio species were isolated only from 16 water samples. further the selective isolation of v. cholerae from nutrient agar without nacl isolated 6 isolates from sewage samples and 3 isolates from pond samples. the distribution of vibrio cholera in the water sample was found to be 9%, distribution of v. alginolyticus was found to be 4% and distribution of v. fluvialis was found to be 3%. in this study, non-sucrose fermenting vibrio species were not isolated from the water samples. however, sucrose fermenting vibrio species was obtained with yellow pigmentation in tcbs agar medium. the yellow pigmented colonies of vibrio isolates recovered from tcbs and even from nutrient agar devoid of sodium chloride provided sufficient evidence of v. cholerae after series of other biochemical tests. this study concludes that yellow colonies (sucrose-fermenting) of vibrio from tcbs agar medium that can grow on nutrient agar without added nacl and which exhibit a positive oxidase reaction can be confidently identified as presumptive v. cholerae. in resource-constrained environments, this simple method can reduce the labor cost, chemicals and time-consuming procedure of performing multiple biochemical and molecular assays for identification. keywords: cholera, lab diagnosis, sunsari, tcbs, vibrio cholerae, water corresponding author, email: interfacebj@gmail.com introduction vibrio is a gram-negative rod-shaped bacterium that is an inhabitant of water sources. the species distribution of this bacterium is dependent on salt concentration and temperature of water [1]. among several species, v. cholerae , v. parahaemolyticus and v. vulnificus are known to be pathogenic. strains of v. cholerae of serogroups o1 and o139 are known to be associated with epidemic potential whereas non-o1 and non-o139 strains are identified with mild diarrheal cases [2]. cholerae symptoms include abrupt onset of watery diarrhea (rice watery stool), nausea, vomiting, and intestinal disorders with abdominal pain [3]. cholera is clinically manifested by rapid loss of water that accounts for dehydration, polydipsia, hollow tear troughs, decreased blood pressure, weak pulse, renal failure, seizures, coma, and death [4]. v. cholerae are known to be associated with several epidemics and pandemics. in underdeveloped nations, the fecal contamination of food-water and poor sanitary habits are associated with cholera epidemic [5]. therefore, the aim of this study was to study the incidence of vibrio species and devise simple method for rapid detection of vibrio cholera from water samples of sunsari, nepal. methods and materials study site the study area was sunsari district (latitude 26° 37' 39.19" n and longitude: 87° 10' 55.82" e) of province no.1, nepal. the district is situated in terai region and covers an area of 1,257 km². sunsari district is situated at an altitude up to 6600 ft. from sea level. study design this study was designed to setup from november 2018 to january 2019. in this study water samples were collected from different regions of sunsari district, nepal. during the study period a total of 100 water nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:interfacebj@gmail.com https://orcid.org/0000-0002-6542-829x mailto:interfacebj@gmail.com https://orcid.org/0000-0002-9397-4511 nepal j biotechnol. 2021dec.9 (2): 33-38 shrestha and shakya ©njb, bsn 34 samples were collected from four available sites (pond, sewage, tap and tube well) of districts rich in water resources. in this study, same frequency of samples was collected from four different places in which 25 water samples were collected from sewage, 25 from pond, 25 from tap and 25 from tube well from random areas of sunsari district to have uniform distribution of different samples. sites of water samples were selected by simple random sampling and lottery method. about 100 ml water was collected in a sterile bod sample bottle and was transported to microbiology laboratory of central campus of technology, dharan on the same day maintained in ice cold chain and processed within 3 hours of collection. societal information the socio-demographic activities of people affecting water sanitation were observed and information were collected from local residents through a semistructured questionnaire. inclusive and exclusive criteria water samples from rivers, ponds, tap and sewage were included in this study. screening v. cholerae from water isolation of v. cholerae from water was performed as described by cdc (1994) [6]. membrane filters technique was most appropriate for clear sample that did not contain debris, mud or silt. clean, non-cloudy and sediment free water from tap and tube well was filtered through membrane filtration technique. in this method, 100 ml of water sample was passed through 0.22 µm membrane filter (himedia, india) attached to suction flask. with the help of a sterile forceps, the membrane filter was inoculated in test tube containing 10ml of sterile alkaline peptone water of ph-8.6 (apw) (himedia, india) and vortexed for 30 sec. the enrichment of bacteria from pond and sewage was performed by inoculating 1 ml of sample water in 9 ml alkaline peptone water. the enriched apw culture tubes were incubated at 37°c for 6 hours and then the topmost layer of enriched aliquot was inoculated on thiosulfate citrate bile salts sucrose (tcbs) agar media (himedia, india) and streaking over the tcbs agar surface was done with the help of sterile inoculating loop. the inoculated culture media were incubated at 37°c for 18-24 hours. after overnight incubation, the vibrio like yellow colony, 2 to 4 mm in diameter, slightly flattened with opaque centers were chosen and cultured on nutrient agar without nacl and incubated at 37°c for 16-24 hours. in routine microbiology tests, the bacterial colonies obtained from tcbs are cultured in nutrient agar with nacl alongside overnight incubation and further the isolated colonies are biochemically tested which requires lengthy cost, reagents, time and labor. most of the biochemical tests require overnight or 18-24 hours long incubation period. in this regard, suspected isolated vibrio culture from tcbs directly grown in na devoid of nacl provides presumptive identification of v. cholerae without further biochemical tests. biochemical test for identification of v. cholerae after incubation, the nutrient agar (na) plate with the growth of colorless, glistening, translucent colonies provided presumptive identification of v. cholerae . for further confirmation, gram staining and biochemical tests like oxidase test, string test, mr test, citrate test, indole test, esculine hydrolysis and arginine dihydrolase activity were performed as described by cdc (1994) [6]. for gram staining and biochemical tests, the culture was taken from nutrient agar without nacl (nonselective media). sucrose fermenting colonies (yellow pigmented) from tcbs which were not recovered in na devoid of nacl were further cultured in nutrient agar with 0.5% nacl and subjected for biochemical tests. quality control v. cholerae o1 el tor strain n16961 was used in this study as positive control. reagents and culture media were regularly monitored for their performance. equipment was standardized, optimized and its performance was checked through pretesting of positive and negative controls. data analysis the data was documented in ms-excel 2010 and was analyzed using spss version 16.0 software. statistical significance was established at p <0.05 with 95% confidence interval. results out of 100 water samples collected, sucrose fermenting vibrio species were isolated only from 16 water samples (9 isolates from raw sewage, 6 isolates from pond devoid of any sewage connections and 1 isolate from public tap water). none of the non-sucrose fermenting vibrio species were isolated from tcbs. further, the selective isolation of v. cholerae from nutrient agar without nacl isolated 6 isolates from sewage samples and 3 isolates from pond samples. the incidence of nepal j biotechnol. 2021dec.9 (2): 33-38 shrestha and shakya ©njb, bsn 35 vibrio cholera in the study sample was reported to be 9%, v. alginolyticus to be 4% and v. fluvialis to be 3%. however, in this study v. cholerae was not isolated from tap and tube well water (table 1). the biochemical tests: oxidase test, string test, citrate test, indole test, mr test, esculine hydrolysis, arginine dihydrolase test confirmed the isolates recovered from na without nacl to be v. cholerae (table 2). the biochemical tests of sucrose fermenting 7 vibrio isolates from tcbs which did not grow in nutrient agar devoid of nacl reported 4 isolates to be v. alginolyticus and remaining 3 isolates to be v. fluvialis. there was significant difference in distribution of vibrio cholera from different water samples (p=0.001). table 2. biochemical tests of isolated v. cholerae . biochemical bacterial isolates test s1 s2 s3 s4 s5 s6 p1 p2 p3 c oxidase test + + + + + + + + + + string test + + + + + + + + + + citrate test + + + + + + + + + + indole test + + + + + + + + + + mr esculine hydrolysis arginine dihydrolase note: isolates s1, s2, s3, s4, s5, s6 were isolated from sewage whereas isolates p1, p2 and p3 were isolated from pond. c is the quality control strain; v. cholerae o1 el tor n16961 strain used in the study table 3. characteristics pertaining to water contamination at study sites. characteristics pond tap water tube well water 1.open human defecation yes no no 2.animal washing and defecation yes no no 3. wastes dumping sites yes no no 4. source of drinking water yes yes yes 5. use of sample site for religious practices yes no no 6. water treatment before drinking no no no figure 1. vibrio sp. on tcbs media with yellow colony, 24mm in diameter, slightly flattened with opaque centers. figure 2. v. cholerae with colorless, glistening, translucent colonies on nutrient agar without nacl. discussion cholera has been a disease of great epidemic potential for the last many centuries and now has become endemic in many countries [7]. cholera outbreaks have been reported frequently from different parts of nepal [8]. v. cholerae 01 strain associated with epidemic outbreak of cholera has been known to establish in aquatic environment posing threat to public health [9]. table 1. vibrio species from culture media. sources (total sample) sucrose fermenters isolates from na without nacl (v. cholerae) isolates from na with nacl (v. alginolyticus) isolates from na with nacl (v. fluvialis) pvalue 1. sewage (s) (25) 9 6 2 1 p=0.0 01 2. pond (p) (25) 6 3 1 2 3. tap water (25) 1 0 1 0 4. tube well (25) 0 0 0 0 total 16 9 4 3 nepal j biotechnol. 2021dec.9 (2): 33-38 shrestha and shakya ©njb, bsn 36 multidrug resistance v. cholerae el tor possessing cholera toxin (ctx) identified with diarrheal illness from three districts from nepal was similar with strains related with cholera outbreak in bangladesh and haiti [10]. v. cholerae and v. mimicus can only grow in media that lacks sodium chloride while other species of vibrio are strictly halophilic in nature [11]. v. cholerae can ferment sucrose in tcbs medium producing yellow colony whereas vibrio mimicus fails to ferment sucrose and hence produce green pigmented colony on tcbs [12]. all the colonies growing on tcbs media with yellow colony are not necessarily v. cholerae. the ability of v. cholerae to grow on culture media devoid of sodium chloride helps in selective isolation and identification of v. cholerae from other vibrio species [13]. in this study the presumptive isolation and identification of v. cholerae was made from media devoid of nacl within two days of sample collection and processing. this study was consistent with the study that has demonstrated that v. cholerae can be selectively isolated from media devoid of sodium chloride [14]. the main principle of this study was the enrichment of vibrio in alkaline peptone water and its selective isolation from tcbs and na without nacl. the biochemical tests like esculine hydrolysis and arginine dihydrolase are efficient techniques for identifying vibrio cholera from aquatic samples [15]. in this study, even arginine dihydrolase test stood key tool for differentiating v. alginolyticus (arginine dihydrolasenegative) from v. fluvialis (arginine dihydrolasepositive). the gram staining, string test and oxidase test along with serogrouping, biotyping and serotyping can be performed minimum from second day of sample processing. however, pcr based methods can provide sensitive result much rapidly within 6-12 hours but conventional methods are only the option in resource limited areas. however, antimicrobial susceptibility test can only be accessed through conventional culture method which is most important in clinical settings. serogrouping, biotyping and serotyping of v. cholerae need to be performed whenever there are reported and outbreak cases which provides evidence for clinical and epidemiological studies. v. cholerae differs from other vibrio species in that it can grow in nutritional broth without the addition of sodium chloride [16]. in this study the identification of v. cholerae from other vibrio species was based on three major trait features like sucrose fermentation, nonrequirement of additional na+ for growth, and presence of oxidase which was in consistent to the findings of other similar study [17]. most sucrose-positive halotolerant or halophilic vibrio were eradicated when grown on nutritional agar without additional nacl. it is also concluded that isolation of v. cholerae from media devoid of nacl is simple and rapid method of isolation and identification which was in agreement to the present study [14]. however, some limiting factors may exists, study reports that some v. cholerae strains, have been reported of not being able to ferment sucrose. these strains would not be detected in conventional studies as only sucrose fermenting vibrio from tcbs are considered for further identification of v. cholerae [18]. some v. cholerae strains which are strictly halophiles might also remain in shadow during isolation through media devoid of nacl. in addition, routine conventional microbiological techniques are not capable of isolating viable but non-culturable (vbnc) state of v. cholerae [19]. in this regard, only the resource rich settings with the polymerase chain reaction provides high specificity and accuracy for identifying vibrio species and can even distinguish its biotypes. according to the study, the prevalence rate of v. cholerae from water samples of bhaktapur was 5% [20] and from kathmandu, the prevalence was 11.11% [21] which were in agreement to the prevalence of the current study. however, the prevalence of v. cholerae was 0.84% in one study from drinking water of kathmandu [22]. the filthy pond with algae growth might be an organism's reservoir [23]. in nepal, the period between mid-june to mid-july had the highest incidence of cholera [24]. in 2012 a.d., diarrhea outbreaks in three districts of nepal were due to transmission of multidrug resistant v. cholerae el tor possessing cholera toxin (ctx) b-7 allele, which is clonal and related closely with v. cholerae associated with cholera in bangladesh and haiti [25]. the increased prevalence and isolation rates of v. cholerae in nepalgunj (banke), dang, and dhangadhi (kailali) might be attributed to contaminated drinking water, poor management of water pipes, sewage, and excreta, and a less sanitary environment [26]. in one study v. cholerae was found in two of the patients from sunsari. in the same study, eight of the thirteen water samples were determined to be unfit for human consumption. contamination of the source of drinking water and the major reasons of such epidemics were unsanitary behaviors. it is, thus, necessary that gastro enteritis outbreaks may be avoided simply by encouraging cleanliness and sanitary practices behaviors involving drinking water and defecation [27]. the presence of heterotrophs and the coliform in the drinking water marketed in eastern nepal is a serious nepal j biotechnol. 2021dec.9 (2): 33-38 shrestha and shakya ©njb, bsn 37 concern for public health [28]. v. cholerae isolated from pond and sewage water samples of sunsari district could pose a severe health hazard to humans who either come in contact with or consume water from the contaminated site. in addition, the lack of safe drinking water, awareness and poor sanitary practices among people of rural areas of sunsari makes them more prone to the infections. water borne diseases among underprivilege family and community remains in shadow until it leads to community burden leading heavy mortality and morbidity. microbiological water surveillance in these rural settings have never gained importance and attention from the concerned authority that alarms the possibility of future outbreak of disease. in this study the possible cause behind the water contamination was also studied. the necessary information was collected from the local residents through questionnaire. it was found that the local villagers of terai observe chhath festival (local ethnic festival that includes fasting, prayers and religious rituals by taking bath in ponds/ rivers water). in this course of ritual, the activity of open defecation results in fecal contamination of water. in this report, the pond of this study sites was even used for many ritual performance, open defecation, animal washing and even for drinking purpose. human activities like disposing human and animal wastes have been identified as the main cause behind deteriorating water resource that leads pond water to be a potent source for cholera transmission [8]. outbreak of cholera in saptari vdc of nepal was associated with use of pond water which was contaminated by v. cholerae [23]. one study reported that diarrheal outbreak in rautahat district of nepal was associated with v. cholerae o1 ogawa serotype, which was result of fecal contamination of drinking water from nearby sewage [29]. hence, these finding addresses the cause and route behind fecal contamination of water which can lead to cholera outbreak. therefore, studies suggest promotion in safe drinking water is fundamental approach to prevent morbidity and mortality induced by cholera in near future [30]. maintenance of safe water chain a key approach for preventing cholera outbreak in near future. conclusions this study concludes that yellow colonies (sucrosefermenting) of vibrio from tcbs agar medium that can grow on nutrient agar without added nacl and which exhibit a positive oxidase reaction can be confidently identified as presumptive v. cholerae. this method can reduce the labor, reagents, cost and time-consuming process of conducting several biochemical and molecular tests in resource limited settings. abbreviations apw: alkaline peptone water bod: biological oxygen demand cdc: centre for disease control and prevention na: nutrient agar nacl: sodium chloride pcr: polymerase chain reaction tcbs: thiosulfate citrate bile salts sucrose vdc: village development committee limitations molecular methods were not performed for identification of bacteria. serotyping, biotyping and antibiotics susceptibility test of isolated pathogens were not done. acknowledgement authors want to thank all the helping hands and department of microbiology, central campus of technology, tribhuvan university, hattisar dharan, nepal. author’s contribution: bks participated in study design, sample collection, sample processing, organism identification, data analysis, intellectual content design and result interpretation. js participated in sample collection, sample processing, and proof-reading manuscript for intellectual content. both the authors drafted the manuscript and agreed for its publication. conflict of interest author declares no conflict of study. funding 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sabina kc, thapa pm, shrestha p, rai sk. diarrheal disease outbreak in gaidatar village of rautahat district, nepal. bmc research notes. 2019; 12(124). doi: doi: https://doi.org/10.1186/s13104-019-4156-9. 30. rahman z, rahman md, rashid mu, monira s, johura ft, mustafiz m, bhuyian si, zohura f, parvin t, hasan k, saif-urrahman km. vibrio cholerae transmits through water among the household contacts of cholera patients in cholera endemic coastal villages of bangladesh, 2015–2016 (chobi7 trial). frontiers in public health. 2018;6(238):1-9. doi: https://doi.org/10.3389/fpubh.2018.00238. https://doi.org/10.4103/1947-2714.104321 https://doi.org/10.1186/1471-2334-14-392 https://doi.org/10.1111/j.1574-6941.2002.tb00934.x https://doi.org/10.1128/aem.68.2.995-998.2002 https://doi.org/10.1128/aem.68.2.995-998.2002 https://doi.org/10.1186/s13104-019-4156-9 https://doi.org/10.3389/fpubh.2018.00238 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 29-34 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 29 nepjol.info/index.php/njb isolation of yeast from soil and different food samples and its characterization based on fermentation sandeep thapa*, rajani shrestha, anjali tibrewal, arjun sharma, yuvraj k.c. college for professional studies, department of biotechnology, kathmandu, nepal abstract yeasts are eukaryotic microorganisms which can also be used for bioethanol production. in this modern era of increasing demand for energy and fuel, the microbial biosynthesis of ethanol has gained importance. in this study, the potential yeasts for ethanol production from pentose and hexose sugars were identified. yeasts were isolated from soil and different food samples. they were identified and characterized based on cell morphology (e.g., mode of cell division and spore shape) and physiology (e.g., sugar fermentation tests). furthermore, quantification of ethanol and cell concentration was performed throughout the fermentation. spot plate count method was followed to determine the viable yeast count whereas potassium dichromate oxidation method was used for determining the ethanol concentration. six different species of yeasts were cultured in three sets of broth for 24, 48, 72, and 96 hours for bioethanol production. the yeasts isolated from black and green grapes relatively synthesized higher concentration of ethanol. key words: yeast, fermentation, ethanol, potassium dichromate oxidation method, yepda. *corresponding author: email: sandipthapa_29@kathmandugenomics.com introduction yeasts are single-celled microorganisms that are classified, along with molds and mushrooms, as members of the kingdom fungi [1, 2]. although yeasts are unicellular organisms, they possess a cellular organization similar to that of higher organisms, including humans. specifically, their genetic content is contained within a nucleus. this classifies them as eukaryotic organisms, unlike their single-celled counterparts, bacteria, which do not have a nucleus and are considered prokaryotes [3, 4]. yeast is widely dispersed in nature with a wide variety of habitats. they are commonly found on plant leaves, flowers, and fruits, as well as in soil. yeasts are also found on the surface of the skin and in the intestinal tracts of warm-blooded animals, where they may live symbiotically or as parasites. yeasts are responsible for several types of infections including oral thrush, vaginitis, urinary tract infection, endocarditis, respiratory syndromes, meningitis, etc. the common "yeast infection" is typically caused by candida albicans. beside infections, yeast is very useful in commercial application. yeast has long been considered to be the organism of choice for the production of alcoholic beverages, bread, and a large variety of industrial products [1, 5, 6]. this is based on the ease with which the metabolism of yeast can be manipulated using genetic techniques, the speed with which it can be grown to high cell yields (biomass), the ease with which this biomass can be separated from products and the knowledge that it is generally recognized as safe (gras). the budding yeast, saccharomyces cerevisiae and other yeast species have long been used to ferment the sugars of rice, wheat, barley, and corn for the production of alcoholic beverages such as beer and wine [7,8]. there are two major types of brewing yeast, top-fermenting ale yeast and bottom-fermenting lager yeast. top-fermenting yeast such as s. cerevisiae rise to the surface during fermentation and are used to brew ales, porters, stouts and wheat beers. in contrast, s. pastorianus, (formerly known as s. carlsbergensis) is a bottomfermenting yeast, used to make lager beer. lager yeasts grow best at lower temperatures. as a result they grow more slowly, produce less surface foam, and therefore typically settle to the bottom of the fermenter [9, 10]. s. cerevisiae or baker’s yeast has long been used as a leavening agent in baking. baker’s yeast ferment sugars present in dough, producing carbon dioxide and ethanol. murcha (locally available yeast) is the complex mixture of various types of microorganisms along with yeast. murcha has been traditionally used in fermentation and nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 29-34 thapa et al. ©njb, biotechnology society of nepal 30 nepjol.info/index.php/njb commonly found in nepal, bhutan and even some parts of india. yeasts are being used in the petrochemical industry where it has been engineered to produce biofuels such as ethanol, diesel and jet fuel precursor. they are also used in the production of enzymes, lubricants and detergents. food additives including colorants, antioxidants, and flavor enhancers can also be produced using the yeast cells. likewise, it is used in the production of pharmaceuticals including antiparasitics, anticancer compounds and bio-pharmaceuticals such as insulin, vaccines, and nutraceuticals. another important characteristic of yeasts being used as model organisms is that they replicate quickly and are easy to manipulate genetically. the doubling time for yeast is about 90 minutes, furthermore, the study of genome and its organization have been completed [11,12]. methodology sample collection four samples were collected from local market and two from department of microbiology, pinnacle academy, lalitpur, nepal and central department of biotechnology, tribhuvan university (tu), nepal. collected samples and subcultures were given different codes for the convenience. a: mango peel; b: black grapes sample; c: banana peel; d: soil sample; e: green grapessubculture from pinnacle academy); f: subculture of tu. sample processing the samples were washed with distilled water and were crushed using mortar and pestle. one gram of finely crushed sample was weighed and serially diluted up to 10-5. isolation and identification of yeast one ml of sample from each dilution was spread on yepda media to isolate yeast. morphological characteristics were studied on the basis of colour, texture, margin, elevation. simple staining was performed to elucidate the morphology and arrangement of yeast cells and budding. subculture of isolated yeast subculture of isolated yeast was done in the yepda plates for further study and preservation. spot plate technique counting of viable yeast was done by spot plate method. 5 µl of serially diluted samples was inoculated with the help of micropipette on the marked area on yepda media. plates were labelled according to the dilution factor and incubated at 37°c for 24 hrs. individual colonies in the most dilute samples were counted and the number of viable cells in the original culture was calculated. preparation of broth three sets of broth (1-xylose, 2glucose, 3xylose + glucose) were prepared for each sample and its fermentative characteristics were studied. inoculation in broth all isolated yeasts from each sample were inoculated in three series of broth and kept at shaker for 4-5 days with regular monitoring. absorbance was taken at 560nm at the interval of 24 hours. smell of the alcohol was also monitored at the same interval. measurement of ethanol concentration using solvent extraction and potassium dichromate oxidation method positive samples with characteristic alcoholic smell were further monitored for estimation of methanol concentration. 1ml of each positive sample was mixed with 1ml tributyl phosphate (tbp) and was centrifuged at 10,000 rpm for 5 minutes and 950µl of upper tbp was transferred to new centrifuge tube. 950 µl of potassium dichromate was added, vortexed for 2-3 minutes and centrifuged at 10,000 rpm for 5 minutes. then upper layer was discarded, lower layer was pipetted out and its absorbance was taken at 595 nm using yepd broth as blank [13, 14, 15]. preparation of standard ethanol solution standard ethanol solution was prepared by mixing different aliquots of 75% ethanol from stock to make up to 5ml by mixing required amount of ethanol and distilled h2o. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 29-34 thapa et al. ©njb, biotechnology society of nepal 31 nepjol.info/index.php/njb table 1: morphological characteristics of isolated yeasts sample morphological characteristics colour shape margin elevation texture consistency a cream oval lobate non smooth mucoid b white round entire elevated smooth mucoid c white oval entire non smooth mucoid d white round entire non smooth mucoid e light –orange oval entire elevated smooth mucoid f cream oval lobate non smooth mucoid results and discussion morphological characteristics of isolated yeasts the morphological characteristics were studied from the colonies isolated on yepda media.(table 1) simple staining of yeast the yeast appeared as round or oval cells that were dark purple in color and budding were also visible under microscope. (table 2) table 2. showing the color and shape of the isolated yeasts counting of viable yeast by spot plate method each row represents a dilution series from a different yeast culture. the same volume of diluted culture is used for each spot. the dilution series was designed so that the most dilute spots contain a small number of individual colonies that can be distinguished from one another, typically less than 15. the colonies were counted respectively. (table 3) determination of growth curve of yeasts absorbance of broth with inoculums added was taken from day first to day fourth at the interval of 24 hours at 565nm. all the readings of three sets of broth of all series of samples with comparable chart are as follows: table 3: table showing the number of yeast by spot plate technique sample dilution factor colony count no of cells /100µl a 103 14 14 × 103 b 104 12 12 ×104 c 105 8 8 × 105 d 104 10 10 ×104 e 104 9 9 ×104 f 105 6 6 × 105 yeast extract, peptone and xylose yeast extract, peptone and glucose yeast extract, peptone, xylose and glucose figure 1: graph showing the absorbance of different samples in the broth containing peptone and xylose from first day up to fourth day. figure 2: graph showing the absorbance of different samples in the broth containing peptone, and glucose from first day up to fourth day. sample staining characteristics shape color a oval shaped purple in color b oval and round purple colored yeast with buds c oval shaped purple colored yeast with buds d round shaped purple in color e oval shaped light purple colored yeast with buds f oval shaped purple in color nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 29-34 thapa et al. ©njb, biotechnology society of nepal 32 nepjol.info/index.php/njb figure 3: graph showing the absorbance of different samples in the broth containing peptone, and xylose +glucose from first day up to fourth day. monitoring of alcoholic smell the flasks were monitored for alcohol production by smelling. there was presence of characteristic alcoholic smell in all the sets of conical flask expect 1 which contains xylose along with yeast extract and peptone (table 4). table 4: absorbance of each broth with respective sample and detection of their characteristic alcoholic smell. sample broth absorbance at 595nm a 1 2 3 b 1 2 0.841 3 0.629 c 1 2 0.714 3 0.542 d 1 2 0.621 3 0.428 e 1 2 0.913 3 0.624 f 1 2 0.357 3 0.601 figure 4. absorbance of standard ethanol solution potassium dichromate testing absorbance of all the positive samples and standard ethanol solution were taken at 595nm. absorbance were taken for those samples only which gave characteristic alcoholic smell. since, there was absence of alcoholic smell in sample a; its absorbance was not taken. comparison with the standard ethanol by comparing the absorbance of sample with standard ethanol solution, it was found that all the isolated yeast were highly fermentative. discussion the different fermentative yeasts were isolated from different samples. the maximum of yeast colonies were obtained from both grape samples, which is mostly used in fermentative purposes. it is also found that when yeast is grown in liquid medium, the culture follows a well established pattern for microbial growth. cultures are usually started by inoculating media with a small number of cells. a lag phase follows the inoculation, during which cells become acclimated to the new environment and begin to condition the media with their own metabolites. lag phase is followed by an exponential, or log phase, when the number of cells increases exponentially. during this phase, the cells are in actively growing stage with high metabolic activities, making this phase a desirable stage for the generation of primary metabolites including ethanol. during the study, six carbon compound glucose, five carbon compound xylose and their mixed culture has been used and were screened for their suitability for fermentation. the broth containing only five carbon compound or pentose sugar xylose didn’t show the fermentative characteristics, which was confirmed by the absence of characteristic alcoholic smell. the broth containing glucose and the other broth containing glucose and xylose has shown the fermentative characteristics. sample e show growth in all the media but however show high growth in the broth containing peptone and glucose and hence it’s highly fermentative. thus, yeasts utilize glucose a major carbon source for fermentation. from the above graph, sample b and c show relatively similar growth in all the media but it shows high growth in medium containing peptone and glucose. thus, sample b and c prefer peptone and glucose as a major carbon source. sample a and b utilize all carbon sources for its metabolism. sample f uses glucose as a major nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 29-34 thapa et al. ©njb, biotechnology society of nepal 33 nepjol.info/index.php/njb source. thus, we can conclude, yeasts utilize six carbon compounds as a fermentative source. the ethanol was extracted from the aqueous phase using the solvent. the ethanol in the solvent phases moved to an acidic aqueous phases and subsequently reacted with dichromate. the ethanol concentration was measured from the increase in green color from orange at 595nm against blank. the solvent extraction in this ethanol measurement is a crucial step because glucose, yeast extract, peptone, and glycerol can cause a change in color through reaction with dichromate. in other words, without the ethanol extraction step, the measured ethanol concentration in the culture medium may be tainted by a direct reaction between the dichromate reagent and other component in the culture medium. many solvents have previously been used for the selective extraction of ethanol, particularly for the ethanol extraction fermentation. primary aliphatic alcohol (eg. n-decanol, ndodecanol) is a representative solvent for extracting ethanol from a culture broth and benzyl alcohol has been used as an alternative to distillation. in this study, however, non alcoholic solvents tbp was investigated in order to find better solvent for the extraction of ethanol from a culture broth. moreover, phase separation after solvent extraction was inhibited when the culture medium was used to prepare the ethanol standard solution. however, tbp made a distinct interface between tbp and water and showed a linear standard ethanol curve in various media [14, 15]. in the measurement of ethanol concentration of the yeast culture, the data from the solvent extractiondichromate oxidation method were similar to those from gas chromatography [16, 17]. therefore, this ethanol assay format is practically useful for the selection of a strain having high productivity, the development of a bioethanol production process, and monitoring and control in alcoholic beverage production. finally, sample e can be used as a yeasts source for fermentation. the further characterization of sample e is essential for the future use acknowledgments the authors are thankful to college for professional studies, kathmandu, nepal for providing financial assistance and encouraging research activities in department of biotechnology. the authors are also thankful to pinnacle academy, lalitpur, nepal and mr. sushil khanal from central department of biotechnology, trubhuvan university, nepal for providing us yeasts samples. references 1. alfenore s, molina-jouve c, guillouet se, uribelarrea jl, goma g, benbadis l: improving ethanol production and viability of saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process. appl microbiol biotechnol, 2002. 60:67–72. 2. alfenore s, cameleyre x, l goma g, molina-jouve c, guillouet se: aeration strategy: a need for very high ethanol performance in saccharomyces cerevisiae fed-batch process. appl microbiol biotechnol, 2004 63:537–542. 3. barnett ja, payne rw, and yarrow d: yeasts: characteristics and identification. 3rd edn. cambridge: cambridge university press. 2000 4. boekhout t, robert v, and smith mt: yeasts of the world. morphology, physiology, sequences and identification. world biodiversity database cd-rom series. eti: university of amsterdam. 2002 5. abouzied m, reddy ca:. direct fermentation of potato starch to ethanol by coculture of aspergillus niger and saccharomyces cerevisiae. appl. environ. microbiol, 1986 52(5):1055-1057 6. 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(allen &mp; umwin, boston, massachusetts. 1987 10. chanchaichaovivat a, ruenwongsa p, panijpan b: screening and identification of yeast strains from fruits and vegetables: potential for biological control of postharvest chilli anthracnose (colletotrichum capsici). biol. contr. 2007 42:326–335. 11. m. raines, advanced yeast culturing kit instruction. booklet (brewers resource, camarillo, california,) 1992 12. martini a: biodiversity and conversation of yeasts. biodiver. conserv. 1992 1(4):324-333. 13. senthilraja p, kathiresan k, saravanakumar k: comparative analysis of bioethanol production by different strains of immobilized marine yeast. j. yeast fungal res. 2011 2(8):113–116. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 29-34 thapa et al. ©njb, biotechnology society of nepal 34 nepjol.info/index.php/njb 14. hyun-beom seo, hyun-joo kim, oh-kyu lee, jihye ha, hyeon-yong lee, kyung-hwan jung: measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process. j indust. microb. biotech. 2009 36(2):285-292 15. pilone gj: determination of ethanol in wine by titrimetric and spectrophotometric dichromate methods: collaborative study. association of official analytical chemists. 1985 68(2):188-90. 16. seo hb, kim hj, lee ok, ha jh, lee hy, jung kh: measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process. j ind microbiol biotechnol. 2009 36(2):285-292. epub 2008 nov 7. 17. martínez-nieto p, vanegas-hoyos m, zapatapineda m, robles-camargo j: hydrolysis of eicchornia crassipes and egeria densa for ethanol production by yeasts isolated from colombian lake fúquene. international journal of environmental, chemical, ecological, geological and geophysical engineering 2013 7(1) http://www.pubfacts.com/author/hyun-beom+seo http://www.pubfacts.com/author/hyun-joo+kim http://www.pubfacts.com/author/oh-kyu+lee http://www.pubfacts.com/author/ji-hye+ha http://www.pubfacts.com/author/hyeon-yong+lee http://www.pubfacts.com/author/kyung-hwan+jung nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 60-65 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 60 nepjol.info/index.php/njb study on phytochemical, antibacterial, antioxidant and toxicity profile of viscum album linn associated with acacia catechu meena kusi1,3*, kanti shrestha2, rajani malla1 1central department of biotechnology, tribhuvan university, kirtipur, nepal; 2nepal academy of science and technology, faculty of science, khumaltar, nepal; 3deerwalk services pvt. ltd., kathmandu, nepal abstract this study focuses on antibacterial, antioxidant and toxic potentials of viscum album linn, commonly known as european mistletoe associated with acacia catechu (khayer in nepali). methanol extract of the aerial parts of the mistletoe was prepared by cold percolation method. the resulting extract was simultaneously subjected to phytochemical screening; anti-microbial activity; anti-oxidant potential and brine shrimp toxicity test. the major biologically active phyto-constituents observed were alkaloids, glycosides, saponins, polyphenols, flavonoids, tannins, terpenoids and cardiac glycosides. upon antibacterial activity screening, the plant extract was found to be highly effective against pseudomonas aeruginosa with the zone of inhibition 16±1mm compared to 17±1mm of chloramphenicol (50 mcg). the antioxidant activity as ec50 value by dpph (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity was found to be 1.58 mg/ml while the ferric reducing capacity was measured to be 282.83±19.55 mg feso4.7h2o eqvt/g dry wt. of the extract during ferric ion reducing antioxidant power (frap) assay. the lc50 value for brine shrimp toxicity assay was found to be 31.62 ppm. this study shows the medicinal value of the mistletoe associated with acacia catechu. further meticulous analysis of this plant might lead to identification of active biomolecules effective as drugs for various ailments. key words: alkaloids, glycosides, polyphenols, flavonoids, tannins, terpenoids, toxicity. *corresponding author email: meena.kusi.917@gmail.com introduction viscum album linn, commonly known as european mistletoe is a semi-parasitic shrub, growing on large variety of woody trees especially on their stems and branches [1]. the plant is distributed along subtropical and temperate himalayas at an altitude of 900-2100m [2]. the most common host plants include salix, populus, acer, malus, crataegus, prunus, sorbus, abies, and pinus [3]. viscum album in nepal has been used as a medicinal herb since very long. traditionally the paste of mistletoe bark is applied to relieve muscular swelling, boils and wounds and even in the management of joint dislocation; the berries act as laxative, tonic, diuretic and aphrodisiac while the plant has been used in complications like enlargement of spleen and tumors [2, 4]. the plant has also been used in case of epilepsy, vertigo, anxiety, exhaustion and hypertension [5]. bussing (2004) has compiled wide use of this plant in various countries as: in ancient greek in spleen diseases and complications associated with menstruation while in argentina; mistletoe has been used as sedative and as stabilizer in bone fractures; in india the mistletoe tea in diabetes; in africa in treatment of various stomach troubles of children including diarrhea. the german commission e monographs, a guide to herbal medicine approved the use of the plant in rheumatism and tremor diseases. the evergreen mistletoe was regarded as a symbol of fertility and good luck and thus was believed to avoid any fertility problems [7]. despite the long traditional use of this herb in medicine, the chemical and biological study of this plant has only been initiated lately. the active chemical constituents reported from the mistletoe are glycoproteinsmistletoe lectin i, ii, and iii; protein viscotoxin; polysaccharidesgalacturonan, arabinogalactan and alkaloids [5]. recently two novel amino-alkaloids namely 4,5,4’-trihydroxy-3,3’iminodibenzoic acid and 4,5,4’,5’-tetrahydroxy-3,3’iminodibenzoic acid have been isolated and characterized [8]. other classes of secondary metabolites from viscum album are flavonoids, phenylpropanoids, triterpenes, phytosterols, cyclic peptides and cyclitols [6]. being a parasitic plant, the phenolic content and the antioxidant capacity of mistletoe have been found to vary depending upon the host trees and the time of harvest [9,10]. new compounds have been also reported in mistletoe growing on different hosts [11]. the organic solvent fraction of mistletoe extract from its twigs and leaves showed good antimicrobial activity against both gram positive and gram negative bacteria [12, 13] .the hexane extract of the plant has shown anti-fungal activity against c. albicans as well [14]. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 60-65 khusi et al. ©njb, biotechnology society of nepal 61 nepjol.info/index.php/njb though the plant contains various cytotoxic toxins there are no reported toxicity to humans. some studies have shown that direct ingestion of the plant parts especially berries might result in mild effects like nausea, vomiting, bloody diarrhea and shock induced hypertension [6] application of recombinant mistletoe lectins are reported to cause some sort of reversible hepatotoxicity [15]. this plant is in high demand by the traditional healers and herbal companies for manufacturing various medicine based on this herb. in nepal herbal drug manufacturers use this plant as an important constituent in various formulations. however the exact chemical compounds or the proven therapeutic applications of this plant have not been stated anywhere. since this plant is parasitic in nature it has been stated that the host plant also plays important role in the active biomedical content of this plant. so, this study of viscum album associated with acacia catechu is unique in itself. the aim of this study is to identify active biomedical components of this plant which is associated with acacia catechu and to assert any associated toxicity. we carried out phytochemical analysis of this plant which was followed by antibacterial activity screening, estimation of antioxidant capacity and toxicity analysis. materials and methods all the procedures were carried out in natural products chemistry laboratory, faculty of science, nepal academy of science and technology, lalitpur, nepal. plant materials: the aerial parts of viscum album linn. associated with acacia catechu were collected from sarlahi district, nepal (1,000 3,300ft). it was shade dried and crushed into powder using a crusher. the resulting powder was stored in plastic bag for further work at room temperature. extraction of phytochemicals: for extraction of phytochemical compounds, cold percolation method followed by intermittent sonication was followed. first the solvent methanol was added in the ratio of 8:1 (plant powder weight in gram) and shaken well then kept on stand for 3 days at room temperature. from day four onwards, the resulting solution was subjected for intermittent sonication (rocker scientific co. taiwan) a continuous cycle of sonication at 20 khz at 60˚c for 15 minutes followed by 2 hour standing at room temperature, till day six. on day six, the supernatant was slowly poured into the round bottom flask through cotton plug filtration which was then evaporated at reduced pressure in rotatory vacuum evaporator (hanshin scientific co., korea). resulting dry extracts were then sealed in small glass containers and stored at 4˚c until use. phytochemical screening: the procedures for phytochemical screening has been followed in reference to the book “phytochemical techniques” and the research paper [16, 17]. mayer’s test and dragendorff’s reagent test were employed for detection of alkaloids while carbohydrates and cardiac glycosides were detected with molisch’s test and fehling’s test. proteins and amino acids were tested by applying biuret test and ninhydrin test. liebermann-burchard’s test gave results for presence of phytosterols. ferric chloride test, alkaline reagent test and magnesium-hydrochoric acid reduction test detected phenols and tannins. bramer’s test was used for specific detection of tannins. liebermann’s burchard test and salkowski’s test detected terpenoids. shinoda test for flavonoids and kellarkilliani test for cardiac glycosides were used. saponins, gums and mucilages were also subsequently detected. antibacterial screening: the antimicrobial activity of the crude extract was studied by agar well diffusion technique [18]. five standard strains of gram negative bacteria (escherichia coli atcc 25922, klebsiella pneumoniae atcc 700603, salmonella typhimurium atcc 14028, serratia marcescens atcc 13880 and pseudomonas aeruginosa atcc 27853) and a single standard strain of gram positive bacterium (staphylococcus aureus atcc 25923) were used for antibacterial assay. the above mentioned strains of bacteria were obtained from national public health laboratory, teku, nepal and institute of medicine, maharajgunj, nepal. a standard antibiotic disc (chloramphenicol, 50mcg) as positive control and dmso as solvent control was used. determination of anti-oxidant activity: dpph (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity: a range of concentration varying from 0-10mg/ml of the sample was taken for the assay. a 50µl of the crude extract was added to 450µl of tris-hcl buffer (0.05m, ph7.4) and 1ml of 0.1mm dpph was added to the resulting mixture and incubated in dark at ambient temperature for 30min. absorbance value of resulting solution at 517nm was recorded [19, 20] nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 60-65 khusi et al. ©njb, biotechnology society of nepal 62 nepjol.info/index.php/njb the free radical scavenging activity (rsa) of the samples was calculated in percentage by using the formula: radical scavenging activity (rsa) = the ec50 values of each extract were calculated by using the formula given below: ec 50 = ferric ion reducing antioxidant power (frap) assay: 10µl of the extract was added to 30µl of distilled water and 300µl of frap reagent; the resulting solution was incubated at room temperature for 5min. then after the intensity of the blue colored complex formed was measured spectrophotometrically using jenway uv-vis spectrophotometer at 593nm. the frap reagent was prepared by mixing 25 ml of acetate buffer (300mm sodium acetate at ph3.6) and 2.5ml of tptz (10mm 2,4,6-tripyridyl-s-triazine in 40mm hcl) with 2.5ml of ferric chloride solution (20mm fecl3.6h2o in distilled water). all the chemicals were prepared fresh and the frap reagent was warmed at 37˚c before use. then the reducing power of each extract was expressed as equivalent to that of 1mm of fe (ii) i.e. frap unit. all the tests were carried out in triplicates (n=3). [2123] brine shrimp lethality assay: crude plant extract was prepared in three different concentrations of 1000ppm, 100ppm and 10ppm. 100ul of each of the extracts of each concentration were taken in wells of micro-titer plate. freshly hatched brine shrimp (artemia salina) nauplii were placed in each well at the rate of 5 in each and the plate was incubated at 22-28˚c for 24hrs. potassium dichromate (k2cr2o7 at 1mg/ml) was used as positive control [24]. the percentage viability of the larvae was observed and the percentage mortality was calculated as: percentage of mortality (pm) = then the lc50 values for each of the extracts were also calculated according to the formula given below: lc 50 = statistical analysis the final data were presented as mean ± standard deviation from the three independent assays. the dpph radical scavenging activity and brine shrimp toxicity values were calculated using microsoft excel 2007 software while these data were exported to graphpad prism v.5.0 for generation of graphs and further analysis. results the dried aerial parts upon extraction with methanol resulted into light brown dry powder. the yield percentage was 26.67% as shown in table 1. table 1: percentage yield and physical characteristics of the crude methanolic extract plant parts used dry wt. taken (gm) wt. of extract (gm) percentage yield (%) characteristics of extract color consistency stem and branches 8.51 2.27 26.67 light brown powdery the qualitative testing of bioactive phytochemicals in the methanolic extract of the aerial parts of viscum album linn. showed rich presence of alkaloids, saponins, phenolics and tannins. however, the flavonoid content in this extract was found to be very low. these phytochemical compounds are known to exert bioactive properties for medicinal plants. other phytochemicals detected were steroids, terpenoids and cardiac glycosides as shown in table 2. table 2: preliminary phytochemical screening of methanolic extract active compounds tests results alkaloids mayer’s test ++ dragendorff’s test ++ carbohydrates and glycosides molisch’s test fehling test saponin +++ protein and amino acids biuret test ninhydrin test phenolics and tannins ferric chloride test ++ alkaline reagent test +++ mg conc. hcl test gums and mucilages tannins braemer’s test +++ steroids liebermann-buchard’s test + terpenoids liebermann-buchard’s test + s ++ flavonoids shinoda test cardiac glycosides kellar-killiani test ++ the anti-bacterial activities of methanolic extract of viscum album were evaluated against 6 bacteria (5 gram negative and one gram positive). the results are nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 60-65 khusi et al. ©njb, biotechnology society of nepal 63 nepjol.info/index.php/njb presented as zone of inhibition by the extract (mm in diameter) against the growth of the test microorganisms. the bacterium pseudomonas aerouginosa was the most susceptible bacterium to the plant extract for which the zone of inhibition was marked to be 16mm while the rest of the microorganisms were not inhibited to significant extent, shown in table 3. chloramphenicol (50mcg) was employed as the positive control which showed significant inhibition to all the test organisms. table 3: anti-bacterial sactivity of the crude methanolic extract the crude methanolic extract was then subjected to in vitro test to evaluate antioxidant activity using two different tests: dpph free radical scavenging assay and frap assay. during dpph assay the ec50 value of the methanolic extract of viscum album was found to be 1.58 mg/ml which was only 50 times lower than that of the standard gallic acid as shown in table 4. table 4: ec50 values of crude methanolic extracts in dpph free radical scavenging assay s.n sample methanolic extract ec50 values (mg/ml) 1. gallic acid 0.039 2. v. album 1.58 the value of frap assay was expressed as mg feso4.7h2o per gm dry wt. of the sample. in table 5, the frap activity value of the plant found to be 282.83±19.55 mg feso4.7h2o/gm dry wt. has been shown. table 5: estimation of antioxidant activity of crude methanolic extracts by frap assay sample methanolic extract frap activity (mg feso4.7h2oeqvt/gm) v. album 282.83±19.55 in the brine shrimp toxicity assay, the degree of lethality was found to be directly proportional to the concentration of the extract. all the brine shrimp larvae were found to be alive after 24 hours in the extract concentration of 10ug/ml while higher concentration was toxic enough to kill all the larvae before 24hrs.(table 6) the plant extract showed significant toxicity which was measured to be an lc50 of 31.62 ppm (table 7). table 6: brine shrimp lethality assay table 7: lc50 values in brine shrimp cytotoxicity assay discussion preliminary phytochemical screening of the aerial parts of v. album linn. associated with acacia catechu, especially the stem parts showed presence of rich bioactive secondary metabolites like alkaloids, cardiac glycosides, saponins, phenolics, tannins and steroids. this result was in positive correlation with findings from bussing (2004) and kunwar (2010). these rich bioactive molecules have potential to be used in various ailments as tannins are capable of binding protein molecules which might have been exploited in treatment of diarrhea and skin bleedings by traditional healers. anabolic steroids are known to retain nitrogen in osteoporosis and various wasting illnesses [25]. alkaloids are known for their inherent toxicity to cells [26]. flavonoids and phenolics are universally known as potent anti-oxidants and antiinflammatory agents and therefore have been found useful against tumors and cancers [27]. regulations of na+/k+-atpase-pumps are assisted by cardiac glycosides while saponins are well known for their immune-modulatory and anti-neoplastic effects [28, 29]. the rich presence of the phytochemicals of medical value thus proves the effective use of this plant in various formulations in ethno medicine. the exciting results from the qualitative test of bioactive phytochemicals continued to show their strong presence in antibacterial activity assay as well. the crude extract was able to form a clear and distinct halo zone of inhibition measuring a diameter of 16mm (table 3). this was quite comparable with that of the positive control used. ps. aeruginosa is considered one of the most rapidly growing bacteria in its resistance to existing antibiotics [30]. this bacterium has been one of the major causes of hospital acquired infections all around the globe [31]. so, the excellent inhibition of this bacterium by v. concentration of viscum album extract (μg/ml) total number of brine shrimp larvae used no. of brine shrimp larvae alive after 24hrs 48hrs 1000 5 0 0 500 5 0 0 100 5 0 0 10 5 5 0 s.n. crude extracts lc50 ppm ± sd 1. v. album 31.62 ± 0.00 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 60-65 khusi et al. ©njb, biotechnology society of nepal 64 nepjol.info/index.php/njb album linn extract could lead to discovery of potent novel antibiotics. although the results against other bacteria were not much promising, an increment of the concentration of the extract could have improved the inhibition ability. since the extract used in this study is crude one, use of refined extract would have given sharper and better results. dpph-free rsa and frap assay exhibit anti-oxidant power based on electron transfer reactions. the progressive fading of the violet color of dpph reagent to yellow indicates the scavenging of free radicals. lower the ec50 value, greater is the antioxidant activity. the ec50 value of viscum album in this study is 1.58 which describes better free radical scavenging ability of this plant. the co-efficient of determination (r2 value) value of 77.30% as shown in figure 1 explains that the radical scavenging activity is in positive correlation with the concentration of the extract used. so, the rsa value can be enhanced further by increasing the purity of the extract. similarly the frap activity has been calculated as 282.83±19.55 which is also very high value which indicates significant reduction potential of the extract. the polyphenols and flavonoids might be responsible for this active anti oxidant activities of this plant. this therefore provides evidence for the use of this plant in treatment of free radicals associated diseases like diabetes. toxicity is yet another important parameter to qualify any drug or drug formulation/preparation regarding safety. preliminary monitoring of the toxicity of natural products has been carried out via brine shrimp toxicity assay. the freshly hatched nauplii between 24-48 hours of hatching are considered highly sensitive to toxins and therefore this stage of brine shrimp larvae are used for toxicity assay. v. album extract was found to have lc50 31.62 ± 0.00 ppm which is below 1000ppm and the standard chart explains that only those plant extracts having lc50 lesser than 1000ppm are practically cytotoxic. this observed toxicity might be attributed to high alkaloid content of the plant. duke, 2010 has mentioned toxicity of the lectin fraction, viscotoxin and the juice of the plant with the lc50 value of 80µg/kg, o.7mg/kg, 32mg/kg of mouse while the capability of mistletoe lectins in inactivating ribosomes thereby halting protein synthesis has been discussed by bussing, 2004. therefore care must be taken while formulating this herb for drug preparation. instead the cytotoxicity of this plant might be exploited for their use as anticancer drugs. however, viscum album linn should be subjected to further rigorous bioassays for confirmation of specific toxicity. conclusion the viscum album aerial parts associated with acacia catechu showed rich presence of bioactive phytochemical constituents like alkaloids, glycosides, saponins, flavonoids and phenolics. the significant inhibition of ps. aeruginosa presents this plant a promise for screening active constituent against its resistant forms. the significant toxicity of the extract demands careful analysis of the plant before use in medical formulations in one hand while in other it also directs its potential use for targeting cancer cells. acknowledgement the authors would like to express sincere gratefulness to university grants commission, sanothimi, bhaktapur, nepal for financial assistance and nepal academy of science and technology, nast, lalitpur, nepal for providing laboratory facilities. references 1. zuber dk: biology and evolution for the european misletoe (viscum album). phd thesis university of zurich, switzerland (eth zurich); 2008 2. nepal dompo: medicinal plants of nepal, his majesty's government of nepal, ministry of forest, department of plant resources, thapathali, kathmandu, nepal; 1970, pp. 86 3. barney cw, hawksworth fg, geils bw: hosts of viscum album. forest pathology, 2007, 28(3): 187-208 4. rajbhandari kr: ethnobotany of nepal, ethnobotany society of nepal 2001, pp. 146-147 5. loeper me: mistletoe (viscum album l.). longwood herbal task force, 1999 6. bussing a: mistletoe: the genus viscum, harwood academic publishers, 2004 7. klein s, rister r, riggins: the complete german commission e monographs: therapeutic guide to herbal medicines, american botatnical council, first editon,1998 8. amer b, juvik oj, dupont f, francis gw, fossen t: novel aminoalkaloids from european mistletoe (viscum album l.). phytochemistry letters. 2012, 5(3): 677-681 9. önay-uçar e, karagöz a, arda n: antioxidant activity of viscum album ssp. album. fitoterapia. 2006, 77(7–8): 556-560 10. vicas s, prokisch j, rugina d, socaciu c: hydrophilic and lipophilic antioxidant nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 60-65 khusi et al. ©njb, biotechnology society of nepal 65 nepjol.info/index.php/njb activities of mistletoe (viscum album) as determined by frap method,. not. bot. hort. agrobot. cluj 2009, 32(2): 112-116 11. luczkiewicz m, 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saravanan d, sundram km, yoga ll: extraction, isolation and characterization of bioactive compounds from plants' extracts. afr j tradit complement altern med. 2011, 8(1): 1-10 18. perez c pm, bazevque p: an antibiotic assay by the agar well diffusion method. acta biologiae et medicine experimentalis 1990,15:113-115 19. gyamfi ma, yonamine m, aniya y: free-radical scavenging action of medicinal herbs from ghana: thonningia sanguinea on experimentally-induced liver injuries. gen pharmacol 1999, 32(6): 661-7 20. hinneburg i, damien dorman hj, hiltunen r: antioxidant activities of extracts from selected culinary herbs and spices. food chemistry. 2006, 97(1): 122-129 21. benzie if, strain jj: the ferric reducing ability of plasma (frap) as a measure of "antioxidant power": the frap assay. anal biochem. 1996, 239(1): 70-6 22. huang d, ou b, prior rl: the chemistry behind antioxidant capacity assays. j agric food chem. 2005, 53(6): 1841-56 23. pfundstein b, el desouky sk, hull we, haubner r, erben g, owen rw: polyphenolic compounds in the fruits of egyptian medicinal plants (terminalia bellerica, terminalia hebula and terminalia horrida): characterization, quantitation and determination of antioxidant capacities. phytochemistry. 2010, 71(10): 1132-1148 24. rahman au, choudhary mi, thomsen wj: bioassay techniques for drug development, harwood academic publishers; 2001, pp. 9-10 25. adachi m, takayanagi r: effects of anabolic steroids on osteoporosis. clinical calcium 2008, 10: 1451-9 26. aniszewski t: biological significance of alkaloids.alkaloids-secrets of life: alkaloid chemistry, biological significance, applications and ecological role. elsevier; 2007:141-180 27. dubios m: bioactive saponins with cancer related and immunomodulatory activity: recent developments: studies in natural products chemistry: bioactive natural products (part l), volume 32; edited by atta-ur-rahaman: elsevier; 2011: 209-230 28. chahar mk, sharma n, dobhal mp, joshi yc: flavonoids: a versatile source of anticancer drugs; pharmacognosy review 2011, 9: 1-12 29. katz am: effects of digitalis on cell biochemistry: sodium pump inhibition. journal of the american college of cardiology 1985, 5: 16a21a 30. cdc: antibiotic resistance threats in the united states 2013. centers for disease control and prevention report 2013, 69 31. kollef mh, napolitano lm, solomkin js, wunderink rg, bae ig, folwer vg, balk ra, stevens dl, rahal jj, shorr af, linden pk, mickek st; health care-associated infection (hat):a critical appraisal of the emerging threat. proceedings of the hai summit; clinical infectious diseases 2008, 47 (suppl 2): s55-s99 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 issn 2091-1130 (print) /issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 15 nepjol.info/index.php/njb effect of lyophilization on infectivity and viral load of adenovirus bimlesh kumar jha1*, birendra prasad gupta 2, prashanna maharjan3, somila kakshapati3, nabin narayan munankarmi,3 1national public health laboratory, teku, kathmandu, nepal 2central department of biotechnology, tribhuvan university, kirtipur, kathmandu, nepal 3biotechnology society of nepal (bsn), kathmandu, nepal abstract freeze drying (lyophilization) performed at temperature and pressure below the triple point is being practiced for the preservation of virus stocks for longer periods. the present study is aimed to lyophilize adenovirus strain to study its effects on infectivity and viral load. in-house adenovirus reference strain (stock virus) was propagated in hep-2 cell line in 25cm2 cell culture flasks. in 24-well plates the serial dilutions of stock virus from 10-1 to 10-7 (100µl inoculum) was inoculated in each well with hep-2 cells for tcid50 titer and viral dna was extracted separately to determine viral load by taqman real time pcr. stock virus was lyophilized in 3 lots and stored at rt (25±2°c) and 4°c separately for 1, 4 and 6 months and subjected to tcid50 (for viral infectivity) and viral load assay (for total viral genome copies). following lyophilisation and storage of adenoviral strains at rt and 4°c separately did not affect significantly on the viral stability, infectivity as well as viral copy number till 4 months. however, storage at rt for 6 months resulted in 1 log reduction in viral copy number. thus, storage of even lyophilized virus stock would necessitate a temperature of at least 4°c for prolonged periods. the present study could successfully lyophilize adenovirus and retain its infectivity over a period of 6 months when stored at rt and 4°c. no significant difference in the infectivity or tcid50 titer was observed in the lyophilized virus as compared to the stock virus. however, the viral load was observed to increase with lyophilization of the virus over 6 months when stored at 4°c which possibly is due to the concentration of the virus on freeze-drying. keywords: adenovirus, lyophilisation, physical stability, formulation *corresponding author email: jhabimlesh7@gmail.com introduction the structure and function of organisms change and get lost with time, as in laboratory cultures. attempts to stop the biological clock have been conjured by ancient and modern minds; and the heart of many such schemes has been experiments with temperature and water content. whereas refrigeration technology provides a means of slowing the rate of deterioration of perishable goods, the use of much lower temperatures has proved a means of storing living organisms in a state of suspended animation for extended periods. adenoviruses (adv), belonging to family adenoviridae, are double stranded deoxyribonucleic acid (dna) viruses that carry dna insert of size 7kb. 52 subtypes of adenoviruses are known to infect humans [1]. adenoviruses are frequent cause of human mucosal surface, infections particularly in pediatric population and can be responsible for ocular and gastrointestinal illnesses in humans. to preserve maximum infectivity for long periods, cell culturegrown adenovirus must be stored frozen at very low temperatures [2]. however, this makes its handling difficult. the removal of water from viable biological material in the frozen state (freeze-drying) provides another means of arresting the biological clock by withholding water, and commencing again by its addition. lyophilization or freeze-drying is a controllable method of dehydrating labile products by vacuum desiccation [3]. it is considered advantageous to freeze dry viruses and vaccines wherever possible in order to reduce their volume for storage in cold, to enable easy handling and transport, and to enhance their keeping quality [4]. materials and methods cell line and virus stock preparation a confluent monolayer (90%) of the hep-2 cell lines were grown in 25mm² presterile disposable nunc tissue culture flask using (full form?)mem with antibiotics (penicillin, streptomycin) and 10% fetal calf serum (fcs). adenovirus reference strain isolated from department of virology was propagated for virus stock preparation in hep2cell line and used for tcid50 assay and viral load. virus titration by tissue culture infectious dose 50 (tcid50) assay hep2 cell monolayer was prepared and virus infection was performed at different dilutions in 10ml sterile nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 jha et al. ©njb, biotechnology society of nepal 16 nepjol.info/index.php/njb tissue culture tubes. cell monolayer was observed daily till the cytopathic effect (cpe) appeared, along with the control wells (mem inoculated) kept under the same condition. tcid50 titer was be calculated by using reed and muench [5]. extraction of viral dna viral dna was be extracted from 200µl of tissue culture lysate using rneasy minikit (qiagen, gmbh, hilden, germany) according to the manufacturer’s instructions. in brief, 200µl of avl buffer containing 20µl of carrier rna were dispensed in 1.5 ml of micro centrifuge tube. 200µl of tissue culture fluid was added to the buffer avl containing proteinase k into micro centrifuge tube and is mixed by vortexing for 15 sec. the suspension was incubated in the dry temperature bath (56°c) for 10 minutes followed by the centrifugation. after adding 200µl of absolute ethanol, the sample was vortexed for 15sec and centrifuged briefly. carefully 620µl of the solution was be applied to the qiaamp mini column with 2ml of collecting tube without wetting the rim. the cap is closed and centrifuged at 6000g (8000rpm) for 1 min. the qiaamp mini column is placed into the 2ml of clean collecting tube and the tube containing filtrate were discarded. the qiaamp mini column were opened carefully and 500µl of aw1 buffer were added to the qiaamp mini column and centrifuged at 10000g for 1 minute. followed by the replacement of new 2ml of collection tube and the tube containing filtrate were discarded. 500µl of the aw2 buffer was added to the column and centrifuged at 20000g for 3 minutes. followed by the replacement of new 2ml of collection tube and the tube containing filtrate were discarded and centrifuged at full speed for 1 minute. finally the qia amp mini column was placed in a sterile, dnaase, rnaase, free 1.5ml of the micro centrifuge tube. the old collecting tube containing the filtrate was discarded. 50µl of ave buffer was added to the qiaamp mini column and were equilibrated to room temperature for 1 min followed by centrifugation at 8000g for 1 min. competent cells preparation, ligation reaction and plasmid isolation dh5-α, a non-pathogenic strain of e.coli was used for the purpose. it were grown overnight in 100ml conical flask containing luria broth (lb). the overnight bacterial culture was diluted 1/200 to 25 ml of soc media in 10x culture volume flasks (25 ml in 250 ml flask). flask containing bacteria was grown to early log phase in shaker incubator. the growth of the culture was measured at an interval of 30 minutes until an od of 0.4 at 600nm were obtained. the cells were collected by centrifugation at 5000g for 5 min in cold centrifuge (4°c) and were kept on ice in all the further steps. the cells were re suspended in 12 ml of 0.1m ice cold cacl2 and placed on ice for minimum 30 minutes. the cells were centrifuged at 5000g for 10 min and the pellet was collected. the pellet was further re suspended in 1/10 volume of 0.1m cacl2. cells were ready to use as competent cells for the transformation experiment. ice cold sterile glycerol was added to final concentration of 10% (v/v) with a gentle mixing. aliquots of 100µl of cells were made and stored at -70°c until further use. the tube containing the pgem-t easy vector and the control insert dna tubes were centrifuged briefly to collect the content at the bottom of the tube. the 2x rapid ligation buffer was vigorously vortexed before use. ligation reaction was prepared and reaction mixture was mixed properly by pipetting and was incubated for 30 min at 4°c for optimal ligation. two µl of the ligation reaction were added to the eppendorf tube containing 100 µl of the competent cells. the transformation tube containing ligation reaction mixture and competent cells were given heat shock at 42°c for 40-50 second and was immediately returned to the ice bucket. 950µl of the soc media containing antibiotic was added to the above tube and were incubated in a shaking incubator for 1.5 hrs at 37°c with shaking (~150 rpm). 100 µl of the transformation culture was plated onto duplicate lb/ampicillin/iptg/x-gal plates. the plates were incubated overnight at 37°c and were screened for blue and white colonies. the white colonies were the desired insert. to facilitate the blue color development, plates were stored at 4°c (after 37°c overnight incubation). finally the white colonies, supposed to contain the vector with the desired insert were selected and were grown in 10 ml of lb containing 100 µg/ml ampicillin, overnight in a shaker incubator at 37°c at speed of 250rpm. the bacterial cells grown in the lb medium by addition of antibiotic were harvested by centrifugation at 6000g for 15 min at 4°c. the cell pellet was resuspended in 0.3 ml of buffer p1. 300µl of buffer p2 was added and mixed thoroughly and vigorously and were incubated at room temperature (15-25°c) for 5 min. 300µl of chilled buffer p3 was added followed by mixing vigorously inverting the tube (8-10 times) and were incubated on ice for 5 min. mixture was nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 jha et al. ©njb, biotechnology society of nepal 17 nepjol.info/index.php/njb centrifuged at 10,000g in a micro centrifuge for 10 min. the supernatant containing the plasmid dna were aspirated out carefully. during the mean time qiagen-tip 20 was equilibrated by applying 1ml of the buffer qbt, and was allowed to empty the column by gravity flow. the supernatant obtained after centrifugation was applied to the qiagen-tip 20 and allowed to enter the resin by gravity flow. the qiagen-tip 20 were washed with 2x2 ml of buffer qc, followed by elution of dna with 0.8ml of buffer qf. the eluted dna was precipitated by adding isopropanol at room temperature. (0.56ml per 0.8ml of elution volume). the mixture was be then mixed and centrifuged immediately at ≥ 10,000rpm for 30 min in a micro centrifuge at 4°c and the supernatant was be decanted carefully. the residual dna pellet were washed with 1ml of 70% ethanol at least thrice by centrifuging the pellet at 10,000rpm for 10 min each. the supernatant was aspirated carefully without disturbing the pellet. the pellet was air dried for 5-10 min, and was re-dissolved in 100µl of te buffer, ph 8.0. virus revival, titration and viral load in lyophilized adenovirus lyophilized virus was revived in sterile conditions using sterile double-distilled water. the virus ampoule was broken with a clean metal rod in one strike at the neck region of the ampoule in the biosafety cabinet. 1ml sterile water were added to the dried virus and mixed by pipetting atleast 20 times. the revived virus was kept at 4°c for 2h so as to acclimatize virus. the virus was aliquoted and further stored at -70°c. to determine the effect of lyophilization on virus titer the lyophilized virus after revival was titrated in hep-2 cell line and virus titer was determined by tcid50 and pfu following the same procedure as described earlier for the stock virus. to determine the effect of lyophilization on adenoviral load the lyophilized adenovirus was be subjected to real time pcr as described earlier and the ct values obtained was extrapolated with standard curve to estimate the viral load. statistical analysis statistical analysis of significance was undertaken by a paired sample t-test using spss/ qq graph pad prism software with a value of p<0.05 for significance. result confirmation of stock virus adenovirus reference strain (kindly obtained from department of virology, pgimer, chandigarh) was successfully propagated in (hep-2) cells. cytopathic effect (cpe) positive cell culture bottles for adenovirus infected cells showed cell grapening, clustering, rounding, clumping and focal dislodging of cells in comparison to control mock infected cells were seen on third to fourth day post inoculation. cpe positive cells were scrapped with a sterile cell scrapper and subjected to immunofluorescence using polyclonal antisera for adv. it showed the characteristic intra nuclear brilliant apple green fluorescence in adeno infected cells. adenovirus pcr was done in virus infected cell line where a fragment of 161bp was amplified from hexon gene of adv genome and visualized by 2% agarose gel electrophoresis (figure 1). figure 1: agarose gel analysis of adenovirus pcr; lane1: 100bp molecular marker (fermentas, usa), lane2: negative control, lane3: adv these culture bottles were preserved at -80ºc deep freezer and subjected to repeated freeze and thaw in three occasions for cell lysis. the supernatant was transferred to a sterile vial and centrifuged aseptically. the clear supernatant occasions for cell lysis. the supernatant was transferred to a sterile vial and centrifuged aseptically. the clear supernatant containing the virus particles was used as positive control for qualitative rt-pcr as well as preparation of standards in quantitative real time rt-pcr. comparison of tcid50 titer of lyophilized virus stored for different time intervals the lyophilized virus was stored at room temperature (rt) and 4°c for 1month, 4 months and 6 months. the virus was revived after these fixed intervals of time and infected in hep-2 to check the infectivity of virus and change in the tissue culture infectious dose 50 (tcid50) nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 jha et al. ©njb, biotechnology society of nepal 18 nepjol.info/index.php/njb titer. it was observed that lyophilized virus was infective till 6 months and there was no change in the tcid50 titer of the lyophilized adv revived after 1month to 6 months of storage at 4°c. in case of lyophilized adv stored at rt the tcid50 titer was observed to reduce from 10-4.5 at 1 month to 10-3.22 at 4 months but it remained constant to 10-3.22 after 6 months of storage (figure 2 and 3). figure 2: tcid50 of lyophilized virus stored at rt and 4°c and revived after 1 month, 4 and 6 months. the value of tcid50 on yaxis corresponds to 10-y of different sample of adenovirus. figure 3: comparison of tcid50 in stock virus and lyophilized virus revived at different time intervals. the value of tcid50 on yaxis corresponds to 10-y of different sample of adenovirus comparison of tcid50 titer of lyophilized virus stored for different time intervals and stock virus the tcid50 titer of the lyophilized adenovirus stored at rt and 4°c for 1, 4 and 6 months was compared with that of the stock virus. the tcid50 titer of lyophilized virus is summarized in table 1 whereas the tcid50 titer of stock virus was observed to be 10-3. table 1: arrangement of data in calculation of tcid50 titer by reed and muench formula virus dilution infected noninfected accumulative values infected non– infected mortality ratio percent 10-1 4 0 14 0 14/14 100 10-2 4 0 10 0 10/10 100 10-3 3 1 6 1 6/7 85.71 10-4 2 2 3 3 3/6 50 10-5 1 3 1 6 1/7 14.28 10-6 0 4 o 10 0/10 0 no significant difference (p, 0.342) was observed in the tcid50 titer or the infectivity of the lyophilized virus stored at 4°c for 1, 4 and 6 months as well as adv stored at rt for 4 and 6 months as compared to the stock virus. the adenoviral load in stock and lyophilized adenovirus was correlated with tcid50 titer as described in the table 2 using spearman correlation coefficient. however, no statistical correlation was observed in the viral load and tcid50 titer. table 2: correlation of tcid50 and viral load in lyophilized and stock virus confirmation of successful cloning hexon region of the viral genome was cloned in pgemt easy vector (chloramphenicol resistance) and transformed in e coli dh5α. cloned plasmid containing the adv hexon insert had a defective lacz gene and those cells are transformed with this vector formed white colony in ampicillin agar plate using jh7 and kw53 primers (figure 4). figure 4: plate showing blue (vectors without inserts) and white (vectors with insert) colonies. stock virus tcid50 viral load by real-time pcr(copies/ml) in 104 10-3 1590 store d at 4°c stored at rt stored at 4°c stored at rt lyophilized adv revived after 1 month 10-4.5 10-3.7 2920 6810 lyophilized adv revived after 4 months 10-3.22 10-4.5 2220 7200 lyophilized adv revived after 6 months 10-3.22 10-3.7 672 2650 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 jha et al. ©njb, biotechnology society of nepal 19 nepjol.info/index.php/njb white colony from the plate, theoretically containing the desired inserts were picked and subjected to plasmid extraction. the plasmids isolated from the white colonies were subjected to pcr to yield 161bp bands in agarose gel. polymerase chain reaction (pcr) resulted in 161 bp amplicon with hexon region specific primer pair (figure 5). figure 5: conformation of successful cloning; lane1: 100 base pair molecular marker (fermentas, usa), lane 2: nc and 3: pc & lane 4: 161 bp band using hexon specific primer in adv clone. comparison of viral load of lyophilized virus stored for different time intervals the effect of lyophilization on viral load of adv over a period of time was studied. no significant difference was observed in viral load of adv stored at 4°c for 1, 4 and 6 months. however, in lyophilized virus stored at rt a significant difference in viral load was observed between the virus stored for 1month and that stored for 6 months at rt (p<0.05) but no difference was observed in viral load of adv stored at rt for 1 and 4 months (figure 6). figure 6: viral load of lyophilized virus stored at rt and 4°c for 1 to 6 months discussion freeze-drying will not reverse the damage incurred prior to formulation and care must be exercised when selecting an appropriate cell type or technique used to culture or purify the cell or its extracts prior to freezedrying. to sustain freeze-drying it is necessary to establish a pressure gradient from a sample (highest pressure), to condenser, and finally vacuum pump (lowest pressure) so that water migrates from the sample as drying progresses. in the present study adenovirus was successfully propagated in hep-2 cells. the lyophilization cycle was standardized and the virus suspension was lyophilized at temperature below -40°c and under 50m torr vacuum. the essence of the formulation exercise should be to minimize freezedrying damage, loss of viability, or activity. this study considers the stability of adenovirus after freeze-drying and storage and also determines its titer and load. the infectivity of the virus was retained when stored for over 6 months at 4°c or rt. we determined the tcid50 titer of the adenovirus lyophilized for 1-6 months and observed no significant difference in the infectivity when stored at 4°c or rt. thus, the cell culture-grown adenovirus could withstand the freeze-drying process, and it should be stored at 4°c and rt to retain maximum infectivity. many culture collections and gene banks insist on high recovery values prior to a protocol being adopted for regular use; 50% viability post thaw has been accepted in some culture collections as a nominal cutoff for adopting maintenance by cryopreservation alone (mcclure et al., 2011). the ability to determine viral titer rapidly and at high accuracy is one of the most important tools desired when working with viruses in the research laboratory [7, 8]. real-time pcr has been shown to be more sensitive than cell culture based techniques for detection of viruses in clinical specimens; it was therefore interesting to investigate whether real-time pcr technology could also be an important tool for rapid and efficient estimation of viral titer [9]. in the present study the viral load of stock and lyophilized adenovirus was determined and compared with each other. the viral load of the lyophilized virus stored at 4°c was observed to increase as compared to the stock virus whereas that stored at rt for 1 and 4 months the viral load increased slightly but at 6 months the viral load decreased as compared to the stock virus. there is evidence that a cryopreservation method yielding high initial recovery values, maintains viability at that level on prolonged storage. titer determined using either by plaque forming unit (pfu) or endpoint dilution (tcid50) assays, methods that are time-consuming and labor intensive [10]. as a complement to these nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 jha et al. ©njb, biotechnology society of nepal 20 nepjol.info/index.php/njb established methods of determination of virus titers, we here describe a real-time quantitative pcr assay that enables rapid recording of viral load values that corresponds to titer measurements of viruses. however, the results presented here showed no significant relationship between real time pcr v/s tcid50. even though the tcid50 technique is time consuming, need expertise but is capable of dealing with the infective/live virus which has the great importance in viral isolation, manufacturing liveattenuated vaccines, viral antigen and viral standards. real time pcr which detects both the infective and non-infective viral particles is highly sensitive and rapid, used most popularly in the viral diagnostic labs and has great importance in the early diagnosis and management by of the patient [9]. it should, however, be taken into consideration that viruses causing persistent infection that consequently would give a positive signal in real-time pcr cannot be converted to viral titer, as this would give a false impression that the virus is able to cause cytopathic effect. our data also demonstrated that using real-time pcr technology is a reliable, rapid and robust method that corresponds to standard quantification methods used to measure viral titers. thus, highly reproducible over a dynamic range of viral concentrations commonly used in research laboratories and could therefore save time in comparison with the traditional cell culture-based viral titration methods. the reproducibility of quantitative real-time pcr was higher than for tcid50, which could be partially explained by the fact that real-time pcr does not require a manual estimation based on visual observations. conclusion the present study could successfully lyophilize adenovirus and retain its infectivity over a period of 6 months when stored at rt and 4°c. no significant difference in the infectivity or tcid50 titer was observed in the lyophilized virus as compared to the stock virus. however, the viral load was observed to increase with lyophilization of the virus over 6 months when stored at 4°c which possibly is due to the concentration of the virus on freeze-drying. competing interest the authors declare no competing interest. authors contribution bimlesh kumar jha designed the experiment. bimlesh kumar jha, birendra prasad gupta, prashanna maharjan, somila kakshapati and nabin narayan munankarmi performed the cloning and transfection experiment. bimlesh kumar jha, prashanna maharjan, somila kakshapati, nabin narayan munankarmi and birendra prasad gupta performed the virus titration experiment and data analysis. bimlesh kumar jha, prashanna maharjan, somila kakshapati nabin narayan munankarmi and birendra prasad gupta wrote the manuscript. acknowledgement we would like to thank national institute of virology (niv) pune for providing us viral stock. reference 1. walls t, shankar ag, shingadia d: adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients. lancet infect dis 2003, 3(2):79-86. 2. croyle ma, cheng x, wilson jm: development of formulations that enhance physical stability of viral vectors for gene therapy. gene ther 2001, 8(17):12811290. 3. nail sl, jiang s, chongprasert s, knopp sa: fundamentals of freeze-drying. pharm biotechnol 2002, 14:281-360. 4. schersch k, betz o, garidel p, muehlau s, bassarab s, winter g: systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins i: stability after freeze-drying. j pharm sci 2010, 99(5):2256-2278. 5. chen s, guo d, guo b, liu j, shen y, xu x, huang w, guo s: investigation on formulation and preparation of adenovirus encoding human endostatin lyophilized powders. int j pharm 2012, 427(2):145-152. 6. mcclure c, cole kl, wulff p, klugmann m, murray aj: production and titering of recombinant adenoassociated viral vectors. j vis exp 2011(57):e3348. 7. xiao x, li j, samulski rj: production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. j virol 1998, 72(3):22242232. 8. aurnhammer c, haase m, muether n, hausl m, rauschhuber c, huber i, nitschko h, busch u, sing a, ehrhardt a et al: universal real-time pcr for the detection and quantification of adeno-associated nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:15-21 jha et al. ©njb, biotechnology society of nepal 21 nepjol.info/index.php/njb virus serotype 2-derived inverted terminal repeat sequences. hum gene ther methods 2012, 23(1):18-28. 9. grigorov b, rabilloud j, lawrence p, gerlier d: rapid titration of measles and other viruses: optimization with determination of replication cycle length. plos one 2011, 6(9):e24135. 10. biacchesi s, skiadopoulos mh, yang l, murphy br, collins pl, buchholz uj: rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression. j virol methods 2005, 128(1-2):192-197. nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1): 1 3 2 4 research article doi: https://doi.org/10.54796/njb.v10i1.226 ©njb, bsn 13 occurrence of purple blotch disease associated with selected garlic varieties and its management through bio-agent, botanicals and fungicides umme habiba akter , fatema begum, m. r. islam, jannatun nahar prinky, mst. rehena khatun department of plant pathology, sher-e-bangla agricultural university, dhaka-1207, bangladesh received: 5 jul 2021; revised: 21 jul 2022; accepted: 26 jul 2022; published online: 30 jul 2022 abstract purple blotch of garlic caused by alternaria porri is recognized as a prominent diseases posing threat to garlic cultivation throughout the world including bangladesh. the experiments were conducted to determine the prevalence of purple blotch disease on garlic varieties in field condition, to test the pathogenicity of isolated causal organism and to find out the suitable management options of the disease. eight garlic varieties viz. bau rashun-1, bau rashun-2, bari rashun-1, bari rashun-2, bari rashun-3, bari rashun-4, local deshi and local indian were explored in prevalence study and nine management option comprising a bio-agent trichoderma harzianum (t1), five botanicals viz. lantana camara (t2), spilanthes paniculata (t3), ocimum sanctum (t4), raphanas raphanistrum (t5) and azadirachta indica (t6), two fungicides mancozeb 80% wp (t7) and sulcox 50% wp (t8) and, an untreated control (t9) were explored in the experiments. bari rashun-3 showed the highest disease incidence (40.00%) and severity (92.00%) of purple blotch disease. isolation, identification of pathogen and pathogenicity test was carried out as well. in case of management, all botanicals and bio-agent were tested significantly beneficial in lessening the disease incidence and severity of purple blotch disease. the results revealed that lantana camara (t2) was found most effective for minimizing the disease incidence (26.67, 26.67 and 33.33%) at 30, 45 and 60 das, respectively while maximum disease incidence was recorded in control (t9) (86.67, 96.67 and 100.00%). t2 also reduced disease severity at 30 das (11.00%) whereas, at 45 das (18.67%) and 60 das (19.33%) t1 performed well against the disease. keywords: purple blotch; alternaria porri; in-vivo; bio-agent; plant extracts and fungicides. corresponding author, email: sumonahabiba31@gmail.com introduction garlic (allium sativum) belongs to alliaceae family is considered as the most demanded and universal spices within the world especially in bangladesh. the position of garlic is second among all the important spices in the world [1]. garlic helps in controlling hypertension, diabetes, cancer, ulcer, rheumatism, germs, fungal and bacterial diseases etc. [2]. according to asfand et al. (2019)[3] the pungent taste of garlic is reduced by the field fresh exfoliated garlic which consist just about 0.8% fiber, 63% water, 7% protein, 28% carbohydrate, 0.2% fat and an excellent amount of sulphur compounds. from the report of faostat, (2021)[4] the united nations food and agriculture organization reported that from 1,634,634 hectares production with 30,708,243 tonnes of garlic globally each year. total production of garlic in the year of 2019 in bangladesh was 466, 389 tonnes from 71734 ha land. though garlic production is enhancing gradually, but due to expanding population rate in the low income country bangladesh, the domestic need cannot be fulfilled. to meet up the demand bangladesh imports enormous amounts of garlic from abroad every year [5]. though garlic has many importance, the yield is below average in many parts around the world. various factors viz. diseases, insects, soil, climatic condition and lack of technical knowledge etc. affect the quality of garlic bulb and the yield greatly. [6]. soil borne diseases are major in garlic. among the fungal diseases, purple blotch caused by alternaria porri (ellis) cif., is a major constraint that leads to considerable loss in yield and quality of garlic [7]. the disease is considered as a crucial disease all over the world including bangladesh [8]. epidemic may cause total failure of the crop in favorable conditions. gupta and srivastava (1993) [9] carried out a study in maharashtra during kharif where extreme loss was noted owing to purple blotch disease. in punjab, haryana and maharashtra purple blotch spotted as serious disease and caused 20-60% loss [10, 11, 12]. the purple blotch disease acting as more dreadful for seed crops in contrast to bulb crops that caused sometimes 100% loss on productivity of onion seed [13, 14, 15]. hence, in existing situation convenient nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: https://nepjb.com/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0003-3361-0473 mailto:mailtosumonahabiba31@gmail.com nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 14 management strategies of purple blotch of garlic has become a turning topic. to control the plant pathogens, farmers around the world need the chemical pesticides with a view to sustain the standard and dismissal of agricultural products [16]. sharma et al. (2012)[17] approximated in her study that because of pests cause 37% of crop loss and 12% crop loss is due to pathogens. on the contrast, issues of environmental pollution and various health complications arose because of the immoderate and the inappropriate use of pesticides over the past decades around the world. patent resistant organisms can be developed by the extreme use of chemical pesticides [18]. however, now a days strict regulation are applied on the implementation of chemical fungicides because of their carcinogenic effects, problems of residual toxicity, environmental pollution and development of fungicide-resistant strains [19, 20]. kumar & palakshappa, (2008) [21] stated that, biological control of plant pathogens through antagonistic microorganisms is proved as an effective, not harmful to the environment and a suitable strategy other than an optimistic alternative of chemical uses. trichoderma sp. is a biological control agent and botanicals have been found to be very effective for several soil borne plant pathogenic fungi. plant extract possess an anti-fungal activity in opposition to a wide range of plant pathogenic fungi. these are less phytotoxic, biodegradable and host metabolism stimulatory. various experiments were undertaken over the past many years to control purple blotch disease through bio-agents, botanicals and fungicides [12, 15, 21, 22, 23, 24, 25]. the current research work was aimed to assess the occurrence of purple blotch disease incidence and severity of selected garlic varieties, to isolate and identify purple blotch disease and pathogenicity test of alternaria porri, and its management using bio-agent, plant extracts and fungicides. materials and methods the experiments were conducted at the central farm of sher-e-bangla agricultural university and in the central laboratory, department of plant pathology, sher-ebangla agricultural university, dhaka, bangladesh (23°41′n latitude and 90°22′e longitudes at the elevation of 8.6 m above the sea level, aez-28) during the rabi season of 2018-2019 and 2019-2020 with three replications consists of 24 units plots in randomized complete block design (rcbd). recommended doses of fertilizer (cowdung @10 tons/ha, triple super phosphate (tsp) @417kg/ha, muriate of potash (mp) @165kg/ha, urea @320kg/ha, gypsum 100kg/ha, zinc oxide @5kg/ha and boric acid @5kg/ha) were applied during field preparation and after sowing of garlic clove and, 2 packets of sevin were applied to control from the attack of ant [26]. source of garlic seeds eight different fresh and disease-free garlic variety seed were collected from three different places. bau rashun1 (v1) and bau rashun-2 (v2) varieties were collected from bangladesh agricultural university, horticulture department, mymensingh. bari rashun-1 (v3), bari rashun-2(v4), bari rashun-3 (v5) and bari rashun-4 (v6) varieties were collected from bangladesh agricultural research institute, joydebpur, gazipur and last two local varieties naming local deshi (v7) and indian local (v8) were collected from siddik bazar, dhaka, bangledesh. disease incidence garlic varieties were assessed on the basis of symptoms appeared on the above ground plants and recorded. for calculation of disease incidence each plant was counted including infected one in the field and then expressed in percentage. for the determination of disease incidence of garlic the following formula was used: [27] % di = number of diseased plants number of total plants observed × 100 disease severity disease severity of purple blotch was assessed using 0-5 scale [28], as follows by randomly selected 10 plants from each plot and final data were calculated for pdi (percent disease index) estimation. grade symptoms description 0 free from infection 1 small sized lesion towards the tip, covering less than 10% leaf area 2 several dark purplish brown patches covering less than 20% leaf area 3 several patches with paler outer zone, covering up to 40% leaf area 4 long streaks covering up to 75% leaf area or breaking of leaves/stems from the center 5 complete drying of the leaves/stems or breaking of the leaves/stems from the base the percent disease index (pdi) was computed according to the formula given by (wheeler, 1969 and islam et al., 2003) [29, 30]. pdi= total sum of numerical ratings number of observations x maximum disease rating 𝑥 100 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 15 yield per hectare yields of harvested garlic bulbs were computed using electric balance after solar drying of bulbs for 10 days. the yields was expressed as kg/hectare. isolation and identification of alternaria porri diseased leaf samples were collected from field, put into brown paper envelope and, taken to the central laboratory, department of plant pathology, sher-ebangla agricultural university, dhaka for isolation. collected diseased leaves were cut into pieces regarding 1 to 1.5 mm with the help of sterilized scalpel, cleaned with sterilized distilled water and disinfected using 0.1 percent mgcl2 solution (30 to 60 seconds). then sterilized cut pieces were washed three times right away with double sterilized distilled water frequently to remove the traces of mercuric chloride and dried with a towel on sterilized filter paper, then placed to petri plates containing 20 ml of autoclaved water agar (agar 20 g with 1000 ml distilled water) in a laminar flow and incubated at 25±1°c for 10 days. after 10 days the growing mycelia on water agar petri plates were transferred to potato dextrose agar media (200g of peeled potatoes, 20 g of dextrose, and 20 g of agar and 1000 ml of distilled water). at 14 days the fungus grew well and sporulated then freshly prepare slide was observed under compound microscope and digital microscope for the identification of the pathogen using relevant literature. after identification of alternaria porri the pure culture was maintained by sub culturing at an interval every 15 days and preserved at low temperature (4⁰c) in refrigerator for future purpose. the observations were equated with the standard measurements following by ellis (1971) [31] for the identification of the pathogens (plate 1). designation of cultured isolates the cultured isolates were designated based on variety and location [32]. for example bau1i1 represents that this isolate was cultured from bau rashun-1 variety. cultural variability of alternaria porri colony diameter was recorded on the 2nd, 7th and 14th days after incubation. the data on radial growth was analyzed statistically [33]. growth per day was calculated by the followed formula: mm/ day = (growth observed on a day growth on previous observation) ∕ 2. morphological variability of alternaria porri fourteen days old cultures of a. porri isolates were studied for morphological variations viz. conidia color, shape, size, colony character and surface structure. pathogenicity test of alternaria porri to prove the association of isolated organism with the disease pathogenicity test was done using koch’s postulates conducted on the agri found onion red varieties [34, 35]. for testing the virulence level of alternaria porri isolates, bari rashun-3 variety was selected. the plants were raised in sterilized plastic pot under greenhouse condition. soil was sterilized for three consecutive days in an autoclave at 20 lbs per sq. inch pressure for one and half an hour. the plastic pots were cleaned entirely with water, rinsed with two per cent formalin and before using it was dried in the sunlight. the fumigated pots were filled with this sterilized soil nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 16 and covered with disinfected polythene sheet to block aerial contamination. after that air dried sandy loam soil and cow dung were mixed thoroughly at the ratio of 4:1 and then filled in earthen pots (20 cm diameter). no chemical fertilizers were used in the pot soil. the conidial suspension (5x105 spores ml-1) was mixed in prepared distilled water from 10 days old culture of a. porri isolates. the garlic plants of 30 days old were inoculated with these spore suspensions after garlic leaves were injured by sterile toothpick. water was sprayed consequently to the plants both before 24 hour and after inoculation the plants were covered with moist polythene bag to keep up high relative humidity (%rh) and also to inhibit natural contamination with other fungal conidia or spores. the inoculation was done on cool evening hours. the inoculated plants were maintained in greenhouse condition. on the 17 days after inoculation the severe symptoms were observed and compared with original symptoms. collection of data on leaf infection after 5 days of inoculation on garlic leaves the size of lesions was recorded on 5th, 7th, 9th, 11th, 13th, 15th, and 17th days after inoculation. size of lesions increased per day was calculated by the formula: leaf infection per day = leaf infection observed on a day−leaf infection on previous observation 2 management of purple blotch of garlic caused by alternaria porri through trichoderma, botanicals and fungicides experimental design the experiment was conducted in a randomized complete block design (rcbd) with three replication and nine treatments with the objective to attain management of purple blotch of garlic caused by alternaria porri. it was conducted in rabi season 20192020 at central farm, sher-e-bangla agricultural university, dhaka-1207, bangladesh. variety and treatments from previous evaluation, most susceptible variety bari rashun-3 was used for the management strategies of purple blotch disease. one bio-agent, five botanicals, two fungicides and a control used as treatments (table 1). preparation of plant extract fresh and healthy leaves of all five test plants were collected from the surrounding field of university for the preparation of plant extract. for the removal of dust material adhering to surfaces the collected leaves were first washed under running tap water and then in distilled water. one hundred grams (100 g) leaves from each sample were then mixed with sterile water (100 ml) at 1:1 (w/w) with the help of mortar and pestle. after through grinding the extract was filtered through muslin cloth and then through whatman filters paper no1. then the extract was passed through sieve filter to eliminate contamination. after that the extract is used as standard plant extract solution of 100% concentration of 1:1 ratio. prepared plant extract was treated at 60°c for 15 minutes for demolition of other microorganism contamination. then after the initial appearance of disease at 30 days after sowing, the foliar spray of botanicals was applied given for 3 times at 15 days interval. the foliar spray was given using hand sprayer at afternoon for better result [36]. table 1. treatments used for management of purple blotch of garlic treatment name common name scientific name / chemical name plant part/media used t1 trichoderma trichoderma harzianum liquid solution t2 lantana lantana camara leaf t3 shormoni spilanthes paniculata leaf t4 tulsi ocimum sanctum leaf t5 bon mula raphanas raphanistrum leaf t6 neem azadirachta indica leaf t7 mancozeb 80% wp ethylene (bis) di thio carbamate powder t8 sulcox 50% wp copper oxychloride powder t9 control preparation of bio-agent and fungicides the bio-agent trichoderma harzianum liquid solution was sprayed 3 times at 15 days interval after the initial appearance of disease at 30 das [36]. fungicidal solutions were prepared following the recommended doses of selected fungicides. the fungicides were mixed thoroughly using required quantity with sterilized water. it was required 2 gm/liter of mancozeb 80% wp and 3 gm/liter of sulcox 50% wp for preparation of solution for recommended concentration. the solutions of the fungicides were sprayed 3 times at 15 days interval at afternoon by hand sprayer [37]. a control treatment was maintained in each block where spraying was done with normal water only. nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 17 parameters observed data were noted on plant height (cm), number of leaves, disease incidence, and disease severity. observations on alternaria porri disease intensity were recorded on randomly selected six plants from the diseased infected leaves. screening was assessed using 0-5 rating scale [28] based on leaf area covered by the pustules. measurement of pdi described before. statistical analysis the data of different characters were statistically analyzed which were obtained from the experiment to observe the significant difference among the treatment by using the mstat-c program. conversions of the data were required when necessary. the mean values of treatments were calculated and analyzed using duncan’s multiple range test. dmrt test were executed to determine the level of significant differences and to separate the means within the parameters at 5% level of probability [38]. results evaluation of selected garlic varieties against purple blotch diseases at field condition small, whitish and sunken like lesions were marked on leaves and stalks initially as the symptoms of purple blotch disease. subsequently watersoaked lesions developed and transferred to brown. while the disease advanced, these lesions expanded and became zonate and turned into purplish color. the border of the lesions turned to purplish red encircling by yellowish brown or pale color margin. upwards and downwards extension were founded in these lesions. infected leaves turned yellow and wilted at advanced stages (plate 2). prevalence of % disease incidence and severity of purple blotch disease among selected garlic varieties significant variation was found at different days after planting in % disease incidence and severity. the results are presented in table 2. disease incidence and severity varied depending on cultivars and climatic condition. the disease incidence varied from 1.47 to 34.44% and 1.47 to 40.00% at 60 das and 90 das, respectively. whereas, the disease severity varied from 36.00 to 70.67% and 64.00 to 92.00% at 60 das and 90 das, respectively. at 60 das, the highest disease incidence (34.44%) and disease severity (70.67%) was noted on bari rashun-3, respectively. statistically similar disease severity (62.67%) was reported on bari rashun-4. on the contrary, the lowest disease incidence (1.47%) was observed on bau rashun-2 which was statistically alike with bau rashun-1 (1.80%). the lowest disease severity (36.00%) was found in bau rashun-1 and bau rashun2 variety that was statistically similar to bari rashun-1 (49.33%) and on local indian variety (44.00%), respectively. table 2. prevalence of % disease incidence and disease severity of purple blotch disease among selected garlic varieties variety (%) disease incidence (%) disease severity 60 das 90 das 60 das 90 das bau rashun-1 1.80 c 1.80 d 36.00 d 68.00 c bau rashun-2 1.47 c 1.47 d 36.00 d 68.00 c bari rashun-1 8.33 b 8.33 c 49.33 b-d 76.00 bc bari rashun-2 8.88 b 15.00 b 56.00 bc 89.33 ab bari rashun-3 34.44 a 40.00 a 70.67 a 92.00 a bari rashun-4 6.67 b 13.89 b 62.67 ab 82.67 ab local deshi 7.78 b 7.78 c 51.33 bc 76.00 bc local indian 6.66 b 7.22 c 44.00 cd 64.00 c cv 14.80 18.89 15.39 9.46 nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 18 at 90 das, the highest disease incidence (40.00%) and disease severity (92.00%) was recorded on bari rashun-3 variety. bari rashun-2 showed statistically similar disease severity (89.33%) with bari rashun-3 along with bari rashun-4 (82.67%). conversely, the lowest disease incidence (1.47%) was observed bau rashun-2 followed by bau rashun-1(1.80%), local indian (7.22%), local deshi (7.78%) and bari rashun-2 (8.88%). local indian was found as less infected variety with lowest disease severity of 64.00% which was statistically similar to bau rashun-1 and bau rashun2(68.00%) respectively. cv = coefficient of variance; in a column mean values having similar letter(s) are statistically similar and those having dissimilar letter(s) differ significantly as per 0.01% level of significance. correlation of purple blotch percent disease severity with yield (t/ha) the yield of the garlic plant affected due to disease severity. correlation analysis was done to find out the assessment of yield loss owing to disease severity. to determine the effect of disease severity on yield of selected garlic varieties correlation of coefficient was considered at 0.01% level of probability. from this correlation, it was estimated that the fresh and dry weight of yield showed negative correlation with disease severity of purple blotch of garlic and became significant at 0.01% level of probability. these values clearly expressed that less disease severity provide higher yield and vice versa (figure 1). figure 1. correlation between % disease severity of purple blotch with yield (t/ha) isolation, identification and pathogenicity of alternaria porri cultural studies of alternaria porri isolated pathogens (alternaria porri) from infected leaves of garlic transferred to water agar thereafter cultured in petri plates on potato dextrose agar (pda). cultured pathogen incubated at 25±1ºc, afterwards sub cultured for future use. the colony appearance and growth of the pathogens were monitored and noted for 14 successive days (table 3). table 3. radial mycelial growth of alternaria porri on pda media isolates radial mycelial growth (mm) 2 dai 7 dai 14 dai bau1 i1 2.80 7.65 9.00 bau2 i2 4.00 6.50 9.00 bari1 i3 3.05 7.00 9.00 bari2 i4 2.90 7.50 9.00 bari3 i5 4.10 7.65 9.00 bari3 i6 2.60 6.45 9.00 bari4 i7 4.10 6.50 9.00 bari4 i8 2.65 7.65 9.00 ld i9 3.10 7.45 9.00 lind i10 4.00 7.65 9.00 in the column bau1 i1= bau1 isolate 1; bau2 i2= bau2 isolate 2; bari1 i3= bari1 isolate 3; bari2 i4= bari2 isolate 4; bari3 i5= bari3 isolate 5; bari3 i6= bari3 isolate 6; bari4 i7= bari4 isolate 7; bari4 i8= bari4 isolates 8; ld i9= local deshi isolate 9 and lind i10= local indian isolate 10 radial mycelial growth of 10 isolate of a. porri from eight different varieties of garlic varied significantly on pda media. alternaria porri is a fast growing pathogen. colony growth of the pathogen appeared after 2 days of incubation; maximum increase (4.10 mm) of colony diameter was recorded in bari3 i5 and bari4 i7 isolates along with isolates bau2 i2 and lind i10 (4.00 mm), bari1 i3 (3.05 mm), ld i9 (3.10 mm). the minimum increment (2.60 mm) of colony diameter was found in bari3 i6 followed by isolates bari4 i8 (2.65 mm), bau1 i1 (2.80 mm) and bari2 i4 (2.90 mm). after 7 days of incubation, the maximum increment (7.65 mm) of colony diameter was recorded in bau1 i1, bari3 i5, bari4 i8 and lind i10 respectively followed by bari2 i4 (7.50mm), ld i9 (7.45 mm) and bari1 i3 (7.00mm). on the other hand, the minimum increase (6.45 mm) of colony diameter was recorded in bari3 i6. after 14 days of incubation all the colonies covered whole petridish which was 9.00mm. morphological studies of alternaria porri regular inspection was done to the pure culture of pathogen alternaria porri under the microscope to determine morphological characteristics of the pathogen viz. color, shape and surface texture. the results of morphological studies of a. porri are shown in table 4, plate 3 and plate 4. almost all the isolates showed fluffy growth appearance on potato dextrose agar. the colony color varied from olivaceous green in bau2 i2 to greyish white in bari1 i3, ashy black in bau1 i1, bari2 i4 and lind i10, off-white in bari4 i8 and black in rest of the isolates. most of the surface texture of the isolates was smooth, cottony black center with whitish to greyish periphery. fresh weight dry weight 60das -0.257 -0.073 90das -0.323 -0.089 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 y ie ld nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 19 table 4 colony characteristics of alternaria porri on pda media isolates colony characteristics color surface texture shape bau1 i1 ashy black velvety smooth regular bau2 i2 olivaceous green fluffy regular bari1 i3 greyish white fluffy regular bari2 i4 ashy black velvety smooth irregular bari3 i5 olivaceous green cottony regular bari3 i6 smoky ash fluffy regular bari4 i7 black fluffy irregular bari4 i8 off white fluffy regular ld i9 dark velvety smooth regular lind i10 ashy black velvety smooth irregular in the column bau1 i1= bau1 isolate 1; bau2 i2= bau2 isolate 2; bari1 i3= bari1 isolate 3; bari2 i4= bari2 isolate 4; bari3 i5= bari3 isolate 5; bari3 i6= bari3 isolate 6; bari4 i7= bari4 isolate 7; bari4 i8= bari4 isolates 8; ld i9= local deshi isolate 9 and lind i10= local indian isolate 10 after 14 days of inoculation all the isolates had suppressed growth on pda media. from the microscopic study of alternaria porri it was found that at first the mycelium of the fungus was hyaline then eventually turned to pale brown through olivaceous brown, smoky ash to black blended black tinge. the hypha of the conidia was septate. colony shape of the isolates was regular with concentric ring sometimes irregular. pathogenicity test of alternaria porri symptoms developed on inoculated plants was recorded from time to time. after 5 days of inoculation, tiny, water soaked, sunken, and whitish lesions on the inoculated leaves were visible. while the disease progressed, the lesions expanded, changed to elliptical to oblong, zonate and transformed reddish brown to purple encircled by pale yellow halo enlarging upwards and downwards. after 15 days of inoculation, chlorosis of the leaves was observed on the plants. the inoculated plants were dried completely after 21 days of inoculation. the symptoms were almost identical to those under field experiment. conidia of a. porri were found under compound microscope from the sectioned diseased leaves (plate 5). efficacy of selected treatment on percent disease incidence and severity of purple blotch of garlic in treated condition in case of % disease incidence, all botanicals and trichoderma were found significantly effective in reducing disease incidence as compared to control and fungicides (table 5). nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 20 the results revealed that lantana camera (lantana leaf) extracts designated as t2 was found most effective in minimizing the disease incidence (26.67%, 26.67% and 33.33%) at all 30, 45 and 60 das, respectively. the second effective treatment was t1 (33.33% and 43.33%) which was statistically alike with t8 (43.33%, 50% and 56.67%) followed by t5 (50%, 53.33% and 56.67%) respectively, whereas the maximum disease incidence was recorded in control t9 (86.67%, 96.67% and 100.00%) at 30, 45 and 60 das. in case of % disease severity, lantana camara (t2) was found most effective in reducing disease severity at 30 das (11.00%) whereas, at 45 das and 60 das (18.67% and 19.33%) t1 (trichoderma harzianum) was found most effective. at 30 das the second effective treatment was 11.33% in t3 and t8 in reducing the disease severity. on the other hand, at 45 and 60 das t2 gave the second lowest severity of (19.33% and 20.67%). all the treatments showed the statistical similar result in reducing disease incidence and severity over control. mean performance of different treatment on growth and yield parameters against purple blotch disease of garlic the results of mean performance of yield parameter due to treatment are presented in the table 6. the data revealed that all the botanical plant extracts, bio-agent and fungicides given significantly better results in comparison to control. the best plant height was noted on t4 treatment (21.55 cm, 29.07 cm and 32.67cm) at 30, 45 and 60 das, respectively in contrast to control. maximum no. of leaf per plant was found on t2, t3, t4, t5, t6 and t9 (4.00) and minimum no. of leaf per plant was found on other remaining treatment (3.67). maximum fresh weight and dry weight was found on t5 (99.33 and 75.00gm/m2) statistically similar with t6 (91.00 and 65.67gm/m2) in contrary, t9 gave poor fresh weight and dry weight of 41.67 and 24.33gm/m2. discussions preliminary symptoms of purple blotch disease were visible on the leaves in the shape of tiny, whitish, table 5. efficacy of selected treatment on percent disease incidence and severity of purple blotch of garlic treatment (%) disease incidence (%) disease severity 30 das 45 das 60 das 30 das 45 das 60 das t1 33.33 cd 43.33 cd 43.33 cd 13.33 c-e 18.67 e 19.33 e t2 26.67 d 33.33 d 33.33 d 11.00 e 19.33 de 20.67 e t3 53.33 b 60.00 b 60.00 bc 11.33 de 22.00 c-e 26.00 de t4 46.67 bc 46.67 b-d 53.33 bc 14.67 b-e 23.33 c-e 26.00 de t5 50.00 b 53.33 bc 56.67 bc 15.33 b-d 26.00 cd 46.67 c t6 56.67 b 56.67 bc 60.00 bc 16.00 bc 26.67 c 30.67 d t7 53.33 b 56.67 bc 70.00 b 18.00 b 46.00 b 55.33 b t8 43.33 bc 50.00 bc 56.67 bc 11.33 de 44.00 b 44.00 c t9 86.67 a 96.67 a 100.00 a 28.67 a 81.00 a 100.00 a cv 16.67 16.50 17.22 15.50 11.91 11.17 cv= coefficient of variance; in a column mean values having similar letter(s) are statistically similar and those having dissimilar letter(s) differ significantly as per 0.01 level of significance. here, t1=trichoderma harzianum; t2=lantana camara; t3=spilanthes paniculata; t4=ocimum sanctum; t5=raphanas raphanistrum; t6=azadirachta indica; t7=mancozeb 80% wp; t8=sulcox 50% wp and t9=control table 6. mean performance of different treatments on growth and yield parameters against purple blotch disease of garlic treatments plant height (cm) no. of leaf per plant fresh weight (gm/m2) dry weight (gm/m2) 30 das 45 das 60 das 30 das 45 das 60 das t1 17.58 c 24.77 bc 28.80 b-d 3.67 a 4.33 cd 5.33 a-c 69.33 c 48.33 c t2 21.55 ab 25.60 bc 29.08 b-d 4.00 a 5.00 a-c 5.67 a-c 64.33 cd 42.00 cd t3 18.48 bc 23.53 c 28.12 cd 4.00 a 4.00 d 4.33 c 62.00 d 39.00 de t4 21.55 ab 29.07 a 32.67 a 4.00 a 5.00 a-c 6.33 a 87.00 b 66.33 b t5 18.70 bc 26.07 b 29.16 a-d 4.00 a 5.33 ab 6.33 a 99.33 a 75.00 a t6 21.55 a 27.20 ab 32.02 ab 4.00 a 5.67 a 6.67 a 91.00 b 65.67 b t7 16.98 c 27.17 ab 30.58 a-c 3.67 a 4.67 b-d 6.00 ab 52.67 e 33.67 e t8 16.72 c 25.24 bc 28.12 cd 3.67 a 5.00 a-c 6.33 a 52.00 e 33.67 e t9 17.97 c 23.20 c 25.77 d 4.00 a 4.00 d 4.67 bc 41.67 f 24.33 f cv 7.46 5.66 6.91 7.42 8.18 15.59 5.96 9.09 cv= coefficient of variance; in a column mean values having similar letter(s) are statistically similar and those having dissimilar letter(s) differ significantly as per 0.01 level of significance. here, t1=trichoderma harzianum; t2=lantana camara; t3=spilanthes paniculata; t4=ocimum sanctum; t5=raphanas raphanistrum; t6=azadirachta indica; t7=mancozeb 80% wp; t8=sulcox 50% wp and t9=control nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 21 sunken like lesions. at the final stage, water soaked large zonate lesions of purplish red color enclosed by yellowish pale brown border was found. the symptoms and advancement of the disease were similar and have been stated by completely different researchers who recorded that white flecks like symptoms appeared at initial stage on older leaves, that enlarged and developed into elliptical to rectangular sunken zonate purple lesions with a yellow to pale brown margin under favorable environmental conditions [39, 40, 41, 42]. the highest disease incidence (34.44% and 40.00%) and severity (70.67% and 92.00%) was found on bari rashun-3 variety at 60 das and 90 das, respectively. on the contrary, the lowest disease incidence (1.47% and 36.00%) was recorded on bau rashun-2 variety. the study was almost similar to patil (1999) [43] throughout survey of purple blotch incidence of garlic he reported, maximum incidence of disease in dharwad and gokak taluks during kharif 1998 and rabi 1998-99. an indian study carried out in the horticulture garden of raichur, where the highest disease severity (49.63%) was noted and least (10.00%) was in neermanvi [44]. yadav (2013) [45] studied on the onion purple leaf blotch (plb) disease in navsari district of gujrat over two rabi seasons where the severity of the disease ranging from 11.29 to 63.73%. relative results were mentioned in [46] wherever, the highest per cent disease index (46.00) were found at the fields of sangreshkoppa village in belgaum district and at hulkund village in belgaum district the least per cent disease index (3.00) of purple blotch disease was recorded. angadi et al. (2018)[47] also attained purple blotch disease from his survey. negative correlation with yield at 0.01% level was found. these values clearly expressed that less infection of disease severity provide higher yield and the more the disease is severe the more the degradation of yield occurred. similar correlation was done by jannatun et al. (2020) [48] wherever leaf height (cm) showed negative correlation with the entire yield parameters considered and at 0.01% levels of probability it become significant. number of leaves showed positive correlation with total yield defining characters except clove diameter (-.859). almost all of the a. porri isolates showed fluffy growth on potato dextrose agar. the colony color varied from olivaceous green in bau2 i2 to greyish white in bari1 i3, ashy black in bau1 i1, bari2 i4 and lind i10, off-white in bari4 i8 and black in rest of the isolates. after 14 days of incubation all the isolates had suppressed growth on pda media. the causal agent, a. porri was isolated and pure culture was cultured on pda media. the isolated pathogen was identified as a. porri following morphological features given by neergaard (1938) [49]. in accordance with chethana (2010), chowdhury (2013) and yadav et al. (2017) [25, 50, 51] potato dextrose agar was the most acceptable culture media for mycelial growth and sporulation of alternaria porri. the cultural characteristics of different isolates of a. porri were examined by shahnaz et al. ( 2013) [52] where she recorded that almost all of the isolates had fluffy growth on pda with colony color varied from pinkish white through dull orange to olivaceous and black with distinct to diffuse patterns of zonation. mohsin et al. (2016) [53] used 27 isolates of alternaria porri which were isolated from diseased leaf samples collected from completely different onion growing regions of bangladesh and later characterized for cultural, morphological and pathogenic variabilities where colony color ranged between light to dark olivaceous and grayish white with irregular, regular with concentric ring and regular without concentric ring shape. isolates impregnated media with color ranged between grey to brown on the reverse of the plates. all botanicals and trichoderma harzianum were found significantly effective in reducing disease incidence and severity compare to untreated control and fungicides. the result revealed that lantana camara designated as t2 was found most effective in minimizing the disease incidence (26.67%, 26.67% and 33.33%) at 30 das, 45 das and 60 das. however, the maximum disease incidence was recorded in control t9 (86.67%, 96.67% and 100.00%). again, lantana camera (t2) was found most effective in reducing disease severity at 30 das (11.00%) contrarily, at 45 das and 60 das (18.67% and 19.33%) t1 (trichoderma harzianum) was found most effective. at 45 das and 60 das t2 gave the second lowest severity of (19.33% and 20.67%). datar (1994) [54] evaluated six plant extracts under field condition and noticed that maximum depletion of purple blotch was attained with leaf extract of. polyalthia longifolia. a field trial were assessed by prasad and barnwal (2004) [55] on stemphylium blight of onion (cv. n-53) during rabi, 1998-1999 and 1999-2000 crop season in bihar where, disease intensity was lowest (38.1% and 38.2%) with 20% leaf extracts of azadirachta indica. the obtained results correspond with [36, 56]. consistent with uddin et al. (2006) [57] after 10 days of sowing disease incidence (19.95 %, 13.63 %) and severity (38.87 %, 34.59 %) were reduced due to the bulb treatment with either dithane m-45 (0.45 %) or rovral 50 wp (0.2 %) followed nepal j biotechnol. 2 0 2 2 j u l ; 1 0 (1):1 3 2 4 akter et al. ©njb, bsn 22 by foliar spraying with the same, and increased seed yield by 64.82 % and 42.18 % respectively. similar results were obtained by [23, 37, 58, 59, 60, 61, 62, 63] where different treatments had inhibitory effect on fungus based on phytochemical present in plants. conclusions the present investigations showed purple blotch highest disease incidence (40.00%) and severity (92.00%) was found on bari rashun-3 variety at 90 das, respectively. on the contrary, the lowest disease incidence (36.00%) was recorded on bau rashun-2 variety. negative correlation was found between disease severity and yield (t/ha) against all identified diseases. in laboratory alternaria porri was isolated from infected leaves. after 14 days of incubation mycelial growth covered the whole petridish by alternaria porri. cultural and morphological variability exits in purple blotch (alternaria porri). all botanicals and trichoderma were found significantly effective in reducing disease incidence and severity compare to control. lantana camara (t2) was found most effective in minimizing the disease incidence (26.67%, 26.67% and 33.33%) at 30 das, 45 das and 60 das. again, lantana camara (t2) was found most effective in reducing disease severity (11.00%) at 30 das and trichoderma harzianum (t1) (18.67% and 19.33%) at 45 das and 60 das against purple blotch disease in compare to control and fungicides. acknowledgements we would like to express cordial gratitude to prof. dr. abdur rahim, horticulture department, bangladesh agricultural university, mymensingh and late arpon haider, scientific officer, bangladesh agricultural research institute, joydebpur, gazipur for providing the different garlic varieties and good cooperation. special thanks to all the teacher and staffs of plant pathology department, sher-e-bangla agricultural university, dhaka, bangladesh for helping throughout the research work. conflict of interest all authors declare that they have no conflict of interest. compliance with ethical standards the present manuscript does not contain any studies with human participants or animals performed by the authors. funding the research work did not receive any funding from any institution or organization. author contributions umme habiba akter conducted the whole research and wrote the manuscript; jannatun nahar prinky and mst. rehena khatun collected the purple blotch samples from field and helped in analyzing the data; fatema begum designed and supervised the research work and helped to correct the manuscript; m. r. islam cosupervised the research work, read the manuscript contributed to the conceptualization, and methodology of the study. references 1. voigt, c. glorious garlic herb of the year 2004. j int herb pp. 1-6. association horticulture committee, virginia state university. 2004. 2. kilgori, m, magaji, m and yakubu, a. effect of plant spacing and date of planting on yield of two garlic (allium sativum l.) cultivars in sokoto, nigeria. am-eurasian j agric environ sci. 2007;2(2): 153157. 3. asfand, r, hidayatullah, raheel, b, arshad, mu, zaffar, a and maouz, i. adaptability studies of garlic (allium sativum) advanced lines. j sci agric. 2019;3: 19-21. 4. faostat. crops. faostat. 2021. http:/ww w.fao.org/faostat/en/#data/qc/ 5. hossain, mm and abdullah, f. forecasting the garlic production in bangladesh by arima model. asian j crop sci. 2015;7(2): 147153. 6. nonnecke, i. vegetable production, new york. 1989; 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[63] rizk, fa, shaheen, am, abd elsamad, eh and ellabban, tt. response of onion plants to organic fertilizer and foliar spraying of some micronutrients under sandy soil condition. j appl sci res. 2014;10(5): 383 – 392. https://ssrn.com/abstract=2812463 nepal j biotechnol. 2 0 2 1 d e c . 9 (2): 29-32 research article doi: https://www.doi.org/10.54796/njb.v9i2.41912 ©njb, bsn 29 sero-prevalance of cryptococcal antigenemia in hiv positive individual having cd4 counts <100 cells/mm3 sundar khadka , samikshya kandel , roshan pandit , rosham manjhi, subhash dhital, jagat bahadur baniya, shravan kumar mishra, raj kumar mahato national public health laboratory (nphl), department of health services, ministry of health and population, teku, kathmandu 44-600, nepal received: 5th nov 2020; revised: 21st sep 2021; accepted: 17th nov 2021; published online: 31st dec 2021 abstract cryptococcus neoformans is one of the foremost common opportunistic infectious agents in people living with acquired immuno deficiency syndrome (aids). it has been reported to cause about 1 million cases of cryptococcal meningitis per year among hiv/aids and 600,000 deaths annually. this study was done to find the prevalence of cryptococcal antigenemia among hiv positive individuals having cd4counts <100 cells/mm3. a cross-sectional study was conducted in the hiv reference unit, national public health laboratory from july to december 2015. the study comprised of 99 hiv positive individuals having cd4counts <100 cells/mm3. cd4 t cell count was performed by flow cytometry (bd biosciences, san jose, ca, usa) and cryptococcal antigen test by latex agglutination assay. the overall prevalence of cryptococcal antigenemia was found to be 18.2%. of the total ninety-nine subjects enrolled in the study, 72 (72.8%) were males and 27 (27.2%) were females. the mean age of the patients was 38 years ranging from 13 to 69 years. higher percentage of female (22.2%) showed cryptococcal infection in our study as compared to male (16.7%). the study concludes higher prevalence of cryptococcal antigenemia among hiv infected individuals and recommends cryptococcal antigen screening to be made mandatory in hiv positive patients having cd4 t cells count below 100/µl. keywords: cd4, crag, cryptococcosis, hiv, aids, lateral flow assay. corresponding author, email: cls.sundar@gmail.com introduction cryptococcus neoformans is one amongst the most common opportunistic pathogens infecting people with advanced human immune deficiency virus (hiv). cryptococcal meningitis and cryptococcal pneumonia are the common form of cryptococcal infection in people living with human immune deficiency virus (plhiv) [1). global data estimates in 2013 shows that 31.8 million people live with hiv among which 1.4 million were reported for acquired immuno deficiency syndrome (aids) related death [2]. cryptococcal meningitis is supposed to cause 15% of aids-related mortality [2]. a study carried out in 2009 had estimated approximately 1 million cases of cryptococcal meningitis [cm) with 600,000 deaths worldwide annually [3]. cryptococcal meningitis represents a contagious form of the cryptococcal disease that necessitates hospitalization and administration of drugs like amphotericin b. the infection is common mainly in immunocompromised hiv infected individuals having cd4 t lymphocytes count less than 100/µl. the infection can be treated easily if diagnosed earlier. however, in several cases the infection is asymptomatic and thus go unnoticed leading to complications that may further lead to death of patients [4]. the world health organization (who) guideline recommends two strategies for the prevention of cryptococcal meningitis; 1) screening of patients with cd4+ t cell counts below 100 cells/mm3 for cryptococcal antigen (crag) and 2) primary prophylaxis with fluconazole for patients with cd4+ t cell counts below 100 [5]. cryptococcal antigen (crag) is a biological marker of cryptococcal infection that can be detected in serum of patients in an average of 21 days (range 5–234 days) before onset of symptoms of meningitis [6]. cryptococcal infection can be identified using several laboratory tests such as latex agglutination tests (lat), enzyme immuno assay (eia), and polymerase chain reaction (pcr); each having different sensitivity and specificity [6]. however, cryptococcal antigen detection using latex agglutination test is performed widely for screening of cryptocococcal infection as it is easy to perform and do not require sophisticated laboratory set up. timely identification of cryptococcosis in plhiv in the aids nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-5110-4446 mailto:cls.sundar@gmail.com https://orcid.org/0000-0001-5170-5500 https://orcid.org/0000-0003-2946-1233 nepal j biotechnol. 2021 dec.9 (2): 29-32 khadka et al. ©njb, bsn 30 stage prevents them from further risk of morbidity and mortality. there are different prevalence rates of cryptococcal antigenemia from different parts of the world [2, 7-9]. however, there is no such study from nepal till date to our knowledge. in this regard we carried out this study with the objective to find out the prevalence of co-infection by cryptococcus neoformans in immunocompromised patients living with hiv in nepal. materials and methods a cross-sectional study was conducted in the hiv reference unit of national public health laboratory (nphl), kathmandu, nepal from july to december 2015. samples were collected from patients visiting nphl for cd4 t cell count. all subjects enrolled in this study were hiv positive patients having cd4 t cells counts less than 100 cells/mm3. a study questionnaire was used to collect demographic data from each patient. about 3 ml of edta whole blood was collected from each patient by trained professionals for the purpose of this study. the whole blood sample was used to determine cd4 t cell count by flow cytometry using the bd fluorescent-activated cell sorter system (bd biosciences, san jose, ca, usa) as per the manufacturer’s instructions. briefly, 20 μl of bd tritest cd3/cd4/cd45 reagent was pipetted into a bd trucount tube containing bead followed by 50 μl of well-mixed anticoagulated whole blood which was then vortexed and incubated in dark at room temperature for 15 minutes for staining of the cells. following staining cells were lysed using bd facs lysing solution and run in the flow cytometer to obtain absolute count. for the purpose of cryptococcal antigen testing plasma sample was separated from edta whole blood, which was then used for antigen detection using cryptococcal latex agglutination test (immuno mycologics inc, usa). the test kit employed is based on antigenantibody agglutination reaction that detects capsular polysaccharide antigen of c. neoformans. the test kit is believed to have higher specificity and greater than 95% sensitivity. the assay procedure involves 5-easy steps with no specimen pretreatment. for test, at first a drop of lateral flow assay (lfa) specimen diluent was added to a disposable test tube. in the second step, 40 µl of the specimen was added to the tube and mixed. afterward, a crag lfa test strip was inserted into the specimen and read at 10 min. the validity of the test was indicated by a single control line and the presence of line in test region indicated a valid positive test [10]. informed consent was from each patient enrolled in this study after explaining them with the objective and outcome of this study. ethical approval was taken from nepal health research council (ref no. 892) before carrying out this study. results a total of ninety-nine hiv positive patients were enrolled in the study during the period of six months. the study comprised 72 males and 27 females which were 72.8% and 27.2% of the total, respectively. the ratio of male to female patients was 8:3. the minimum and maximum age of study population was 13 years and 69 years, respectively with the mean age of 38 years. the mean cd4 t cell count was 60.4 that range from 2 to 98 cells/mm3. of the total patients enrolled in the study, 18 (18.2%) were found positive for cryptococcal antigen. the positive to negative ratio for cryptococcal antigen was observed to be 2:11. the prevalence rate of cryptococcal antigen among male patients was found to be 16.7% while that for female patients was found to be 22.2%. in total, the prevalence rate was 18.2%. the prevalence rate was observed higher among female patients as compared to male patients table 1. table 1. prevalence of cryptococcal antigen among hiv patients (n=99) sex crag positive crag negative total male 12 (16.7%) 60 (83.3%) 72 (100%) female 6 (22.2%) 21 (77.8%) 27 (100%) total 18 (18.2%) 81 (81.8%) 99 (100%) discussion this study highlights the sero-prevalence of cryptococcal antigen in hiv positive individuals having cd4 counts less than 100 cells/mm3. hiv positive patients having cd4 count < 100 cells/mm3 is referred to be in the aids stage. these patients are in immunocompromised state and are at higher risk of opportunistic infection. cryptococcus neoformans is a fungus widely present in the environment and are able to infect immunocompromised host. thus, hiv infected individuals in aids stage are highly susceptible for opportunistic infection by several other agents beside c. neoformans. in this study, we have estimated the prevalence rate of opportunistic infection by c. neoformans in immunocompromised hiv positive individuals. the prevalence of cryptococcosis was 18.2 % among 99 hiv positive patients having cd4 count < 100 cells/mm3 in this study, which shows that a significant proportion of plhiv has cryptococcus infection in nepal. this finding is comparable to a study by micol et al. (2007) and oyella et al. (2012) but, much higher than nepal j biotechnol. 2021 dec.9 (2): 29-32 khadka et al. ©njb, bsn 31 the study by lakoh et al. [9, 11, 12]. in one study done by osazuwa et al. the prevalence rate was just 12.7% among 150 art naïve populations, which is less as compared to our study [13]. art naïve refers to a stage before receiving antiretroviral therapy (art). it is believed that persons in art have lower risk of opportunistic infection. however, the data in our study is much higher as compared to study by osazuwa et al. the difference in data might be due to the difference in exposure to the causative agent of cryptococcosis. the ratio of female to male hiv patients in our study was 2:11. the number of males 72 (72.8%) were higher than the number of females 27 (27.2%). the data is in contrary to the study of geda et al. that reported higher prevalence of hiv infection in females [14]. this may be due to differences of exposure as well as social stigma. higher number of hiv-infected males as compared to females in nepal might be another reason for the difference in enrollment of males and females in our study [15]. fungal culture is the gold standard for the detection of cryptococcus neoformans. however, some literature suggest that the latex agglutination test for cryptococcal antigen has 100% sensitivity and 96% specificity compared to culture [10]. our study shows that there is high prevalence of cryptococcal antigenemia among immunocompromised hiv positive individuals in nepal. though our national guidelines do not recommend cryptococcal screening and primary prophylaxis mandatory for every hiv infected individuals having cd4 t cell count less than 100 cells/mm3, this study recommends testing for cryptococcal infection at least by latex agglutination assay to be made as a routine test for such cases. this may be helpful in preventing morbidity and mortality caused due to meningitis among hiv infected individuals. conclusion the study revealed that 18.2 % of people living with hiv in aids stage in nepal are seropositive for the cryptococcal antigen. furthermore, in absence of gold standard test available for detection of cryptococcal infection, serological antigen screening by latex agglutination assay is helpful to detect the cases of cryptococcosis among immunocompromised hiv infected individuals. abbreviations hiv: human immunodeficiency virus aids: acquired immuno deficiency syndrome art: antiretroviral therapy cd4: cluster of differentiation 4 crag: cryptococcal antigen lat: latex agglutination tests consent for publication not applicable competing interests all the authors declare that they have no any competing interests. author’s contributions sk, sd, skm, and rkm were responsible for study design, supervision of work, and guidance. sk, sk, rp, rm, sd and jbb performed laboratory work and data analysis. sk, sk, rp, rm and sd prepared the draft and wrote the manuscript. finally, all the authors read the manuscript and approved for publication. acknowledgment we would like to acknowledge the all staffs of hiv reference unit, national public health laboratory, nepal for their valuable support in completing this work ethical approval and consent ethical approval was taken from nepal health research council (ref no. 892) before carrying out this study references 1. osazuwa f, dirisu jo, okuonghae pe, ugbebor o. screening for cryptococcal antigenemia in anti-retroviral naïve aids patients in benin city, nigeria. oman medical journal. 2012;27(3):228. 2. rajasingham r, smith rm, park bj, jarvis jn, govender np, chiller tm, et al. global burden of disease of hiv-associated cryptococcal meningitis: an updated analysis. the lancet infectious diseases. 2017;17(8):873-81. 3. park bj, wannemuehler ka, marston bj, govender n, pappas pg, chiller tm. estimation of the current global burden of cryptococcal meningitis among persons living with hiv/aids. aids. 2009;23(4):525-30. 4. smith rm, nguyen ta, ha htt, thang ph, thuy c, xuan lien t, et al. prevalence of cryptococcal antigenemia and costeffectiveness of a cryptococcal antigen screening program– vietnam. plos one. 2013;8(4):e62213. 5. who. guideline for the diagnosis, prevention and management of cryptococcal disease in hiv-infected adults, adolescents and children. world health organization (who); 2018. 6. saha dc, xess i, biswas a, bhowmik dm, padma m. detection of cryptococcus by conventional, serological and molecular methods. journal of medical microbiology. 2009;58(8):1098-105. 7. letang e, müller mc, ntamatungiro aj, kimera n, faini d, furrer h, battegay m, tanner m, hatz c, boulware dr, glass tr. cryptococcal antigenemia in immunocompromised human immunodeficiency virus patients in rural tanzania: a preventable cause of early mortality. inopen forum infectious diseases 2015 apr 1 (vol. 2, no. 2). oxford university press. 8. ezenabike c, ashaka os, omoare aa, fadeyi a, salami ak, agbede oo. cryptococcal antigen among hiv1-infected individuals in north-central nigeria. current medical mycology. 2020; 6(2):43. nepal j biotechnol. 2021 dec.9 (2): 29-32 khadka et al. ©njb, bsn 32 9. lakoh s, rickman h, sesay m, kenneh s, burke r, baldeh m, et al. prevalence and mortality of cryptococcal disease in adults with advanced hiv in an urban tertiary hospital in sierra leone: a prospective study. bmc infectious diseases. 2020;20(1):1-7. 10. crag® lfa cryptococcal antigen lateral flow assay for the detection of cryptococcal antigen. immy. 11. micol r, lortholary o, sar b, laureillard d, ngeth c, dousset j-p, et al. prevalence, determinants of positivity, and clinical utility of cryptococcal antigenemia in cambodian hiv-infected patients. jaids journal of acquired immune deficiency syndromes. 2007;45(5):555-9. 12. oyella j, meya d, bajunirwe f, kamya mr. prevalence and factors associated with cryptococcal antigenemia among severely immunosuppressed hiv-infected adults in uganda: a crosssectional study. journal of the international aids society. 2012;15(1):1-7. 13. osazuwa of, dirisu o, okuonghae e. cryptococcal antigenemia in anti-retroviral naive aids patients: prevalence and its association with cd4 cell count. acta medica iranica. 2012:344-7. 14. geda n, beyene t, dabsu r, mengist hm. prevalence of cryptococcal antigenemia and associated factors among hiv/aids patients on second-line antiretroviral therapy at two hospitals in western oromia, ethiopia. plos one. 2019;14(12):e0225691. 15. government of nepal mohap. national hiv strategic plan 20162021. in: control ncfaas, editor. nepal2017. sero-prevalance of cryptococcal antigenemia in hiv positive individual having cd4 counts <100 cells/mm3 abstract introduction materials and methods results discussion conclusion abbreviations consent for publication competing interests author’s contributions acknowledgment references nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:55-59 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 55 nepjol.info/index.php/njb prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection among the patients attending outpatient department of seti zonal hospital, dhangadi, nepal tek raj awasthi1, narayan dutt pant2*, puspa raj dahal3 1department of microbiology, siddhanath science campus, kanchanpur , nepal 2department of microbiology, grande international hospital, dhapasi, kathmandu, nepal 3department of microbiology, trichandra multiple college, kathmandu, nepal abstract involvement of multidrug resistant bacteria in causing community acquired infection is very serious public health issue. the main objective of our study was to determine the prevalence of multidrug resistant bacteria in causing community acquired urinary tract infection. in this study we cultured the 384 mid stream urine samples collected aseptically from the patients attending outpatient department of seti zonal hospital and having no past history of hospitalization. the organisms isolated were identified by using conventional biochemical tests and antimicrobial susceptibility testing was performed by kirby bauer disc diffusion technique. out of total 384 samples 98 (25.52%) samples showed significant bacterial growth. the most prevalent bacterium isolated was escherichia coli. 42.86% of the bacteria isolated were found to be multidrug resistant (mdr). the involvement of such large numbers of multidrug resistant bacteria in causing community acquired urinary tract infection is very serious issue and cannot be neglected. and some abrupt initiatives should be taken by the responsible authorities to improve or at least avoid the further worsening of the situation. key words: pyelonephritis, bacteriuria cystine lactose electrolyte deficient (cled) agar, clsi *corresponding author email: ndpant1987@gmail.com introduction urinary tract infection (uti) is the presence of multiplying bacteria within the urinary tract. the presence of significant numbers of bacteria in aseptically collected urine is the indication of urinary tract infection. despite the presence of different host defense mechanisms against microbial infection in urinary tract, uti is present as one of the commonest bacterial infections [1]. it is an important global health problem affecting millions of peoples annually, from all age groups. women are more vulnerable to uti due to presence of shorter urethra and its proximity to perianal region [2]. uti in men is less common in comparision to that in women but may be very serious when occurred [1]. people with defects which cause the retention of the urine are at high risk of getting uti. further increased rate of uti is seen in patients with catheters or tubes placed in urinary tract and patients with problems with the body’s natural defense mechanisms. the most common cause of uti is escherichia coli. other bacteria include the other members of the enterobacteriaceae, pseudomonas aeruginosa, staphylococcus saprophyticus, enterococcus spp. etc [2]. multidrug resistant bacteria are defined as the bacteria which are resistant to three or more than three different structural classes of the antibiotics [3]. the involvement of multi drug resistant bacteria in causing uti has created a serious problem for its early proper management. for the proper management of the infections hence to prevent the possible complications like chronic pyelonephritis, chronic renal failure; timely appropriate treatment as suggested by urine culture and sensitivity report is essential. but in most of the part of nepal including the farwestern region the urine culture and sensitivity is not performed due to lack of resources and competent manpower. studies on the prevalence of uti due to multidrug resistant bacteria have been carried out frequently in different parts of nepal but no such studies have been carried out in farwestern region. hence the prevalence of uti caused due to multidrug resistant bacteria is unknown in this region of nepal. further the antimicrobial susceptibility patterns of different bacteria for commonly used antibiotics are not also clear in this region. therefore the suspected patients of uti are treated just on the basis of guess most of times mailto:ndpant1987@gmail.com nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:55-59 awasthi et al. ©njb, biotechnology society of nepal 56 nepjol.info/index.php/njb resulting in treatment failure and development of more drug resistance among the bacteria. hence this study will present the clear picture of antimicrobial susceptibility patterns of different bacteria for commonly used antibiotics and the involvement of the multidrug resistant bacteria in causing uti in far western region of nepal. seti zonal hospital is the referral center for whole farwestern region. antimicrobial susceptibility pattern of the bacteria isolated from the patients (having no past history of hospitalization) with uti attending the outpatient department of seti zonal hospital was determined and prevalence of uti due to multidrug resistant bacteria was calculated. materials and methods a cross sectional study was conducted among the patients suspected of urinary tract infection visiting outpatient department of seti zonal hospital from june 2013 to december 2013. the samples were collected from only those patients who do not have past history of hospitalization. total 384 midstream urine specimens collected aseptically were cultured on cystine lactose electrolyte deficient (cled) agar by using semi-quantitative culture technique. the colonies of the bacteria from the samples with significant growth (≥105cfu/ml) after 48 hrs of aerobic incubation at 37o c were isolated and were identified up to species level with the help of colony morphology, staining reactions and conventional biochemical tests. the common biochemical tests used were oxidase test, catalase test, urease test, sulphide indole motility test, citrate utilization test, triple sugar iron test, lysine decarboxylase test, methyl-red voges proskauer test, coagulase test etc. during isolation and identification purity plate culture was used for quality control. antimicrobial susceptibility testing was performed according to the clinical laboratory standard institute (clsi 2013) guidelines by modified kirby-bauer disc diffusion method using mueller hinton agar (mha). the diameter of each zone of inhibition (in mm) was measured and results were interpreted with the help of zone size interpretive chart. control strains e. coli (atcc 25922) and staphylococcus aureus (atcc 25923) were used for the standardization of the antibiotic susceptibility testing. analysis for multidrug resistant bacteria: based on susceptibility patterns of isolates, bacteria resistant to ≥3 classes of antibiotic were considered as multi drug resistant (clsi 2013). data analysis the data obtained was entered into ms excel and analyzed using spss version 11.0. p-values <0.05 were considered statistically significant. results out of total 384 midstream urine samples, 98 (25.52%) samples showed significant bacterial growth. 29/154 (18.83%) males and 69/230 (30.00%) females had significant bacteriuria. the association of significant bacteriuria in male and female patients was found to be statistically significant (p<0.05). among 98 significant bacteriuria cases, 6 different microorganisms were isolated. among these isolates, e. coli (53.06%) was found to be the most predominant organism followed by klebsiella pneumoniae (21.43%), pseudomonas aeruginosa (12.24%), proteus vulgaris (7.14%), staphylococcus aureus (4.08%) and proteus mirabilis (2.04%) (table no.1). table no.1: different bacteria isolated from urine of the patients. susceptibility of gram negative bacteria towards different antibiotics among the common antibiotics used against all gram negative bacteria, the most effective antibiotic was found to be gentamicin (57.45%) followed by ceftriaxone (51.06%), nitrofurantoin (45.75%), cotrimoxazole (32.98%), ofloxacin (29.79%), nalidixic acid (15.96%), and ampicillin (5.32%) (table no. 2). name of the bacteria numbers isolated (%) escherichia coli 52 (53.06%) klebsiella pneumoniae 21 (21.43%) pseudomonas aeruginosa 12 (12.24%) proteus vulgaris 7 (7.14%) staphylococcus aureus 4(4.08%) proteus mirabilis 2 (2.04%) nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:55-59 awasthi et al. ©njb, biotechnology society of nepal 57 nepjol.info/index.php/njb table no.2: susceptibility of gram negative bacteria towards different commonly used antibiotics. antibiotics numbers of sensitive gram negative bacteria (%) gentamicin 57.45 ceftriaxone 51.06 nitrofurantoin 45.75 co-trimoxazole 32.98 ofloxacin 29.79 nalidixic acid 15.96 ampicillin 5.32 among the 4 gram positive bacteria all isolates were staphylococcus aureus. all of them were susceptible to gentamicin and 3(75.00%) were susceptible to ceftriaxone. half of the s. aureus isolates were susceptible to ampicillin, co-trimoxazole and ofloxacin each. 1(25.00%) isolate was found to be susceptible to nalidixic acid and nitrofurantoin each. multidrug resistance among various bacteria out of 98 isolates, 42 (42.86%) were found to be mdr. 48.08% of the e. coli, 19.05% of the k. pneumonia, 50% of the p. aeruginosa, 85.71% of the p. vulgaris and 25% of the staphylococcus aureus were found to be mdr. no isolates of p. mirabilis were mdr (table no. 3). table no.3: multidrug resistance among different bacteria. name of bacteria number of multi drug resistant bacteria (%) escherichia coli 48.08 klebsiella pneumoniae 19.05 pseudomonas aeruginosa 50 proteus vulgaris 85.71 staphylococcus aureus 25 proteus mirabilis 0 discussion out of total 384 urine samples 98 (25.52%) samples showed significant growth. similar type of result was obtained by sharma et al (27.3%) [4]. females are more prone to uti than males. in the present study also, same fact was observed where the rate of growth positivity was found to be 30.00% (69/230) in females and 18.83% (29/154) in males. our findings were consistent with the findings by shrestha et al [5] who reported the culture positivity of 29.8% in females and 15.2% in males. in a similar study by baral et al. [6] the growth positivity was 33.5% among female patients and 23.7%, in male patients. this higher growth positivity seen in females was found to be statistically significant (p<0.05) and is due to their anatomical structure (short urethra and proximity to anal orifice) leading to easy access for enteric bacteria. among the total 98 bacterial isolates, 94(95.92%) were gram negative bacilli and only 4 (4.08%) were found to be gram positive cocci. the results were in the favor of the findings of shrestha et al [5] and karki et al [7]. in the study by shrestha et al [5] among the total 80 bacterial isolates, 75 (93.8%) were gram negative bacilli and only 5 (6.3%) were gram positive cocci. similarly karki et al [7] found 91.1% of the isolates from urine to be gram negative bacilli and 8.8% of them to be gram positive cocci. among 98 significant bacteriuria cases, 6 different species of the bacteria were isolated. among these isolates, e. coli 52 (53.06%) was found to be the most predominant organism followed by klebsiella pneumoniae 21(21.43%), pseudomonas aeruginosa 12(12.24%), proteus vulgaris 7(7.14%), staphylococcus aureus 4(4.08%) and proteus mirabilis 2(2.04%). in a similar study done by khanal et al, out of 41 isolates isolated from mid stream urine samples 8 different species were isolated among which e. coli (65.85%) was found to be most predominant organism followed by klebsiella pneumoniae (9.75%) [8]. the high prevalence of e. coli in causing uti also resembled with the studies by raza et al in kathmandu, nepal [9] and patel et al in india [10]. e. coli can bind to the glycoconjugate receptor (gal α 1-4 gal) of the uroepithelial cells of human urinary tract with its unique virulence determinant, the p pilus (gal-gal receptor) so as to initiate the infection [11]. as in the study by gautam et al [12] all the gram positive isolates were staphylococcus aureus and were obtained from female patients only. antibiotic resistance is a serious public health concern and is associated with prolonged hospitalization, high health-care cost, increased morbidity and mortality. in our study, gentamicin (57.45%) was found to be the most effective antibiotic against gram negative bacteria followed by ceftriaxone (51.06%) and nitrofurantoin (45.75%). in a similar study carried out by jha and bapat at sukhraraj tropical hospital, kathmandu, nepal 92.5% of urinary isolates were found to be susceptible to gentamicin [13]. in a study by khanal 50% of gram negative organisms were sensitive to ceftriaxone and 58.34% of the gram nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:55-59 awasthi et al. ©njb, biotechnology society of nepal 58 nepjol.info/index.php/njb negative isolates were sensitive towards nitrofurantoin [8]. on the other hand, in our study ampicillin was found to be the least effective drug against gram negative bacteria (5.32% sensitive). resistance to penicillins may be determined by the organisms due to the production of penicillin destroying enzymes (βlactamase). 15.96%, 29.79% and 32.98% of the gram negative bacilli were susceptible to nalidixic acid, ofloxacin and cotrimoxazole respectively. these results are in accordance with the results of shrestha [5]. similar findings were also given by gautam et al [12] and sharma et al [4]. among gram positive cocci gentamicin was found to be 100% effective where as nalidixic acid and nitrofurantion were found least effective with 25% of the isolates being sensitive to each antibiotic. the exposure of the bacteria to antibiotic causes selective pressure causing the killing of susceptible bacteria allowing the resistant ones to survive. the rapid development of the antibiotic resistance among the bacteria is attributed to the haphazard use of antibiotics [14]. the problem of the drug resistance among bacteria is more prevalent in developing countries due to lack of awareness and lack of effective implementation of the policy that regulates the use of antibiotics. out of 98 isolates, 42.86% were found to be multi drug resistant. the finding of the present study was supported by the results of the study done by khanal [8] and upadhaya et al [15] noted the mdr causing uti to be 56.09% and 48% respectively. in our study 48.08% of the e. coli, 19.05% of the k. pneumonia, 50% of the p. aeruginosa, 85.71% of the p. vulgaris and 25% of the staphylococcus aureus were found to be mdr. similar findings were obtained in the study done by tuladhar et al [16] in a hospital in kathmandu, where mdr bacterial strains were detected in 35.21% with the most predominant mdr bacterium being e. coli followed by klebsiella spp. increasing haphazard use of antibiotics and sales of substandard drugs are responsible for development of multi drug resistance among the bacteria [17]. due to development of drug resistance against commonly used antibiotics among the bacteria the therapeutic options have become limited. the isolation of the multi drug resistance bacteria from the patients with no past history of hospitalization indicates that the infection was community acquired. the involvement of such large number of multidrug resistant bacteria in causing community acquired urinary tract infections is very serious issue and cannot be neglected. some abrupt initiatives should be taken by the responsible authorities to improve or at least avoid the further worsening of the situation. conclusion involvement of multidrug resistant bacteria in causing large numbers of community acquired infections is a very serious public health concern. some necessary initiatives should be taken immediately to control the situation. competing interests the authors declare that they have no competing interests. references 1. leigh da, smith gr, easman csf: topley and wilson's principles of bacteriology, urology and immunology, bacterial diseases, 8th edition, frome and london; butter and tanner ltd 1990,3:197-214. 2. forbes ba, sahm df, weissfeld as: baily and scott's diagnostic microbiology, 11th edition. mosby inc, usa; 2002. 3. tuladhar nr: a report: surveillance of multiple drug resistant (mdr) bacterial infection among the patients attending to different out patient departments (opd) and hospitalized patients in tribhuvan university teaching hospital; 2001. 4. sharma ar, bhatta dr, shrestha j, banjara mr: antimicrobial susceptibility pattern of escherichia coli isolated from urinary tract infected patients attending bir hospital. nepal j science and technology 2013, 14(1): 177-184. 5. shrestha p: study of bacteria causing urinary tract infection and their antimicrobial resistance trend at national public health laboratory. msc thesis. tribhuvan university, central department of microbiology; 2007. 6. baral p, neupane s, marasini bp, ghimire kr, lekhak b, shrestha b: high prevalence of multidrug resistance in bacterial uropathogens from kathmandu, nepal. bmc research notes 2012,5:38. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:55-59 awasthi et al. ©njb, biotechnology society of nepal 59 nepjol.info/index.php/njb 7. karki a, tiwari br, pradhan sb: study of bacteria isolated from urinary tract infection and their sensitivity pattern. j nepal med assoc 2004,43:200203. 8. khanal s: a study on microbiology of urinary infection at tribhuvan university teaching hospital, kathmandu, nepal. msc thesis. tribhuvan university, central department of microbiology; 2006. 9. raza s, pandey s, bhatt cp: microbiological analysis of the urine isolates in kathmandu medical college teaching hospital, kathmandu, nepal. kathmandu univ med j 2011,36(4):295-7. 10. patel s, taviad pp, sinham, javadekar tb, chaudhari vp: urinary tract infections (uti) among patients at g.g. hospital & medical college, jamnagar. national j community medicine 2012, 3(1): 138-41. 11. johnson jr: virulence factors in escherichia coli urinary tract infection. clin microbiol rev 1991,4:80128. 12. gautam k, pokhrel bm: prevalence of urinary tract infection at kanti children’s hospital. j chitwan medical college 2012, 1(2):22-5. 13. jha n, bapat sk: a study of sensitivity and resistance of pathogenic microorganisms causing uti in kathmandu valley. kumj 2005,3:123-9. 14. dalhoff a: global fluoroquinolone resistance epidemiology and implictions for clinical use. interdisciplinary perspectives on infectious diseases 2012, 12:37. 15. upadhyay g, shakya g, upadhyaya bp, shrestha s, ansari s, ghimire p et al: comparative evaluation of urine isolates among kidney transplanted and other uti suspected patients visiting national public health laboratory, (nphl) teku, nepal. int j biomedical and advance research 2013,4(6):36975. 16. tuladhar nr, banjade n, pokhrel bm, rizal b, manandhar r, shrestha s, shah a and chaurasia s. antimicrobial resistant bacterial strains from inpatients of tribhuvan university teaching hospital kathmandu. j inst med 2003, 25:19-26. 17. gautam r, chapagain ml, acharya a, rayamajhi n, shrestha s, ansari s, upadhaya g, nepal hp. antimicrobial susceptibility patterns of escherichia coli from various clinical sources. jcmc 2013,3:14-7. 18. baral r, timilsina s, jha p, bhattarai nr, poudyal n, gurung r, khanal b, bhattachary sk. study of antimicrobial susceptibility pattern of gram positive organism causing uti in a tertiary care hospital on eastern region of nepal. health renaissance 2013, 11(1): 119-24. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:6-9 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 6 nepjol.info/index.php/njb detection and quantitation of aflatoxin f or the diagnosis of aspergillus flavus geeta rajbhandari shrestha1* and amin udhin mridha2 1amrit campus, tribhuvan university, kathmandu, nepal. 2university of chittagong, chittagong, bangladesh abstract aflatoxins are the potent mycotoxins produced by aspergillus flavus, which is hepatotoxic causing hepatocellular carcinoma. a. flavus produces sufficient amount of aflatoxin b1 under favourable environments. inhalation of spores and use of aflatoxin b1, contaminated food by aspergillus spp., could transfuse the toxins in the blood streams. the presence of these toxins in body fluid can be detected by immunological assays and which provides an effective technique for the diagnosis of the disease caused by a. flavus. aflatoxins producing strain of a. flavus were screened in aflatoxin producing medium. production of aflatoxin b1 by a. flavus was studied in different parameters such as incubation periods, temperatures, ph variations, sucrose concentration in yeast extract sucrose medium and different natural media such as par-boiled rice, corn and groundnuts. the detection of toxins was done by tlc using silica gel (merk) coated plates and confirmative test was done by association of official analytical chemists (aoac) method. presence and quantization was done by enzyme linked immunosorbent assay (elisa) technique. highest amount of aflatoxin b1 was reported 68.56 ng/ml by elisa in synthetic medium (yeast extract sucrose) with 2% sucrose, ph 5.5, on 14th days of incubation, at 28±10c (p-value 0.05). similarly, highest amount was recorded in groundnuts (121.20ng/g) by elisa and (500ng/kg) by tlc methods. elisa is one of the most efficient methods used for detection and diagnosis of human diseases cause due to exposure of aflatoxin b1 and a. flavus. key words: aflatoxin, aspergillus flavus, elisa, aoac, tlc *correspondence author: email: ga.gi.cha@hotmail.com introduction mycotoxins are secondary metabolites produce by different fungi under certain environmental conditions. mycotoxins cause acute kidney failure (ochratoxin), damage of central nervous system (tremogenic mycotoxin) and damage the upper respiratory tract. aflatoxins are the most potent and naturally occurring mycotoxins produce by aspergillus flavus and a. parasiticus. many factors affect the growth of fungi and contamination aflatoxins on foods and feeds. different factors affecting aflatoxins contamination include the climate of the region, the genotype of the crop planted, soil type, minimum and maximum daily temperatures, and daily net evaporation. contamination of toxins can occur at any time of growth of plant, pre and post harvesting periods and storage conditions [1]. it was found that high doses (>6000mg) exposure of aflatoxins may cause acute toxicity with lethal effect and prolonged exposure to small doses is carcinogenic [2]. mainly aflatoxin b1 (aftb1) is the potent carcinogenic toxins to some animals and humans [3], which is one of the leading cause of cancer deaths worldwide [4]. when animals are exposed to through the contaminated feeds, the toxin is converted into aflatoxinm1 and contaminate in their milk. it is one of the sources of contamination aflatoxins of dairy products [5]. the exposure of aflatoxins causes many diseases such as hepatocellular carcinoma (hcc), impaired growth, immune suppression etc. exposure of aflatoxins and the risks of toxic doses are more prevalent in the poor nations worldwide in both urban–rural areas, however more strongly in the rural populations [6]. these diseases are more common in most of the developing countries. it was estimated that 550,000– 600,000 new hcc cases worldwide each year, of which about 25,200–155,000 were due to aflatoxins exposure. most of the people from developing countries such as sub-saharan africa, southeast asia, and china including nepal are suffering from hcc. their prevalence accounted largely due to aflatoxins contamination in food [7]. it is essential to control contamination of food and feeds for minimization of outbreak due to aflatoxins. the presence of aftb1 biomarker reflects the formation of the reactive metabolite and the level of dna damage in the livers. enzyme linked immunosorbent assay (elisa) is one of the diagnostic test, depends upon nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:6-9 shrestha and mridha ©njb, biotechnology society of nepal 7 nepjol.info/index.php/njb protein content, is used to determine aflatoxins qualitatively and quantitatively in the samples [8]. elisa and a monoclonal antibody against afb1, afb1 bound to albumin can be used to detect aflatoxins in urine and blood samples. the presence of aflatoxins residues adducts, and metabolites are assayed directly in tissues, fluids and excreta [9] and analyzed. the method is easy and inexpensive that developed with the necessary reliability, accuracy, and sensitivity to bring immunoassay technology. materials and methods aspergillus flavus was isolated from the atmosphere of kathmandu by gravity plate method. lyophilized aflatoxins producing strains of a. flavus was obtained from united states department of agriculture and used as the reference to compare the aflatoxin b1 production with that isolated from air. aflatoxins standard was purchased from sigma co. the different strains of a. flavus producing aflatoxins were screened on aflatoxin producing medium (apm) as described by donald et al. (1981) [10]. extraction of aflatoxin b1 was carried out as the method described by abarca et al, (1988) and chu (1987) [11, 12]. detection of aflatoxin b1 was done by thin layer chromatography (tlc) as the method described by using pre-coated silica gel plates (merck) [13]. the confirmation test was done by association of official analytical chemists (aoac) method [14]. quantization of aftb1 in different samples were carried out by elisa in various parameters such as incubation periods (7th, 9th, 11th, 13th, and 15th, days), ph variations (4.5, 5.5 and 6.5), temperatures (240, 280 and 320c), and concentrations of sucrose (1.5%, 2% and 2.5%) in yeast extract sucrose medium (yes) and different media like; synthetic low salt medium (sls), coconut medium (cm), natural media such as par-boiled rice, corn and groundnut described by davis et al.1966 [15]. for the study of production of aflatoxin b1, three replicas were maintained in each parameter. the experimental results from this study were analyzed by spss 16.0 [10, 12]. results the highest amounts of aftb1 (68.56 ng/ml) was recorded in yes medium with 5.5 ph, 2% sucrose and after 14 days of incubation at 28 ±1°c (figure 1a). the various temperatures have significant effect on the production of aftb1 (figure 2b). however, sucrose concentrations different and ph have no significant the effect, since p-values are more than 0.050 (figure 2a and figure 2b). figure 1: effect of a. incubation periods b. temperature on production of aflatoxin b1 figure 2: effect of a. sucrose concentration and b. ph in yes medium on the production of aflatoxin b1 spearman's rho test showed that there is no significant linear relation between two variables incubation period (time in days) and production of aftb1 (ng/ml) as the p-value is more than 5% level of significance (table 1). table 1: analysis of aflatoxin b1 produced by a.flavus; tlc and elisa method in various incubation periods. note: ++ denotes for intensity of fluorescence kruskal-wallis test showed that there is no significant effect of a particular medium on the production of aftb1 (the p-value of 0.998 is more than 5% level of significance). moreover, each of six medium has equal effect on it. however, the highest no. incubation periods* aflatoxin b1 tlc elisa ng/ml 1 7 + 2.6 2 9 ++ 6.46 3 11 +++ 24.76 4 13 +++ 68.56 5 15 +++ 58.8 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:6-9 shrestha and mridha ©njb, biotechnology society of nepal 8 nepjol.info/index.php/njb level (121.20ng/g) of alfatoxin b1 was obtained in natural medium; groundnuts. note: yes = yeast extract medium, sls= synthetic low salt medium, cm= coconut medium figure 3: effect of different media on the production of aflatoxin b1.in different incubation periods at 28 ±1°c. discussion aflatoxins produced by food borne aspergillus flavus and a. parasiticus cause several human diseases such as hepatocellular carcinoma (hcc), or liver cancer. contamination of aflatoxins can occur at any stage of food production from pre-harvest to storage [16]. its contamination is determined by the most commonly used method; thin layer chromatography (tlc). the thin layer chromatography method was used for screening and quantification. aflatoxins production is low during early stages of growth that increases with the decline of growth and the maximum production was recorded after the stationary phase of growth, which soon decline in its production (table 1 and figure 1). similarly, west et al. (1973) reported that the aflatoxin level falls after reaching a maximum, which might be due to non-specific chemical mechanism for degradation of the toxin [17]. smith and moss (1985) also reported that very low aflatoxins was produced during the phase of vigorous growth in a laboratory and when some nutritional factor runs out and limits growth toxins biosynthesis occurs rapidly [18]. factors that affect aflatoxins contamination include the climate of the region, the genotype of the crop planted, soil type, minimum and maximum daily temperatures, and daily net evaporation. wilson and payne (1994) and fandohan et al. (2005) reported that many factors affect the growth of aspergillus fungi and the level of aflatoxins production in food [16, 19]. it is essential to reduce aflatoxins contamination of food by providing unsuitable condition to produce toxins. aftb1 production is also promoted by stress or damage to the crop due to drought prior to harvest, insect activity, poor timing of harvest, heavy rains at harvest and post-harvest, and inadequate drying of the crop before storage [20]. humidity, temperature, and aeration during drying and storage are also important factors for aflatoxins contamination. the most effective method of detection and quantification of aflatoxins contamination in foods and feeds is elisa by which a very low amount of it can be detected [12]. in this study the highest amounts of aftb1 (68.56 ng/ml) was recorded in yes medium with 5.5 ph, 2% sucrose and after 14 days of incubation at 28 ±1°c. the study on the effect of different parameters on aflatoxin b1 production showed that temperature has significant effect (p= 0.050). however, different ph and sucrose concentrations have no significant effect, since p= > 0.050. this method can be employed for the detection and quantification of aflatoxins exposure of human and causes of hepatocellular carcinoma due to aspergillus flavus from urine, blood samples. groopman et al (1994) showed that aflatoxin metabolites in urine reflect recent exposure (i.e. 2-3 days) whereas the measurement of aflatoxins albumin adducts in blood reflects exposure over a longer period (i.e. 2-3 months) [21]. qian et al (1994) studies showed correlation of aflatoxins intakes to biomarker levels and to disease [22]. conclusions the spores of a. flavus under the favourable environmental conditions produce sufficient amount of aftb1 that effect on human health. aftb1 had been detected and quantified by tlc and elisa. aflatoxin b1 could use as a marker for the diagnosis of various diseases caused by aspergillus species, especially a. flavus. more research is needed to determine aflatoxins levels in biological specimens that are associated with adverse health effects. acknowledgement we are highly obliged to unesco for providing fellowship to execute the work in china and very much thankful to prof. dr. zhang yizeng, the head of the department of molecular biology, sichuwan university, prof. dr. guangian wang, dept. of sanitary technology, school of public health, west china university of medical science, chengdu, china. we are grateful to dr. peterson sw, microbiologist, united states department of agriculture, midwest area, and national center for agriculture utilization research for providing lyophilized aflatoxins producing strains of a. flavus. references 1. wilson dm, payne ga: factors affecting aspergillus flavus group infection and aflatoxin contamination of the crops. the toxicology of b nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:6-9 shrestha and mridha ©njb, biotechnology society of nepal 9 nepjol.info/index.php/njb aflatoxins: human health, veterinary, and agricultural significance. dl eaton and j d groopman. san diego, ca, academic press, inc. 1994: 309-325 2. groopman jd and donahue kf: aflatoxin, a human carcinogen: determination in foods and biological samples by monoclonal antibody affinity chromatography. j of assoc. of off. anal. chem. 1988, 71: 861-867. 3. iarc: some naturally occurring substances: food items and constituents. iarc monographs on evaluation of carcinogenic risk to humans 1993: 56 4. who: the global burden of disease: 2004 update. geneva: world health organization; 2008. [accessed 27 april 2010]. 5. strosnider h, azziz-baumgartner e, banziger m, bhat rv, breiman r, brune m: workgroup report: public health strategies for reducing aflatoxin exposure in developing countries. environ health perspect, 2006, 114:1898–1903. 6. plymoth a, viviani s, hainaut p: control of hepatocellular carcinoma through hepatitis b vaccination in areas of high endemicity: perspectives for global liver cancer prevention. cancer lett 2009, 286(1):15–21 7. liu y, wu f: global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment environmental health perspectives, 2010, 118(6): 818-824. published online 2010 feb 19 doi: 10.1289/ehp.0901388. 8. ozaslan m, caliskan i̇, kilic ih, karagoz id: application of the elisa and hplc test for detection of aflatoxin in pistachio, scientific research and essays 2011, 6(14):2913-2917. 9. bennett jw, klich m: mycotoxins. clinic microbio 2003, 16(3):497–516. 10. donald t, wicklow ol, shotwell la, gordon : use of aflatoxin producing ability medium to distinguish aflatoxin producing strains of a.flavus. appl.eviron. micribiol. 1981, 41: 697-699. 11. abarca ml, bragulat mr, bruguera mt and cabanes fj: comparison of some screening methods for aflatoxigenic moulds. mycopathologia, 1988 104: 75-79. 12. chu fs: current immunochemical method of aflatoxin in groundnuts and groundnut products. in aflatoxin contamination of groundnut. icrisat. india: proc int works. 1987 :6-9. 13. ren p, ahearn dg, crow sa: comparative study of aspergillus mycotoxin production on enriched media and construction material. j ind microbiol biotechnol 1999, 23:209-213 14. horwitz e: official methods of analysis of the association of official analytical chemists, aoac, washington d.c. 1975: 462467. 15. davis nd, diener ul, eldridge: production of aflatoxins b1 and g1 by aspergillus flavus in semisynthetic medium. app. microbiol. 1966, 14(3): 370-380. 16. wilson dm, payne ga: factors affecting aspergillus flavus group infection and aflatoxin contamination of the crops. the toxicology of aflatoxins: human health, veterinary, and agricultural significance. dl eaton and j d groopman. san diego, ca, academic press, inc. 1994: 309-325. 17. west s, wyatt rd, hamilton pb: improved yield of aflatoxin by incremental increases of temperature. applied microbiol 1973, 25:10181019. 18. smith je, moss mo mycotoxins formation, analysis and significance. john e. smith & sons chicherster,new york. brisbane tokonto. singapore, 1985. 19. fandohan p, zoumenou d, hounhouiganb dj, marasasc wfo, wingfieldd mj, helle k: fate of aflatoxins and fumonisins during the processing of maize into food products in benin. int j food microbiol 2005, 98(3): 249-59. 20. hell k, cardwell kf: the influence of storage practices on aflatoxin contamination in maize in four agroecological zones of benin, west africa. j stored prod res 2000. 36(4): 365-382. 21. groopman jd, wogan gn, roebuck bd, kensler tw: molecular biomarkers for aflatoxins and their application to human cancer prevention. cancer res 1994, 1(54) (suppl 7):1907-1911 22. qian gs, ross rk, yu mc, yuan jm, gao yt, henderson be, wogan gn, groopman jd: a follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in shanghai, people's republic of china. cancer epidemiol biomarkers prev 1994, 1:3-10. microsoft word editorial.docx the  future  science     the  current  thrust  of  scientific  discourse  in  biological  science  is  centered  on  exploration  of  the  intrinsic   factors  directly  involved  in  facilitating  the  life,  and  determining  other  ancillary  factors  maintaining  it  in   proper  order  for  conglomeration  into  the  concrete  life  system.  one  of  the  trademark  breakthroughs  has   been   the   venterian   artificial   life,   in   which   whole   life   was   created   artificially   mediated   by   genetic   reconstitution  in  scaffold  of  cellular  system.  similarly,  rapid  interest  and  flux  of  cutting  edge  research  is   centered   to   understanding   and   thus   devising   mechanism   to   control   the   metabolic   and   physiological   profile   to   prevent   various   diseases   such   as   alzheimer’s   disease,   parkinson   disease,   melanomas,   sarcomas  etc.  in  this  effort  whole  consortium  of  scientific  community,  academicians  and  researchers  are   putting   their   hands   together   for   creation   of   panoramic   globe,   where   there   is   cure   for   every   kind   of   diseases  or  disorders.     one  of  the  most  important  aspects  associated  with  the  fast  moving  technological  intervention  has  been   co-­‐ordination,   co-­‐operation   and   integration   of   scientific   communities,   for   which   the   current   communication  technology  is  responsible.  due  to  amalgamation  of   ideas  and  knowledge  of  scientists   across  the  cross-­‐country  boundaries,  the  scientific  pulse  is  rapidly  increasing.  but  still   in  this  phase  of   transition  of  world,  it  can  be  felt  that  some  patch  on  the  globe  is  still  undermined  and  to  some  extent   this  part  is  unaware  of  the  technological  charisma.  but,  this  part  too  has  some  crucial  contribution  when   conclusion  has  to  be  summed  up  taking  into  consideration  of  the  whole  globe  as  a  single  system.   the   crux   of   technological   impetus   has   been   the   utilization   of   natural   resources,   so   the   developed   community  is  moving  forward  for  the  world  with  flux  of  artificial  stuffs.  the  current  research  in  top  tier   economic  colossus  is  pivoted  on  automation  with  robotics,  complementation  and  supplementation  of   requisites  with  artificial  stuffs,  and  designing  of  efficient  strategies  to  curtail  the  natural  requirements.   but,   regarding   the   sustainability   it   is   still   questionable   in   terms   of   rationality.   in   contrast,   on   the   backdoor   we   can   realize   that   undermined   patch   of   world   is   still   furbished   with   enormous   natural   resource   in   terms   of   flora   and   fauna.   it   is   unquestionable   that   we   can   go   for   sustainable   use   of   the   natural  resources  rather  than  purging  all  our  effort  in  artifice  creation.  it  is  more  rational  to  devise  the   strategic  approaches  to  maintain  the  sustainable  utilization  of  the  natural  resources  available.  for  that   the   idea   from   the   developed   community   should   be   mingled   with   the   natural   resources   available   in   hotspots  patches  of  globe,  to  create  a  utopian  era.  hence,  the  future  science  should  be  be  natural   with  sustainability!   dipesh dhakal executive editor nepal journal of biotechnology nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 2-5 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 2 nepjol.info/index.php/njb accumulation of poly-hydroxy-butyric acid (phb) by bacillus strain isolated from paddy field of kathmandu university premises deepak upreti, naresh prasad sapkota, bibek aryal, sangita shakya* department of biotechnology, kathmandu university, dhulikhel, nepal abstract in this study, the effect of applying nutrient limitation on the production of poly-hydro-oxy-butyric acid (phb) from soil bacteria was examined. phb is a biodegradable polymer which provides a reserve of carbon and energy. phb was extracted by chloroform dispersion method. the amount of synthesized phb was determined as crotonic acid by spectrophotometry. we found that nitrogen limiting condition stimulated phb accumulation. the highest level of phb accumulation was observed in dnb-6 strain which accumulated 31 % of the dry mass at 20 % glucose concentration. the probabilistic identification of bacteria by pibwin software version 1.9.2 showed that the strain dnb-6 was close in nature to bacillus cereus. keywords: polyhydrooxybutyrate, cell dry weight, nutrient limitation, biodegradable, bioplastic. *corresponding author email: sangita@ku.edu.np introduction poly-hydro-oxy-butyric acid (phb) is a member of a polymer belonging to a group polyhydroalkanoate (pha). a wide variety of prokaryotic organisms have been shown to accumulate this polymer, including numerous heterotrophic and autotrophic aerobic bacteria, photosynthetic anaerobic bacteria, gliding bacteria, actinomycetes species, cyanobacteria [1]. phb is a biodegradable thermoplastic which provides a reserve of carbon and energy and accumulates as intracellular granules when grown under nutrient limiting conditions [2]. three distinct enzymatic reactions involved in the phb biosynthetic pathway. the initial reaction involves condensation of two acetyl-coa molecules to form acetoacetylcoa, which is catalyzed by ß-ketothiolase encoded by phba. this step is followed by the reduction of acetoacetyl-coa by an nadphdependent acetoacetyl-coa dehydrogenase encoded by phbb. then, the (r)-3hydroxybutyrlcoa monomers are polymerized into phb by phb synthase encoded by phbc (figure 1) [3]. the synthetic plastics are made from petrochemicals which are not renewable. they do not readily biodegrade and often the collection and transport of this waste is difficult and expensive. due to the non biodegradable characteristics of petrochemicalsderived plastic materials much interest has been created in the development of biodegradable plastics. biodegradation is chemical degradation of materials brought about by the action of naturally occurring microorganisms such as bacteria and algae [4]. biodegradable plastics produced from renewable sources are considered a potential substitute for conventional petrochemical plastics because of their biodegradability and non-toxicity characteristics [1, 57]. phb is one of the potential raw materials for producing biodegradable plastic. the phb production capacities of bacteria have been investigated for possible application in industry. however, the use of phb in industrial applications has been hampered mainly by their high production cost compared to petrochemical-based polymers [8,9]. the condition for bacterial phb production can be met in soil, due to its heterogeneous nature. it may become a limiting factor for bacterial growth especially in some nitrogen poor (carbon-rich) sites. soil bacillus species have been shown to accumulate phb during the sporulation of bacterial growth. phb production by the isolate has been favoured by the glucose concentration [10]. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 2-5 upreti et al. ©njb, biotechnology society of nepal 3 nepjol.info/index.php/njb this study makes an effort to evaluate the phbproducing efficiency of bacillus species isolated from soil, located in the paddy field of kathmandu university premises under nutrient limited conditions. the polymer has been extracted from a selected bacillus species strain and characterized partially. materials and methods isolation and purification of soil bacteria the soil sample for the isolation of bacteria was collected during the months of march to may. soil samples were collected from five different areas of paddy field of kathmandu university premises. fifteen soil isolates were named as (dnb-1 – dnb-15) and further screened randomly to check their phbproducing efficiency. each gram of the sample was suspended in 9 ml of sterile distilled water and shaken vigorously for 2 min. the sample was heated at 80 °c for 10 minutes in water bath. after heating, dilution of 10-3, 10-4, 10-5, 10-6 g/ml were prepared from the soil suspension for plate counts and spread on nutrient agar medium. after incubation at 30 °c for 2448 hours, serially diluted plates were picked up and examined microscopically. as standard practice, plates having 30-300 colonies were chosen for isolating the single bacterial colony. the spore morphology, gram staining, motility and several biochemical tests were carried out to characterize the bacteria. the isolate was identified on the basis of comparison of these characters with those described in bergey’s manual of determinative bacteriology [11]. screening for phb production to test the production of phb, seed culture of isolated colonies were grown in 10 ml nutrient broth and incubated for 24 hours at 30 °c. later, the seed culture was transferred to 50 ml in 250 ml erlenmeyer flasks with a 2% (v/v) inoculums and incubated at 30 °c with vigorous orbital shaking at 150 rpm. the culture was grown to a stationary phase and the cultures were examined by fluorescence microscopy after staining with nile red. it has been proved that nile red is an excellent vital stain for the detection of intracellular lipids. the cells were flame-fixed on slide glass, then a drop of 0.1 µg/ml nile red solution was added to the smear. after being heated over a flame for 1 second, the smear was covered with a cover-glass and examined under the fluorescence microscope. nile red staining gives strong fluorescence [12]. phb analysis dry cell mass was treated with a dispersion containing chloroform and 80% sodium hypochlorite in water at a 3:1 ratio. the mixture was then incubated at 30 °c for 1 hour and then centrifuged at 10,000 g for 10 minutes. three different layers were formed. the upper phase was a hypochlorite solution, the middle phase contained non-phb cell material and undisrupted cells, and the bottom phase was chloroform containing phb. the upper phase was removed first with a pipette. the chloroform layer was also drawn using a pipette. the phb was then extracted from the chloroform layer by slowly introducing the chloroform into ten volumes of ice cold methanol with continuous stirring. chemical determination of phb chloroform extract was dried at 40 °c and 10ml of concentrated sulphuric acid was added. then they were heated at 100 °c in a water bath for 20 min. after cooling, the sample was transferred to a silica cuvette and the absorbance at wavelength of 235 nm is measured against a sulphuric acid as a blank. results of the fifteen samples evaluated for phb production, dnb-6 isolate accumulated the maximum phb (31.91%) of dry cell weight. the isolate was identified as a member of genus bacillus when biochemical and morphological test was matched to bergey’s manual of determinative bacteriology. citrate utilization, growth pattern, carbohydrate utilization and number of other tests as summarized in the (table 1, figure 2). when the results of these tests were inserted in pibwin software version 1.9.2, the strain dnb-6 was identified to close in nature to bacillus cereus with id score of 0.76. a b figure 2 a, b: gram staining and endospore test. a: gram staining of dnb-6, b: endospore of strain dnb-6. growth curve analysis the growth curve was plotted under nitrogen deficient media (ndm), phosphate deficient media (phdm), and potassium deficient media (pdm), sulfur deficient media (sdm) (figure 3). nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 2-5 upreti et al. ©njb, biotechnology society of nepal 4 nepjol.info/index.php/njb table 1: pbb production and characteristics of dnb-6 it was observed that the dnb-6 showed good growth at nitrogen deficient media (figure 3). the growth curve was plotted up to 72 hours in 6 hours interval of time at 20% glucose concentration (figure 3). figure 3: comparison of growth curve of dnb-6 under nitrogen deficient media (ndm), phosphate deficient media (phdm), potassium deficient media (pdm) and sulfur deficient media (sdm) at 20% glucose concentration. phb yield analysis production of phb was maximum at 48 hours (figure 4) when dnb-6 strain was subjected to nitrogen limiting at 20% glucose concentration. figure 4: time profile of phb accumulation under nitrogen deficient media at 20 % glucose concentration (1: 24 hr, 2: 48 hr, 3: 72hr and 4: 96hr). the intensity of fluorescence was checked at different time interval under nitrogen limitation by fluorescence microscope (figure 5a, 5b, 5c, 5d). time profile of growth and phb production accumulation have indicated that maximum phb production during starting of stationary phase and degraded during late stationary phase. a b c d figure 5: phb intensity under fluorescent microscope. a: phb intensity at 24 hours of strain dnb-6, b: phb intensity at 48 hours of strain dnb-6, c: phb intensity at 72 hours strain dnb-6 d: phb intensity at 96 hours of strain dnb-6. discussion growth curve analysis showed that phb was a growth associated product and its accumulation is significantly increased when all cultures reached from exponential phase to till-stationary phase. different intensity of phb obtained at different time also coincided with the phb yield at different time. the analysis of phb yield was carried out in nitrogen deficient media because the growth curve as well as phb intensity observed under this condition was better than in other conditions. the bacteria were harvested at 24 hours, 48 hours, 72 hours and 96 hours. the yield percentage of phb was maximum at 48 hours at 20% glucose concentration. this reflected that by this time the bacteria had already entered the stationary phase. it was observed that the bacteria start accumulating phb granules in its stationary phase (anupam et al., 1999). this high yield of polymer could be because of using glucose as the carbon source. bacillus cereus when grown on structurally unrelated carbon such as fructose, sucrose and gluconate has produced interesting other polymer beside phb [13]. there was variability in phb accumulation at different time profile. the highest phb producing efficiency was found in isolate dnb-6. the maximum phb yield was 31.91% of dry weight under nitrogen deficient media at 20% glucose concentration. the recovery process was carried by chloroform dispersion method. the phb yield was parallel to the bacterial growth. in conclusion, soil bacterial isolate found were capable of accumulating phb as an energy material. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1: 2-5 upreti et al. ©njb, biotechnology society of nepal 5 nepjol.info/index.php/njb the present study showed that the maximum phb production was under nitrogen limitation condition which agrees well with other study that suggests phb production was maximum under nutrient limiting condition. acknowledgements this research was supported by kathmandu university, department of biotechnology. author contributions assistant prof. sangita shakya designed and supervised the project. deepak upreti prepared the manuscript. deepak upreti, naresh prasad sapkota and bibek aryal share the equal amount of contributions in this work. references 1. lara lm, huisman gw: metabolic engineering of poly (3-hydroxyalkanoates): from dna to plastic. microbiol mol biol rev 1999, 63: 21-53. 2. choi j, lee sy: process analysis and economic evaluation for poly (3-hydroxybutyrate) production by fermentation. bioprocess engineering 1997, 17: 335-342. 3. stockdale h, ribbons dw, dawes ea: occurrence of poly-ß-hydroxybutyrate in the azotobacteriaceae. j bacteriol 1968, 95: 1798-1803. 4. kyrikou i, briassoulis, d: biodegradation of agriculture plastic films: a critical review. j polym environ 2007, 15: 125-150. 5. wang f, lee yl: poly (3-hydroxybutyrate) production with high productivity and high polymer content by a fed-batch culture of alcaligenes latus under nitrogen limitation. appl environ microbiol 1997, 63: 3703-3706. 6. singh m, patel s, kalia v: bacillus subtilis as potential producer for polyhydroxyalkanoates. microb cell fact 2009, 8: 38. 7. ojumu tv, yu j, solomon bo: production of polyhydroxyalkanoates, a bacterial biodegradable polymer .a j biotechnol 2004, 3: 18-24. 8. byrom d: polymer synthesis by microorganisms: technology and economics. trends biotechnol 1987, 5: 246-250. 9. chio j, lee y: factors affecting the economics of polyhydroxyalkanotate production by bacterial fermentation. appl microbiol biotechnol 1999, 51: 1321. 10. anupam m, banerjee r, paul ak: accumulation of poly (3-hydroxybutyric acid) by some soil streptomyces. curr microbiol 1999, 39: 153-158. 11. holt jg: bergey’s manual of determinative bacteriology. lippincott williams and wilkins baltimore; 1994. 12. greenspan p, mayer ep, fowler sd. nile red: selective fluorescent stain for intracellular lipid droplets. j cell biol 1985, 100: 965-973. 13. stockdale h, ribbons dw, dawes ea: occurrence of poly-ß-hydroxybutyrate in the azotobacteriaceae. j bacteriol 1968, 95: 1798-1803 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:22-28 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 22 nepjol.info/index.php/njb heat shock protein 70 (hsp70) expression in antimony susceptible/resistant clinical isolates of leishmania donovani mahendra maharjan1,2* and rentala madhubala2 1central department of zoology, tribhuvan university, kirtipur, kathmandu, nepal 2 school of life sciences, jawahar lal nehru university, new delhi, india abstract pentavalent antimonials have long been the first line of defence against leishmaniasis, but resistance has been reported in different parts of the world. pentavalent antimony is reduced into trivalent form in the cells and is a potential inducer of hsp70 in l. donovani. expression profile of hsp70 in antimony susceptible and resistant l. donovani isolates were characterized by southern blot, northern blot and western blot analysis. hsp70 gene copy number, gene expression and hsp70 protein expression was found uniform in both antimony sensitive and resistant clinical isolates. in laboratory condition, leishmania cells respond to antimonial drug stress by three fold over expression of the hsp70 protein. the observed results indicated that hsp70 play important role in stress tolerance against antimonial drug without differential expression in antimony sensitive and resistance clinical isolates of l. donovani. keywords: pentavalent antimony; hsp70; visceral leishmaniasis; drug resistance; pfge; hybridization *corresponding author email: mmaharjan@cdztu.edu.np introduction visceral leishmaniasis is a protozoan parasitic disease caused by leishmania donovani. leishmaniasis constitutes a major public health problem with increasing pattern of disease burden [1,2]. it is a neglected tropical disease (ntd) which affects mainly the poor population groups, primarily in rural areas. unfortunately, there is still lack of effective, affordable and easy-to-use drugs for leishmaniasis treatment. since vaccine against leishmaniasis is still under development, the control lies solely on chemotherapy [3]. however, emergence of drug resistance in parasitic protozoa is becoming a major public health problem. heat shock proteins (hsps) are highly conserved proteins found in both prokaryotic and eukaryotic cells. hsp70 is a molecular chaperone which plays an important role in protein folding and assembly of polypeptides within the cell. when cells are exposed to the stressed condition, the proportion of misfolded proteins (mfps) suddenly increases and the cell reacts by synthesizing hsps to assist those proteins in refolding. the stress response is controlled primarily at the transcription level by a heat shock factor (hsf) [4]. it has been shown that transcript level of hsp70 increased in l. major and in l. infantum, in response to elevated temperature conditions [5, 6, 7]. besides elevated temperature, metals are also known to be important stress inducers in the cells. antimonial compounds; pentostam and glucantime are still the drug of choice in the treatment against all forms of leishmania infections [8, 9]. since, antimonial drug resistance is becoming a common problem in many leishmaniasis endemic regions, extensive studies have been carried out to investigate the mechanism of drug resistance in the laboratory mutants. one of the mechanisms of resistance is postulated to be hsp70. l. tarentolae cells transfected with hsp70 gene showed resistance to sbiii or arsenite [10]. this suggests that hsp70 has a role in antimony resistance mechanism. in the present study, we have characterized the expression of heat shock protein 70 in antimony sensitive and resistant clinical isolates of l. donovani. materials and methods parasite and culture condition promastigotes of indian leishmania donovani clones ag83-s (mhom/in/1983/ag83), ge1 (mhom/in /80/ ge1f8r) and three untyped strains 2001, ns2, and 41 were isolated from patients with visceral leishmaniasis (vl) and were routinely cultured at 22c in m-199 medium (sigma, usa) supplemented with 10% heat inactivated fetal bovine serum (fbs, gibco/brl, life technologies, scotland, uk) and 13 mg/ml penicillin and streptomycin (sigma, usa) [11]. nepal journal of biotechnology. dec. 2015 vol. 3, no. 1:22-28 maharjan and madhubala ©njb, biotechnology society of nepal 23 nepjol.info/index.php/njb clinical isolates obtained from vl patients who responded to sag chemotherapy were designated as sag-s (sag sensitive) whereas vl patients that did not respond to sag were designated as sag-r (sag resistant). accordingly, sag sensitive isolate used in this study is 2001-s whereas the four sag-r isolates are 41-r, ns2-r, and ge1-r. the sag-resistant isolates were maintained in the absence of drug pressure in vitro. the isolates have been passage through hamsters or balb/c mice to retain their virulence and importantly their chemo-sensitivity profiles have remained unchanged. cloning of heat shock protein 70 (hsp70) gene from l. donovani. a 1962-bp dna fragment was amplified from the genomic dna using a sense primer with a flanking bamhi site, 5’cgcggatccatgacattcgacggcgccatc-3’, at position 1-21, and the antisense primer with a flanking hindiii site, 5’ – cccaagcttttagtcgacctcctcgaccttgg – 3’, including the stop codon, at position 1939-1962. polymerase chain reaction (pcr) was performed in 50 µl reaction volume containing 100 ng of genomic dna, 25 pmol each of gene-specific forward and reverse primers, 200 µmol of each dntp, 2 mm mgcl2, 5u taq dna polymerase (mbi fermentas) and 5% dmso. the pcr conditions were as follows: 940c for 10 min, 940 c for 45 sec, 620c for 30 seconds, 720c for 2 min and 35 cycles. final extension was carried for 10 min at 720. a single 1.9 kb pcr product was obtained and cloned into the bamhi hindiii site of pet-30a (figure 1) vector (novagen). the recombinant construct was transformed into bl21 (de3) strain (figure 2) of escherichia coli and subjected to automated sequencing.. expression and purification of hsp70 protein. expression of hsp70 protein from the construct pet30a ldhsp70 was induced at od of 0.7 with 50 µm, 100 µm and 500 µm isopropylthiogalactoside (iptg) (sigma) at 37c for different time periods. all the steps of purification were carried out at 4c as mentioned in general materials and methods. figure 1: restriction map of vector pet-30a(+).the hsp70 pcr product (1.9 kb) was cloned into the bamhi and hindiii restriction site of pet-30a vector. figure 2 : cloning strategy of heat shock protein (hsp70) in pet-30a expression vector protein determination protein concentration was determined by bradford’s method using bovine serum albumin (bsa) as standard [12]. antibody production the purified recombinant hsp70 protein (20 µg) was subcutaneously injected in mice using freund’s complete nepal journal of biotechnology. dec. 2015 vol. 3, no. 1:22-28 maharjan and madhubala ©njb, biotechnology society of nepal 24 nepjol.info/index.php/njb adjuvant followed by two booster doses of recombinant hsp70 protein (15 µg) with incomplete adjuvant at twoweek intervals to produce the polyclonal antibody against the recombinant hsp70 protein. the mice were bled two weeks after the second booster dose and sera was collected for western blot analysis. isolation of genomic dna and total rna. genomic dna was isolated from ~ 2 x 109 cells from 10-15 ml culture (mid log phase promastigotes) as described in general materials and methods. 5 µg of genomic dna was digested overnight with hindiii and subjected to electrophoresis in 0.8% agarose gels. the gel was run at a constant voltage of 30v overnight. total rna was isolated from 2  108 promastigotes using tri reagenttm (sigma) according to manufacturer’s instructions. the isolated rna was stored in diethyl pyrocarbonate (depc) treated water in small aliquots at –800c. for northern blot analysis, 15 g of total rna was fractionated by denaturing agarose gel electrophoresis and transferred to nylon membrane following the standard procedure. pulse field gel electrophoresis (pfge) leishmania chromosomes were separated by pfge. low melting agarose blocks, containing embedded cells (108 log phase promastigotes/ml) were electrophoresed in a contour clamped homogenous electric field apparatus (chef driii, bio-rad) in 0.5  tbe with buffer circulation at a constant temperature of 14ºc. saccharomyces cerevisiae chromosomes were used as chromosomal size markers. pulse field gel electrophoresis (pfge) running conditions were as follows: initial switch time, 60 s; final switch time, 120 s; run time, 24 h; current 6v/cm; including angle 120º. the transfer of pfge separated chromosomes from agarose gels to nylon membrane was achieved by capillary method as described by [13]. hybridization of northern, southern and pfge blot pre-hybridization was done at 65°c for 4 hours in a buffer containing 0.5 m sodium phosphate; 7% sds; 1mm edta (ph 8.0) and 100 µg/ml sheared denatured salmon sperm dna. the blots were hybridised with denatured α-[p32]-dctp-labelled dna probe (pcr probe described for the l. donovani hsp70 coding region) at 106cpm/ml. the probe was labeled by random priming (neb blot®kit, new england biolabs, inc.). membranes were washed sequentially as follows: 2 ssc, 0.1% sds; 1 ssc, 0.1% sds; 0.5 ssc, 0.1% sds; 0.2 ssc, 0.1% sds for 10 min each at 65°c until the non-specific counts had substantially reduced. membranes were air-dried and exposed to imaging plate. the image was developed by phosphorimager (fuji film fla-5000, japan) using image quant software. western blot analysis late log phase promastigotes (1  108) were harvested and the resultant cell pellet was lysed by sonication and cell supernatants were prepared by centrifugation at 20,000 x g. 50 µg of protein from each cell line were fractionated by sds-polyacrylamide gel electrophoresis and blotted on nitrocellulose membrane using electrophoretic transfer cell (biorad). western blot analysis was done using the ecl kit (amersham pharmacia biotech) according to the manufacturer’s protocol. polyclonal antibody to purified recombinant l. donovani hsp70 generated in mice was used for the western blot analysis. autoradiograms were analyzed by using model fla 5000 imaging densitometer (fuji, japan). the results shown are from a single experiment typical of at least three giving identical results. results heat shock protein 70 (hsp70) sequence analysis the gene encoding the heat shock protein 70 kda (hsp70) nucleotide sequence was retrieved from embl sequence data bank under the accession no. x52314. in order to clone the full length gene encoding hsp70, polymerase chain reaction (pcr) was performed using specific oligonucleotides. the sense primer was 5'-cgcggatccatg acattcgacggcgccatc-3’ at position 1-21 and the antisense primer with a flanking hindiii site, 5’cccaagcttttagtcgacctcctcgaccttgg3’ including the stop codon at position 1939-1962. genomic dna from l. donovani ag83 (mhom/in/1983/ag83) promastigotes was used as a template. a single full length 1962-bp pcr product was obtained. the pcr fragment was purified from the gel and double digested with bamh1 and hindiii restriction enzymes. the digested product was ligated with pet30a cloning vector at 160c overnight. the ligated product was transformed into dh5α competent cells. the positive clones were confirmed by colony pcr. further, the cloned hsp70 pcr fragment was confirmed by sequencing. a single open reading frame consisting of 1962-bp was isolated. no variation in the coding sequence was found from the sequence described earlier. nepal journal of biotechnology. dec. 2015 vol. 3, no. 1:22-28 maharjan and madhubala ©njb, biotechnology society of nepal 25 nepjol.info/index.php/njb over-expression and purification of fulllength l. donovani hsp70 in e. coli in order to overexpress and purify recombinant protein, the encoding l. donovani hsp70 sequence was cloned inframe in pet-30a expression vector. the resultant pet-30a -ldhsp70 construct was transformed into e. coli and protein over expression was induced using iptg (figure 3a and 3b) as described in general materials and method. a protein with a molecular weight that matched the estimated 70 kda according to amino acid composition of l. donovani hsp70 with his6 tag and s-tag present at its n-terminal end was also induced. the recombinant protein was purified by ni2+-nta affinity chromatography column purification of his-tagged l. donovani hsp70 by metal affinity chromatography (figure 3c) yielded ~200 µg of purified protein from 1litre bacterial culture. figure 3: overexpression and purification of l. donovani hsp70 protein. (a) coomassie blue staining of sds–page showing overexpression of full-length l. donovani hsp70 protein in e. coli. the pet-30a bacterial extract before induction (lane 2 and 6) and after induction (lanes 3-5 and 7-9) of two positive clones at 1, 2, and 3 h, respectively with 1 mm iptg. arrow shows the induced recombinant hsp70 protein. broad range protein mw marker (mbifermentas) was used to identify the size of the recombinant protein (lane 1). ( b) the pet30a bacterial extract before induction (lane 1), molecular marker (lane 3) and three hour after induction (lane 2, 4, 5 and 6) with 50 m, 100 m, 200 m and 500 m iptg respectively. (c) purification profile of soluble form of hsp70 protein by ninta affinity bead affinity chromatography resin. lane 1 flow through, lanes 2 and 3, washes with 20 mm and 60 mm immidazole, lane 4-7 elutes using 100 mm, 150 mm, 200 mm, and 250 mm immidazole, lane 8 nibeads, lane 9blank. all the purification fractions were run along with the molecular weight protein marker (lane m). polyclonal antibody production and western blotting western blot using size-fractionated parasite protein, antiserum could detect a band of anticipated l. donovani hsp70 size ~70 kda in promastigote extracts. in antimony resistant l. tarentolae mutants, hsp70 was reported to increase more than 4-fold compared to the wild-type cells [10]. the overexpression of hsp70 remained stable in l. tarentolae mutants after several hundreds of passages without drug [10]. to test the link between hsp70 expression and resistant phenotype in antimony resistant l. donovani field isolates, we performed western blot analysis using polyclonal antibody. a western blot using equal amount of parasite protein (50 µg each) extract from antimony sensitive (ag83-s and 2001-s) and resistant (ge1-r, 41-r and ns2-r) field isolate did not show any differential expression of hsp70 protein (figure 4). figure 4. western blotting using anti-his-hsp70 antibody raised against ldhsp70 in balb/c mice. hsp70 protein in fractionated extracts obtained from sag-s and sag-r cultures harvested after 48 h of growth. the same blots were reprobed with antibody against βtubulin protein to normalize the loading on to each lane of the gel. southern blot hybridization of hsp70 gene the copy number of hsp70 gene varies considerably among various leishmania species [6, 10, 14]. to determine hsp70 gene copy number in l. donovani field isolates, southern blot analysis was performed using 1.9 kb hsp70 pcr product as a probe as described under general materials and methods. hindiii which digests outside the gene producing a 10 kb restriction fragment (expected size) was used to check the copy number of the gene. the probe hybridized to a single fragment of 10 kb in all the field isolates (figure 5) indicating that hsp70 exists as a single copy gene in these strains. further, we checked the chromosomal localization of hsp70 gene by southern blot hybridization of pfge blot. the hsp70 probe hybridized to a single chromosomal band of 1.2 mb region that corresponds to the chromosome number 28 of the l. infantum genome database (figure 6). nepal journal of biotechnology. dec. 2015 vol. 3, no. 1:22-28 maharjan and madhubala ©njb, biotechnology society of nepal 26 nepjol.info/index.php/njb figure 5. southern blot analysis of hsp70 gene in sag–s and sag–r leishmania donovani field isolates. total genomic dna of leishmania strains were isolated and digested with hindiii restriction enzyme. the blot was probed with 1.9 kb full length hsp70 gene. the blot was reprobed with α-tubulin to monitor the amount of digested dna layered on the gel. figure 6. pfge analysis of sag-s and sag-r l. donovani isolates indicating chromosomal localization of hsp70 gene. chromosomes of l. donovani isolates were separated by chef and southern blots were hybridized with the hsp70 probe. the size of hybridizing band was identified using chromosomes of s. cerevisae as marker figure 7. expression analysis of hsp70 gene in l. donovani northern blot analysis of mrna from sag-s and sag-r isolates (log phase culture). 15 µg of total rna was loaded per lane, transferred and hybridized with hsp70 probe. a α-tubulin probe and was used to monitor the amount of rna layerd on the gel. the rrna stained with ethidium bromide was used to normalize the rna loading. northern blot hybridization of hsp70 gene since, we couldn’t find any difference in hsp70 protein expression and copy number of gene in between sags and sag-r l. donovani field isolates, we checked transcript level of hsp70 gene expression. northern blotting of sag-s and sag-r l. donovani field isolates revealed a single transcript of ~ 3.5 kb in all the strains (figure 7). densitometric analysis of hybridizing bands corresponds to the internal control α-tubulin showed similar expression of hsp70 gene in between sag sensitive and resistant field isolates. induction of hsp70 by sbiii in promastigote of l. donovani field isolate leishmania cells respond to sbiii exposure by increasing hsp70 protein [10]. induction of hsp70 was found more than four (>4) fold in l. tarentolae and l. infantum cells when incubated for 24 hours at ic50 concentration of the drug [10]. to test whether strain were incubated with 15 µm (ic50 concentration of ag83-s) sbiii for 24 hours. a time dependent increase of hsp70 protein expression in l. donovani field isolates was observed. protein expression increased by more than > 1.7 fold at 12 hours and more than three (>3) fold after 24 hours compared to the cells incubated without drug (figure 8a and 8b). trivalent antimony could induce the expression of hsp70 protein in l. donovani field isolates, we used polyclonal antibody raised against hsp70 antigen in balb/c mice. promastigotes of l. donovani (ag83-s) discussion physiological role of the hsp70 as a molecular chaperon has been well described [15]. induction of hsp70 can protect cells by binding to misfolded proteins in variety of adverse conditions [16]. besides its physiological cytoprotective role an increased expres figure 8. effect of sbiii on hsp70 protein expression at different time points. (a) western blot analysis of hsp70 in l. donovani promastigotes after treatment with 15 µm sbiii for 0 h, 12 h and 24 h. the same blot was reacted with antibody against β-tubulin protein to normalize the loading on to each lane of the gel. (b) densitometric scanning of the western blot in a. the bands were quantified by scanning on a densitometer and fold difference in signal intensities relative to the zero control were plotted. nepal journal of biotechnology. dec. 2015 vol. 3, no. 1:22-28 maharjan and madhubala ©njb, biotechnology society of nepal 27 nepjol.info/index.php/njb expression of hsp70 protein in antimony resistant l. tarentolae and l. infantum laboratory mutants and the increased expression of hsp70 was stable after hundreds of passages without the drug [10]. if the linkage between hsp70 overexpression and antimony resistant phenotype observed in those laboratory mutants is also true for l. donovani field isolates, hsp70 gene could be one of the important biomarker for monitoring antimony resistance in the field conditions. with this hypothesis we have cloned the hsp70 gene in bacterial expression vector, purified the recombinant protein and raised the polyclonal antibody against hsp70 antigen in balb/c mice. we checked the hsp70 protein expression in two antimony sensitive (ag83-s and 2001-s) and three natural antimony resistant (41-r, ck2-r and ns2-r) l. donovani clinical isolates using western blotting. we found constitutive expression of hsp70 protein in both sag-s and sag-r clinical isolates. no difference in hsp70 protein expression was observed in antimony sensitive and resistant field isolates. further, we characterized the hsp70 gene in sag-s and sag-r l. donovani indian field isolates to check the chromosomal localization and copy number of the gene. the copy number of hsp70 gene has been reported to be considerably varied among different trypanosomatids such as single copy in l. braziliensis, 2 copies in l. tarentolae, 4 copies in l. major, 6 copies in l. infantum, 7 copies in l. amazonensis and t. cruzi [5, 6, 10, 14, 17, 18]. in l. donovani indian field isolates, the copy number of hsp70 gene was found to be single copy. no difference in gene copy number was observed in between sag-s and sag-r field isolates. to further verify the hsp70 gene amplification, we performed southern blot hybridization of pfge blot. hsp70 probe hybridized to a highly amplified single band at the 1.2 mb region which corresponds to the 28 chromosome of the l. infantum genome database. transcript level of the hsp70 gene has been shown to be transiently induced by environmental stress such as exposure to heat shock or heavy metals [19]. during heat shock, hsp70 transcription rate increases rapidly and then decline slowly [20]. increased hsp70 transcript level has been shown in leishmania when cells were exposed to heat [6]. the transcription mechanism has shown to be controlled at the posttranscriptional level specifically by the 3’untranslated region of the gene in contrast to most other organisms [7, 21]. similar regulation has also been described in the related parasite t. brucei [5]. increased expression of hsp70 has been reported in antimony resistant l. tarentolae and l. infantum mutants compared to wild type cells [10]. the increased expressions of hsp70 in laboratory mutant were found to be stable after hundreds of passages without the drug [10]. earlier studies indicated that hsp70 overexpression in antimony resistant mutants was the stable phenotype. to check whether similar mechanism is operating in field condition, we performed northern blot hybridization of hsp70 gene in between natural antimony resistant l. donovani clinical isolates to that of the antimony sensitive parasites. no difference in hsp70 transcript level was found between sag-s and sag-r clinical isolates. arsenite and sb iii have been described as the known inducer of the hsp70 in leishmania [10, 22, 23]. by gene transfection experiment, it has been shown that hsp70 contribute first line of defense against sb iii [10] suggesting the role of hsp70 in antimonial drug resistance. in the present study, we checked hsp70 protein expression in promastigotes of l. donovani (ag83-s) in presence of sb iii. interestingly, we could find considerable over expression of hsp70 protein when cells were incubated in ic50 concentration of sb iii. protein over expression was about 1.7 fold at 12 hours that increased up to 3 fold at 24 hours time period. the observed results suggest that leishmania cells respond to antimonial drug stress by over expression of the hsp70 protein. acknowledgements the authors thank university grants commission, new delhi, india for providing jrf/srf to mahendra maharjan to conduct this study. references 1. desjeux p: the increase in risk factors for leishmaniasis worldwide. trans r soc trop med hyg 2001, 95: 239-243. 2. desjeux p: leishmaniasis: current situation and new perspectives. comp immunol microbiol infect dis 2004, 27: 305-318. 3. brandonisio o and spinelli r: immune response to parasitic infections--an introduction. curr drug target. immune endocr metabol disord 2002, 2: 193199. 4. pockley ag: heat shock proteins in health and disease: therapeutic targets or therapeutic agents? expert rev mol med 2001, 3: 1-21. nepal journal of biotechnology. dec. 2015 vol. 3, no. 1:22-28 maharjan and madhubala ©njb, biotechnology society of nepal 28 nepjol.info/index.php/njb 5. lee mg, atkinson bl, giannini sh, van der ploeg lh: structure and expression of the hsp 70 gene family of leishmania major. nucleic acids research, 1988, 16(20):9567-9585. 6. quijada l, soto m, alonso c, requena jm: analysis of post-transcriptional regulation operating on transcription products of the tandemly linked leishmania infantum hsp70 genes. j biol chem 1997, 272(7):4493-4499. 7. quijada l, soto m, alonso c requena jm: identification of a putative regulatory element in the 3'-untranslated region that controls expression of hsp70 in leishmania infantum. mol biochem parasitol. 2000, 110(1):79-91. 8. guerin pj, olliaro p, sundar s, boelaert m, croft sl, desjeux p, wasunna mk, bryceson ad: visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. lancet infectious diseases, 2002, 2(8):494-501. 9. haimeur a, brochu c, genest p, papadopoulou b, ouellette m: amplification of the abc transporter gene pgpa and increased trypanothione levels in potassium antimonyl tartrate (sbiii) resistant leishmania tarentolae. mol biochem parasitol 2000, 108(1):131-135. 10. brochu c, haimeur a, ouellette m: the heat shock protein hsp70 and heat shock cognate protein hsc70 contribute to antimony tolerance in the protozoan parasite leishmania. cell stress chaperones 2004, 9(3):294-303. 11. bhattacharjee h, carbrey j, rosen bp, mukhopadhyay r: drug uptake and pharmacological modulation of drug sensitivity in leukemia by aqp9. biochemical and biophysical research communications 2004, 322(3):836-841. 12. bradford mm: a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analytical biochemistry, 1976, 72:248-25,. 13. sambrook j, fritsch ef, maniatis t: molecular cloning: a laboratory manual, cold spring harbour press, 1989. 14. zurita ai, rodriguez j, pinero je, pacheco r, carmelo e, del ca, valladares b: cloning and characterization of the leishmania (viannia) braziliensis hsp70 gene, diagnostic use of the cterminal fragment rlb70(513-663). journal of parasitology 2003, 89(2):372-378. 15. hartl fu, hayer-hartl m: molecular chaperones in the cytosol: from nascent chain to folded protein. science 2002, 295(5561):1852-1858. 16. morimoto ri, santoro mg: stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. nature biotechnology 1998, 16(9):833-838. 17. bock jh, langer pj: sequence and genomic organization of the hsp70 genes of leishmania amazonensis. molecular and biochemical parasitology 1993, 62, no. 2, pp. 187-197. 18. requena jm, lopez mc, jimenez-ruiz a, de la torre jc, alonso c: a head-to-tail tandem organization of hsp70 genes in trypanosoma cruzi. nucleic acids research, 1988, 16:1393-1406. 19. milarski kl, morimoto ri: expression of human hsp70 during the synthetic phase of the cell cycle. proceedings of the national academy of sciences 1986, 83(24):9517-9521. 20. theodorakis ng, morimoto ri: posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mrna stability. molecular and cellular biology, 1987, 7(7):4357-4368. 21. brandau s, dresel a, clos j: high constitutive levels of heat-shock proteins in humanpathogenic parasites of the genus leishmania. biochemical journal, 1995, 10:225-232. 22. del razo lm, quintanilla-vega b, brambilacolombres e, calderon-aranda es, manno m, albores a: stress proteins induced by arsenic. toxicology and applied pharmacology, 2001 177(2):132-148. 23. lawrence f, robert-gero m: induction of heat shock and stress proteins in promastigotes of three leishmania species. proceedings of the national academy of sciences, 1985, 82(13):4414-4417. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 issn 2091-1130 (print) / issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 40 nepjol.info/index.php/njb molecular markers for septoria leaf spot (septoria lycopersici speg.) resistance in tomato (solanum lycopersicum l.) bal k. joshi1*, frank j. louws2, g. craig yencho3, bryon r. sosinski3, consuelo arellano4, dilip r. panthee3 1genebank-narc, khumaltar, po box 3055, kathmandu, nepal 2department of plant pathology, north carolina state university, north carolina 3department of horticultural science, north carolina state university, raleigh, north carolina. 4department of statistics, north carolina state university, raleigh, north carolina. abstract marker assisted selection (mas) has not been initiated in tomato (solanum lycopersicum l.) for septoria leaf spot (sls) resistance caused by septoria lycopersici speg due to lack of molecular markers. we studied the inheritance of sls resistance and identified molecular markers linked to sls resistance using bulked segregant analysis (bsa) in a segregating f2 population. tomato inbred lines, nc 85l-1w (2007), susceptible to sls and nc 839-2(2007)-1, resistant to sls were used to develop the segregating population. a total of 250 f2 plants, and 10 plants each of p1, p2 and f1 were grown at the mountain horticultural crops research and extension center (mhcrec), mills river nc in the summer of 2009. disease severity was scored using a scale of 0 to 5, where 0 = no disease and 5 = complete development of disease. dna was extracted from 2-3 week old plants and parental lines were screened with a total of 197 random amplified polymorphic dna (rapd) primers, of which 34 were polymorphic. two dna bulks, called resistant bulk (rb) and susceptible bulk (sb) were prepared from the f2 individuals. the rb and sb consisted of 8 individuals each with disease scores of 0, and 4.0 or 4.5, respectively. the segregation ratio of resistant and susceptible plants in f2 generation fit the expected mendelian ratio of 3:1 for a single dominant gene. five rapd markers were linked to the sls disease reaction, of which two were linked to susceptibility and three to the resistance. subject to verification in independent populations, these markers may be useful for mas of sls resistance in tomato. key words: bulked segregant analysis, resistant bulk, septoria leaf spot, solanum lycopersicum, susceptible bulk *corresponding author e-mail: joshibalak@yahoo.com introduction tomato (solanum lycopersicum l., 2n = 2x = 24) is one of the most important vegetable crops worldwide. among the foliar diseases of tomato, septoria leaf spot (sls) caused by septoria lycopersici speg is one of the most devastating diseases. it occurs worldwide including canada and northeast america. it can cause complete defoliation leading to a significant crop loss under favorable environmental conditions, particularly in humid regions during periods of heavy rainfall, frequent dew or over-head irrigation [1,2]. although fungicides are effective to control this disease, breeding for resistance is preferred by tomato growers due to the costs involved in the management of the disease and their associated environmental hazards. however, because sls is relatively easy to control with fungicides this disease has not been an important breeding priority in the past [3,4]. it has been reported that resistance to sls is controlled by a single dominant gene [3]. while the majority of the source of resistance lines belongs to wild species including s. peruvianum, s. glandulosum and s. pimpinellifolium, the highest degree of resistance was found in s. habrochaites [1, 4]. in this study, 22 out of 700 accessions, mostly from s. habrochaites and s. peruvianum, had a score of 2.0 and 3.9 when scored on a scale of 0 to 9, where 0 = no disease and 9 = severe disease. the resistance was found to be associated with small fruit size and late maturity[5]. useful levels of resistance have also been found in s. pennelli, s. pimpinellifolium, s. chilense, and s. lycopersicum var. cerasiforme. breeding lines of interspecific crossing with s. habrochaites accessions have shown high level of resistance. however, these interspecific lines had one or more undesirable horticultural traits such as nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 41 nepjol.info/index.php/njb indeterminate growth habit, late maturity, small fruits or low yield. breeding for sls resistance was not a priority for tomato breeders for a long time. however, sls has become a major problem in canada and northeastern america [6,7,8] and north carolina(nc) (randy gardner, personal communication). the level of intensity of the disease has become so high that it may be even more severe than early blight (randy gardner, personal communication). because of the magnitude of the problem, breeders at cornell university have begun to introgress sls resistance into tomato breeding lines and nc state is following suit. as discussed above, sources of sls resistance are available but resistance is linked with horticulturally unacceptable traits. one of the ways to mitigate this problem is to use molecular markers. molecular markers linked to the gene(s) of interest can be used to select the plants that are genetically similar to the recurrent parent possessing the desired horticultural traits. however, due to lack of molecular markers linked to the sls resistance in tomato, marker assisted selection (mas) has not been initiated for sls resistance. michelmore et al. (1991) developed a rapid and simple pcr based method, which was called bulked segregant analysis (bsa), to identify single genes linked to a trait [9]. using this approach, they identified random amplified polymorphic dna (rapd) markers linked to the downy mildew resistance gene in lettuce. for bsa, any kind of mapping population (e.g. recombinant inbred lines (ril), backcross (bc), f2 or double haploid (dh) that are segregating for a trait of interest can be used. many disease resistance genes have been identified in tomato using rapd following the bsa approach. for example, de giovanni et al. (2004) identified rapd marker linked to the ol-2 gene conferring resistance to powdery mildew using bsa in f2 population [10]. stevens et al. (1995) and chague et al. (1996) identified rapd markers linked to the sw-5 gene, resistance to tomato spotted wilt virus (tswv) [11,12]. smiech et al. (2000) used bsa in an f2 segregating population and found five primers that distinguished resistant and susceptible bulks. in this study, we used bsa technique to identify rapd markers linked to sls resistance in tomato using an f2 population [13]. as explained by michelmore et al. (1991), this is an appropriate starting point for molecular studies of disease like sls in tomato [9]. material and methods plant materials two tomato inbred lines, nc 85l-1w (2007) (referred onward as nc 85l) and nc 839-2(2007)-1 (referred onward as nc 839) were used to produce an f2 population in the greenhouse. nc 85l, was used as a female and is susceptible to sls (susceptible parent, sp) and nc 839, was the male and is resistant to sls (resistant parent, rp). the source of resistance in nc 839 traces back to la3707, a s. pimpinellifolium line (randy gardner, personal communication). a total of 250 f2 plants, and 10 plants each of sp, rp and f1 were grown at the mountain horticultural crops research and extension center (mhcrec), mills river, nc. among f2 plants, data could not be recorded from 16 plants, which were used as missing points. therefore, we used observations from 234 f2 plants for data analysis. the fruits of nc 85l were mini-roma type with dark red color whereas nc 839 was a grape tomato with light red fruit color (table 1). the nc 85l selection was made for late blight and early blight resistance in the disease nursery at waynesville, nc and the nc 839 selection was made at mills river for outstanding fruit and plant type along with sls resistance. field evaluation seeding was done on june 1, 2009 in 30.5 x 45.5 cm trays containing peat moss and vermiculite. trays were kept in the greenhouse at an average temperature of 21.1oc. twelve-day old seedlings were transplanted in a 12.7 x 24.4 cm 50-cell tray. six-week old seedlings were transplanted in the field with siltyloam soil with a row-to-row and plant-to-plant spacing of 150 cm and 45 cm, respectively. the beds were raised and covered with black plastic. other recommended cultural practices were followed as described in the southern us 2009 vegetable crop handbook [14]. a total of 280 plants consisting of 10 plants each of sp, rp and f1, and 250 f2 plants were planted in a hotspot for sls at the mhcrec, mills river, nc in summer of 2009. data scoring and analysis disease severity was scored at 60 days after transplanting (august 17, 2009). individual disease rating scores were based on visual assessment of nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 42 nepjol.info/index.php/njb table 1. parental description along with their partial pedigree and coefficient of parentage in the population used for tagging septoria leaf spot resistance gene in tomato. severity. the following scoring criteria were developed based on [15,16] and used in this study: 0 = no disease symptoms 0.5 = less than 10% leaf area with symptoms 1 = 10-20% leaf area with symptoms 1.5 = 20-30% leaf area with symptoms 2 = 30-40% leaf area with symptoms 2.5 = 40-50% leaf area with symptoms 3 = 50-60% leaf area with symptoms 3.5 = 60-70% leaf area with symptoms 4 = 70-80% leaf area with symptoms 4.5 = 80-90% leaf area with symptoms 5 = 90-100% leaf area with symptoms for the inheritance study, we grouped the segregating plants into resistance groups with scores from 0 to 2, and susceptible groups with a score from 2 to 5. scores of parental lines and f1 were an average of individual plants. frequency of different score categories was estimated for f2 populations using sas v.9.1 for segregation analysis and frequency distribution. skewness was estimated using sas v.9.1. frequency data were analyzed by 2 to test the goodness of fit for a single dominant gene using sas v.9.1 [17]. dna extraction, quantification and dilution dna was extracted from 2-3 weeks old plants following the method of fulton et al. (1995). approximately 100 mg of young leaves from 2-3 week old tomato seedlings were collected from the greenhouse in 1.5 ml eppendorf tubes.[18] the tubes were dipped into liquid nitrogen and the samples were ground by glass rod. after adding 200 µl microprep buffers, samples were incubated in a 65oc water bath for about 60 min and filled with chloroform/isoamyl (24:1) solution. samples were then centrifuged at 10,000 rpm for 5 minutes. the aqueous phase was pipetted out into a new microcentrifuge tube and 2/3 to 1 times the volume of cold isopropanol was added to precipitate the dna. after centrifuging this sample at 10,000 rpm for 5 minutes, the dna pellet remaining was separated and washed with 70% ethanol. the dry dna pellet was re-suspended in 100 µl of te buffer and stored at -20oc. the concentration of dna was determined by nanodrop (nanodrop 1000, thermo scientific, de, usa). working solutions of dna samples with a concentration of 20 ng/ µl were prepared from original dna samples in te buffer. rapd screening and bulked segregant analysis a total of 197 10-mer random amplified polymorphic dna (rapd) primers were used to screen parental lines using 20 ng dna template. primers polymorphic to parental lines were then used to screen resistant and susceptible bulks. amplification reactions were performed in 10 l reaction volume containing 1x buffer (10 mm trishcl ph 8.3, 50 mm kcl, 1.5 mm mgcl2), 200 m of each dntp, 0.2 m primer and 1 u taq polymerase. about 15 l mineral oil was overlaid on the reaction mixture. dna amplifications were performed in thermal cycler (eppendorf, ny) using the following cycling condition: one cycle of 92oc for 3 min; 45 cycles of 92oc for 30 seconds, 42oc for 1 min and 72oc for 30 seconds; one cycle of 72oc for 8 min followed by holding at 4oc. bulked segregant analysis (bsa) was performed following the method of michelmore et al. (1991) [9]. two dna bulks, called resistant bulk (rb) and susceptible bulk (sb) were prepared from f2 individuals. the rb consisted of 8 individuals with disease score of 0 and the sb contained 8 individuals with the score of 4 or 4.5 (figure 1). dna bulks were parent maturity fruit characters septoria leaf spot reaction pedigree common pedigree cop nc 85l-1w (2007) early mini roma type, dark red susceptible nc051(x)18//0463/9722(x)-18 nc0179(x)-118-4, nc215e1(93), nc9722(x)-18, nc051, nc03220, la3707 0.23 nc 839-2 (2007)-1 average grape type, light red resistant nc051(x)-18//cb25(x)18-3/9722(x)-18/0464 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 43 nepjol.info/index.php/njb prepared by pooling equal amounts of dna of eight resistant and eight susceptible f2 individual plants for rapd analysis. pcr was run with polymorphic primers on the bulked samples using the same reaction conditions as described above. pcr was repeated for at least two times for those primers that were polymorphic between bulks. gel electrophoresis all rapd pcr products were analyzed in 2% agarose gels containing ethidium bromide in tbe buffer (40 mm tris-borate, ph 8.0, 1 mm edta) with a 100 bp ladder. electrophoresis was run at 135 v for 2 hr. gels were rinsed with water to enhance contrast and photographed under uv light. rapd fragments were scored as 1 for presence and 0 for absence. bands size was estimated based on the 100 bp dna ladder. simple statistics based on the dna bands were calculated using ms excel 2007. figure 1. frequency distribution of 234 f2 individuals derived from nc 085l-1w(2007) x nc839-2(2007)-1 based on the score of infestation of septoria leaf spot in tomato at mills river, nc in 2009. figure shows the bulked segregant analysis method adopted in this study and schematic representation of rapd band linked to resistance gene. the average phenotypic values of the parents and f 1 are shown by arrow. sp = septoria leaf spot susceptible parent, nc 85l-1w(2007). rp = septoria leaf spot resistant parent, nc 839-2(2007)-1. r = resistant. s = susceptible. p = parent. b = bulk. results segregation of resistance severity of sls infestation was assessed in 234 individual plants at 60 days after transplanting, based on the percentage of total leaf area infected. only 234 plants out of 250 were scored as some of the plants were dead or malformed. no symptoms were observed in the resistant parent, but the susceptible parent had intermediate levels of sls infection (figure 1). comparing the sls scores of f2 individuals with their parents, it was clear that transgressive segregation was found towards susceptibility. the distribution of disease reaction was highly left-skewed (figure 1). this suggests that the susceptible parent may also have contribution to resistance. based on the distribution of f2 individuals, we found two distinct groups of resistant and susceptible plants. this allowed us to readily perform bsa to identify the linked markers. about fifty percent of the f1 plants had a disease score of 0 indicating that resistance to sls was incomplete dominant. among 234 f2 individuals, 164 were resistant (0-2 score) and 70 were susceptible (2-5 score). the segregation ratio of resistant and susceptible plants fit the expected ratio of 3:1 for a single dominant gene (2 = 3.014, p > 0.05) which indicated that the inheritance of resistance to sls was based on a single dominant gene in the present study. rapd markers and bulked segregant analysis out of the 197 rapd primers used to screen parent lines, 34 (17.26%) were polymorphic (data not shown). a total of 176 bands with a maximum fragment size of 1500 bp and minimum fragment size of 100 bp were amplified using 34 primers. among these fragments, 84 were polymorphic between parents. the 34 polymorphic rapd primers were used to screen the resistant and susceptible bulks and 11 exhibited polymorphisms between resistant and susceptible bulks (data not shown). a total of 87 bands were amplified by 34 rapd primers. among these bands, 34 and 20 bands were polymorphic between the parents and bulks, respectively. the size of bands ranged from 150 to 2000 bp. five primers were linked to sls reaction (table 2). f2 rb (8 individuals with 0 score) sb (8 individuals with 4-4.5 score) sp rp rb sb x nc 85l nc 839 f1 rapd analysis band linked to resistance gene 93 24 19 28 20 20 19 10 1 0 1 1.5 2 2.5 3 3.5 4 4.5 f re q u e n c y score of septoria leaf spot in f2 population f1 (1.1)rp sp skewness = 2.46 n = 234 nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 44 nepjol.info/index.php/njb one band of each of two primers, namely mrtomr-121 and mrtomr-031 (figure 2) was found only in the susceptible parent nc 085l and the susceptible bulk. similarly, one band of each of three rapd primers (mrtomr-022, mrtomr-117 and mrtomr-121) was amplified only in the resistant parent nc 839 and the resistant bulk. amplified band sizes linked to susceptibility were 800 and 600 bp whereas those linked to resistance ranged from 600 to 1000 bp (figure 3). six primers were not linked to any of the loci (figure 4). these primers distinguished only the parents and not the bulks. some of the amplified bands were only found in bulks but not in either parent (figure 4). this may be due to recombination in f2 population. table 2. polymorphic bands of rapd markers linked to either resistance or susceptible genes of tomato to septoria leaf spot. marker sequence pbn size, bp sp rp rb sb mrtomr022 agggc cagc 1 1000 0 1 1 0 2 800 0 1 0 0 3 600 1 0 1 1 7 250 1 0 1 1 8 150 1 0 1 1 mrtom r-031 gggac gtcgc 1 1100 0 1 1 1 3 600 1 0 0 1 mrtom r-117 ccgaa caatc 2 850 0 1 1 0 mrtom r-118 tgcttg gggg 3 800 1 0 0 0 4 750 0 1 0 0 5 650 1 1 1 1 6 600 0 1 1 0 mrtom r-121 ggcgtc gtaa 1 1100 0 0 0 1 3 900 1 1 0 0 4 850 1 1 0 1 5 800 1 0 0 1 6 650 1 1 1 0 7 420 1 0 0 0 8 380 1 0 0 1 discussion resistance to sls in tomato was found to be controlled by a single incomplete dominant gene in this study. andrus and reynard (1945) also reported that sls resistance was dominant and named it the se gene.[1] however, wright and lincoln. (1940) have reported recessive gene conferring resistance to the sls in the field observation in the past studies.[19] the differences observed in the inheritance of resistance in the present study from the past studies might be due to use of different figure 2. electrophoresis pattern of dna fragments generated by rapd markers (a. mrtomr-031, b. mrtomr-121). polymorphic band (i.e. linked to susceptible) between parents, and between resistant and susceptible bulks are indicated by arrow. sp = susceptible parent, nc 085l. rp = resistant parent, nc 839. rb = resistant bulk. sb = susceptible bulk. m = marker. figure 3. electrophoresis pattern of dna fragments generated by rapd marker (a. mrtomr-022, b. mrtomr-117 and c. mrtomr-118). polymorphic band (i.e. linked to resistance) between parents and between resistant and susceptible bulks are indicated by arrow. sp = susceptible parent, nc 085l. rp = resistant parent, nc 839. rb = resistant bulk. sb = susceptible bulk. m = marker. sources of resistance. the susceptible parent used in this study did not appear completely susceptible suggesting that there may be its allelic difference in the expression of resistance. in fact both parents, nc nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 45 nepjol.info/index.php/njb figure 4. rapd marker (a. mrtomr-130) showing polymorphic band (indicated by arrow) only to parents, i.e. band with unlinked loci and rapd marker (mrtomr-146) showing band (indicated by arrow) only in two bulks. sp = susceptible parent, nc 085l. rp = resistant parent, nc 839. rb = resistant bulk. sb = susceptible bulk. m = marker 85l and nc 839 have a coefficient of parentage (cop) of 0.23 (table 1) indicating that they have common parentage. this fact has been confirmed based on their common pedigree (randy gardner, personal communication). based on the field screening of the f2 population with 197 rapd primers, we identified three rapd markers linked to resistance alleles and two rapd markers linked to susceptible alleles. through the bulking of the extreme individuals segregating in the f2 population we were able to rapidly tag the markers associated with chromosomal segment that has a role in reaction to sls in tomato. for bsa consisting of eight individuals in each bulk, five primers yielded different banding patterns, which were useful markers in sls screening in tomato. bands of two of these markers were only present in susceptible parent and bulk, and bands of three markers were present only in resistant parent and bulk. therefore, these bands were considered associated either susceptible allele or resistant allele. tagging of resistance genes using bsa is very fast, which facilitates the screening of new alleles of resistance for a particular disease, especially for one that does not have background information available such as sls in tomato. the two parental lines used in this study are closely related to each other (cop=0.23). however, we found rapd to distinguish these parents at the molecular level. rapds are multi locus-based markers. therefore, the primers identified might be from the same regions of the chromosome. for example, mrtomr-022 produced a 1000 bp band and mrtomr-118 produced a 600 bp band. the band produced by mrtomr-118 might be the part of the band produced by mrtomr-022. the disadvantages associated with rapds include the fact that they anneal in multiple sites, and they are dominant in nature, and sensitive to reaction conditions, which may limit their use directly in mas. therefore, these rapd markers need to be converted to sequence characterized amplified region (scar) or cleaved amplified polymorphic sequence (caps), which are much more useful for mas. through bsa, marker development and mas has been used for the selection of resistance to a number of diseases in tomato. for example, de giovanni et al. (2004) identified rapd marker linked to the ol-2 gene conferring resistance to powdery mildew.[10] a single rapd marker, opu31500 with 1500 bp in size was detected in the susceptible bulk, which was converted into a caps marker. stevens et al. (1995) and chague et al. (1996) identified rapd markers linked to the sw-5 gene conferring resistance to tomato spotted wilt virus (tswv).[11][12] among the four rapd markers, two were tightly linked to sw-5 gene. linkage analysis mapped these markers within a distance of 10.5 cm from sw-5. czech et al. (2003) have used mas using a co-dominant marker through bsa for developing tswv resistant tomato.[20] smiech et al. (2000) used bsa in f2 segregating population and found 5 primers that distinguished resistant and susceptible for tswv. [13] a pcr-based co-dominant marker, tightly linked to mi was developed using the information from bsa [21](williamson et al. 1994). in light of these past reports, the five rapd primers identified in the present study may be informative to develop co-dominant markers for sls resistance breeding. rapd markers identified here needs to convert into scar or caps marker for mas of resistance to sls in tomato. the mas is cost effective and more reliable for screening, because it does not need to have a pathological evaluation and can genotype at any growth stage. molecular markers linked to the sls resistance in tomato may also have a potential nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 46 nepjol.info/index.php/njb role on gene pyramiding. to our knowledge, there are no any molecular markers reported associated with sls resistance in tomato. molecular markers identified in this study are novel, and provide enough background to develop different group of markers (scar or caps) which may be useful for speeding up the tomato breeding program aiming to improve sls resistance. references 1. andrus cf , reynard gb: resistance to septoria leaf spot and its inheritance in tomatoes. phytopathology 1945,35:16-24. 2. delahaut k , stevenson w: tomato disorders: early blight and septoria leaf spot. the university of wisconsin 2004, madison, wi 3. barksdale th , stoner ak: resistance in tomato to septoria lycopersici. plant disease reporter 1978,62:844-847. 4. locke sb: resistance to early blight and septoria leaf spot in the genus lycopersicon. phytopathology 1949,39:829-836. 5. poysa v and tu jc: response of cultivars and breeding lines of lycopersicon spp. to septoria lycopersici. canadian plant disease survey 1993, 73:9-13. 6. mutschler ma, sm zitter, d dejong, ta zitter and k ivors: performance of hybrids combining genetic control to early blight and late blight with and without resistance to septoria leaf spot. tomato disease workshop, 2011, ithaca, ny. 7. zitter ta, sm zitter and ma mutschler: comparing the performance of early blight and septoria leaf spot resistant materials in the presence and absence of fungicides. tomato disease workshop, 2011a, ithaca, ny. 8. zitter ta, sm zitter, ma mutschler and sp mckay: using host resistance and reduced-risk fungicides to control early blight and septoria leaf spot on tomato, 2010. plant disease management report, 2011b, cornell university. 9. michelmore rw, paran i and kesseli rv: identification of markers linked to diseaseresistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. proceedings of national academic of sciences, usa 1991,88:9828-9832. 10. de giovanni c, orco p dell', bruno a, ciccarese f, lotti c and ricciardi l. identification of pcr-based markers (rapd, aflp) linked to a novel powdery mildew resistance gene (ol-2) in tomato. plant science 2004, 166:41-48. 11. stevens mr, lamb em and rhoads dd:mapping the sw-5 locus for tomato spotted wilt virus resistance in tomatoes using rapd and rflp analysis. theoretical and applied genetics 1995,90:451-456. 12. chague v, mercier jc, guenard m, decourcel a and vedel f:identification and mapping on chromosome 9 of rapd markers linked to sw5 in tomato by bulked segregant analysis. theoretical and applied genetics 1996: 92:10451051. 13. smiech m, rusinowski z, malepszy s and niemirowicz-szczytt k: new rapd markers of tomato spotted wilt virus (tswv) resistance in lycopersicon esculentum mill. acta physiologiae plantarum 2000, 22:299-303. 14. kemble jm (ed):southeastern vegetable crops handbook. auburn university, auburn, al 2009. 15. tu jc and poysa v: a brushing method of inoculation for screening tomato seedlings for resistance to septoria lycopersici. plant disease 1990, 74:294-297. 16. winstead nn , kelman a: inoculation technique for evaluating resistance to pseudomonas solanacearum. phytopathology 1952, 42:628-634. 17. sas institute inc:the sas system, version 9.1.3 for windows, 9th ed. sas institute, cary, nc 2007. 18. fulton tm, chunwongse j and tanksley sd: microprep protocol for extraction of dna from tomato and other herbaceous plants. plant molecular biological reporter 1995,13:207-209. 19. wright v and lincoln re:resistance to defoliation diseases of tomato. purdue agricultural experiment station 1940, pp 42-43. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:40-47 jhoshi et al. ©njb, biotechnology society of nepal 47 nepjol.info/index.php/njb 20. czech as, szklarczyk m, gajewsk z, zukowska e, michalik b, kobylko t and k strzalka:selection of tomato plants resistant to a local polish isolate of tomato spotted wilt virus (tswv). journal of applied genetics 2003, 44:473480. 21. williamson vm, ho jy, wu ff, miller n and kaloshian i: a pcr-based marker tightly linked to the nematode resistance gene, mi, in tomato. theoretical and applied genetics 1994, 87:757-763. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:10-14 issn 2091-1130 (print) /issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 10 nepjol.info/index.php/njb doxorubicin induced histomorphometric changes in testes of albino rats surendra kumar sah1, saroj khatiwada2*, deepak chaudhary1, chandra bhushan jha3, soumya bhattacharya3 1department of anatomy, nobel medical college, biratnagar, nepal 2department of biochemistry, modern technical college, lalitpur, nepal 3department of human anatomy, b p koirala institute of health sciences, dharan, nepal abstract anticancer drugs like doxorubicin have been found to affect male gonads thereby leading to infertility. this study was conducted to evaluate the effects of doxorubicin over short, mid and long term on testes of male albino rats. sixty male albino rats aged 6-8 weeks were taken for study. the rats were randomly divided into 3 groups of experimental (each group containing 10 rats) and 3 groups of control (each group containing 10 rats). the experimental groups were given a single dose of doxorubicin i.e. 10 mg/kg body weight intra-peritoneally and sacrificed after 3 different duration for each group (second week, eighth week and sixteenth week). all rats under 3 control groups were given a single intra-peritoneal dose of 2.5 ml/kg body weight normal saline and sacrificed with their respective experimental groups. significant difference in diameters (p=0.029) and cross-sectional area (p=0.028) of seminiferous tubules was observed between short term experimental and short term control rats. for both between midterm experimental and midterm control group, and between long term experimental and long term control group, a significant difference in right testis weight (p<0.001 for both), left testis weight (p<0.001 for both), volume of testis (p<0.001 and p=0.038), diameter (p<0.001 for both) and area (p<0.001 for both) of seminiferous tubules was observed. as compared to short term experimental group, midterm experimental group and long term experimental group had significantly lower right testis weight (p<0.001 for both), left testis weight (p<0.001 for both), diameter of seminiferous tubule (p<0.001 for both) and cross-sectional area of seminiferous tubule (p<0.001 both). cross-sections of the seminiferous tubules of all the control groups had normal architecture. however, there was progressive destruction of seminiferous tubules structure across the experimental groups. doxorubicin has deleterious effect on seminiferous tubules of albino rat testis. key words: doxorubicin, histomorphometry, seminiferous tubule, albino rat *corresponding author email: khatiwadasaroj22@gmail.com introduction doxorubicin, also known as hydroxyd-aunorubicin, is a drug used in cancer chemotherapy. it is highly effective in many human tumors and is currently the first line anti-cancer drug in many chemoresponsive tumors such as ovarian cancers, breast cancers and lymphomas [1]. the clinical use of doxorubicin can be viewed as double edged sword. on one hand, doxorubicin plays an undisputed key role in the treatment of many neoplastic diseases; on the other hand, chronic administration of doxorubicin induces organ toxicity particularly testicular injury [2]. previous investigations indicated that doxorubicin has the ability to induce mutations and chromosomal aberrations in normal and malignant cells in addition to a wide variety of toxic side effects on organs like testes [3]. doxorubicin is said to alter sperm development, production, structural integrity and motility rates in association with increased cellular apoptosis in spermatogonia and spermocyte [4]. the reduction of oxidative dna damage by antioxidants has been evaluated as a chemotherapeutic approach for reducing damage caused by chemotherapy agents such as doxorubicin [5]. damage to the testicular germinal epithelium is a potential side effect of cancer therapy, and is of particular concern in case of men of reproductive age having tumors with high cure rates [6]. thus it seems important to assess the effects of doxorubicin on various components of male reproductive system and particularly in testis where sperm production takes place. different studies have been carried out to investigate the effects of doxorubicin on testis, however there are only few studies utilizing stereological tool to elucidate histomophometric nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:10-14 sah et al. ©njb, biotechnology society of nepal 11 nepjol.info/index.php/njb evidences of doxorubicin mediated derangement of the testis and evaluate midterm and long term effects of doxorubicin [7]. considering this we conducted the present study among albino rats to find short, mid and long term effects of doxorubicin on histomorphometric characters of testis as compared to controls. methods the study was conducted at the department of anatomy of b p koirala institute of health sciences, dharan, nepal to find the short, mid and long term effects of doxorubicin on testis of albino rat as compared to controls. sixty healthy albino rats aged 6-8 weeks and weighing 150-200 gm were obtained from the animal house of anatomy department and used in study. they were housed in well ventilated room at controlled ambient temperature (25±5 0c) with a 12 hour alternating light-dark cycle and provided standard rodent diet and water throughout the study period. the experimental work was carried out as per research and ethical guidelines of nepal health research council (nhrc) for the care and use of animals in health research in nepal and the research protocol was approved by the institute review board of b p koirala institute of health sciences. the rats were randomly divided into 3 groups of experimental (each group containing 10 rats) and 3 groups of control (each group containing 10 rats), such that weight do not vary by more than 10% of average weight of study population. the experimental groups were given a single dose of doxorubicin i.e. 10 mg/kg body weight intraperitoneally and sacrificed after 3 different duration for each group. group 1 animals, which were investigated for short term effect, were sacrificed on 14th day (2nd week). group 2 animals (studied for midterm effects) were sacrificed on 56th day (8th week), and group 3 animals (studied for long term effects) on 112th day (16th week). all rats under 3 control groups (control 1, control 2 and control 3) were given a single intra-peritoneal dose of 2.5 ml/kg body weight normal saline and sacrificed with their respective experimental groups (at second, eighth and sixteen weeks respectively). comparison was done among 3 groups of experimental rats in terms of effect of doxorubicin, and as well as between each matched experimental and control group. all rats were weighed at the time of euthanasia. animals were anesthetized with ether soaked in cotton and their testes were fixed by in vivo perfusion technique as mentioned below. rats were kept on the dissecting tray with their ventral surface facing upward and all four limbs pinned. the abdomen was opened to expose the abdominal aorta and inferior vena cava. a 18 guage needle was inserted on the abdominal aorta and tied with thread to keep it in constant position. the needle was attached to clean flask tube, connected with two bottles containing bouin’s fluid and physiological saline separately. then the chest was opened and the right atrium was nicked by a scissor or knife (scalpel) to permit the drainage of blood. thereafter, the physiological saline was perfused to flush out the blood. the fixative bouin’s fluid was then transfused with the help of three ways stopcock. approximately 200 ml of each fluid was perfused. perfusion was stopped when clear fixative drops started oozing out from the snout of the animal. after completion of perfusion, the testes were isolated from the scrotum with the help of scalpel and forceps and weight was measured followed by volume measurement by the application of water displacement method and post fixed for 24 hours by the same fixative. after 18 hour fixation in bouin’s fluid, the tissue was processed and histological slides were prepared from vertical sections from the polar and the equatorial regions of each testis. micrometer was used for quantitative measurement of parameter; diameter of seminiferous tubules, and interstitial spaces, germ cells, sertoli cells and leydig cells were observed qualitatively in histological slides. diameter (d) of each seminiferous tubule was measured by two directions and mean of two was taken as a diameter of that seminiferous tubule. the cross-sectional area of the seminiferous tubules were determined by formula; area (a)=πd2/4. two slides per rat were observed for both control and experimental groups. statistical analysis was done in spss version 19. all data were expressed as mean ±sd. independent t test and one way anova was applied to test for statistical significance at 95% confidence interval. a nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:10-14 sah et al. ©njb, biotechnology society of nepal 12 nepjol.info/index.php/njb value of p<0.05 was considered statistically significant. results it was observed that mean body weight consistently decreased among the short term and long term rats. on the other hand, there was increase in body weight among the midterm rats in comparison to its initial body weight, as well as in controls. various anatomical parameters in experimental groups and controls are shown in table 1. no significant difference in right testis weight (p=0.184), left testis weight (p=0.179), and testicular volume (p=0.388), but significant difference in diameters (p=0.029) and cross-sectional area (p=0.028) of seminiferous tubules, were observed between short term experimental and short term control rats. however, a significant difference in right testis weight (p<0.001), left testis weight (p<0.001), volume of testis (p<0.001), diameter (p<0.001) and area (p<0.001) of seminiferous tubules was observed between midterm experimental and midterm control group. similarly significant difference in right testis weight (p<0.001), left testis weight (p<0.001), volume of testes (p=0.038), and diameter (p<0.001) and area (p<0.001) of seminiferous tubules was observed between long term experimental and long term control group. as compared to short term experimental group, midterm experimental group and long term experimental group had significantly lower right testis weight (p<0.001 for both), left testis weight (p<0.001 for both), diameter of seminiferous tubule (p<0.001 for both) and cross-sectional area of seminiferous tubule (p<0.001 both). however, no significant difference were observed among midterm and long term experimental group in terms of testis weight, volume, diameter and crosssectional area of seminiferous tubules. figure 1 (a, b, c and d) shows the cross-section of seminiferous tubules of control and various experimental groups. it was observed that the crosssections of the seminiferous tubules of all the control groups were moderately circular or oval in outline with normal seminiferous epithelium and numerous spermatozoans within their lumen. the interstitial cells were also normal and prominent. rats that were given doxorubicin showed progressive degenerations in their seminiferous tubules lumen. group 1 rats showed a little reduction in the basal epithelial layer resulting in the apparent detachment of the basement membrane. the rats of group 2 (midterm experimental group) showed vacuolization of the cells, reduction in the number of spermatozoa. lumen of the seminiferous tubules was significantly vacant. the interstitial spaces were distinct. group 3 rats (long term experimental group) showed marked variation in its architecture. there was much testicular atrophy and most of the spaces were occupied by the interstitial cells. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:10-14 sah et al. ©njb, biotechnology society of nepal 13 nepjol.info/index.php/njb figure 1. a, b, c and d show the cross-section of seminiferous tubules of control (all control groups), short term experimental group, midterm experimental group and long term experimental group respectively. discussion doxorubicin is one of the most widely used anticancer agents in the clinic despite its dose– limiting side-effects. in the present study, we assessed the effects of a single dose (i.e. 10mg/kg body weight of animal) of the drug on male albino rats. present study shows that weight of the rats that were given doxorubicin decreases in comparison to the control groups. the decrease in body weight may be due to loss of appetite in experimental rats. we however observed that, the midterm experimental rats showed increase in body weight. the increase in body weight in midterm might be due to the reversible metabolic changes caused by the drug. after some period of time the body regains its metabolic function and then it again decreases due to the long term effect of the drugs. in the present study significant difference in testicular weight among the midterm experimental and the midterm control groups, and among the long term experimental and the long term control groups was observed, which is similar to the findings of saalu et al [7]. we also observed progressive decrease in testis weight from short term to long term experimental group. previous study done by howell et al. also reported that administration of doxorubicin can decrease the testicular weight of the rats. this could be due to the severe parenchymal atrophy in the seminiferous tubules following the doxorubicin administration [8]. however study by prahalathan et al. did not report any significant testicular weight changes with doxorubicin treatment [9]. in the present study, we found that the diameter and cross-sectional area of seminiferous tubules decreases with the time in the rats given doxorubicin. as compared to short term experimental group, midterm experimental group and long term experimental group have decreased diameter and cross-sectional area of the seminiferous tubules, which may be due to more effect of the drugs on exposure to long time. our findings are in accord to previous study by saalu et al [7]. study by saalu et al. reported that doxorubicin chemotherapy induces morphological and morphometric impairments of testes of rats and progressive worsening of testicular derangement with time following a single dose of doxorubicin treatment [7]. recent study done in india showed that treatment with doxorubicin alone caused decrease in body weight, sperm count and serum testosterone and increase in serum level of lactate dehydrogenase (ldh), creatine phosphokinase (ck), and glutamic oxaloacetate transaminase (got) [10]. in the present study, we observed deterioration in seminiferous tubules architecture in experimental group, and the architecture was more impaired in long term experimental group than midterm and short term experimental rats. the study also demonstrates that testicular cytotoxicity caused by doxorubicin provokes germ cell depletion in the seminiferous epithelium of the rats. saalu et al. showed that even a single dose of doxorubicin progressively causes testicular derangement which may be due to its capacity to generate intracellular free radicals and reactive oxygen species [7]. single higher doses or multiple lower doses of doxorubicin have been referred to be toxic to the stem spermatogonia, diminishing their survival [11]. the alteration of the measurements of testicular volume and weight, tubular diameter, seminiferous epithelium height and volume densities of tubular lumen and seminiferous epithelium can give information about the testicular damage degree as a consequence of germ cell death [12]. franca et al. nepal journal of biotechnology. d e c . 2 0 1 5 vol. 3, no. 1:10-14 sah et al. ©njb, biotechnology society of nepal 14 nepjol.info/index.php/njb reported that when a massive germ cell loss occurs, it is followed by a sharp decline in testicular morphometric parameters [13]. in general, germ cell death caused by anticancer drugs, including doxorubicin, culminates with a reduction of morphometric parameters [14]. many of doxorubicin dose-limiting toxicities occur due to its generation of toxic oxygen species, resulting in oxidative stress. in summary, present study reveals deteriorative effect of doxorubicin on seminiferous tubules of rats. the study has however, several limitations. first, we did not assess the ultrastructural changes in testis caused by doxorubicin. also, sperm count was not done in this study. the body weight of the experimental rats fluctuated with the duration. the exact causes for the fluctuation of body weight were not identified in this study. competing interests none author’s contribution sks, sk, dc, cbj and sb designed the study. sks, sk and dc conducted the experiments. sks and sk wrote the manuscript. dc, cbj and sb reviewed manuscript. all authors read and approved the final version of manuscript. acknowledgements we kindly acknowledge b p koirala institute of health sciences for the support in the study. references 1. atessahin a, karahan i, turk g, gur s, yilmaz s, ceribasi ao: protective role of lycopene on cisplatin induced changes in sperm characteristics, testicular damage and oxidative stress in rats. reprod taxicol. 2006 21(1):42-7. 2. rubin p, louis sc, lawrence bm, paul o. late effect of cancer treatment on normal tissues. springer verlog, berlin, heidelberg. 2008 1:109-30. 3. yagmurcaa m, erdoganb h, irazc m, songurd a, ucare m, fadillioglu e: caffeic acid phenethyl ester as a protective agent against doxorubicin nephrotoxicity in rats. clin chim acta. 2004 348(1-2):27-34. 4. sawada t, tamada h, mori j: secretion of testosterone and epidermal growth factor in mice with oligozospermia caused by doxorubicin hydrochloride. andrologia.1994 26(3):151-3. 5. quiles jl, huertas jr, battino m, mataix j, ramíreztortosa mc: antioxidants nutrients and adriamycin toxicity. toxicology. 2002 180(1):79-95. 6. kobayashi h, urashima m, hoshi y, uchiyama h, fujisawa k, akatsuka j: testicular morphological changes in children with acute lymphoblastic leukemia following chemotherapy. pediatrics international. 1996 38:640-3. 7. saalu lc, enye la, osinubi aa: an assessment of the histomorphometric evidences of doxorubicin-induced testicular cytotoxicity in wistar rats. int j med med sci. 2009 1(9):370-4. 8. howell st, shaler sm: testicular function following chemotherapy. hum reprod. 2001 7:363-9. 9. prahalathan c, selvakumar e, varalakshmi p: lipoic acid ameliorates adriamycininduced testicular mitochondriopathy. reprod toxicol. 2005 20:111-6. 10. patil l, r. balaraman r: effect of melatonin on doxorubicin induced testicular damage in rats. 2009 1(3):879-84. 11. jahnukainen k, hou m, parvinen m, eksborg s, söder o: stage-specific inhibition of deoxyribonucleic acid synthesis and induction of apoptosis by antracyclines in cultured rat spermatogenic cells. biol reprod. 2000 63(2):482-7. 12. vendramini v, sasso-cerri e, miraglia sm: amifostine reduces the seminiferous epithelium damage in doxorubicin-treated prepubertal rats without improving the fertility status. reprod biol endocrinol. 2010 8:3. 13. franca lr, russel ld: the testis of domestic animals. in: martínez-garcia f, regadera j, eds. male reproduction: a multidisciplinary overview. churchill communications, madrid. 1998 :198-219. 14. stumpp t, sasso-cerri e, freymuller e, miraglia sm: apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase. anat rec. 2004 279:611-22. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 doi: https://doi.org/10.3126/njb.v8i1.30208 research article ©njb, bsn 36 standardization of ayurvedic drugnyctanthes arbor-tristis, hippophae salicifolia, ocimum tenuiflorum and reinwardtia indica and combined herb-herb rinki kumari1 , g.p. dubey1, 2 1advanced centre for traditional and genomic medicine, institute of medical science, banaras hindu university, varanasi, uttar pradesh -221 005, india 2genome foundation, kalwari, sikrara, jaunpur, uttar pradesh 222131, india article history:received: 4 oct 2019; revised: 11 dec 2019; accepted: 25 dec 2019; published online: 31 jul 2020 abstract the herbal medicines have reached extensive acceptability as therapeutic agents for various clinical diseases due to global demand. therefore, standardization is the essential and initial step to drug development. it is for the establishment of consistent biological activity, a consistent chemical profile and biomarker identification. it improves the safety and efficacy of herbal medicine to provide the best herbal medicine to society and increase popularity rather than non-standardized extracts. in addition, it is essential to practice or maintain a quality assurance program for the production and manufacturing of herbal medicine that includes the basis of organoleptic characters and photomicrographs, physicochemical, proximate analysis phytochemical evaluation and quality control analysis and order to assess the quality of drugs, based on the concentration of their active principles. who has provided specific guidelines for the assessment of the safety, efficacy and quality of herbal drugs as a prerequisite for global harmonization and of utmost importance. in the present study, the herbal extracts were cleaned, dried in the shade and powdered by passing through the sieve as per the method described in the standard protocol. an overview covering various techniques employed in the extraction and characterization of nyctanthes arbortristis, hippophae salicifolia, ocimum tenuiflorum and reinwardtia indica, standardization is reported in this study. the obtained data would be very significant for future clinical aspects, as the bioactive molecules present in the extracts may exhibit synergistic effect with other bioactive compound and show a better therapeutic value. thus, this study provides standardized and therapeutically potential data of active polyherbal formulations for the different ailments. keywords: physicochemical, polyherbal formulation, single, herb-herb combination, flavonoids. corresponding author, email: rinkiv3@gmail.com introduction in recent years, standardization is the essential parameter to assess the quality of any type of drugs and more important for the quality assessment of herbal formulations to justify their acceptability in the modern medical system. it plays a crucial role to assess the quality, purity, safety and efficacy of some drugs including herbal medicine. standardization is more important in the drug discovery area due to lack of scientific evidence such as pharmacognostic evaluation, phytochemical study, and pharmacological evaluation of the polyherbal formulation. nowadays attention has been directed towards the utilization of ayurvedic plants in the prevention and management of clinical diseases so that the trend of using herbal medicines has increased in a tremendous mode in the last decade [1]. consequently, the world health organization (who) has taken a broader step in the clinical system and also in phytotherapy. sponsorship and encouragement of studies substantiating parameters of related to standardization of herbal medicines of ayurveda and ancient system are under principal consideration of who for new drug discovery [2]. ayurvedic practitioners have developed several herbal medicines by using single herb or herb-herb combinations or as a polyherbal formulation, which were used in ancient traditional medical practice for many years. these medicines offer several options to modify the progress and symptoms of various diseases [3-6]. globally, both types of mailto:rinkiv3@gmail.com https://orcid.org/0000-0003-4720-795x mailto:joshibalak@yahoo.com nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 37 commercial and non-commercial single herbal medicine or polyhedral formulations have been developed, but many polyherbal formulations lack scientific evidence such as pharmacognostic, phytochemical and pharmacological evaluation [4]. nyctanthes arbor-tristis (n. arbor-tristis) linn. is known as parijat belonging to the oleaceae family. the therapeutic utility of n. arbor-tristis has been described in various classical texts of ayurveda for the management of various mental and physical disorders. in sushruta sutra sthana; the yoga prepared out of n. arbor-tristis, along with other drugs is used for a good sleep, for the management of diabetes, stomach disorders, epilepsy and many more [3-6]. n. arbor-tristis is used in combination with other drugs for the management of ksharaagada and also for the purification of poisons. according to sushruta uttar tantra, n. arbor-tristisis was also given for the management of various diseases [7-11]. the word hippophae (latin word) ‘hippo’ meaning horse and ‘phaos’ means ‘shine’, and hippophae salicifolia known as sea buckthorn (sbt). it was used as a medicinal plant in tibet as early as 900 ad. h. salicifolia have has been used as a herbal medicine for many years globally not only as therapeutic but also as prophylactic and health promotive agents. recently, sea buckthorn elaeagnaceae, a unique and valuable plant has gained worldwide attention, mainly for its medicinal, nutritional potential and its edible fruit which is rich in vitamins, often made into jam. in greece, h. salicifolia leaves and twigs were used to feed animals, which resulted in weight gain and a shining coat, especially in horses. it has a rich history of use in treating numerous medical conditions. many of its pharmacological effects have been recorded in classics such as sibuyidian from the tang dynasty and jing zhu ben cao from the qing dynasty [12-14]. ocimum tenuiflorum, also known as, vishnu priya, holy basil, or tulsi (also spelt tulasi), is an indian aromatic plant and belongs to the family lamiaceae, and cultivated throughout the southeast asian tropics. tulsi (sanskrit:-surasa) has been used for thousands of years in ayurveda for its diverse healing properties.). from ancient times, tulsi extracts were used as ayurvedic remedies for a variety of clinical ailments. it is mentioned in the charaka samhita, an ancient ayurvedic text in sutrasthan for skin disorders [15], in shwashara mahakashaya [16], for nadisweda in vatakapha disorders (neuro-muscular disorders) [17], in harita varga for hikka (hiccough), kasa (cough), vishavikara (toxins), swas (asthma), parshvashula (pain in chest) and durgandhanashaka (for fragrance) [18]. in vimanasthanatulsi has been described for the treatment of worn disorders and panchakarma [19, 20], katuskandha [21]. reinwardtia indica dumort. (r. indica dumort) is also known as basanti and belong to as flax family [22]. the medicinal power of r. indica mainly depends on phytochemical constituents that have great pharmacological significance. r. indica extract is widely used by local communities for different purposes such as for tongue wash, to bring about an increase in lactation period, a remedy against skin diseases, as an anti-infection agent etc. [23-26] as well as in the development of several polyherbal formulations. the selection of these four herbs was based upon its activity like an antidepressant, antioxidant and anti-inflammatory. this study reports on the standardization of polyherbal formulation, based on organoleptic assessment, photomicrographs, physicochemical properties, proximate examination phytochemical assessment and quality control investigation. materials and methods plant material leaves of n. arbor-tristis, o. tenuiflorum and r. indica were collected from the forests of india. fruits of h. salicifolia was collected from himachal pradesh (leh & ladakh). these plant extracts were identified, authenticated and voucher specimens of the plants have been deposited (accession no.: sh2010, lh-2008, sh-2008 & sh-2008) in the herbarium for further reference. fresh plant materials of these plants were taken for the medicinal purpose used for microscopic study. the collected materials of the plants were washed with water and dried in the shade at room temperature and sieved. the dried parts were grinded to a coarse powder. the powdered drug was stored in an airtight and light-resistant container for the study. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 38 chemicals all the chemicals (toluene: ethyl acetate, petroleum ether, silica gel, chloroform, ethanol, methanol, naoh, hcl, hno3 and nutrient agar, salmonella shigella agar (ssa) and potato dextrose agar (pda) media etc. used, were of analytical grade and were obtained from e. merck limited india and hi-media laboratories, mumbai, india. organoleptic evaluation various sensory parameters of the plant material (such as colour, odour, size, shape, and taste) were studied by organoleptic evaluation [27]. pharmacognostic studies pharmacognostic analysis for each sample were carried out according to the standard procedural methods. sample preparation each sample was preserved in a fixative solution. the fixative used was faa (5 ml formalin + 5 ml acetic acid + 90 ml 70% ethyl alcohol). the materials were left in the faa for more than 48 hours. dehydration process was followed by a graded series of tertiary-butyl alcohol as per the schedule by sass 1940 [28]. after dehydration, paraffin infiltration was carried out till the supersaturation of tertiary-butyl alcohol was achieved. following supersaturation, the materials were transferred to pure paraffin wax two times and the materials were cast into paraffin blocks. sectioning the embedded specimens were sectioned with the help of a rotary microtome. the thickness of the section was 10 to 12 µm which were then/further stained with toluidine blue as per the method by o’brein et al. [29]. photomicrographs the transverse section was photomicrographs using zeiss axio trinocular microscope attached to zeiss axio cam camera under bright field. descriptive terms of the anatomical features are as given in the standard anatomy books [30]. physico-chemical evaluation the physico-chemical evaluation of prepared sections/samples of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica and a mixture of the herb were done by testing loss of weight on drying at 1050c, total ash, acid insoluble ash and watersoluble ash. preparation of samples the fresh fruit of h. salicifolia, leaf of n. arbor-tristis and root of r. indica and whole plant of o. tenuiflorum were washed, air-dried in 25ºc for three days in the absence of sunlight and grinded into a coarse powder. these samples were used for physico-chemical studies and proximate analysis [31]. loss on drying at 105c/moisture content to determine the weight loss in drying, 10 g of each herbal extract was placed in a tarred evaporating dish after accurately weighing it. the extract was then dried at 105c for 5 hrs and weighed. after drying, tarred evaporating dish was cooled in desiccators for 30 mins and then weight was taken. the % of loss on drying at 105 c = the difference in weight after heating weight of the sample taken x 100 2.0 g of the powdered extract was incinerated in a tarred silica dish at a temperature not exceeding 450c until free carbon was left, then cooled and final weight was taken. the percentage of ash was calculated concerning the weight of the sample. the % of total ash = weight of ash obtained weight of sample taken x 100 estimation of acid insoluble ash the ash obtained as the above method was boiled for 5 mins with 25 ml of dilute hydrochloric acid and collected the insoluble matter on an ash-less filter paper (whatman 41), washed with hot water and ignited to constant weight. the percentage of acid-insoluble ash concerning the air-dried drug was calculated. the % of acid insoluble ash = weight of acid insoluble residue weight of the sample taken x 100 estimation of water-soluble ash: the incinerated ash of the sample was dissolved in 25 ml of dilute hydrochloric acid and made up to 50 ml with water and boiled. the suspension was filtered with the help of the whatman filter paper and the residue was collected. it was kept in a weighed silica crucible and maintained in a muffle furnace for 6 hrs at 450-650ºc. the crucible was taken out and cooled at room temperature and nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 39 weighed. the percentage of ash obtained was thus calculated. the % of water − soluble ash = weight of ash weight of the sample taken x 100 estimation of sulphated ash: the substance was ignited with concentrated sulphuric acid, which decomposed and oxidized organic matter, leaving a residue of inorganic sulphates. a large silica crucible was ignited to constant weight and approximately 5 g of the sample was weighed, and moistened by adding sulfuric acid. this was then heated gently at first and more strongly later so that volatile matter could be removed. further, it was again ignited more strongly to remove the cool carbon, remoistened with sulphuric acid and then re-ignite to achieve constant weight. finally, the percentage of sulphated ash was calculated. the % of sulphated ash = weight of ash weight of the sample taken x 100 determination of extractable matter in alcohol (cold maceration method) 5 g of samples were macerated with 100 ml of alcohol in a stoppered flask with frequent shaking during the first 6 hrs and allowed to stand for 18 hrs. it was filtered after 24 hrs. 25 ml of the filtrate was evaporated in a tarred dish at 105°c and weighed. alcohol soluble extractive values were calculated. the experiment was repeated twice, and the average value was taken [32]. the % of alcohol − soluble extractive = weight of extract × 4 weight of the sample taken x 100 determination of extractable matter in water (cold maceration method) about 5.0 g of coarsely powdered air-dried material was accurately weighed in a glass stopper conical flask and macerated with 100 ml of distilled water specified for the plant material for 6 hrs with frequent shaking, then allowed to stand for 18 hrs. it was then filtered instantly, taking care not to lose any solvent. the extracted matter was dried at 105c for 6 hrs, cooled in desiccators for 30 mins and then weighed. the percentage of the extractable matter was calculated. the % of water − soluble extractive = weight of extract × 4 weight of sample taken x 100 proximate analysis estimation of fibers the fiber content was estimated by the method of raghuramulu et al. [33]. about 5 g of moisture and the fat-free sample were weighed into a 500 ml beaker and 200 ml of boiling 0.255 n (1.25% w/v) sulphuric acid was added. the mixture was boiled for 30 mins, but the volume was kept constant by the addition of water at frequent intervals. finally, the mixture was filtered through a muslin cloth and residue was washed with hot water until it was free from acid. the material was then transferred to the same beaker, and 200 ml of boiling 0.313 n (1.25%) naoh was added. after boiling for 30 mins (keeping the volume constant as before) the mixture was filtered through a muslin cloth. the residue was washed with hot water until it was free from alkali, followed by washing with some alcohol and ether. it was then transferred to a crucial, dried overnight at 800-100ºc and weighed (we). the crucial was heated in a muffle furnace at 600ºc for 2-3 hours; then it was cooled and weighed again (wa). the difference in the weights (we-wa) represents the weight of fiber. fiber content = {100 − (moisture + fat)} × (we − wa) weight of sample taken estimation of total carbohydrate (anthrone method) the total carbohydrate content was estimated by the method of hedge et al. [34]. 100 mg of the sample was weighed into a boiling tube, hydrolyzed by keeping it in a boiling water bath for three hours with 5.0 ml of 2.5 n hcl and cooled at room temperature. then, the material was neutralized with solid sodium carbonate until effervesce was seen and the final volume made to 100 ml. this was centrifuged, at 5000 rpm for 10 min and the supernatant was collected. from that, 0.2 to 1.0 ml was taken and the standard was prepared from the working standard. 1.0 ml of water serves as a blank made up the volume to 1.0 ml in all the tubes with distilled water, then added 4.0 ml of anthrone reagent, heated for eight minutes in a boiling water bath, cooled rapidly and read the green to dark green colour at 630 nm. the carbohydrate content of the sample was compared with the standard curve. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 40 estimation of protein (modified lowry’s method) the total protein content was estimated by the method from raghuramulu et al. [33]. 5 g extracts was kept 3 separate tubes with 50 ml of water by overnight cold percolation method. in separate tubes, 0.5 ml of each; extract, blank and standard, were taken in duplicate and 0.5 ml of alkaline copper reagent was added, mixed and allowed to stand undisturbed for 10 minutes. then 2 ml of phenol reagent was added to each tube; mixed immediately and placed at room temperature for 5 mins and absorbance of samples and standards were taken at 615 nm against a blank. the protein content of the sample was calculated by comparing with the standard curve. estimation of fat 5 g of extract was placed in a soxhlet fitted with a condenser. 90 ml of petroleum ether (boiling point 4060°c) was taken in a 150 ml round bottom flask and boiled for 6 hrs. the extract was taken in a preweighed conical flask, and petroleum ether was evaporated on a water bath. the traces of petroleum ether were removed using a vacuum pump [35]. phytochemical test phytochemical analysis for each sample’s crude extract and her-herb combination were carried out according to the standard procedure methods [36, 37]. preparation of hydroalcoholic extracts the dried powder of plant parts was exclusively removed by hydroalcoholic cold permeation technique. 10 g of dried powder of each plant were taken in 100 ml of oil ether in a cone-shaped jar, applied with cotton fleece stopper and after that kept on a turning shaker at 120 rpm for 24 hrs. after 24 hrs, it was seived through eight layers of muslin fabric, centrifuged and the supernatant was collected and air-dried under decreased strain to acquire the dried buildup. oil ether was dissipated from the powder. this dry powder was then taken exclusively in 100 ml of every dissolvable, for example, ethanol (et), 75% et, half et, 25% et and water and after that it was kept on a revolving shaker at 120 rpm for 24 hrs. at that point, the system pursued was equivalent to above, and the deposits were weighed to get the extractive yield of the considerable number of concentrates and were put away in hermetically sealed containers at 4ºc [38]. the phytochemical studies were performed for testing the different chemical groups present in the drug. for this, 10% (w/v) solution of extract was taken, unless otherwise mentioned in the respective individual test. general screening of various extracts of the plant material was carried out for the qualitative determination of the groups of organic compounds present in them as per sethi 1966 [39]. test for alkaloids dragendorff’s test: for this test, 2 g of alcoholic and aqueous extract of the drug were dissolved in 5 ml of distilled water, followed by the addition of 2 m hydrochloric acid until an acid reaction occurs, then 1 ml of dragendorff’s reagent added. subsequently, an orange or orange-red precipitate was produced immediately. test for flavonoids shinoda’s test: in a test tube containing 2 g of alcoholic extract of the drug, 5-10 drops of dilute hydrochloric acid as added, followed by the addition of a small piece of magnesium. in the presence of flavonoids, a pink, reddish-pink or brown color was produced. test for triterpenoids liebermann-burchard’s test: for this 2 ml of acetic anhydride solution was added to 1 ml of petroleum ether extract of the drug in chloroform followed by 1 ml of conc. sulphuric acid. a violet ring was formed indicating the presence of triterpenoids. test for resins dissolved the extract was dissolved in acetone and solution was poured into distilled water. turbidity indicated the presence of resins. test for saponins in a test tube containing about 2 g extracts of the drug was added a drop of sodium bicarbonate solution, followed by shaking the mixture vigorously and left for 3 minutes. honeycomb like forth was formed. test for steroids liebermann-burchard’s test: in this test, 2 ml of acetic anhydride solution was added to 1 ml of petroleum ether extract of the drug in chloroform followed by the addition of 1 ml of conc. sulphuric nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 41 acid. a greenish color was developed, which turned to blue. salkowski reaction: for this test, 1 ml of conc. sulphuric acid was added to 2 ml of chloroform extract of the drug carefully, from the side of the test tube. the red color was produced in the chloroform layer. test for tannins added a few drops of 5% fecl3 solution in few grams of extract and a green color indicated the presence of gallotannins while brown color tannins. test for starch 0.015 g of iodine and 0.075 g of potassium iodide were dissolved in 5 ml of distilled water and few grams of extract of the drug was added. the blue color was produced. test for glycosides for the detection of glycoside in an extract sample, on paper spray, solution no. 1 (0.5 % aqueous sol. of sodium metaperiodate) was sprayed & waited for 10 minutes. after that spray solution no. 2 [0.5 % benzidine (w/v) was sprayed in solution of ethanol – acetic acid (4:1)], presence of glycoside was detected by formation of white spot with a blue background. estimation of total phenolics many natural antioxidants are polyphenolics in nature e.g. flavonoids, chalcones, aurones and phytoalexins. these polyphenolic compounds exhibited anti-inflammatory, antioxidant and analgesic activity. hence, the amount of total phenol present in the air-dried plant, as well as hydroalcoholic extracts, were estimated using folin-ciocalteau reagent [40]. detection of numerous microbial masses in herbs the plant material and plant extract obtained were subjected to microbial analysis. a total 1 g of the sample was taken, and 99 ml of sterile distilled water was added for preparing the serial dilution. the samples in the flask were kept in a mechanical shaker for a few minutes to obtain uniform suspension of microorganisms and diluted to 1:100 or 10-2. from that 1 ml of the 10-2 dilution was transferred to 9 ml of sterilized distilled water. this was 1:1000 or 10-3. this procedure was repeated up to 10-6 dilution. 0.1 ml of serially diluted samples were inoculated into the sterile plate containing nutrient agar, salmonella shigella agar (ssa) and potato dextrose agar (pda) medium by spread plate method. nutrient agar (na) and ssa plates were incubated at 37ºc for 24 hours, and pda plates were incubated at room temperature for 3-5 days. the bacterial and fungal colonies were counted using a colony counter. salmonella spp, shigella spp can be counted using ss agar medium. enterobacter spp. were identified by enterobacteriaceae kits. test for heavy metal toxicity sample collection the samples were cleaned and dried under shade. the dried samples were grinded to powder form in an agate mortar-pestle, were labeled and stored in pre-cleaned polyethylene bottles for further analysis. calibration of instruments: more than three working standard solutions of analyses were prepared, covering the concentration range as recommended by the manufacturer of the instrument for the elements to be determined. before the analysis of samples, the instruments were calibrated with a prepared working standard solution. the calibration curves were obtained for concentration vs absorbance and were statically analyzed. cadmium, lead and arsenic analysis (flame aas/graphite furnace): the digested sample was subjected to analysis of cadmium, lead and arsenic by aas flame/graphite furnace with specific instrumental conditions as given by instruments manufacturer. the solutions were introduced into flame. the mean of the three readings was recorded against the standard calibration curve obtained from concentration vs absorbance. after calibrating the instrument with prepared working standard, 10 ml of digested sample was pipette out to a specific container of mercury hydride system analyzer followed by adding 10 ml 1.5 % of hcl as diluents for each sample and blank. the digested samples were run through the reaction flask containing reluctant (3% nabh4 in 1 % of naoh) to quartz cell without heating against the calibration curve obtained from concentration vs absorbance. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 42 fluorescence analysis fluorescence is an essential parameter of pharmacognostical evaluation, exhibited by various chemical constituents present in plant material and convert nonfluorescent into fluorescent derivatives by reagents. it was observed under ordinary and ultraviolet light according to the procedure of kokoshi et al. [41]. for this, 10 mg of the extract of each plant was taken in a glass slide and treated with various reagents for the presence of their fluorescence characteristics under the ultra‐violet lamp at 254 nm and 366 nm. high-performance thin layer chromatography (hptlc) hptlc analysis was carried out by sethi 1996 [39]. in this technique, 5µl each of the extract was applied on a precoated silica gel 60 f254 on aluminium plates to a bandwidth of 8mm using linomat 5 tlc applicator. the plates were developed in toluene-ethyl acetate (9:1) and the developed plates were visualized and scanned by tlc scanner 4 win cats software version 1.4.6.2002 under uv 254, 366. after derivatization in vanillin-sulphuric acid was sprayed as reagent at 620 nm, rf colour of the spots and densitometry scan were recorded. hptlc plates of hydraulic extracts of plants separate mobile phases viz. ethylacetate: dichloromethane: formic acid: acetic acid: water (10:2.5:1:1:0.5) were examined. rf value, numbers of peaks, peak area and the peak height of plants extract in separate mobile phases viz. nethylacetate: dichloromethane: formic acid: acetic acid: water (10:2.5:1:1:0.5) were also analyzed under 254 nm and 366 nm respectively. further studies was done by the help of standards for quantitative estimation and identification of the ingredients peak. present hptlc fingerprinting data can help in authentication and identification of formulation in the performed solvent system and extract. statistical analysis statistical analysis was done by using graphpad prism version 5.02. one-way analysis of variance (anova), with tukeypost-test, was used for statistical analysis of collected data. a probability value of p<0.05 was considered significant, and p<0.01 was considered highly significant. all the data are expressed as mean ± sd (standard deviation). results the organoleptic properties of herbs showed the colour revealed was light brown for leaves of n. arbor-tristis, o. tenuiflorum and r. indica, yellow for fruits of h. salicifolia, have a characteristic odour, bitter taste and moderately fine texture and combined herb-herb color was yellowish-brown. pharmacognosy is the study of medicinal plants, produced from natural sources and analysis of their biological, chemical, biochemical, and physical properties, or it is the study of crude drugs based on their shape, size, color, and texture and cut surface morphology. microscopic features of entire transverse section of the leaf of n. arbor-tristis. figure 1: microscopy of n. arbor-tristis leaf. figure 1a represents microscopy of the leaf ts through midrib where col=collenchyma; e=epidermis; gt=ground tissue; me=mesophyll; ph=pholem; sg=starch grains; t=trichome; xy=xylem. figure 1b represents microscopy of the leaf ts through lamina where cu=cuticle; le=lower epidermis; mer=meristele; pal=palisade; spp=spongy parenchyma; t=trichome; ue=upper epidermis. the transverse section of leaf passing through the mid-rib convexly projects on the lower side, and slightly grooved with a shallow central elevation on the upper side. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 43 leaf lamina demonstrated an epidermis of digressively stretched cells on the two surfaces, bigger on the upper surface, secured by striated fingernail skin, mesophyll separated into 2 or 3 layers of palisade cells, 5 to 7 layers of approximately masterminded, fairly isodiametric supple parenchyma, rosette gems of calcium oxalate present in a couple of cells, stomata more on the lower surface, anisocytic where anomocytic type likewise occur on the two surfaces (figure 1a & figure 1b). midrib: show a single layered epidermis, 2 or 3 layered collenchyma on both surfaces, 4 or 5 layered parenchyma, mostly devoid of chloroplasts, central zone occupied by the vascular bundles differentiated into xylem towards ventral side and phloem towards dorsal side, phloem consisting of sieve tubes, companion cells and phloem parenchyma, xylem consisting of radial rows of vessels with xylem parenchyma. the total thickness of the leaf ranges from 158.08– 295.36 µm, the mean value being 248.46 µm. the thickness of mesophyll ranges from 110.24–249.60 µm with the mean as 197.91 µm. the ratio of height to width of upper epidermal cells speaks for their barrel-like shape. the height vis-à-vis width ratio of the lower epidermal cells reveals that the cells are more or less square shaped. petiole: in the transverse section of the petiole leaf of n. arbour-tristis have a specific feature, there are two projections adjacent to the ventral groove, epidermis single-layered, cells cubical covered by a thick cuticle, inner walls of epidermal cells adjoining the cortex much thickened, hairs absent. hypodermis layer composed of collenchyma 2 or 3 layered and a broad zone of more or less rounded parenchyma cells present with intercellular spaces. it has a few rosette crystals of calcium oxalate. resin canals are also present on the dorsal side of each vascular bundle except in the vascular bundles occurring projecting arms. in this section, vascular bundles 5 to 7 in number (figure 2a & figure 2b). microscopic features of entire transverse section of the fruit of h. salicifolia: the ts of the fleshy tissues of the fruit or hypanthium reveals various structures. vascular tissues are oriented longitudinally through the central portion of the fruit flesh. viewed crosssectional (figure 3), the vascular tissues are surrounded by large parenchyma cells with round, oval, and teardrop shapes and contain large quantities of red pigments, which are likely carotenoids. long narrow cells shaped like sclereid tissues, and containing green pigments, are found on either side of the vascular and storage tissues. these groups alternated around the middle circumference of the mesocarp. 2a 2b 2c nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 44 figure 2: the transverse section of the petiole leaf of n. arbour-tristis. figure 2a represents transverse section of petiole, 2b, 2c 2d represents enlarged portion of petiole figure 3: transverse section of fruit from h. salcifolia microscopic features of the entire transverse section of the leave of o. tenuiflorum. leaf: transverse section of the leaf had a pot shape midrib and a flimsy lamina with uneven lower epidermis appended at the sidelong sides of its upper side leaving a curved focal dorsal. midrib comprises of an emanating curve of xylem and phloem. both upper and lower epidermis demonstrated straightforward, covering, uniseriate trichomes just as sessile short-stalked, glandular trichomes. powder of the air-dried leaves of this plant was seen under the magnifying instrument. the various glandular basic trichomes of the normal length of 101 μm were found. petiole: indicates to some degree cordate framework, comprising of single-layered epidermis made out of flimsy walled, oval cells having various covering and glandular trichomes; covering trichomes multicellular 1-8 celled long, rarely somewhat reflexed at tip; glandular trichomes short, sessile with 1-2 celled stalk and 2-8 celled inflatable formed head, estimating 22-27 in diameterr; epidermis pursued by 1 or 2 layers and 2 or 3 layers of meager walled, stretched, parenchyma cells towards upper and lower surfaces individually; three vascular groups arranged midway, center one bigger than the other two; xylem encompassed by phloem. midrib: epidermis, trichomes and vascular bundles similar to those of petiole except cortical layers reduced towards the apical region. lamina: epidermis and trichomes similar to those of petiole; both monocytic and diacytic type of stomata present on both surfaces, slightly raised above the level of epidermis; palisade single layered followed by 4-6 layers of closely packed spongy parenchyma with chloroplast and oleoresin (figure 4a & figure 4b). 2d 4b nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 45 figure 4: transverse section of leaf from o. tenuiflorum. figure 4a represents transverse section of leaf from o. tenuiflorum and figure 4b, 4c, 4d represents enlarged section of leaf. the physicochemical characteristics of the extracts were determined as per who guidelines [34]. the physico-chemical estimation of the drug is an important parameter in adulteration or improper handling of drugs [35]. it can serve as a valuable source of information and provide appropriate standards to establish the quality of plant material for further study. equally important in the evaluation of the loss on drying, ash value, water soluble ash, sulphated ash, acid insoluble ash value and water-soluble extractive value and alcohol soluble extractive value determination. the low value of moisture content could prevent bacteria, fungal or yeast growth [40, 41, 42]. this value varies within fairly wide limits and is, therefore, an important parameter for the purpose of evaluation of crude drugs [43]. therefore, percentage on the loss on drying, ash value, water-soluble ash, sulphated ash, acid insoluble ash value and water soluble extractive value and alcohol soluble extractive value calculated. results for the physicochemical parameters are given in table 1. ash of any organic material contains non-volatile inorganic components. the ash content indicates that the seed, is rich in mineral elements. controlled incineration of crud drugs result in an ash residue composed of inorganic matter such as metallic salts and/or silica. therefore, the kind of care that must be taken in the plant drug is important. total ash value was noted in (table 1). the result showed that the negligible amount of acid-insoluble siliceous matter is present in these extracts. percentage of total ash value, watersoluble ash, sulphated ash, acid insoluble ash in these extracts accessed falls within the margins as per. the ayurvedic pharmacopoeia of india, (2001), which states that the total ash, water-soluble ash, sulphated ash acid insoluble ash content of the samples tested should in a limited range[44].the water-soluble extractive value indicates the presence of sugar, acids and inorganic compounds, table 1. result reveals that the sugar, acids and inorganic compounds were presented with normal range in these extract. the alcohol-soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, glycosides and flavonoids table 1. the alcoholsoluble extractive content of these extracts falls within the margins as per the ayurvedic pharmacopoeia of india (2001), which states that the alcohol-soluble extractive content of the samples tested should be more than 3% [44, 45]. table 1: the physicochemical analysis of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica. physicochemical parameters n. arbor-tristis h. salicifolia o. tenuiflorum r. indica combined herbs loss on drying 4.59±0.05 9.87±0.09 4.87 ±0.04 4.64±0.05 5.98±0.05 ash value 19.64±0.07 10.35±0.03 8.61±0.06 5.38 ±0.05 10.99±0.05 water soluble ash 9.29±1.6 4.64±0.7 3.76±0.4 2.12±0.5 4.95±0.25 sulphated ash acid 10.82±1.0 3.67±0.6 2.64±0.2 2.09±0.3 4.80±0.52 insoluble ash value 2.11±0.9 2.09±0.6 1.53±0.3 1.11±0.1 1.71±0.47 water soluble extractive 4.69±0.3 15.26±0.4 8.52±0.3 8.31±0.3 9.19±0.32 alcohol soluble extractive 6.38±0.03 6.27±0.02 10.29±0.3 3.64±0.01 6.64±0.09 value are mean ±sd for 5 different preparations 4c 4d nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 46 next parameter, proximate analysis reveals the nutritive content of each extract and combined extract. the proximate analysis revealed that h. salicifolia is a very good natural source of protein and all calculated values showed in (table 2). in the proximate analysis, the combined herbal extract possesses the nutritive content of four herbs and found the study presence of carbohydrate, protein, fat and crude fiber. according to the results of the energy values which were based on the carbohydrate, fat and fiber content in these extracts were high. thus, it may be used as an alternative for food as well as medicinal. these results correlate with kocchar et al. [46]. preliminary phytochemical analysis of polyherbal formulation is of significant importance as scientist need to understand the change upon extraction of different portion of four different medicinal showed the presence of alkaloids, glycoside, flavonoids, phenol, steroids, saponins, tannins, terpenoids and these results are presented in table 3. the investigation showed that n. arbor-tristis contains phenolic compounds, resins, tannins, starch, glycosides and alkaloids. flavonoids were absent. the h. salicifolia results revealed the presence of carbohydrate, amino acids, alkaloids, flavonoids, phenolic compounds and terpenoids present in the extracts whereas starch and steroids were absent. the results reveal the presence of medicinally active constituents like tannins, alkaloid, terpenoids, steroids and flavnois, phlobatannins, glycosides in the leaves of o. tenuiflorum while saponins were absent in this plant. in r. indica, active constituents like steroids and flavnois, phlobatannins, glycosides were present except terpenoids, tannins and alkaloid. the phytochemical analysis revealed that combined herb shows the strong presence of flavonoids, phenolic compounds, alkaloids and tannin. these values are in accordance with the results obtained by mowl et al. and iwalokun et al. [47, 48]. quality control analysis: numerous microbial load in herbsthe plant material and plant extract obtained was subjected to microbial analysis and result reveals that the level of total microbial count and total yeast and mold count were not more than 1000 cfu/ml in the extracts of n. arbor-tristis, h. salicifolia, o. tenuiflorum, and r. indica and combined herb. the values were found to be within the limit of who standards. also, results showed absence of various pathogens like salmonella, e.coli, s. aureus and pseudomonas (table 4). heavy metal toxicity evaluation in the extracts of polyherbal formulation: the growth of medicinal plants not only need nutrients for normal plant growth, but also can selectively uptake and accumulate some trace elements which are good and may also be toxic for human health if there not within the limits. table 2: proximate composition of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica. proximate parameters n. arbor-tristis % w/w h. salicifolia % w/w o. tenuiflorum % w/w r. indica % w/w combined herbs carbohydrate 9.48±1.96 3.6±0.53 24.71±1.49 29.71±1.85 16.87±1.62 protein 15.02 ±0.96 26.31±2.02 5.01±0.09 12.01±2.52 11.68±1.39 fat 2.10±0.36 2.03±0.02 3.26±0.56 2.16±0.23 2.38±0.29 crude fiber 15.03±0.02 14.11±0.36 13.20±0.29 16.19±0.69 14.63±0.34 values were expressed as mean ± s.d for 5 different preparations table 3: phytochemical analysis of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica. parameters n. arbor-tristis h. salicifolia o. tenuiflorum r. indica combined herb alkaloids (+) (+) (+) (-) (+++) flavonoids (-) (+) (+) (+) (+++) terpenoids (+) (+) (+) (+) (++++) resins (+) (+) (+) (+) (++++) saponins (+) (+) (-) (+) (+++) steroids (+) (-) (+) (+) (+++) tannins (+) (+) (+) (-) (+++) starch (+) (-) (+) (+) (+++) glycosdes (+) (+) (+) (+) (++++) total phenolics (+) (+) (+) (+) (++++) nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 47 the results revealed the presence of heavy metal and inorganic compounds in the extracts of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica and also in combined herb and the values were found to be within the limit of who standards (table 5 and table 6, respectively). heavy metal and inorganic elements are present within the permissible limits. fluorescent analysis in the present study dried powder extract of each plant treated with various chemical reagents (several solvents and chemicals) showed characteristic fluorescence at 254 nm and 366 nm wavelength as shown in table 7a, table 7b, table 7c, table 7d and table 7e . high performance thin layer chromatography (hptlc) of hydroalcoholic extracts of h. salicifolia, n. arbor-tristis, o. tenuiflorum, r. indica tlc was done for the separation of different active constituents which are present in the extracts of h. salicifolia, n. arbor-tristis and o.tenuiflorum. the developed spots was visualized at various nm. in figure 5a from 1 to 3 track of the plate was of leaves extract and the fourth track was of standard nyctanthoside. from the picture obtained at 254 nm assumed that standard nyctanthoside has rf 0.47 in track 4 and similarly, obtained purple band from track 1 to 3 match with track 4 (figure 5a). figure 5a & 5b: tlc profile of test solution of n. arbortristis leaf. in figure 5a, 1-3:test solution, 4: n. arbortristis standard. figure 5b represents photo documentation report of hydro-alcoholic extract of n. arbor-tristis at 254 nm and 366 nm. table 4. microbiological analysis of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica. nmt=not more than, a=n. arbor-tristis, b=h. salicifolia, c=o. tenuiflorum, d=r. indica, e=combined herb plant extracts total microbial count cfu/ml total yeast and mold count cfu/ml pathogens salmonella e.coli s. aureus pseudomonas a nmt 1000 nmt 1000 absent absent absent absent b nmt 1000 nmt 1000 absent absent absent absent c nmt 1000 nmt 1000 absent absent absent absent d nmt 1000 nmt 1000 absent absent absent absent e nmt 1000 nmt 1000 absent absent absent absent table 5. heavy metal content of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica. a=n. arbor-tristis, b=h. salicifolia, c=o. tenuiflorum, d=r. indica, e=combined herb plant extracts heavy metals (specification-(ppm/ml)) lead (> 10 ) cadmium(>0.3 ) arsenic (>5) mercury(>0.5) chromium (>10) a 0.036 0.017 0.054 0.0010 0.024 b 0.042 0.019 0.051 0.0011 0.027 c 0.039 0.018 0.052 0.0013 0.028 d 0.049 0.015 0.059 0.0011 0.019 e 0.041 0.017 0.054 0.0011 0.024 table 6: inorganic elements of n. arbor-tristis, h. salicifolia, o. tenuiflorum and r. indica. note: a=n. arbortristis, b=h. salicifolia, c=o. tenuiflorum, d=r. indica, e=combined herb; al=aluminum, co=copper, ca=calcium, fe=iron, mg=magnesium, k=potassium, na=sodium, mn=manganese, zn=zinc, ni=nickel plant extracts inorganic compound al co ca fe mg k na mn zn ni a 32.03 0.153 0.26 4.69 0.049 0.184 0.226 0.099 0.578 0.453 b 35.78 0.192 0.22 4.703 0.043 0.192 0.217 0.094 0.679 0.441 c 36.23 0.157 0.23 4.56 0.049 0.189 0.239 0.089 0.745 0.563 d 34.15 0.195 0.29 4.26 0.042 0.163 0.215 0.062 0.697 0.438 e 34.54 0.174 0.25 4.55 0.045 0.182 0.224 0.086 0.674 0.473 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 48 table 7a: fluorescence analysis of n. arbor-tristis leaf treatment visible light under uv light short wavelength (254 nm) the long wavelength (365 nm) powder brown light brown green powder + methanol brown light brown yellowish green powder + 70% ethanol brown light brown green powder + pet. ether light brown light green green powder + 50% h2so4 brown greenish brown brownish powder + 50% hcl dark brown green green black powder + 1n naoh (aq.) light brown dark brown brownish black powder + 1n naoh (alc.) light brown dark green greenish black powder + 50% hno3 brown light brown light green powder + 5% koh brown purplish green dark purplish green powder + ammonia brown green black powder + picric acid yellowish brown green dark brown table 7b: fluorescence analysis of h. salicifolia fruit treatment visible light under uv light short wavelength (254 nm) the long wavelength (365 nm) powder yellow light yellow yellowish brown powder + methanol yellow light yellow yellow powder + 70% ethanol yellow light yellow yellow powder + pet. ether light yellow light yellow yellow powder + 50% h2so4 light yellow greenish yellow yellow powder + 50% hcl dark yellow greenish yellow colourless powder +1n naoh (aq.) light yellow green fluorescence yellowish brown powder + 1n naoh (alc.) light yellow brownish yellow light greenish yellow powder + 50% hno3 yellow greenish yellow yellow powder + 5% koh yellow brown greenish yellow powder + ammonia yellow light yellow yellow powder + picric acid yellowish brown brownish yellow light greenish yellow table 7c: fluorescence analysis of o. tenuiflorum leaf treatment visible light under uv light short wavelength (254 nm) the long wavelength (365 nm) powder brown dark brown powder + methanol brown green black powder + 70% ethanol yellowish brown green black powder + pet. ether brown green black powder + 50% h2so4 yellowish brown green black powder + 50% hcl brown green black powder + 1n naoh (aq.) yellowish brown green black powder + 1n naoh (alc.) brown green black powder + 50% hno3 yellowish brown green black powder + 5% koh brown green dark brown powder + ammonia yellowish brown green black powder + picric acid brown green black nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 49 table 7d: fluorescence analysis of combined herb-herb treatment visible light under uv light short wavelength (254 nm) the long wavelength (365 nm) powder brown brown brown powder + methanol brown brown brown powder + 70% ethanol yellowish brown brown brown powder + pet. ether dark brown light brown light brown powder + 50% h2so4 dark brown light brown light brown powder + 50% hcl brown yellowish green dark red powder + 1n naoh (aq.) brown yellowish green dark brown powder + 1n naoh (alc.) green green reddish orange powder + 50% hno3 green yellowish brown orange red powder + 5% koh brown reddish brown reddish brown powder + ammonia yellowish brown light orange light orange powder + picric acid brown yellowish brown orange table 7e: fluorescence analysis of r. indica leaf treatment visible light under uv light short wavelength (254 nm) the long wavelength(365 nm) powder yellow brown yellow powder + methanol brown greenish brown brownish powder + 70% ethanol light brown green green black powder + pet. ether light brown dark brown brownish black powder + 50% h2so4 brown dark green greenish black powder + 50% hcl brown light brown light green powder + 1n naoh (aq.) brown purplish green dark purplish green powder + 1n naoh (alc.) green green brownish green powder + 50% hno3 green green pale yellow powder + 5% koh brown green black powder + ammonia yellowish brown green dark brown powder + picric acid brown green black further, the sample track (track 1, 2, 3) and standard track (track 4) were scanned at 254 nm showed the same rf value 0.47 for nyctanthoside in both the track (table 8). finally, this nyctanthoside band in all this track which came at 0.47 rf were scanned at 366 nm. the spectral pattern for nyctanthoside in extract matched with the standard track. thus the presence of nyctanthoside was confirmed by overlaying the uv spectra at 366 nm and 254 nm (figure 5b) figure 5c, 5d & 5e: hptlc profile of hydroalcoholic of h. salicifolia with standard quercetin as developed in ethylacetate: dichloromethane: formic acid: acetic acid: water (10:2.5:1:1:0.5). figure 5c represents fruit extract of h. salicifolia and figure 5d represents quercetin standard. figure 5e represents photo documentation report of hydro-alcoholic extract of h. salicifolia at 254 nm and at 366 nm. fe=fruit extract; st=standard. table 8: rf value and color of peak of fruit extract and standard nyctanthic acid in toluene:ethyl acetate (8.0:2.0) at 366 nm s.no rf value colour of the band 1. 0.17 pink 2. 0.32 blue 3. 0.37 brown 4. 0.47 purple (nyctanthic acid) 5. 0.57 grey 6. 0.63 green 7. 0.70 blue 8. 0.77 blue nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 50 the extracts of dried powder of fruits of h. salicifolia was subjected to hptlc analysis and illustrated the number of active compounds. the quercetin content determined by hptlc method in fruits of h. salicifolia reported in (table 9) and showed 4 peaks at 254 nm and 8 peaks at 366 nm. at 366 nm, h. salicifolia (ha) extract showed 8 peaks and rf value matches with quercetin standard, so this confirms the presence of quercetin in the extract which is a flavonoid (figure 5c, 5d, 5e). the hptlc fingerprints of hydroalcoholic extracts of o. tenuiflorum showed 6 peaks at 254nm (table 10a) and 9 peak at 366 nm (table 10b) (shown in figure 6). hptlc fingerprint of o. tenuiflorum shows six different peaks at maximum rf values of 0.08, 0.32, 0.46, 0.54, 0.61 and 0.94. the peak at rf value of 0.46 is noticed to have an area of about 26650.2, which is the highest among the other peaks obtained and similar to eugenol. hptlc fingerprint of r. indica points out eight different peaks at starting rf values of -0.02, 0.14, 0.19, 0.26, 0.35, 0.43, 0.58 and 0.74 (table 11 and figure 7). among those peaks, the peak with rf value of 0.58, exhibits a larger area of 4786.3. the extract shows the presence of saponin in hydroalcoholic extract of r. indica at rf :0.69. figure 6: hptlc profile of extract of ocimum tenuiflorum with standard eugnol. (a) profil at showed 6 peak at 254nm ;(b) profil at showed 9 peak at 366 nm and (c)photo documentation report of hydro-alcoholic extract at 254nm and at 366nm le –leaf extract;st-standard. table 9: rf value and peak area of fruit extract and standard qurecetin in ethylacetate: dichloromethane: formic acid : acetic acid : water (10:2.5:1:1:0.5) at 366 nm. rf (max) area(au) -0.01 2256.7 0.06 247.9 0.10 142.1 0.53 494.5 0.57 426.9 0.77 195.8 0.82 127.4 0.94 7453.1 (fe) 0.96 8189.5(qurecetin standard) table 10a. rf value, no. of peaks, peak area and height of hydroalcholic extract of o. tenuiflorum in toluene: ethyl acetate at 254 nm. peak start rf start height max rf max height height % end rf end height area area % 1 0.01 302.6 0.01 324.0 47.62 0.03 0.6 2776.3 17.53 2 0.18 0.2 0.21 12.7 1.87 0.25 4.7 357.8 2.26 3 0.31 8.5 0.35 11.7 1.72 0.41 2.2 557.8 3.52 4 0.75 2.2 0.78 10.4 1.53 0.80 6.7 326.6 2.06 5 0.81 7.1 0.89 166.0 24.39 0.91 152.6 6422.5 40.55 6 0.91 152.9 0.92 155.6 22.87 0.97 71.1 5398.9 34.08 table 10b. rf value, no. of peaks, peak area and height of hydroalcholic extract of o. tenuiflorum in toluene: ethyl acetate at 366 nm. peak start rf start height max rf max height height % end rf end height area area % 1 0.00 0.2 0.02 265.5 31.82 0.06 157.6 6275.7 18.02 2 0.06 157.9 0.07 162.0 19.41 0.16 76.6 9120.9 26.19 3 0.16 76.7 0.18 92.8 11.12 0.24 31.6 3444.2 9.89 4 0.26 31.4 0.27 39.2 4.69 0.33 18.3 1545.7 4.44 5 0.36 16.3 0.47 67.7 8.12 0.55 12.6 5161.8 14.82 6 0.56 14.7 0.62 41.4 4.97 0.66 24.9 2258.7 6.49 7 0.66 24.9 0.71 59.5 7.13 0.79 6.5 3032.2 8.71 8 0.83 15.9 0.89 55.1 6.60 0.93 31.1 2453.6 7.05 9 0.93 31.4 0.95 51.2 6.14 0.99 1.6 1527.8 4.39 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 51 table 11: rf value, no. of peaks, peak area and height of hydroalcholic extract of r. indica in toluene: ethyl acetate at 254nm peak start rf start height max rf max height height % end rf end height area area % 1 0.00 0.2 0.02 265.5 31.82 0.06 157.6 6275.7 18.02 2 0.06 157.9 0.07 162.0 19.41 0.16 76.6 9120.9 26.19 3 0.16 76.7 0.18 92.8 11.12 0.24 31.6 3444.2 9.89 4 0.26 31.4 0.27 39.2 4.69 0.33 18.3 1545.7 4.44 5 0.36 16.3 0.47 67.7 8.12 0.55 12.6 5161.8 14.82 6 0.56 14.7 0.62 41.4 4.97 0.66 24.9 2258.7 6.49 7 0.66 24.9 0.71 59.5 7.13 0.79 6.5 3032.2 8.71 8 0.83 15.9 0.89 55.1 6.60 0.93 31.1 2453.6 7.05 figure 7: hptlc profile of hydroalcoholic of r. indica with standard saponin. photo documentation report of hydro-alcoholic extract of r. indica at 254 nm and at 366 nm le; leaf extract; st-standard. discussion in the current period, there is a need for logical assessment of natural formulation for better treatment of the clinical issues in a clinical manner. the bioactive compound of every, one of the four restorative plants exhibits inside homegrown concentrate and in joined herb-herb (polyherbal definition) that may result in all the more dominant for mental issue treatment. a few reports demonstrated that the utilization of a poly-natural plan has the greatest useful power when contrasted with a single herb. yet different definitions couldn't be valuable because of the absence of legitimate institutionalization. in india, ayurvedic pharmacopeia is not progressively sufficient to guarantee the quality and virtue of plant extracts’ use as a herbal drug. since the concentrates from assembling points are not in a condition that could impact appropriately. along these lines, for the experimentally demonstrated, customary medications and natural plans are to be institutionalized for guaranteeing and defending the best quality, immaculateness, and genuineness of the homegrown medications convert into powerful medication [49]. along these lines, institutionalization is the easiest and least expensive approach to the investigation of homegrown medication through synthetic, different strategies, morphological, minute examination and afterward thin layer chromatography examination as well as initiating to assigning the right id of the source plant extract [48]. as in the present investigation there is no comparable work accessible on these restoratively strong plant extricate and consolidated herb-herb. this examination work was embraced to mastermind the norms profile for building up its credibility. in this way, the results of the above discoveries will fill in, as a promising hotspot for setting down the pharmacopeia principles profile of n. arbortristis, h. salicifolia, o. tenuiflorum and r. indica and consolidated herb-herb for future investigations and research for treatment of mental malady. conclusion the present study was taken up in the view to standardize these herbals in accordance with who norms and standard laboratory procedures. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):36-53 kumari and dubey ©njb, bsn 52 formulations were investigated for their organoleptic characters, physicochemical parameters, hplc analysis and phytochemical parameters etc. the heavy metal quantity was also found within the standard limit as given by the regulatory authorities hence, the pharmacognostic parameter of the plant extracts will be helpful in further preclinical and clinical study. the presence of phytochemicals also indicated that it can be used to develop the new lead phyto-molecule in the treatment and management of lifestyle disease or disorder. the finding of this study can be used for evaluating the quality and purity of these plants as a polyherbal formulation for clinical application. author contributions all authors have equal contributions. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. funding the authors declared that no grants were involved in supporting this work. acknowledgments the creators stretch out their true gratitude to the department of science and technology, ministry of health, government of india for supporting this examination. the authors would like to thank, dr. r.c. satish kumar, m.d.(ayu) m.b.a (h.m.), interdisciplinary institute of indian system of medicine (iiism), sri ramaswami memorial university (srm) university, kattankulathur chennai, india for identification and authentication of plants. the authors are appreciative to similarly, grateful to srm university, chennai, india for the offering help in recognizable proof of bioactive atoms and factual investigation and input on the outcomes. ethical approval and consent not applicable. reference 1. aslam ms, ahmad ms, mamat as, ahmad mz, salam f. antioxidant and wound healing activity of polyherbal fractions of clinacanthus nutans and elephantopus scaber. evidence-based complementary and alternative medicine. 2016 jan 1;2016. https://doi.org/10.1155/2016/4685246 2. chaudhary a, singh n. contribution of world health organization in the global acceptance of ayurveda. journal of ayurveda and integrative medicine. 2011 oct;2(4):179. https://doi.org/10.4103/0975-9476.90769 3. 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https://doi.org/10.5897/ajb2007.000-2254 49. verma s, singh sp. current and future status of herbal medicines. veterinary world. 2008 nov 1;1(11):347. https://doi.org/10.5455/vetworld.2008.347-350 race for excellence science and technology has always been frontiers for prosperity and well-being of human life. the fundamental backbone of scientific clues and technological intervention relies on course and discourse of the natural attributes within the nature itself; taking into consideration of the physical, chemical, biological and computational prospect. nature is no doubt the greatest scientist, researcher and expertise, who timely challenges us, evokes us and motivates us for innovations, creations and constructive implications. biological sciences and allied research is on the hype in current scenario, because it is direct or indirect (through model animals) discourse of aspects pertaining to human at genetic, biochemical or molecular level. utilizing the science of biology, chemistry, physics, engineering, and information technology to develop products and services of great promise is the threshold of current scientific research. with different technological intervention and theological implications, the scientific community has not only been able to manipulate organisms but also manipulate life and materials at the atomic level for benefit of human civilization. human capacity has been exemplified by cutting edge innovations and implications for better life. human has been able to unravel from bottom of sea, through the surface of earth, and beyond the atmosphere into the space. but, as one goes on unraveling the intricate aspects of nature, you face with new meshwork of mysteries. this is the charm and vigor of science and technology that makes the things just moving on forward and ahead. the scientific community: the team leaders, associates, and assistants in academia and industries are enormously involved in breaking through these mysteries, modulate them and utilize them for worthy outputs. with this manifestation of the hypothesis to final accomplishment, the current science and technology is moving rapidly and efficiently. but, the story starts from here, the deeper we go the wider the horizon becomes; accomplishing one landmark is starting point for a longer race to compete with nature. it is race for excellence; nature versus the scientific nurture, zeal, passion and commitment. are you ready to go is the call? if yes, get set ready and go! if not then wait and let others to go before you but you have to move on. it is something like, you make a choice or the choice will make you! be ready with the strategy! we heard the voice, culminated our energies and got on the track for the journey! before, saying “here, it is”; we are thankful to all the stakeholders and bolstering hands on our effort. dipesh dhakal executive editor nepal journal of biotechnology nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 doi: https://doi.org/10.3126/njb.v8i1.30209 review article ©njb, bsn 54 regeneration technique of bamboo species through nodal segments: a review meena maiya suwal, janardan lamichhane, dhurva prasad gauchan department of biotechnology, school of science, kathmandu university, dhulikhel, kavre, nepal. article history:received: 23 nov 2018; revised: 7 jul 2020; accepted: 21 jul 2020; published online: 31 jul 2020 abstract micropropagation is an alternative technique to propagate at large scale plants to meet global plant demand. various researchers have worked on the micropropagation technique to regenerate bamboo species by using nodal segments from years. contamination, browning, necrosis, and acclimatization with physiological stress are the extreme problems of the micropropagation technique. but, many numbers of papers have been published on micropropagation of the bamboo species through nodal segments as explants. the proliferation of the bamboo shoots is dependent on the season of collection, size of explants, the position of explants, diversity of plants, concentration and combination of plant growth regulators, most adequate culture medium, environmental condition of the equipment, handling, and individual species. bamboo is a monocarpic fast-growing, tall perennial grass and having the high potential to generate economic and social benefits. it helps to maintain land patterns and control soil erosion. the long life cycle of the bamboo produces a huge amount of seeds but unfortunately, mostly, they are non-viable. so, bamboos are propagated from vegetative by cutting and air layering. however, these methods are only for a small scale and they also tend to destroy large mother plant stocks and difficult to be transported. so, the in vitro propagation technique is useful to obtain large progenies from desired genotypes. mostly, bap and tdz growth hormones are widely used for shoot multiplication and iba, naa and iaa are used for root initiation as per developed protocols in tissue culture for large scale production. this review intends to explore an overview of the recent literature reports to summarize the importance of micropropagation by using nodal segments of bamboo species and factors influencing it. keywords: micropropagation, nodal segments, bamboo species, plant growth regulators, shooting and rooting corresponding author, email: gauchan@ku.edu.np introduction bamboo is an attractive plant species for various purposes due to its versatile utilization with high economic potentiality and its accelerated growing capacity at short period [1, 2]. it is perennial strong woody giant grass [1-5] having a unique complex branching system of rhizomes with root. it belongs to subfamily bambusoideae of the poaceae family and subsequently divided into three tribes [3, 4, 5, 6]. it can grow from approximately 75 to 400mm per day [7]. it is distributed up to 4000 m. a. s.l. in temperate to the zone of all continents except europe [8]. it prefers to grow at a minimum of 100 cm annual rainfall with high atmospheric humidity in steep hillsides, road embankments, gullies, or on the banks of ponds and streams [9]. it occupies 3% of the forest in the tropical, subtropical, and temperate zone of the world but while, in asia; it covers 10 % of the forest. there are 120 genera and 1641 species found in the world [10, 11 and 12]. tropical asia has rich bamboo diversity with up to 60 genera and 1000 species [13]. however, nepal has only 12 genera and 65 species of bamboos distributed in the tropical, subtropical, and temperate zone [14]. since the ancient period, bamboo species are widely used as a renewable source due to its versatile nature [15, 16]. according to hsiung (1988), more than 4000 traditional uses and 1500 commercial applications have been known to bamboo. it’s multipurpose potentiality from cradles to coffin and as “green gold” [17]. similarly, it is used for making paper, pulp, food, scaffolding, textiles, plywood, boards, raw materials for construction, fencing, clothes, reinforcing fibers, etc, and bio-energy applications [10]. also, it is used as an alternative source of energy and helped to prevent soil erosion due to the closely woven mat of intertwining roots and rhizomes [17, 18] with its high agro-climatic suitability. mailto:gauchan@ku.edu.np http://orcid.org/0000-0002-3728-5076 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 55 it is perennial plants with monocarpic nature having vegetative and reproductive cycle range from 3 to 120 years [19], sporadic flowering with the recalcitrance of seeds which made difficult for identification and floral characterization of bamboo. mostly, the classification and identification of bamboo is done based on the vegetative pattern but it is not reliable due to influence of ecological factors an easily practicable because bamboo can produce a limited number of seeds after a long period of its life span which has short viability period only 3 to 6 months [17, 18, 19] and suffering from insects and rodents. hence, the bamboo is regenerated through the vegetative method by planting of rhizome offset, branch & culm cuttings, nodal macro-cutting and layering [20, 21] but for this process, it needs a large mass of bamboo plant stocks. this leads to the destruction of entire clumps and the gradual depletion of bamboo resources. again, it is not easily available sufficiently due to seasonal dependency and low rooting capacity so it is highly expensive [17]. similarly, it is difficult to transport and handle due to heavyweight and long length [22, 23]. also, there is more chance of liable desiccation before rooting [24]. moreover, yield production of bamboo is hardly possible [22-27] through conventional and non-conventional methods. therefore, it is necessary to apply other techniques of plant tissue culture because it is highly demanded the industry to agriculture. in vitro propagation of bamboo plant tissue culture is the only advanced technique that can be applied to solve the challenges of speedy and mass propagation of the bamboo species [27]. for the conservation of bamboo and to fulfill the growing demand of the markets, micropropagation is the alternative method that provides rapid mass multiplication of bamboo along with disease-free plants as well as the same clone [25-32]. micropropagation is not only ensuring the supply of quality planting material regularly but it also helps in conserving of germplasm of bamboo [25]. different types of explants viz. seed, seedlings, inflorescences, root, culm, mature clumps, nodal segment, meristem domes or leaves, etc [33, 34, 35] are used for bamboo micropropagation. in bamboo, both juvenile and mature plants can be considered as explants [36]. the nodal segments containing is considered as more effective explants for in vitro culture because of resumed food materials. due to the presence of highly active meristematic tissue in nodal segments, it develops into new plantlets [37]. the response of explants depends on the physical condition of plants, the health of mother plants, collection of the season from field and size of explants, and its position in mother plants [38]. using nodal explants for organogenesis reduces the chance of somaclonal variation [27]. there is so much research conducted on micropropagation of bamboo through nodal explants [27-47] however, the developed protocols are either insufficient or not applicable because the protocols are only limited to the research which is not applicable for industrial mass production. it is intended to explore the suitable protocols on the micropropagation technique for mass bamboo propagation. this review intense to give an overview of the recent literature reports to summarizes the importance of micropropagation by using nodal segments of bamboo species and factors that influence it. collection of explants nodal explants were collected from january to february, march to april, may to june, july to august), september to october, and november to december [32, 41, 48]. the establishment of explants in culture was directly related to the collection of explants seasons and plants species which influenced in pure culture. it was dependent on external factors i.e. contamination and concentration of the hormones in media [49]. explants collected during february-march and september-october showed maximum bud break [48]. it was reported that the rainy season was better for bud break in dendrocalamus strictus [49] bambusa tulda [40] gigantochloa atroviolacea [50] with the high rate of contamination [40, 51, 52]. the explants collected from november to january (winter months) produced only 35-45% bud break response during the culture of bambusa vulagaris by [53]. spring and summer season (february to june) was a comparatively better period for the collection of explants [37]. however, negi and saxena [29, 30] have obtained high aseptic cultures along with a 90% bud response from july to october in bambusa balcooa. according to negi and saxena (2011) and mehta et al. (2011), the best collection of explants was in july for culture initiation in bambusa nutans. but singh et al. (2012b) reported the collection of nodal explants at pre-monsoon induced maximum nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 56 bud break in dendrocalamus asper. ramanayake et al. (1995) observe that the influence of seasons on bud break was countable in d. giganteus and berberis vulgaris. it was observed that seasonal effects on bud initiation and found that february to march is a good period for obtaining auxiliary buds for cultural development [54]. moreover, shivabalan et al. (2014) have established a new culture form explants of b. balcooa collected in december. in other seasons, the establishment of pure culture is difficult due to the high scale of contamination suffering from the variable pathogen in the summer and rainy season. better establishment of pure aseptic cultures and bud response of explants depends on the ratio of contamination, handling, physiological state of explants, species, type of explants, and the season of collection [24, 43, 52, 55, 56, 57, 58, 59]. different aged of the mother stock plants were also used for culture initiation. the new culm (1 year) and lateral branched (frequently actively branched) of 2 to 5 years old bamboo can be used as explants [25, 43]. but patel et al. (2015) have established in vitro culture from 2 to 30 years old explants and also sharma et al. (2012) used nodal explants from the current year’s growth mature and healthy clumps of bambusa nutans. similarly, nodal explants of 40 years old b. nutans; 30 years old b. balcooa [60] and 10 years old b. tulda were used for multi proposed [59, 61]. the aged of the explants could not effectually determine in the initiation of the shoot in vitro. furthermore, there may be some result influenced by the age of the plants during the experiment but there was no impact reason behind it. it depended on the composition of media, concentration of hormones, contamination rate, and condition of the culture [31]. also, various aged of the explants can be established successfully in vitro culture through single nodal segments from bamboos. position of the nodal explants to date, only limited researchers have mentioned the position of nodal explants in mother plants. nodal segments from the healthy mature mother plant with disinfected lateral branches and were more effective for the initiation of the culture [62]. chowdhury et al. (2004) recorded that the 1st and 2nd position from the base of secondary branches of d. strictus was the best for regeneration in micropropagation. but, according to mudoi et al. (2008, 2014), the 5th to 7th position of the b. balcooa, b. nutans and b.tulda explants from mother stock culm was best for maximum regeneration in vitro culture rather than below 5th position because the base explants can excaudate phenolic compound which resulted in browning problem on shoots. a similar result was also illustrated in the report of devi and sharma (2009) in which the top position of explants showed a low frequency of bud break in comparison to the basal and mid culm nodes in arrundinaria callosa munro. middle node explants of culm were very effective resulted in vitro propagation of the b. vulgaris, [63]. another experiment revealed that the auxiliary branch of explants from healthy mother stock was found to be good for regeneration of the new plants such as in d. hamiltonii [1, 64], d. asper [65], d. giganteus [56]. similarly, sharma and sarma (2013) reported that young lateral buds also showed the bud break in b. tulda. therefore, it is stated that the top and the base portion of the nodal segment in culm bamboo can hardly regenerate in vitro propagation of bamboo. surface sterilization the size of 2.5 mm explants was more effective than a smaller size (5-7 mm) to initiate the culture within a short period because of high endogenous hormonal effect [37, 41]. for the initiation of the culture, the explants were surface sterilized by treating different kinds of chemicals for a certain time to avoid the contamination. it was reported that explants treated in 70% ethanol for the 30s to 1 min [25, 29, 30, 31, 33 39] followed running tap water after washing in 4-6 drops of detergent (tween 20/tween 80) for 30 mins. [29, 30, 33, 68] reduced the rate of contamination. it was reported that 0.1% mercuric chloride (hgcl2) was found more effective than other surface sterilants (sodium hypochlorite, potassium hypochlorite, hydrogen peroxide, etc.) for various species of bamboo micropropagation [29,31, 33, 35, 36, 38, 39, 6571]. so it is suggested that 0.1% mercuric chloride was more effective for the disinfection of the explants because the high concentration of the hgcl2 retarded the growth of plants due to the impact of chemical in the internal tissues [72]. wei et al. (2015) have reported that the treatment of explants on 0.1% hgcl2 at lower duration enhanced the survival rate of explants and frequency of bud break. when treatment time was increased at the same concentration of hgcl2 in d. strictus, the bacterial and fungal contamination was decreased [36]. similarly, for the establishment of a pure culture of bamboo, different researchers have nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 57 used antiseptics like savlon, tempol, streptomycin sulphate, gentamicin, cetavelon, etc [29, 40, 42, 60] and fungicide like bavistin, benomyl, mancozeb, carbendazim, tetracycline, etc [37, 41, 42. 49, 53, 56]. culture media according to chang and ho (1997), the nutritional composition is greatly varied in culture media which depends on the types of tissues and plant species. so, for the establishment of the culture, the proper media play a major role in the growth and development of the plants. it is difficult to consider a unique media for all types of plants in tissue culture. not only for getting maximum auxiliary bud breaking, but ms [78] medium has also been widely practiced for more superior responsive bud proliferation and further multiplication of bamboo in comparison to other media such as sh (schenk and hildebrandt 1972), b5 (gamborg et al. 1968) and nn (nitsch and nitsch 1969), wp medium (lloyd and crown 1980) [34-122]. but kabade (2009) observed that the wpm media is suitable for shoot induction from nodal segments of b. bambos. similarly, shirgurkar et al. (1996), singh et al. (2001), ogita et al. (2009), and negi and saxena (2011) reported that the half-strength rather than full strength ms medium was better for successful in vitro culture in bamboo [123-124]. the physical condition of the media is also a factor that influences to grow plant tissue under in vitro culture. several researchers reported that the proliferation and shoot multiplication of the bamboo was successfully obtained under in vitro culture on semi-solid/solid ms media [34, 35, 36, 39, 40-48]. mostly, 0.8% agar was widely used as a gelling agent to solidified/semi-solidified the media which influenced the plant metabolism [32-39, 76]. some researchers also used phytagel (0.2%) or gelrite (0.20.35%) to agar which influenced high bud breaking in b. wamin [72] and shoot proliferation in b. oldhamii [77]. similarly, it was noted that dwarf and a lower number of shoots per explant in ms solid media due to leaching and browning problems [79]. similarly, sharma and sarma (1998) have also observed leaching of phenolic exudates and poor growth of shoot in ms agar gelled medium. several reporters mentioned that liquid ms media was also observed more suitable than ms semi-solid/ solid media for proliferation and multiplication of the shoots in bamboo species [34, 43, 78]. the high rate of shoot initiation has observed in the liquid medium compared to agar gelled medium means attributed to easy availability and faster uptake of nutrients in liquid medium [81]. when culture initiated in liquid media generally shoots were grown faster and less required hardening time [82]. plant growth regulators the chemical substances which influenced either promote (positive) or inhibit (negative) the growth of the plant are plant growth regulators (pgr). the low quantity of pgr can change the morphological structure of plants. natural and synthetics phytohormones are widely used in tissue culture. mostly cytokinins and auxin are used for callogenesis and histogenesis of bamboo. different concentration of the 6–benzyl aminopurine (bap), 6–benzyl adenine (ba), napthalene acetic acid (naa), indole 3-butyric acid (iba), indole acetic acid (iaa), zeatin (zn), kinetin (kn), thidiazurn (tdz) with the supplement of 3% sucrose and 100 mg/l myo-inositol was used on the micropropagation of the bamboo. the growth regulators hormones used by researchers in table 1. there were various factors affect the initiation of an aseptic culture of explants. the rate of percentage on bud break was varied with different concentrations of plant growth hormones, condition, physiological status of explants, size, the position of the explants, age of mother plants, the health of mother stock and collection season of explants. mostly, bap and ba were widely used for micropropagation of bamboo because it might be cost-effective and autoclave nature [36], and ultimately bap and ba showed successful results when in cooperated within ms media for bud breaking, proliferation, and multiplication of shoots of several bamboo species. but arya et al. (2003) could not obtain an axillary bud break in b. tulda in the presence of bap only. venkatachalam et al. (2015) have reported that 85% bud break was obtained separately or a combination of different concentrations of bap, naa, and kn with a supplement of additivesincooperation with ms solid media. moreover, similar combined effects of two cytokinins (bap and kn) in different concentrations have found successfully result in bud breaking and shoot initiation of b. arundinancea retz. wild [84]. however, in shoot initiation experiment, different researchers have tried auxin (naa, iaa) along with different combinations of cytokinins (bap, kn, and tdz and also suggested that increased levels of bap and kn retarded in bud initiation [23, 57, 58, 62, 78]. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 58 table 1. micropropagation from nodal segments/explants bamboo species plant growth regulators results ref bud breaking shoot multiplication rooting arundinaria callosa munro liquid ms + bap(13.3 μm/l) liquid ms + bap (13.3 μm/l) + iba (1.0 μm/l) 1/2 ms liquoid + iba ( 25 μm) + bap ( 0.05 μm/l) shoot multiplication and rooting 70 b. arundinacea bap (5.0 mg/l) bap (5.0 mg/l) naa (3.0 mg/l) mass multiplication 79 b. arundinacea retz. willd ms + bap(3.0 mg/l ) +kn (0.5 mg/l) ms +bap (3.0 mg/l) + kn (0.5 mg/l) ½ ms+ iba ( 2.0 mg/l) + kn (0.5 mg/l) mass multiplication 94 b. balcooa bap (11.25μm/l) +kn (4.5 μm/l) 1/2 ms+ iba (1.0 μm/l) in vitro regeneration 67 b. balcooa bap (1.0 mg/l) bap (1.0-5.0 mg/l) 1/2ms+naa(1-3mg/l) + iba(1 -5. mg/l) mass multiplication 112 b. balcooa ms +bap (4.4 µm/l)+naa (0.53µm/l) ms + bap (4.4 µm/l)+ naa (0.53µm/l) ms + naa (16.11 µm/l) mass multiplication and rooting 49 b. balcooa roxb ms+ citirc acid (25mg/l) + ascorbic (50 mg/l) +bap (3.5 mg/l) ms+ bap (3 mg/l)+ naa (0.5 mg/l) ms +naa ( 4 mg/l) mass multiplication and rooting 51 b. balcooa ms+tdz (0.1 mg/l)+ gelrite (2g/l) ms + tdz (0.1 mg/l) ms + tdz ( 0.01 mg/l) + 2,4-d (0.5 mg/l). mass multiplication and rooting 85 b.balcooa ms + bap (1 mg/l) ms+ bap (1 mg/l) ms+ bap (1 mg/l) + naa (3mg/l) mass multiplication and rooting 61 b.balcooa ms+ bap (4 mg/l) liquid ms + bap (4 mg/l) ms liquid+ iba (1mg/l) mass multiplication and rooting 68 b.balcooa liquid ms + bap (1 mg/l) ms+bap (1.0-5.0 mg/l) ½ ms+ naa (3 mg/l)/ iba (5 mg/l) mass multiplication and rooting 112 b.balcooa liqiud ms + bap (11.25 μm/l) + kn (4.5 μm/l) liquid ms + iba (1 μm/l) ½ ms liquid) + iba (1 μm/l) mass multiplication and rooting 98 b.balcooa ms+ bap (4.4 μm/l) + kn (2.32 μm/l)+ gelrite (0.2% w/v) liquid ms + bap (6.6 μm/l)+kn ( 2.32 μm/l)+ coconut water (2.5% (v/v) 1/2 ms +iaa (5.71 μm/l)+ iba (4.9 μm/l)+naa (5.37 μm/l) mass multiplication and rooting 34, 35 b.balcooa ms + bap (3 mg/l) ms + bap (5 mg/l) ms +naa (4.5 mg/l) mass multiplication and rooting 124 b. bambos ms + bap (4.4 μm/l) ms+ bap (4.4 μm/l) + kn (1.16 μm/l) ms+ iba (9.80 μm/l) mass multiplication and rooting 49 b.bambos ms + bap (4.4 µm/l)+kn ( 1.16µm/l) ms + bap (4.4 µm/l)+kn ( 1.16 µm/l) ms + iba (9.80µm/l) mass multiplication and rooting 49 b.bambos … bap (5.0 mg/l) naa (3.0 mg/l) micropropagat-ion 48 b. edulis bap (1mg/l)/ bap(1mg/l)+ naa(1mg/l) tdz (0.01 mg/l) tdz (0.01 mg/l) micropropag-ation and invitro flowering 85 b.glaucescenswilld ms + ba (5 μm/l) liquid ms+ ba(5 μm/l) + kn 15 μm/l) ms+ iba (25 μm/l) mass multiplication and rooting 99 b. nutans wall ex. munro ms+ bap (1.0 mg/l) ms +bap ( 0.5 mg/l) + naa (0.1 mg/l). ms+ naa (2.0 mg/l) mass multiplication and rooting 29 b. nutans wall ex. munro liquid ms+ bap (1 mg/l) ms+ bap (1.0-5.0 mg/l) ½ ms +naa(3.0 mg/l)/ iba (5.0 mg/l) mass multiplication and rooting 112 b. nutans wall ex. munro ms+ ba (2.22 μm/l) liquid ms+ ba (2.22 μm/l) ms+ iba (49.0 μm/l) mass multiplication and rooting 97 b.nutans ms + bap (4.44 µm/l)+ 2,4-d (4.2 µm/l) + 3% sucrose ms + tdz (6.49 µm/l)+ naa ( 0.74 µm/l) ms +naa (16.11 µm/l) +2% sucrose mass multiplication and rooting 58 b. nutans wall ex. munro ms+ ba (4.4 μm/l + kn (2.32) liquid ms + ba (13.2 μm/l) + kn (2.32 μm/l) + iba (0.98 μm/l) ½ ms+iba (9.8 μm/l) + iaa (2.85 μm/l)+ aa (2.68 μm/l) mass multiplication and rooting 34, 35 b. nutans wall ex. munro ms + bap (5.0 µm/l) ms + bap (5.0 µm/l) ms + iba (10.0 µm/l) shoot multiplication and rooting 119 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 59 b. oldhamii munro ms+ tdz (0.45 μm/l)+ gelrite (2.2 g/l) liquid ms + tdz (2.27 μm/l) ms basal + naa (10.74-26.85 μm/l) shoot multiplication and rooting 85 b. pallida liquid ms+ ascorbic acid (50 mg/l) + citric acid (25 mg/l) + cysteine (25 mg/l)+ naa 1.34 μm/l+ tdz (1.125 μm/l) liquid ms+ naa (1.34 μm/l)+ bap ( 4.44 μm/l) ½ ms+ 2% sucrose +1% glucose + 0.6% agar after treatment of iba (0.5 μm/l) for 30 min mass multiplication and rooting 111 b. pallida ms+ ba (1 mg/l) + gelrite (2.5% ) ms+ ba (3 mg/l) ms+ naa ( 2.0 mg/l) mass multiplication and rooting 88 b. salarkhanii liquid ms+ bap (1 mg/l) ms+ bap (1.0-5.0 mg/l) ½ ms+ naa (3 mg/l)/iba(5 mg/l) mass multiplication and rooting 112 b. tulda ms+ ba (1.0 mg/l) semi-solid ms+ ba (1.0 mg/l) ms+ naa(5 mg/l) mass multiplication and rooting 88 b. tulda semi-solid ms+ ba (10 μm/l) + iaa ( 0.1 μm/l) ms (l) + glutamine (100 μm/l ) + iaa (0.1 μm/l) + bap (12 μm/l) ms liquid medium + 40 μm/l coumarin mass multiplication and rooting 47 b. tulda ms+ bap (3mg/l) liquid ms+ kn (2mg/l) + bap (3mg/l) ½ ms+ iba (3mg/l)+ coumarin 10 mg/l + 3% sucrose mass multiplication and rooting 46 b. tulda ms (liquid) + bap (8.8 µm/l)+ kn(4.46 µm/l) + 2% sucrose ms (liquid) + bap(8.8µm/l)+kn ( 4.46 µm/l) + 2% sucrose ms (liquid) + iba (18.8 µm/l) + 2% sucrose mass multiplication and rooting 122 b. tulda liquid ms + bap (2.5 mg/l) + kn (1mg/l) + 8% coconut water liquid ms+ bap (2.0 mg /l) + kn (1.0 mg/l)+ 8% coconut water ½ ms + iba (0.2 mg/l) mass multiplication and rooting 98 b. ventricosa bap (4.44 μm/l) bap (4.44μm/l) naa (5.4 μm/l) + bap (0.44 μm/l) in vitro regeneration 53 b. ventricosa ms+ ba (22.2 μm/l) ms+ ba (22.2 μm/l) + tdz (0.23 μm/l)+ naa (0.27 μm/l) ms + naa(2.7 µm/l) +iba( 4.9 μm/l)+ ba (4.4 µμ/l) mass multiplication and rooting 78 b.vulgaris modified ms+ bap(2 mg/l) modified ms+ bap (2 mg/l) modified ms+ iba (20 mg/l) mass multiplication and rooting 113 b. vulgaris liquid ms+ bap (1 mg/l) ms+ bap (1.0-5.0 mg/l) ½ ms +naa (3.0 mg/l)/iba ( 5.0 mg/l) mass multiplication and rooting 112 b. vulgaris liquid ms+ bap (1 mg/l) ms+ bap (1.0-5.0 mg/l) ½ ms+ naa (3 mg/l) / iba(5 mg/l) mass multiplication and rooting 112 b. vulgaris ms+ bap (2.0 mg/ l) ms +bap (4.0 mg/l) ½ ms+ iba (3.0 mg/l) mass multiplication and rooting 64 b. wamin ms (l) + bap (5.0 mg/l) semisolid ms+ bap (2.0 mg/l) +kn (0.8 mg/ l) ½ ms+ iba (7.5 mg/l) 8 80 d. asper bap ( 0-2.0 mg/l + cw (0-20.0 mg/l) bap (5.0 mg/l) naa (0.5 mg/l) in vitro culture 2,96 d. asper ms+ bap ( 5 mg/l) ms (l) bap ( 5 mg/l) +ads ( 40 mg/l) ms (liquid) + iba (1 mg/l) shoot multiplication and rooting 103 d.asper ms+ ba (0.1-15 mg/l) ms (l) + iba (3 mg/l) ms + iba (10 mg/l) shoot multiplication and rooting 25 d. asper bap (3.0 mg/l) bap (1.0-4.0 mg/l naa (2.0 mg/l) or iba (10.0 mg/l) shoot multiplication and rooting 48 d.asper ms + bap (15 μm/l) ms + bap (10 μm/l) + ads (75 μm/l)+ table sugar 3% ½ ms +iba ( 5 μm/l) + naa (5 µμ/l) shoot multiplication and rooting 21 d. asper ms+bap (8.86 μm/l)+ ads (13.5 μm/l) ms+bap (8.86 μm/l) + ads (13.5 μm/l) ms+ iba (14.76 μm/l)+ naa (3.67 μm/l) shoot multiplication and rooting 59 d.asper {schult. & schult.f.} backer ex k. heyne) ms+ bap (15 μm/l) ms + bap (10 μm/l)+ ads (75 μm/l) ½ ms + iba (5 µμ/l) + naa (5 μm/l) shoot multiplication and rooting 66 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 60 d.asper ms+ ba (3 mg/l) liquid ms + ba (3 mg/l) + ads (50 mg/l) ms (liquid) + iba (1.0 mg/l) shoot multiplication and rooting 2 d.brandisiikurz. ms (l) + ascorbic acid (25mg/l) +citric acid (12.5mg/l)+cystei ne (12.5mg/l) + glutamic acid (50 mg/l) + tdz (0.25 mg/l)+ naa (0.25 mg/l) ms (liquid) + naa (0.25 mg/l) + bap (2.5 mg/l) ½ ms (l)+ naa (1mg/l) shoot multiplication and rooting 86 d. giganteus bap (2.0 -5.0 mg/l) kn (10.0 µm/l) + bap (0.5 µm/l) … mass multiplication 73 d.giganteus bap (30.0 µm/l) bap (20.0 µm/l) iba (25 µm/l) + bap (0.05 µm/l) rapid multiplication 48 d.giganteus .. iba+tdz+ coumarin root induction 105 d. giganteus munro semi-solid ms+ bap (2 mg/l) + kn (0.1 mg/l)+ benlate (1g/l) ms (liquid) + bap(6 mg/l)+ kn (1 mg/l) + 8% (v/v) coconut water ½ ms+ iba (3 mg/l) + 10 mg/l coumarin shoot multiplication and rooting 106 d.hamiltoniinees et arn. ex munro ms+ 2% sugar followed by ms+ bap (8 μm/l)+ naa (1 μm/l) ms+ bap (8 μm/l)+ naa (1 μm/l) ms+iba (100 μm/l) followed by growth regulator free media shoot multiplication and rooting 102 d.hamiltonii arn. ex munro ms+ tdz (3.0 μm/l) ms+ tdz (1.5 μm/l) + ascorbic acid (56.0 μm/l) ½ ms+ iba (25.0 μm) + choline chloride (36.0 μm/l) shoot multiplication and rooting 62 d. longispathus kurz. ms+ bap (12 μm/l)+ kn (3 μm/l) ms (l) + bap (15 μm/l)+ iba (1 μm/l) + coconut water (10%) ½ ms+ iba (1μm/l)+ iaa (1μm/l) +coumarin(68 μm/l). shoot multiplication and rooting 28 d. membranaceus ms +bap (1-5mg/l naa (0.5 g/l) bap (1-5mg/l) + naa ( 0.5 mg/l) naa ( 3.0 mg/l) / iba (10.0 mg/l) mass multiplication 73, 79 d. membranaceus ms + bap (4.4 µm/l) + kn ( 1.16 µm/l) ms + bap (4.4 µm)+ kn ( 1.16 µm/l) 1/2 ms + naa (5.37 µm/l) + bap( 4.4 µm/l) mass multiplication and rooting 49 d. strictus ms + bap (2.0-5.0 mg/l) bap (2.0-5.0 mg/l) mass multiplication 73 d. strictus nees white medium liquid ms+ ba (0.5 mg/l)+ kn (0.5 mg/l)+ coconut water (200 ml/l) solid ms+ iba (0.25 mg/l) shoot multiplication and rooting 1 d.strictus nees ms (l)+ ba (0.5 mg/l)+ ads (15 mg/l) ms (l)+ iba (0.5 mg/l)+ ads (15 mg/l) 1/2ms liquid + iba ( 0.25 mg/l) shoot multiplication and rooting 45 d.strictus nees ms + bap (2 mg/l) ms + bap (4 mg/l) + ads (15 mg/l) ms+iba (5mg/l) shoot multiplication and rooting 75 d.strictus nees ms+ iaa (0.5 mg/l)+ ads (15 mg/l ) ½ ms+ iba (1 mg/l)+naa(1mg/l)+ 2,4-d (0.5mg/l)+ phloroglucinol (1mg/l) shoot multiplication and rooting 56 d.strictus nees ms+ bap (4 mg/)+ tdz (0.25 mg/l) ms+ bap (4mg/ l)+ tdz (0.25 mg/ l) liquid ms+ bap (2.5 mg/ l)+ iaa ( 5 mg/ l) shoot multiplication and rooting 31 d.strictus nees ms+ bap (4 mg/l) ms + bap (4 mg/l) ms+ naa (3 mg/l) shoot multiplication and rooting 44 d.strictus (roxb.) nees ms+ 2,4-d (5 mg/l) -----------ms + 2,4-d (0.5 mg/l) callus initiation shoot multiplication and rooting 31 melocanna baccifera ms+ bap (3 mg/l) liquid ms+ kn (2mg/l) + bap (3mg/l) ½ ms+ iba (3 mg/l)+ coumarin (10 mg/l)+ 3% sucrose, shoot multiplication and rooting 46 thamnocalamusspat hiflorus (trin.) munro ½ strength ms ms medium + bap ( 5.0 μm/l) + iba (1.0 μm/l) ½ ms+ iba (150 μm/l) shoot multiplication and rooting 91 thyrsostachysoliveri liquid ms+bap (1 mg/l) ms+ bap (1.0-5.0 mg/l) ½ ms + naa (3.0 mg/l)/ iba (5.0 mg/l) shoot multiplication and rooting 112 nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 61 contamination: bottleneck for culture establishment surface and systemic contamination is the major incidence in tissue culture of bamboo because it has large intercellular spaces and vessel cavities at the cut end which accumulated contaminating agents deeply. therefore, various bacterial and fungal contaminations have been serious problems obtained in bamboo in vitro culture. according to ramirez et al. (2009), several bacteria like xanthomonas, pseudomonas, agrobacterium and erwinia and bacillus sp. by cruzmartin et al. (2007) are contaminating agents in some species of bamboo tissue culture while pantoea agglomerans and p. ananatis by nadha et al. (2012) are the main bacterial contaminates in the nodal segments of g. angistifolia. to overcome this problem, there is either incorporation of antibiotics in media or explants treatment in antifungal. surface sterilization of all pieces of equipment including explants has been done with care to escape contamination [35-40]. yasodaha et al. (2008) have used streptomycin and kanamycin during the culture initiation stages. the combinations of bavistin with streptomycin [55], streptomycin sulphate and tetracycline hydrochloride [68], bacteriocin [88] gentamycin [89] are used successfully to reduce contamination. when there is not directly used of tap water for washing explants during surface sterilization, it helps to minimize the contamination [38]. bavistine (cardazamine) and mancozebas antifungal and gentamycin as antibacterial [25, 41-45] are used as anti infections. also, ali et al. (2009) have been found that the combination of the antibiotics streptomycin, rifampicin and ciprofloxacin with the fungicide bavistine is successfully disinfecting in the micropropagation of nodal explants of b. tulda, b. balcooa, b. bamboos, and d. asper. oprin et al. (2004) found that rinsing explants with acetone for 3-4 times with a bleaching solution help to disinfect the explants. during surface sterilization of the explants, it is useful to apply the pre-treatment technique with the combination of the standard disinfected compounds. it is proved by jimenez et al. (2006) when they pretreated the explants in extran, agrimycine, and benomyl before surface sterilization in sodium hypochlorite and plant preservative mixture. the contamination was reduced from 2% to 11% in gaudua angustifolia. ramanayake et al. (1995) have suggested that it can be controlled systemic fungal contamination in b. vulgaris by supplement of benomyl in the culture medium. similarly, the other major problem for the bamboo in vitro propagation is browning and necrosis of the shoots in the initiation of culture, shooting stage and rooting stage due to phenolic compounds exudation [25] and increase the production of polyphenol oxidase by wounding of the tissue. it converts browning into blackish, and later on, it dies because the increased production of polyphenol is phytotoxic to the explants [45]. according to huang et al. (2002) and oprins et al. (2004), the browning of the plants depends on the species, age and position of the tissue, age of mother plants, the season of explants collection, used nutrient media and used sterilizing agents. because of the exudation phenolic compound by the plant itself, there was encounter browning problem during shoots multiplication which decreased the multiplication rate. but huang et al. (2002) observed that the browning of bamboo is in higher ph values of 7 and 8 while in the acidic nutrient media with standard ph 5.7 has a relatively low browning rate. such a problem is solved by the supplement of some additives along with plant growth regulators in the media [46]. the browning is occurred according to species, tissue or organ, and nutrient medium in vitro [47]. different types of antioxidants (ascorbic acid, cysteine, activated charcoal, citric acid, adenine sulphate, polyvinylpyrrolidone (pvp) ) with various concentrations are either substituted into the media reduced the browning problem or soaking the explants in a liquid solution of those mentioned [73,74] to reduce the browning percentage in multiple shoots. waikhom and louis (2014) have shown that the addition of nacl and silicon in ms media significantly enhances the activities of antioxidant enzymes. but some researchers have succeeded to overcome serious browning and leaching problem by frequently transfer the clumps to the fresh medium without the incorporation of any antioxidants with media and treatments [74]. huang et al. (2002) found in b. oldhamii, d. latiflorus, and p. nigra browning control when they used pvp, activated charcoal, ascorbic acid, cysteine, ferulic acid, and thiourea. shoot multiplication the size and number of propagules have a vital role in the shoot multiplication. three to four propagules for each culture to multiply was observed effective nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 62 [29-31] than individual propagule cultured [38]. furthermore, bap was extensively used on in vitro multiplication of different bamboos shoot [86, 87, 124]. a higher concentration of bap was an impact on decreasing numbers and length of shoots. kn alone is not a significant response in shoot multiplication in b. balcooa, which results in the clumps dried and browning. incorporation between kn and bap was found effective in shoot multiplication [88]. similar to the synergistic effect of kn and ba on the shoot multiplication rate was reported in b. balcooa [59]; b. glaucescens [89]; d. giganteus [40]; b .tulda and m. baccifera [38]. similarly, tdz has been reported an effective cytokinin for shoot proliferation [82, 90]. the effect of coconut water on bamboo shoot multiplication has been reported by different researchers. saxena and bhojwani (1993) have found that the addition of 10% coconut milk (cm) as an additive in the media is better for shoot proliferation and multiplication in d. giganteus. ramanayake et al. (2001) reported that a high level of sucrose (4%) adversely affected the shoot multiplication in d. giganteus. there are 3% sucrose is widely used in tissue culture. devi and sharma (2009) have found iba was superior over naa for shoot multiplication in arundinaria callosa munro. the use of iaa and naa in conjunction with ba and kn was found to increase the length of shoots but lowered the multiplication rate [29]. further, gibberellic acid (ga) was effective and enhances for multiplication of shoot in b. vulgaris [63]. rathore et al. (2009) only one researcher who has accounted for that the combined effect of naa and bap was effective for shoot multiplication of b. balcooa and b. bambos. rooting in vitro rooting is the bottleneck for the researchers in bamboo. generally, naa, iaa, and iba are used individuals or combined for root initiation. these three hormones were more suitable for rooting in d. asper [84, 91,92,93,94, 97, and 98]. clusters of 3-5 shoots were effective for transferring into rooting medium [29, 51, 58, 96, 99, 123, 124]. full strength ms and half-strength ms media with the supplement of rooting hormones were frequently practiced for in vitro rooting. arya et al. (2008) reported 80-90% of roots obtained in ms medium with supplementing of naa or iba within 5 weeks of transferring while working in d. asper and d. falcatum. but singh et al. (2012b) have observed that 100% rooting in d. asper by using a combination of iba and naa. whereas for b. tulda and b. balcooa a two-step treatment of 7 days on liquid ms medium supplemented with iba and then transferred in vitro shoots to basal ms medium (pulse treatment) without any rooting hormones, was followed for in vitro rooting [43]. the combination of iba and naa for rooting has been also reported by islam and rahman (2005); arya et al. (2006) and rathore et al. (2009) in many important bamboo species. some studies proved that iba was found to be the most favorable root inducer compared to naa and iaa on several bamboos such as drepanostachym falcatum [40], oxytenanthera abyssinica [94], d. hookeri [95], d. hamiltonii [92]); melocanna baccifera [100]. on rooting medium shoots also elongated and good root and shoot system developed in 5-7 weeks. saxena (1990) working on b. tulda has reported supplementing of coumarin in rooting media resulted in better root induction and elongation. similarly, ramanayake and yakandwala (1997) working on d. giganteus and sood et al. (2009) on d. hamiltonii observed a high frequency of rooting when iba was used in combination with coumarin. in the case of d. strictus, up to 90%, rooting was found in medium containing iba [38]. however, well-developed roots with healthy shoots were observed in half-strength ms medium supplemented with naa [36]. negi and saxena (2011) have a document that the highest rooting frequency was obtained on ½ ms media with supplemented of iaa, iba and naa in b. nutans and also similar result obtained by kapoor and rao (2006) who reported that 100% rooting in ½ ms media containing the optimal concentration of ba and naa in b. bambos. sanjaya et al. (2005) have achieved in vitro rhizome in p. stocksii with continued subculturing of rooted plantlets on medium containing ½ strength of major salts within the addition of iba, ba, ascorbic acid, citric acid, cysteine and glutamine in different concentration. chowdhury et al. (2004) obtained in vitro rhizome in d. strictus culturing in rooting media that have ½ strength major salts and iba. again, several researchers observed half-strength ms supplemented with naa and iba was better than that of the full strength of ms media [21, 25, 29, 78] in various bamboo species. islam and rehman (2005) have accounted that the couple of naa and iba nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 63 were suitable for the rooting of bamboo. but iba was found better than naa in b. arundinacea and d. giganteus for rooting by. again, the iba supplemented medium showed only poorly developed roots in b.nutans and b. balcooa [29]. but ravikumar et al. (1998) reported that iba supplement in ms media was more effective to root induction in d. strictus and d. asper respectively which was also supported by bag et al. (2000) in the result of thamnocalamus spathiflorus. singh et al. (2012b) reported that the combination of iba and naa was a synergistic effect for rooting in d. asper in place of single use. but mudoi and borthakur (2009) observed a combination of naa and bap was found most effective in rooting of b. balcooa which was also confirmed by goyal et al. (2015) in d. strictus. the limited number of the report was available for rooting in iaa. kapruwan et al. (2014) reported iaa for in vitro rooting in d. strictus. the effect of growth regulators on rooting was varied species to species [95]. negi and saxena (2011) have practiced successfully rooting in full strength ms liquid media with the supplement of iaa, iba, and naa. and the high concentration of cytokine introduced for rooting might have resulted in cell death and cell cultures became yellowing leaves and reduced root mass in intact plants [97-100]. hardening the transfer of in vitro propagated plantlets form lab to land is another big nutshell of micro-propagation [22]. the plant developed in vitro is unable to survive in vivo directly due to lack of adaptation and proper hardening [43] however it has well-developed roots. to overcome the bottleneck of hardening, researchers have followed various hardening procedures. in general, the healthy and well-rooted plantlets are washed to free from the rooting medium and transferred to the pot containing growth supporting composition such as soil, sand, soil rite, perlite, cocopeat, agro peat, vermiculite, compost, farmyard manure, etc either alone or in various ratios [22]. most researchers have used mention substrate in 1:1:1 ratio or modified. some researchers have described the primary hardening and secondary hardening to obtained maximum numbers of plantlets. like, in vitro plantlets were transferred to ½ strength ms liquid medium without plant growth regulators and vitamins for hardening in d. asper [65], b. nutans [109], and d. hamiltonii [54]. then when plantlets transferred to polybags 1:1:1 composition of sand: farmyard manure: soil they obtained high rate plantlets. the mortality percentage of plants is increased when the direct transfer of in vitro propagated plantlets to the external environment because of their inability to survive against biotic and abiotic stresses [91]. but negi and saxena (2011) obtained a 95.83% success rate by directly hardening in 2:1 mixture of soil: agro peat in b. nutans. similarly, several workers reported on hardening of in vitro plants in the mixture of soil: sand: compost cocopeat (1:1:1) in b.nutans [87, 110]; soil: sand: cow dung(1:1:2) in b. balcooa [25] and b. nutans (sharma and sarma2014); soil mixture of peat, perlite, and vermiculite (1:1:1) in b. oldhamii [77], perlite, soil, and farmyard manure (1:1:1) in d. strictus [36]; 3:1 ratio of coco peat and vermicompost (3:1) in b. balcooa [43]. without other substrates composition with soil, in vitro plantlet was acclimatized successfully in b. bambos [22, 29, 33, 41, 42, 62, 65] ba6benzyl adenine, bap6benzyl aminopurine, tdzthidiazuron, ibaindole -3butyric acid, iaaindole -3-acetic acid, naaαnaphthalene acetic acid, adsadenine sulphate, knkinetin, ms-murrashige and skoog media, cwcoconut water. conclusion as a fast-growing and high potential economic development of the country, the demanding bamboo has enhanced the depleting rootstock of bamboo rapidly. bamboo has a high capacity to carbon sequester and it is the mitigation of climate change and environments. similarly, it is an alternative source of the forest. so that it has a great role in conservation biology and is become a priority concern. with knowledge of the awareness of the conservation biology and environment however people have to fulfill the enormous demands of the markets, they have to exploit the limited resources. harvesting from the resources means that a large scale of bamboo plantlets are necessary highly through micropropagation to fill up the gap of plant stocks. mass yield production protocols along with factors influence on it are discussed in the review. several protocols are reported by several researchers. nodal explants are better explants for micropropagation technique with the supplement of proper plant growth regulators in ms media at nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 64 decontamination condition, the proper season of explants collection, and appropriate position of the nodal segment in mother plant stocks. ba/ bap is the best cytokine for bud initiation and shoots multiplication bamboo species. in vitro rooting is specific for bamboo species. iba is a more effective rooting hormone for bamboo in comparison to other naa and iaa. but several studies indicated that a couple of iba and naa is also effective for rooting in in vitro. it is also reported that the incorporation of naa, iba, and iaa is also suitable for rooting in some bamboo species. sand alone is restricted for hardening but the combination of different substrates varies, the ratio is appreciable for hardening the in vitro rooted plants. the more relevant protocols have to develop by addressing those issues properly. future research must be focused to generate a large scale of bamboo plants. author’s contribution mms, jl and dpg were equally contributed to preparing framework of literature, conceptualization, review and editing the final draft. mms was involved in writing original draft of this review article. jl was project coordinator and dpg was project in charge. all authors read and approved the final manuscript. competing interest the authors declare that they have no competing interest, which includes personal, financial, or any other kind of relationship with people or organizations that could inappropriately affect this review. acknowledgments it is our radiant sentiment to place on record our best regards as well as the deepest sense of gratitude to the department of biotechnology, school of science, kathmandu university, and president of tarai madhesh chure conservation broad for providing us financial assistance. abbreviations benzyl amino purine (bap), 6 –benzyl adenine (ba), naphthalene acetic acid (naa), indole 3-butyric acid (iba), indole acetic acid (iaa), zeatin (zn), kinetin (kn), thidiazurn (tdz), ms (murashige and skoog), plant growth regulators pgrs, coconut water (cw), adenine sulphate (ads) etc. references 1. godbole 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contamination nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):54-68 suwal et al. ©njb, bsn 68 during in vitro propagation of guadua angustifolia kunth. pharmacognosy magazine. 2012 apr;8(30):93. https://doi.org/10.4103/0973-1296.96547 123. chang wc, ho cw. micropropagation of bamboos. inhightech and micropropagation v 1997 (pp. 203-219). springer, berlin, heidelberg. https://doi.org/10.1007/978-3-66207774-0_13 124. thapa n, gauchan dp, suwal mm, bhuju s, upreti a, byanju b, lamichhane j. in vitro assessment of bambusa balcooa roxb. for micropropagation. journal of emerging technologies and innovative research. 2018;5(12). nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 6 3 7 4 review article doi: https://doi.org/10.3126/njb.v9i1.38669 ©njb, bsn 63 plant and plant associated microflora: potential bioremediation option of indoor air pollutants y.h.k.i.s. gunasinghe1 , i.v.n.rathnayake1 , m.p.deeyamulla2 1department of microbiology, faculty of science, university of kelaniya, kelaniya sri lanka 2department of chemistry, faculty of science, university of kelaniya, kelaniya sri lanka received: 17 oct 2020; revised: 10 jul 2021; accepted: 18 jul 2021; published online: 31 jul 2021 abstract indoor air pollution is a significant problem today because the release of various contaminants into the indoor air has created a major health threat for humans occupying indoors. volatile organic compounds (vocs) are pollutants released into the environment and persist in the atmosphere due to its low boiling point values. various types of indoor activities, sources, and exposure to outdoor environments enhance indoor vocs. this poor indoor air quality leads to adverse negative impacts on the people in the indoor environment. many physical and chemical methods have been developed to remove or decompose these compounds from indoors. however, those methods are interrupted by many environmental and other factors in the indoor atmosphere, thus limit the applications. therefore, there is a global need to develop an effective, promising, economical, and environmentally friendly alternatives to the problem. the use of the plant and associated microflora significantly impact reducing the environmental voc gases, inorganic gases, particulate matter, and other pollutants contained in the air. placing potted plants in indoor environments not only helps to remove indoor air pollutants but also to boost the mood, productivity, concentration, and creativity of the occupants and reduces stress, fatigue, sore throat, and cold. plants normally uptake air pollutants through the roots and leaves, then metabolize, sequestrate, and excrete them. plant-associated microorganisms help to degrade, detoxify, or sequestrate the pollutants, the air remediation, and promote plant growth. further studies on the plant varieties and microorganisms help develop ecofriendly and environmentally friendly indoor air purifying sources. keywords: plants, microorganisms, voc, air pollution, biological remediation corresponding author, email: kaviisugunasinghe@gmail.com introduction people spend the bulk of their lifetime indoors, either in residential or public areas. number of pollutants in the indoor air are higher than the outdoor air; hence poor air quality in these indoor environments will lead to several health issues. today, it has become one of the biggest environmental threats [1]. therefore, most studies have been disclosed the connection between indoor air pollution and associated adverse health effects [2,3]. continuous exposure of individuals to poor indoor air quality can lead to "sick building syndrome" (sbs); health problems such as headache, fatigue, eye and skin irritation, or respiratory illnesses, etc. [4]. in 2012, world health organization (who) reported that indoor air pollution by households cooking over coal, wood, and biomass stoves caused about 4.3 million deaths worldwide [5]. indoor air contaminants are generated through several sources such as occupational activities, household products, chemical reactions indoors, pets, materials, underground garages, and outside air sources [6,7]. particles, biological agents, radon, asbestos, and gaseous contaminants such as co, co2, nox, sox, aldehydes, and volatile organic compounds (voc) are released as main indoor air contaminants from the sources as mentioned above [8]. removing the pollutant generating sources from indoors, increasing the ventilation rates, improving air distribution and cleaning the indoor air, etc. are the primary air purifying principles at indoors. increasing the ventilation rate is the easiest way to reduce indoor air pollutants. however, it is usually affected by outdoor weather and external pollution condition [9]. other current strategies used to remove indoor air pollutants are filtration, electrostatic precipitator with ionization, adsorption, ozonization, photolysis, photocatalysis etc. [8]. among the above mentioned treatment strategies, some are very much expensive and complex methods. however, biological purification is a simple, low cost, and environmental friendly technique. therefore has been investigated in many studies [10,11]. this review covers the potential use of plant and plant associated microflora for indoor air pollutant removal and degradation. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-1293-5562 https://orcid.org/0000-0002-1293-5562 https://orcid.org/0000-0003-3476-7018 https://orcid.org/0000-0002-3085-4280 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 64 indoor air quality an average person needs 30 lb of air per day to live. however, he needs only 1.360 kg (3 lb) and 0.680 kg (1.5 lb) of water and food per day [12]. it indicates why air becomes the foremost necessary thing for the survival of humans and other living beings. according to the u.s. national institute for occupational safety and health (niosh) reports in 2007, the average total vocs concentration in air samples could reach 2.90 mg m−3[13]. inadequate building ventilation is the leading cause of the high level of pollutant content indoors [14], and high pollutant content also causes severe public health threats [1]. humans spend most of their time indoors, thus more researches are focused on indoor air quality and related studies. ambient air is often contaminated with high amounts of indoor air pollutants like particulate matter (pm), vocs like benzene, toluene, ethylbenzene, xylene, polyaromatic hydrocarbons (pahs), formaldehyde, and inorganic pollutants as sulfur dioxide (so2), nitrogen oxides (nox), carbon monoxide (co), carbon dioxide (co2) and ozone (o3). although many of those compounds are outdoor air pollutants, can also be found indoors in higher amounts than outdoors [15]. benzene is a ubiquitous trace element in indoor air [16], and its indoor concentration is higher than outdoors. a safe level for benzene exposure cannot be recommended. pahs presence in the atmosphere is typically attached to air particles and present as complex mixtures. therefore, the composition of pah may vary from site to site. however, who (2000) reported that 8.7×10-5 ng/m3 of pahs have a risk for lung cancers. exposure of 0.01 mg/m-3 naphthalene is described as a safe level. still, long term inhalation can cause respiratory tract lesions leading to inflammation and malignancy of animals. formaldehyde exposure of 0.36 mg/m-3 for 04 hours causes sensory irritations of the eyes in humans [17]. furniture, carpets, construction materials, sprays, cleaning, restoration activities, and surrounded industries are the foremost sources of the various volatile organic compounds, aliphatic and aromatic hydrocarbons, alcohols, and aldehydes, and chlorinated compounds [6,7,18,19]. inorganic gaseous pollutants, so2, nox, co, and co2 are generated through the combustion of fossil fuels, gas fired appliances (stoves and ovens), kerosene heaters, tobacco smoking [7,20,21], and outdoor sources exposure [22]. potential health hazards the presence of toxic volatiles and other pollutants in indoor air can cause various illnesses in humans. the european environmental agency has shown that indoor air quality is one of the priority considerations in children’s health [23]. prevalence of sbs is higher in buildings with air conditioners than in natural ventilation systems [24]. typically this has been reported in offices, schools, aged care homes, and apartments like building-associated environments [2]. sbs is often associated with various symptoms such as headache and nausea, nasal congestion (runny nose, stuffy nose, shortness of breath, wheezing, sneezing, sinus, chest tightness, and chest congestion), throat problems (dry throat, sore throat, hoarseness), eye problems (dry eye, itching, tearing, blurry vision, burning eyes, sore eyes, and problems with contact lenses), fatigue (sleepiness, or drowsiness and unusual tiredness,), chill and fever, muscle pain (aching muscles or joints, pain or stiffness in the lower back, pain or stiffness in the upper back, and pain or numbness in shoulder/neck), and even neurological symptoms (feeling depressed, difficulty remembering or concentrating, and tension or nervousness), dry skin, and dizziness as well [25]. apart from these illnesses, sometimes poor indoor air conditions also cause adverse health effects like respiratory tract illnesses, lung cancers, and heart diseases [26]. potential harmful effects of benzene, toluene, xylene, and formaldehyde exposure were summarized below (table 01). prevalence of illnesses due to indoor air contaminants depends on factors like individual sensitivity to the contaminant, concentration of the contaminant, current physical health state of the individual, and also duration of exposure to the contaminant [27]. according to the international agency for research on cancer (iarc), benzene is a toxic chemical proven as a carcinogen [28]. benzene can cause most hematological diseases, such as acute and chronic lymphocytic leukemia, acute and myeloid leukemia, non-hodgkin’s lymphoma, multiple myeloma, and aplastic anemia even at the low dose of exposure [29– 31]. the safe level for benzene exposure is still unknown, but the european union recommended in 2000 that the benzene concentration in the ambient air should not exceed 5 µg m-3 [32]. impure indoor air with particulate matter (pm≤10 µm) is often correlated with cardiovascular or respiratory disorders, and recently it is revealed that exposure to pm during the period of pregnancy or early life may cause autism spectrum disorder (asm) [33,34]. these potential health hazards associated with poor indoor air quality highlight the need to review indoor air pollution and purification methods more seriously. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 65 how to avoid indoor air pollutants? many strategies can be used for the reduction of indoor air pollutants. those are supported by several efforts, such as removing the pollutant source from indoors, enhancing the ventilation rate, improving indoor air distribution, and cleaning [37]. many industries have taken steps to scale down the usage of possible sources of indoor air pollutants during their product manufacturing cycle. current strategies applied to remove or reduce indoor air pollutants are filtration, electronic precipitator with ionization, adsorption, ozonation, photolysis, and photocatalysis [8,38]. manipulation of filtration is suitable for particle removal in indoor air [39]. however microbial colonization on the filters will hinder the filtration. during electrostatic precipitation, by generating an electrical field, charged particles of air can be trapped. however, there is a risk of generating hazardous charged particles. removing air pollutants using adsorption might be a highly specific technique, which is used as a post-treatment. the problem associated with oxidizing the pollutant may be the generation of unhealthy toxic products. researchers are still proposing strategies to address this case with nonadverse impacts. membrane separation, enzymatic oxidation, botanical purification, biofilters, and biotrickling filters are number of those strategies. out of those plants and plant associated microflora, lowering the toxicity of contaminants in indoor environments is becoming a popular alternative as an economical air restoration technology [38]. indoor pollutant removal capability of plants plants remove voc, through aerial plant parts and plant associated microflora. growing media and plant roots are also capable of removing voc in the air. recent studies showed that plants are one of the best air pollutant absorbing and metabolizing agents [40]. plant volatile organic matter removal or degradation rate and efficiency rely upon the plant species, light, temperature, growing media, and voc (concentration, identity, and voc mixture effects). stomata, cuticle, and adsorption to the plant wax layer are the critical voc removal sites of the aerial plant parts. after entering into the leaf, the compound often undergoes degradation, storage, excretion, and translocation to alternative plant elements. microorganisms present in the plant pot soil and plant root also can remove voc from indoor air [41]. these plant pollutant removal and degradation strategies have been confirmed using several plant species using radiolabeling [42,43]. several studies on plants with 14c labeled aromatic hydrocarbons revealed that aromatic rings of those hydrocarbons were cleaved during their metabolic transformations and utilization of aromatic hydrocarbons under sterile conditions [44]. plants can sink air pollutants through their large surface area of foliage and canopies because it provides a surface for the pollutant substances. also, plant leaves can sorb several gaseous substances as nutrients or as micronutrients [45]. the plant uses processes like complexation, precipitation, and oxidation-reduction to detoxify or utilize those substances as nutrients. these plant and atmospheric interactions result in the reduction of these harmful particulate substances and voc’s [46]. vocs removal and degradation capability of many indoor and outdoor plant species have been recorded in the literature. as reported in the literature, table 1 potential health hazards benzene, toluene, xylene, and formaldehyde exposure. vocs limit of exposure (µg m-3) potential health hazards ref short term long term toluene 15,000 (8h) 2,300 (one day average) short-term exposure – eye, nose, and throat irritation, dizziness, headaches, and feelings of intoxication. long term exposure –neurological effects including reduced scores in tests of short-term memory, attention, and concentration [35] benzene no safe level of exposure recommended . no safe level of exposure recommended carcinogenic chemical (group1) to humanscause adult acute myeloid leukaemia. positive associations have been observed for non-hodgkin lymphoma, chronic lymphoid leukaemia, multiple myeloma, chronic myeloid leukaemia, acute myeloid leukaemia in children lung cancer [36] xylene 100 (1year) irritation to the lungs, throat, and nose. severe inhalation exposure can cause dizziness, headache, confusion, liver and kidney damage, heart problems, and coma [35] formaldehyde 100 (30 min) 10 (1year) sensory irritation of eyes, nose, and throatexposure-dependent discomfort, lachrymation, sneezing, coughing, nausea, and dyspnoea. human carcinogenic chemical. long-term exposure linked to nasal cancer. [36] nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 66 plant species and their potential in removing or detoxifying toluene (table 2), benzene (table 3), xylene (table 4), and formaldehyde (table 5) removal or degradation are summarized below. however, there could be some deleterious effects like impairment of plant physiological activity and plant injuries due to these chemicals. chronic exposure to higher concentrations of air pollutant substances can affect plant photosynthesis, vitality, and productivity. this stress makes the plant more susceptible to diseases and insect infections [47] table 2 plant species and their potential for toluene removal. plant species results ref zamioculcas zamiifolia toluene uptake per unit area of z. zamiifolia plant leaf at 72 h of exposure 0.93±0.02 mmol m−2 [48] hemigraphis alternate, hedera helix, hoya carnosa, asparagus densifloru tradescantia pallida, fittonia argyroneura removal efficiency of toluene and total voc by twenty-eight selected ornamental plants varied substantially among the species tested, range of pollutant removal, toluene 1.54 9.63 µg m–3 m–2 h–1 total voc 5.55 -44.04 µg m–3 m–2 h–1. [6] o. microdasys, d. dermensis time taken for the complete removal of 2 ppm toluene from an airtight chamber was 55 h and 120 h, respectively for o. microdasys and d. dermensis plants. [49] dieffenbachia maculate, spathiphyllum wallisii asparagus densiflorus toluene removal rate constant ranged from 3.4 to 5.7 l h−1m−2 leaf area when exposed to 20.0 mg m−3 of toluene [50] hedera helix ,spathiphyllum wallisii syngonium podophyllum, cissus rhombifolia toluene (initial 1 μl l–1 ) removal efficiencies of h. helix -220.2 ± 31.8 ng m–3 h–1 cm–2 s. podophyllum, 161.6 ± 19.2 ng m–3 h–1 cm–2 s. wallisii 203.7 ± 24.3 ng m–3 h–1 cm–2 lowest efficiency c. rhobifolia. 85.7 ng m–3 h–1 cm–2 [51] herbs aloysia triphylla, brittonz melissa officinalis mentha piperita , mentha piperita mentha suaveolens ,mentha suaveolens pelargonium graveolens, plectranthus tomentosus rosmarinus officinalis ,salvia elegans herbaceous foliage plants begonia maculata ,davallia mariesii farfugium japonicum, fittonia verschaffeltii hedera helix philodendron spp. soleirolia soleirolii woody foliage plants ardisia crenata , ardisia japonica ardisia pusilla, cinnamomum camphora schefflera elegantissima, eurya emarginata , ilex cornuta, ligustrum japonicum, pinus densiflora, pittosporum tobira, rhododendron fauriei efficiency of toluene removal ranged from 378 to 16.6 µg m–3 h–1 m–2 [52] fatsia japonica, draceana fragrans volatile toluene and xylene removal efficiencies were increased as the plant’s root zone volume increased. [53] schefflera actinophylla, ficus benghalensis toluene and total xylene (m, p, o) removal efficiency of leaf area over a 24h period in s. actinophylla, 13.3 μg m−3 m−2 f. benghalensis 7.0 μg m−3 m−2 [54] phoenix roebelenii purification capability (pa) increased with an increase in room temperature from 21 to 26°c , reaching a range of 15–35 (v/h) initial toluene 1.5 ppm, pa for toluene was 6.5 (v/h) [55] azalea indica time taken to remove 339 mg m-3 of toluene 76 h [56] epipremnum aureum, spathiphyllum removal rate for tvoc was 74%, and 68%respectively [57] epipremnum aureum, davallia fejeensis epipremnum aureum plant had a positive impact on mixed voc(decane, toluene, 2 ethylhexanol, benzene, octane, xylene, αpinene) filtration than davallia fejeensis [58] nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 67 diversity of plant associated microflora microbial reservoirs like soil, rhizosphere, phyllosphere, anthosphere (external environment of flower), spermosphere (the exterior of germinating spores), and carposphere (external area of the fruit) indicate plant microbial relationships [77]. diverse groups of bacterial taxa namely proteobacteria, acidobacteria, actinobacteria, bacteroidetes, verrucomicrobia, planctomycetes, cloroflexi, firmicutes, and gemmatimonatedes are present as root endophytes [78,79]. among those, a representative amount of taxa have been derived from the soil environments [80]. plant root microbiota is mostly transferred horizontally. however, bacteria can sometimes be transferred via seeds by relocating microorganisms to proliferating plants [81,82]. the narrow layer of soil on plant roots has high microbial diversity, it’s one of the most complex ecosystems and is called as a rhizosphere [83]. root exudate containing organic acids, phenolic compounds, plant growth regulators, sugars, sterols, vitamins, amino acids, fatty acids, and nucleotides ensures good microbial growth around roots [84,85]. plant root endophytes enter into tissues through passive mechanisms (root cracks or emerging points of lateral roots) or active mechanisms [86]. aerial plant tissues are different in ecology from belowground parts; however, it’s a good source for phyllosphere and endosphere bacteria. normally endophytes spread systemically to the leaves, fruits, and stems via the xylem. in addition, endophytes enter plant tissues through aerial plant parts; as fruits and flowers. phyllosphereic bacterial community is highly dependent table 3 plant species and their potential for benzene removal. plant species results ref howea forsteriana, spathiphyllum floribundu, dracaena deremensis , spathiphyllum sensation, dracaena marginata, epipremnum aureum , scheflera actinophylla from seven potted plant species, benzene removal was ranged from 12-28 ppm day-1. [59] dracaena deremensis, spathiphyllum wallisii benzene removal per leaf area of dracaena deremensis 606 ± 155 mg m−3 d−1 m−2 spathiphyllum wallisii 686 ±73 mg m-3 d-1 m-2; howea forsteriana 537± 69 mg m-3 d-1 m-2. [60] zamioculcas zamiifolia benzene uptake per unit area of z. zamiifolia leaf was 0.96± 0.01 mmol m−2 [48] crassula portulacea, hydrangea macrophylla, cymbidium, ficus microcarpa var. fuyuensis, dendranthema morifolium, citrus medica var. sarcodactylis, dieffenbachia amoena, spathiphyllum, nephrolepis exaltata, dracaena deremensis removal of benzene was in the range of 22.1561.3 µg m-2 min-1 [61] superior removal efficiency hemigraphis alternate, hedera helix tradescantia pallida, asparagus densifloru hoya carnosa intermediate removal efficiency ficus benjamina, polyscias fruticose, fittonia argyroneura, sansevieria trifasciata guzmania spp., anthurium andreanum, schefflera elegantissima benzene removal efficiency of hemigraphis alternata -5.54 µg m–3 m–2 h–1 tradescantia pallida3.86 µg m–3 m–2 h–1 hedera helix 3.63 µg m–3 m–2 h–1 fittonia argyroneura -2.74 µg m–3 m–2 h–1 asparagus densiflorus,2.65 µg m–3 m–2 h–1 hoya carnosa 2.21 µg m–3 m–2 h–1 [6] dracaena deremensis opuntia microdasy removal rates of 2 ppm of benzene from the test chambers by o. microdasys -3.2 mg/ m3 d1 d. dermensis 1.46 mg/ m3d1 [49] hedera helix, spathiphyllum wallisii syngonium podophyllum, cissus rhombifolia highest removal efficiency -s. wallisii. medium level removal efficiency s. podophyllum and h. helix lowest removal efficiency c. rhombifolia [51] chamaedorea seifrizii, scindapsus aureus sansevieria trifasciata, philodendron domesticum ixoraebarbata craib, monster acuminate epipremnum aureum, dracaena sanderiana highest benzene uptake d. sanderiana 10.00 ±1.04 mmol of benzene at 72 h crude wax 46 % and stomata 54 % [62] syngonium podophyllum benzene removal 25 ppmv from the test chambers within 7 days [63] epipremnum aureum, davallia fejeensis epipremnum aureum plant had a positive impact on mixed voc (decane, toluene, 2 ethylhexanol, benzene, octane, xylene, αpinene) filtration than davallia fejeensis [58] nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 68 table 4. plant species and their potential for xylene removal. plant species results ref alternanthera bettzickiana,drimiopsis botryoides, aloe vera, chlorophytum comosum, aglaonema commutatum, cordyline fruticose, philodendron martianum, sansevieria hyacinthoides, aglaonema rotundum, fittonia albivenis, muehlenbeckia platyclada, tradescantia spathacea, guzmania lingulata, zamioculcas zamiifolia, cyperus alternifolius best xylene removing plant zamioculcas zamiifolia 88% xylene removal within 72 hours. xylene uptake was 0.81 ±0.01 mmol m−2 leaf area as [64] zamioculcas zamiifolia at 72 h of xylene exposure, z. zamiifolia leaf uptake about 0.86±0.07 mmol m−2 per unit area. [48] d. deremensis o. microdasys time taken for complete removal of 2 ppm xylene from the airtight chamber of o. microdasys and d. dermensis plants were respectively 47 hours and 98 hours. [49] xora coccinea, muraya paniculat, ficus benjamina, euphorbia milii, adenium obesum, millingtonia hortensis, dalbergia cochinchinensis, pterocarpus indicus, phyllanthus acidus, cassia fistula, b. buttiana, gardenia jasminoides, ehretia microphyllalam uptake of xylene by b. buttiana plant parts stems 53.1±1.9% epicuticular waxes 32.3±0.9% plant stomata 14.6±0.0% [65] fatsia japonica draceana fragrans volatile toluene and xylene removal efficiencies were increased as the plant’s root zone volume increased. [53] schefflera actinophylla ficus benghalensis toluene and total xylene (m, p, o) removal efficiency leaf area over a 24-h period was in s. actinophylla13.3 μg m−3 m−2 and 7.0 μg m−3 m−2 f. benghalensis 13.0 μg m−3 m−2 and 7.3 μg m−3 m−2 [54] phoenix roebelenii purification capability (pa) increased with an increase in room temperature from 21 to 26 °c, reaching a range of 15– 35 (v/h) [55] epipremnum aureum spathiphyllum removal rate for tvoc -74% odor 68%. [57] epipremnum aureum davallia fejeensis epipremnum aureum plant had a positive impact on mixed voc (decane, toluene, 2 ethylhexanol, benzene, octane, xylene, αpinene) filtration than davallia fejeensis [58] table 5. plant species and their potential for formaldehyde removal. plant species results ref osmunda japonica, selaginella tamariscina, davallia mariesii, polypodium formosanum, psidium guajava, lavandula spp.,pteris dispar, pteris multifidi, pelargonium spp formaldehyde removal 86 plant species were analyzed and osmunda japonica showed the best 6.64 µg m–3 formaldehyde/cm2 of leaf area over 5 h [66] hedera helix, chrysanthemum morifolium dieffenbachia compacta epipremnum aureum 90% removal by -hedera helix, chrysanthemum morifolium, dieffenbachia compacta, epipremnum aurenum (from the initial amount of 1.63 ppm within 24 hours). [67] fatsia japonica ficus benjamina time interval required to reduce 50% of benzene from the initial concentration (2 µl l-1) f. japonica 96 min f. benjamina.123 min [68] tillandsia velutina the plant decreased formaldehyde concentration by 22.51 % in 12 h [69] phoenix roebelenii purification capability (pa) increased with an increase in room temperature from 21 to 26 ℃, reaching a range of 15– 35 (v/h) [55] schefflera arboricola nephrolepis exaltata these plants reported a high air purification ability [70] fatsia japonica reducing rate, 225 μg m−3 the first 2 h around 80 μg·m−3 for the final 3 h. [71] epipremnum aureum removal rate for [57] nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 69 spathiphyllum tvoc 74% odor 68%. nicotiana tabacum transgenic plants increase formaldehyde removal by 20 % [72] chlorphytum comosum aloe vera epipremnum aureum formaldehyde removal efficiencies; spider plant-soil system at the light intensities of 90%, 92%, and 95% were respectively 80 µmolm−2s−1, 160 µmol m−2 s−1, and 240 µmol m−2 s−1 in the daytime. [73] aglaonema commutatum, spathiphyllum floribundum, commutatum, agave potatorum, dracaena fragrans, d. reflexa, cordyline fruticose, gasteria gracilis, d. angustifolia , d. sanderiana, d. deremensis, sansevieria trifasciata, a.commutatum , alocasia macrorrhiza, s. trifasciata, aloe nobilis, scindapsus aureus, d. amoena, a.commutatum, scindapsus pictus, philodendron sodiroi, syngonium podophyllum , asparagus setaceus, aloe aristata, chlorophytum comosum, philodendron martianum , zamioculcas zamiifolia, philodendron selloum scindapsus aureus, asparagus setaceus, s. trifasciata, c. comosum, a. commutatum, a. commutatum , a. commutatum, s. pictus, g. gracilis, and p. sodiroi reported a high formaldehyde purification capabilities with less damages. [74] chamaedorea elegans initial formaldehyde concentration 14.6 mg m-3 maximum formaldehyde elimination capacity of 1.47 mg/m2h [75] hedera helix hedera helix reported a 70% reduction of the required time to reach 0.5 ppm of gaseous hcho when compared with natural dissipation [76] on environmental factors such as temperature, humidity, and air pollutants [87,88]. plant associated microflora plays a crucial role in voc degradation by increasing the bioavailability of vocs to plants via the production of biosurfactants and the formation of biofilms [89]. these microbial associations with plants increase the ability of microorganisms to metabolize large numbers and varieties of organic compounds, together with improving plant strength of voc remediation. therefore, many studies have focused on the ability of microbial air remediation and its potential applications. role of microflora during air pollutant removal and degradation plant associated microbial flora helps the growth and development of the plant by enhancing the availability of nutrients through the production of siderophores, organic acids, and plant growth promoters (indole acetic acid (iaa)). it helps the plant’s survival in biotic and abiotic stress conditions. as an example, during stressful conditions, ethylene is produced from 1aminocyclopropane-1-carboxylate (acc). bacteria can produce 1-amino cyclopropane-1-carboxylatedeaminase and degrades acc into ammonia and α-ketobutyrate and lowers the amount of acc inside the plant resulting in the reduction of ethylene production and stress [10,90,91]. they not only support phytoremediation; through the detoxification, degradation, and sequestration of the contaminants, but also promote plant growth [92]. phyllosphere bacteria facilitate the absorption of pollutants into the plants. endophytes and phyllosphere bacteria can degrade absorbed pollutants by detoxification, transformation, or sequestration [93]. in soil pollution, root endophytes can decrease phytotoxicity by enhancing the pollutant accumulation inside the plant [94]. biological nitrogen fixation of rhizobium bacteria incorporate carbon and nitrogen into the soil. these plant root nodule associated bacterial flora provide nutrients to plants. natural behaviors of bacteria improve the nutrient availability to the plant and the environmental tolerance [95] through remediation of organic and metal contaminants by absorbing, accumulating, detoxifying, and degrading those pollutants [94]. plant associated microflora detoxifies the pm, which the host plant absorbs. pm activates reactive oxygen species (ros) that adversely affect bacteria, but bacteria have mechanisms to detoxify ros toxicity [96,97]. microorganisms have degradation pathways to degrade and reduce the phytotoxicity of pollutants. therefore it reduces the evapotranspiration of volatile pollutants [93]. in some cases, plants produce biogenic volatile organic compounds. thus voc degrading microorganisms should present in the phyllosphere. however, a limited number of studies are available about phyllosphere microflora since they are transient flora that occupies the phyllosphere temporarily, and the diversity changes depending on various factors. therefore, the study of this transient flora is somewhat difficult. many root nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 70 associated voc degrading microflora are used to treat groundwater and soil and air remediation [10,92,98,99]. air-remediation through soil is somewhat different; there are trapped air and moisture inside the soil particles. once soil contains low moisture conditions air particles with pollutants penetrates through the soil so that the soil microflora can degrade those pollutants. after the water is supplied to the soil, cleaned air is released into the atmosphere. this is how soil and rhizosphere microflora contribute to removing indoor air pollution [59]. microbial pollutant degrading capabilities are enhanced when they are associated with plants [100]. air pollution due to inorganic pollutants (nox, sox, and o3, etc.) also remediated through the microorganisms. it is a well understood fact that chemoorganotrophic bacteria (nitrogen producers, sulfur depositors, photosynthetic bacteria) use these inorganic compounds to generate energy. ozone is a toxic compound to bacteria, and it is used as a bactericidal agent. therefore the use of bacteria in detoxifying ozone is difficult [96,97]. metabolic activities of bacteria in bioremediation of air pollutants several aromatic compounds have become significant air pollutants. their persistence and widespread occurrence throughout the environment are facilitated by the thermodynamic stability of the benzene ring [101]. microorganisms adapted to use these pollutants as their carbon sources through their catabolic pathways [102]. during aerobic respiration of microbes, oxygen is the final electron acceptor, and it provides energy yield to the cell. in addition, oxygen helps to activate the substrates via oxygenation reactions [103]. most of the pseudomonas sp. are aerobic therefore, many studies have been conducted on its ability to degrade many environmental contaminants aerobically [104]. bacterial biodegradation of voc relies on the type of degrading enzymes and the microorganisms [105]. in the aerobic catabolic funnel, most of the peripheral pathways involve oxygenation reactions which are carried out by monooxygenases and hydroxylating dioxygenases and generate dihydroxy aromatic compounds such as catechol, homogentisate, protocatechuate, gentisate, homoprotocatechuate, hydroquinone, and hydroxyquinol. these intermediate compounds are the substrates for ring cleavage enzymes. these enzymes use oxygen to open the aromatic ring between the two hydroxyl groups like ortho cleavage, catalyzed by intradiol dioxygenases or proximal to at least one of the two hydroxyl groups (catalyzed by extradiol dioxygenases, and meta cleavage) [102]. according to murray (1972) and williams (1974), pseudomonas putida mt-2 strain utilizing toluene also grown on the substrates like 1,2,4-trimethylbenzenemethyltoluene, m-xylene, and p-xylene and oxidize all these substrates to corresponding benzylalcohols, benzaldehydes [106,107]. subsequently, the above products were mineralized by meta-cleavage pathways. p.mendocina kr1, ralstonia picketti pko1, and burkholderia vietnamiensis g4 reported degradation of benzene as well as toluene using toluene-4monooxygenase (tmoa), toluene 3-monooxygenase (tbua1), and toluene 2-monoocygenase (toma), respectively [108–110]. nitrosomonas europea produced amminomonooxygenase enzyme, which activates by ammonia and oxidize btex compounds [111]. bacterial mobile genetic elements like plasmids and transposons contain genes responsible for these catabolic activities. once bacteria are exposed to the contaminated environment, they facilitate the horizontal gene transformation and rapid adaptation to utilize the pollutants [104]. bacterial natural adaptations and pollutant remediation is a slow and time-consuming process. however, their utilization for in-situ bioremediation of polluted sites, and biotransformation of toxic compounds into non-toxic compounds such as fine chemicals and other value added products, development of in-situ high sensitive biomonitoring devices such as biosensors are the techniques that can be used to enhance the remediation process [112–114]. conclusion several methods have been proposed to reduce the indoor air pollution caused by various chemicals released into the air due to anthropogenic activities occurring indoors. although chemical and physical methods are available, most of them have issues in efficiency, short-life span, high cost, need for recovery systems, high maintenance demand, and secondary pollutants generated during voc removal. use of plants and their associated microflora provides a solution to these issues as an economical and environmentally friendly alternative. this review provides an overview of the use of ornamental plants and their associated microflora in removing the air pollutants indoors. according to the literature zamioculcas zamiifolia, spathiphyllum wallisii, sansevieria trifasciata, hedera helix, and ficus benjamina plants can be suggested as the effective plants for benzene, toluene, nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):6 3 7 4 gunasinghe et al. ©njb, bsn 71 xylene, and formaldehyde removal. microbial associations with plants benefit in voc remediation because it increases the microbial capability in metabolizing large numbers and varieties of organic compounds. also microflora influence the plant strength during voc remediation. more laboratory and field studies are needed to increase the efficiency in using plants for indoor air purification as well as to understand their mechanisms of air purification. acknowledgment this research was supported by the accelerating higher education expansion and development (ahead) development oriented research (dor) grant of the ministry of higher education, sri lanka funded by the world bank author’s contribution the authors confirm contribution to the paper as follows: study conception and design: y.h.k.i.s.gunasinghe, i.v.n.rathnayake, and m.p.deeyamulla; draft manuscript preparation: y.h.k.i.s.gunasinghe; review, and editing the final draft: y.h.k.i.s.gunasinghe, i.v.n.rathnayake; all authors read and approved the final manuscript. competing interest the authors declare that they have no competing interest, which includes personal, financial, or any other kind of relationship with people or organizations that could inappropriately affect this review. ethics approval not applicable. references 1. kirchner s, derbez m, duboudin c, elias p, gregoire a, lucas jp, et al. indoor air quality in french dwellings. indoor air 2008 [internet]. 2008. p. paper-id. available from: 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an  overview  of  computational  approaches  in  structure  based  drug  design   dipali singh*, anushree tripathi, gautam kumar 1department of bioinformatics, indian institute of information technology-allahabad, india. *corresponding address: indian institute of information technology-allahabad, 211012, india. email: ibi2010002@iiita.ac.in, dipali.d4@gmail.com short title: computational approaches in sbdd abstract drug design is a costly and difficult process. drug must fulfill several criteria of being active, nontoxic and bioavailable. the conventional way of synthesizing drugs is a monotonous process. but computer aided drug design is a proficient way to overcome the tedious process of conventional method. drugs can be designed computationally by structure or target based drug designing (sbdd). this review summarizes the methods of structure based drug design, usage of related softwares and a case study that explores to find a suitable drug (lead) molecule for the mutated state of h-ras protein in order to prevent complex formation with raf protein. keywords: computer aided drug design, structure based drug design, ras-protein introduction traditionally, new drugs were generated from plants and other natural products through accidental observations and discoveries. leads for new drug were generated from screening of organic compounds. increasing information on the three dimensional structure of the biological target has paved path for structure based drug design. the rapid progress in the field of genomic, proteomic, and structural biology has increased the opportunities for future drug lead discovery. the antihypertensive drug, captopril, an angiotensin-converting enzyme (ace) inhibitor was the first success story in structure based drug design [1]. kubinyi has reviewed success stories of structure based drug design in the search for new, potent and selective hiv protease inhibitors, thrombin inhibitors, neuraminidase inhibitors and integrin receptor antagonists [1]. anderson in his review paper mentioned that two of the first drugs to reach the market using sbdd were amprenavir and nelfinavir developed against hiv protease [2]. structure nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     54   based drug design can successfully contribute to the discovery process at different stages. it can be used at a very early stage at which no leads are available [3]. 1. overview of the process proteins 3d structures are generally used by sbdd to assist for the development and design of new lead (drug compounds). the overall process of sbdd (figure 1) would be divided mainly into two parts: a. docking ligands proteins are flexible molecules and they adjust their shape to place bound ligands through rotation of bonds. sbdd allows to dock ligand/drug molecules into protein active sites and to visualize the movement that occurs in amino acid side chains. b. lead optimization lead optimization is a technique of refining 3d structures of drug molecules and it promotes the binding of drug to protein active sites. in this technique, researches gradually modify the structure of the drug compound by docking every specific structure of a drug compound in active site of protein, and calculating their extent of interactions. ! !"#$%&'()*#$+,$-'.&/'&.#0123#4$5.&6$5#3)6*! !   fig.1: the outline of structure-based drug design 2. design process a. choice of drug target the target should be closely linked to cause of human disease and binds to a small molecule, generally a protein, in order to carry out a function. drug target are usually protein having a well-defined binding pocket. sbdd against rna targets with well-defined secondary structure has also been effective [2]. after the identification of target, structure can be determined following any of the methods: 1. x-ray crystallography 2. nuclear magnetic resonance spectroscopy (nmr) 3. computational methods (modelling) 4. atomic force field microscopy (afm) 3. drug design methods once identification of structure and target site is completed, there are number of ways to develop lead based on the structure of the target nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     55   which can be categorized as computer aided versus experimental. in experimental method, high-throughput screening is performed with combinatorial chemistry and thousands of molecules are tested for biochemical effects. computer-aided methods can be classified into 3 categories. a. database searching and docking methods b. de novo drug design methods c. ligand binding scoring functions a. database searching and docking methods widely used computational docking methods are dock, concord, autodock, flo98 and flexx. dock systematically attempts to fit each compound from a database to the target structure’s binding site in such a way that in the database, three or more atoms of the molecule overlap with a set of predefined site points in the target binding site [7]. the default method for site point generation involves creating an inverse surface of the binding site. this is specified by the set of overlapping spheres that fill the binding site and touch the molecular surface at two points. the sphere centers (for all spheres with radii within a specified range) are used as site points. concord is based on the combination of geometry rules and optimization methods. it selects lowest energy conformer of the molecule then scores on grid using different energy functions. on the basis of precalculated values for protein, each match is scored on a grid throughout the binding site of target molecule [7]. b. de novo drug design methods structure based drug designing methods rely exclusively on ligand optimization approach based on the study of protein active site properties. there are three important categories of computational methods for the de novo design of structure based ligands: fragment positioning methods, molecule growth methods, and fragment methods coupled to database searches [6]. fragment positioning methods basically these methods are based on the selection of structures of individual functional groups or fragments from predefined library which fill the active site of enzyme [5]. two well-known programs which predict energetically favorable binding site positions for chemical fragments are grid and mcss (multiple copy simultaneous search). grid calculates protein interaction energies for functional groups on a grid surrounding the target structure. it includes non-bonded interaction like hydrogen bonding, electrostatic and van der waals. it is mainly useful for modifying existing lead compounds. limitation of grid is that the sphere probe must be nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     56   capable of making hydrogen bonds and must not be in a linear arrangement [6]. in mcss method, the probes are fully flexible and individual atoms are represented by charmm potential energy function. the de novo drug designing approach involves three steps [6]. initially it uses fragment positioning method. secondly, clustering and connecting the optimally placed molecular fragments to form chemically sensible candidate ligands. finally, depicts the binding of proposed compounds with another and to existing drugs. molecule growth methods a fragment is fitted in the binding site of the target structure while ligand molecule is successively built by bonding a further fragment to it. there are various molecule growth methods are available, including smog (small molecule growth), growmol, groupbuild and genstar. smog uses simple model for ligand-protein interactions as well as a knowledge-based potential. a large number of structures are statistically analyzed by an efficient monte carlo molecular growth algorithm that generates molecules through the adjoining of functional groups directly in the binding region [7]. growmol generates ligand structures from a library of atom as well as small functional group types and is scored based on its chemical complementarities with nearby atoms to the binding site of the target. groupbuild is similar to growmol, it uses a predefined library of chemical fragments and scores candidate fragment positions depending on force field to get candidate small molecule ligands fragment by fragement. genstar generates chemically reasonable structure which fills active site of enzyme. the proposed molecules provide good steric contact with the enzyme and also exist in low energy conformation. these structures consist of sp3 hybridized carbons which are grown sequentially, but which can also branch or form rings. atoms are grown from predocked inhibitor core. for each new atom generated by the program, several hundred candidate positions representing a range of reasonable bond lengths, bond angles, and torsion angles are considered. then, each candidate is scored, with a simple enzyme contact model. from the highest scoring cases, positions are chosen at random. duplicate structures may be removed applying variety of criteria. energy of compounds may be minimized and displayed using standard modeling programs. fragment methods coupled to database searches it is an integrated approach for fragment nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     57   positioning methods and database searching techniques either to extract those existing molecules from a database that can be docked with the preferred fragments in their most favorable positions into the binding site or for de novo design. hook generates a database of molecular skeletons without involving functional groups on the database molecules and then fit molecular skeletons into the target binding site such that two mcss functional group minima can be hooked by using docking. then, it undergoes geometrical superposition of two designated hooks in the skeletal molecules and finally using inverted lennardjones type contact potential, the fit of the skeleton in the binding site in two functional group minima, is scored. after validating scores, secondary searches are carried out to attach additional mcss minima to the skeleton, if fit is acceptable [6]. caveat is comparatively faster method due to consideration of interaction between the skeletal molecule and the binding site in post processing step. it is similar to hook in that it involves searching of a database of threedimensional structures of small cyclic molecules to connect optimally placed fragments in the binding site. in the database, specific bonds of each molecule are represented as vectors, and the molecule is specified as a set of pairwise combinations of bond vectors. it finds matches between pairs of bond vectors from the fragments of the query molecules and the database molecules [6]. c. ligand binding scoring functions the ligands binding scoring functions are major determinant of the accuracy of scoring functions that ranks the lead compounds. factors which contribute to ligand binding include hydrophobic effect, dispersion interactions, hydrogen bonding, other electrostatic interactions and solvation effects. with increasing complexity, the various approaches for estimating binding affinities include scoring functions based on statistical analysis of known structure of protein ligand complexes, physicochemical properties, force field calculations, force field calculation with added solvation corrections and free energy perturbation (fep) calculations. smog pseudo energy function is a scoring function based on statistical analysis of high resolution x ray structure. currently knowledge based, regression based and first principle based methods have been developed to rank lead compounds [8]. 4. case study in the ras subfamily, mainly k-ras, h-ras and n-ras codes for those proteins which are nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     58   made up of 189 amino acids with molecular weight 21kda protein [9]. guanosine nucleotide binding protein or g-proteins works in form of signaling switches with two states that are active and inactive. usually it is bound to the nucleotide gdp in the inactive state. on the other hand, it is bound to gtp in the active state. guanine nucleotide exchange factors (gefs) and gtpase activating proteins (gaps) are mainly used in exchanging the bound nucleotide. ras is associated with an intrinsic gtpase activity in which it can hydrolyze bound gtp into gdp. but, due to its less efficiency, rasgap is needed which is formed by binding of ras and gap and stabilizes the ras catalytic residues by releasing inorganic phosphate and ultimately leads to ras molecule in gdp bound state for ras inactivation. it has been found that mutations in the ras family of proto-oncogenes are very commonly observed in 20% to 30% of all human tumors [10].the inappropriate activation of the gene affects malignant transformation, proliferation and signal transduction [11], due to which, the mutated ras p21 has a structure that disables its ability to bind with gtpase activating protein (gap) and creates an autophosphorylation site, keeping the ras p21 in the gtp-bound activate state and contributing to a malignant cell phenotype [12, 13]. in this context, target-based drug discovery is considered to be highly potential. the mutated h-ras is perceived to be an important target to treat colorectal and pancreatic cancer. a suitable drug (lead) molecule can be searched for the mutated state of h-ras protein in order to prevent complex formation with raf protein. a. materials and methods the protein structures of h-ras p21 mutant (pdb id 521p) and of ras-binding domain (pdb id-1wxm) were taken from protein data bank http://www.rcsb.org/pdb/home/home.do. there were two methods used to predict potential binding site. in the first approach, screening of ligand molecules was carried out through blast search engine by submitting the mutated hras (pdb id: 521p) protein sequence to drugbank database: http://redpoll.pharmacy.ualberta.ca/drugbank/dr ugblast.htm. the drugbank search showed trifluoroethanol, s-oxymathionine and isopropanol as active ligands. in a second approach, chembank ligand entries were downloaded from ligand in sdf format and entries of ligand was used for virtual screening and docking into effectors region of mutated h-ras by using discovery studio/ligandfit program to identify active nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     59   potential drugs. the ligand fit docking algorithm produced 10 different hits of ligand, such as ys035, nizatidine, leuhistin, 3aminopropanesulphonic acid, guanidine, acetamide, methoxamine, urea, aluminum fluoride and hydroxyurea from two different binding site cavities that were encompassed in effectors region of mutated h-ras. 5. results and discussion leaving all the other molecules, the 3aminopropanesulphonic acid was docked with energy of -0.009 kcal/mol and hydroxyurea with -3.014 kcal/mol. these two ligand molecules were also found to obey the lipinski’s rule of five. this rule evaluates drug ability, or finds a chemical compound with some particular pharmacological properties that can make it an orally active drug in humans. this result correlates well with earlier experimental results [14, 15] and it depicts that the identified binding conformations of these inhibitors are reliable and produce anti-tumor effects in a variety of solid tumor [16] and leukemia. 3-aminopropanesulfonic acid is a synthetic gammaaminobutyric acid (gaba) analog. hydroxyurea is an antineoplastic agent that produces anti-tumor effects in animals and man in a various forms of solid tumor and would be an effective drug to inhibit function of mutant h-ras p21 protein, which will be able to arrest cell growth and cancer cell proliferation. from this study and previously reported experimental data in literature, we observe that hydroxyurea and 3aminopropanesulphonic acid would be an effective drug to inhibit function of mutant hras p21 protein, which will in turn arrest the process of cell growth and proliferation of the cancer cell [17]. it was earlier reported that the oral administration of hydroxyurea to 20 patients with chronic myelogenous leukemia, resulted in the decrease count of white blood cell [18]. conclusion the major goal of structure-based drug design is to develop an efficient process that involves a high resolution crystal structure of validated biological target molecules and reliably generates an easily synthesized, high affinity small molecule with desirable pharmacological properties. new advancement in the field of structural genomics, proteomics and bioinformatics will enhance variety of approaches for structure based drug design. acknowledgement we are thankful to dr. pritish varadwaj for the nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     60   informative lecture on computer aided drug design course. we are also thankful to all the faculties of department of bioinformatics, indian institute of information technology, allahabad, india for introducing and enlightening us to the bioinformatics field. references 1. kubinyi h: structure-based design of enzyme inhibitors and receptor ligands. current opinion in drug discovery and development 1998, 1(1):4-15. 2. amy ca: the process of structurebased drug design. chemistry & biology 2003, 10: 787-797. 3. shravanti k et al: a review on structure based drug design of protein tyrosine phosphatase 1b inhibitors for target for obesity and type 2 diabetes mellitus. journal of pharmacy research 2010, 3(12): 29392940 4. sistla r, ghadiyaram c, srinivasan nc and subramanya hs: a structure based strategy for new drug discovery. innovations in pharmaceutical technology 2006,august 30, 20:18-23 5. sergio hr, mark am. groupbuild: a fragment-based method for de novo drug design. j.med. chem 1993, 36: 1700-1710 6. robert sd, eugene is. smog: de novo design method based on simple, fast, and accurate free energy estimates. 1. methodology and supporting evidence. j. am. chem. soc 1996, 118: 11733-1174 7. diane jm: computational approaches to structure-based ligand design. pharmacology & therapeutics 1999, 84: 179–191. 8. holger g, gerhard k: statistical potentials and scoring functions applied to protein–ligand binding, current opinion in structural biology. elsevier 2001, 11(2):231-235. 9. valencia a, chardin p, wittinghofer a, sander c: the ras protein family: evolutionary tree and role of conserved amino acids. biochemistry 1991, 30: 4637-4648. 10. bos j: ras oncogenes in human cancer: a review. cancer res 1989, 49 (17): 4682-4689. 11. lodish h, berk a, zipursky sl, matsudaira p, baltimore d, darnell j: chapter 25, cancer. molecular cell biology (4th ed.). san francisco: w.h. freeman 2000. isbn 0-7167-3706-x. 12. bos j: the ras gene family and human carcinogenesis. mutat res. 1988, 195(3): 255-71 13. henson es, gibson sb: surviving cell death through epidermal growth factor (egf) signal transduction pathways: implications for cancer therapy. cell signal 2006, 18: 20892097. 14. young cw, schochetman g, karnofsky da: hydroxyureainduced inhibition of deoxyribonucleotide synthesis: studies in intact cells. cancer res nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  53–  61                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved     61   1967, 27: 526-534. 15. akerblom l, ehrenberg a, graslund a, lankinen, reichard p, thelander l: overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3t6 cells. proc natl acad sci usa 1981, 78: 2159-2163. 16. schwartz hs, garofalo m, sternberg ss, philips fs: hydroxyurea: inhibition of deoxyribonucleic acid synthesis in regenerating liver of rats. cancer res 1965, 25:1867-1870. 17. krackoff ih, savel h, murphy ml: phase ii studies of hydroxyurea (nsc32065) in adults: clinical evaluation of cancer. cancer chemother rep1964, 40: 53-5. 18. kennedy bj: hydroxyurea therapy in chronic myelogenous leukemia. cancer 1972. 29: 1052-1055. microsoft word paper 4 deswal author copy.doc nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   62 review article models  of  t  cell  antigen  receptor  activation:  the  puzzle  still  remained   sumit deswala,b adepartment of molecular immunology, max planck institute of immunobiology and epigenetics and faculty of biology iii, university of freiburg, stübeweg 51, 79108 freiburg, germany bspemann graduate school of biology and medicine (sgbm), university of freiburg, germany corresponding address: max planck institute of immunobiology and epigenetics, stübeweg 51, 79108 freiburg, germany. e-mail: deswal@immunbio.mpg.de, tel.: +49-761-5108-314, fax: +49-761-5108-423 abstract t cell antigen receptor (tcr) is a protein-complex expressed on all t cells of the immune system and is responsible for the activation of t cells when infectious agent is presented by an antigen presenting cell (apc) in the form of peptides bound to the major histocompatibility complex (pmhc). despite numerous studies it is not clear what biochemical changes upon binding of antigen ligand to the extracellular domains of tcr leads to activation of intracellular signaling (a process known as tcr triggering). this review summarizes possible biochemical mechanisms for tcr triggering and discusses their comparative limitations and advantages in explaining various experimental observations. keywords: t cell antigen receptor, activation, model introduction t cell antigen receptor (tcr or tcr-cd3) is a protein complex expressed exclusively in t cells and is composed of the variable tcr α and β chains and the constant cd3γ, cd3δ, cd3ε and cd3ζ chains (figure 1). cd3γ and cd3δ chain are glycoproteins each of which form a heterodimer with non-glycosylated cd3ε chain. along with the cd3ζ-ζ homodimer, these chains associate with the tcrαβ heterodimer to generate the full tcrcd3 complex [1]. the tcr-cd3 is responsible for activation of a t cell upon antigen encounter which then help in the activation of b cells by releasing helper cytokines (helper t cells) or kill the target cell directly by the secretion of cytotoxic effector molecules such as granzymes, perforin and granulysin. antigenic ligand binding to the tcr leads to the phosphorylation of immunoreceptor nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   63 tyrosine-based activation motifs (itams) of the cd3 complex by the src family protein tyrosine kinase lck, resulting in the membrane targeting and activation of another kinase, zap-70. once activated, zap-70 phosphorylates several substrates, including the transmembrane adaptor protein, linker for activation of t cells (lat). phospho-lat serves as an important point of divergence for signals initiated from the tcr by recruiting several effector molecules to the plasma membrane thus initiating multiple pathways essential for full t cell activation [2]. ! " # $% &'& # ++ + -itam -v c v c - fig.1: the tcr-cd3 complex. v and c represent variable and constant domains respectively for the tcr α and β. the hypervariable region that binds to the pmhc complex is shown in red color. although the downstream signaling mechanisms of t cell activation have been explored to quite detailed extent, the basic mechanism of how ligand binding to extracellular domains of tcrαβ leads to downstream signaling remained unclear. several models of tcr triggering have been proposed based on the experimental findings, but none of the models so far could explain every aspect of tcr triggering. broadly, these models can be grouped in three major processes which involve aggregation, conformational change or segregation (figure 2). this review summarizes the proposed models of tcr triggering and discusses their comparative abilities for explaining various experimental observations (table 1). 1. antigen induced clustering several forms of clustering have been proposed as the triggering mechanism for the tcr. these include homodimer, heterodimer and the pseudodimer models as discussed below. nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   64 b c d e f lck tcr pmhc lck lcklck binding tcr lck cd45 pmhc lck lck lck lcklck binding lipid raft lck tcr pmhc co-receptor lck tcr pmhc lck tcr pmhc lcklck cd45 tcr pmhc binding ! lck lck tcr pmhc lck pullbinding tcr pmhc binding a g h clustering models conformational change models segregation models fig.2: various models of tcr triggering. a) homodimer model, b) heterodimer model, c) pseudo-dimer model, d) membrane binding model, e) kinetic deformation model, f) permissive geometry model, g) kinetic segregation, h) lipid raft mediated segregation model. full description of each model is given in the main text. nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   65 table 1: abilities of different models to explain various experimental observations. observation     triggering  model       homodimer   heterodimer   pseudodimer   membrane binding   kinetic deformation   permissive geometry   kinetic segregation   lipid raft   pre-­‐clustered   tcr   no   yes   yes   yes   yes   yes   yes   yes   coreceptor   independent   triggering   yes   no   no   yes   yes   yes   yes   yes   triggering  by   soluble   monomer  pmhc   no   yes   no   yes   no   no   no   yes   triggering  by   anti-­‐cd3   antibodies   yes   no   no   yes   no   yes   no   no   activation  by   pervanadate   no   no   no   no   no   no   yes   yes   inhibition  by   truncated  cd45   no   no   no   no   no   no   yes   no     inhibition  by   elongated  pmhc   no   no   no   no   no   no   yes   no     triggering  in   absence  of  self   pmhc   yes   yes   no   yes   yes   yes   yes   yes   a. homodimer model clustering of tcr–cd3 complexes following tcr engagement could lead to enhanced phosphorylation, for example by increasing the proximity of associated lck molecules, resulting in the activation of the second receptor in the cluster by transautophosphorylation (or this enhanced proximity of kinases might lead to their transphosphorylation which enhance their activity to phosphorylate the receptor). this model is supported by the observations that artificial ligands such as pmhc tetramer and the antitcrαβ and anti-cd3 antibodies that crosslink the tcr lead to its activation whereas fab fragment of these antibodies or the monomeric pmhc in solution fail to activate the tcr [3]. also the observations that mhc class i [4, 5] and mhc class ii [6] exist in pre-formed clusters, support the homodimer model. however, since several studies have now shown the existence of pre-clustered tcrs on the t cell surface [7, 8], it is difficult to imagine that clustering alone could trigger the nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   66 tcr. in fact in a study by schamel and colleagues [9], both clustering and conformational change were shown to be required for tcr triggering (permissive geometry model, as discussed later). b. heterodimer model in the heterodimer model [10], coreceptor cd8 or cd4 binding to the same pmhc complex as the tcr brings the coreceptorassociated lck kinase into proximity with tcr–cd3 itams and their phosphorylation. further it has been proposed that to fully activate the tcr, a pmhc ligand need to interact with a tcr–cd8 pair with a threshold time to induce stable zippering between the membrane-proximal domain of cd8 and the connecting peptide motif in the tcrα [11]. this allows stable association of lck with the cd3 complex and results in complete phosphorylation of the cd3 itams. a low-affinity pmhc ligand that interacts with a tcr–cd8 pair with less than threshold time induces incomplete zippering and therefore allows only transient lck association and partial cd3 phosphorylation. however, the observations that t cells can develop and function normally in the mice lacking both cd4 and cd8 suggest that coreceptors are not absolutely required for tcr triggering and t cell activation [12, 13]. also the fact that anticd3 antibodies and their f(ab’)2 fragments that do not engage the co-receptors can still activate the tcr [14, 15], indicates that coreceptors are not required for the tcr triggering itself, though they might be important for full activation of a t cell. c. pseudo-dimer model only a few mhcs carry high affinity agonist peptides for the tcrs, whereas a great majority of mhcs carry endogenous selfpeptides. on the basis of the crystallographic studies which showed that the cd4 tail associating with lck was far from their own tcr/cd3 complex, the pseudodimer model postulates that two tcrs are brought together by binding low-affinity self or high-affinity agonist pmhc ligands and that the cd4 associated with a tcr engaging the agonist pmhc complex can assists in phosphorylation of neighboring tcr (engaged to low affinity self peptide) by the associated kinase lck [16]. thus according to this model, a ‘pseudodimer’ consists of one foreign and one self-antigen engaged receptor linked via cd4 molecule is the primary unit of tcr signal initiation module. 2. conformational change model several studies have proposed conformational nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   67 change as a requirement for tcr triggering [17, 18]. however, how the ligand binding to tcrαβ ectodomains leads to a conformational change in the cytoplasmic tails of cd3 subunits is not clear. the situation is complicated by the fact that the crystal structure of the fully assembled tcr-cd3 complex is not known, although individual subunits have been crystallized partially [1922]. some of the important models for the mechanism of conformational change in tcrcd3 are discussed below. a. membrane binding model according to this model, cytoplasmic tails of cd3ε [23, 24] and cd3ζ [25] associate with the lipids present in the plasma membrane of a t cell. thereby, the itams are buried in the plasma membrane, making them inaccessible for phosphorylation by the kinases. ligand binding leads to release of these cd3 chains from the plasma membrane, making them accessible to kinase and hence the phosphorylation. b. kinetic deformation model several line of evidence have proposed that the mechanical effects (such as pulling or shearing) of pmhc binding to the tcr leads to a piston-like displacement of the tcr–cd3 complex [26, 27]. this induces a change in the conformation of the cd3 cytoplasmic domains, allowing itam phosphorylation. c. permissive geometry model based on the experimental evidence for the existence of tcr-cd3s in a pre-clustered form [7] and the fact that pmhc monomers in solution fail to trigger tcr[28, 29] while pmhc dimer or higher oligomer can induce tcr triggering, the permissive geometry model was proposed[30]. according to this model, before ligand binding, the mono and multivalent tcr-cd3s exist in an autoinhibited state where itams are inaccessible for phosphorylation by the kinase. simultaneous dimeric (or higher order) ligand binding to the two tcr-cd3 complexes leads to a scissor-like movement in the two tcrcd3 complexes leading to exposure of their cytoplasmic tails, thus making the itams accessible for phosphorylation. 3. segregation model in a resting t cell, phosphorylation of the tcr-cd3 is kept in check by the active phosphotases. this is supported by the fact that pervanadate, a phosphatase inhibitor, nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   68 treatment leads to strong phosphorylation of the tcr-cd3 itams and also initiates the downstream signaling [31]. thus any process that favors the kinase-phosphtase balance towards kinase, can lead to tcr-cd3 phosphorylation and the signaling. redistribution of the tcr-cd3, kinases and phosphatases in which phosphatase is segregated from the tcr can interfere with the dephosphorylation. there are two mechanisms proposed for such ligand dependent re-distribution, which are discussed below. a. kinetic segregation kinetic segregation model was proposed based on the topological view of the cell surface molecules at the t cell–apc interface [32, 33]. the tight intercellular contact causes the segregation of the molecules by sizes of their ectodomain resulting in physical separation of tcr from the large inhibitory tyrosine phosphatase cd45, leading to stable phosphorylation of tcr–cd3 itams by lck. though sounding good in the context of a tcell – apc contact, the model fails to explain the activation of t cells by soluble antitcrαβ and anti-cd3 antibodies and the pmhc tetramer [14, 34]. b. lipid raft mediated segregation model lipid rafts are the detergent (such as triton x100, brij-series, np-40 or chaps)-resistant membrane microdomains which are enriched in glycosphingolipids, cholesterol and lipidmodified proteins such as the gpi-anchored proteins. also these microdomains are enriched in double-acylated (myristoylated, palmitoylated) src-family kinases and the important signaling molecules such as the coreceptors cd4 and cd8 and the adaptor protein lat. lipid raft mediated segregation model postulates that pmhc engagement results in partitioning of the tcr–cd3 complex into lipid rafts enriched in lck and deficient in cd45 [35, 36]. partial support for this model comes from the observations that palmitoylation-deficient (and raft excluded) mutants of lck and lat are functionally defective. in addition, artificial targeting of other cytoplasmic molecules, such as shp-1 [37], cd45 [38] or plcγ [39], to membrane rafts has marked functional effects on tcrinduced signaling. a connecting peptide in the tcrα chain and the cd3δ chain were identified as the critical sites essential for effective raft association. mutations in these components interfere with tcr signaling [40]. the mechanism of directing association of the engaged tcr complex with lipid rafts is not clear but might be based on co-engagement of the raft resident cd4/cd8 coreceptors. nepal  journal  of  biotechnology.    jan  2012,  vol.  2,  no.  1:  62  –  71                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   69 conclusion analysis of various proposed mechanisms reveals that probably a combination of these mechanisms is responsible for tcr triggering. existence of pre-clustered tcr molecules rules out the homodimerization as a requirement for tcr triggering. it is possible that cd3 itams are inaccessible for phosphorylation by the kinase in the resting state and some sort of conformational change is necessary for exposing the itams, but additional studies are required to find out what mechanism is responsible for such conformational change. segregation models argue the balance of kinase mediated phosphorylation and phosphatase mediated dephosphorylation shifts in favour of kinase upon ligand binding. though such 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  signaling.   european   journal   of   immunology   2002,  32(9):2578-­‐2587.     39. veri   mc,   debell   ke,   seminario   mc,   dibaldassarre   a,   reischl   i,   rawat   r,   graham   l,   noviello   c,   rellahan   bl,   miscia   s   et   al:   membrane   raft-­‐dependent   regulation   of   phospholipase   cgamma-­‐1   activation   in   t   lymphocytes.   molecular   and   cellular   biology   2001,  21(20):6939-­‐6950.     40. werlen   g,   hausmann   b,   palmer   e:   a   motif   in   the  alphabeta  t-­‐cell  receptor  controls  positive   selection   by   modulating   erk   activity.   nature   2000,  406:422-­‐426.     nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 1 nepjol.info/index.php/njb assessing the role of potential biomarkers in antimony susceptible and resistant clinical isolates of l. donovani from india mahendra maharjan1,2*, swati mandal1, rentala madhubala1 1school of life sciences, jawaharlal nehru university, new delhi, 110067, india. 2 central department of zoology, tribhuvan university, kathmandu, nepal. abstract failure of antimonial drugs, the mainstay therapy for leishmaniasis has become an escalating problem in the treatment of indian leishmaniasis. using 14 clinical isolates from both visceral (vl) and post-kala-azar dermal leismaniasis (pkdl) patients, we have examined the role of atp-binding cassette transporter (abc transporter) gene, multidrug resistant protein a (mrpa) and two building blocks of the major thiol, trypanothione namely, ornithine decarboxylase gene (odc) (a rate limiting enzyme in the polyamine biosynthesis) and γ glutamylcysteine synthetase (γ-gcs) (a rate limiting enzyme in glutathione biosynthesis) in antimony resistance. amplification of these three genes was observed in some but not all clinical isolates. increased expression of the three rnas as determined by real-time pcr was observed in all sag-r clinical isolates. significant increase in cysteine and glutathione levels was observed in the resistant isolates. our studies report the underlying mechanism of antimony resistance in the clinical isolates. key words: abc transporter, ornithine decarboxylase, γglutamylcysteine synthetase, antimony resistance. *corresponding author email: maharjan.m@gmail.com introduction the protozoan parasite leishmania is the causative agent of kala-azar and is responsible for a variety of clinical manifestations. it causes a wide spectrum of diseases ranging from the simple self healing cutaneous form to the debilitating visceral form. visceral leishmaniasis (vl) is caused by l. donovani in the indian subcontinent. pentavalent antimonials (sbv) are the first line of drug used in the treatment against all forms of leishmanial infections [1,2]. resistance to this drug is becoming a major barrier in the treatment of vl in many endemic regions particularly in india [3]. kala-azar transmission in india is thought to be anthroponotic and postkala-azar dermal leismaniasis (pkdl) patients are considered to serve as a source for new outbreaks [4]. the post-kala-azar dermal leismaniasis (pkdl) is a sequel to vl in india and sudan; the disease develops months to years after the patient’s recovery from vl [5]. the mechanism of action of sodium antimony gluconate (sag) remained an enigma for more than 60 years of its effective use against all forms of leishmaniasis. it is generally agreed that pentavalent form (sbv) is reduced to a more toxic trivalent form (sbiii) which constitutes the active form of the drug against the parasite [6]. molecules possibly implicated in reduction of sbv to sbiii include host and parasite thiols and two newly discovered parasite enzymes thioldependent reductase (tdr1) and arsenate reductase (acr2) [7-9]. a loss of drug activation is reported to lead to resistance [10]. the route of entry of sbv into leishmania cells is still unknown but sbiii has been reported to be transported in leishmania through aquaglyceroporin (aqp1) [11,12]. recent evidence has suggested that part of the mode of nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 2 nepjol.info/index.php/njb action of sbv could be in depleting the cells of its reduced thiols [13]. trypanothione (tsh), a major reduced thiol of leishmania is a n1, n8 bisglutathione spermidine conjugate. it is thought to bind to the active reduced form of the metal. these metal-trypanothione conjugates are either sequestered into an intracellular organelle by the abc transporter mrpa or extruded outside the cell by an efflux pump [13-16]. a number of candidate genes associated with increased thiol concentration have been described in leishmania laboratory mutants. resistance was induced in these mutants in vitro in the presence of antimony related metals such as arsenic or antimony [2,17,18]. however, till date it remains unclear as to whether similar mechanisms can be extrapolated to clinical isolates from geographical zones with a high incidence of primary antimony resistance. to address this question, we have characterized both the vl and the pkdl isolates from india and report that diverse mechanisms of resistance are operative in these isolates. this work aims at characterizing possible biomarkers for monitoring antimonial resistant visceral leishmaniasis and post-kala-azar dermal leismaniasis in the field isolates. in the present study, we report the role of thiols and also assessed the role of abc transporter (mrpa), ornithine decarboxylase (odc) and γglutamylcysteine synthetase (γ-gcs) genes as potential biomarkers for monitoring antimonial resistance in indian leishmaniasis. materials and methods parasite and culture conditions promastigotes of leishmania donovani clones, ag83 (mhom/in/80/ag83), 2001, mc4, mc7, mc8 and mc9 were isolated from patients with vl and strains, rk1, ms2, nr3a, rmp8 (hm/in/rmp-8), rmp-19 (hm/in/rmp-19), rmp142 (hm/in/rmp-142), rmp155 (hm/in/rmp-155) and rmp240 (hm/in/rmp-240) used in the present study were isolated from patients with pkdl [19]. clinical isolates obtained from vl and pkdl patients who responded to sag chemotherapy were designated as sag-s (sag-sensitive) whereas isolates from vl and pkdl patients who did not respond to sag were designated as sag-r (sag-resistant). sag-sensitive strains, ag83-s, 2001-s, mc7-s, rk1-s, ms2-s and the nine sag-r isolates, mc4-r mc8-r, mc9-r, nr3a-r, rmp8-r, rmp19-r, rmp142-r, rmp155-r and rmp240-r have been characterized earlier [20]. clinical history of the patient infected with the strain rk1-s showed that the interval between the cure of vl and the onset of pkdl was 2.5 years where as in the case of pkdl patients infected with the strains, ms2s and nr3a-r, the interval between the cure of vl and the onset of pkdl was 7 and 11 years respectively. the interval between the cure of vl and the onset of pkdl for the remaining isolates is not known. the clinical isolates were maintained in vitro in the absence of the drug pressure. promastigotes were routinely cultured at 220c in modified m-199 medium (sigma, usa) supplemented with 10% heat-inactivated fetal bovine serum (fbs; gibco/brl, life technologies scotland, uk) and 0.13 mg/ml penicillin and streptomycin. this study has the approval of the institutional level ethics committee. dna and rna manipulations chromosomes of the clinical isolates were separated by pulse field gel electrophoresis (pfge) in which low melting agarose blocks, containing embedded cells (108/ml log phase promastigotes) were electrophoresed in a contour clamped homogeneous electric field apparatus (chef driii, bio-rad) as reported earlier [20]. mid log phase promastigotes (~2 x 109 cells) of all the field isolates were used for isolation of genomic dna. 5 µg of genomic dna was digested with hindiii enzyme and subjected to electrophoresis. total rna was isolated from promastigotes (2 x 108 cells) using rnaeasy plus mini kit (qiagen). standard protocols were followed for southern hybridization [21]. dna probes used in the present study included a 400-bp mrpa fragment (released from plasmid pm12 that was nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 3 nepjol.info/index.php/njb digested with bamhi and psti), a 2.3-kb γgcs fragment (derived from plasmid pspαhygroαγgcs digested with hindiii and xbai), a 2.0-kb odc-full length probe (derived from plasmid pspαhygroα-odc digested with hindiii and xbai) and a 1.6-kb 5’-ptr1 probe derived from plasmid (psp72-y-hygro-5’-ptr1). cdna synthesis and real time rtpcr total rna was isolated from 108 leishmania cells in the mid-log phase of growth using the rneasy plus mini kit (qiagen) as described by the manufacturer. quality and quantity of the rna were determined using the rna 6000 nano lab chip kit on the bio-analyzer 2100 (agilent technologies). the sequences of the primers for mrpa are forward 5’gcgcagccgtttgtgcttgtgg-3’ and reverse 5’-ttgccgtacgtcgcgatggtgc-3’ and for the glyceraldehyde 3-phosphate dehydrogenase (gapdh) control forward 5’gaagtacacggtggaggctg and reverse 5’cgctgatcacgaccttcttc primers. the sequences of the primers for odc are forward 5’gatggtgcgcccttactttgc-3’and reverse 5’-ttccatctccagcgggttgtcc-3’, and for the γgcs, forward 5’cattggctggcgcgttgagttc-3’ and reverse 5’-atgtgcgcggcccatattctcg -3’ primer. complementary dnas from promastigotes were synthesized from 500 ng of total rna using the accusuperscript high fidelity rt-pcr kit (stratagene, la jolla) and oligo (dt)18 primers following manufacturer’s instructions. real-time pcr was performed in triplicate in 25 l volumes using quantifast sybr green pcr master mix (qiagen) in an applied biosystem 7500. reactions were run using the following thermal profile: initial denaturation at 95°c for 5 min followed by 40 cycles with denaturation at 95°c for 30 s, annealing at 62°c for 1 min and extension at 72°c for 20 s. the pcr was followed by a melt curve analysis to ascertain that the expected products were amplified. the relative amount of pcr products generated from each primer set was determined based on the threshold cycle (ct) value and amplification efficiencies and was normalized by dividing the values by the relative amount of the gapdh gene used as a control. transfection and overexpression of the mrpa and γ-gcs gene episomal leishmania expression vectors, pglneoluc containing luciferase encoding dna and neomycin phosphotransferase selectable marker, psphygro-gcs containing coding sequence for heavy subunit of gcs with hygromycin phosphotransferase as selectable marker and pgem72f-neo-mrpa containing mrpa coding dna and neomycin phosphotransferase as selectable marker were obtained as gifts from prof. marc ouellette, centre de recherche en infectiologie du centre de recherche du chul, universite laval, quebec, canada. twenty µg of each construct was transfected into l. donovani promastigotes by electroporation. electroporation was done with a single pulse with the following parameters 450 v, 500 f (bio-rad). transfectants were selected for resistance to either g418 (40 μg/ml) or hygromycin b (80 μg/ml) as described earlier [22]. chemosensitivity profiles of sag-s and sag-r strains in an amastigote macrophage model stationary phase leishmania promastigotes expressing the luciferase gene (pglneoluc) and psphygro-gcs containing coding sequence for heavy subunit of gcs with hygromycin phosphotransferase as selectable marker or pgem72f-neo-mrpa containing mrpa coding dna and neomycin phosphotransferase as selectable marker were infected into j774a.1 macrophages. macrophage cell line j774a.1 (american type culture collection) was maintained at 37c in rpmi1640 medium (sigma) containing 10% heat inactivated fetal bovine serum. briefly, j774a.1 murine macrophages (1 x 105 cells/ petridish) were infected with 1 x 106 promastigotes (expressing the luciferase gene (pglneoluc) in m199 media with 10% fbs. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 4 nepjol.info/index.php/njb after 3 h, the non-internalized parasites were washed off and sag was added at different concentrations (10 – 100 g/ml). after 5 days of drug exposure, plates containing adherent macrophages were washed and luciferase activity was determined [22]. the 50% inhibitory concentration (ic50) was determined from the graph representing different concentrations of sag plotted against relative light units (rlu) produced by luciferase expressing parasites. thiol analysis thiols were derivatized with monobromobimane and separated by highperfomance liquid chromatography (hplc) as reported earlier [18]. statistical analysis data was analyzed by the student’s ttest. the data is represented as mean ± s.d. the results are representative of three independent experiments. a p value of < 0.05 was considered statistically significant. results intracellular thiol levels in sag-s and sag-r clinical isolates resistance to antimonials in clinical isolates is not well defined. understanding the mechanism of resistance to antimony in clinical isolates of l. donovani will aid in the development of biomarkers for antimony resistance and this in turn will enable the clinicians to monitor the treatment of the patients. with this background in mind, we used the previously characterized sag-s and sag-r, vl and pkdl clinical isolates for the present study [20]. a total of fourteen field isolates were used to assess the putative role of the abc transporter mrpa, ornithine decarboxylase and γglutamylcysteine synthetase in antimony susceptible and -resistant clinical isolates of l. donovani from india. as reported earlier, sag-s isolates, ag83-s, 2001-s, mc7-s, rk1-s and ms2-s coming from sag responsive patients had ic50 values 6.2 ± 1.8, 0.9 ± 0.12, 8 ± 3.3, 0.01 ± 0.02 and 4.75 ± 0.12 µm respectively whereas the sag-r isolates, mc4-r, mc8-r, mc9-r, nr3ar, rmp8-r, rmp19-r, rmp142-r, rmp155-r, rmp240-r coming from the sag-unresponsive patients had ic50s that were 2 to >10 fold higher than that of the sensitive isolate, ag83-s.(20) rk1-s was the most sensitive of all the isolates with an ic50 of 0.01 ± 0.02 µm. intracellular thiol levels were quantified in the sag-s and sag-r isolates (figure 1). sag-r isolates maintained significantly higher levels of cysteine and glutathione as compared to the sag-s isolates. cysteine levels in the sag resistant strains mc4-r, mc8-r, mc9-r, nr3ar, rmp8-r, rmp19-r, rmp142-r, rmp155-r and rmp240-r were ~1.7, ~1.6, ~1.5, ~2.7, ~2.0, ~1.8, ~1.8, ~2.0 and ~1.7 -fold higher respectively when compared to the sag-s isolate, 2001-s (figure 1a). similarly gsh levels showed significant increase in the sag-r isolates, mc4-r (~2.7-fold), mc8-r (~3.6 -fold), mc9-r (~3.0 -fold), nr3a-r (~2.0 fold), rmp8-r (~3.2 -fold), rmp19-r (~2.5 -fold), rmp142-r (~3.1 -fold), rmp155-r (~2.4 -fold) and rmp240-r (~3.0 -fold) when compared to the sag sensitive isolate, 2001-s (figure 1b). a sag-s strain, rk1-s had glutathione levels that were ~2.1 -fold higher when compared to the sag sensitive isolate, 2001-s. interestingly, no significant difference was observed in the trypanothione levels between the sag-s and sag-r isolates (figure 1c). similar observation was made in our earlier study using a small set of clinical isolates [23]. gene copy number and expression profiling of the abc transporter mrpa in sag-s and sag-r clinical isolates our earlier work on the clinical kala azar l. donovani isolates from india showed mrpa overexpression as an important sag resistance factor [23]. to further validate, the role of mrpa gene in antimony resistance phenotype, we checked the amplification of mrpa gene by southern blot hybridization. southern blot hybridization of total genomic dna digested with hindiii followed by hybridization with nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 5 nepjol.info/index.php/njb figure 1: intracellular levels of thiols, cysteine, glutathione and trypanothione in sag-s and sag-r l. donovani vl and pkdl clinical isolates. a: cysteine levels b: glutathione levels c: trypanothione levels. thiols were derivatized with monobromobimane and separated by high-perfomance liquid chromatography (hplc). each value is the mean  sd of triplicates from two independent experiments. a indicates p <0.01; b indicates p < 0.001; c indicates p < 0.0004 when compared to 2001-s respectively mrpa specific probe was done. sag-sensitive and sag-resistant field isolates showed a single hybridizing fragment of 11-kb indicating that mrpa gene exist as a single copy gene in all the isolates (figure 2a). quantitation of the southern blot hybridization signal was done using imagequant 5.2 (molecular dynamics) and the fold difference in dna copy number of the isolates with the ag83-s or 2001-s was calculated. amplification of mrpa gene was observed in the resistant isolates, mc4-r, mc8r, mc9-r, rmp19-r, rmp142-r, rmp155-r and nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 6 nepjol.info/index.php/njb figure 2: a: southern blot analysis of mrpa and ptr1 genes in sag-s and sag-r l. donovani vl and pkdl clinical isolates. total genomic dna of isolates were digested with hindiii, electrophoresed, blotted and hybridized with a mrpa specific probe of 400-bp and 6 kb 5’-ptr1 specific probe. the size of the hybridizing bands was determined using hindiii digested lambda dna. the blot was rehybridized with α-tubulin probe to monitor the amount of digested dna layered on the gel. quantitation of southern blot was done by image-quant software 5.2 (molecular dynamics) and the fold difference in dna copy number of mrpa is presented below each blot. b: southern blot analysis of the pulse field gel electrophoresis of sag-sensitive (sag-s) and sagresistant (sag-r) isolates of leishmania donovani chromosomes. agarose blocks containing chromosomal dnas of promatigotes of l. donovani clinical isolates were prepared and subjected to pulsed field gel electrophoresis for 24 hours run time and hybridized with a mrpa specific probe of 400-bp. c: real time rt-pcr expression analysis of mrpa gene in l. donovani clinical isolates. mrpa rna expression ratios in promastigotes of sag -resistant isolates are relative to the sag-sensitive isolate (2001-s). the graph represents mean of three independent experiments performed from three different rna preparations. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 7 nepjol.info/index.php/njb figure 3: a: characterization of odc gene in sag-s and sag-r isolates l. donovani vl and pkdl clinical isolates. genomic dnas were isolated and digested with hindiii and hybridized with a full-length odc specific probe, derived from the l. donovani odc gene. the sizes of the hybridizing bands were determined using hindiii digested lambda dna marker. the blot was rehybridized with α-tubulin probe to monitor the amount of digested dna layered on the gel. b: real time rt-pcr expression analysis of odc in l. donovani clinical isolates. odc rna expression ratios in the sagresistant isolates are relative to the sag-sensitive isolate, 2001-s. results are mean of three independent experiments performed from three different rna preparations. rmp240-r (figure 2a). no amplification was observed in the resistant isolates, nr3a-r and rmp8-r (figure 2a). the amplification observed in the resistant isolates was further analyzed by pfge (figure 2b). none of the resistant isolates showed circular amplification as was observed in our earlier studies with limited number of isolates [23]. interestingly, two sensitive pkdl isolates, rk1-s and ms2-s and one resistant pkdl isolate, nr3a-r had the presence of mrpa on two chromosomes as indicated by their characteristic migration in pfge (figure 2b). we had earlier reported co-amplification of pterin reductase gene (ptr1) with mrpa in the clinical isolates [23]. in the present study, coamplification of ptr1 gene with mrpa gene was not observed in any of the clinical isolates as nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 8 nepjol.info/index.php/njb figure 4: a: southern blot analysis of the -gcs gene in the sag-sensitive and the sag-resistant leishmania donovani vl and pkdl field isolates. total genomic dna was isolated and digested with hindiii. the digested dna was electrophoresed, blotted and hybridized with -gcs probe. the sizes of the hybridizing bands were determined using hindiii digested λdna. the blot was rehybridized with α-tubulin probe to monitor the amount of digested dna layered on the gel. b: real time rt-pcr expression analysis of -gcs gene in l. donovani clinical isolates. -gcs gene expression ratios in the sag-resistant isolates are relative to the sagsensitive isolate, 2001-s. results are mean of three independent experiments performed from three different rna preparations. determined by southern-blot analysis using ptr1-specific probe (figure 2a). comparison of mrpa gene expression in sag-s versus sag-r field isolates was done to verify if there is any correlation between mrpa gene expression and sag susceptibility profile of the clinical isolates. total rna from promastigotes of the clinical isolates was isolated and complementary dnas were synthesized. realtime -pcr using quantifast sybr green pcr master mix (qiagen) with mrpa (gene specific) and gapdh (internal control) primers was performed. up-regulation of mrpa expression was observed in the resistant clinical isolates. mrpa expression in the resistant strains, mc4r, mc8-r, mc9-r, nr3a-r, rmp8-r, rmp19-r, nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 9 nepjol.info/index.php/njb rmp142-r, rmp155-r and rmp240-r was ~4-, ~4.2-, ~9.6-, ~7.2-, ~6.1-, ~6.8-, ~6.0-, ~7.5 and ~4.4fold more respectively when compared to the expression in the sensitive isolate, 2001-s (figure 2c). gene copy number and expression profiling of the ornithine decarboxylase (odc) gene in sag-s and sag-r clinical isolates. ornithine decarboxylase (odc) is the ratelimiting enzyme of the polyamine biosynthetic pathway. in addition to mrpa, overexpression of the odc gene has been reported in the antimony resistant mutants [23-25]. these observations prompted us to determine if amplification of the odc gene occured in our clinical isolates. southern-blot analysis of the total genomic dna digested with hindiii and hybridized with the odc specific probe was done. sag-s and sag-r isolates showed a single copy of the odc gene (figure 3a) with the exception of three pkdl isolates, rk1-s, ms2-s and nr3a-r. interestingly, two copies of the odc gene were observed in these three pkdl isolates (figure 3a). comparison of odc gene expression in sag-s versus sag-r field isolates was done to verify if there is any correlation between the gene expression and sag susceptibility profile of the clinical isolates. real-time pcr with the odc (gene specific) and the gapdh (internal control) primers was performed. up-regulation of the odc expression was observed in the resistant clinical isolates. odc expression in the resistant strains, mc4-r, mc8-r, mc9-r, nr3a-r, rmp8r, rmp19-r, rmp142-r, rmp155-r and rmp240-r was ~3.6-, ~7.4-, ~3.1-, ~2.7-, ~2.3-, ~3.3-, ~3.5-, ~2.8 and ~3.0fold more respectively when compared to the sensitive isolate, 2001-s (figure 3b). sag-s isolate, rk1-s was an exception since it was the only sensitive isolate that showed 2.9-fold up-regulation when compared to the reference strain, 2001-s and also in comparison with all other sag-s isolates (figure 3b). no uniform correlation was observed between gene amplification and odc gene expression in these isolates. gene copy number and expression profiling of the γ-glutamylcysteine synthetase (γ-gcs) gene in sag-s and sag-r clinical isolates in addition to mrpa and odc, another locus that has been reported to be amplified in the antimony-resistant isolates is the gsh1 gene coding for the heavy subunit of -gcs. -gcs is the rate limiting enzyme for gsh synthesis [14,23]. we performed southern blot analysis of the total genomic dna digested with hindiii and hybridized with the -gcs specific probe. southern blot analysis of the -gcs gene showed two copies in a resistant isolate mc4-r but in all other isolates, -gcs probe hybridized to a single hybridizing fragment of 10-kb (figure 4a). sag-s isolate, rk1-s was again an exception since it showed amplification of the -gcs gene (figure 4a). comparison of gcs gene expression in sag-s versus sag-r field isolates was done to verify if there is any correlation between gene expression and sag sensitivity profile of the clinical isolates. real-time pcr using -gcs (gene specific) and gapdh (internal control) primers was performed. up-regulation of -gcs expression was observed in the resistant clinical isolates. -gcs expression in the resistant strains, mc4-r, mc8-r, mc9-r, nr3a-r, rmp8-r, rmp19-r, rmp142-r, rmp155-r and rmp240-r was 4.3-, 5.2-, 6.4-, 5.5-, 3.0-, 5.0-, 5.3-, 4.2 and 8.8fold more respectively compared to the expression in the sensitive isolate, 2001-s (figure 4b). in the present study though upregulation of -gcs was observed in all sag-r isolates, interestingly one sag-s isolate, rk1-s showed 5.5-fold up-regulation of gcs expression when compared to a sag-s isolate, 2001-s (figure 4b). l-butathione-sulfoxamine (bso), an inhibitor of -gcs was used to compare its effect on the sag-s isolate, rk1-s and 2001-s. rk1-s promastigotes overexpressing -gcs were ~5 fold more resistant to bso when compared to the ic50 of promastigotes of the 2001-s, the ic50 being 6.5 ± 0.5 mm and 1.3 ± 0.3 mm respectively. nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 10 nepjol.info/index.php/njb figure 5: over-expression of mrpa and γ-gcs gene in a sensitive l. donovani isolate. a: transfection of mrpa in sag-sensitive isolate leads to resistance to antimony in amastigotes. ag83-s (-■-) was transfected with pgl-neoluc, ag83-s + ldmrpa (-□-) was transfected with pgem72f-neo-mrpa + pglneoluc and sensitivity to sag in the j774a.1 line was measured as described in materials and methods. each data point represents the mean  sd of three determinations. northern blot analysis (insets in a) of vector transfected controls and mrpa transfected lines was performed as described under methods section b: transfection of γ-gcs gene in sagsensitive isolate leads to antimony resistance in intracellular amastigotes. ag83-s (-■-) transfected with pgl-neoluc, ag83-s + γ-gcs (-□-) transfected with psphygro-gcs + pglneoluc and sensitivity to sag measured as described in materials and methods. each data point represents the mean  sd of three determinations. northern blot analysis (insets in b) of vector transfected controls and γ-gcs transfected lines was performed as described under methods section. overexpression of mrpa or -gcs in an antimony-sensitive isolate conferred increased expression and resistance to antimony to determine whether overexpression of mrpa and -gcs conferred antimony resistance in a sensitive isolate, we transfected mrpa or -gcs constructs into a sag-s isolate, ag83-s. these mrpa and -gcs recombinant parasites were co-transfected with pgl-neoluc, encoding luc. intracellular amastigotes over-expressing mrpa (ic50, 32.3 ± 6.4 g/ml) were 3.6-fold more resistant to sag when compared to amastigotes of the parent strain transfected with the control vector (ic50, 9 ± 0.5 g/ml) (figure 5a). intracellular amastigotes over-expressing gcs were 3.3-fold resistant to sag when compared to the sensitive l. donovani the ic50 value being (29.7 ± 1.5 g/ml) respectively (figure 5b). in our previous studies we had demonstrated that the odc overexpressors exhibited significant resistance to pentostam compared to the wild type cells (26). intracellular amastigotes over-expressing odc (ic50, > 80 g/ml) were >8.8-fold more resistant to sag when compared to amastigotes of the parent strain transfected with the control vector [26]. discussion currently, chemotherapy is the only effective way to control leishmania infection. pentavalent antimonials are the mainstay of therapy in the treatment of visceral leishmaniasis [27]. increase in resistance to sag has led to an upsurge in therapeutic failure and in the absence of limited chemotherapeutic alternatives, it is extremely necessary to identify biomarkers for monitoring antimony resistance. trivalent form of the antimonial drug (sbiii) is the prodrug that is formed by conversion of pentavalent antimony (sbv) by a putative metalloid reductase present in the macrophages [28]. antimonial resistance in both laboratory mutants and clinical isolates has been associated with (a) decreased uptake of the drug through aquaglyceroporin (aqp1) that codes for the protein responsible for sbiii transport nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 11 nepjol.info/index.php/njb [11,12,20,29] (b) over expression of odc and γgcs enzymes of the trypanothione biosynthetic pathway [2,25] and (c) increased expression of the abc transporter mrpa, which sequesters sbiii-thiol conjugate [16,23]. we had earlier reported decreased uptake of antimony in all nine sag-r isolates used in the present study. down-regulation of sbiii influx pump; aquaglyceroporin (aqp1) was observed in seven out of the nine resistant isolates strains, mc8-r and nr3a-r were an exception since they showed up-regulation of aqp1 gene expression [20]. the abc transporter gene mrpa causes drug sequestration in leishmania promastigotes and amastigotes selected for sbiii resistance [17,30]. increased copy number of mrpa has been reported in sbiii resistant mutant [2]. our earlier studies using limited number of sag-s and sag-r clinical isolates showed amplification of mrpa gene as part of an extrachromosomal circle [23]. in the present study, we found distinct correlation between copy number of mrpa gene and antimony sensitivity in a majority of the isolates (figure 2a). however, two sag-r strains, nr3a-r and rmp8-r were an exception. we have reported earlier that abc transporter gene mrpa was amplified in three out of four resistant vl isolates as part of the extrachromosmal circle and coamplifcation of ptr1 along with mrpa suggested amplification of the h locus in sag-resistant clinical isolates [23]. in the present study, co-amplification of ptr1 gene with mrpa gene was not observed in any of the clinical isolates. chef gel analysis of the sag-s and sag-r isolates did not show any circular amplification in any of the sag-r isolates. mrpa gene expression in sag-s versus sag-r field isolates showed correlation between mrpa gene expression and sag susceptibility profile of the clinical isolates. the present observation validated our earlier results where we have shown correlation between mrpa gene expressions with the antimony resistant clinical profile in the field conditions [23]. in our previous study amplification of odc gene was noted in the resistant isolates but not that of the γ-gcs [23]. in another study on l. donovani isolates from nepal, expression of γ-gcs and odc was significantly decreased in the resistant isolates [31]. in the present study we observed increased expression of odc gene and γ-gcs in all sag-r isolates. it has been reported earlier that abc transporter mrpa, confers resistance to antimonials by sequestration of metal thiol conjugates in an intracellular organelle located close to the flagellar pocket [16]. this model has been demonstrated in promastigotes of l. tarentolae, in amastigotes and also in clinical isolates from india [16,23,30]. these observations clearly highlighted the importance of intracellular thiol in mrpa mediated efflux of the antimony. an increase in cysteine and glutathione levels were reported in antimony resistant l. donovani clinical isolates [23]. in the present study also, we observed increase in cysteine and glutathione levels in all sag-r isolates. however, no change in trypanothione levels were observed in sag-r isolates in comparison to sag-s isolates. similar observation was made in our analysis of the mode of action of antimony in clinical isolates and earlier studies in l. infantum resistant to sb(iii) [23]. it was pointed out that antimony possibly depleted trypanothione by efflux of sbtrypanothione conjugate [16]. it is possible that the efflux system is increased in the resistant isolates thereby leading to increased trypanothione efflux. this would explain the constant levels of trypanothione in the present study in the resistant isolates. rk1-s though a sag-s isolate, was an exception to the mutlifactorial antimony resistant mechanisms reported in clinical isolates and also in the lab based resistant isolates. clinical history of the rk1-s patient showed that the interval between the cure of vl and the onset of pkdl was 2.5 years where as ms2-s and nr3a-r, pkdl isolates the interval between the cure of vl and the onset of pkdl was 7 and 11 years respectively. it will be interesting to look at the nepal journal of biotechnology. d e c . 2 0 1 6 vol. 4, no. 1: 1-13 maharjan et al. ©njb, biotechnology society of nepal 12 nepjol.info/index.php/njb pkdl isolates with known clinical history in order to determine if this interval has a role to play in antimony susceptibility/resistance. the data presented here in vl and for the first time in pkdl isolates, establishes the relevance of overexpression of γ–gcs and odc and an increased expression of mrpa that may be responsible for an increased efflux of thiol-sb-iii conjugate. our data further confirms that resistance mechanisms present in the laboratory strains can be found in the clinical isolates. further work is presently going on the laboratory to get a global overview of the resistant mechanism by proteomics approach in order to find other possible resistant determinants. it will further help in proper selection of therapeutic regimen and improve treatment strategy against leishmaniasis. acknowledgements the clinical isolates used in this study were kindly provided by dr. mitali chatterjee (institute of post graduate medical education and research, kolkata, india) and dr. sarman singh, (division of clinical microbiology, all india institute of medical sciences, new delhi, india). funding this work is supported by a grant from the department of biotechnology to dr. rentala madhubala. mahendra maharjan and swati mandal are supported by a fellowship from the university grants commission, government of india transparency declarations: none to declare. references 1. guerin pj, olliaro p, sundar s, boelaert m, croft sl, desjeux p, wasunna mk, bryceson ad: visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. lancet infect dis. 2002, 2(8):494501. 2. haimeur a, brochu c, genest p, papadopoulou b, ouellette m: amplification of the abc transporter gene pgpa and increased trypanothione levels in potassium antimonyl tartrate (sbiii) resistant leishmania tarentolae. mol biochem parasitol. 2000, 108(1):131-135. 3. sundar s, more dk, singh mk, singh vp, sharma s, makharia a, kumar pc, murray hw: failure of pentavalent antimony in visceral leishmaniasis in india: report from the center of the indian 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parasitol. 2000, 110(2): 195-206. 23. mukherjee a, padmanabhan pk, singh s, roy g, girard i, chatterjee m, ouellette, m, madhubala r: role of abc transporter mrpa, gammaglutamylcysteine synthetase and ornithine decarboxylase in natural antimony-resistant isolates of leishmania donovani. j antimicrob chemother. 2007, 59(2): 204-211. 24. grondin k, haimeur a, mukhopadhyay r, rosen bp, ouellette m: co-amplification of the gammaglutamylcysteine synthetase gene gsh1 and of the abc transporter gene pgpa in arsenite-resistant leishmania tarentolae. embo j. 1997, 16(11): 30573065. 25. haimeur a, guimond c, pilote s, mukhopadhyay r, rosen bp, poulin r, ouellette m: elevated levels of polyamines and trypanothione resulting from overexpression of the ornithine decarboxylase gene in arsenite-resistant leishmania. mol microbiol. 1999, 34(4): 726-735. 26. singh s, mukherjee a, khomutov ar, persson l, heby o, chatterjee m, madhubala r: antileishmanial effect of 3-aminooxy-1aminopropane is due to polyamine depletion. antimicrob agents chemother. 2007, 51(2): 528-534. 27. den boer ml, alvar j, davidson rn, ritmeijer k, balasegaram m: developments in the treatment of visceral leishmaniasis. expert opin emer. drugs. 2009, 14(3): 395-410. 28. sereno d, cavaleyra m, zemzoumi k, maquaire s, ouaissi a, lemesre jl: axenically grown amastigotes of leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action. antimicrob agents chemother. 1998, 42(12): 3097-3102. 29. maharjan m, singh s, chatterjee m, madhubala r: role of aquaglyceroporin (aqp1) gene and drug uptake in antimony-resistant clinical isolates of leishmania donovani. am j trop med hyg. 2008, 79(1): 69-75. 30. el fk, messier n, leprohon p, roy g, guimond c, trudel n, saravia ng, papadopoulou b, legare d, ouellette m: role of the abc transporter mrpa (pgpa) in antimony resistance in leishmania infantum axenic and intracellular amastigotes. antimicrob agents chemother. 2005, 49(5): 1988-1993. 31. decuypere s, rijal s, yardley v, de ds, laurent t, khanal b, chappuis f, dujardin jc: gene expression analysis of the mechanism of natural sb(v) resistance in leishmania donovani isolates from nepal. antimicrob agents chemother. 2005, 49(11): 4616-4621. http://www.ncbi.nlm.nih.gov/pubmed/?term=persson%20l%5bauthor%5d&cauthor=true&cauthor_uid=17101681 http://www.ncbi.nlm.nih.gov/pubmed/?term=heby%20o%5bauthor%5d&cauthor=true&cauthor_uid=17101681 http://www.ncbi.nlm.nih.gov/pubmed/?term=chatterjee%20m%5bauthor%5d&cauthor=true&cauthor_uid=17101681 http://www.ncbi.nlm.nih.gov/pubmed/?term=madhubala%20r%5bauthor%5d&cauthor=true&cauthor_uid=17101681 microsoft word paper 3 two colum final.docx nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         26     original  research  article   lovastatin  production  by  aspergillus  fischeri  under  solid  state  fermentation  from   coconut  oil  cake   parvatham madhu latha1*,, pallem chanakya2, manipati srikanth3 1,* nri college of pharmacy, pothavarappadu, agiripalli mandal, vijayawada rural, andhra pradesh. 2centre for biotechnology, department of chemical engineering, au college of engineering (a), andhra university, visakhapatnam, andhra pradesh. 3nri academy of medical sciences, china kakani, mangalagiri mandal, guntur, andhra pradesh *corresponding author: srimani.nri@gmail.com, chanakya.pallem@gmail.com abstract: the main aim of the present investigation was to optimize the fermentation parameters that enhance the maximum production of lovastatin by aspergillus fischeri using coconut oil cake as the solid substrate under solid state fermentation. the maximum yield of lovastatin (14.77 mg/g dry substrate) using coconut oil cake as the substrate was achieved with the following optimized process parameters: fermentation time (7 days), initial moisture content (60% v/w), inoculum volume (2ml of five day old culture), initial ph (5.0), incubation temperature (30ºc), lactose (1% w/v) and malt extract (1% w/v). keywords: lovastatin, aspergillus fischeri, coconut oil cake, fermentation parameters, optimization introduction: lovastatin also called as mevinolin, monacolin k and mevacor, a kind of fungal metabolite, serves as one of the competitive inhibitors of enzyme hydroxymethylglutaryl coenzyme a reductase (hmg-coa), which catalyzes the rate-limiting step in cholesterol biosynthesis, resulting in lowered blood cholesterol [1,2]. in addition, lovastatin has been used in the biomedical applications such as, in treating coronary heart diseases, renal diseases, alzheimer’s disease, bone fractions [3]. moreover, it has been indicated as a potential therapeutic agent for the treatment of various types of tumors because of its capability to suppress tumor growth in vivo through inhibition of the synthesis of nonsterol isoprenoid compounds [4, 5]. commercial production of lovastatin has been carried out by submerged fermentation using different microorganisms [6, 7]. in recent nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         27   years, researchers have shown an increasing interest in solid state fermentation (ssf) as it is a potential alternative of submerged fermentation because economical and cheap substrates can be utilized in ssf for production of value-added products, and also requires less energy, and low capital costs. now days, ssf has been exploited for the production of highly potent drugs on large scale. but still, few reports are available on the microbial production of lovastatin under ssf [8-10]. in the present study, we evaluated the feasibility of ssf process for lovastatin production by aspergillus fischeri and optimized different process parameters that maximize the lovastatin yield. materials and methods microorganism and inoculum preparation: the fungal strain aspergillus fischeri ncim 509 used in this study was procured from the culture collection centre of national collection of industrial microorganisms (ncim), ncl, pune. the microbial strain was maintained on potato dextrose agar (pda) slants containing (g/l) dextrose 20.0 and agar 15.0. inoculated slants were grown in an incubator at 28°c for 5 days. after that, the slants were preserved at 4°c and sub-cultured once every four weeks (monthly). inoculum preparation: inoculum was prepared by adding 10 ml of sterilized 0.1% tween 80 solution to a wellsporulated a. fischeri slant. the spore surface was scrapped with an inoculating loop to suspend the spores in the solution and the obtained spore suspension was used as the inoculum for the fermentation process. solid state fermentation (ssf): coconut oil cake obtained from the local market of vijayawada, andhra pradesh was used as the substrate in the present study. five grams of coconut oil cake was dried and grounded to required particle size having both coarse and fine particles in 1:1 ratio (w/w). the substrate was taken in a 250ml erlenmeyer flask and moistened with distilled water to maintain the moisture content of 50% (v/w) and autoclaved at 121ºc (15lb) for 1520 min, cooled to room temperature and inoculated with 2 ml of the 5 day spore suspension (spore concentration of about 107-108 per ml) of a. fischeri under aseptic conditions. the contents of the inoculated flasks were mixed thoroughly and incubated at desired temperature in an incubator for desired length of fermentation time. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         28   optimization of process parameters: various process parameters that influence the lovastatin production during ssf were optimized over a wide range. the strategy adopted for standardization of fermentation parameters was to evaluate the effect of an individual parameter and incorporate it at the standard level before standardizing the next parameter. the process parameters optimized in the present study include fermentation time (24-192h), initial moisture content (40-100% v/w), inoculum volume (0.5-4ml), initial ph (4-10), incubation temperature (26-40˚c), carbon sources (1% w/v) and nitrogen sources (1% w/v). all the experiments were done in triplicate and the mean values of the lovastatin yield was reported. lovastatin extraction: after the fermentation time, the fermented flasks were dried at 60°c for 2 hrs. to this 20 ml of methanol was added to the flasks to extract lovastatin by keeping them in an orbital shaker at 180 rpm for 2 h. the residue was filtered with filter paper and then centrifuged at 1500 rpm for 15 min. the supernatant obtained was collected and analyzed for quantitative determination of lovastatin [10]. quantitative analysis of lovastatin: to 1 ml of the obtained supernatant, 1 ml of 1% trifloroacetic acid was added and incubated for 10 min (lactonization of hydroxyl acid form of lovastatin).from the solution, 0.5 ml was taken and diluted to 10100 ml in methanol and its absorbance was read at 238 nm by using uv-visible spectrophotometer [11]. concentration of lovastatin (mg/g) = concentration of lovastatin (mg/ml) x dilution factor (df) amount of substrate taken (g) results and discussion selection of solid substrates in ssf for lovastatin production: solid substrates employed in ssf processes are heterogeneous natural raw-materials which are generally insoluble in water and play a dual role-supply of nutrients to the microbial culture growing and anchorage for the growing cells. in the present study, coconut oil cake was used as the solid substrate for both the fungal sporulation and lovastatin production by aspergillus fischeri. maximum nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         29   lovastatin yield of 6.81 mg/g dry substrate was obtained. effect of fermentation time: the effect of fermentation time on lovastatin production was studied by incubating the fermentation flasks from 24-192 h. the maximum lovastatin yield of 7.43 mg/g of dry substrate was achieved at 168 h of fermentation time (fig.1). the lovastatin yield increased from 24-168 h which explained that lovastatin is a kind of secondary metabolite and its accumulation in mycelia seems growth related and slightly decreased from 168-192h. the lovastatin yield decreased after seven days (168 h) of fermentation which may be due to the onset of death phase of microorganism and nutrient depletion. fig.1: effect of fermentation time on lovastatin production effect of initial moisture content to investigate the influence of initial moisture content of the substrate, fermentation was carried out under various initial moisture content levels (40, 50, 60, 70, 80, 90 and 100(% v/w)) of coconut oil cake, which was adjusted with moistening media. maximum lovastatin yield (8.04 mg/g dry substrate) was achieved at 60% (v/w) initial moisture content ( fig. 2.). as the moisture content in the fermentation medium increases, the air present in the void volume decreases, resulting in poor oxygen availability for the process without forced aeration and with low moisture content, the available oxygen is sufficient but the water content is not enough to support good metabolic activity and dissipation of heat generated and may account for lower lovastatin production. the same 60% (v/w) moisture content was also observed for both aspergillus flavipes and aspergillus terreus under ssf [8, 10]. 0   1   2   3   4   5   6   7   8   24   48   72   96   120   144   168   192   y ie ld o f l ov as ta ti n (m g/ g) fementation time (h) nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  84  –  xx                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   . fig. 2: effect of moisture content on lovastatin production effect of inoculum volume fermentation was carried out with different inoculum volumes (0.5-4ml) of five day old a. fischeri culture for a period of 144h to study its effect on lovastatin production. the maximum yield of lovastation (8.97 mg/g dry substrate) was obtained with 2ml of a.fischeri culture as shown in fig. 3. with the further increase in inoculum volume, the yield of lovastatin had decreased which might be due to the depletion of the available nutrients in the production medium, yielding poor mycelia growth, thus promoting less product formation. with low inoculum volume, the yield is also low due to the insufficient microbial culture to form mycelia and produce lovastatin. 0   1   2   3   4   5   6   7   8   9   40   50   60   70   80   90   100   y ie ld o f l ov as ta ti n (m g/ g) moisture content (% v/w) nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  84  –  xx                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   . fig. 3: effect of inoculum volume on lovastatin production effect of initial ph: experiments were performed to the optimum ph in order to maintain the favorable conditions for increased production of lovastatin. this was established by carrying out the fermentation by varying the ph from 4-10. the profound effect of initial ph of the fermentation on lovastatin production was as shown in fig. 4. maximum lovastatin yield (9.71 mg/g dry substrate) was recorded at ph 5.0. a further increase in ph resulted in gradual decrease of lovastatin production due to the denaturation or inactivation of the microbial strain, because ph strongly influences the transport of various components across the cell membrane which support the cell growth and product formation, and most of the fungi are active in the ph range of 3.5-7 and also lower ph avoids the contamination by other microbes. the result was in accordance with the lovastation production using aspergillus terreus under ssf [8]. 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 3.5 4 y ie ld o f l ov as ta ti n (m g/ g) inoculum volume (ml) nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  84  –  xx                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   . fig. 4: effect of ph on lovastatin production. effect of temperature fermentation was carried out at various temperatures from 26-40°c to study their effect on enzyme production (fig. 5). results indicated that maximum lovastatin production (10.88 mg/g dry substrate) was obtained when ssf was carried out at 30°c. however, lovastatin production reduced gradually above the optimal incubation temperature of 30°c. with further increase in temperature, more heat is accumulated in the medium during mesophilic aerobic ssf, which leads to poor heat dissipation thus reducing the oxygen level and thereby reducing the growth of microorganism, as lovastatin is growth related product. these results are coinciding with those previously reported for lovastatin production by aspergillus terreus and monascus ruber [10,13]. 0   2   4   6   8   10   12   4   5   6   7   8   9   10   y ie ld o f l ov as ta ti n (m g/ g) ph nepal  journal  of  biotechnology.    dec.  2011,  vol.  2,  no.  1:  84  –  xx                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   . fig. 5: effect of temperature on lovastatin production effect of carbon source to determine the effect of incorporation of additional carbon sources on lovastatin yield, different carbon sources namely glucose, maltose, sucrose, xylose, lactose, galactose and soluble starch at 1% (w/v) added to the basal solid state fermentative medium of a. fischeri exerted a considerable effect on the biosynthesis of lovastatin (fig. 6). maximum lovastatin production was promoted by lactose (12.02 mg/g dry substrate) followed by maltose (11.11 mg/g dry substrate) and sucrose (10.47 mg/g dry substrate). the enhanced production of lovastatin by the incorporation of lactose to the medium might be attributed to the positive influence of lactose as a cometabolic agent for enhanced microbial metabolite biosynthesis. fig. 6: effect of different carbon sources on lovastatin production. 0   2   4   6   8   10   12   26   28   30   32   34   36   38   40   y ie ld o f l ov as ta ti n (m g/ g) temperature (˚c) 0   2   4   6   8   10   12   14   y ie ld o f l ov as ta ti n (m g/ g) carbon source nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         34   effect of nitrogen source nitrogen source can be an important limiting factor in the production of microbial metabolites [14]. the supplementation of additional nitrogen sources (either organic or inorganic) such as sodium nitrate, ammonium sulphate, urea, peptone, tryptone, yeast extract, malt extract and beef extract at 1% (w/v) had shown a profound impact on the production of lovastatin by a. fischeri under ssf ( fig. 7). among the various nitrogen sources evaluated, malt extract in the fermentation medium promoted enhanced fungal growth and consequent lovastatin production of 14.77 mg/g dry substrate was achieved followed by yeast extract (12.36 mg/g dry substrate) and sodium nitrate (11.03 mg/g dry substrate). fig. 7: effect of different nitrogen sources on lovastatin production conclusion: the current investigation was mainly focused on the evaluation of the potentiality of aspergillus fischeri for utilization of coconut oil cake as the substrate for the production of lovastatin under solid state fermentation. the yields obtained in the present study have to be further increased for its industrial importance. it has proved the feasibility of solid state fermentation as a promising technique in exploiting cheaply available agro-residual wastes as substrates for the large-scale production of microbial metabolites of biotechnological importance ultimately leading to an effective solid waste management. 0 2 4 6 8 10 12 14 16 y ie ld o f l ov as ta ti n (m g/ g) nitrogen source nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         35   references: 1. alberts aw: lovastatin and simvastatin-inhibitors of hmg coa reductase and cholesterolbiosynthesis: cardiology 1990, 77:1421. 2. alberts aw, chen j, kuron g, hunt v, huff j, hoffman c, rothrock j, lopez m, joshu h, harris e, patchett a, monaghans r, currie s, stapley e, alberts sg, hensens o, hirshfield t, hoogsteent j, liescht j, springeri j: mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme a reductase and a cholesterol-lowering agent. proc. natl. acad. sci. 1980, 77 (suppl. 7): 3957-3961. 3. seenivasan a, subhagar s, aravindan r, viruthagiri t: microbial production and biomedical applications of lovastatin. ind. j. of pharm. sciences 2009, 701-709. 4. jones kd, couldwell wt, hinton dr, su yh, he dk, anker l, law re: lovastatin induces growth inhibition and apoptosis in human malignant glioma cells. biochem. biophys. res .commun. 1994, 205:1681-1687. 5. newman a, clutterbuck rd, powles rl, millar jl: selective inhibition of primary acute myeloid leukemia cell growth by lovastatin. leukemia 1994, 8:2022-2029. 6. casas lopez jl, sanchez perez ja, fernandez sevilla jm, acien fernandez fg, molina grima e, chisti y: production of lovastatin by aspergillus terreus: effects of the c: n ratio and the principal nutrients on growth and metabolite production. enzyme microbiol technol. 2003, 33(suppl. 2-3): 270-277. 7. sitaram kumar m, jana sk, senthil v, shashanka v, vijayakumar s, sadhukhan ak: repeated fed batch process for improving lovastatin production. process biochem. 2000, 36 (suppl. 4): 363-368. 8. valera hr, gomes j, lakshmi s, gurujara r, suyanarayan s, kumar d: lovastatin production by solid state fermentation using aspergillus flavipes. enzyme microbiol technol. 2005, 37 (suppl. 5): 521-526. 9. xu bj, wang qj, jia xq, sung ck: enhanced lovastatin production by solid state fermentation of monascus ruber. biotechnol and bioprocess engg. 2005, 10 (suppl. 1):78-84. 10. pie-lian wei, zhi-nan xu, pei-lin cen: lovastatin production by aspergillus terreus in solid-state fermentation. j. zheijan univ. 2007, 9:1521-1526. 11. mielcarek j, naskreni m, grobelny p: photochemical properties of simvastatin and lovastatin by radiation. j. thermal analysis calorimetry 2009, 96:301-305. 12. pansuriya rc, singhal rs: response surface methodology for optimization of production of lovastatin by solid state fermentation. brazilian j. microbiol. 2010, 41:164-172. 13. panda bp, javed s, ali m: optimization of fermentation parameters for higher lovastatin nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  26  –  36                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved         36   production in red mold rice through co-culture of monascus purpureus and monascus ruber. food bioprocess technol. 2008, 53:342-346. 14. chandrasekaran m, lashmanaperumalsamy p, chandramohan d: combined effect of environmental factors on spoilage bacteria. fish technology (india) 1991, 28: 146-153.             microsoft word bsn rapd rice_joshi_final two column.docx nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   16 original research article genetic  relationship  among  nepalese  rice  landraces  and  cultivars  based  on   rapd  markers bal k. joshi*, hari p. bimb, david kansakar and ekta ghimire biotechnology unit, narc, po box 1136 kathmandu, nepal * corresponding author: email: joshibalak@rediffmail.com abstract genetic information of any genotype is necessary to manage and utilize them in conservation and breeding program. a total of 28 rapd markers were used to relate the genetic structure among 50 nepalese rice genotypes consisting of 29 landraces, 12 breeding lines and 9 released cultivars. some of them are aromatic and blast resistance. only four primers (p41, p60, p109 and p141) amplified the dna of these genotypes with scorable bands. primer 60 produced the highest number of bands (8). the highest number of present bands (6) was shown by primer 41 in 10 rice genotypes. grouping of these genotypes based on the adaptation to agro-climatic zone was not observed, probably due to low percentage coverage of genome by four primers. most of the genotypes grouped in two clusters. kali marsi and ir-24 formed separate individual cluster. mansara and jarneli were the most similar landraces (0.96). churenodhan and pranpyuri were the most closely related with masuli. only one genotype nr-285-18 has fallen in the first quadrant by principal component (pc) analysis and the fourth quadrant was empty. the highest contribution in pc1 was from the second band of primer 41. this rapd information can be used for selecting lines and for blast resistance breeding. key words: genetic distance, rice, rapd introduction nepal is rich in rice genetic resources [1, 2]. knowledge on genetic diversity contributes significantly for the better management and utilization of these resources. diversity analysis with the help of molecular markers provides reliable information which can be utilized for breeding purposes. rapd (randomly amplified polymorphic dna) [3] markers though dominant markers, provides fast, reliable and cost effective determination of genetic diversity in plant varieties, breeding lines and accessions [4-6]. in rapd, a single random primer is added to the template dna and subjected to polymerase chain reaction (pcr). this simple but effective method of revealing polymorphism is cheap and nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   17 universally applicable [7, 8]. the indica and japonica cultivars are classified into separate groups by cluster analysis using rapd [5]. we studied the genetic diversity of rice particularly adapted to mid and high hills using rapd markers to support for effective management and utilization of rice genetic resources. materials and methods a. plant materials and plant dna extraction the rice genotypes analyzed are given in table 1. a total of 50 rice samples consisting of landraces, breeding lines and released cultivars were used. dna was extracted employing the modified ctab method of [9]. b. dna amplification for rapd analysis 28 decamer primers were tested (table 2). amplification was carried out in a 10 µl reaction volumes consisting of 10mm tris-hc1 ph 8.3, 2mm mgc12, 0.2mm dntps, 1mm primer, 0.35 unit of taq dna polymerase and 1 ng of total dna as template. the amplification reaction was carried out in ptc-100 thermocycler (mj research, usa). the first cycle consisted of denaturation of template dna at 93.5oc for 1 min, primer annealing at 36oc for 2 min and primer extension at 72oc for 3 min. in the next 44 cycles, the three steps of first cycle were repeated. in the last cycle it is hold at 72oc for 7 min and then at 4oc for 3 min. pcr products were separated on a 1.8% agarose gel using tae buffer. the gels were run for 2.5-3 hr at 70 v and stained with ethidium bromide. dna fragments were visualized under uv light and photographed using gel doc system. only the four primers amplified the dna of test lines. polymorphisms were scored for the presence or absence of bands on a 1/0 basis and data analyzed using the ntsys-pc software [10]. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   18 table 1. rice landraces, cultivars and breeding lines used in this study. s.n. genotype collection site altitude, m collection year remarks 1 krishnabhog achham 1000 1985 landrace 2 thapachini bajura 1768 1995 landrace 3 tauli bhojpur 1219 1987 landrace 4 tunde dhan dailekh 1400 1995 landrace 5 rato dhan dadeldhura 1585 1995 landrace 6 hansraj dadeldhura 1128 1995 landrace 7 mansara dadeldhura 1128 1995 landrace 8 chureno dhan dang 2120 1985 landrace 9 anpjhutte gorkha 1981 1988 landrace 10 jarneli gulmi 2000 1998 landrace 11 bhuwa dhan humla 1970 1985 landrace 12 jhul dhan humla 1350 1985 landrace 13 pahele kaski 1075 1998 landrace 14 radha-7 kaski 1040 1998 released 15 pakhe lamjung 1920 1988 landrace 16 pranpyuri lamjuing 1996 1988 landrace 17 madise lamjung 1524 1988 landrace 18 kali marsi mugu 2600 1985 landrace 19 ghaiya dhan mugu 2380 1985 landrace 20 dhokro mugu 2350 1985 landrace 21 maine pokhreli mustang 1400 1985 landrace 22 lekali dhan myagdi 1800 1985 landrace 23 hanse sallyan 1200 1992 landrace 24 pale dhan sindupalchok 1500 1985 landrace 25 bageri dhan solukhumbu 1707 1989 landrace 26 jethobor tanahun 1250 1988 landrace 27 pokhara masino tanahun 1250 1988 landrace 28 chananchur udaypur 1829 1989 landrace 29 lalshar udaypur 1829 1989 landrace 30 nr10315-145 abd, khumaltar breeding line 31 nr10286-6 abd, khumaltar breeding line 32 manjushree-2 abd, khumaltar released 33 nr10375-20 abd, khumaltar breeding line 34 khumal-11 abd, khumaltar released 35 nr10353-8 abd, khumaltar breeding line 36 nr285-18 abd, khumaltar breeding line 37 nr10276-15 abd, khumaltar breeding line 38 nr10414-25 abd, khumaltar breeding line 39 nr10414-34 abd, khumaltar breeding line 40 taichung-176 abd, khumaltar released 41 jumli white abd, khumaltar landrace 42 chandan nath-1 abd, khumaltar released 43 chandan nath-3 abd, khumaltar released 44 nr10276-9 abd, khumaltar breeding line 45 nr10285-29 abd, khumaltar breeding line nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   19 s.n. genotype collection site altitude, m collection year remarks 46 sabitri nrrp, hardinath released, br 47 ir-24 nrrp, hardinath released, br 48 a57-115-8 nrrp, hardinath breeding line, bdi (3 gene pyramid) 49 co39 nrrp, hardinath breeding line, bs 50 masuli nrrp, hardinath released, bs note: abd , agriculture botany division. nrrp, national rice research program. b, blast. r, resistant. s, susceptible. di, differential line. table 2. details of rapd primers used in this study. s.n. primer sequence band scored remarks 1 p36 gggggtcgtt 2 p40 ggcggactgt 3 p41 gagtgcgcag 6 rice genome 4 p42 ccggactgag 5 p48 gaaggcgcgt 6 p52 ggcaccacca 7 p60 catcggccct 8 8 p109 tggccactga 3 9 p141 gtgatcgcag 7 operon tech 10 p142 caatcgccgt 11 p144 cagcacccac 12 p165 ctgacgtcac 13 p169 agtcgacgcc 14 p181 acggacgtca 15 p189 tgggtccctc operon tech 16 p191 ctgcgctgga 17 p194 aggcccgatg 18 p197 gaccccggca 19 p198 gcctggttac 20 p202 cgcagacttg tag 91:65-667.’95. lentil 21 p205 gccgtgaagt tag 91:65-667.’95. lentil 22 p209 ggcgtcgggg tag 91:65-667.’95. lentil 23 p217 gggttgccgt tag 85:937-95.’93.vicia faba 24 p222 gtcacccgga tag 85:937-95.’93.vicia faba 25 p225 agtggtcgcg 26 p232 gcgcattaga bio/tec 10:686-690. conifer 27 p270 agccagtttc tag 85:190-196.’92. brassica 28 p292 caaacggcac tag 86:788-794.’95. alfalfa nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   20 results and discussion a. primers and genetic similarity among the 28 rapd primers, only four primers (p41, p60, p109 and p141) amplified the genomic dna of test lines (table 2). the percentage of primers that amplified the dna was very low. these four primers showed polymorphism. we considered only those primers that could amplify the dna of all samples with scorable bands (figure 1, 2). most of the primers did not work probably due to the old or not related to rice genome or poor quality of template dna. polymorphism percentage of the tested rapd primers are 90.0 in the study of [11] and 67 in [12]. in their study, with selected primers, sufficient polymorphism is detected to allow identification of individual varieties. rapd analyses offer the greatest chance of detecting small genetic differences, since a larger component of the genome can be scanned than in other systems [8, 13]. primer 60 produced the highest number of bands (8). the highest number of present bands (6) was shown by primer 41 in 10 rice genotypes. the genetic similarity ranged from 0.00 to 0.96. mansara and jarneli were the most similar landraces (0.96). the second most similar landraces were tunde dhan and krishnabhog. ir-24 showed the zero similarity coefficients with all genotypes. the zero similarity coefficient of kali marshi with thapachini, krishnabhog and tauli indicates the most genetic dissimilarity. the similarity index between chandannath-1 and lalshar was also zero. a57-115-8 showed the zero similarity index with chandannath-3. two blast susceptible varieties, mansuli and co-39 have mostly the similar coefficients with all tested genotypes. fig.1. rapd polymorphism of different rice genotypes with primer 141 m            1            2          3          4          5            6          7          8          9        10      11      12        13      14        15      16        17   a b nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   21 (m, marker; sample a: 1, kali marshi; 2, ghaiya dhan; 3, dhokro dhan; 4, maine pokhreli; 5, lekali dhan; 6, hanse; 7, pale dhan; 8, bageri dhan; 9, jethobor; 10, pokhara masino; 11, chananchur; 12, lalshar; 13, nr10315-145-2-3; 14, nr10286-6-3-2-2; 15, manjushree-2 ; 16, nr10375-20-1-2; 17, khumal 11. sample b: 1, nr10353-8-2-1; 2, nr28518-3-2-3-1; 3, nr10276-15-2-3-3-2; 4, nr10414-25-2; 5, nr1041434-2-3; 6, taichung-176; 7, jumli white; 8, chandhannath-1; 9, chandhannath-3; 10, nr10276-9-3-3-3-2; 11, nr10285-29-3-1; 12, sabitri; 13, ir-24; 14, a57-115-8; 15, co 39; 16, masuli;17, check3 from jumla, 2 from humla and 3 mugu). fig.2. rapd polymorphism of different rice genotypes with primer 41 (m, marker; sample a: 1, *krishnabhog; 2, *thapachini; 3,tauli; 4,tunde dhan; 5,rato dhan; 6, *hansraj; 7, mansara; 8,chureno dhan ; 9, anpjhutte; 10,jarneli ; 11, bhuwa dhan; 12, jhuldhan; 13, *pahele; 14,radha-7; 15,pakhe ; 16, pranpyuri; 17, madise. sample b: 1, kali marshi; 2, ghaiya dhan; 3, dhokro dhan; 4, maine pokhreli; 5, lekali dhan; 6, hanse; 7, pale dhan; 8, bageri dhan; 9, *jethobor; 10, *pokhara masino; 11, chananchur; 12, lalshar; 13, nr10315-145-2-3; 14, nr10286-6-3-2-2; 15, manjushree-2 ; 16, nr10375-20-1-2; 17, khumal 11. sample c:1, nr10353-8-2-1; 2, nr28518-3-2-3-1; 3, nr10276-15-2-3-3-2; 4, nr10414-25-2; 5, nr10414-34-2-3; 6, taichung176; 7, jumli white; 8, chandhannath-1; 9, chandhannath-3; 10, nr10276-9-3-3-3-2; 11, nr10285-29-3-1; 12, sabitri; 13, ir-24; 14, a57-1158; 15, co 39; 16, masuli;17, check3 from jumla, 2 from humla and 3 mugu). * aromatic rice. b. cluster analysis the dendrogram generated by the rapd analysis showed four distinct groups (figure 3). ir-24 and kali marsi formed the separate individual cluster. most of the genotypes fell in two clusters. grouping of these genotypes based on the adaptation to agro-climatic zone was not observed, probably due to low percentage coverage of genome by four primers. mansara and jarneli were the most similar landraces followed by nr-10276-9 and nr-10285-20. churenodhan and pranpyuri were the most m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 a b c nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   22 closely related with masuli. the three blast resistance genes pyramided rice genotype, a57-115-8 was genetically near with anpjutte, tauli and thapachini. fig.3. clustering of 50 rice genotypes based on rapd markers. c. principal component analysis a scatter plot was drawn based on the similarity coefficients among the 50 rice genotypes (figure 4). all genotypes except nr-285-18 fell in the second and third quadrant. only one genotype nr-285-18 has fallen in the first quadrant by principal component analysis and the fourth quadrant was empty. the highest contribution in pc1 was from the second band of primer 41 (table 3). considerable overlapping among the various samples is evident, which suggests that genetic variation among them is rather narrow. nevertheless, some rice samples appeared separate from the overlapping ones e.g. aromatic rice like pahele, jethobor, maine pokhreli, pokhara masino, and hansraj. the level of coefficient 0.00 0.24 0.48 0.72 0.96 krishnabhog tundedhan jumliwhite pokharamasino jhuldhan chandannath-1 nr10276-15 sabitri hansraj mansara jarneli bhuwadhan nr10315-145 pahele nr10414-25 taichung-176 ratodhan nr10276-9 nr10285-29 chandannath-3 radha-7 madise pakhe ghaiyadhan mainepokhreli paledhan dhokrodhan hanse chananchur lekalidhan jethobor bageridhan manjshree-2 nr10353-8 nr10414-34 thapachini tauli anpjhutte a57-115-8 churenodhan pranpyuri masuli lalshar nr10286-6 nr10375-20 khumal-11 nr285-18 co39 kalimarsi ir-24 nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   23 distinctness versus overlapping was in good concordance with that of the cluster. this preliminary genetic information could supplement for breeding and conservation works based on morphological markers. for increasing the value of genetic information derived from rapd markers, number of primers should be increased. choudhury et al. [14] suggest that a set of 10 primers can be employed for an initial assessment of genetic diversity in a large number of collections. because of multilocus nature of rapd, its use is considered more suitable for fingerprinting and genetic diversity measurement. table 3. eigen vectors of rapd primers based on 50 rice genotypes. primer band pc1 pc2 pc3 p41 1 -0.363 0.203 -0.123 2 -0.374 0.217 0.019 3 -0.300 0.370 0.046 4 -0.371 0.247 -0.012 5 -0.299 0.240 0.078 6 -0.148 0.174 0.536 p60 1 -0.225 -0.284 -0.022 2 -0.252 -0.357 -0.071 3 -0.167 -0.200 -0.327 4 -0.096 -0.053 -0.172 5 -0.097 -0.153 0.029 6 0.197 0.268 -0.034 7 0.124 0.071 0.083 8 0.065 0.063 -0.037 p141 1 0.085 0.124 -0.060 2 -0.157 -0.257 0.472 3 -0.112 -0.321 0.372 4 -0.083 -0.143 0.198 5 0.068 0.016 0.048 6 -0.045 -0.090 0.059 7 -0.009 0.003 0.032 p109 1 -0.202 -0.097 -0.221 2 -0.199 -0.135 -0.230 3 -0.180 -0.175 -0.157 eigenvalue 1.062 0.629 0.396 proportion 0.243 0.144 0.090 cumulative 0.243 0.386 0.477 nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   24 fig.4. scatter plotting of 50 rice genotypes based on four rapd markers. 0-1-2-3-4 2 1 0 -1 -2 pc i p c ii masu lico 3 9a 5 7 -1 1 5 -8 ir-2 4 sab itri n r1 0 2 8 5 -2 9 n r1 0 2 7 6 -9 ch an d an n ath -3 ch an d an n ath -1ju mliwh ite taich u n g -1 7 6 n r1 0 4 1 4 -3 4 n r1 0 4 1 4 -2 5 n r1 0 2 7 6 -1 5 n r2 8 5 -1 8 n r1 0 3 5 3 -8 k h u mal1 1 n r1 0 3 7 5 -2 0 man ju sh ree-2 n r1 0 2 8 6 -6 n r1 0 3 1 5 -1 4 5 lalsh ar ch an an ch u r po k h aramasin o jeth o b o r bag erid h an paled h an h an se lek alid h an main epo k h reli d h o k ro d h an g h aiy ad h an k alimarsi mad ise pran p y u ripak h e rad h a-7 pah ele jh u ld h an bh u w ad h an jarn eli a n p jh u tte ch u ren o d h an man sara h an sraj rato d h an tu n d ed h an tau li th ap ach in i k rish n ab h o g nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  16  –  25                                                                                                                      biotechnology  society  of  nepal  (bsn),  all  rights  reserved   25 references 1. joshi bk: rice gene pool for mid and high hills and its conservation in nepal. in agricultural research for enhancing livelihood of nepalese people. proceedings of 2nd sas-n convention, 30 july 1 aug 2003; kathmandu. edited by joshi bk, joshi sl, paudyal kp2004:252-264. 2. joshi bk: rice gene pool for tarai and inner tarai areas of nepal. nepal agric. res. j. 2005, 6:10-23. 3. williams jgk, kubelik ar, livak kj, rafalski ja, tingey sv: dna polymorophisms amplified by arbitrary primers are useful as genetic markers. nucleic acids res. 1990, 18:6531-6535. 4. fuentes jl, escobar f, alvarez a, et al. analysis of genetic diversity in cuban rice varieties using isozyme, rapd and aflp markers. euphytica 1999, 109:107-115. 5. mackill dj: classifying japonica rice cultivars with rapd markers. crop sci. 1995, 35:889-894. 6. virk ps, newbury hj, jackson mt, pordlloyd bv: the identification of duplicate accessions within a rice germplasm collection using rapd analysis. theor. appl. genet. 1995, 90:109-155. 7. karp a, kresovich s, bhat kv, ayad w, hodgkin t: molecular tools in plant genetic resources conservation: a guide to the technologies. ipgri technical bulletin no. 2. ipgri, rome; 1997. 8. ren f, lu br, li s, hunag j, zhu y: a comparative study of genetic relationships among the aa-genome oryza species using rapd and ssr markers. . theor. appl. genet. 2003, 108:113-120. 9. sul iw, korban ss: a highly efficient method for isolating genomic dna from plant tissues. plant tissue culture biotechnol. 1996, 2:113-116. 10. rohlf fj: ntsys-pc. numerical taxonomy and multivariate analysis system. version 1.8. new york: exerter software; 1993. 11. ravi m, geethanjali s, sameeyafarheen f, maheswaran m: molecular marker based genetic diversity analysis in rice (oryza sativa l.) using rapd and ssr markers. euphytica 2003, 133:243-252. 12. ko hl, cowan dc, henry rj, et al. random amplified polymorphic dna analysis of australian rice (oryza sativa l.) varieties. euphytica 1994, 80:179-189. 13. morell mk, peakall r, appels r, preston lr, lloyd hl: dna profiling techniques for plant variety identification. . aust. j. exp. agric. 1995, 35:807-819. 14. choudhury pr, kohli s, srinivasan k, mohapatra t, sharma rp: identification and classification of aromatic rice based on dna fingerprinting. euphytica 2001, 118:243-251. 7 nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 55‐58  55  biotechnology society of nepal (bsn), all rights reserved   brief communication   biotechnology growth partnership: a potential  flagship program in s&t  raju adhikari and benu adhikari  nrn skill, knowledge and innovation (ski) exchange task force, non resident nepali association (nrna), www.nrn.org.np  correspondence author:   raju adhikari  e‐mail: r_adhikari@hotmail.com  background  nepal  is a  land  locked country with very few mineral  resources.  however,  she  is  vastly  rich  in  her  diverse,  native, high altitude flora and fauna from the tropical  to subalpine region; well known for their high value in  medicinal  application.  these  natural  resources  have  not  been  exploited  to  their  full  potential  due  to  the  lack  of  necessary  expertise  and  required  technical  facilities  needed  for  their  full  scientific  investigation.  nepal  is  primarily  an  agricultural  country  and  the  agriculture  contributes  to  approximately  38%  of  nepal’s  31  b$  gdp.    in  the  past  decade,  there  were  many  initiatives  by  government  research  agencies,  private sectors and foreign agencies such as usaid and  rockefeller foundation to promote biotechnology and  natural product research activities in nepal. however,  due  to  lack  of  policies  and  collaborations  at  an  institutional level, most of the r&d work had remained  focused  only  in  the  tissue  culturing  of  few  plants  (medicinal/horticulture/ornamental)  and  fruits/ vegetables so far.   nepal academy of science and technology (nast) and  the ministry of s&t have strongly emphasized the need  to promote innovation and collaboration in the above  stated priority areas whilst working in partnership with  overseas  countries.  the  nepal  government  has  announced a new national plan for the biotechnology  research by focusing on altitude medicinal plants and  setting  up  a  national  biotechnology  research  and  development  centre  to  speed  up  research  and  encourage  private  sector  participation.  the  plan  recommends  biotechnology  courses  and  scholarships  in nepal’s universities and provides financial incentives  in tissue culture and genetic technologies to select and  breed  improved  crop  varieties,  usage  of  microbial  cultures  to  manage  industrial  waste,  biosensors  for  monitoring  soil  and  air  pollutants,  and  scientific  assessment of the country's bio diverse floras for use in  pharmaceutical and cosmetic industries.  biotechnology  research  globally  and  especially  in  countries  like  usa,  uk,  european  union,  australia,   china  and  india  have  a  proven  track  record  of  commercial  success.    australia  has  signed  an  international  biotechnology  growth  partnership  initiative with a few countries and such collaborations  are expected to induce a great benefit to the economy  of both countries through sharing of ideas, innovation  and  resources.  nepal  academy  of  science  and  technology  (nast)  and  nepal  agriculture  research  council (narc) have shown interest in developing such  partnerships with australia and overseas organizations  to help nepal biotechnology industry in agriculture and  health sectors. recently, nrna and nast have signed  a memorandum of understanding (mou) in s&t field to  work  jointly  in  national  priority  areas  such  as  biotechnology and environment. this will allow nast,  nrna and other scientific institutions in nepal to work  jointly on collaborative projects and submit proposals  raju  adhikari  is  currently  working  as  a  principal  research  scientist  at  csiro  material  science  and  engineering, vic, australia and is the chair of ski task  force. dr benu adhikari,  is a member ski task force  and  a  senior  lecturer  at  university  of  ballarat,  vic  australia  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 55‐58  56  biotechnology society of nepal (bsn), all rights reserved   to  interested  overseas  biotechnology  companies  and  research organizations for funding.  potential project area  nepal’s high altitude herbal medicinal plants are native  and unique to geo‐climatic regions and have potential  niches  for  the  global  market.  nepal  currently  trades  about  36  different  types  of  products  derived  from  herbal plants and the total revenue earned is reported  to  be  approximately  12  million  nrs  (us  $240,000).  around 98% raw products are exported to dabur india,  a  leader  in  manufacturing/marketing  herbal,  nature‐ based products  in asia. due to the  lack of any patent  protection alongside the governments weak policy on  farming  and  trade  of  medicinal  plants,  the  revenue  source from these herbal medicinal plants are mostly  from  the  selling  of  raw  products.  in  nepal  ayurvedic  industries  are  also  selling  mostly  the  raw  materials  without  any  value  added  research  input  and  thus  contributing very little to the country’s economy (ntfp  trade bulletin, 2005)  biotechnology growth partnership could be initiated as  a  long  term  collaborative  project  in  the  above  mentioned  areas.  the  project  could  aim  to  commercially exploit high altitude flora such as yarsha  gumba (cordyceps sinensis) (figure 1) by carrying out  research tailored to value add by conducting rapid high  throughput bioassay screenings against certain specific  target  diseases  and  developing  appropriate  formulations  for  its  use  as  a  common  medicines  and  health  products  [1‐9].  a  medicinal  herb  of  illustrious  history  known  as  himalayan  viagra  has  been  well  documented  in  the  literature  for  its  use  as  a  drug  to  boost  immune  system,  anti‐ageing  and  aphrodisiac  properties. only 3 species of the caterpillar fungus are  reportedly found in nepal at a reserve ~ 4,900 m with  50%  collection  coming  from  dolpa  region  alone.  the  raw materials collected in this manner are exported to  tibet,  china  and  other  countries.  government  had  legalized  the  trade  of  raw  material  in  2001  and  imposed  a  royalty  of  $280/kg  whereas  the  global  market price currently stands between us$ 3, 000/kg  for  the  lowest  quality  to  over  us$18,  000/kg  for  the  highest  quality  larvae.  the  project  will  also  aim  to  establish  a  national  germplasm  bank  of  flora  of  high  medicinal values to protect their genetic diversity.   similarly  in  food  biotechnology,  yak  cheese  could  be  used as a valuable resource due to  its  low fat (26%),  high  protein  (26%  )  and  non  conjugated  linoleic  acid  (clas) content compared to cow cheddar cheese (see  table  1).  the  above  fatty  acid  is  reported  to  posses  anticancer  and  anti  type  t2  diabetes  properties  [10‐ 15).  a  project  proposal  to  address  its  large  scale  production,  assess  scientific  effectiveness  and  quality  control could help diary food industries and bring huge  economic benefit to the farming community.   furthermore,  in  a  country  like  nepal,  where  large  quantity  of  fruits  are  wasted  due  to  lack  of  infrastructure  (processing,  storage,  transportation),  a  project focused to converting these fruits into powders  without  compromising  their  nutritional  value  could  help food industry in a big innovative way. a research  team  led  by  a  nrn  stf  team  member  dr.  benu  adhikari (university of ballarat, victoria, australia) has  developed  some  breakthrough  technologies  in  this  area.   there  are  many  other  potential  areas  in  agriculture  where such collaboration could be  initiated. ski takes  this  opportunity  to  invite  our  nrn  professionals  who  are working in the biotechnology field to come forward  and  contribute  in  this  new  venture  initiative  of  s&t  stream.  path to market  the collaboration initially could focus on high altitude  native  medicinal  plants  in  order  to  develop  common  drugs  and  cosmetic  products  for  pharmaceutical  industries. these types of products serve an advantage  as they do not require stringent regulatory criteria and  are an easy path to the market. the products based on  developed formulation from the above project can be  manufactured  and  marketed  as  a  common  drug  and  can be sold under the patent/trade rights to guarantee  its claim. nepal’s commitment will be to provide raw  materials  and  manpower  and  conduct  preliminary  screening  research  while  advanced  testing  and  formulation  matrix  could  be  carried  out  in  australia.  the clinical trials and manufacturing of products could  be  carried  out  in  nepal  which  will  help  develop  necessary  infrastructure  for  the  production  of  such  value added products and clinical trial facilities  in the  long  run  on  low  cost.    the  patent  royalties  will  be  shared and 10% of profit from the sale of such product  can be reinvested in r&d to develop further advanced  technologies  or  to  extend  research  in other  potential  areas. the  life of the above flagship project could be  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 55‐58  57  biotechnology society of nepal (bsn), all rights reserved   approximately 3‐5 years to allow patent protection and  development work.  approach  nrn ski team  is working closely with nepal academy  of science and technology in nepal to develop a joint  collaborative  project  proposal  to  explore  fund  from  potential investors or overseas s&t organisations. the  s&t organisations in nepal will provide commitment to  raw  materials,  manpower  and  facilities  to  conduct  preliminary  research  work  and  the  advanced  testing  and  formulation  development  work  could  be  carried  out  overseas.  final  formulation  trials  and  manufacturing  of  such  value  added  products  will  be  carried out in nepal (cheap resources) to help develop  research  infrastructure.  the  patent  will  be  shared  depending  upon  the  nature  of  the  agreement  and  nepal  will  get  the  dollar  value  from  the  royalty  obtained  from  the  sale  of  such  products.    nrna  will  lobby nepal government to provide tax incentives and  other  flexibility  for  establishment  of  any  spin‐off  company  arising  from  such  an  investment.  a  general  scheme of research approach is given in figure 2.   figure 1. (a) dolpa community collecting yarsha  gumba,   figure 1. (b) a raw specimen of yarsha gumba   fatty acid  yak cheese  cow‐cheddar cheese  cis‐9, trans‐11 cla  2.01±0.07  0.48±0.07  trans‐9, cis‐11 cla  0.13±0.01  0.03±0.01  trans‐10, cis‐12 cla  0.038±0.001  0.009±0.001  trans‐11, trans‐13 cla  0.039±0.001  0.018±0.001  trans‐9, trans‐11 cla + trans‐10, trans‐12 cla  0.057±0.003  0.031±0.003  table 1. a comparison of cheese composition of yak and cow’s milk  figure 2. a general  scheme of approach in  flagship projects  5 yrs  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 55‐58  58  biotechnology society of nepal (bsn), all rights reserved   expected outcome  the  above  conceived  research  programs  will  bring  innovation  and  expertise  to  nepal  and  help  in  conducting  applied  research  activities  to  asses’  the  scientific  effectiveness  of  the  above  mentioned  resources  and  value  add  the  product  for  their  commercial  exploitation.    the  above  mentioned  research  streams  will  generate  new  patents  and  publications.  the  revenue  will  be  generated  by  licensing the technology or through spin off companies  with  the  royalties  that  can  be  shared  mutually.  such  collaborative  research  programs  will  build  strong,  longer term partnership between nepal and overseas  institutions  in  the  field  s&t  and  extend  the  collaborations  further  in  other  areas  of  commercial  potentiality. nepal as well as the collaborating partner  country stands to win from these ventures.   the s&t  stream will also explore and help conduct a feasibility  study to establish a national biotechnology centre of  excellence, lobby with the government to formulate a  biotechnology  policy  and  help  establish  a  separate  biotechnology department under ministry of s&t.  risk factor  the  current  political  instability,  lack  of  collaborative  agreements  and  the  absence  of  commercial  focus,  poor  governance  and  funding  of  s&t  projects,  long  term  commitment,  poor  private  sector  participation  and  the  absence  of  clear  s&t  plan  are  some  of  the  factors  that  may  undermine  confidence  of  stake‐ holders.  because  of  these  reasons  the  government  needs  to  provide  better  initiatives  and  assurance  to  protect such investment by brining a special legislation  in s&t investment. the s&t organisations in nepal are  interested  to  enter  into  such  a  partnership  program  and  by  signing  mou  with  nast;  nrna  has  shown  its  commitment  to  provide  a  helping  hand  and  work  in  partnership to achieve the above goals.   references  adhikari,  m.  k.,  2008.  the  medicinal  fungi  from  nepal.  pp.  107‐118. in: jha, p. k., karmacharya, s. b., chhetri, m. k.,  thapa,  c.  b.,  shrestha,  b.  b.,  eds,    medicinal  plants  in  nepal: an anthology of contemporary research, ecological  society, kathmandu, nepal.  bok, j. w. lermer, l., chilton, j., 1999. antitumor sterols from  the mycelia of cordyceps sinensis. phytochemistry, 51, 891‐ 898.  chen,  y.  q.,  hu,  b.,  xu,  f.,  zhang,  w.,  zhou,  h.,  qu,  h.  l.,  2004.  genetic variation of cordyceps sinensis, a fruitbody  producing  entomopathogenic  species  from  different  geographical  region  in  china.  fems  microbioliological  letttter, 230, 153‐158.  dong, c.‐h., yao, y.‐j.2008. in vitro evaluation of antioxidant  activities  of  aqueous  extracts  from  natural  and  cultured  mycelia of cordyceps sinensis. lwt, 41, 669‐677.  koh,  j.  h.,  kim,  j.  m.,  chang,  u.  j.,    sub,  h.  j.,  2003.  hypocholesterolemic  effect  of  hot  water  extract  from  mycelia of cordyceps sinensis. biol. pharm. bull. 26, 84‐87.  koh, j. h., yu, k.w., shu, h. j., choi, y. m., ahn, t.s., 2002.  activation  of  macrophages  and  the  intestinal  immune  system by an orally administrated decoction from culture  mycelia  of  cordyceps  sinensis.  bioscience,  biotechnology  and biochemistry, 66 (2), 407‐411.  li,  s.  p.,  li,  p.,  dong,  t.  t.  x.,  tsim,  k.  w.  k.,  2001.  anti‐ oxidation  activity  of  different  types  of  natural  cordyceps  sinensis and cultured cordyceps mycelia. phytomedicine, 8,  207‐212.  shrestha,  b.,  lee,  w.‐h.,  han,  s.‐k.,  sung,  j.‐m.,  2006.  observations  on  some  of  the  mycelia  growth  and  pigmentation characteristics of cordyceps militaris isolates.  mycobiology, 34, 83‐91.  zhu,  j.  s.,  halpern,  g.  m.,  jones,  k.,  1998.  the  scientific  rediscovery  of  an  ancient  chinese  herbal  medicine:  cordyceps  sinensis:  part  i.  j.  alt.  comp.  med.  4  (3),  289‐ 303.  fatty  acid  composition  of  yak  (bos  grunniens)  cheese  including  conjugated  linoleic  acid  and  trans‐18:1  fatty  acids; mamun m. or‐rashid, nicholas e. odongo, bhishma  subedi, pralhad karki and brian w. mcbride, department of  animal and poultry science, university of guelph, guelph,  ontario, canada n1g 2w1; asia,  agric. food chem., 2008,  56 (5), pp 1654–1660.  the big cheese "nepal is perfect for cheese production. you  couldn't ask for more, nepali times, " from  issue #427  (28 nov 2008 ‐ 04 dec 2008).   high  hill  yak  cheese  production  in  nepal:  an  analysis  of  privatization  policy  incorporating  the  impacts  of  market  failures  or  agro‐industries  in  developing  countries,  by  luke  a.  colavito,  virginia  polytechnic  institute  and  state  university, july 8, 1997.  yak cheese: exotic, but healthy? cheese from grass‐fed yaks  may  have  better  fatty  acid  profile  than  cheese  from  grain‐fed  cows,  study  shows, by  miranda  hitti,  webmd  health news, webmed, march 14,2008.  yak cheese is a miracle; http://news.softpedia.com/news/ yak‐cheese‐is‐better‐for‐the‐heart81087.shtml   microsoft word paper 5 patel.docx nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   37 original research article isolation  and  characterization  of  bacterial  endophytes  from  lycopersicon  esculentum   plant  and  their  plant  growth  promoting  characteristics     hardik a. patel1, rajesh k. patel1*, sunil m. khristi1, kruti parikh1, geetha rajendran2 1 ashok & rita patel institute of integrated study and research in biotechnology and allied sciences (aribas), new vallabh vidyanagar, gujarat-388121, india 2 sultan qaboos university, muscat, department of biology, sultanate of oman corresponding author: rkpatel46@yahoo.com abstract the study was designed to isolate and characterize bacterial endophytes from root and stem of lycopersicon esculentum plant which was collected form different region of gujarat. total 18 isolates of endophytic bacteria were selected in which, all the endophytic bacteria produced one or the other different characteristics involved in plant growth promotion. they either produced phytohormones like indole acetic acid, siderophore, protease, pectinase, organic acid showed antifungal activity, chromium tolerance and solubilized phosphate. four of the strains among the 18 showed maximum positive results of plant growth promoting regulators (pgpr) test and among them best probable isolate was selected and thus its 16srdna was amplified and sequenced. only hr7 endophyte of tomato turned out to be pseudomonas aeruginosa. it’s a gram negative coccobacili, sporeforming motile bacilli and show maximum pgpr activity. the results of our present studies indicated that above strains might be endophytic and therefore, were associated with the plant growth. keywords: lycopersicon esculentum, endophytic bacteria, pgpr, iaa, 16srdna introduction there are many endophytic and epiphytic bacteria are directly or indirectly involved in plant growth and development. endophytic bacteria live in plant tissues without causing substantive harm to the host or gaining any benefit other than a noncompetitive environment inside the host. it has recently been demonstrated that bacterial endophytes may also have beneficial effects on host plants, such as growth promotion and biological control of pathogens [10, 25, 28]. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   38 some studies have indicated that the plant growth-promoting potential of endophytes is higher than that of rhizosphere microbes [23,31], but the role of bacterial endophytes in plant growth are not yet fully understood. most of these microorganisms are not pathogenic to the host plant. moreover, the association between the plant and its endophytes is very often mutualistic. in 1926, endophytic growth was recognized as a particular stage in the life of bacteria, described as an advanced stage if infection and as having a close relationship with mutualistic symbiosis [22]. since then, endophytes have been defined as microorganisms that could be isolated form surface-sterilized plant organ [15]. although the presence of bacterial endophytes in plants is variable and, occasionally transient [32], they are also often capable of eliciting drastic physiological changes that modulate the growth and development in the plant [8]. the utilization of endophytic and epiphytic bacteria in agriculture production depends on our knowledge of the bacteria-plant interaction and our ability to maintain, manipulate and modify beneficial bacteria population under field condition [14]. many pgprs are known to promote plant growth by a variety of mechanisms: fixation of atmospheric nitrogen that is transferred to the plant, production of siderophores that chelate iron and make it available to the plant root, solubilization of minerals such as phosphorus, and synthesis of phytohormones [12]. pgpr have been reported to directly enhance plant growth by the production of plant growth regulators, and improvements in plant nutrient uptake [12,16] or indirectly by the production of metabolites like antibiotics, siderophores etc that decrease the growth of phytopathogens [12]. pgprs can be of two different types when associated with host tissue that is endophytes or epiphytes; otherwise they can be even rhizospheric bacteria that are present in the root adhering soil. the aim of the present study was to isolate and characterize the endophytic bacteria associated to root and stem part of lycopersicon esculentum, to evaluate different characteristics involved in plant growth promotion. result revealed that four of the strains showed maximum positive results of pgpr test and its 16srdna was amplified and sequenced. materials and methods isolation of endophytic bacteria form lycopersicon esculentum endophytes strains were isolated from root and stem of lycopersicon esculentum plant (table 1). roots and stem part of plant were thoroughly nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   39 washed with sterile n-saline (0.85%) and cut down in 1 cm long pieces through sterile forceps with the help of alcohol. the pieces were transferred to sterile n-agar plate and incubated for 24 hrs at 300c. morphological and physiological characterization of endophytic isolates for the present study, total 18 endophytic bacteria were isolated whose systemic morphological characters done which includes: size, shape, margin, elevation, consistency, opacity, pigmentation, was done by systematic microbiology [3]. gram’s staining bacterial suspension was prepared in sterile distilled water and from this suspension a smear was prepared on clean & dry glass slide, air dried and then heat fixed. the smear was treated with 1% crystal violet for 1-2 min. gram’s iodine was applied for 30 sec. to 1 min. smears were then decolorized with 10% alcohol. the counter stain, saffranin was then applied for 45-60 seconds. the stained slide were washed with tap water, air dried & observed under oil immulsion. for motility test, the culture was inoculated into the edward’s and ewing motility agar stab medium by stabbing the medium right into the center of agar. the entire depth of the medium was punctured. the medium was incubated at 28 ± 2ºc for 24 hrs. after incubation, it was observed for the turbid growth across the line of inoculation, which indicates motile organisms. for antibiotic assay, top agar (1.5%) was prepared and autoclaved. it was cooled to 45º c, 100µl of culture was added to this and overlaid preset n-agar plates. using sterile forceps, disc containing the antibiotic of interest was placed on the agar and incubated at 28±2ºc for 48 hrs. estimation of plant growth promoting properties (i) detection of siderophore production: this was performed by a method described by schwyn and neilands [27], which involved the use of chrome azurol s containing indicator plates. siderophore detection was performed by mixing equal volumes of chrome azurol s (cas) assay solution and the culture supernatant. colour change from blue to yellowish orange was indicative of presence of siderophore. two percentage of overnight grown culture was inoculated in magnetotactic bacterium magnetospirillum magneticum amb-1 (amb) and grown for 48 hrs. then the culture was centrifuge at 8000 rpm for 20 min and the supernatant was examined for the presence of siderophore by cas solution. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   40 (ii) evaluation of endophytes for chromium tolerance: isolated strains were tested for resistance to cr (vi) by plate dilution method using yeast extract mannitol agar (yema) medium. in a plate dilution method, agar plates amended with k2cr2o7 at 50-500 µg/ml were inoculated with 48 hrs grown cultures and incubated at 28 ± 2ºc for 72hrs. the lowest concentration of cr (vi) inhibiting on yema plates was defined as minimum inhibitory concentration [35]. (iii) phosphate solubilization ability: the phosphate solubilizing ability of the cultures were examined by growing the cultures on pikovskaya’s agar plate and looking for the zone of clearance after incubating at 28 ± 2°c for 4872h. (iv) antifungal activity: the spores of fungal cultures (fusarium oxysporium, alternaria,, trichoderma and rhizoctonia solani) grown on potato dextrose agar (pda) blocks were placed in the centre of pda plates and the bacterial cultures were streaked at four ends of the plate. this was incubated at 28 ± 2°c for 48-96 hrs and examined for zone of growth inhibition. (v) protease production: it is indicated by casein degradation, which was determined by observing clearing zones in nutrient casein agar plate. all isolated culture was streak on nutrient casein agar plate and incubated at 28 ± 2°c for 24-48 hrs. next day flood the plate with frazier’s reagent to detect clear zone around the colony. (vi) indole 3-acetic acid (iaa) production test: iaa in presence of fecl3 develops pink color. this fact is utilized in determination of iaa. different mineral acids like hydrochloric acid, perchloric acid, phosphoric acid, nitric acid and sulphuric acid can be used to develop the color. fecl3 –hclo4 reagent is the most sensitive and shows least interference by other iodole compounds like, tryptophan, skatole, acetyletryptamine etc. loopful of each culture was inoculated in luria broth (lb) 2ml containing 50µg/ml tryptophan and incubated at 28oc for 24 hrs on shaking condition, centrifuged at 9000 rpm for 15min, 2ml of supernatant was taken in fresh tube and 2-3 drops of orthophosphoric acid was added. a quantity of 4ml of reagent (1ml of 0.5 m fecl3 in 50 ml of 35% hclo4) was added to this aliquot and incubated for 25 min at rt. absorbance was measured at 530 nm. auxin quantification values were recorded by preparing standard calibration curve made by using iaa standard in the range of 10-100 µg/ml. iaa stock solution was prepared as 100 µg/ml in 50% ethanol. standard graph of iaa concentration was plotted against o.d 530 and nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   41 the concentration of iaa in samples used was calculated. organic acid production: it was studied by growing the cultures in calcium carbonate agar plate and observing for a clear zone around the colony. chitinase production: it was observed by spotting the culture on chitin agar plate and observing zone of clearance after incubating at 28 ± 2°c for 4872h. chitinase activity (degradation of β1,4nacetylglucosamine polumer) were tested in a minimal medium. there were clear zones were detected after 5 days incubation period at 30°c. pectinase production: it was detected by spotting the culture on pectin agar plate and observing zone of clearance after incubating at 28 ± 2°c for 4872h. 16s-rdna sequencing of pgpr isolates well isolated colonies (2-3 colonies) of the culture whose 16s-rdna had to be amplified were suspended in 20µl-30µl of sterile distilled water. the suspension was heated at 95ºc for 20 min and centrifuged at 9000 rpm for 1min. the supernatant was used as template dna in the pcr system [26]. the 16s-rdna gene fragment was amplified using universal eubacterial fulllength primers. the amount of dna taken for amplification was 10ng. primer sequences forward primer (pf) 5’ aga gtt tga tcc tgg ctc ag 3’ reverse primer (pr 5’ acg gct acc ttg tta cga ctt 3’ the pcr components and conditions (to set a system of 30 µl) used for amplification. amplifications were performed in eppendroff gradient thermal cyclers programmed for 30cycles. the pcr thermal cycle consist of an initial denaturation step of 3 min at 94°c, then 30 sec at 94°c for denaturation, 30 sec at 57°c for primer annealing and in last step primer extension done by 2 min at 72°c. steps 2, 3, 4 repeated for 30 cycles followed by a final extension of 10 min at 72°c. the amplified products were then examined by an aliquot of the dna (2µl) was analyzed on a 1.0 % agarose gel along with 500bp ladder and stained with ethidium bromide (0.5µg/ml). the gels were visualized under uv light in a transilluminator and photographed subsequently. sequence analysis: the product was sequenced and matched with the already available sequences in the gene bank by uploading the obtained sequence in its fasta format in nucleotide sequence match available at the online tool of rdp database project ii. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   42 results and discussion isolation of endophytic bacteria we have isolated endophytic bacteria from the lycopersicon esculentum (tomato) plants from different field areas on the nutrient agar (na) medium. colonies showing different morphological characteristics on the nutrient agar plates were selected for further characterization. about 18 strains were isolated. the number of isolates, the source of their plant and field from where the samples were procured are mentioned in the table 1. table1: bacterial endophytes isolates form lycopersicon esculentum sample location no. of isolate s name of the isolates 1 aau (anand) 5 hr 1 hr 2 hr 3 hr 4 hr 5 2 mansa (gandhina gar) 7 hr 6 hr 7 hr 8 hr 9 hr 10 hr 11 hr 12 3 gana (anand) 6 hr 13 hr 14 hr 15 hr 16 hr 17 hr 18 morphological and physiological characterization in this work all the 18 isolates strains were picked on the basis of different morphological characteristics. the morphological characteristics of the final four short-listed isolates are shown in table 2. gram’s staining and motility result showed that out of 18 isolates tested 9 were gram negative coccobacilli, 5 were gram positive bacilli and only 4 were gram negative cocci. this indicated that majority (50%) of the bacteria in our studies belonged to gram negative coccobacilli strains followed by 22.22% gram positive bacilli and gram negative cocci seemed to be the most uncommon one constituting only 27.77% of the total isolates. while in case of motility 66.66% were motile and remaining 43.44 % were non-motile (table 3). antibiotic assay the endophytic isolates were also checked for their sensitivity (s) and resistance (r) against antibiotics like ampicillin, gentamycin, spectinomycin, tetracycline. the result of the antibiotic assay of the rhizospheric isolates is tabulated (table 4). nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   43 table 2: morphologyical and physiological characteristics of 18 isolates colony character size shape margin elevation texture opacity pigmentation hr1 medium round entire raised smooth transparent no pigmentation hr2 medium round entire flat smooth transparent no pigmentation hr3 small round entire slightly raised smooth transparent yellow pigmentation hr4 small round entire slightly raised smooth opaque yellow pigmentation hr5 small round entire flat rough transparent no pigmentation hr6 small round entire slightly raised smooth transparent yellow pigmentation hr7 medium round entire flat smooth opaque pitch pigmentation hr8 medium round entire raised smooth opaque yellow pigmentation hr9 medium irregular irregular flat rough opaque white pigmentation hr10 medium round entire flat smooth transparent yellow pigmentation hr11 small round entire raised smooth transparent yellow pigmentation hr12 medium irregular irregular flat rough opaque white pigmentation hr13 small round entire flat rough transparent no pigmentation hr14 medium round entire flat smooth transparent yellow pigmentation hr15 medium irregular irregular flat rough opaque white pigmentation hr16 medium round entire flat smooth transparent yellow pigmentation hr17 medium irregular irregular raised rough transparent golden yellow pigmentation hr18 small irregular irregular flat rough opaque white pigmentation siderophore production assay of siderophore production performed by cas agar plate method in which following isolates hr1, hr3, hr4, hr7, hr18 showed production of siderophore. so further estimation of siderophore was performed to determine which types of siderophores are produced, either cathecolate or hydroxymates type of sideophore. unfortunately we could not obtain the result. iaa production test all the isolates were tested for their iaa production. after 24 hrs of incubation with tryptophan all the strains exhibited a significant amount of iaa production. the production of iaa by isolates indicated that the tested strains nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   44 table 3: the gram nature and motility of the 18 isolated strains. isolates from tomato plant gram’s nature motility hr 1 gram –ve cocco bacilli + hr 2 gram –ve cocco bacilli + hr 3 gram –ve cocco bacilli + hr 4 gram –ve cocco bacilli + hr 5 gram –ve cocco bacilli + hr 6 gram –ve cocco bacilli + hr 7 gram –ve cocco bacilli + hr 8 gram –ve cocco bacilli + hr 9 gram -ve cocci hr 10 gram -ve cocci hr 11 gram +ve bacilli + hr 12 gram +ve bacilli hr 13 gram +ve bacilli + hr 14 gram +ve bacilli hr 15 gram +ve bacilli hr 16 gram -ve cocci + hr 17 gram –ve cocco bacilli + hr 18 gram –ve cocco bacilli + + : indicates motile organism, : indicates non-motile organism. utilized tryptophan as a precursor for growth and produced iaa, the primary auxins in the majority of plant species as a plant growth promoter. data indicated that all the bacterial endophytes from plant were able to produce iaa in the presence of tryptophan (table 5). production of iaa is widespread among bacteria-plant associated. several bacteria having the ability to anabolise indole-3-acetic acid (iaa) with supplemented ltryptophan have been isolated from the plant surfaces. bacterial iaa producers (bips) have the potential to interfere with any of these processes by input of iaa into the plant's auxin pool [1]. patten and glick [20,21] have shown that bacterial iaa stimulates the development of the root system of the host plant and brandi and lindow (1998) have studied the contribution of iaa for bacterial epiphytic fitness, observation supported by the investigation of other workers [12,20,2,9,33]. chromium tolerance of the endophytic strains almost 17 out of 18 isolates from lycopersicon esculentum tolerated a chromium concentration upto 500µg/ml. one of the isolate hr11 tolerated upto 300µg/ml, wheres all the isolates showing tolerance above 450µg/ml. there are reports of certain bacillus spp. tolerating upto nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   45 table 4: antibiotic assay of isolated strains isolates ampicillin streptomycin tetracycline chloramphenicol hr1 10 16 18 23 hr2 r 19 13 22 hr3 14 21 19 24 hr4 r 9 15 r hr5 r 17 r 14 hr6 r 9 r 13 hr7 r 18 r 17 hr8 r 11 r 8 hr9 28 29 27 38 hr10 r r 7 19 hr11 7 16 18 21 hr12 r 13 11 19 hr13 r r 9 16 hr14 13 21 15 14 hr15 8 r 11 14 hr16 r r 8 18 hr17 r 12 r r hr18 21 20 19 24 resistance microorganismr, number mentioned is zone of inhibition in mm 550 µg/ml [35] and bacilli spp. is a well known pgpr strain. all the standard strains except r. leguminosarum and s. meliloti showed very less tolerance to chromium. both the strains r. leguminosarum and s. meliloti are well known for their pgpr activity in leguminous plants. a rhodococcus erythropolis mtcc 7905 strain has been shown to be resistant to 300 mg l-1 of cr6+ isolated from metal contaminated soil samples from a site near indian himalayan region has been reported to reduce substantial amounts of cr6+ to cr3+ as well as showed to have plant growth promotion of pea (pisum sativum) in the presence of toxic cr6+ concentration [30]. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   46 table 5: indole acetic acid production by endophytic bacterial isolates isolates od at 530nm hr1 0.061 hr2 0.020 hr3 0.050 hr4 0.241 hr5 0.199 hr6 0.067 hr7 0.057 hr8 0.114 hr9 0.056 hr10 0.097 hr11 0.181 hr12 0.007 hr13 0.029 hr14 0.270 hr15 0.112 hr16 0.094 hr17 0.166 hr18 0.079 phosphate solubilisation phosphorous is one of the most important plant nutrient and a large portion of inorganic phosphate applied to soil as fertilizer is rapidly immobilized [19,24]. endophytic bacteria possess the capacity to solubilize immobilized mineral phosphates. in this study all the 18 isolates were tested for their phosphate solubilizing activity on pikovasky agar medium. it was interesting to note that out of 18 endophytic isolates, 8 showed phosphate solubilisation activity (table 6). result revealed that majority of the pgpr strains do have phosphate solubilizing activity and such organisms play a major role in plant growth promotion [24]. table 6: phosphate solubilization by endophytic bacterial isolates isolate no. growth on pv zone (mm) hr 1 full growth 20 hr 2 full growth hr 3 full growth 21 hr 4 no growth hr 5 no growth hr 6 no growth hr 7 full growth 17 hr 8 no growth hr 9 no growth 21 hr10 no growth hr 11 less growth 8 hr 12 less growth 11 hr 13 no growth hr 14 no growth hr 15 no growth hr 16 no growth hr17 full growth 31 hr 18 less growth 9 mm zone of clearance (pink colour zone) organic acid production out of the 8 endophytic isolates showing phosphate solubilization, all 8 showed organic acid production. the isolates number hr7, hr8, hr9, hr10, hr14, hr15 showed slight organic acid production by forming a very thin zone of clearance on the plates of pikovasky with methyl red as ph indicator dye. this gave the pink coloured zone that indicated shift in ph change from alkaline to acidic. some isolates like hr4, hr7, hr13, hr16 were unable to solubilize phosphate and also did not produce organic acid. this could be because the amount nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   47 of organic acid produced might be very less to do so (table 7). chitinase production in the present study none of the strain revealed a clear zone, but 5 isolates out of 18 showed growth on the chitin agar plate, remaining 13 strains did not show any growth (table 8). biological control of plant pests and diseases is much more attractive than chemical treatment methods due to its greater specificity and less harmful impact on the environment [34,35]. major component of fungal cell is chitin. thus organism having the ability to produce chitinase might have antifungal property. pectinase production for pectinase production, 17 out of 18 isolates of lycopersicon esculentum revealed the production of pectinase. the strains showing table 7: isolates showing organic acid production sample no. isolate no. production of organic acid zone (mm) pink colour of zone sample 1. hr 1 medium 13 +++ hr 2 medium 10 ++ hr 3 very less 8 ++ hr 4 no hr 5 no sample 2. hr 6 very less 7 hr 7 medium 11 ++ hr 8 medium 13 hr 9 medium 10 hr 10 very less 9 hr 11 medium 12 + hr 12 very less 8 ++ hr 13 no sample 3. hr 14 medium 11 hr 15 very less 8 hr 16 no hr 17 medium 11 +++ hr 18 medium 13 + + : 1.0 mm zoc (zone of clearance), ++: 1.2 mm zoc, +++: 1.4 mm zoc, -: no zone production of pectinase on pectin agar plate are listed in table 9. maximum research indicated that pectin methyl esterase (pme) (ec 3.1.1.11) catalyzes the hydrolysis of methyl-ester groups of cell wall pectins. it has been found in all plant tissues and in some of plant cell wall-degrading microorganisms or insects [5,6] and has been implicated in a number of processes including cell growth [18], fruit ripening [11, 29], abscission and senescence [17], pathogenesis [7] and cambial cell differentiation [13]. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   48 table 8: isolates showing chitinase production isolates chitinase production hr1 hr2 + hr3 hr4 + hr5 hr6 hr7 + hr8 hr9 hr10 hr11 hr12 hr13 hr14 + hr15 hr16 hr17 hr18 + + : positive : negative table 9: isolates showing pectinase production isolates pectinase production hr1 + hr2 + hr3 + hr4 + hr5 + hr6 + hr7 + hr8 + hr9 + hr10 + hr11 + hr12 + hr13 + hr14 + hr15 + hr16 hr17 + hr18 + + : positive : negative 16s-rdna sequencing of pgpr isolates colony pcr all the plant growth promoting results when compiled together showed one strain (hr7) showed maximum positive features and thus the 16srdna of the strain was amplified using universal full length primers. an amplicon of 1.5kb was obtained and sent for sequencing to bangalore genei, pvt, ltd india. the sequence obtained was matched with the online available sequences in rdp (ribosomal database project ii) bioinformatics tool. multiple sequence alignment phylogenetic analysis blast (basic local alignment search tool) search was done for partial 16s rdna of the isolates hr7 by submitting queries to ncbiblast (http://blast.ncbi.nlm.nih.gov/blast.cgi.) and homologous sequences obtained by standard nucleotide-nucleotide blast (blastn) were aligned with the different 16s rdna isolates after sequencing and various related sequence were retrieve after blasting the partial sequence of the isolates obtained after sequencing. accession no. of the related species was retieved and multiple sequence alignment (fig 1) was performed using clc free protein workbench 5.0. evolutionary tree for the same nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   49 data was obtained by neighbor joining method with bootstrap values (expressed as percentages of 100 replications) as shown in (fig 2). except hr7 other do not give the sequencing results. accession no. of some isolates used for multiple alignment with hardikseq-1(hr7) isolate were, jf423918, jf281099, hq995502, hq268732, hq202541, hq202540, hq259948, fm995816, fm995815, fm995811, fm995802, fm995800, fm995798, fm995797, fm995796. fig. 1: multiple sequence alignment for the partial 16s rdna sequence of hardik seq-1 (hr7) isolate with other related species retrieve after blast, resulted in versatile coloring scheme that highlighted the conserved sequence in aligned sequences. fig. 2: phylogenetic tree of partial 16s rrna genes of hardik seq-1(hr7) islolates from closely related of resistant bacteria obtained after blast. the tree was constructed based on partial 16s rrna sequences of the isolates and the reference strains. bootstrap values (expressed as percentages of 100 replications) are shown at branch points. bootstrap values over 50% are shown. the scale bar 0.500 indicates 50% nucleotide sequence substitution nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  37  –  52                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   50 precisely, the research concluded that endophytic bacteria isolated form lycopersicon esculentum produced one or the other different characteristics involved in plant growth promotion. they either produced phytohormones like indole acetic acid, siderophore, protease, pectinase, organic acid showed antifungal activity, chromium tolerance and solubilized phosphate. only hr7 endophyte of tomato turned out to be pseudomonas aeruginosa, it is a gram negative coccobacili, sporeforming motile bacilli,which showed maximum pgpr activity. it may be concluded that the above strains may be endophytic and was associated with the plant probably because they may benefit the plant by stimulating its growth. acknowledgments this research was undertaken with support from the ashok and rita patel institute of integrated study and research in biotechnology and allied sciences, new vallabh vidyanagar (managed by the charutar vidya mandal). references 1. andreae wa, van ysselstein mwh: studies on 3-indole acetic acid metabolism. vi. 3-indole acetic acid uptake and metabolism pea roots and epicotyls. plant physiol.1960, 35: 225232. 2. bastián f, cohen a, piccoli p, luna v, baraldi and bottini, v: production of indole-3-acetic acid and gibberellin a1 and a3 by acetobacter diazotrophicus and herbaspirillum seropedicae in chemically-defined culture media. plant growth regul 1998 24: 7–11. 3. bergey's manual of systematic bacteriology 2009, 2nd edition. published by springer, new york. 4. brandi mt, lindow se: contribution of indole-3-acetic acid production to the epiphytic fitness of 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soil. agron sustain dev. 2007, 27: 145-153. microsoft word review article 3 joshi author copy revised.docx nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   72 review article association  mapping  for  improvement  of  quantitative  traits  in  plant  breeding   populations   umesh r. rosyara1 and bal k. joshi2 1  south  dakota  state  university,  plant  science  department,  brookings,  south  dakota,  usa   2  biotechnology  unit,  narc,  khumaltar,  po  box  1135  kathmandu,  nepal   *correspondence  author  recent  address:  joshibalak@yahoo.com   abstract dna-based molecular markers have been extensively utilized for mapping of genes and quantitative trait loci (qtl) of interest based on linkage analysis in mapping populations. this is in contrast to human genetics that use of linkage disequilibrium (ld)-based mapping for fine mapping of qtls using single nucleotide polymorphisms. ld based association mapping (am) has promise to be used in plants. possible use of such approach may be for fine mapping of genes / qtls, identifying favorable alleles for marker aided selection and cross validation of results from linkage mapping for precise location of genes / qtls of interest. in the present review, we discuss different mapping populations, approaches, prospects and limitations of using association mapping in plant breeding populations. this is expected to create awareness in plant breeders in use of am in crop improvement activities. kew words: association mapping, plant breeding, dna marker, quantitative trait loci introduction the development and use of molecular markers for the detection and exploitation of dna polymorphism in plant and animal systems is one of the most significant developments in the field of molecular biology and biotechnology. of mapping techniques, linkage based mapping is popular in mapping genes in self and cross pollinated crop species. the objective of such genetic mapping is to identify simply inherited markers in close proximity to genetic factors affecting quantitative traits (quantitative trait loci, or qtl). this localization relies on processes that create a statistical association between marker and qtl alleles and processes that selectively reduce that association as a function of the marker distance from the qtl. when using crosses between inbred parents to map qtl, we create in the f1 hybrid complete association between all marker and qtl alleles that derive from the same parent. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   73 recombination in the meioses that lead to doubled haploid, f2, or recombinant inbred lines reduces the association between a given qtl and markers distant from it. unfortunately, arriving at these generations of progeny requires relatively few meioses such that even markers that are far from the qtl (e.g. 10 cm) remain strongly associated with it. such long-distance associations hamper precise localization of the qtl. one approach for fine mapping is to expand the genetic map, for example through the use of advanced intercross lines, such as f6 or higher generational lines derived by continual generations of outcrossing the f2 [1]. in such lines, sufficient meioses have occurred to reduce disequilibrium between moderately linked markers. when these advance generation lines are created by selfing, the reduction in disequilibrium is not nearly as great as that under random mating. the central problem with any of the above approaches for fine mapping is the limited number of meioses that have occurred and (in the case of advanced intercross lines) the cost of propagating lines to allow for a sufficient number of meioses. an alternative approach is association mapping (am), taking advantage of events that created association in the relatively distant past. assuming many generations, and therefore meioses, have elapsed since these events, recombination will have removed association between a qtl and any marker not tightly linked to it. f2 / bc pedigree association mapping 4 0 10 2 recombinant inbred lines near isogenic lines positional cloning intermated recombinant inbreds r es ea rc h ti m e (y ea rs ) 1 5 1 1 x 10 4 1 x 10 7 resolution (bp) a lle le n um be r fig.1: schematic comparison of various methods for identifying nucleotide polymorphism trait association in terms of resolution, research time and allele number. bc, backcross. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   74 am, also known as association analysis (aa) or linkage disequilibrium mapping is a method that relies on linkage disequilibrium to study the relationship between phenotypic variation and genetic polymorphisms [2]. thus linkage mapping counts recombination between markers and the unknown genes whereas association mapping measure correlation between marker alleles and trait allele in a population (linkage disequilibrium). association mapping allows for much finer mapping than standard bi-parental cross approaches. time requirement and resolution of association mapping is compared with other types of mapping approaches (figure 1). linkage disequilibrium linkage disequilibrium (ld) is the nonrandom combination of alleles at two genetic loci, which in random mating populations is mostly generated by mutation and genetic drift, and decays by recombination. the trend of ld decay is shown in graphs with different recombination fractions (figure 2). therefore, ld will be observed between two loci if they are in tight linkage or if the haplotype is recent (hedrick, 2005). mutations are rare events hence, it is expected that most mutations happened many generations ago and should be in linkage equilibrium with other loci, unless they are very closely linked. while significant ld in random mating populations is evidence of tight linkage, population perturbations like migration, inbreeding, and selection can build up ld among loosely linked or even unlinked loci. therefore, the characteristics of the population under study must be recognized when conducting aa or am and interpreting its results. studies have shown that ld levels vary both within and between species (for detail, [2]. for example, ld extends less than 1000 bp [3] for maize landraces and roughly 2000 bp for diverse maize inbred lines [4], but can be as high as 100 kb for commercial elite inbred lines [5]. ld decay can also vary considerably from locus to locus. for example, significant ld was observed up to 4 kb for the y1 locus (encoding phytonene synthase), but was seen at only 1 kb for psy2 (a putative phytonene synthase) in the same maize population [6]. beside the outbred maize, many ld studies have also been carried out in other plant species [7-12]. a variety of mechanisms generate linkage disequilibrium, and several of these can operate simultaneously. the two most common mechanisms include populations expanding from a small number of founders and through admixture. the haplotypes present in the founders will be more frequent than expected under equilibrium. three special cases are noteworthy. first, genetic drift affects gametic phase disequilibrium (gpd) by this mechanism in that a population nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   75 experiencing drift derives from fewer individuals than its present size. second, by considering an individual with a new mutation as a founder, we see that its descendants will predominantly receive the mutation and loci linked to it in the same phase. linked marker alleles will therefore be in gpd with the mutant allele. finally, an extreme case arises in the f2 population derived from the cross of two inbred lines. here, all individuals derive from a single f1 founder genotype and association between loci can be predicted based on their mapping distance. second, gametic phase disequilibrium arises in structured populations when allelic frequencies differ at two loci across subpopulations, irrespective of the linkage status of the loci. admixed populations, formed by the union of previously separate populations into a single panmictic one, can be considered a case of a structured population where sub-structuring has recently ceased. fig. 2. decay of linkage disequilibrium with time for four different recombination fractions (θ). for unlinked loci, θ = 0.5 and ld decays rapidly within a small number of generations. for closely linked loci, the decay in ld is extremely slow. d, coefficient of linkage disequilibrium (source: [13]). methods for association mapping multi-parent advanced generation intercross in the advanced intercross [1], f2 individuals are intermated for several generations before mapping. the successive rounds of recombination cause ld to decay and the precision of qtl location to increase. this approach has now been extended to include populations with multiple parents, to take into account information from multiple linked markers [14, 15] and to prioritize candidate polymorphisms [16, 17]. the multiparent advanced generation intercross (magic) was first proposed and applied to mice [14] and is described as heterogenous stock. recently more successes are described [18]. in both crops and animals, an advantage of the method is that a population can be established containing lines that capture the majority of the variation available in the gene pool. although it might take several years before these populations are suitable for fine mapping, they are cheap to set up and their value as mapping resources increases with each generation. in plants, magic can be used to combine coarse mapping with low marker densities on lines derived from an early generation, with fine mapping using lines derived from a more advanced generation of crossing and a higher marker density. if such nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   76 populations were established now, they would be well placed to exploit the advances in genomics technology and reduction in genotyping and sequencing costs predicted to occur in the next few years [16, 19, 20]. the transmission disequilibrium test the ability to map qtl in collections of breeders’ lines, old landraces or samples from natural populations has great potential. in these populations, ld often decays more rapidly than in controlled crosses. furthermore, phenotypic data often already exist, saving time and money. the challenge is to distinguish qtl–marker associations arising from ld between closely linked markers from spurious background associations. the first and most robust method of achieving this was the transmission disequilibrium test (tdt) introduced by richard spielman and colleagues in 1993 [21]. the tdt provides a way of detecting linkage in the presence of disequilibrium [21]. neither linkage alone nor disequilibrium alone (i.e. between unlinked markers) will generate a positive result so the tdt is an extremely robust way of controlling for false positives. at its simplest, multiple families consisting of two parents and a single progeny are collected, as shown in figure 3. starting from such trios, different models have been evolved since then and some new models allow nuclear families [22] to extended family [23] for quantitative trait analysis in addition to qualitative traits. the test of association for extended families allows use of available genotypic and phenotypic data from family of any size and structure. different possible types of families that can be analyzed are shown in figure 4. figure 3. the transmission disequilibrium test in the simplest case, progeny are selected for an extreme phenotype and transmissions to the progeny from heterozygous parents counted. in the case shown, the a allele is transmitted to affected offspring four times out of five the single progeny in each family is usually selected for an extreme phenotype. in human genetics this typically means they are affected by the disease under study. parents and progeny are genotyped, but only parents heterozygous at the marker locus are included in the analysis. from each parent, one allele must be transmitted to the progeny and one is not transmitted. over all families, a count is made of the number of transmissions and nontransmissions. in the absence of linkage between qtl and marker, the expected ratio of transmission to nontransmission is 1:1. in the presence of linkage it is distorted to an extent that depends on the strength of ld nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   77 between the marker and qtl. the distortion is tested in a chi-squared test. power depends on the strength of ld and on the effectiveness of selection of extreme progeny in driving segregation away from expectation. this elegant test is extremely robust to the effects of population structure, but is susceptible to an increase in false positive results generated by genotype error and biased allele calling [24]. this risk can be reduced by modeling genotype errors and missing data in the analysis [25-27] or by comparing the transmission ratio for extreme phenotypes with that for control individuals or for the opposite extreme. the tdt has been extended to study haplotype transmissions, quantitative traits, the use of sib pairs rather than parents and progeny, and information from extended pedigrees. tdt and other family-based association tests are reviewed elsewhere [28]. fig. 4. different type of families for association mapping elite inbred lines three way cross four way cross grand parents parents offspring a. extended pedigree b. nuclear family pedigree c. trios nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   78 in crops, parental and progeny lines are usually separated by several generations of gametogenesis rather than by one. in this case, the tdt is still valid, but might no longer be so robust, the process of breeding might itself distort segregation patterns. a family-based association test that is applicable to plant breeding programs has recently been proposed [29]. the authors point out that for candidate gene studies, this method is more cost effective than the alternative methods described below given that no additional control markers are required. however, some power will be lost because only progeny derived from f1s known to have a heterozygous marker genotype are informative. genomic control population structure arising from recent migration and population admixture will generate ld between a trait and markers distributed over the whole genome. this can be detected by studying whether the distribution of the test statistic for association, estimated empirically from a set of genomewide distributed markers, differs from the expected null distribution. this is the basis of genomic control (gc) [30, 31]. to estimate the empirical distribution accurately would require many markers. however, all that is required is to estimate the mean test statistic and compare it with its expected value (1.0 for a 1 degree of freedom chi-squared test) for which only ~50 markers are needed [32]. if the average chisquared at a set of 50 control markers is much greater than 1.0, population structure is indicated. for any candidate marker, the nullhypothesis is now no longer absence of association between it and the trait. rather, it is that there is no association above the background level resulting from population structure. to test for this, we simply divide the observed chi-squared between the candidate and trait by the average chisquared at the control markers and look up the p-value of the adjusted chi-squared in the usual manner. gc is valid for any single degree of freedom test. preferably, the control markers should loosely match the test marker in allele frequency, but this is not crucial [31]. for quantitative traits, the difference between trait means for each marker class is usually tested in a t test. provided the number of observations is reasonably large, t2 is distributed as a 1 degree of freedom chi-squared and gc can still be carried out. more recent work has suggested that greater accuracy is achieved by treating the test statistic as an f test with one degree of freedom (df) in the numerator and degrees of freedom in the denominator equal to the number of control loci [33]. more sophisticated versions of gc are available. with large numbers of candidate polymorphisms to test, the majority are not expected to be genuinely associated with the trait. in this case, procedures and software are nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   79 available in which, in effect, the candidate markers act as their own controls. gc has also been extended to control for bias in accuracy of genotyping between dna samples from different origins [34] and to tests with >1 df [35]. gc also corrects for unknown kinship among collections of lines [30]. the presence of related lines can greatly increase the frequency of false positives. for many crop datasets this will be the greatest source of bias. the correction of the false positive rate using gc comes at a cost: power is always decreased. this loss of power can be great in cases of extreme population subdivision. furthermore, because loci can vary in their differentiation between populations, the uniform adjustment of gc might be insufficient for some candidate polymorphisms and overcorrect at others. structured association structured association (sa) provides a sophisticated approach to detecting and controlling population structure [36-38]. again, additional markers are required, randomly distributed across the genome. just as for gc, recent migration and population admixture are assumed to generate ld among unlinked and loosely linked markers that have yet to decay fully. however, we expect the parental populations themselves to be in linkage equilibrium. by trial and error one could allocate the individuals in our sample to parental populations such that disequilibrium within populations was minimized. one could then include information on population membership in the test of association. this is the approach taken for sa. first individuals are allocated to populations, then this information is used to control for population membership in the test of association [36-38]. to allocate individuals to populations we need to know in advance how many populations there are. if unknown, this can be estimated: the allocation process is repeated for different possible numbers and the best fitting selected. nevertheless, deciding on population number can be problematic. the computer program structure [37] uses computationally intensive methods to partition individuals into populations. many individuals or lines will not belong uniquely to one, but will be the descendents of crosses between two or more ancestral populations. structure also estimates the proportion of ancestry attributable to each population. following allocation of individuals to populations, the test for association is carried out in a model fitting exercise. here, the principle is that variation attributable to population membership is accounted for first, using estimates of population membership from structure, and then the presence of any residual association between the marker and phenotype is tested. for example, to test for association between a quantitative trait and a microsatellite, the trait is first regressed on the nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   80 estimated coefficients of population membership and then on the marker – coded as a factor as if in an analysis of variance [39]. sa is effective in detecting and adjusting for the presence of population structure, but does not deal with consanguinity within populations. recently, ed buckler’s group introduced a method in which population membership is estimated using structure and kinship among varieties is estimated empirically from a second set of control markers [40]. the analysis takes into account both population structure and the correlation between individuals that results from their relationships. this method is implemented in the software tassel. association mapping in plant breeding populations scientific plant breeding is a recent activity that normally involves a narrow genetic pool, such that breeding populations can be traced back to relatively few original parents, normally landraces, within a relatively small number of generations (e.g. [41, 42]. under this scenario, mutations play a minor role and most of the observed ld is expected to reflect the haplotypes of the original parents. moreover, because there were few opportunities for recombination between the time of introduction of a parent and the present, ld in some plant breeding populations may not reliably indicate tight linkage. between unlinked loci, ld can be caused by simultaneous selection of combinations of alleles at different genes, including epistasis, and by population structure [43]. both phenomena should be common in plant breeding populations. selection should affect ld in parts of the genome related to traits that are relevant for the breeding program. this source of distortion should be taken into consideration in the interpretation of results of aa in a casespecific manner. in contrast, population structure is expected to affect the pattern of ld over the whole genome and must be controlled a priori for correct association analysis [38]. most of the literature on aa refers to human populations or theoretical panmitic populations. there is limited information and discussion about applications of this technique to plant breeding. as the information generated by qtl studies accumulates, a method is needed to convert efficiently that information into practical tools for plant selection. association analysis can be an effective approach for closing the gap between qtl analysis and marker-assisted selection. the objective of this review paper is to discuss potential applications of association mapping for plant breeding populations. plant breeding populations include basically three types germplasm bank collections, synthetic populations, and elite lines. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   81 choice of populations for am in plant breeding programs in a plant breeding program, three main types of populations could be considered for implementation of am: germplasm bank collections, elite breeding materials, and synthetic populations. the application of aa differs among those populations in several aspects (table 1). for efficient integration of am with other methods currently in use, material that is routinely generated and evaluated should be used for both purposes. in the case of germplasm banks, core collections are expected to represent most of the genetic variability with a manageable number of accessions [44], and thus are suitable for genetic studies. in the case of elite materials, the sample could be composed by lines and checks evaluated in regional trials. for synthetic populations, the evaluation unit should be also the association unit (or closely related to it), whether it is an individual or a family. germplasm bank core collections samples representing the genetic diversity of a species are attractive for am because of the wide allele diversity encompassed. methods of selection of core collections often involve genotyping unlinked markers to compute genetic distances, thus providing information about population structure. the process of selection of a minimum sample with maximum variation has a normalizing effect that is expected to reduce population structure and ld between unlinked loci, thus creating a situation favorable for association analysis [45]. a difficulty likely to occur in this type of material is related to genetic heterogeneity within samples. landraces and natural populations often consist of open-pollinated varieties or mixtures of genotypes, and the dna extraction, genotyping, and phenotyping schemes must account for this variability. core collections are useful materials for am of qualitative traits, such as disease resistance or special quality characteristics (color, aroma, etc). studies focusing on domestication-related traits such as seed dormancy, shattering, or inflorescence type also could require wide phenotypic variation, beyond the limits of cultivated germplasm [46]. conversely, the broad genetic variability of those collections normally make them unsuitable for analysis of quantitative traits because part of the accessions would be unadapted to growing conditions and prevalent diseases, resulting in poor precision of trait measurement. common ancestors of distantly related individuals occurred many generations ago; therefore, ld is expected to have decayed to short genetic distances. for this reason, am in core collections will probably require candidate genes or major qtl mapped within narrow confidence intervals [47]. compared with linkage-based fine mapping and nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   82 table 1. comparison of different types of populations for association analysis. depends on the collection, conservation and sampling schemes aspects of association mapping germplasm bank elite material synthetic populations samples entries of a core collection inbred lines and cultivars individual plants or progenies sample turnover static gradually substituted ephemeral source of phenotypic data phenotypic screenings replicated yield trials evaluation for recurrent selection type of traits high heritability and domestication traits low heritability, yield depends on the evaluation scheme level of ld low high intermediate population structuring medium high low allele diversity among samples high low intermediate allele diversity within samples variable 1 allele 1 or 2 alleles resolution of am high low intermediate and increasing power of am low high intermediate and decreasing application of significant markers marker-assisted backcross marker-assisted selection incorporation in selection index depends on the collection, conservation and sampling schemes. for diploid species. (source: [45]. positional cloning [48] the am approach would offer the advantage of simultaneously detecting the effect and screening the germplasm for useful alleles. significant markers would be useful for introgression of the new variation into elite germplasm through marker-assisted backcrossing [49], while markers used for population structure inference could be used to speed up the recovery of the recurrent parent genome. theoretical projections indicate that the use of two markers per chromosome for selection against the donor genotype could shorten the transfer by about two generations [50] elite lines and cultivars maximum relative efficiency of markerassisted selection compared with phenotypic selection is expected when heritability is low and markers capture a significant portion of the variation for the trait [51]. elite lines are desirable materials for am of low heritability traits, including yield, yield components, and tolerance to abiotic stresses because elite lines are genetically stable and are well adapted to normal growing conditions. in plant breeding programs, there is normally a large body of phenotypic data accumulated for nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   83 elite lines and cultivars from replicated field experiments over locations and years. use of those data for am requires statistical models accounting for covariances introduced both by experimental design (years, locations, replicates) and polygenic effects. moreover, those data are often unbalanced because new lines are included in field trials each year, while other lines are discarded. maximum likelihood solutions of mixed-effects models yield minimum-variance unbiased estimates of allele effects from unbalanced data, taking into account the correlation structure of the data [52]. mixed-effects models were used to analyze plant height, disease resistance, and grain moisture in maize [53] and grain size and milling quality in wheat [45]. population structure can be prominent in elite material because it is common for closely related lines to be admitted to advanced trials. if pedigrees are known, the relationships among the lines can be determined [41] and used to control for polygenic effects [54]. in this case, it is not essential to estimate population structure through unlinked markers, although there may still be interest in marker data as a genetic fingerprint for variety protection [55] and for purity control of seed production. a typical elite plant breeding pool is derived from few founders in the recent past, and is submitted to intense selection. for those reasons, ld is expected to be high in this material, and the first experimental results confirm this expectation [3, 5]. although am in elite lines may not offer much improved resolution compared with qtl analysis in biparental mapping populations, there are at least two important advantages: a substantially higher level of polymorphism and detection of favorable alleles directly in the target population. elite lines are natural candidates for crossing to generate the next round of breeding, and significant markers could be used for marker-assisted selection in the progeny. synthetic populations although the potential of synthetic populations for am is largely unknown, they might be the plant breeding materials that best approximate the assumption of random mating because synthetics are normally designed and maintained to minimize inbreeding. population structure is expected to be mild or absent, which is an important advantage of synthetics for am. if the experimental material represents a single intermating population, the power of am is maximized and the risk of false associations is minimized [56]. nevertheless, population structure can still occur because of differences in flowering time, plant height, and other traits that may lead to assortative mating. genotypic information could be useful in all phases of population breeding. in the choice of nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   84 parents to form the population, knowledge of the genetic distance among lines would be useful to achieve a compromise between high means for agronomic traits and high allelic variability. by genotyping samples of subsequent cycles with unlinked markers, breeders can monitor changes in allele diversity, effective population size, and population structure [57, 58]. the allele diversity of synthetic populations depends on the number and divergence of parents and the intensity of selection applied. the level of ld in synthetic populations is expected to be high in the initial generations, such that a genome scan could detect large chromosome segments associated with traits, and trace them back to parental haplotypes. in subsequent generations, the decay of ld by recombination would favor increasingly refined mapping. however, synthetic populations are often submitted to recurrent selection, a breeding scheme consisting of successive cycles of evaluation, selection, and recombination [59]. intense selection could build up ld by favoring allelic combinations or by promoting genetic drift [6]. for this reason, populations subjected to mild or no selection would be preferred for am. [60] developed a population for association analysis from the illinois high/low oil populations, with 10 generations of recombination without selection. a short time frame is a fundamental characteristic of plant breeding populations for am, compared with natural populations. therefore, in plant breeding populations, the most significant association does not necessarily indicate the position of the gene [45]. in the long term, linkage becomes the major factor defining the association between qtl or gene and marker, and only closely linked markers remain in high ld; however, the time required to achieve this situation is longer than most breeding programs have been in existence. for this reason, am in plant breeding programs should be considered a method of detection of markers for indirect selection, rather than a method for finemapping qtl [45]. to alleviate this problem, the breeder should use methods like recurrent selection, which maximizes the heterozygosity and the opportunities for recombination. the resolving power of ld mapping depends on how rapidly ld decays with genetic distance. this varies between populations of landraces, wild progenitors and modern cultivars as a result of the diverse history to which crop plants have been subjected since their domestication [61]. in some populations, ld will decay so rapidly that they are best suited for fine mapping, whereas in others the decay might be so slow that whole genome scans are practical. in crops where collections of contemporary, historical and wild material exist, selection of different sets of lines might permit both fine and coarse mapping [61]. however, in most crops, marker density is nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   85 currently too low for genome scans. before attempting these, power calculations should demonstrate that, given the rate of decay of ld in the population to be studied, the density of markers and their allele frequency distribution are adequate to detect linked qtl accounting for specified proportions of the phenotypic variation. population size is also important. an ld mapping experiment will almost always have lower power than a family based linkage mapping experiment of equivalent size: if 100 lines are just sufficient for a family based linkage mapping study, they will be too few for ld mapping. for these reasons this is believed that the best use of ld mapping is to refine the location of qtl identified in family based linkage mapping and candidate gene studies. while linkage mapping methods offer a high power to detect qtl in genome-wide approaches, association mapping methods have the merit of a high resolution to detect qtl [4]. linkage and association analysis are thought to be complementary to each other in terms of providing prior knowledge, crossvalidation, and statistical power [62]. so if both analyses are done this is expected to help in proper location of qtls. longer term, prospects for high-throughput genotyping and sequencing might make whole-genome scans by ld mapping more feasible. the challenge is to identify and create the appropriate populations so that computational, analytical and profiling advances can be rapidly harnessed by the crop science community. for plant breeding application, at current situation am could be useful for validating location of qtl of interest and identifying favorable allele for marker aided selection. once a genetic marker has been demonstrated to be associated with a phenotypic trait of interest, it can be used as a selection target to obtain an indirect response in the trait. in recurrent selection, markers could be used to store information acquired from phenotypic evaluations, which can be used for selection in later cycles. likewise, in pedigree breeding, markers could carry information about yield potential from the phase of replicated field trials to the phase of single-plant selection, when evaluation of yield cannot be made with reasonable precision. conclusion with the availability of high density maps in a number of crop plants, the whole genome sequences in model plants like arabidopsis and rice, and the sequences of gene rich regions in crops like sorghum, maize and wheat the association mapping tool have future for increasing applications. even though most of plant breeders’ populations could not be used for fine mapping as such the association mapping could be helpful in identification of favorable alleles for marker aided selection and cross validation of results of linkage based mapping. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  72  –  89                                                                                                                    biotechnology  society  of  nepal  (bsn),  all 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grade and cheap substrate by bacillus subtilis as microbial cell factory niranjan koirala1,2⸸ , sareeta khanal1,3⸸, sujan chaudhary3,4 , sagar gautam5, shiv nandan sah3 , prince subba3, najat marraiki6, gaber el-saber batiha7 1department of natural products research, dr. koirala research institute for biotechnology and biodiversity, kathmandu 44600, nepal 2laboratory of biotechnology, faculty of science and technology, university of macau, macau sar 999078, china 3department of microbiology, central campus technology, dharan 56700, tribhuvan university, nepal 4department of botany, amrit science campus, kathmandu 44600, tribhuvan university, nepal 5department of microbiology. trichandra multiple campus,ghantaghar, kathmandu 44600, tribhuvan university, nepal 6department of botany and microbiology, college of science, king saud university, riyadh 11451, saudi arabia 7department of pharmacology and therapeutics, faculty of veterinary medicine, damanhour university, damanhour 22511, albeheira, egypt ⸸both the authors contributed equally to this work and share the first authorship received: 15th oct 2020; revised: 19th nov 2021; accepted: 20th dec 2021; published online: 31st dec 2021 abstract bio-surfactants are surface-active molecules which are produced by the wide range of microbes including bacteria, fungi, moulds, and yeast. this study was conducted to identify bio-surfactants by bacillus subtilis combined with use of cheap substrates and industrial wastes (mustard cake, whey and soya cake) which are found locally in nepal. bacillus subtilis, one of the most potential bio-surfactants producer; was isolated from soil sample of hydrocarbon contaminated site. isolates were grown in a minimal salt media (msm) with 10% (v/v) mustard oil cake, whey and soya cake separately. the presence and potential of surfactant was determined by the oil spreading technique, emulsification index (%e24) and surface tension measurement. it was revealed that the surface tensions of cell free extract were 54.41, 60.02 and 56.64 mn/m for from mustard cake, whey and soya cake respectively as compared to distilled water (72.09) at 25oc. the emulsification index values was found to be highest in engine oil from the bio-surfactant extracted from mustard cake, soya cake and whey respectively. similarly, mustard oil showed the lowest value of emulsification index. the highest emulsification activity was shown in mustard oil i.e. 1.13 from the cell free extract from mustard oil and lowest in engine oil i.e., 0.07, by the extract from soya cake medium, when measured in spectrophotometer at 540 nm. in conclusion, strain of bacillus subtilis was found to be the potential surface active agent producers on the mustard oil cake, which can be useful medium for various environmental, food, medicinal and industrial processes. keywords: bacillus subtilis; bio-surfactants; emulsification index; hydrocarbons; surface tension corresponding author, email: koirala.biochem@gmail.com introduction surfactants are the compounds capable of reducing surface tension between any two substances of same or different phase. surfactants can be either chemically synthesized or obtained biologically. when surfactants are produced on living surfaces, mostly microbial cell surfaces or excreted extracellularly, they are termed as bio-surfactants. such bio-surfactants generally contain hydrophobic and hydrophilic moieties[1]. surfactants are characterized by their capacity to alter the surface and interfacial properties of a liquid, which allows the formation of micro emulsions, which further allows oils and related substances to become solubilized in water or vice-versa [2]. such properties of surfactant enable a wide range of industrial applications including emulsification, detergency, foaming capacity, lubrication, moisture retention, solubilization, and phase dispersion [3]. in recent years, there has been a steady increase in the interest in bio-surfactants as they have numerous advantages over the chemical surfactants. few of such advantages include lower toxicity, higher biodegradability, higher foaming, better environmental compatibility and effective properties even at extreme condition of temperature, ph and salinity[4]. in light of such advantages, bio-surfactants can be used for oil recovery, treatment of oil spillage and bioremediation, in foods, cosmetics and pharmaceuticals. moreover, due to their biodegradability they can safely be used in the nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-7777-1191 mailto:koirala.biochem@gmail.com https://orcid.org/0000-0002-8300-4570 https://orcid.org/0000-0003-3300-8191 https://orcid.org/0000-0002-7817-425x nepal j biotechnol. 2021 dec.9 (2): 21-28 koirala et al. ©njb, bsn 22 environment without the risks observed of some of their chemically synthesized counterparts. as a result, biosurfactant occupies an advantageous position both for research and industrial production [5].the most prevalent bacterial species capable of producing surfactant belong to the genera are pseudomonas, , micrococcus flavobacterium, bacillus, acinetobacter, achromobacter, arthrobacter, klebsiella, aeromonas, alkaligenes, streptococcus sp, corynebacteriumsp, moraxella, and proteobacteria [6]. microorganism utilize the substrate i.e. hydrocarbons to produce bio-surfactant and often minimalize them and produce ecologically harmless products [7]. bacillus is one of such genus of bacteria associated with the production of vital biosurfactant. they are gram positive rod shaped aerobic spore former bacterium commonly found in soil. members of the genus are considered as a suitable group for research as well as synthesis of bio-surfactants industrially mainly due to their capacity to produce various metabolites including surface active ones [8]. they not only produce good bio-surfactants, but are also capable of growing under facultative or anaerobic conditions, and have also been reported to be nonpathogenic, which permits their use in wide range industries, apart from environmental applications [8]. among the many species, bacillus subtilis is one of the most potential species capable of producing a wide range of extracellular metabolites including surfactant. surfactin, a lipopeptide bio-surfactant produced from b. subtilis, is able to reduce the surface tension of water to 25 mn/m and interfacial tension of water/hexadecane up to <1 mn/m which is among the best result given by any class of bio-surfactants. most bio-surfactant can lower surface tension of water from 72 to35 mn/m and the interfacial tension of water/ hexadecane from 40 to 1 mn/m [9]. the cosmopolitan distribution of b. subtilis, ability to form resistant spores and extreme tolerance ability of the bacteria makes it a suitable candidate for further research and industrial application for the purpose. hydrocarbons are commonly used as the substrate for the production of bio-surfactants. it has been postulated that the biological function of surface-active compounds is related to hydrocarbon uptake, and therefore a spontaneous release occurs with these substrates[10, 11]. a wide range of hydrocarbon rich substrate has been experimented and studied for the commercial application for bio-surfactant production. however, there have been difficulties in industrial production and commercialization of the product due to the higher manufacturing cost. the cost of substrate account for 1030 % of total production cost of bio-surfactant which makes the overall production cost higher that chemical surfactants [12].this is mainly due the use of different chemically synthesized media for production. consequently, chemical surfactant holds a higher position in the market. thus, the practical and possible way to win over the market of chemical surfactant is to reduce the manufacturing cost of bio-surfactant which can then be made available in the market at lower cost. reduction in cost of substrate by using lower grade and cheap substrate and using more efficient microbes could significantly reduce the manufacturing cost [13]. present study aims for the production of bio-surfactant by b. subtilis on the industrial wastes, cheaper and low grade substrates (mustard oil cake, soya cake and whey) which are found locally and abundantly in different areas of nepal. materials and methods this work was conducted at the central campus of technology, hattisar, dharan. the eight soil samples from auto-mobile workshop (garage) were collected form dharan-8 by using simple random sampling method. all samples were collected in aseptically dried and clean plastic bags and were transported to laboratory as soon as possible. bacterial isolation the test organism (bacillus subtilis) was isolated form a soil sample, by heating test samples at 80o c/ 10 min by using water bath to destroy all the vegetative cells. then 1g of soil was weighed and was suspended in 9 ml sterile distilled water. serial dilution of soil sample was done up to104. nutrient agar with 1% soluble starch was prepared onto which an aliquot (0.1 ml) of 102 to 104 dilutions were inoculated and spread plate method was followed. the plates were incubated aerobically at 37o c for 48 hours [14]. colonies that showed big, creamy, wrinkle, and spreading colonies were picked and sub cultured on nutrient agar broth and on nutrient agar slant. gram staining and endo-spore staining were carried out for the presumptive identification of the isolates[14]. identification the test isolate was identified by using standard microbiological techniques as described in bergey’s manual of systematic bacteriology (1986). different biochemical tests viz; catalase, citrate, urease, indole, starch hydrolysis, methyl red voges-proskauer, sugar fermentation test (lactose, mannitol, sucrose), triple nepal j biotechnol. 2021 dec.9 (2): 21-28 koirala et al. ©njb, bsn 23 sugar iron (tsi) and sim were carried out to identify the isolates. substrate preparation the medium used for the experiment was a minimal salt medium supplemented with 10% substrate (mustard oil cake, soya cake and whey). it was prepared by dissolving 1.73 g dipotassium phosphate, 0.68g potassium dihydrogen phosphate, 0.1 g magnesium sulphateheptahydrade, 0.33 g ferrussulphate, 4 g sodium chloride, 1 g ammonium nitrate, 0.02 g calcium chloride and 5 g glucose[15]. inoculation and incubation one ml of 24 hours broth culture of the isolate was pipetted into each flask. all six flasks were plugged with cotton and allowed to stand in water bath shaker for 5 days. temperature of the water bath shaker was maintained at 37o c. after 5 days all flask were taken out from shaker and centrifugation was done to obtain cell free supernatant [15]. oil spreading technique the oil displacement test was performed according to the protocol described by walter (2010). a petri-dish (150 mm diameter) was filled with 40 ml of distilled water. 15 ml of weathered crude oil was added. the crude oil will form a thin oil layer on the water surface. then, 10 ml of free cell culture supernatant was carefully placed on the center of the oil film. if there were microbial surfactants present in the supernatant, the oil was displaced and a clearing zone was formed [16]. determination of emulsification activity 0.5 ml of the extracted supernatant was added to 7.5 ml of 1m tris-hcl buffer and 0.1 ml of oil (kerosene, mustard oil, sunflower oil and engine oil). the mixture was vigorously vortexed and allowed to stand for 1 hour. absorbance was measured at 540 nm 5 times at an interval of 1 hour. after obtaining the absorbance emulsification activity was calculated by calibrated graph [17]. measurement of emulsification index (e24) the bacterial broth was centrifuged and was studied for its emulsifying ability by a modified method of [18]. two ml cell-free broth was pipetted into the screw cap test tube, and 3 ml of oil (kerosene, mustard, engine and sunflower) was then added. the mixture was vortexed at high speed for 2 min and left at room temperature. the result was observed after 24 h for the stability of emulsion. photograph 4 shows the test tubes left for the calculation of emulsification index. the total volume of the mixture, volume of emulsified, and volume of nonemulsified phase was observed [3]. the emulsification index (e24) was calculated by the equation: e24 = heightofemulsionlayer totalheight x100 the method was followed at the end of each day to obtain reading for 5 days. surface tension measurement surface tension was determined at room temperature i.e. 30oc by drop number method by using stalagmometer[19]. basically, the weight of a drop of a liquid falling after passing through a capillary tube of uniform radius is approximately proportional to the surface tension of the liquid. for two liquids of surface tension ϒ1 and ϒ2, d1 and d2 are the densities and n1 and n2 are the number of drops made by the same volume of liquids then the surface tension is calculated as ϒ1 ϒ2 = 𝑑1𝑋𝑛2 𝑛1𝑋𝑑2 pyknometer was used to determine the density of a liquid and stalagmometer was used to determine the number of complete drops made by a liquid [20]. results bacterial isolation and identification the test organism (bacillus subtilis) was isolated from a soil sample of automobile workshop. presence of bacillus subtilis was confirmed based on the macroscopic examination of colonies, microscopic examination and the different chemical tests which were performed as given in table 1. the microscopic examination of the isolates after gram staining is shown in photograph 1 and the biochemical test for the conformation of the isolates is shown in photograph 2. table 1. biochemical tests for confirmation of bacillus subtilis biochemical test bacillus subtilis properties catalase positive oxidase positive citrate positive indole negative mr (methyl red) negative vp (vogesproskauer) positive urease negative starch hydrolysis positive gelatin hydrolysis positive sucrose acid production lactose negative gram staining positive endospore staining positive (central spore) nitrate reduction positive arabinose positive arabitol positive glucose positive glycerol positive nepal j biotechnol. 2021 dec.9 (2): 21-28 koirala et al. ©njb, bsn 24 emulsification activity (ea) figure 1. the emulsification activity of cell free broth extracted from mustard cake figure 2. the emulsification activity of cell free broth extracted from whey figure 3. the emulsification activity of cell free broth extracted from soya cake this study recorded the emulsification activity was observed highest in mustard oil with the cell free extract from mustard oil cake whereas engine oil showed the lowest (figure. 1). emulsification activity in mustard oil from extract from mustard oil cake was found to be 1.13, 0.99, 0.81, 0.95 and 0.76 at the time interval of 1, 2, 3, 4 and 5 hour respectively. however, ea of engine oil was found to be 0.13, 0.06, 0.08, 0.07 and 0.07 at the time span of 1, 2, 3, 4 and 5 hour respectively (figure 1). similarly, ea by extract from whey was also found to be higher of mustard oil. emulsification value of mustard oil was found to be 0.69, 0.58, 0.46, 0.53 and 0.42 at the time span of 1, 2, 3, 4 and 5 hour respectively (figure 2). the ea by the extract from whey was followed by sunflower oil, kerosene oil and engine oil with the lowest emulsification activity (figure 2). ea in mustard oil was again found dominating in the extract from the soya cake followed ea in sunflower oil, kerosene oil and engine oil (figure 3) respectively. the emulsification activity of cell free broth extracted from mustard cake, whey and soya cake are shown in figure 1, 2 and 3 respectively. emulsification index (e24) the emulsification index revealed highest in engine oil by the extract from mustard oil cake whereas mustard oil showed the lowest values (table. 1). corresponding e24 in engine oil by extract form mustard oil cake substrate was found to be 22.09, 28.55, 29.96, 32.85 and 38.82 in the time span of 1, 2, 3, 4, and 5 day respectively while e24 in mustard oil was 1.43, 5.57, 7.27, 9.32 and 12.21 in 1-5 day respectively. similarly, e24 in engine oil with extract from whey was found to be highest with 11.40, 14.48, 23.60, 33.45 and 36.30 in the time span of 1, 2, 3, 4 and 5 day respectively. emulsification index of extract from the whey in different medium was followed by sunflower, kerosene and mustard oil. similar result was found with the extract from soya cake. emulsification index of biosurfactants obtained from mustard oil, whey and soya cake are listed in table 1, 2 and 3 respectively. table 2. emulsification index of bio-surfactants obtained from mustard oil cake time (day) kerosene oil sunflower oil mustard oil engine oil 1 4.44 19.28 1.43 22.09 2 5.80 20.01 5.57 28.55 3 9.26 20.81 7.27 29.96 4 12.50 22.74 9.32 32.85 5 17.86 23.26 12.21 38.82 table 3. emulsification index of bio-surfactants obtained from whey. time (day) kerosene oil sunflower oil mustard oil engine oil 1 0.85 16.40 2.79 11.40 2 3.27 18.38 5.43 14.48 3 6.69 19.62 6.63 23.60 4 9.36 20.08 8.48 33.45 5 11.61 21.29 10.33 36.30 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1 2 3 4 5 a b so rb a n ce ( 5 4 0 n m ) time (hrs) kerosine oil engine oil sunflower oil mustard oil 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 1 2 3 4 5 a b so rb a n ce ( 5 4 0 n m ) time (hrs) mustard oil kerosine oil engine oil sunflower oil 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 1 2 3 4 5 a b so rb a n ce ( 5 4 0 n m ) time (hrs) mustard oil kerosine oil engine oil sunflower oil nepal j biotechnol. 2021 dec.9 (2): 21-28 koirala et al. ©njb, bsn 25 table 4. emulsification index of bio-surfactants obtained from soya cake. time (day) kerosene oil sunflower oil mustard oil engine oil 1 1.77 16.79 3.73 17.76 2 4.90 18.68 4.74 22.01 3 8.54 20.50 6.31 25.80 4 9.08 21.64 7.57 32.70 5 12.84 22.20 8.06 37.05 the emulsification index was found to be increasing every day and the maximum result was obtained at day 5. the comparative study of the emulsification index of three substrates with different oils is given in figure 4. figure 4. bar diagram for comparative study of emulsification index of three substrates with different oil at maximum level (5 days). surface tension the surface tension of water at 30oc was 71.34 dyne/cm. the result calculated from cell free broth of mustard oil cake, whey and soya cake were found to be 54.41, 60.02 and 56.64 dyne/cm respectively (table 4). photograph 3 shows the cell free broth extracted using different substrates. table 5. the surface tension of cell free broths cell free broth surface tension (dyne/cm) mustard cake 54.41 whey 60.02 soya cake 56.64 photograph 1. gram stain of b. subtilis photograph 2. biochemical test (citrate) photograph 3. bio-surfactants extraction (cell free broth) photograph 4. emulsification index of hydrocarbons. discussion bio-surfactants, compounds with such wonderful advantages over chemical ones, have not yet been commercialized significantly as a result of high production cost. since the cost of substrates, chemical ones used more widely, account for 10-20% of the 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 mustard cake whey soya cake e m u ls if ic a ti o n i n d e x kerosene oil engine oil mustard oil sunflower oil i) sunflower oil ii) engine oil iii) kerosene oil i) mustard ii) whey iii) soya nepal j biotechnol. 2021 dec.9 (2): 21-28 koirala et al. ©njb, bsn 26 production cost of bio-surfactants, the production cost is higher [12]. the possible measure to minimize the cost is to reduce the cost of substrate i.e. use of cheap substrates preferably natural substrates like mustard oil cake, soya cake and whey. present study was performed with the objective of identifying the utility of the substrates mentioned above for production of bio-surfactants using bacterial species isolated from oil contaminated soil. from the total 4 soil samples, one isolate of bacillus subtillis was isolated based on its morphological and bio chemical characteristics based on the study of banat et al. [21] and used for the production of bio-surfactant. however, the study couldn’t identify the exact strain as a result of less resources for genetic analysis. the isolate was then accessed for its capacity to produce biosurfactant in mustard oil cake, soya cake and whey. luckily it was able to grow on all the substrate used. the oil spreading technique proved that the isolate has the ability to produce the surfactin as it spread the oil when cell free broth was poured on it. the result is comparable to the study of banat et al.[22]. the produced extracellular metabolites were then compared by calculation of emulsification activity and emulsification index on 4 different oil medium; kerosene oil, mustard oil, engine oil and sunflower oil. the emulsification activity was found to be highest for mustard oil while lowest for engine oil. this shows that the bio-surfactants extracted possessed lower ability to emulsify the mustard oil but has higher to engine oil. since, the habitat of microbial used in the study was from automobile workshop, the organism showed higher ability to emulsify the engine oil as the organism might had adapted to the oil contaminated soil. this study revealed that the emulsification index on all tested oils mediums was found a little higher by the biosurfactant produced in mustard oil cake as compared to soya cake and whey. this might be due to the ability of b. subtilis to utilize nitrogen from various sources for cell multiplication and biosynthetic pathway [23]. this point was also supported by patel and desai [24] where optimum c/n ratio would be a favorable factor in mustard oil cake for the production of surfactants by b. subtilis. bio-surfactant produced from the respective substrate has the highest emulsification index value of 38.82, 37.05 and 36.05% (table 1, 2 and 3) respectively in engine oil while they have the lowest emulsification index value of 12.21, 10.33 and 8.06% (table 1, 2 and 3) respectively in mustard oil. similarly, the emulsification index in sunflower was also found higher value than in the mustard oil. this difference of emulsification index in different medium can be attributed to biochemical properties of the medium. sunflower oil contains the long chain of mono and polyunsaturated fatty acids (91.49 ± 1.91) compared to mustard oil (86.80 ± 3.07) [25]. a similar prediction based on the length of hydrocarbon chain can be made about the higher value of emulsification index in engine oil. however, any solid research is lacking regarding the topic and is open for speculations. the emulsification index in each of the oil medium is found to be increasing gradually with increase in time in case of each of the bio-surfactant extracted. the fifth day showed the maximum value of emulsification index for each of the cell free extracts, which is quiet obvious as the index is found to increase with time in most of the previous similar studies. with increase in time, the emulsion gradually gets back together forming a separate layer as before being vortexed. this study has also revealed that the surface tension of the extracted biosurfactants from the substrate mustard cake, whey and soya cake was found to lie within the normal range as prescribed by [26, 27] many similar studies have identified the bacterial strain as well as the possible substrates of bio-surfactant production. makkar and cameotra[28] have reported the studies on bio-surfactant production by bacillus strains under thermophilic conditions on sucrose and molasses as substrate. al-bahry et al. has reported production of bio-surfactant by bacillus subtilis b20 using date molasses and its possible application in enhanced oil recovery [29]. nevertheless, the mustard oil cake, an agro-industrial waste, could be the potential substrate for the commercial bio-surfactant production as suggested by present study. although the potential of the crude bio-surfactant produced is lower than expected, it cannot be denied that higher potential could have been achieved with better resources. moreover, the extracted crude surfactant was also able to give satisfying result despite of being crude. however, the study could have been considered more achieving if the purity of the crude extract could have been increased. conclusion the result of this study showed that the strain of bacillus subtilis isolated from mustard cake, whey and soya cake was found to be the potential surface active agent producers which are useful tools for various environmental, food and industrial processes. the organism isolated from oil contaminated area showed greater ability to produce bio-surfactants. furthermore, nepal j biotechnol. 2021 dec.9 (2): 21-28 koirala et al. ©njb, bsn 27 each of the substrates obtained from industrial and household waste were found to show great potential as substrate for the production of bio-surfactant. among them, the mustard oil cake substrate is cheap and best among the three studied substrate for bio-surfactant production. author’s contribution conception, data acquisition, analysis and drafting were done by sk, sc, ps, and sg. experimental work was performed by sk, sg, sc, and ps. writing and preparation of manuscript was performed by sc, sg, and sk. analysis and interpretation of data and critical revision of manuscript was done by ps, sns, ge-sb and nm. supervision by nk and funding acquisition was done by ge-sb, nm and nk. final approval of manuscript was done by all the authors. acknowledgement we want to express our sincere gratitude to the helping hand members/staffs of laboratory for making our work more convenient. the authors also appreciate the researchers’ supporting project number rsp-2020/201 from king saud university, riyadh, saudi arabia. we want to thank all the researchers who have previously worked in the realm of this topic. conflict of interest the authors declare that there is no conflict of interest with present publication. declaration we hereby declare 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makkar rs, cameotra ss. biosurfactant production by a thermophilic bacillus subtilis strain. journal of industrial microbiology and biotechnology. 1997 jan 1;18(1):37-42. https://doi.org/10.1038/sj.jim.2900349 29. al-bahry sn, al-wahaibi ym, elshafie ae, al-bemani as, joshi sj, al-makhmari hs, al-sulaimani hs. biosurfactant production by bacillus subtilis b20 using date molasses and its possible application in enhanced oil recovery. international biodeterioration & biodegradation. 2013 jul 1;81:141-6. https://doi.org/10.1016/j.ibiod.2012.01.006 https://doi.org/10.3389/fmicb.2019.00302 https://doi.org/10.1016/j.biortech.2006.05.032 https://doi.org/10.1038/sj.jim.2900349 https://doi.org/10.1016/j.ibiod.2012.01.006 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1):24-41 review article doi: https://doi.org/10.3126/njb.v9i1.38647 ©njb, bsn 24 avian/bird flu: a review: h5n1 outbreaks in nepal dhiraj shrestha1, balkrishna bhattachan2, hiramani parajuli3, and sujata shrestha4 * 1department of microbiology, shi-gan international college of science and technology, kathmandu, nepal. 2biotechnology society nepal, bsn bulletin, bhaktapur, nepal. 3department of microbiology, tri-chandra multiple college, kathmandu, nepal. 4department of laboratory, central jail hospital, kathmandu, nepal. received: 11 dec 2019; revised: 03 feb 2021; accepted: 26 feb 2021; published online: 31 jul 2021 abstract avian/bird flu is a viral disease of birds, caused by avian influenza virus (aiv). a highly pathogenic avian influenza (hpai) h5n1 has breached the barrier of species to humans and other animals escalating the pandemic threat. if the h5n1 evolves to a human-to-human transmissible virus retaining its pathogenicity, it can trigger an influenza pandemic. h5n1 has a mortality rate of about 60%, varying with strains. meaningful antigenic alteration in hemagglutinin (ha) and/or neuraminidase (na) results in recurring pandemics. the hpai h5n1 subtype alone has outreached more than 77 nations around the world since the first human case and death was reported in 1997. wild and migratory birds are the aiv reservoirs. poultry is primarily impacted by incidents and outbreaks of the disease. a wide range of serological and molecular methods have substantially aided in the identification of bird flu in humans. candidate vaccines have been developed, yet are not ready for widespread use. oseltamivir (brand name: tamiflu) is the preferred drug for the management of human influenzalike illness (ili). surveillance, mass awareness, and pandemic preparedness abiding who recommendations are of paramount importance for the prevention of bird flu outbreaks. keywords: avian influenza virus (aiv), avian flu, bird flu, h5n1, nepal corresponding author, email: ssuju.046@gmail.com introduction the word ‘influenza’ has italian origin meaning ‘influence’; from the belief that epidemics were due to the influence of the stars [1]. it became a term for a particular disease after the 1743 epidemic. but, the virus was isolated from a human only in 1933 [2, 3]. avian influenza virus (aiv), better known as bird flu, is a type-a influenza virus of the orthomyxoviridae family. theoretically, owing to the combination of antigens, hemagglutinin (ha) and neuraminidase (na), type-a influenza comprises thousands of distinct antigenic subtypes [4]. to date, 18 subtypes of ha and 11 subtypes of na have been identified, while two extra subtypes of ha and na have been identified in bats [5, 6]. world organisation for animal health (oie) defines aiv as “an infection of poultry by any influenza a virus, including by subtypes h5 and h7” [7]. oie requires notification for all low-pathogenic avian influenza (lpai) virus outbreaks, i.e. h7 and h5 subtypes as they can mutate into highly pathogenic avian influenza (hpai) viruses, as documented in some poultry outbreaks. non-h5 and non-h7 lpai are not deemed notifiable [8]. in certain poultry, such as ducks, some hpai virus (e.g., h5n1) have been shown to cause no illness. hpai has been associated with aiv subtypes h5 and h7, including the viruses h5n1, h7n7, and h7n3. human infections have varied from moderate (h7n3, h7n7) to severe and lethal (h5n1) infections [9]. the hpai h5n1 virus has raised concerns around the world as it threatens poultry, especially chickens; also it has shown the potential to pass from poultry to humans and caused severe infection and death [10]. the first reported human infection of h5n1 occurred in 1997 in hong kong [11]. the virus has been endemic in several countries after the re-emergence of h5n1 in asia, africa, the pacific region, europe, and the middle east in 2003, and continues to cause poultry outbreaks [12]. in dhaka, the presence of the h5n1 virus was confirmed in march 2007 [13]. the first case of aiv was identified at a remote non-commercial poultry farm in kakarvitta, eastern nepal, on january 16, 2009 in nepal [14, 15]. more than 256 h5n1 outbreaks has been witnessed in poultry since 2009 [16]. incidence and prevalence climate change can bring a quick ecosystem shift which alters the evolution and ecology of infectious diseases including aiv. this ecosystem shifts had played a nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0002-3521-2279 mailto:ssuju.046@gmail.com nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 25 cumulative effect in the outbreak of the influenza pandemic in the past century [17]. furthermore, studies show an increase in temperature of freshwater in temperate and arctic regions had a persisting impact altering the suitable habitat variation in prevalence rate and viral spreading [18]. several studies have posited agricultural system, poultry trade, availability of water temperature, salinity as factors resulting in an alteration of aiv ecology [19, 20]. studies have shown that the occurrence of new aivs is supported by either point mutation, partial genes recombination, or genetic reassortment of the whole genome. most new strains of aiv evolved due to point mutations while genetic reassortment performs a key task in the genesis of h5n1 and h7n9 strains [21]. the pathogenicity of aiv is associated with efficient virus replication. host immune responses and the genetic markers are determinants for efficient virus replication [22]. the occurrence of human influenza a (h5n1) often parallels with massive outbreaks of avian h5n1 influenza a, while avian outbreaks in 2004 and 2005 have seldom led to human infection [23]. different types of studies based on geographical distribution and outbreak of aivs revealed that prevalence is related to the migratory route and stages of the itinerary of migrating birds [24, 25]. studies have concluded that asia is the prevalent continent for the avian influenza virus due to a unique ecosystem of various lakes, wetlands, creeks, and rivers that constitute migratory bird wintering areas [26]. due to consequence and the risk represented to human health, aiv is of great concern and are widely studied in recent times. wild birds of genera antidae (ducks, geese, and swans) and laridae (gulls, terns, and kittiwakes) have been in focus for decoding the epidemiological links between hosts and transmissions of aiv [27]. etiology the pandemic influenza virus has its origins in avian influenza [28]. its genomic material is composed of eight segmented negative strand rnas. the influenza a virus shows external spikes when examined with an electron microscope. various techniques can distinguish two kinds of spikes. the ha molecules made up one type and the na molecules the other. there are roughly 5 times as many ha spikes as na spikes [29] (figure 1). the 18 different subtypes of ha and 11 different subtypes of na exist allowing for 198 potential different viral strains. as of 2019, only 131 subtypes have been detected in nature [30]. figure 1. schematic diagram of the influenza a virus with the virus components. note: na=neuraminidase, ha=hemagglutinin, m1=matrix protein-1, m2=matrix protein2, ss rna=single stranded rna, pb2=polymerase basic-2, pb1=polymerase basic-1, pa=polymerase acidic, np=nucleoprotein, ns1=non-structural protein-1, ns2=nonstructural protein-2. genomic variation genetic analyses have indicated that since 1997 the h5n1 virus has evolved into multiple genotypes [31]. whether the genetic modifications in the ha protein are the result of immune escape or are related to host adaptation is not clear [32]. hence, the best h5n1 epitopes is difficult to predict to target with vaccines without understanding the antigenicity of emerging strains. as h5n1 viruses have expanded their geographical and host ranges, it has become increasingly important to determine acquired features that permit successful human-to-human transmission. the transmission of h5n1 viruses to humans has been ineffective, arising either by direct interaction with infected poultry or through ingestion of undercooked meat or blood of infected birds [33]. in influenza a viruses, antigenic drift is a continuous mechanism where point mutations occurs during replication of the viral genome. this mechanism is apparent in all the influenza a virus gene products; however, it is most pronounced in the ha and na glycoproteins’ antibody-binding sites. these mutations cannot be repaired because of the lack of a proofreading mechanism, and the resulting aggregation of variations in the amino acid sequence transforms these antigenic sites in such a way that they are less detectable by the host antibody response. thus, these virus strains are naturally selected as host antibodies no longer no longer neutralized them, and they lead to increased viral fitness [34, 35]. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 26 antigenic variation both the ha and the na undergo antigenic variation [36]. antigenic shift is a major change in antigenicity, while antigenic drift is a minor change sufficient to spark a new outbreak. in the human population, pandemics happened in 1957 (asian influenza) and 1968 (hong kong influenza); both are associated with the presence of antigenically transformed ha molecules. the ha antigenic variation is known to be more important quantitatively than that of the na. the human pandemic virus of 1918 (spanish influenza) was the common ancestor of human and classical swine h1n1 influenza virus [37, 38]. epidemiology avian flu or bird flu was initially known as fowl plague, and later fowl plague virus was discovered as an influenza virus. the outbreak of avian flu was first documented in 1878 in italy. later on, in 1924 and 1929 two massive outbreaks in poultry were recorded in the united states [39]. since the first reported human case and death by the influenza virus in 1997 in hong kong, the eruption of the disease has been documented from wild and domestic birds including humans. hpai h5n1 alone has been reported from over 77 countries [40, 41]. till the date, a total of 860 human cases have been reported since 2003 with more than 50% deaths by h5n1[42]. since 2013, 1,568 human cases and 616 deaths were reported worldwide attributed to the novel h7n9 [43]. geographically the disease is globally prevalent dominating the asia continent showing the higher outbreaks in china, vietnam, india, taiwan, israel, japan, and south korea [26]. host range, transmission, and spread aiv is found distributed in a wide variety of hosts. predominantly free-flying water birds, like geese, ducks, shorebirds, and gulls, are major reservoirs of aivs. besides this, aiv can infect both wild and domestic birds like chicken, turkeys, partridges, pheasants, quails, pigeons, and ostriches [44]. however, the aivs primarily associated with transmission in chickens but not in ducks were found to be adapted to ducks by acquiring genes from duck influenza viruses [45]. aiv is essentially a disease of birds, but evidence has shown that the infection can be transmitted in cats, dogs, eagles, ferrets, hamsters, horses, humans, macaques, marine mammals, mice, minks, pigs, and tigers; but the zoonotic infection had been only reported in china [46]. the transmission and spread of disease were primarily related to poultry contact, yet human-to-human transmission is also possible [47]. moreover, the outbreak of avian flu is also associated with the vicinity of water source and 97.5% of reported cases showed the proximity of water source [26]. virus replication the influenza a virus genome consists of segmented rna encoding 10 proteins [48]. these 10 viral proteins are necessary for a successful infection cycle within immuno-competent hosts. on the surface of the host cells, the ha protein binds to sialic acid, enabling the virus to enter [49]. the host cell endocytoses the virus particles. the endosome maturation lowers the ph triggering a ha conformational change. this results in the membrane fusion of the endosome and the virion. the viral matrix protein-2 (m2) acts as an ion channel that further lowers the ph of the virus particle. this aids in the dissociation of the matrix protein-1 (m1). the virus ribonucleoproteins (vrnps), including polymerase basic1 (pb1), polymerase acidic (pa), and polymerase basic-2 (pb2), are released into the cytosol [50]. for viral replication and transcription, the vrnps are then moved into the host cell nucleus. the nuclear export protein (nep) and m1 traffic out freshly produced vrnps into the cytoplasm and then to the plasma membrane after replication, transcription, and protein synthesis. several viral proteins, including m1 and m2, contribute to budding. finally, in both the viral and host cell membrane, na removes sialic acid from glycoproteins. this stops ha and host cells from interacting, releasing new infective virus particles [1]. nonstructural protein 1 (ns1) counteracts the innate host-cell defense within the infected cell, including interferon, for efficient virus replication [51]. new proteins generated by cotranscription or co-translation with the host are being continuously identified [52]. pathogenesis virulence factors comprise the highly cleavable ha that several cellular proteases can activate, a specific substitution in the polymerase basic protein-2 enhances viral replication [53]. non-structural protein-1 substitution confers improved resistance to interferons and tumor necrosis factor-alpha inhibition [54]. the h5n1 viruses potently induce proinflammatory cytokines in macrophages, the most notable being tumor necrosis factor-alpha, which may contribute to the unusual severity in humans [55]. with antigenicity change [56], constellation of internal gene, expanded range of avian species [57], enhanced pathogenicity [58], and increased environmental stability, these viruses keep nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 27 evolving. despite extensive exposure to infected poultry, comparatively low frequencies of h5n1 infection in humans suggest that the species barrier to acquisition of this avian virus is important [33]. disease in birds avian flu has been reported in many wild and domestic birds. chickens, ducks, swans, gooses, quails, crows, storks, etc. have been known to have been infected with aiv. it is understood that h5n1 infections show distinct clinical presentations in chickens and ducks. the infection is characterized by clinical signs and high mortality in chickens, whereas the infection is generally asymptomatic in ducks. this leads to an underestimation of the prevalence of the disease [59]. infected ducks can thus help to sustain and spread the virus “silently” to other vulnerable hosts [60]. the h5n1 infection in ducks has been considered a threat to the poultry flock and public health due to its silent nature [57]. thus, monitoring and surveillance based on ducks is substantial for aiv control [61]. since ducks may become infected and co-infected with multiple aivs, reassortment of the viral genes is likely [62]. spatial evidence on hpai outbreaks in southeast asia has shown that scavenging ducks lead to hpai outbreaks in domestic poultry [63]. evidence shows that ducks play a major role in the transmission of hpai since they do not exhibit the disease symptoms. characterization of virus in 1996, the hpai h5n1 virus was found in gundong province, china which was thought to have originated from the h5 virus in migratory birds through both drift and shift mutation mechanisms [64]. since then the influenza virus in human cases has evolved and is classified in a different list based on hemagglutinin phylogeny known as a clade. this list of the clade is updated for continuous change [65]. there are 13 major clades in the list distinguished by the hemagglutinin gene of the h5n1 representative subtype of hpai. these clades are determined by sequencing and measuring the average pairwise nucleotide distance (apd) [66]. these clades were further split by measuring the within-clade average pairwise distance to determine the sub-clade resulting in more than 32 clades or sub-clades [67]. clade 0 comprises viruses that were first identified to cause human infections in gundong province, china 1996 (a/goose/guandong/1/96 linage). in the early phase of epidemic (2004-2005), clade 1 viruses predominated in vietnam, thailand, and cambodia, and clade 2.1 viruses are endemic in indonesia. the clade 2.1.3.2b was found to be restricted to a certain geographical area i.e. java, indonesia [68]. a major outbreak of h5n1 disease in migratory birds has been associated with clade 2.2 and the virus is distinct from z genotype virus and was first detected in russia, kazakhstan and later spread, causing avian disease in africa, central asia, europe, the middle east, south asia, and human disease in western africa, asia, and the middle east resulting pandemic in 77 countries [69]. in southern china, clade 2.3 has been dominant and has also been reported in adjacent countries. later on, clade 2.3.2.1c was detected in canada and austria. clade 7 was restricted to china and vietnam in the year 2006-2009 [70]. the pattern of viral replication the characterization of virologic course of aiv h5n1 is yet to be done. many studies reported prolonged viral replication. aiv can be detected in nasopharyngeal or lower respiratory sites ranging from 1 to 16 days. nasopharyngeal replication has been lower in humans as compared to lower respiratory tract replication [71]. aiv has also been documented to replicate in the gastrointestinal tract [72, 73]. the invasive infection has been documented in humans [72]. the ha cleavage site’s polybasic amino acid sequence is linked with visceral dissemination in birds [33]. host immune response (innate type) despite widespread exposure to infected poultry, the comparatively low disease frequencies in humans illustrate the species barrier of aiv [33]. h5n1’s ability to infect birds or humans tends to be partially determined by the ha binding specificity. generally, has of human strains of influenza virus preferentially bind sialic acids bound to the terminal galactose of the oligosaccharides on the cell surface by α 2,6 linkage (sa α 2,6), which is abundant in human respiratory epithelia [74]. the ha of avian strains, on the other hand, binds preferentially to α 2,3-linked sialic acids (sa α 2,3). these linkages are common in the avian intestinal tract [75]. for host cell infection and the dissemination and virulence of influenza viruses, for host cell infection and the dissemination and virulence of influenza viruses, ha interaction with sialylated glycans on the cell surface is important [76]. mutations altering the specificity of the receptor binding of avian viruses may be essential for avian to human crossover, and for enabling direct human-to-human transmission [77]. pathogenesis of disease is contributed by the innate immune response [33]. elevated blood nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 28 levels of interleukin-6, interleukin-8, interleukin-1 b, soluble interleukin-2 receptor, tumor necrosis factor (tnf), tnf-a, interferon-g, monokines induced by chemokines interferon-inducible protein 10, interferon-g, and monocyte chemoattractant protein 1 were observed in the fatal cases [71, 78]. such responses can be attributed to the sepsis syndrome, acute respiratory distress syndrome (ards), and multiorgan failure. corticosteroids are used to minimize such responses. individuals developing specific humoral immune responses can survive [33]. transmission transmission of aiv occurs through direct contact via the inhalation of droplets and droplet nuclei. transmission also occurs perhaps through indirect (fomite) contact and self-inoculation onto the upper respiratory tract or conjunctival mucosa [79]. the data is consistent with bird-to-human, potentially environmental-to-human, and limited human-to-human transmission to date. animal to human transmission is mainly attributed to exposure to live ill poultry and butchering of birds [80]. in addition to plucking and preparing infected birds, handling of fighting cocks, handling poultry notably asymptomatic infected ducks, and eating of undercooked poultry or duck’s blood has also been proposed for transmission of aiv. also, feeding tigers and leopards in zoos with raw, infected chickens has been proposed for transmission of aiv [81]. human-to-human transmission has also been manifested in recent years. but no case of human-to-human transmission through aerosols of small particles or through social contact has been reported. still, severe illness has been reported in clinicians exposed to an infected patient. the survival of h5n1 in the environment outlines the possible mode of transmission. direct intranasal or conjunctival inoculation, and oral intake of contaminated water during swimming is the possible mode of transmission. also, contamination of hand and subsequent selfinoculation is the possible mode of transmission. the use of untreated poultry feces as fertilizer has increased the risk of environmental transmission [82]. clinical features incubation: the incubation period of h5n1 infection is 2–4 days after exposure to infected, sick, or dead poultry [83]. but incubation time can be prolonged up to 8 days [33, 84]. the degree of virus shedding during this time is still unknown [85-87]. initial symptoms the most common and initial symptoms that appear after h5n1 infection is respiratory distress. milder cases show uncomplicated flu-like illness [88]. pneumonia is often of viral origin with no bacterial superinfection, but may differ from cases to cases [78]. crackles on examination of the chest are also seen [89]. other frequently occurring symptoms include a high fever with >38°c, sore throat, cough, shortness of breath, rhinorrhoea, etc. [33]. some cases suffer from diarrhea, vomiting, and abdominal pain apart from typical clinical manifestation [90]. conjunctivitis or upper respiratory infections are not common [87]. further complication includes multi-organ failure like renal disorder, cardiac malfunction, and pulmonary hemorrhage. reye’s syndrome, pneumothorax, ventilator-associated pneumonia, and ards are also seen in some cases [33]. though central nervous system involvement is rare there are cases where convulsion and progressive coma are reported leading to the death if not treated on time [90, 91]. two patients in vietnam were detected with encephalitis only [92]. clinical course a patient infected with h5n1 has a rapid clinical course, developing progressive lower respiratory tract disease and viral pneumonia. mechanical ventilation may be needed depending upon the severity of the case [33]. ards following the multi-organ failure develops in 68% of patients within six days of disease onset [91]. the average duration from onset of disease to hospital admission is four days and from onset to death in critical cases is nine days [93]. few cases of seropositive patients but without typical manifestation are also reported [94]. mortality/morbidity the mortality rate of h5n1 is significantly high and is at an alarming rate accounting for about 60% [42]. but the rate may increase up to 73% in the youth of the age group 10-19 years followed by 18% in the age group 50 years [95]. the mortality rate was even 90% in severe cases [96]. most of the death cases are frequent with late admission [97, 98]. diagnosis specimen for upper respiratory tract infections nasal swab/nasal wash polyester or dacron swab is recommended with an aluminum or plastic shaft. the dry swab is inserted in the internal nares below the inferior turbinate. it is then allowed to absorb secretion for some time and is rotated nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 29 around the area before the withdrawal. the tip is then broken and dipped in viral transport media (vtm) vial. for nasal wash, physiological saline is used [99]. the patient is advised to close the pharynx by saying the letter “k” and the saline is introduced in nostrils one at a time with the head tilted backward and later the saline is collected in a cup or dish by tilting the head forward. the obtained wash is then mixed with the vtm [100]. throat swabs a throat swab is considered to be the high yielding specimen for upper respiratory tract infections and should be obtained within three days after symptoms appeared for the best result [100]. by using the tongue depressor or blade, polyester or dacron swab is gently rubbed several times in the posterior pharynx. the tip is then broken and dipped in a vtm vial [99]. nasopharyngeal secretions a catheter with a mucus trap is inserted in nostrils. by use of a vacuum, the catheter is withdrawn with a rotating motion. after that, the catheter is flushed with a transport medium in a 1:2 ratio [99]. specimen for lower respiratory tract infections an endotracheal aspirate or bronchoalveolar lavage is the best specimen for lower respiratory tract illness. for increasing the potential of viral isolation, multiple specimens can be taken from multiple respiratory sites for at least two consecutive days [72]. blood samples both acute and convalescent serum samples are recommended if feasible. within the first 3-5 days after the onset of symptoms, acute serum samples should be taken. convalescent serum samples collected after 3-4 weeks would be of great use if collected in conjunction with acute samples [99, 101]. other specimens it may be either plasma or rectal swab in diarrhea for the detection of viral. spinal fluid/ tap is also preferred in the case of meningitis [100]. autopsy specimens along with para-mortem biopsies are also recommended [28]. transportation and storage of specimens vtm contains balanced salt solutions, a protein stabilizer, bovine albumin, and a spectrum of antibiotics to minimize bacterial and fungal growth. non-phosphate based vtm is selected if the specimen is only suggested for the pcr test. the collected specimen is placed in vtm and refrigerated at 4°c or with refrigerated packs for transportation to the designated laboratories [100]. the clinical specimens should be well labeled and sent to the laboratory as soon as possible. if specimens cannot be processed, then it should be either stored at 4°c or frozen at ≤-70°c. samples should not be repeatedly freezed0 and thawed [102]. in the case of viral antigen detection by immunofluorescence staining, specimen processing should be done as soon as possible not more than 1-2 hours after collection [83]. a guide for field operations is recommended regarding the collection, storage, processing, and even transportation of samples for h5n1 detection [100]. laboratory diagnosis since most of the respiratory infections show similar manifestations, h5n1 can only be diagnosed in account with endemic areas and contact with dead or infected poultry or with confirmed cases. direct detection of virus or viral antigen virus isolation or reverse transcriptase pcr (rt-pcr) virus isolation is considered to be the gold standard method and can be carried out in the laboratory either by inoculation of embryonated eggs or by using cell lines like mardin-darby canine kidney (mdck). the obtained viral isolate can be later used for the study of pathogenicity, antiviral resistance, and dna sequencing and analysis. but culture needs a biosafety level 3 (bsl3) which is not available at ease. so, rt-pcr is used as the first diagnostic tool in contrast to culture [85, 86]. real-time pcr or quantitative pcr (qpcr) qpcr methods are preferred to conventional rt-pcr. there is a panel of qpcr assays covering even the specific detection of different na genes and ha subtypes like h5, h3, and h1. h5 and n1 specific primers are used as they exclude false-negative results due to mutation in genes [72, 103]. loop-mediated isothermal amplification (lamp) tests are no longer in use [104, 105]. serology the antigen detection for the detection of viral antigens by using specific monoclonal antibodies against h5 and n1 direct immunofluorescence and enzyme immunoassay (eia) are commonly used. the eia method is considered simple, convenient, sensitive, and even applicable as point of care testing (poct). the sensitivity of eia is 1,000-folds low compared to viral isolation. thus, subtype-specific diagnostic methods like rt-pcr should be carried out simultaneously [106]. antibody detection since seroconversion is delayed and needs paired sera, antibody detection provides a retrospective scenario of infection. it can be done by detecting a four-fold rise nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 30 while comparing acute and convalescent sera. hemagglutination inhibition (hi) assay is conventional yet the preferred method. but hi has low sensitivity for the detection of subtype-specific antibodies [107-109]. the use of horse erythrocytes while detecting antibodies against h5n1 shows effective results [110]. one of the studies done in 1997 during the hong kong outbreak reported similar results and reported that the microneutralization test is more effective, reliable, and sensitive as compared to hi [111]. western blot analysis with recombinant h5 is also done for the conformation [112-114]. hematological profile it includes leukopenia, relative lymphopenia, and even thrombocytopenia. disseminated intravascular coagulation occurs but is rare [33, 84]. biochemical parameters levels of liver enzymes are elevated, along with lactate dehydrogenase and creatinine kinase, serum glutamate oxaloacetate transaminase (sgot) and serum glutamate pyruvate transaminase (sgpt) [85, 86]. others blood culture, sputum culture, and csf analysis are carried out in case of serious complications. chest x-ray reveals effusions, multifocal consolidation, lymphadenopathy, and even shows diffuse ground-glass appearance as in the case of ards [71, 78]. differential diagnosis diagnosis of aiv should be differentiated with atypical pneumonia, community-acquired pneumonia (cap), corona-virus disease 2019 (covid-19), endemic respiratory infections, hantavirus pulmonary syndrome, middle east respiratory syndrome (mers), pediatric pneumococcal infections, pneumococcal infections (streptococcus pneumoniae), respiratory syncytial virus infection, seasonal influenza, and severe acute respiratory syndrome (sars) [83, 94, 115]. definition of exposures to poultry and humans human exposure definition differs from the environments and the situation (table 1). table 1. definition of exposures to poultry and humans [116]. exposure to live poultry in occupations poultry-related exposure at workplace (e.g., persons engaged with poultry raising, trafficking, selling, and quarantine) within 2 weeks of the start of symptoms exposure to poultry at live bird markets visiting a live poultry or bird wholesale or retail market within 2 weeks before the start of symptoms exposure to sick or dead poultry close or direct physical contact with infected or dead poultry or poultry products (e.g., meat) or feces within 2 weeks before the start of symptoms exposure to backyard poultry close or direct contact with poultry raised in the backyard within 2 weeks before the start of symptoms any exposure to poultry close or direct or indirect contact to healthy, or sick, or dead poultry (including birds-e.g., chickens, ducks, geese, pet birds, pigeons) in live bird markets, or backyards, or farms, or neighborhoods, or consumption of improperly processed poultry products exposure through patient contact a patient with a history of close contact within 2 weeks before the start of symptoms with a person with a confirmed or suspected influenza h5n1 virus infection (at any time from the day before the onset of symptom to death, or during the period during which the patient was hospitalized) case definitions [117, 118] for prompt diagnosis and treatment, who has defined h5n1 infection as: person under investigation a person who is decided to be investigated for possible h5n1 infection by assigned public health authorities suspected h5n1 case a person with fever (>38ºc) exhibiting unexplained acute lower respiratory distress along with cough, shortness of breath, or dyspnoea. and in the 7 days prior to symptom onset, one or more of the following exposures: a) contact with a person within 1-meter distance during caring, speaking, or touching whether s/he is a suspected, probable, or confirmed h5n1 case. b) exposure either to poultry in an area suspected or confirmed with h5n1 infections not more than a month or wild animals during handling, slaughtering, de-feathering, etc. and even contact with the contaminated feces. c) intake of poultry products raw or undercooked in an area where animal or human h5n1 infections is reported or confirmed in the past month. d) close contact with an animal infected with confirmed h5n1 other than poultry or wild birds (e.g. cat or pig) e) handling animal or human samples in a laboratory or other facility suspected of having the h5n1 virus. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 31 probable h5n1 case (notify who) probable definition 1: a person who satisfies the conditions for a suspected case and additional one of the following criteria: a) infiltrates or proof of acute chest radiograph pneumonia, with proof of respiratory failure (hypoxemia, severe tachypnea) or b) confirmed laboratory influenza a infection but inadequate evidences in laboratory. probable definition 2: a person dying from an unidentified acute respiratory illness who is known to be epidemiologically linked to a probable or confirmed case of h5n1 by time, place, and exposure. confirmed h5n1 case (notify who) a person who satisfies the condition of a suspected or probable case and either of the following findings in a national, regional, or international laboratory of influenza whose h5n1 test results are recognized as confirmatory by who: a) h5n1 virus isolation; b) h5 pcr positive by tests using two separate targets of pcr, e.g. influenza a and h5 ha specific primers; c) on test of an acute serum sample (collected 7 days or less after the start of symptom) and a convalescent serum sample, the h5n1 neutralization titer must be at least fourfold rise. the convalescent neutralizing antibody titer must also be at least 1:80; d) in a single serum sample collected at day 14 or later after the start of symptom a microneutralization h5n1 antibody titer must be at least 1:80 and a positive in a different serological assay, for e.g. titer of at least 1:160 in a horse red blood cell hi or western blot specific to h5. management/treatment hospitalization h5n1 can be a serious disease in humans that needs hospitalization, isolation, and intensive care [119]. antiviral agents the primary therapy is the use of antiviral medication. the who and cdc guidelines (2015) recommend use of a neuraminidase inhibitor [92]. amantadine amantadine interferes with m2 protein and affects the release of infectious viral nucleic acid [120]. it is found to be effective for both prophylaxis and short-term treatment [121]. since most of the h5n1 virus shows resistant patterns to amantadine or rimantadine, combination therapy with oseltamivir is recommended [92]. rimantadine (brand name: flumadine) it inhibits uncoating affecting viral replication. but h5n1 develops resistance to it [92]. oseltamivir (brand name: tamiflu) it affects the receptor of host cells towards viral ha [92]. but the spread of oseltamivir-resistant h5n1 from 2007 to 2009 surprised the influenza community [122]. failures of treatment have been reported in single-drug oseltamivir regimens due to resistance. there are current researches on the relative success of high-dose and/or extended oseltamivir treatment courses [83]. if the use of high-dose tends to be more effective, it will affect the supply of antiviral drugs in the case of a large epidemic, in addition to medical considerations for patients who are moderately or seriously ill [92]. zanamivir (brand name: relenza) in an animal model, it shows better results but has not been tested in humans. but in severe cases, inhaled zanamivir may not be able to reach the distal airways [83, 92]. uricosuric agents probenecid can be used as adjunctive therapy [92]. but no study has been carried out to confirm the suitable dose. studies are still going on other drugs like arbidol and peramivir [123]. immunomodulators low dose corticosteroids are used to treat acute lung injury (ali)/ards caused due to h5n1 [83, 124]. but the long term and high dose use may result in serious complications increasing the probability of opportunistic infections [92]. other immunomodulators used are nonsteroid anti-inflammatory drugs (nsaids), antipyretics, growth hormones, etc. but the use of such intervention modalities has not been proven any significant benefits [125, 126]. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 32 miscellaneous supportive therapy in a critical case these include oxygen therapy, ventilatory support [127], and non-ventilatory treatments for ali/ards [128]. prevention and control immunization based on different studies, three vaccines of h5n1 were licensed as sanofi pateur's vaccine, (monovalent killed vaccine approved by the us food and drug administration (fda), united states), glaxo smith kline's vaccine-prepandrix (approved by the european union), and csl limited's vaccine-panvax (approved by australia) [129-131]. even though none of the vaccines are available for the civilians yet [132]. since the egg inoculation method is not applicable, tissue or cell culture and recombinant viruses are used for the production of vaccine and the adjuvants like aluminum hydroxide can be used to increase immunogenicity [133]. the inactivated vaccines produced by using the h5n1 was found to be immunogenic with high doses of ha [134]. but due to rapid mutation, immunization is not quite effective and practicable [135]. for travelers, prophylaxis by antiviral is not recommended, instead avoiding contact with dead birds and poultry, use of properly cooked food, and if needed use of n95 respirator mask, goggles while traveling in outbreak areas is recommended [136]. disease management for preventing outbreaks the most effective way to prevent an outbreak is to avoid the source of exposure. people should avoid contact with the wild as well as suspected domestic birds and even should avoid contact with contaminated feces or surface. active surveillance and strengthening biosecurity done in poultry workers, animals, and the environment in suspected sites contribute to a great extent. [136]. one health concept or collaboration of different sectors play a vital role in early detection and prevention. proper supervision of animal transportation, inspection on the border while transporting, and provision of isolation stations on different sites play a vital role [137]. skilled manpower in the field of veterinary to help with identifying the diversity and emerging disease will help in early warning [136]. stamping-out, proper carcass disposal, and surveillance and monitoring of animal health are needed [138]. minimizing the occupational risk of exposure and vaccination of exposed poultry workers and animals should be prioritized [136]. pandemic preparedness pandemic preparedness can be implemented effectively only if it is planned by a collaboration of global, federal, and state or local levels. it requires the involvement of public health personnel, different health care professionals, researchers, and even private sectors to prepare effective measures for pandemic situations. thus, the instant response becomes possible that will help to tackle the pandemic scenario in a relevant way [139]. pandemic preparedness includes mass surveillance, continuous monitoring, early diagnosis, formulation of triage policies [140], development and distribution of different medical interventions like therapeutics, personal protective equipment (ppe), the rapid response of health care system, and proper interaction [139]. precautionary measures for general publics as a general precaution, avoiding contact with suspected wild birds, contaminated poultry products, contaminated water, and proper hand hygiene is recommended for the public. besides, awareness programs on the public level regarding the transmission and different preventive measures should be made available by the government [136, 138]. hospital infection control specific measures taken in a hospital setting not only protect the health care workers but also guide them to control the infection that in turn prevents the spread of disease in the community. health workers must ensure the environment cleaning, proper disinfection and must be trained about occupational hazards and various modes of virus transmission [141]. n95 masks are more effective than multiple surgical masks. pre-exposure prophylaxis should be considered if there is evidence of transmission of the virus or likely to be at risk of exposure [33, 142]. chemoprophylaxis by oseltamivir is recommended for a person at high risk. the appropriate dose is 75mg of oseltamivir, a single dose for 7-10 days [143, 144]. household and close contacts there are many routes of transmission for the h5n1 virus as such there are different protective measures to avoid the infection. proper handwashing plays a significant role in avoiding the self-inoculation of the virus, thus preventing the disease [145]. besides, households with illness should follow extra specific measures of postexposure chemoprophylaxis that include oseltamivir [146, 147]. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 33 table 3. major events related to avian influenza in nepal [16, 42, 150-154]. time major events 2004 surveillance of influenza started from jhapa, eastern nepal. oct 2005 first report of serologic evidence of aiv infection (h9n2) in poultry in nepal. 2009 domestic ducks seropositive for antibodies against h5 and h9, but not h7. 2009 with the deployment of real-time pcr (qpcr) at the national public health laboratory (nphl), a molecular diagnostic assay-based influenza surveillance was initiated. jan 2009 the first case of h5n1 detected in the jhapa district, eastern nepal jun 2009 during the 2009 pandemic influenza virus outbreak, detection and molecular characterization of pandemic influenza virus a h1n1 in a human specimen obtained at tribhuvan international airport. oct 2009 in kathmandu valley, the community spread of pandemic h1n1 2009 was identified. apr 2010 highly equipped national influenza centre was established at nphl. apr 2011 for the isolation and characterization of influenza and parainfluenza viruses, the madin darby canine kidney (mdck) cell line was successively cultured and propagated at nphl. jun 2011 from a clinical specimen obtained and stored at nphl, the influenza virus was isolated and characterized successively. nov 2011 the nphl isolated and characterized a total of 28 influenza viruses and sent them to the who collaborating centre (whocc), national institute of infectious diseases, japan. mar 2019 the first human death by h5n1 in nepal. avian influenza in the context of nepal epidemiology and outbreaks history nepal had not observed hpai h5n1 until 2009, although adjoining india and china reported several episodes of outbreaks. in jhapa, a district bordering india and close to bangladesh, the first hpai outbreak was identified in january 2009. although nepal has undergone sporadic hpai outbreaks since january 2009, until january 2012, no clinical outbreaks were identified in the capital city of kathmandu. as per the national agriculture census 2011, nepal has a population of domestic poultry of around 53.6 million birds (table 2). the 45% of this poultry are commercial birds and 55% are backyard birds. in chitwan and kathmandu valley, the majority of the layers and broilers are raised. there were an estimated 58 hatcheries and 800 poultry producers in nepal [148]. most of the parent stocks are imported to nepal from foreign countries. occasional hpai outbreaks has occurred in some of these stocks. in most of these countries, parent flocks are, however, subjected to strict biosecurity measures and national hpai surveillance. it is extremely unlikely that parent stocks are contaminated with the hpai virus. the import from tibet is negligible. however, smuggling from india poses a greater risk of hpai infected parent stock across the border. also, there are minimal biosecurity practices across the industry, and migratory wild birds also use the same bodies of water used by domestically raised ducks. with arrays of wildlife reserves and national parks, migratory birds and wild birds often visit the water bodies in nepal [149]. tfigure 2: map of nepal showing risk districts and wild water bird zones. level of risk as represented by a different color: black color represents the 27 high-risk districts, blue color represents the 18 medium-risk districts, white color represents the 32 low-risk districts, 6 green bubbles represent the wild water bird zones [figure adapted from reference 149]. based on the national hpai surveillance plan, kathmandu and chitwan are identified as high-risk disease areas in nepal (figure 2). the high-risk designation is based on the higher commercial poultry density, the higher poultry influx from other districts, table 2. poultry population and products in nepal [148]. poultry population fowl 45,171,185 ducks 376,916 laying hens 7,907,468 laying ducks 174,978 meat weight in metric tonnes chickens 40,346 ducks 217 eggs numbers in 1000 hens 788,310 ducks 13,060 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 34 the larger free-ranging ducks, the larger natural and manmade ponds and lakes visited by migratory birds annually, the existence of live bird markets, and the lower bio-security of commercial poultry farms [16, 153]. since the first outbreak of 2009, 256 outbreaks have been reported to oie by nepal. outbreaks peaked during 2012 followed by silent 4 years. outbreaks have been resurgent in recent years. most of the infected population includes commercial broilers and commercial layers (table 4) (figure 3) [16]. recently epidemiology and disease control division, ministry of health and population confirmed the first human death by h5n1 on 2nd may 2019. the 21 years old male was admitted on 24th march 2019 and died on 29th march 2019. it has been the first and the only case of identified human death by h5n1 infection in nepal [42, 154]. isolation and study of influenza in human/birds/poultry national influenza centre at national public health laboratory (nphl) is currently recognized by who and so is a member of the who global influenza surveillance network. initially, influenza viruses were detected in suspected cases by rapid diagnostic test (rdt), but now qpcr is employed. the center is primarily focused on collecting appropriate clinical specimens from patients, storing, transporting, and processing. initial identification of virus type and subtype is done and isolates are forwarded to the who collaborating centre for reference and research on influenza and alert the who global influenza programme [152]. figure 3. map showing avian influenza outbreak clusters since 2009 in nepal and its periphery [figure adapted from reference 155]. to date, 256 outbreaks have been reported by nepal to oie. besides these notifications, several surveillance studies have been done in a bird population in nepal. the first report of serologic evidence of aiv infection in poultry in nepal was reported in october 2005 which was later determined to be the h9n2 subtype. this was clear evidence of the introduction of the virus in nepal before 2005. the antibodies to influenza a were detected in the table 4: avian influenza outbreak history in nepal [16]. years total outbreaks new outbreaks district species affected population cases killed and disposed 2019 15 14-feb makwanpur birds commercial layers 9,769 41,125 28-feb kathmandu birds layers 2,600 2,600 06-nov bhaktapur birds commercial broilers 1,535 3,961 17-mar kathmandu birds house crow 200 0 2018 3 03-may chitwan birds commercial layers 1,500 12,091 20-may kathmandu birds ducks 270 240 2017 4 17-feb kaski birds chicken and ducks 98 297 01-mar sunsari birds commercial layers 3,650 2,550 02-mar sunsari birds swans and storks 15 0 2016 0 0 0 2015 0 0 0 2014 1 13-feb sunsari birds commercial layers 570 1,430 2013 0 0 0 2012 210 27-aug lalitpur birds commercial layers 2,500 0 2011 13 10-nov bhaktapur birds chicken and ducks 88 308 2010 8 26-jan kaski birds chicken and ducks 153 11,128 25-oct chitwan birds commercial poultry 66 11,437 2009 2 08-jan jhapa birds poultry 14 24,689 total 256 23,028 111,856 nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 35 chicken while ducks and pigeons were still serologically negative for the virus. this also outlines the absence of hpai and notifiable h5 and h7 subtypes until that time. this also proved the brewing adaptation and introduction of another influenza a viruses in nepal [150]. h5 subtype was not detected in 2007 by the rtpcr test in suspected dead birds [156]. similarly, domestic ducks were found seropositive for antibodies against h5 and h9 but not against h7 in 2009. though none of the seropositive ducks were symptomatic for hpai or lpai virus infection [151]. h9n2 was reported in a fecal sample of migratory birds in southern nepal. however, h7n9 and other hpai viruses were not detected [157]. different studies reported pandemic h1n1 in nepal [158, 159]. pandemic h1n1 detected in nepal was the lineage of the novel influenza h1n1 virus (a/california/07/2009) [159]. hpai virus has been a major threat to the nepalese poultry industry since its outbreak in india in 2003. a substantial number of customers temporarily stopped buying chicken meat and switched to other sources of meat. the loss to entire value chain actors was estimated at nrs 1,154 million per year ($1=nrs. 73.06, the average exchange rate from 2001 to 2010) [160] from 2001 to 2010 [161]. since the 2009 outbreak, the impacts have spilled across the country. the economic loss is even devastating. loss of more than nrs 4.5 million has been attributed to the pokhara bird flu outbreak alone, the third outbreak in nepal. the commercial and backyard farmers including butchers on a small scale are primarily affected by the outbreak. nepal government had tried to compensate for the loss during the outbreak, but the compensation offered is lower as compared to the gate price of the poultry and products [161, 162]. outbreak preparedness in nepal a pandemic imposes an increased pressure on infrastructures to contain the disease, be it a developed nation or a developing nation. developing nations face much more stress in resource mobilization which in the case is already scarce. but in global context, pandemic help to better understand the disease and effects of preventive measures. pandemic preparedness includes the holistic approach from virologists, epidemiologists, animal and human health professionals, military and paramilitary forces, press, media, and administrations. continuous coordination among all parties is warranted for the execution of swift countermeasures in case of a new pandemic outbreak. but as is the case in most developing countries, this co-ordination is still lacking in nepal [163]. besides this, aiv is dynamically evolving and adapting to newer hosts so there is still the need to establish tests that are quick and easy to use to characterize new influenza strains [164]. cross country movements of birds and products should strictly follow oie recommendations. farms should be designed to minimize the interaction with wild bird populations and there should be maximum biosecurity practices [165167]. public awareness level about the disease in nepal different studies reported that most of the nepalese population were aware of aiv and that poultry workers were at risk of infection. televisions, radios, newspapers, and social media had been conveying the message to the general population about aiv. however, on preventive measures, only hand washing was widely accepted by most folks even though the majority of the population was familiar with most of the preventive behaviors. thus, there is a strong degree of acceptance of many specific government regulation policies in public. but the implemented control measures were not considered sufficient even though preventive measures are frequently conveyed by the government (table 4) [168, 169]. policy in controlling outbreaks in nepal between 2007 and 2011, the world bank-funded the aiv surveillance and awareness campaign as the avian influenza control project (aicp) in nepal. since 2011, nepal has continued the aicp with its internal resources. the management program focuses largely on outbreakassociated culling of poultry in tandem with washing and disinfection. control policy is mainly focused on the table 4: excerpt of the preventive measures released by the ministry of health and population, nepal in the public interest [154]. frequent hand washing using soap. personal hygiene and cleanliness. environmental cleanliness. avoid close contact with sick poultry and wild birds. immediately visit nearby health institutions on suspicion of ill birds or persons in the vicinity. keep children far away from dead and sick birds. safe handling and personal safety should be considered while preparing poultry meat. the handler should clean hands and tools after preparing meat. consume only well-prepared poultry meat (cooked to at least 70ºc). don’t do drugs without consultation with physicians. avoid close contact with dead birds or their droppings. bury the dead birds carefully if found. nepal j biotechnol. 2 0 2 1 j u l ; 9 (1): 2 4 4 1 shrestha et al. ©njb, bsn 36 outbreak-related culling of poultry in conjunction with cleaning and disinfection. however, despite of current aicp, the continuing incidence of outbreaks has raised concerns about the effectiveness of the money expended. poultry and product export from nepal is negligible. but domestic demand is nearly self-sufficient, though parent stocks and vaccines are imported. thus, it is crucial to preserve the poultry industries as a pandemic can wipe out the industry owing to the small geography of the country. thus, aicp alone cannot be fully relied upon, instead, the vaccination control program can be implemented along with bio-security, as proven effective in pakistan [170]. nevertheless, there is always a possibility of the circulation of the asymptomatic virus and the future spread of infection [171]. control and management strategies in nepal the control and management of aiv are primarily focused on outbreak zone/s in nepal. once the outbreak is identified, the area is sealed and poultry production is banned for 45 days. surveillance is intensified in the proximity of epidemic regions and other regions at higher risks. the suspected poultry are quarantined and culled. since the first outbreak, 111,856 poultry have been killed to control the further spread. thus, occasional outbreaks in different areas have seriously hampered the poultry industry (table 2). the control and management strategies are thus targeted to outbreak areas only after the outbreak has been identified, rather than focusing on surveillance and adopting preventive measures in the outbreak prone and high-risk areas. the control measures were adopted by nepal to curb aiv outbreaks, as reported to oie (table 5). table 5. measures taken by nepal to curb aiv outbreaks (as reported to oie). control of movement in the country surveillance outside containment and/or protection zone surveillance within containment and/or protection zone screening traceability quarantine official destruction of animal products official disposal of carcasses, by-products, and wastes stamping out disinfection vaccination prohibited no treatment of affected animals conclusion more than 20 years ago, h5n1 in humans was first identified. the reservoirs and key sources of human h5n1 infections are infected birds. human-to-human transmission is very low and initial manifestations of illness are non-specific so detailed histories along with possible travel in endemic areas should be considered for case detection. outbreaks have been recurrent in recent years in nepal. commercial rapid antigen tests are insensitive and only suitable for screening. confirmatory diagnosis can only be done by molecular techniques. oseltamivir has been warranted for the treatment of severe cases. the understanding of epidemiology, natural history, and human h5n1 disease control is still inadequate; thus, warranting the need for co-ordination in clinical and epidemiological research among institutions globally. author’s contribution bb and hp compiled the literature review. ds and ss prepared the initial draft. ds finalized the draft. all authors read and approved the final draft. competing interest the authors declare no conflict of interest or competing interest. acknowledgements none references 1. dictionary m-w: influenza. in: merriam-webster dictionary. merriam-webster corporation; 2019. 2. smith w, andrewes ch, laidlaw pp: a virus obtained from influenza patients. the lancet 1933, 222(5732):66-68. https://doi.org/10.1016/s0140-6736(00)78541-2 3. verhoeyen m, fang r, jou wm, devos r, huylebroeck d, 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2004. 166. dharma k, chauhan rs, kataria jm, mahendran m, tomar s: avian influenza: the current perspectives. j immunol immunopathol 2005, 7:1-33. 167. dharma km, mahendran m, tomar s: pathogens transmitted by migratory birds: threat perceptions to poultry health and production. int j poult sci 2008, 7:516-525. https://doi.org/10.3923/ijps.2008.516.525 168. neupane d, khanal v, ghimire k, aro ar, leppin a: knowledge, attitudes and practices related to avian influenza among poultry workers in nepal: a cross sectional study. bmc infectious diseases 2012, 12(1):76. https://doi.org/10.1186/1471-2334-12-76 169. sah jk, chiluwal s, yadav sk, jha d: a study on knowledge and preventive practices related to avian influenza among higher secondary school students of rajbiraj municipality, nepal. al ameen j med sci 2017, 10(4):276-280. https://doi.org/10.15406/mojph.2016.04.00091 170. naeem k: the avian influenza h7n3 outbreak in south central asia. avian diseases 2003, 47:31-35. 171. ellis tm, leung cy, chow mk, bissett la, wong w, guan y, malik peiris j: vaccination of chickens against h5n1 avian influenza in the face of an outbreak interrupts virus transmission. avian pathology 2004, 33:405-412. https://doi.org/10.1080/03079450410001724012 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 doi: https://doi.org/10.3126/njb.v7i1.26957 ©njb, biotechnology society of nepal 82 nepjol.info/index.php/njb. original research article screening of methicillin resistant staphylococcus aureus (mrsa) from wounds in pediatric patients visiting tertiary care in hospital ganesh bhandari1, basant pokhrel1, yogesh oli2, archana katuwal2, netra lal bhandari1* 1department of chemistry, tri-chandra multiple campus, tribhuvan university, kathmandu, nepal 2department of microbiology, tri-chandra multiple campus, tribhuvan university, kathmandu, nepal abstract the extent of methicillin-resistant staphylococcus aureus (mrsa) in children is still unknown. the collected wound pus samples were processed. identification of s. aureus was done according to standard microbiological procedures as per the clinical laboratory standards institute (clsi) guidelines (2016). the antibiogram of the isolates was carried out by the kirby-bauer disc diffusion technique. mrsa was determined by measuring the zone of inhibition (zoi) surrounding to cefoxitin disc, with resistance defined as zoi of ≤ 21 mm. out of 357 bacterial culture-positive samples, 278 (77.87 %) were s. aureus isolates, among them 102 (36.69%) were found to be mrsa. the percentage of mrsa isolates was found high in male children and inpatients with 61.76 % and 73.52% respectively. all the mrsa isolates were susceptible to gentamicin (79.41%), whereas (91.17%) were resistant to penicillin. the distribution of mrsa in inpatients 75 (73.52%) is higher than that of outpatients 27 (26.74%). this study shows that the mrsa occurrence is prevalent in pediatric patients. keywords: staphylococcus aureus, mrsa, antibiotic susceptibility test, disc-diffusion, methicillin resistant *corresponding author email: netra.tu.edu@gmail.com introduction staphylococcus aureus (s. aureus) is one of the important human pathogen known over the past several decades. it is the cause of hospital and community-acquired infections [1]. it is one of the versatile nosocomial pathogens worldwide causing skin infections to life-threatening systemic illnesses including pneumonia, osteomyelitis and endocarditis [2, 3]. s. aureus is one of the common and important gram-positive hospital-acquired organisms. it can cause infection; most commonly at sites of lowered host resistance such as damaged skin or mucosal membranes, infections like pimples, boils and abscesses to severe systemic infections like bacteremia, endocarditis, pneumonia and toxic shock syndrome [4, 5]. s. aureus resistant to oxacillin, methicillin and a few others related antibiotics are all known under the generic term methicillin-resistant s. aureus. the penicillin-resistance s. aureus (prsa) is typically coincides with the emergence of mrsa [6]. mrsa is a global public health problem, associated with considerable morbidity and mortality, causing both hospital and communityacquired infections [7]. among the many species of antibiotic-resistant bacteria, mrsa is one of the most important causes of antibiotic treatment failure, increased morbidity and mortality. s. aureus has a remarkable ability to colonize the skin and mucous membranes. hospitalized patients and health-care workers have higher colonization rates than the general population [8]. studies revealed that nearly 6% hospital personnel and nearly 3% outpatients carry mrsa [9-12]. the prevalence of mrsa has varied from hospital to hospital in various countries. several types of research and studies conducted in nepal about s. aureus and their antibiotic susceptibility pattern suggest the gradual emergence of mrsa in hospitals. however, only a few reports related to mrsa are available in nepal which is carried out in different hospitals at different periods. in a bacteriological study carried out at tribhuvan university teaching hospital (tuth), the prevalence of mrsa was found to be 23.5% [13]. a similar study carried out at kanti children’s nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 83 nepjol.info/index.php/njb. hospital showed 31.4% of mrsa isolates and 11.7% from tuth [14]. rajbhandari et al. (2002) reported 54.9% strains of mrsa [15]. similarly, kumari et al. (2008) reported 26.14% mrsa strains in a study carried out in a tertiarycare hospital in eastern nepal [16]. sanjana et al. (2010) also reported 39.6% methicillin-resistant s. aureus isolates at the college of medical sciencesteaching hospital, chitwan. tiwari et al. (2009) reported mrsa isolates were resistant to co trimoxazole was 77% and 81.7% (ansari et al. 2014). pant et al. (2018) reported 82.6% mrsa resistance to chloramphenicol [17-20]. the prime focus of the study is the distribution and prevalence of s. aureus and mrsa in wound samples collected in the microbiology laboratory of international friendship children’s hospital as well as on its antibiotic sensitivity pattern. the study will also demonstrate the present scenario of mrsa in children and the sensitivity pattern of different antibiotics used against it. this is useful for future planning and policy making in health care centers and hospitals in order to combat the spreading of infectious diseases. materials and methods sample collection and transportation clinical specimens were collected, stored, transported and processed in the laboratory immediately after collection by following standard microbiology guidelines [21]. the sterile swab was moved across the surface of wounds in a zigzag motion and pus aspirates were collected aseptically by puncturing the wound using a sterile syringe. the accurate, rapid microbiological diagnosis of mrsa and s. aureus infections begins with proper specimen collection and rapid transportation to the laboratory. to make sure the collection of the best possible specimen, the health care workers (hcws) must be properly trained, and the patient must be provided with clearly presented and fully understood instruction for sample collection. specimen processing pus samples received in the microbiology laboratory were processed as per routine standard microbiological procedures described by the established guideline for the isolation and identification of s. aureus [22]. culture of specimen all the pus samples were inoculated on blood agar (ba) and mac-conkey agar (ma) plates and incubated aerobically at 37 ̊ c for 24 to 48 hours in the incubator. if no growth was observed, it was reported as growth negative. the suspected s. aureus isolates (on the basis of haemolysis produced on ba) were inoculated in to mannitol salt agar (msa) to check if the isolates ferment mannitol or not. photograph of s. aureus culture on blood agar and msa is pictured in figure 1. figure 1. photograph of s. aureus culture on (a) blood agar (b) mannitol salt agar (msa) figure 2. photograph of antibiotic sensitivity test of s. aureus on mha. a: gentamicin (30mcg), b: chloramphenicol (30mcg), c: cefoxitin (30mcg), d: ciprofloxacin (5mcg), e: penicillin (10mcg) b a nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 84 nepjol.info/index.php/njb. figure 3. flow chart representing isolation and identification of mrsa from pus samples identification of the isolates the growth was identified as s. aureus by using conventional biochemical methods as per standard microbiological techniques described in bergey's manual of determinative bacteriology [23]. it comprises colonial morphology, staining reactions and various biochemical properties such as catalase-positive, coxidase negative, fermentative, voges-proskauer positive, etc. preservation of isolates isolates in pure culture were preserved in glycerol 20% (v/v) in brain heart infusion broth (bhi) at –20 ̊ c to -70 ̊ c and recovered by sub culturing in brain heart infusion broth at 37 ̊ c for 24 hrs followed by further subculture on nutrient agar. antimicrobial susceptibility testing of s. aureus antibiotic susceptibility testing of isolates was assessed by the modified kirby-bauer disk diffusion method as recommended by the clinical and laboratory standard institute using mueller hinton agar (mha) [22]. using a sterile wire loop, a single isolated colony of which the sensitivity pattern is to be determined was touched and inoculated into a nutrient broth (nb) tube and was incubated for 2-4 hours. after incubation in a good light source, the turbidity of the suspension was matched with the turbidity of the standard of mac farland 0.5 (prepared by adding 0.6ml of 1% w/v barium chloride solution to 99.4ml of 1% v/v solution of sulphuric acid) [24]. using a sterile swab, a plate of mueller hinton agar was inoculated with bacterial suspension using carpet culture technique and plates were taken for incubation at 37 ̊ c for 18-24 hours. the result was interpreted as whether the organism was sensitive or resistant to the tested antimicrobial agents. photograph of antibiotic sensitivity test of s. aureus on mha is shown in figure 2. detection of mrsa mrsa detection was done by the cefoxitin disc (30µg) diffusion test. as per clsi (2015) guideline, a lawn culture was made on mueller hinton agar (mha) supplemented with 4% nacl and cefoxitin disc from the suspension of turbidity equivalent to 0.5 mc farland standards from overnight growth in nutrient agar and incubated for 24 hours aerobically at 35 ̊ c. after incubation, the plates were examined for zone of inhibition. isolates exhibiting zone of inhibition ≥ 22 mm were considered as sensitive, whereas those showing zone of inhibition ≤ 21 mm were considered as resistant and were reported as mrsa. purity plate the purity was used to ensure that the inoculation used for the biochemical tests was. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 85 nepjol.info/index.php/njb. table 2. gender wise distribution of patients (p > 0.05) male female total culture positive 196(54.9) 161(45.09) 357(54.9) culture negative 162 (45.2) 131 (44.8) 293(45.1) total 358 (55.1) 292 (44.9) 650 (100) pure culture and also to see whether the biochemical tests were performed in aseptic condition or not. thus, while performing biochemical tests, in biochemical media, the same inoculum was sub-cultured on one half of nutrient agar (na) medium before (pre purity) inoculation and another half after (post purity) inoculation. the maintenance of aseptic condition is indicated by the growth of the same organism in pure form in both pre and post halves of the medium. the chart below represents the isolation and identification of mrsa from pus samples. all the raw data collected in the microbiology laboratory were statistically analyzed by using computer-based software program spss version 20. the flow chart representing isolation and identification of mrsa from pus samples is shown in figure 3. results and discussion bacterial growth in different pus samples and gender wise distribution of patients out of 650 total samples, 550 samples were pus swab and 100 samples were pus aspirate. among total pus swabs 303 (55.09%) were culture positive and 247 (44.91%) showed no growth. moreover, among total pus aspirates, 54 (54%) were culture positive and the remaining 46 (46%) showed no growth as shown in table 1. out of total cases, 358 (55.07%) were male patients and 292 (44.92%) were female patients. the growth was found to be higher in male patients 196 (54.9%) than in female patients161 (45.09%). the association between gender and growth of bacteria was not statistically significant (p-value > 0.05) is presented in table 2. among 650 pus samples, the majority i.e. 352 (54.15%) were in-patients who were admitted to the different departments in the hospital and 298 (45.23%) were from opd. a total 221 (62.78%) of inpatient department (ipd) and 136 (45.36 %) of out patient department (opd) showed growth. such distribution pattern of growth on the basis of the department was statistically significant (pvalue <0.05) is as in table 3. distribution of s. aureus in pus sample among the culture-positive specimens, 278 (77.87%) s. aureus isolates were obtained while remaining 79 (22.12 %) were other bacterial isolates which include both gram-positive coagulase negative staphylococcus species (cons) and gram-negative bacteria (e. coli, pseudomonas aeruginosa, klebsiella pneumonia and enterobacter aerogens) as shown in pie chart in figure 4. distribution of clinical isolates of s. aureus according to age and gender of the patient among 278 s. aureus isolates, a higher number of isolation was found in male 169 (60.79%) as compared to female 109 (39.20%) patients. in the male, the maximum number of s. aureus isolates (n=87, 51.47 %) was observed in the patient with an age group of 1-5 and in females, the maximum table 1. bacterial growth in different pus samples types of sample growth (no. %) no growth (no. %) total pus swab 303(55.09%) 247(44.91%) 550 pus aspirate 54 (54%) 46 (46%) 100 total 357 (54.92) 293 (45.08) 650(100) table 3. distribution of bacterial growth on the basis of hospital department organism ipd {n (%)} opd {n (%)} total {n (%)} p-value culture positive 221 (62.78) 136 (45.36) 357 (54.92) culture negative 131 (37.21) 162 (54.36) 293 (45.07) p < 0.05 total 352 (54.15) 298 (45.84) 650 (100) nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 86 nepjol.info/index.php/njb. figure 4. distribution of s. aureus in pus sample. number of s. aureus isolates (n=55, 50.45 %) was observed in the same 1-5 age group patients. likewise, the least number of s. aureus isolates (n=12, 7.10%) was observed in 11-15 age group patient in male and female least number of s. aureus isolates (n=9, 8.25 %) was observed in the same 11-15 age group patients is shown in table 4. table 4. distribution of clinical isolates of s. aureus according to age and gender of the patient age group(in years) gender total male female no. % below 1 1-5 6-10 11-15 32 87 38 12 18 55 27 9 50 142 65 21 17.98 51.07 23.38 7.55 total 169(60.79%) 109(39.2%) 278 100.0 comparison of clinical isolates of s. aureus in inpatients and outpatients among 278 s. aureus isolates, 177 (63.66 %) isolates of s. aureus were from inpatients whereas 101 (36.33%) s. aureus were from outpatients. similarly, among 79 growths other than s. aureus isolates, 47 (59.49 %) and 32 (40.50%) cases of cultures other than s. aureus were found from inpatients and outpatients respectively. a comparison of clinical isolates of s. aureus in inpatients and outpatients is pictured in figure 5. distribution of mrsa among s. aureus and in different age groups out of 357 culture-positive isolates, 278 (77.87 %) were s. aureus. out of total s. aureus; 102 (36.69%) were mrsa and 176 (63.30 %) were mssa (methicillin-sensitive staphylococcus aureus). out of 169 (60.79%) s. aureus isolates among male patients, 63 (61.76%) were found to be mrsa. likewise, among 109 (39.20 %) s. aureus isolates among female patients, 39 (38.23 %) were found to be mrsa. such distribution of mrsa based on gender was not statistically significant (p-value; > 0.05) is given in the pie chart in figure 6. figure 5. comparison of clinical isolates of s. aureus in inpatients and outpatients figure 6. distribution of mrsa among s. aureus out of 278 s. aureus isolates, altogether 102 (36.69%) and 176 (63.30%) were identified as mrsa and mssa respectively by cefoxitin disc diffusion test which constituted 75 (73.52%) mrsa and 101 (57.38%) mssa in admitted patients. similarly, in outpatients, 27(26.47%) mrsa and 75(42.61%) mssa were observed. the association in the mrsa occurrence and inpatients was statistically significant (p = 0.024) is in table 5 77.87% 22.12% s. aureus others 59.49% 40.50 % 63.66% 36.33% 0 20 40 60 80 100 120 140 inpatient outpatient others s. aureus 63.30 % 36.69% mssa mrsa table 5. distribution of mrsa in outpatients and inpatients inpatients (no. %) outpatients (no. %) total (no. %) p-value mrsa mssa 75(73.52%) 101 (57.38%) 27 (26.47%) 75 (42.61 %) 102 (36.69%) 176 (63.30%) p < 0.05 total 176 102 278 (100.0) nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 87 nepjol.info/index.php/njb. antibiotic susceptibility pattern of s. aureus isolates the antibiotic susceptibility pattern showed that the highest number of isolates were resistant to penicillin (n=250, 89.92%), ciprofloxacin (n=178, 64.02%), ofloxacin (n=162, 58.27%), cotrimoxazole (n=153, 55.04%), erythromycin (n=112, 40.28%), cefoxitin (n=102, 36.69%) and cloxacillin (n=98, 35.25%). the highest number of penicillin resistant s. aureus developed the ability to inactivate the βlactam ring of penicillin by producing plasmid encoded βlactamase [25]. similarly, highest number of mrsa isolates were susceptible to gentamicin (n=264, 94.96%) is due to the combination βlactam and amino glycosides which increases bacterial killing invitro and in animal model of endocarditis, followed by chloramphenicol (n=255, 91.72%), cefotaxime (n=236, 84.89%) and amikacin (n=236, 84.89%), cloxacillin (n=180, 64.74%), cefoxitin (n=176, 63.30%), erythromycin (n=166, 59.7%) is as shown in table 6. antibiotic susceptibility pattern of mrsa and mssa isolates the isolates of s. aureus were broadly categorized into two groups: mrsa and mssa. both the groups of s. aureus showed marked variation in the sensitivity pattern against common antibiotics. it was observed that, for mrsa, the most effective antibiotic was cefotaxime (n=83, 81.37%) followed by gentamicin (n=81, 79.41%), amikacin (n=77, 75.49%), chloramphenicol (n=76, 74.50%). similarly, highest resistance of mrsa was with penicillin (n=93, 91.17%) followed by ciprofloxacin (n=85, 83.3%) and cotrimoxazole (n=77, 75.49%) is in table 7. conclusion mrsa has acquired resistant to previously effective antimicrobials including in the penicillin, methicillin in comparison over the past 50 years. in hospital infected patients and health carriers are also important source of nosocomial s. aureus [26, 27]. out of 278 s. aureus isolates isolated from pus samples, 102 (36.69%) were methicillin-resistant (mrsa). from this study it can be concluded that mrsa infection in pediatric patients is still one of the most threatening infections in hospitals of nepal. the rate of mrsa occurrence was found to be higher in inpatients compared to outpatients and the male patient than female patients. the highest number of mrsa isolates was found in 1-5 age table 6. antibiotic susceptibility pattern of s. aureus isolates antibiotics used total no of s. aureus isolates =278 sensitive % resistant % amikacin ciprofloxacin cotrimoxazole cefoxitin erythromycin cloxacillin cefotaxime penicillin ofloxacin chloramphenicol gentamicin 236 100 125 176 166 180 236 28 116 255 264 84.89 35.97 44.96 63.30 59.7 64.74 84.89 10.08 41.72 91.72 94.96 42 178 153 102 112 98 42 250 162 23 14 15.10 64.02 55.04 36.69 40.28 35.25 15.10 89.92 58.27 8.27 5.03 table 7. antibiotic susceptibility pattern of mrsa and mssa isolates antibiotics used mssa(n=176) mrsa(n=102) sensitive % resistant % sensitive % resistant % amikacin 163 92.61 13 7.38 77 75.49 25 24.50 ciprofloxacin 91 51.70 85 48.29 17 16.60 85 83.33 cotrimoxazole 93 52.84 83 47.15 25 24.5 77 75.49 cefoxitin 176 100.0 0 0.0 0 0.0 102 100.0 erythromycin 114 64.77 62 35.22 59 57.84 43 42.15 cloxacillin 151 85.79 25 14.20 76 74.50 26 25.49 cefotaxime 161 91.47 15 8.52 83 81.37 19 18.62 penicillin 35 19.88 141 80.11 9 8.82 93 91.17 ofloxacin 88 50.00 88 50.00 26 25.49 76 74.50 chloramphenicol 165 93.75 11 6.25 76 74.50 26 25.49 gentamicin 167 94.88 9 5.11 81 79.41 21 20.59 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 88 nepjol.info/index.php/njb. group patients. the most effective antibiotic against mrsa in pediatric patients was found to be gentamicin. similarly, chloramphenicol, cefotaxime were also found to be effective against mrsa infections whereas penicillin was found to be the least effective antibiotic to treat mrsa infections where 89.92 % of s. aureus isolates showed resistance towards this antibiotic. acknowledgements authors would like to thank kanti children's hospital (kch), maharajgunj, kathmandu, nepal for laboratory facilities. thanks also go to department of microbiology and department of chemistry, tri-chandra multiple campus, tribhuvan 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fishbain j, eberly b, viscount h, et al: rates of carriage of methicillin-resistant and methicillinsusceptible staphylococcus aureus in an outpatient population. infect control hosp epidemiol. 2003 24 (6): 439–444. 13. pokharel j, rawal k: bacteriological study at t.u teaching hospital, kathmandu. nepal j institute med. 1993, 15: 217-221. 14. lamichhane r, adhikari rp, sherchand jb: study of methicillin-resistance staphylococcus aureus (mrsa) isolated from different clinical samples. a dissertation submitted to the central department of microbiology, tribhuvan university, kirtipur, nepal 1999. 15. bhandari rr: prevalence and antibiotic sensitivity pattern of methicillin-resistance staphylococcus aureus (mrsa) in bir hospital. a dissertation submitted to the central department of microbiology, tribhuvan university, kirtipur, nepal 2002. 16. kumari n, mohapatra tm, singh yi: prevalence of methicillin-resistant staphylococcus aureus (mrsa) in a tertiary-care hospital in eastern nepal. j 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informational supplement, document m100–s25. wayne, usa 2105, 35 (3). 23. bergey dh, buchanan re, gibbons ne: bergey’s manual of determinative bacteriology. williams and wilkins usa 1974. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 82-89 bhandari et al. 2019 ©njb, biotechnology society of nepal 89 nepjol.info/index.php/njb. 24. cheesbrough m: microbiological tests district laboratory practice in tropical countries, part-ii. low price edition, cambridge university press, india 2006, 01-267. 25. lencastre dh, oliveira d, tomasz a: antibiotic resistant staphylococcus aureus: a paradigm of adaptive power. curr opin microbiol. 2007 10(5): 428-435. 26. lelievre h, lina g, jones me: emergence and spread in french hospitals of methicillinresistant staphylococcus aureus with increasing susceptibility to gentamicin and other antibiotics. j clin microbiol. 1999 37: 34523457. 27. pant j, rai sk, singh a, lekhak b, shakya b, ghimire g: microbial study of hospital environment and carrier pattern study among staff in nepal medical college teaching hospital. nmcj. 2006 8: 194199. 6 nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 49‐54  49  biotechnology society of nepal (bsn), all rights reserved   brief communication   in vitro cultivation and regeneration of solanum  melongena(l.) using stem, root and leaf explants.  bishnu pada ray, lutful hassan and smreeti kana sarker  biotechnology laboratory, department of biotechnology, bangladesh agricultural university (bau), mymensingh.  abstract  the treatment combinations was bap (0, 2.0, 3.0 and 4.0 mg/l) and naa (0, 0.1, 0.5, and 1.0 mg/l). the rate of  callus formation varied  in different treatments. the highest amount of callus (48.66%)  was produced on ms  medium containing 2.0 mg/l bap and 0.5 mg/l naa from stem and 8.2 days required for callus induction. the  number  of  shoot  regenerated  through  callus  from  stem  containing  2.0  mg/l  bap  and  0.5  mg/l  naa  was  3.4  (23.287%) and days required for 38.8 days.     key words: regeneration, bap, naa.    correspondence author:  e‐mail: bpray2003@yahoo.com  introduction  brinjal (solanum melongena l.), belongs to the family  solanaceae, is one of the most popular, palatable and  nutritious  vegetable  crop  in  bangladesh.  it  is  thought  to  be  originated  in  indian  subcontinent  with  the  secondary  centre  of  origin  in  china  (zeven  and  zhukovsky, 1975). brinjal  is cultivated throughout the  entire  tropics  and  sub‐tropics.  it  has  higher  calorie,  iron, phosphorus and riboflavin than tomato. brinjal is  the second most important vegetable crop after potato  in respect of total acreage (million ha) and production  (370,000 mt) in bangladesh (bbs, 2003). it also plays a  vital  role  in  the  national  economy  as  a  cash  crop.  brinjal  is  highly  susceptible  to  different  insects,  pests  and  diseases  that  exert  a  deleterious  effect  on  yield,  market quality, storability and international germplasm  distribution.  the  seed‐borne  pathogens  of  previous  years  can  be  perpetuated  over  the  generations  with  symptoms expressed. to overcome this situation, plant  tissue culture offers an efficient method for pathogen  free  materials  and  germplasm  preservation  of  plants.  the potential value of tissue culture in plant breeding  has been widely recognized, and it is generally used as  useful  tool  for  crop  improvement.  regeneration  of  valuable economic plants through tissue culture based  on the principle of totipotency,  individual plant cell  is  capable  of  regenerating  new  plantlets.  anwar  et  al.  (2002)  cultured  the  aborigine  leaf  explants  on  ms  media containing iaa, ba (benzyl adenine), iba, naa or  2,4‐d at 2 mg/l. naa produced greenish, fast‐growing  callus. 2, 4‐d induced early callus production from the  petiole, while ba induced green callus production from  the upper surface of the  lamina. the addition of naa  or  iba  at  0.5  mg/l  in  ba  supplemented  medium  increased  the  mass  production  of  callus  and  shoot  regeneration. the regeneration efficiency of the plant  decreased in ms medium supplied with kinetin (2 mg/l)  and naa (0.5 mg/l).     the  seeds  of  brinjal  cv.  jhumki  were  collected  from  bangladesh  agricultural  research  institute  (bari),  joydebpur, gazipur and stem, root and leaf were used  for  establishment  of  culture.  healthy  seeds  of  brinjal  cv.  jhumki  were  collected  from  the  bari.  the  seeds  were  then  washed  thoroughly  in  running  tap  water.  the  instruments  like  scalpels,  forceps,  needles  etc.  were  sterilized  inside  the  laminar  air  flow  cabinet.  other requirements like petridishes, distilled water and  glassware were sterilized by an autoclave. the surface  sterilization of these seeds was carried out by dipping  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 49‐54  50  biotechnology society of nepal (bsn), all rights reserved   and flaming method under a laminar air flow cabinet  and  others  were  rinsed  in  70%  ethyl  alcohol  for  one  minute,  and  then  thoroughly  washed  with  sterilized  distilled  water.  the  alcohol  treated  seeds  were  sterilized  with  0.1%  hgcl2  solution  for  8‐10  minutes,  few drops tween‐20 per 100 ml was also added at that  time.  the  seeds  were  then  washed  5‐6  times  with  sterilized distilled water. the seeds were then ready for  placement into the media. sterilized seeds were placed  into  seed  germination  medium  in  petridish.  six  seeds  were  placed  in  each  petridish.  the  culture  was  then  incubated  in dark till the germination of seeds. these  were  then  transferred  to  16  hours  light  for  normal  seedling growth. ms (murashige & skoog, 1962) basal  medium  with  different  concentrations  and  combinations of bap (0, 2.0, 3.0 and 4.0 mg/l) and naa  (0,  0.1,  0.5  and  1.0  mg/l)  were  used.  six  pieces  (2‐3  mm) of stem segments were arranged horizontally on  each petridish  and  gently pressed  into  the  surface  of  the  sterilized  culture  medium  with  various  concentrations  and  combinations  of  hormones  like  naa  and  bap.  the  petridish  was  covered  and  sealed  with  para  film.  leaf  segment  from  each  germinated  seedling  were  cut  into  small  pieces  using  sterilized  scalpel under a laminar air flow cabinet. six pieces of  leaf  segments  were  arranged  on  each  petridish  and  gently pressed into the surface of the sterilized culture  medium.  the  petri  dishes  were  covered  and  sealed  with para film. root tip segments (0.5mm) were placed  on  a  sterilized  petridish  under  a  laminar  air  flow  cabinet.  the  petridish  was  covered  and  sealed  with  para film.  plant regeneration from induced calli of brinjal through  ms  medium  supplemented  with  different  combinations of hormones was used. stem,  leaf, root  segments  were  used  as  explants  to  observe  their  callusing response. thirty explants were  inoculated  in  each  treatment.  among  the  explants  used,  stem  was  comparatively  more  responsive  for  callus  induction  than other explants. the combined effect of explants  and different combinations of bap and naa on callus  induction has been presented in table 1.stem showed  the  highest  callusing  mean  (8.363)  whereas  leaf  segments  gave  callusing  mean  (6.950)  and  root  segments had the  lowest callusing mean (6.688). the  highest callusing was obtained in 2.0 mg/l bap (8.567)  and 0.5 mg/l naa (9.333). also minimum days (9.725)  were  required  for  callus  induction  from  stem.  days  required for callus  induction from 2.0 mg/l bap were  10.350 and days required for callus induction from 0.5  mg/l were 10.367. in case of stem, among the different  combination  of  ms  media  containing  2.0  mg/l  bap  +  0.5 mg/l naa and 4.0 mg/l bap + 0.5 mg/l naa showed  better  callus  induction  i.e.  14.600  and  11.600  respectively  out  of  30  cultured  explants  in  fig.  2  and  fig.  3.  on  the  other  hand,  in  case  of  leaf  the  combination of 2.0 mg/l bap + 0.5 mg/l naa showed  better callus induction i.e. 13.4. the explants cultured  on ms medium without hormones did not produce any  callus.  it  was  also  found  that  calli  were  induced  in  medium supplemented with bap and naa which  is  in  support  of  the  results  obtained  by  jayasree  et  al.  (2001). the percentage of callus induction was highest  in ms media containing 2.0 mg/l bap + 0.5 mg/l naa  from stem i.e. 48.666% followed by callus induction in  figure 1: seed germination from brinjal cv. jhumki on  ms media without  hormones at 7 days   figure 2. callus induction from brinjal cv. jhumki on ms  media with hormones (bap and naa) at 22 days   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 49‐54  51  biotechnology society of nepal (bsn), all rights reserved   explants  were  cultured  on  ms  media  supplemented  with different combinations and concentrations of bap  (0, 2.0, 3.0 and 4.0 mg/l) and naa (0, 0.1, 0.5, and 1.0  mg/l).  the  highest  amount  of  callus  (48.66%)  was  produced on ms medium containing 2.0 mg/l bap and  0.5  mg/l  naa  from  stem  and  8.2  days  required  for  callus formation. the growth of callus was faster on ms  media supplemented with 2.0 mg/l bap and 0.5 mg/l  naa  from  the  stem.    maximum  number  of  plant  regeneration through callus from stem containing 2.0  mg/l  bap  and  0.5  mg/l  naa  were  3.4  (23.287%)  and  from  leaf  containing  2.0  mg/l  bap  and  0.5  mg/l  naa  were 1.6 (11.94%).  acknowledgement  all  praises  are  due  to  almighty  god  ,for  bestowing  mercy upon me and for imbibing confidence on me to  materialize  the  research  work  .the  author  deem  it  a  proud  privilege  to  express  his  deep  gratitude,  over  indebtedness  sincere  appreciation  to  his  reverend  teacher  and  supervisor  foreign  professor  dr.  lutful  hassan,  dept.  of  genetics  &  plant  breeding  ,bangladesh  agricultural  university  ,  mymensingh  for  his  constant  supervision,  untiring  assistance  ,  scholastic  ,continuous  impression  and  constructive comments. the authors also wish to thank  usda  project  for  providing  fund.  this  paper  is  supported  by  bangladesh  association  for  biotechnology (babt).   leaf. the combination of 2.0 mg/l bap + 0.5 mg/l naa  required  8.2  days  for  callus  induction  from  stem  explants.  on  the  otherhand,  the  combination  of  2.0  mg/l bap + 0.1 mg/l naa needed 10.8 days for callus  from  root  explants.  so,  callus  induction  from  stem  required minimum days. among the supplements, the  highest  regeneration  potentiality  observed  from  2.0  mg/l bap (0.717) and 0.5 mg/l naa (0.667). but there  was  no  regeneration  ability  without  hormones.  the  combined effect of different combinations of bap and  naa in ms medium on plant regeneration from stem,  leaf and root of brinjal cv. jhumki have been presented  in  table  2.  various  combinations  of  supplements  showed  significant  variation  in  regeneration  ability.  among the used combinations, 2.0 mg/l bap + 0.5 mg/l  naa showed the highest regeneration of plantlets from  stem  (3.400).  the  regeneration  of  plantlets  was  (1.6)  from  leaf  in  2.0  mg/l  bap  and  0.5  mg/l  naa  combinations.  root  showed  lowest  regeneration.  the  percentage(i.e. 23.28%) of regeneration was recorded  the highest in ms media containing 2.0 mg/l bap + 0.5  mg/l  naa  from  stem  and  days  required  for  regeneration  was  minimum  (38.8  days).  the  percentage  (11.94%)  of  regeneration  was  the  highest  in 2.0 mg/l bap + 0.5 mg/l naa from leaf. i.e. 1.6 and  percentage  of  regeneration  from  root  is  the  lowest.  plant regeneration from leaf in 2.0 mg/l bap + 0.5 mg/l  naa combination required minimum days (46.2 days).  from  the  above  discussion,  we  found  that  the  best  shoot  regeneration  was  recorded  from  media  supplemented with 2.0 mg/l bap + 0.5 mg/l naa in fig.  4.  figure 3. callus induction from brinjal cv. jhumki on ms  media with hormones (bap and naa) at 22 days.   figure 4. direct regeneration from brinjal cv. jhumki on  ms medium supplemented with 2.0 mg/l bap + 0.5 mg/l  naa   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 49‐54  52  biotechnology society of nepal (bsn), all rights reserved   treatment combinations  no. of explant showing  callus induction  % of callus induction  days required for callus  induction explants  treatments  bap (mg/l)  naa (mg/l)     stem  0  0  0.000 r  0.000 r  0.000i  0.1  7.000 jklmn  23.333 jklmn  10.400 abcdef  0.5  6.600 klmno  22.000 klmno  10.44 abcdef  1.0  7.400 hijklm    24.066 hijkim  10.600 abcdef  2.0  0  6.200 lmno  20.660 lmno  10.400 abcdef  0.1  8.600 fghi  28.666 fghi  9.600 ebcdefg  0.5  14.600 a  48.666 a  8.200 gh  1.0  9.600 defg  32.000 defg  9.600 efg  3.0  0  6.800 klmno  22.660 klmno  11.200 abcde  0.1  9.400 defg  31.330 defg  10.800 abcde  0.5  10.200 de  34.000 de  9.800 defg  1.0  9.200 defg  30.666 defg  10.600 abcdef  4.0  0  6.600 klmno  22.000 klmno  11.400 abcde  0.1  10.000 def  33.333 def  11.200 abcde  0.5  11.600 c  38.666 c  10.000 cdef\g  1.0  10.000 def  33.333 def  11.400 abcde     leaf  0  0  0.000 r  0.000 r  0.000 abcde  0.1  5.800 nop  19.333 nop  11.000 abcde  0.5  6.000 mno  20.000 mno  10.800 abcde  1.0  9.200 defg  30.666 defg  10.600 abcdf  2.0  0  4.600 pq  15.330 pq  11.000 abcde  0.1  7.400 ijklm  24.660 ijklm  10.200 bcdef  0.5  13.400 b  44.660 b  8.600 fh  1.0  9.800 def  32.666 def  10.800 abcde  3.0  0  4.400 q  14.666 q  10.800 abcde  0.1  6.400 klmno  21.333 klmno  10.800 abcde  0.5  6.200 lmno  20.660 lmno  11.200 abcde  1.0  10.000 def  33.333 def  10.800 abcde  4.0  0  4.400 q  14.666 q  11.400 abcde  0.1  7.600 hijk  25.333 hijk  11.600 abcde  0.5  7.400 ijklm  24.661 ijklm  11.600 abcde  1.0  8.600 fghi  28.660 fghi  11.200 abcde     root  0  0  0.000 r  0.000 r  0.000 i  0.1  4.400 q  14.666 q  12.200 ab  0.5  6.400 klmno  21.333 klmno  12.400 a  1.0  6.600 klmno  22.000 klmno  11.800 abcd  2.0  0  4.200 q  14.000 q  12.000 abc  0.1  6.600 klmno  22.000 klmno  11.400 abcde  0.5  9.600 defg  32.000 defg  10.800 abcde  1.0  8.200 ghij  27.330 ghij  11.600 abcde  3.0  0  5.400 opq  18.000 pq  11.400 abcde  0.1  7.800 hijk  26.000 hijk  11.000 abcde  0.5  10.600 cd  35.330 cd  10.200 bcdef  1.0  8.800 efgh  29.330 efgh  11.600 abcde  4.0  0  4.400 q  14.666 q  7.600 h  0.1  6.800 jklmno  22.660 jklmno  11.800 abcd  0.5  9.400 defg  31.330 defg  10.400 abcdef  1.0  7.800 hijk  26.00 hijk  11.400 abcde  table 1. the effect of bap and naa in ms medium on callus induction from different explants.  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 49‐54  53  biotechnology society of nepal (bsn), all rights reserved   table 2. the effect of bap and naa in ms medium on plant regeneration from different explants.   treatment combinations  no. of plants regenerated through  callus  % of regeneration  days required for regeneration  explants  bap (mg/l)  naa (mg/l)  stem  0  0  ‐  ‐  ‐  0.1  ‐  ‐  ‐  0.5  ‐  ‐  ‐  1  ‐  ‐  ‐  2  0  0.200 cd  3.222 cd  39.200 g  0.1  0.600 cd  6.976 cd  39.800 g  0.5  3.400 a  23.287 a  38.800 g  1  0.600 cd  6.25 cd  39.000 g  3  0  0.200 cd  2.94 cd  39.400 g  0.1  0.800 c  8.510 c  39.800 g  0.5  0.800 c  7.843 c  39.800 g  1  0.600 cd  6.521 cd  39.600 g  4  0  0.400 cd  6.060 cd  40.000 g  0.1  0.600 cd  6.000 cd  40.000 g  0.5  0.400 cd  3.448 cd  39.800 g  1  0.400 cd  4.00 cd  39.600 g  leaf  0  0  ‐  ‐  ‐  0.1  ‐  ‐  ‐  0.5  ‐  ‐  ‐  1  ‐  ‐  ‐  2  0  0.400 cd  8.695 cd  48.800 cd  0.1  0.600 cd  8.108 cd  48.000 de  0.5  1.600 b  11.940 b  46.200 f  1  0.600 cd  6.122 cd  49.000 cd  3  0  0.400 cd  9.090 cd  48.800 cd  0.1  0.400 cd  6.25 cd  48.400 cde  0.5  0.600 cd  9.677 cd  47.400 e  1  0.400 cd  4.00 cd  48.200cde  4  0  0.200 cd  4.545 cd  49.000 cd  0.1  0.400 cd  5.361 cd  49.000 bc  0.5  0.400 cd  5.405 cd  50.200 c  1  0.400 cd  4.651 cd  49.200 bc  root  0  0  ‐  ‐  ‐  0.1  ‐  ‐  ‐  0.5  ‐  ‐  ‐  1  ‐  ‐  ‐  2  0  ‐  ‐  ‐  0.1  ‐  ‐  ‐  0.5  0.200 cd  2.083 cd  60.200 a  1  0.200 cd  2.439 cd  60.000 a  3  0  ‐  ‐  ‐  0.1  0.200 cd  2.564 cd  59.600 a  0.5  0.400 cd  5.128 cd  59.800 a  1  0.200 cd  2.272 cd  59.600 a  4  0  ‐  ‐  ‐  0.1  0.200 cd  2.947 cd  60.400 a  0.5  0.200 cd  2.127 cd  59.400 a  1  0.400 cd  5.128 cd  60.000 a  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 49‐54  54  biotechnology society of nepal (bsn), all rights reserved   references  anwar, s; d. sabana; s. a. siddiqui; a. shahzad and s.  din. (2002). clonal     propagation of brinjal, solanum  melongena,  through  young  petiolated  leaf  culture.  bionotesx, 4(3): 61.  bbs.2003.  statistical  yearbook  of  bangladesh.  bangladesh bureau of statistics. ministry of planning,  government of the people’s republic of bangladesh.    dhaka, bangladesh. p. 150.  jayasree,  t.;  v.  paban  ;  m.  ramesh;  a.v.  rao  and  k.  j.m. reddy. (2001). somatic embryogenesis from leaf  cultures of potato. plant cell tissue organ cult., 64 (1):  13‐17.  murashige, t. and f. skoog. (1962). a revised medium  for rapid growth and bioassays with tobacco  tissue  cultures. physiol. plant., 15: 473‐497.  zeven, a. c. and p. m. zhukovsky.(1975). dictionary of  cultivated  plants  and  their  centre  of  diversity.  wageningen, netherlands.p. 219  nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 doi: https://doi.org/10.3126/njb.v7i1.26951 ©njb, biotechnology society of nepal 50 nepjol.info/index.php/njb. original research article nematode fauna associated with kiwi (actinidia delicosa, chev.) plants in machchhegaun, kathmandu, nepal bijay chhetri1* 1central department of zoology, tribhuvan university, kirtipur, kathmandu, nepal 1amrit campus, tribhuvan university, lainchaur, kathmandu, nepal abstract plants harbor many trophic groups of nematodes in them. the plant production is also determined by the occurrence of nematodes adjacent to the rhizosphere of plants, such as parasitic, free-living etc. altogether 40 samples from the 30 cm away from the kiwi plants were examined to detect the nematode distribution in kiwi plants in machchhegaun, kathmandu, nepal. total 10 genera of nematodes including both free living/beneficial and parasitic were identified. overall, 880 individuals of 10 nematodes genera belonging to four orders were found to be linked with kiwi plants, among them the highest report was of order dorylaimida (40.91%) (dorylaimus spp., cephalobus spp., eucephalobus spp. and discolaimus spp.) which was followed by mononchida (36.36%) (iotonchus spp., parahadronchus spp. and mononchus spp.), rhabditida (18.18%) (rhabditis spp. and mesorhabditis spp.) and tylenchida (4.55%) (helicotylenchus spp.). no any published data about study of plant nematodes was found from study area. so, these four order of plant nematodes have been reported for the first time associated with kiwi plants from machchhegaun in nepal. the result specified no significant distribution of nematodes in all kiwi plants. proper management of manures and kiwi plants treatment is recommended for more production of kiwi fruits in study area by nitrogen reduction, phosphorus reduction, odor reduction, energy recovery and adding value to manure techniques. keywords: kiwi, nematode, herbivorous, soil, vine *corresponding author email: bjme686@gmail.com introduction kiwi (actinidia delicosa) belongs to the order ericales and family actinidiaceae, which normally ripens within 25 weeks after the appearance of first flowers [1]. the kiwi fruits are beneficial to the human health because it contains vitamin a, b, c, e and k, copper, potassium, fiber and folate [2]. the demand of kiwi in the worldwide market is hiking due to these nutrients available in kiwi fruit, which is mainly beneficial for the treatment of many diseases like cancer and heart disease, children and pregnant women [2]. nematodes are classified into free-living and parasitic [3, 4]. some soil nematodes increase the plant production, whereas some lower the production by causing harm to the plants. harmful nematodes are more disadvantageous to the crop production and cause massive loss to the farmers [5]. about 100-157 us$ crop production is lost worldwide per year from the nematodes [6]. the crop rotation techniques are being practiced by farmers to lower the crop loss from disease and parasites [7, 8]. however, this technique won’t be conceivable to perennial plants like kiwi. plant parasitic nematodes constitute one of the most important groups of organism inhabiting the soil and in and around the roots of plants. plant parasitic nematodes lower the plant production by damaging crops. the most abundant plant parasitic nematodes fall under the orders like tylenchida, dorylaimida and aphelenchida [9]. furthermore, rhabditis spp., mesorhabditis spp., cephalobus spp. and eucephalobus spp., etc. are the free-living nematodes, which are common in the plants, and benefit plants by providing nutrients through decaying organic matters [10]. likewise, aphelenchus spp. is one of the fungivorous nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 51 nepjol.info/index.php/njb. nematodes, which also turn organic matters into inorganic form [9]. however, despite of being least abundant predatory nematode in most of the soil contents, mononchus spp. feeds indiscriminately both free-living and plant parasitic nematodes [10]. the kiwi fruit production is declining, which may be due to the effects of nematodes such as xiphinema spp., pratylenchus spp., meloidogyne spp., helicotylenchus spp., paratylenchus spp., psilenchus spp. heterodera spp., tylenchus spp. and paratrichodorus spp. [11]. in nepal, till date very less research specified the occurrences/ distribution of soil nematodes in kiwi plants. therefore, the present study aims to lessen this research gap. materials and methods study area the present study was carried out on the kiwi plants at machchhegaun, kathmandu, nepal on march to april 2017. geographically, machchhegaun lies in between 27 ̊ 47 n latitude and 85 ̊ 79 e longitude, at 1531 m of elevation [12]. the study area covers 1.6 hectors of land, and 10 farmers are commercially farming kiwi plants in the study area. two hundred fifty kiwi plants are planted in 16000 m2 of machchhegaun, kathmandu. preliminary survey preliminary survey was done to acqire general information about study area. interaction with concerned person was done to collect information about kiwi plants farming in machchhegaun. soil sampling total 40 kiwi plants randomly selected to collect kiwi plants related soil nematodes. about 20 cm was dug on the side of kiwi plants, and 500 gm of soil sample was taken. the soil was kept in plastic bags and tagged and brought to the lab of central department of zoology for further processing. processing of soil samples processing soil samplesin laboratory, the chunks were smashed and pebbles were removed from soil samples by hand picking method. processing of samples were done by cobb’s sieving and decantation techniques [13] (mesh size: 53µm and 200 mm) with continuous wash with fresh water. the nematode suspension acquired from soil sample was transferred to baermann’s funnel [14]. isolation of nematodes from soil the funnel was left for 24 hours for the movement of nematodes to the bottom of the funnel. counting of nematode species total 10 ml of nematode suspension was prepared and 1 ml out of 10 ml suspension was taken into a counting chamber for counting of nematodes and result obtained was multiplied by 10. killing and fixation of nematodes the killing and fixing of nematodes were done by seinhorst’s process [15] by using 4% formaldehyde heated at 60-70 ͦ c. dehydration of nematodes dehydration of nematodes was done by keeping them in a desiccator for about 1 week [16]. mounting and sealing the slides of nematodes were prepared by using glycerin, purified wax and hot plate. observation and identification the slides were then observed under the low power and high power magnification. i.e. 10× (eye piece) and 10×, 20× and 40× (nose piece) from left to right side of the slide. the nematodes were identified up to genus level by comparing the characters of observed nematodes with available taxonomic keys [17]. statistical method the distribution of kiwi nematodes was analyzed in r program 3.4.1 version [18]. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 52 nepjol.info/index.php/njb. results isolation and identification of different types of nematodes found in rhizosphere of kiwi plant. nematode species belonging to four orders were found in the present work. among them dorylaimida (40.91%) (dorylaimus spp., cephalobus spp., eucephalobus spp. and discolaimus spp.) order was significantly higher, which was followed by order mononchida (36.36%) (iotonchus spp., parahadronchus spp. and mononchus spp.), rhabditida (18.18%) (rhabditis spp. and mesorhabditis spp.) and tylenchida (4.55%) (helicotylenchus spp.). out of 40 soil samples, all soil samples were positive (100%) for the prevalence of free living nematodes. however, plant parasitic nematodes were documented only from 50% soil samples. there was high prevalence of iotonchus spp. (22.73%) and low prevalence of helictylenchus spp., mononchus spp., mesorhabditis spp. and discolaimus spp. (4.55%). the number of iotonchus spp. was highest (22.73%) followed by rhabditis spp. (13.64%), dorylaimus spp. (13.64%), eucephalobus spp. (13.64%), parahadronchus spp. (9.09%), cephalobus spp. (9.09%), mononchus spp. (4.55%), mesorhabditis spp. (4.55%) helichotylenchus spp. (4.55%) and discolaimus spp. (4.55%) (table 1). there was significant difference (χ2cal> χ2tab) between the total number of nematode species in samples studied in machchhegaun study area (p< 0.05). figure 1: tropic groups of soil nematodes. different tropic groups of nematodes were selected for the study. in the study area predatory nematodes (54.55%) (mononchus spp., parahadronchus spp., iotonchus spp., discolaimus spp. and dorylaimus spp.) comprised more than half of the soil nematode community in study area. bacterivores (40.91%) (rhabditis spp., mesorhabditis spp., cephalobus spp. and eucephalobus spp.) and herbivores (4.54%) (helicotylenchus spp.) were also represented (figure 1). discussion many nematodes endeavor kiwi plants for getting nutrition and for reproduction, such as pratylenchus vulnus (walnut root lesion nematode) table 1: genus wise nematodes identified in rhizosphere soil of kiwi plant. s.n. nematodes frequency (n) absolute frequency (af %) total no. of each genus of nematode mean density pvalue 1 helicotylenchus spp. 20 50 40 1 2.2×10-16 2 iotonchus spp. 40 100 200 5 3 dorylaimus spp. 40 100 120 3 4 parahadronchus spp. 20 50 80 2 5 mononchus spp. 18 45 40 1 6 rhabditis spp. 24 60 120 3 7 cephalobus spp. 20 50 80 2 8 eucephalobus spp. 20 50 120 3 9 mesorhabditis spp. 10 25 40 1 10 discolaimus spp. 8 20 40 1 total 313 880 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 53 nepjol.info/index.php/njb. and meloidogyne spp. (root knot nematode), both belonging to order tylenchida is the main pest to kiwi plants [19]. among four orders of nematodes, order dorylaimida (dorylaimus spp., cephalobus spp., eucephalobus spp. and discolaimus spp.) was dominant and order tylenchida (helicotylenchus spp.) was least found, probably due to the regular supplying of organic matters to the kiwi plants. this assumption can be supported by the occurrence of nematodes belonging to orders dorylaimida, mononchida, rhabditida and tylenchida in central horticulture centre, kirtipur, nepal [20]. kiwi plants harbor several trophic groups of nematodes, namely, bacterivorous (rhabditis spp., mesorhabditis spp., etc.) and different stages of insects and animal parasites like deladinus female, eudiplogaster larva, sphaerularia spp. and mermis spp., etc. [21]. among the identified nematodes in the kiwi plants the free-living species such as iotonchus spp. rhabditis spp., dorylaimus spp., eucephalobus spp., parahdronchus spp., cephalobus spp., mononchus spp., mesorhabditis spp. and discolaimus spp. were dominant, probably due to the regular supplying of organic matters to the kiwi plants. this assumption can be supported by the occurrence of free-living nematodes iotonchus spp. and rhabditis spp. in central horticulture centre, kirtipur, kathmandu [20], established grasslands [22, 23, 24]. the occurrence of predatory nematodes (mononchus spp., parahadronchus spp., iotonchus spp., discolaimus spp. and dorylaimus spp.), were probably supported by the occurrence of numerous freeliving nematodes. the dominance of predatory nematodes in study area may be due to the use of organic manure like cattle dung and plant leaves in 20:80 ratio on the rhizosphere soil of kiwi plants. the composition of the soil nematode community depends on the vegetation present, as well as on soil type, season, soil moisture level, amount of soil organic matter, and many other factors. because they are responsive to so many different factors, it is believed that nematodes may be useful bio-indicators of the condition of the soil environment [25]. the identified herbivorous nematode (helicotylenchus spp.) in the kiwi plants was less dominant, probably due to the presence of freeliving nematodes like iotonchus spp. rhabditis spp., dorylaimus spp., eucephalobus spp., parahdronchus spp., cephalobus spp., mononchus spp., mesorhabditis spp. and discolaimus spp. which graze on bacteria, fungi and other phyto-parasitic nematodes, thus they control the populations of harmful micro-organisms [20, 26]. generally, plant parasitic nematodes can be found in less number in those plants where fungivorous eucephalobus spp. are common are more common in homogeneous crop residues such as in maize (zea mays) [27]. the predatory nematodes such as mononchus spp., parahadronchus spp., iotonchus spp., discolaimus spp. and dorylaimus spp. were highest in kiwi plants of this study area which might be due to the high number of bacterivorous nematodes (rhabditis spp., mesorhabditis spp., cephalobus spp. and eucephalobus spp.). these genera are supposed to be common food of the predatory nematodes. increase of predatory nematodes cause declines in prey populations, which in turn prevents further increase of the predator population [28]. out of all identified nematodes from rhizosphere of kiwi plants, the bacterivorous nematodes like rhabditis spp., mesorhabditis spp., cephalobus spp. and eucephalobus spp. were dominant, probably due to the regular supplying of organic matters to the kiwi plants. nematode occurrences can be supported by nematodes present in central horticulture centre, kirtipur, kathmandu [20] and litter present in the soil [29]. conclusion the presence of predatory nematodes and bacterivore nematodes almost in equal number and also high number of herbivorous nematode species in machchhegaun study area may be due to the use of organic manure like cattle dung and plant leaves as fertilizer in the rhizosphere soil of nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 54 nepjol.info/index.php/njb. kiwi plants. therefore, regular use of manures and treatment of kiwi plants are recommended for increased yield of kiwi fruits. conflict of interest declared none acknowledgements i heartily thank prof. dr. arvind kumar keshari and my thesis supervisor late mr. ashok bahadur bam for their kind cooperation for genus identification. i am also thankful to central departments of zoology and environmental science for providing lab for this research. furthermore, lastly, i would like to thank the farmers of machchhegaun for research permission in their farm. references 1. wilson eh, yichang ar, ferguson eh: wilson, yichang and the kiwifruit. http://arnoldia. arboretum.harvard.edu/pdf/articles/115 5.pdf. 15 april, 2017. 2. beutel ja: kiwifruit production in california. division of agriculture and natural resources. 1990. 3. blaxter ml, ley pd, garey jr, liu lx, scheideman p, vierstraete a, et al: a molecular evolutionary framework for the phylum nematoda. nature. 1998 392: 71–75. 4. blaxter mm, dorris m, de ley p: patterns and processes in the evolution of animal parasitic nematodes. nematologica. 2000 2: 43–55. 5. singha s, singh b, singh ap: nematodes: a threat to sustainability of 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actahortic.1992.297.70. 12. nepal census 2001: nepal’s village development committees. digital himalaya. archived from the original on 2008-10-12. retrieved 2008-09-03. 13. cobb na: estimating the nema population of soil. us dep agric tech circ. 1: 48. coetzee, v. 1967. species of the genus iotonchus (nematoda: mononchidae) occurring in southern africa. nematologica. 1918 13(3): 367377. 14. baermann g: eineeinsache method zurauffindung von ankylostomum (nematoden) larven in erdproben. genee sk. tijdschr. nederlands-indië. 1917 57: 131-137. 15. seinhorst jw: killing of nematodes for taxonomic study with hot f.a. 4:1. nematologica. 1966, 12:178. 16. seinhorst jw: a rapid method for the transfer of nematodes from fixative to anhydrous glycerin. nematologica. 1959, 4: 67-69. 17. smart gc, nguyen kb: illustrated key for the identification of common nematodes in florida. university of florida gainesville, florida, 1988. 18. r core team. r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url https://www.r-project.org/, 2017. 19. uc ipm: uc pest management guidelines. http://ipm.ucanr.edu/pmg/r430200111.html. 02 novemmber, 2019 20. chhetri b, subedi jr: nematodes associated with kiwi plants in central horticulture centre kirtipur, kathmandu, nepal. int j appl agri sci. 2019, 5(3): 71-74; doi: 10.11648/j.ijaas.20190503.12 21. freckman dw: nematodes in soil ecosystems. university of texas press, austin, tx., 1982. 22. reyes ps, domingo j: mononchid nematodes from spain. description of iotonchus rotundicaudatus sp. n. and observations on i. zschokkei (menzel, 1913) altherr, 1955. rev nematol. 1991, 14(3): 353-360. 23. čerevková a: diversity and distribution of nematode communities in grassland in relation to its establishment age and utilization. nova science publishers, inc., 2011, 1-15. 24. el-borai k, fahiem: plant parasitic nematodes in subtropical and tropical agriculture. 2nd edition. 2005. 25. wallace hr: nematode ecology and plant disease. edward arnold, london, uk. 1973, 266(1415): 163–171. http://www.digitalhimalaya.com/collections/nepalcensus/form.php?selection=1 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 55 nepjol.info/index.php/njb. 26. fao: food and agriculture organization of the united nations. the importance of soil organic matter. viale delle terme di caracalla, 00100 rome, italy, 2005, 64. 27. mcsorley r, frederick jj: nematode population fluctuations during decomposition of specific organic amendments. j nematol. 1999, 31(1): 37–44. 28. djigal d, brauman a, diop t, chotte jl, villenave c: influence of bacterial-feeding nematodes (cephalobidae) on soil microbial communities during maize growth. soil bio biochem. 2004, 36: 323–331. 29. xue q: nematode diversity of soil and litter in mount hamiguitan, the philippines, with morphological and phylogenetic analysis of selected species. m.sc. thesis. department of biology, ghent university, ghent, b-9000, belgium, 2013 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 56 nepjol.info/index.php/njb. photo plate figure 2: entire body of helicotylenchus sp. through 10×40x magnification. figure 3: entire body of helicotylenchus sp. through 10×100x magnification. figure 4: anterior region of mononchus sp. through 10×40x magnification. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 57 nepjol.info/index.php/njb. figure 5: posterior region of mononchus sp. through 10×40x magnification. figure 6: anterior region of iotonchus sp. through 10×40x magnification. figure 7: posterior region of iotonchus sp. through 10×40x magnification. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 58 nepjol.info/index.php/njb. figure 8: anterior region of rhabditis sp. through 10×40x magnification. figure 9: posterior region of rhabditis sp. through 10×40x magnification. figure 10: anterior region of dorylaimus sp. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 59 nepjol.info/index.php/njb. figure 11: posterior region of dorylaimus sp. through 10×40x magnification. figure 12: anterior region of cephalobus sp. through 10×40x magnification. figure 13: posterior region of cephalobus sp. through 10×40x magnification. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 60 nepjol.info/index.php/njb. figure 14: anterior region of eucephalobus sp. through 10×40x magnification. figure 15: posterior region of eucephalobus sp. through 10×40x magnification. figure 16: entire body of discolaimus sp. through 10×40x magnification. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 61 nepjol.info/index.php/njb. figure 17: posterior region of discolaimus sp. through 10×40x magnification. figure 18: anterior region of parahadronchus sp. through 10×40x magnification. figure 19: posterior region of parahadronchus sp. through 10×40x magnification. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 50-62 chhetri 2019 ©njb, biotechnology society of nepal 62 nepjol.info/index.php/njb. figure 20: anterior region of mesorhabditis sp. through 10×40x magnification. figure 21: posterior region of mesorhabditis sp. through 10×40x magnification.. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 57-61 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 57 nepjol.info/index.php/njb isolation of bacillus spp. bacteria from soil for production of cellulase amika ahmed manzum and md arafat al mamun* pilot plant research lab., centre for advanced research in sciences, university of dhaka, bangladesh abstract cellualse is one of the most important enzymes used in textile, detergent, paper, food and feed industries. therefore, a study was undertaken to isolate bacillus bacteria having the potential to produce cellulase from soil samples. 24 soil samples were analyzed and 54 presumptive bacillus isolates were isolated after heating the soil samples at 80°c for 10 min. among them 45 isolates showed enzyme activity ranging from 0.003 to 0.17 u/ml in test tubes containing 5 ml medium composed of (g/l) glucose 0.5 gm, peptone 0.75 gm, feso4 0.01 gm, kh2po4 0.5 gm, and mgso4 0.5 gm at 120 rpm, 37 °c and ph 7. among them 1rw, 2ws, 3yr, 4wt, 6 rr, and 9ss showed 0.17, 0.15, 0.14, 0.15, 0.147 and 0.14u/ml enzyme activities, respectively. production of cellulase by these isolates was further scaled up to shake culture containing 50 ml medium similar to that used in test tube culture. among the isolates 1 rw showed the maximum activity. this 1 rw was identified by api kit and showed that 59 % belongs to bacillus licheniformis strain (51% confirmation) or bacillus subtilis (31% confirmation). further gene analysis is required to confirm the species. the genetic improvement study will make the isolate a good source of cellulase. keywords: cellulase, bacillus, isolation, api kit, shake culter. *corresponding author email: arafat@du.ac.bd introduction: microbial cellulases have applications in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture [1]. in food industry, cellulases have an important application for extraction and also clarification of juices from fruits and vegetables [2]. it is also used in the conversion of cellulosic wastes into glucose [3] and the enzymatic saccharification of lignocellulosic materials for bio-fuel production [1]. cellulases are successfully used in textile industries for finishing of cellulose-based textiles in wet processing; in paper industries for deinking of paper wastes and in animal feed industries for pretreatment of silage and grain to increase its nutritional value [1]. cellulase can be produced by various fungal species such as aspergillus, rhizopus, trichoderma, fusarium, neurospora, penicillium, etc. [4]. production of cellulases by some bacteria such as cellulomonas, cellvibrio, pseudomonas sp, bacillus, and micrococcus has also been reported [5]. however, bacteria with comparatively higher growth rate can potentially be used in cellulase production. nevertheless, production of cellulase by bacteria is not widely observed. sometimes bacteria are preferred for large scale production of enzyme because most of them produce large amount of thermostable extracellular enzymes, which are active at a wider ph range [6]. since each microbial strain is unique in their molecular, biochemical, metabolic and enzyme production properties, new strains with higher activity and unique properties are always searched to meet the demands at a commercial level [7]. among bacteria, the bacillus strains are most important industrial enzyme producer as they have potential for production of large amount of extracellular enzymes [8].therefore in this study an investigation was undertaken to isolate bacillus bacteria capable of producing cellulase enzyme. materials and methods screening and isolation of bacillus spp. generally the bacillus spp. are spore former. therefore, the major interest was isolation of the spore forming rod shaped bacteria. for this purpose the 10 g soil sample was diluted in 90 ml sterile normal saline and heated at 80°c for 10 min to eliminate vegetative cells [9]. cellulaseproducing bacteria were isolated by the dilution spread plate method using cmc agar media in which cmc was the sole carbon source. the plates were incubated at 37°c for 48 hours. gram staining of the bacteria was performed. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 57-61 manzum and mamun ©njb, biotechnology society of nepal 58 nepjol.info/index.php/njb selection of cellulolytic bacteria the colonies found on the cmc agar plates were collected and a quantitative assay method was used to determine the cellulase activity of the selected bacterial isolates in liquid medium containing (g/l) glucose 0.5 gm, peptone 0.75 gm, feso4 0.01 gm, kh2po4 0.5 gm, and mgso4 0.5 gm in test tubes [5]. the cellulase activity of each culture was measured by determining the amount of reducing sugars liberated using a dns method [10]. bacterial identification the assumed bacillus sp. was identified using biomerieux api 50 chb/e kit. bacterial suspension was made with medium and each tube of the strip was then inoculated with the bacterial suspension. the bacteria fermented the carbohydrates to acids which decreased the ph and it was detected by the change in color of the indicator. the identification software identified the strain using the biochemical profile made up from the results. the colony selection, inoculum preparation, inoculation, incubation and interpretation of results were performed according to the manual provided by biomerieux. the presumptive strain was determined by the software (apiweb tm) based on the results found by the api kit [11]. a positive test corresponding to acidification was indicated by the phenol red changing to yellow, except the esculin test (tube no. 25) where a change in color from red to black was observed as positive. enzyme production medium production medium contained (g/l) glucose 0.5 gm, peptone 0.75 gm, feso4 0.01 gm, kh2po4 0.5 gm, and mgso4 0.5 gm. 50 ml of media were taken in 100 ml conical flasks. the flasks were sterilized in an autoclave at 121°c for 15 min and after cooling, the flasks were inoculated with overnight grown bacterial cultures. the inoculated media were incubated at 37°c and 120 rpm in a shaker incubator for 48 h. after fermentation, the culture media were centrifuged at 6000 rpm for 10 min and the supernatant were used as enzymes. enzyme assay cellulase activity was measured following the method of miller (1959) [10]. briefly, a reaction mixture composed of 0.5 ml of crude enzyme solution plus 1.0 ml of 1% carboxymethyl cellulose (cmc) in citrate buffer (ph 5.2) was incubated at 50°c in a shaking water bath for 30 min. the reaction was terminated by adding 3 ml of dns reagent. the color was then developed by boiling the mixture for 15 min. od of samples was measured at 540 nm against a blank containing all the reagents except the crude enzyme. one unit of cellulase is the amount of enzyme necessary to produce 1 µmol reducing sugar per min under the standard assay conditions. result and discussion for rapid screening of cellulolytic bacteria, agar media containing 0.5 % cmc as sole carbon source are flooded with congo red after incubation. cellulolytic colonies can be detected by observing a surrounding pale orange to clear zone against red background. the cellulolytic bacteria can be screened directly on such plate, but replica plating (each colony is inoculated onto multiple plates) from master plate is recommended because flooded reagents interfere the isolation [12]. another procedure reported where grams iodine was used instead of congo red. since there is poor correlation between enzyme activity and size of halo the platescreening methods using dyes are not quantitative [12]. in this study the colonies were directly selected and used for quantitative analysis of cellulase activity. bacillus spp. are moderately human friendly microorganisms. various species are being used for the production of numerous industrial products such as enzymes [13], gamma polyglutamic acid [14], bacteriocin [15], biopesticides [16], waste management [17], probiotics [18] etc. one strain of bacillus may produce different kinds of valuable products [19]. therefore in this study the main target was the isolation of bacillus spp. and the production of cellulase enzyme form the isolates. since bacillus is a spore former the isolation of this organism involved the heating of the samples at 80 °c for 10 min so that the vegetative cells were eliminated keeping the spores stable. then gram positive rods were selected as presumptive nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 57-61 manzum and mamun ©njb, biotechnology society of nepal 59 nepjol.info/index.php/njb bacillus. 24 soil samples were investigated. after the heat treatment the bacterial load was found to be lower. there were found in different types of colonies such as wrinkled large, smooth small and smooth large colonies. all bacteria showed gram positive rod shaped results. initially these organisms were grown in media in test tubes at 37 °c and 120 rpm in shaking incubators. the isolates produced very low amount of enzyme showing in the table 1. figure 1. biochemical test result of isolated bacillus sp on api kit among the isolates 1rw, 3yr, 6rr, 9ss, 2ws and 4wt were selected for further experiment to find out whether these strains are stable at shake flask culture. in shake flask culture, the 1 rw showed the maximum activity among the isolates. this type of result (enzyme activity 0.5 to 0.9 u/ml) was reported for environmental strains such as bacillus spp. [19-21] and cellulomonas spp. [22]. in this study, the isolate 1rw was further identified by the api kit method. the results showed that the isolate belonged to the bacillus genera and might be bacillus licheniformis. the figure 1 showed the biochemical reaction pattern of the isolate. reaction 1, 4, 5, 6, 11, 12, 13, 17, 18, 19, 24, 25, 26, 27, 28, 31, 32, 35, 36, 37 became yellow which indicated positive results. tube number 25 designated for esculine turned black in color. using the software it was determined that the isolate belonged to bacillus licheniformis 51 % and bacillus subtilis 31 %. the b. licheniformis and b. subtilis are genetically related [23]. these two strains are used to produce various microbial products. to remove the confusion further gene sequencing would be done. the natural strain normally produces a lower yield. therefore further genetic improvement through mutation will be performed for 1rw. the bacteria are more suitable to produce enzyme than fungi because bacterial cellulases are more resistant to alkaline and thermophilic conditions and are good candidates for using in laundries [24]. among the bacteria, the bacillus spp. are more suitable because they have high growth rates, able to secrete enzymes in extracellular media and table 1. enzyme production by the presumptive bacillus isolates. isolate enzyme activity (u/ml) isolates enzyme activity (u/ml) isolates enzyme activity (u/ml) isolate enzyme activity (u/ml) 1 rw 0.17 1 sl 0.074 2 ws 0.15 2 yr 0.124 3 yr 0.14 3 ws 0.11 4 wt 0.15 5 rw 0.012 5 ss 0.13 6 rr 0.147 6 ws 0.1 7 rw 0.026 7 sl 0.11 6 ws 0.1 7 rw 0.026 7 sl 0.11 7 ss 0 8 rw 0 8 sl 0.09 9 rw 0 9 sl 0 9 ss 0.14 10 sl 0.13 10 rw 0.016 10 ss 0.13 11 rw 0 11 sl 0 12 sl 0.135 12 rw 0.015 13 rw 0.002 14 sw 0 14 ww 0.018 15 wp 0.04 15 rw 0.05 15 sw 0.007 15 ss 0.11 16 rw 0 16 sw 0.035 17 rw 0.007 17 sw 0.035 17 ss 0.11 18 rw 0.004 18 ss 0.11 19 rw 0.0034 19 sl 0.018 20 rw 0.0013 21 rw 0.008 21 ss 0.01 22 rw 0.032 22 ss 0.04 22 ssp 0.045 23 rw 0.044 23 rs 0.055 24 rw 0.05 24 ss 0.043 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 57-61 manzum and mamun ©njb, biotechnology society of nepal 60 nepjol.info/index.php/njb generally regarded as safe [8]. to improve the enzyme production yield, optimization of medium and fermentation conditions as well as genetic improvement of natural strains, are required [25]. conclusion in this study, bacillus sp. has been isolated and identified using api 50 chb/e kit. this bacteria is capable of producing cellulase in submerge culture. the bacillus isolate would be a good source of cellulase if further investigations in terms of medium optimization and 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its utilization in industry: a review. advances in enzyme research 2016, 4: 44–55. http://dx.doi.org/10.4236/aer.2016.42005 25. singh v, haque s, niwas r, srivastava a, pasupuleti m and tripathi ckm: strategies for fermentation medium optimization: an in-depth review. front microbiol 2017, 7: 2087. doi: 10.3389/fmicb.2016.02087 3 nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  14   biotechnology society of nepal (bsn), all rights reserved   original research article  analysis of katg ser315thr mutation in multidrug resistant  mycobacterium tuberculosis and slc11a1 polymorphism in  multidrug resistance tuberculosis in central development  region of nepal using pcr‐rflp technique: a pilot study  raunak shrestha1, rubin narayan joshi1, kriti joshi1, bal hari poudel2 and bhupal govinda  shrestha1  1 department of biotechnology, kathmandu university, dhulikhel, nepal.  2 genetics nepal pvt. ltd., lalitpur, nepal.  abstract  ser315thr mutations in genes encoding the mycobacteria catalase‐peroxidase (katg) has been associated with  the major resistance to isoniazid (inh) in mycobacterium tuberculosis (mtb). also g/c polymorphisms in int4  region of the solute carrier family 11 member 1 gene (slc11a1) and susceptibility towards tuberculosis (tb) has  been demonstrated worldwide. 24 drug resistant mtb culture positive samples and 24 whole‐blood samples  were collected from different tb patients of central development region of nepal in 2009. a polymerase chain  reaction (pcr) ‐ restriction fragment length polymorphism (rflp) assay was carried out in order to investigate  ser315thr  katg  mutation  and g/c  polymorphism  in  int4  region.  4  (16.67%)  samples  out  of 24  mtb  culture  samples demonstrated the ser315thr katg mutation whereas none of the 24 whole blood samples were found  to  contain  g/c  polymorphism  in  int4.  though  no  significant  correlation  could  be  found  between  int4  polymorphism  and  tb  susceptibility,  overall  scenario  of  nepal  cannot  be  drawn  from  this  data.  molecular  diagnostic technique such as pcr‐rflp can be used  in a robust scale to carry out base  line studies  in the tb  population of nepal.    key words: multi‐drug resistance, tuberculosis, pcr, rflp    correspondence author:  e‐mail: sraunak@gmail.com  introduction  during the last decades, incidence of tuberculosis (tb)  has increased in many countries and more people have  the disease now than at any other time in history. it is  estimated that tb still kills more people globally than  any  other  infection  (who,  2009).  the  situation  has  worsened  by  the  emergence  of  antibiotic‐resistant  strains of mycobacterium tuberculosis (mtb) (musser,  1995). the strains resistant to the two most important  anti‐tb  drugs,  rifampin  (rif)  and  isoniazid  (inh)  are  commonly defined as multidrug resistant (mdr). drug  resistance  in m. tuberculosis  is attributed primarily to  the  accumulation  of  mutations  in  the  drug  target  genes;  these  mutations  either  leading  to  an  altered  target or to an alternation in effective titration of the  drug (rattan et al., 1998).   in  2008,  there  were  an  estimated  8.9–9.9  million  incident cases of tb, 9.6–13.3 million prevalent cases  of  tb  (who,  2009)  and  390,000–510,000  cases  of  mdrtb  (who,  2010).  among  the  incident  tb  cases  globally,  3.6%  (95%  confidence  interval  (ci):  3.0–4.4)  are estimated to have mdr‐tb. almost 50% of mdr‐tb  cases worldwide are estimated to occur  in china and  india. in 2008, mdr‐tb caused an estimated death of  150,000 people across the globe (who, 2010).   who  estimated  the  prevalence  of  all  types  of  tuberculosis  cases  for  nepal  to  be  67,546  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  15   biotechnology society of nepal (bsn), all rights reserved   (240/100,000). the proportion of mdr‐tb in nepal was  2.9%  (95%  ci:  1.8‐4.3)  among  new  cases  and  11.7%  (95%  ci:  7.2‐17.7)  among  retreatment  cases  (ntc,  2009).  in  2009,  with  the  assistance  of  who,  national  tb  center in collaboration with national anti‐tuberculosis  association  (nata)  and  german  nepal  tuberculosis  project  (gentup)  had  conducted  a  surveillance  of  extremely drug‐resistant tuberculosis (xdr‐tb) among  the  registered  mdr  tb  patients.  the  study  showed  a  prevalence  of  5%  of  xdr‐tb  cases  among  mdr  tb  cases registered (ntc, 2009).  isoniazid  (inh)  is  one  of  the  important  first‐line  tuberculosis  drugs.  mtb  is  highly  susceptible  to  inh,  with a minimum inhibitory concentration (mic) of 0.03 ‐0.06 mg ml‐1. inh inhibits the biosynthesis of cell wall  mycolic  acids  (long  chain  α‐branched  ß‐hydroxylated  fatty  acids),  thereby  making  the  mycobacteria  susceptible  to  reactive  oxygen  radicals  and  other  environmental factors (rattan et al., 1998). it is a pro‐ drug  that  requires  activation  by  the  mtb  catalase‐ peroxidase enzyme (katg) to its active form. mutation  of  the  katg  gene,  which  leads  to  loss  of  or  reduced  catalase‐peroxidase  activity,  is  a  major  mechanism  of  inh  resistance  in  mtb  (musser,  1995).  although  various  mutations  in  the  katg  gene  have  been  reported  in  inh‐resistant  isolates,  the  most  common  mutation  is the ser315thr mutation, which  is present  in approximately 50–90% of all inh‐resistant isolates, is  associated  with  relatively  high‐level  resistance to  inh  (mokrousov et al., 2002).   more  than  one‐third  of  the  global  population  is  infected  with  mtb.  however,  only  10%  develop  the  clinical  disease.  many  factors  contribute  to  the  immune  response  against  tuberculosis.  the  solute  carrier  family  11  member  1  gene  (slc11a1,  formerly  known  as  nramp1:  natural  resistance  associated  macrophage protein 1)  is one of the candidate genes  for susceptibility to human tuberculosis (takahashi et  al., 2008). the gene is located on human chromosome  2q35 and has 15 exons spanning about 14 kb (marquet  et  al.,  2000).  the  gene  encodes  a  transmembrane  protein  expressed  exclusively  in  macrophages/ monocytes  and  polymorphonuclear  leukocytes.  the  protein acts as a transporter for divalent cations fe2+,  zn2+  and  mn2+  and  has  pleiotropic  effects  on  macrophage activation (goswami et al., 2001).  studies  have  demonstrated  an  association  between  three  different  polymorphisms  (int4,  d543n  and  3’utr)  in  slc11a1 and pulmonary tb (taype et al., 2006).  in the present study, ser315thr katg mutation  in the  mdr‐tb  culture  samples  and  g/c  polymorphisms  in  int4  region  of  slc11a1  of  tb  patients  were  studied  using  polymerase  chain  reaction  (pcr)  ‐  restriction  fragment length polymorphism (rflp) technique.  materials and methods  study samples. 24 mtb culture (all afb stain positive)  samples were obtained from gentup, kathmandu. the  cultured  samples  comprised  of  both  pulmonary  and  extra‐pulmonary origin. drug susceptibility testing was  also carried out  in gentup and all of the 24 samples  were  graded  as  mdr‐tb.  24  whole  blood  samples  of  mdr‐tb  patients  were  also  obtained  for  slc11a1  analysis. the samples were collected from the central  development  region  of  nepal.  however,  we  were  unable  to  obtain  the  clinical  history  of  the  test  subjects. further molecular analysis was carried out in  a bsl‐iii laboratory at genetics nepal pvt. ltd., lalitpur,  nepal.  genomic  dna  isolation  of  m.  tuberculosis.  dna  isolation from cultured samples was done using sorpo  clean™  genomic  dna  extraction  kit.  a  loop  full  of  bacterial  colonies  from  the  culture  samples  were  suspended  in  200µl  of  sterile  water.  a  spin  column  extraction  was  carried  out  as  prescribed  by  the  manufacturer.  human  genomic  dna  isolation.  the  whole‐blood  samples  from  human  test  subjects  were  collected  in  edta  coated  vial  and  immediately  centrifuged  at  5000rpm/5min.  plasma  was  separated  and  200µl  of  figure 1: prevalence of mdr‐tb in nepal.   (source: ntc, 2009)  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  16   biotechnology society of nepal (bsn), all rights reserved   whole‐blood  was  taken  as  sample  for  further  processing. sorpo clean™ genomic dna extraction kit  was used for genomic dna isolation. all the other steps  were same as described above.  genomic  dna  visualization:  the  extracted  dna  was  visualized in a 0.8% agarose (amresco®) gel.  pcr‐rflp  analysis:  amplification  of  the  200  bp  fragment with katg codon 315 (the fragment  in katg  from  nucleotide  positions  904  to  1103;  http:// genolist.pasteur.fr/tuberculist)  was  performed  in  tpersonal  thermocycler  (biometra®)  with  primers  katg1f and katg4rb (mokrousov et al., 2002) (table 1)  in  25µl  of  a  pcr  mixture  (0.4  μm  of  each  primers,  2.5mm  of  dntp  mix,  1u  of  recombinant  taq  dna  polymerase (fermentas®) and 2.5 mm of mgcl2) under  the  following  conditions:  initial  denaturation  at  94⁰c  for 5 min; 30 cycles of 94⁰c for 1 min, 56⁰c for 45 sec  and 72⁰c for 45 sec; and a final elongation at 72⁰c for 5  min.  the  amplified  fragment  was  assessed  by  electrophoresis  in a 2% agarose gel and cleaved with  restriction  enzyme  mspi  (hpaii)  (fermentas®)  as  per  the  instructions  of  the  manufacturer.  the  restriction  fragments  obtained  were  electrophoresed  in  a  2%  agarose gel and were visualized under ultra‐violet (uv)  light on a transilluminator (biometra®). m. tuberculosis  h37rv was taken as the reference strain for this study.  pcr‐rflp assay was designed to detect the katg codon  mutation agc(ser) à acc(thr), which leads to the inh  resistant  phenotype.  this  mutation  creates  an  additional mspi site (ccgg) and thus can be detected  by use of this restriction endonuclease. as a result, the  longest  fragment  size  in  the  wild  type  katg  product  would  be  153  bp  and  in  ser315thr  katg  mutant  the  longest  fragment  would  be  132  bp  which  could  be  easily  resolved  in  a  2%  agarose  gel  while  the  shorter  bands  (21  bp  ‐  8  bp)  cannot  be  resolved  in  the  gel  (figure 2).   similarly, 514 bp of int4 region of slc11a1 gene was  amplified using primers int4f and int4r (taype et al.,  2006) (table 1).   a 25µl of a pcr mixture (0.4 μm of  each primers, 2.5mm of dntp mix, 1u of recombinant  taq  dna  polymerase  and  2.5  mm  of  mgcl2)  was  prepared and the reaction was carried out under the  following  condition:  initial  denaturation  at  95⁰c  for  5  min; 30 cycles of 94⁰c for 1 min, 61°c for 1 min and  72⁰c for 1 min; and a final elongation at 72⁰c for 4 min.  the  amplified  fragment  was  assessed  by  electrophoresis in a 2% agarose gel. to analyze the g/c  polymorphisms  in  int4,  the  amplified  products  were  cleaved  with  restriction  enzyme  apai  (fermentas®)  as  per  the  instructions  of  the  manufacturer.  the  restriction  fragments  obtained  were  electrophoresed  in a 2% agarose gel and were visualized under uv light  on a transilluminator.   results:  genomic  dna  visualization:  all  the  extracted  dna  samples  displayed  a  positive  band  when  they  were  electrophoresed in 0.8% agarose gel. (figure 3 and 4)  pcr‐rflp analysis: all the 24 mtb dna samples were  observed to have a positive amplification for katg gene  fragment. an amplicon of 200 bp size were observed in  a 2% agarose gel (figure 5).  in the electrophoresis of  the  katg  amplified  product  digested  with  mspi,  4  (samples  015,  016,  020  and  024)  out  of  24  samples  were  obtained  at  132  bp  region  indicating  the  occurrence of ser315thr mutation whereas rest of the  20 samples were obtained at 153 bp region indication  no  ser315thr  mutation  (figure  6).  gel  picture  of  samples 015 and 016 are not shown.  all the 24 human dna samples from whole blood had a  positive amplification at 514 bp region for int4 (figure  7).  when  these  amplicons  were  subjected  to  apai  digestion, all of the 24 samples were observed at 514  bp  region  indicating  no  g/c  polymorphisms  in  int4  region of slc11a1 (figure 8).   discussion  south asia holds around 40% of the global tb burden  and  nepal  has  no  different  scenario.  the  millennium  development  goal  (mdg)  aims  to  eliminate  tb  as  a  public health problem (1 case per million population)  by 2050. nepal has made satisfactory progress towards  mdg. however, the challenges to address the growing  burden of tb and mdr‐tb population are yet far away.  nepal still lacks the health care facilities and access to  modern  diagnostic  technologies  is  very  poor  in  the  area (basnet et al., 2009). although directly observed  treatment,  short‐course  (dots)  strategy  has  significantly contributed towards the treatment of tb,  a  lack  of  rapid  and  sensitive  rapid  and  sensitive  methods  of  detection  is  a  major  hindrance  to  the  ongoing battle against the disease.  afb  microscopy  is  the  primary  screening  tool  for  tb  and  culture  of  mycobacteria  is  still  regarded  as  the  “gold  standard”  in  tb  diagnosis.  but  these  days  new  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  17   biotechnology society of nepal (bsn), all rights reserved   target gene region  primer  sequence  katg  katg1f  5'‐agctcgtatggcaccggaac‐3’  katg4rb  5'‐aacgggtccgggatggtg‐3'  int4  int4f  5'‐gtctgccatctctactaccctaaggtg‐3’  int4r  5'‐catgtccctctaggtatgtgctatcag‐3'  table 1. primer sequence for katg and int4  figure  2.  schematic  illustration  of  the  katg  200‐bp  fragment  amplified  with  primers  katg1f  and  katg4rb.  the  vertical  line  represents  the  mspi  restriction  site  (ccgg).  in  the  wild‐type  katg  pcr  product  there  is  no  mspi  restriction site at 315th codon (agc) and the  longest fragment  is of 153 bp. but  in the mutant katg pcr product  there is an addition of mspi restriction site at 315th codon (acc) and it gives 132 bp band as the longest fragment.   figure  3.  visualization  of  the  extracted  mtb  dna  from  cultured sample  in a 0.8% agarose gel. 020‐024 are the  dna  samples,  pc  is  the  positive  control  and  nc  is  the  negative control. the bands are very close to the loading  well indicating the larger fragment of genomic dna.   figure 4. visualization of the extracted dna sample from  human whole blood in a 0.8% agarose gel. 001‐005 are  the  dna  samples,  pc  is  the  positive  control  and  nc  is  the  negative  control.  the  bands  are  very  close  to  the  loading  well  indicating  the  larger  fragment  of  genomic  dna.   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  18   biotechnology society of nepal (bsn), all rights reserved   figure 5. pcr product of katg gene fragment visualized  in a 2% agarose gel. 020‐024 are the mtb samples, pc is  the positive control and ntc is the no template con‐ trol. all the samples have positive band amplification at  200 bp region.   figure 6.mspi digested product of katg gene fragment  visualized  in  a  2%  agarose  gel.  020‐024  are  the  mtb  samples, pc is the positive control and ntc is the no  template  control.  samples  021,  022,  and  023  has  a  band  size  of  153  bp  indicating  no  mutation  at  315th  codon  position  where  as  sample  020  and  024  has  a  band sized 132 bp indicating seràthr mutation at 315th  codon position of katg gene.   sensitivity and specificity.  the present study  looks at the resistance  in the katg  ser315thr  towards  a  popular  anti‐tb  drug  isoniazid.  this  is  just  one  among  many  possible  mutations  in  katg accounting for inh resistance. 4 (16.67%) samples  molecular  diagnostic  tools  are  promoted  globally  mostly in the detection of mtb, drug monitoring and in  mdr‐tb diagnosis (pai et al., 2006). this is due to many  advantages  of  molecular  genomic  tools  over  conventional diagnostic methods that  lack the speed,  figure 7. pcr product of int4 gene fragment visualized  in  a  2%  agarose  gel.  001‐005  are  the  human  dna  samples, pc  is the positive control and ntc  is the no  template  control.  all  the  samples  has  positive  band  amplification at 514 bp region   figure 8. apai digested product of int4 gene fragment  visualized in a 2% agarose gel. 001‐002 are the human  dna samples, pc is the positive control and ntc is the  no template control. all the samples were observed at  514 bp region indicating no g/c polymorphisms in int4  region of slc11a1 gene   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  19   biotechnology society of nepal (bsn), all rights reserved   out  of  24  mtb  culture  samples  demonstrated  ser315thr katg mutation (i.e. these samples were inh  resistant).  it  indicates  that  there  could  possibly  be  significantly  high  anti‐tb  drug  resistant  population  if  screened in a larger population. one of the drawbacks  of  this  study  is  that  other  mutation  in  various  genes  (inha,  oxyr,  ahpc)  that  contributes  to  the  inh  resistance  were  not  studied.  studies  have  reported  that even if there is ser315ther katg mutation, it does  not necessarily account to inh resistance and have an  overall high level of catalase activity (guo et al., 2006).  both  the  catalase  activity  and  drug  sensitivity  information could not be obtained.   the association between slc11a1 polymorphisms and  susceptibility  to  tuberculosis  has  been  described  in  many  studies,  which  showed  positive  association  in  some, while no association in others (takahashi et al.,  2008).  a  study  showed  that  nramp1  polymorphisms  may be associated with progression to severe forms of  pulmonary tuberculosis rather than with susceptibility  to m. tuberculosis infection (zhang et al., 2005). all the  int4  amplicons  when  subjected  to  apai  digestion,  bands of 514 bp were observed. if the int4 region had  g/c polymorphisms then restriction site(s) would have  been additionally created and the rflp bands of <500  bp size would have been observed.  this indicates that  no  g/c  polymorphism  was  present  in  the  amplified  figure 9. percentage distribution of katg ser315thr mu‐ tation   sample id  afb stain  mtb culture  mdr‐tb  katg pcr ampli‐ con size (bp)  mspi digested  product size  (bp)  ser315thr katg  mutation  mtb‐001  positive  positive  positive  200  153  negative  mtb‐002  positive  positive  positive  200  153  negative  mtb‐003  positive  positive  positive  200  153  negative  mtb‐004  positive  positive  positive  200  153  negative  mtb‐005  positive  positive  positive  200  153  negative  mtb‐006  positive  positive  positive  200  153  negative  mtb‐007  positive  positive  positive  200  153  negative  mtb‐008  positive  positive  positive  200  153  negative  mtb‐009  positive  positive  positive  200  153  negative  mtb‐010  positive  positive  positive  200  153  negative  mtb‐011  positive  positive  positive  200  153  negative  mtb‐012  positive  positive  positive  200  153  negative  mtb‐013  positive  positive  positive  200  153  negative  mtb‐014  positive  positive  positive  200  153  negative  mtb‐015  positive  positive  positive  200  132  positive  mtb‐016  positive  positive  positive  200  132  positive  mtb‐017  positive  positive  positive  200  153  negative  mtb‐018  positive  positive  positive  200  153  negative  mtb‐019  positive  positive  positive  200  153  negative  mtb‐020  positive  positive  positive  200  132  positive  mtb‐021  positive  positive  positive  200  153  negative  mtb‐022  positive  positive  positive  200  153  negative  mtb‐023  positive  positive  positive  200  153  negative  mtb‐024  positive  positive  positive  200  132  positive  table 2. relationship between afb stain, mtb culture and pcr‐rflp results  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 14‐21  20   biotechnology society of nepal (bsn), all rights reserved   region of int4. the present study shows no significant  association between g/c polymorphism int4 region of  slc11a1 with tb. with the limited number of samples  and failure to study d543n and 3’utr region, the role  of  slc11a1  in  susceptibility  towards  tb  cannot  over‐ ruled.  as  who  claims  one‐third  of  the  world’s  population  to  be  infected  with  latent  form  of  tb,  slc11a1  polymorphisms  can  be  accredited  to  be  a  helping factor for tb susceptibility or progression.  this study has considered m. tuberculosis h37rv as a  reference  strain  but  other  clinical  strain  could  also  have been prevalent. this fact remained as a challenge  sampleid  mdr‐tb  int4 pcr amplicon size  (bp)  apai digested product of  int4 (bp)  g/c polymorphism in int4  slc‐001  positive  514  514  negative  slc‐002  positive  514  514  negative  slc‐003  positive  514  514  negative  slc‐004  positive  514  514  negative  slc‐005  positive  514  514  negative  slc‐006  positive  514  514  negative  slc‐007  positive  514  514  negative  slc‐008  positive  514  514  negative  slc‐009  positive  514  514  negative  slc‐010  positive  514  514  negative  slc‐011  positive  514  514  negative  slc‐012  positive  514  514  negative  slc‐013  positive  514  514  negative  slc‐014  positive  514  514  negative  slc‐015  positive  514  514  negative  slc‐016  positive  514  514  negative  slc‐017  positive  514  514  negative  slc‐018  positive  514  514  negative  slc‐019  positive  514  514  negative  slc‐020  positive  514  514  negative  slc‐021  positive  514  514  negative  slc‐022  positive  514  514  negative  slc‐023  positive  514  514  negative  slc‐024  positive  514  514  negative  table 3. relationship mdr‐tb and g/c polymorphism in int4 region of slc11a1  to  our  study  as  no  base‐line  study  on  mtb  strain  genotyping  has  been  conducted  for  nepalese  mtb  isolates till date.  conclusion  the  findings  in  this  study  suggests  that  still  more  people could be properly diagnosed for the actual drug  resistance  mutations  utilizing  molecular  genomic  diagnostic tools. further study with a greater number  of  mdr‐tb  patients  is  needed  to  get  the  actual  scenario of tb population of nepal.   reference  world health organization. global tuberculosis  control. a short update to the 2009 report. 2009    world health organization. multidrug and extensively  drug‐resistant tb (m/xdr‐tb) 2010 global report on  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resistance.  expert  rev  mol diagn. 2006 may;6(3):423‐32.  basnet r, hinderaker sg, enarson d, malla p, mørkve  o.  delay  in  the  diagnosis  of  tuberculosis  in  nepal.  bmc public health. 2009 jul 14;9:236.  guo h, seet q, denkin s, parsons l, zhang y. molecular  characterization of isoniazid‐resistant clinical isolates  of mycobacterium tuberculosis from the usa. j med  microbiol. 2006 nov;55(pt 11):1527‐31.  zhang w, shao l, weng x, hu z, jin a, chen s, pang m,  chen  zw.  variants  of  the  natural  resistance‐ associated  macrophage  protein  1  gene  (nramp1)  are  associated  with  severe  forms  of  pulmonary  tuberculosis. clin infect dis. 2005 may 1;40(9):1232‐ 6. epub 2005 mar 23.  nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):1-11 doi: https://doi.org/10.3126/njb.v8i1.30204 research article ©njb, bsn 1 genetic diversity in finger millet landraces revealed by rapd and ssr markers bal krishna joshi1 , darbin joshi2, surya kanta ghimire3 1national agriculture genetic resources center, khumaltar, kathmandu, nepal 2cimmyt, kathmandu, nepal 3agriculture and forestry university, rampur, chitwan, nepal article history:received: 10 apr 2020; revised: 19 jun 2020; accepted: 30 jun 2020; published online: 31 jul 2020 abstract genetic diversity assessment is the preliminary work for the development of variety and conservation of diversity. finger millet is a very important crop in nepal however, its genetic potential has not been fully utilized. genetic diversity was assessed in forty landraces of finger millet using 9 rapd and 5 ssr markers. these landraces were collected from kaski and dhading districts. none of single primers of these rapd and ssr could separate all 40 landraces. the average number of bands were 6.33 and 7.8 per rapd and ssr primers respectively. mean polymorphism information content was of 0.314 for rapd and 0.37 for ssr. primer opa-4 produced the highest number of bands and the lowest numbers of bands were produced by opa-16. among the ssr primers, ssr-06 produced the highest number of polymorphic bands and ugep-53 produced the lowest bands. rapd based dendrogram has generated four clusters and ssr based dendrogram has generated two clusters. in both dendrogram and principal component analyses, purbeli landrace was found unique locating separately in the cluster and scatter plot. nei's genetic distance produced by rapd and ssr primers was similar that is 0.327 by rapd and 0.296 by ssr markers. genetic distance produced by ssr markers was higher than distance produced by rapd marker. these landraces were from two districts and therefore have shown intermediate diversity. these molecular marker-based findings should would be more useful if we could link with agromorphological traits. inclusion of large number of landraces collected from different areas are required to get higher level diversity in addition to associate genetic diversity with geographical sites. groupings of these landraces could be useful for selecting landraces in breeding program as well as planning conservation program. keywords: finger millet landraces, dna fingerprint, genetic diversity, dna marker corresponding author, email: joshibalak@yahoo.com introduction finger millet [eleusine coracana (l.) gaertn] is found mostly in warm temperate regions of the world from africa to asia and in australia [1]. it is the primary food for millions in central africa and asia including southern parts of nepal. the consultative group on international agricultural research (cgiar) has estimated that 10% of the area under different millets is covered by finger millet [2]. finger millet is the fourth important cereal crop grown in nepal commonly known as kodo [3]. it is grown from terai to high hill of nepal [4]. finger millet is multipurpose cereal crop and its grains and flour are mostly used in preparation of many traditional products like dhedo, roti, haluwa, paniroti, sada parautha, bharuwa parautha, khole, buniya, haluwa, sel, pancake, pizza, doughnuts, namkins, chowmins, pastry momo, cheese balls, chocolates, birthday cake, biscuits, peanut cookies and alcoholic beverages like jandh, rakshi, chhyang, tumba, which have religious and cultural importance in many ethnic communities [5]. along with this importance of finger millet, different landraces of finger millet are being grown across the country [6-7]. knowledge on genetic diversity is important to manage and use of finger millet landraces and genotypes properly. different molecular markers have been widely used in many plant species including finger millet for identification, genetic diversity analysis, phylogenetic analysis, population studies and genetic linkage mapping. dna marker technology has been applied to a wide range of crop species including maize [8] panicum millet [9], genus elusine [10] and finger millet [11]. das and misra [12] reported the efficiency of rapd (random amplified polymorphic dna) markers in nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:joshibalak@yahoo.com https://orcid.org/0000-0002-7848-5824 nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 2 investigating genetic relationships at the molecular level, which is important for germplasm conservation and varietal identification. muza et al. [13] reported a diversity of 26 germplasm lines of finger millet from africa and india based on the southern blot hybridization patterns. salimath et al. [10] reported that the molecular diversity of 20 finger millet accessions by using isozyme, rflp (restriction fragment length polymorphism) and rapd. dida et al. [14] also developed a first genetic map of finger millet by using rflp, aflp (amplified fragment length polymorphism), est (expressed sequence tag) and ssr (simple sequence repeat) markers. the map span was 721 cm on the a genome and 787 cm on the b genome and cover all 18 finger millet chromosomes. they developed a set of 82 ssr markers specific for finger millet by small-insert genomic libraries generated using methylation sensitive restriction enzymes and among them, 31 ssrs were mapped. comparative analysis of this map with rice genetic map was a novel attempt that reported high level of conserved co-linearity between the finger millet and rice genomes [14]. diversity at landrace and trait levels have been reported on nepalese finger millet landraces [3,4,6,7,15–18]. this finger millet diversity has been poorly utilized in breeding and conservation [19] and only so far six varieties have been released for general cultivation [20]. due to many factors diversity of finger millet is at risk of losing from the fields [4,20]. study on genetic diversity helps to accelerate breeding and conservation works. thus, the study was carried out to assess the genetic diversity and genetic relatedness of finger millet landraces collected from different eco-geological regions of nepal, and to strengthen breeding and conservation of finger millet diversity. materials and methods finger millet landraces a total of 40 landraces of finger millet were collected from local initiatives for biodiversity, research and development (li-bird), pokhara (table 1). li-bird (an ngo working in different parts of nepal) has collected these landraces from two districts (kaski and dhading) of nepal. table 1. landraces of finger millet collected from different regions for this study sn landrace site and district 1 setothulo kaski 2 kalo ghudo-2 pumdibhumdi-6, kaski 3 bachuwa-1 kaski 4 thulo kalo-1 kaski 5 mangsire-2(k) kaski 6 chimte dhading 7 thulo kalo-2 kaski 8 kholse kaski 9 setosano kaski 10 kalo ghudo-3 kaskikot-5, kaski 11 setojhyapa kaskikot-2, kaski 12 tori pane kaskikot-2, kaski 13 seto kaskikot-2, kaski 14 mangsire-1 dhikur pokhara-2, kaski 15 kalo ghude-1 dhikur pokhara-2, kaski 16 mangsire-1 chimkeswori-3, kaski 17 katike-2 chimkeswori-2, kaski 18 kaile chimkeswori-3, kaski 19 khukur kane-2 chapkot-6, kaski 20 dalle chapkot-9, kaski 21 khukur kane-1 chapkot-9, kaski 22 chamare dhading 23 champate dhading 24 setokodo dhading 25 mangsire-2(d) dhading 26 katike-1 dhading 27 jhyape seto-1 kaski 28 pumdeli kaski 29 dalle kodo-1 kaski 30 purbeli kaski 31 seto usro-2 kaski 32 kalo ghude-1 kaski 33 dhudekodo kaski 34 usrokodo kaski 35 dalle kodo-2 kaski 36 gairegaule kaski 37 kalo ghude-2 kaski 38 mangsire kaski 39 raikare kaski 40 setobhachuwa kaski k: kaski district , d: dhading district isolation of genomic dna the dna was extracted from the leaf using the modified ctab (cetyl trimethylammonium bromide) method [21]. young leaves (2 g) were collected from the 8 days old plants. the leaves were then cut into small pieces with scissors, lyophilized in liquid nitrogen and stored at -40°c. the lyophilized leaves were grounded to a fine powder nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 3 using a pestle and mortar and transferred to a 2 ml centrifuge tube. about 600 µl of warm ctab buffer (65°c) was added and the contents were mixed well on a rotating shaker and incubated for 1 h at 65°c in a water bath with occasional mixing. the tubes were taken out, cooled to room temperature; and 600 µl of chloroform-isoamyl alcohol (24:1) was added and mixed gently by inverting. the tubes were centrifuged at 12000 rpm for 12 min. the aqueous phase was transferred to a new 1.5 ml tube (approx. 400 µl), to which an equal volume of chloroformisoamyl alcohol was added and mixed gently for 5-6 times. the solution was then centrifuged at 12000 rpm for 2 min. the aqueous phase (approx. 300 µl) was then transferred to 1.5 ml centrifuge tube. on the same tube 600 µl of chilled isopropanol was added for the precipitation of dna. the precipitated dna was carefully taken out into new tube with the help of a pipette. the dna was washed with 70% ethanol, and dried for more than 1 hour and finally dissolved in 50 µl of the 1x te buffer (1 mm edta, 10 mm tris-hcl, ph 8.0) and stored at -20ºc. dna concentration was estimated by q5000 uv vis spectrophotometer (quawell). working template dna solution was prepared with the concentration of 50 ng/ µl using 1x te buffer. rapd primers and amplification rapd analysis was performed by using 10-mer primer from operon technologies (alameda, ca, usa). nine arbitrary rapd primers were tested for amplification (table 2). dna amplification was performed in 20 µl reaction volume containing 1.5 mm mgcl2, 10 mm dntps, 0.8 picomole of primer, 1.5 u taq dna polymerase and 50 ng template dna. pcr amplification was performed in a thermo cycler (multigene optimax, labnet international, inc.) with the following cycling conditions: denaturation at 94ºc for 1 min, annealing at 37ºc for 1 min and elongation at 72ºc for 2 min for 40 cycles after an initial denaturation for 5 min at 94ºc. after finishing of amplification 12 µl aliquots of amplification products were loaded in a 1% (w/v) agarose gel (bioneer) for electrophoresis in 1x tae buffer (bioneer). gels were stained with ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of uv light within gel doc system (uvdi, major science). there was standard 1 kb ladder (promega) for comparing band size. table 2. rapd and ssr primers used in this study s n primer sequence repeat motifs tm (oc) ref a. 10-mer rapd primer 1 opa 03 agtcagccac 38.6 [22] 2 opa-16 agccagcgaa 38.6 [23] 3 opa 08 gtgacgtagg 38.6 [23] 4 opa-04 aatcgggctg 42.7 [22] 5 opa 13 cagcacccac 42.7 [24] 6 opc-06 gaacggactc 38.6 [25] 7 opc-14 tgcgtgcttg 48.6 [26] 8 opb-01 gtttcgctcc 46.8 [27] 9 opa-10 gtgatcgcag 42.7 [28] b. ssr primer 1 ssr-06 f: gcctcgagcat catcatcag r: caacctgcact tgcctgg 55 [29] 2 ssr-08 f: ttccctgtta agagagaaatc r: tgtatttggtg aaagcaac 55 [29] 3 ugep-10 f: aaacgcgatga attttaagctc r: ctatgtcgtgt cccatgtcg (ga)19 60 [30] 4 ugep-53 f: tgccacaact gtcaacaaaag r: cctcgatggcc attatcaag (ag)26 60 [30] 5 ugep-1 f: ttcagtggtga cggaagttct r: ggctccatga agagcttgac (tc)11 60 [31] ssr primer and amplification eight pairs of ssr primers were tested for amplification and among them only five primers were found good for dna profiling (table 2) based on readable and reproducibly of the bands. ssr amplification was performed in 20 µl reaction volume containing 50ng genomic dna, 10 µl of 2x green gotaq® reaction buffer (ph 8.5), 400μm datp, 400μm dgtp, 400μm dctp, 400μm dttp and 3 mm mgcl2 (promega) and 10 picomole of each forward and reverse primer. these components were gently mixed and centrifuged prior to adding 2 drops of mineral oil. the amplification was performed in a thermo cycler (multigene optimax, labnet international, inc.). the cycling conditions were 1 cycle of 95°c for 5 min followed by 30 cycles of 95°c for 60 sec, 55°c for 1 min 30 sec, 72°c for 2 min, and finally 1 cycle of 72°c for 10 min. nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 4 after finishing amplification 12 µl aliquots of amplification products was loaded in a 1% (w/v) agarose gel (bioneer) for electrophoresis in 1x tae buffer (bioneer). after completion of electrophoresis, gels were stained with ethidium bromide (0.5 µg/ml for 30 min) and visualized under exposure of uv light within gel doc system (uvdi, major science). there was standard 100 bp ladder (promega) for comparing size of the band. gel image and molecular data analysis gels were processed and adjusted brightness and contrast in ms picture manager before scoring. ladder were labelled and gel band scoring scale (figure 1) was developed in ms powerpoint. based on this gel scoring scale, all bands of both rapd and ssr profiles were scored for the presence (1) or absence (0) and developed separate binary matrix. molecular weights of bands were estimated using gelanalyzer 19.1. number of amplified bands, polymorphism information content (pic) and genetic distance were estimated. pic was estimated following the method of roldan-ruiz et al. [32] for rapd scores and of smith et al. [33] for ssr scores. genalex 6.5 software was used to estimate these parameters. cluster and principal component analyses were applied separately for rapd and ssr profiles. additional cluster analysis was done combining both rapd and ssr data. figure 1. gel band scoring scale used in ms powerpoint for gel image analysis one by one to determine robustness of the dendrogram, the data were bootstrapped with 1000 replications. mantel test [34] was applied for estimating correlation between ssr and rapd coefficients matrices. data were processed in ms excel and analyzed using genalex 6.5 (for estimating pic and genetic distance) and ntsyspc 2.0 (for cluster and principal component analysis). results rapd and ssr profiles have generated different bands for each of 40 finger millet landraces. as an example, profiles of 40 landraces based on opa-10 rapd primer and ugep-10 ssr primer are given in figure 2 and figure 3 respectively. none of single primers of these rapd and ssr could separate all 40 landraces. ssr profiles indicated that some loci are heterogenous, producing 4 distinct bands. finger millet is tetraploid self-pollinated crops, therefore, majority of loci have two bands showing homozygosity for that loci. figure 2. rapd profiles of 40 finger millet landraces amplified by opa-10 primer. m, 1kb ladder and lane number represent the sequence number of table 1 figure 3. ssr profiles of 40 finger millet landraces amplified by ugep-10 primer. m, 100 bp ladder and lane number represent the sequence number of table 1 http://biopublisher.ca/index.php/mpb/article/html/896/policy#ckwx nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 5 genetic diversity based on rapd markers nine rapd primers generated 57 different bands fragments, with average of 6.33 bands per primer and 73.68% polymorphic bands. primer opa-4 produced the highest number of bands and the lowest numbers of bands were produced by opa-16 (table 3). the polymorphic information contents range from 0.15 in primer opa-13 to 0.45 in opa-08. mean pic value was 0.314±0.174. primer opa-04 amplified the maximum number of bands. opc-14 produced 8 bands, opa-03, opa-08 and opa-10 each produced 7 bands, opb-01 showed 6 bands, opa-13 and opc-06 each showed 5 bands and opa16 produced the lowest numbers of bands (3). table 3. rapd and ssr primers with number of amplified bands, polymorphism information content (pic) and standard deviation s n rapd marker bands amplified polymorphism information content (pic) sd rapd marker 1 opa-03 7 0.27 0.23 2 opa-16 3 0.28 0.20 3 opa-08 7 0.45 0.08 4 opa-04 9 0.40 0.15 5 opa-13 5 0.15 0.18 6 opc-06 5 0.27 0.21 7 opc-14 8 0.33 0.20 8 opb-01 6 0.42 0.05 9 opa-10 7 0.26 0.23 mean 6.33 0.314 0.174 ssr marker 1 ssr-06 15 0.32 0.18 2 ssr-08 8 0.34 0.09 3 ugep-53 8 0.49 0.00 4 ugep-10 1 0.35 0.16 5 ugep-1 7 0.37 0.16 mean 7.8 0.37 0.12 the phylogenetic analysis based on upgma method separated 40 finger millet landraces into four major groups (figure 4). the nei’s genetic distance obtained by the rapd markers were ranged from 0.027 in between dalle kodo-2 and seto bhachuwa to 0.853 in between dale kalo-2 and purbeli. first group consisted of twenty-two late flowering landraces, and second, third and fourth group consisted of seven, eight and three landraces respectively according to mid to late flowering. second and third groups were mid maturing landrace and forth as the early maturating landraces. scatter plot of these landraces was well distributed among the principal coordinates with four major groups (figure 5). variance accounted by principal components i, ii and iii were 33.28%, 21.71% and 15.67% respectively. kukurkane-2 and purbeli landraces have scattered separately. genetic diversity based on ssr markers five ssr primers generated 39 different bands fragments, with average of 7.8 bands per primer and 87.18% polymorphic bands. primer ssr-06 produced the highest number of polymorphic bands and ugep-53 produces the lowest bands during amplification. the polymorphic information contents range from 0.32 in primer ssr-06 to 0.49 in ugep-53 with mean pic value of 0.37± 0.123. ssr-6 showed the maximum number of bands (15) followed by ssr-08 (8), ugep-10 (8), ugep-07 (7) and ugep-01 (1). the phylogenetic analysis based on upgma grouping separated 40 finger millet landraces into two major groups (figure 6). first group consisted of twenty-four landraces and second group comprised of sixteen landraces. the upgma grouping had further made two sub groups from each main group. these landraces were not grouped according to their collection district. scatter plots of these landraces have been found well distributed among the principal coordinate that also separated two major groups (figure 7) which corresponds with cluster analysis (figure 6). this first principal component accounted for 47.02%, second components accounted for 15.53% and third one had explained 14.67% of total variation. purbeli and mangsir-2(d) were scattered separately. comparison between rapd and ssr primers nine rapd markers produced 57 bands with 6.33 number of bands per primers and five ssr markers produced 39 bands with average number of 7.8 bands per primer (table 4). nei's genetic distance produced by rapd and ssr primers was similar that is 0.327 by rapd markers and 0.296 by ssr markers. genetic distance produced by ssr markers was higher than distance produced by rapd marker. mantel test indicated the lower correlation (r=0.30) between rapd and ssr base coefficient matrices. nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 6 figure 4. dendrogram of 40 finger millet landraces based on nei’s genetic distance of 9 rapd markers using upgma methods with bootstrap of 1000 replications figure 5. scatter plot of 40 finger millet landraces based on 9 rapd markers nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 7 figure 6. dendrogram of 40 finger millet landraces using upgma methods with bootstrap of 1000 replications based on nei’s genetic distance estimated from five ssr markers figure 7. scatter plot of 40 finger millet landraces using 5 ssr markers nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 8 figure 8. dendrogram showing 40 finger millet landraces based on nei’s genetic distance calculated from 9 rapd and 5 ssr markers profile using upgma methods with bootstrap of 1000 replications. table 4. comparison between the rapd and ssr markers in 40 finger millet landraces parameter rapd marker ssr marker number of primers used 9 5 number of bands obtained 57 39 polymorphic bands, n 42 35 monomorphic bands, n 15 4 mean number of bands per primer 6.33 7.80 mean genetic distance 0.32 0.29 highest genetic distance 0.80 0.93 lowest genetic distance 0.027 0.04 correlation coefficient between two matrices 0.30 cluster analysis based on both rapd and ssr markers correlation between rapd and ssr marker based coefficients was low therefore, cluster analysis was done combining both rapd and ssr profiles. three clusters were observed (figure 8). there were 21 landraces in cluster i and 18 landraces in cluster ii. purbeli landrace make a separate cluster indicating unique landrace compare to other landraces. kalo ghudo-3 and tori pane landraces were very closely related. discussion rapd and ssr markers are being commonly used for diversity assessments. majority of the findings of genetic diversity have not been greatly utilized particularly in nepal for further breeding and conservation works. it is very common that diversity is generally revealed by different dna markers. we have used finger millet landraces from just few farmers of two districts. these landraces therefore have shown intermediate diversity. landraces generally possesses intra level diversity but such diversity could not generally be estimated through one sample per landrace, therefore, population level study might be more appropriate for revealing intra landrace diversity. all markers have sown polymorphism producing different band sizes. these findings are similar to other many studies. das et al. [24] found that amplification generated by opa-13 was of 12 bands with the size range from 300 to 3000 base pairs. opa-4 produced the highest nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 9 number of bands (8-9), and opa-13 was 100% polymorphic. the genetic similarity and group analysis based on similarity coefficient indicated two major groups, first major group had one genotype and a second major group contained 29 genotypes. babu et al. [22] found the maximum number of bands in opa-04. babu et al. [22] reported the diversity of 32 finger millet genotypes using 50 rapd markers and reported a total 529 loci of which 479 loci (91%) were polymorphic and informative to differentiate the accessions. sharma et al. [35] used the opa-13 for the variability study of blast pathogen in rice and finger millet. panwer et al. [29] found 10 amplified bands with 60% polymorphism by ssr-06 and 15 amplified bands with 40% polymorphism by ssr-08 in finger millet with pic value of 0.523 and 0.511 respectively. arya et al. [30] found the similar result while using upeg10 and upeg-53 ssr markers for assessing genetic diversity and population structure in indian and african finger millet where upeg-10 produced two bands and upeg-53 produced five bands with pic value of 0.2392 and 0.6681 respectively. nethra et al. [31] used 35 ssr primers to find degree of genetic diversity in finger millet in which ugep-1 produced 2 bands with pic value of 0.16, ugep-10 amplified 3 allelic bands with pic of 0.53 and ugep-53 produced 3 allelic bands with pic value of 0.51. dida et al. [36] reported the population structure of 79 finger millet accessions with 45 ssr markers and identified significant difference of plant architecture and yield among asian and african sub-population. salimath et al. [10] experimented with three different dna marker techniques, viz., rflp (8 probe-3enzyme combination), rapd (18 primers) and issr (6 primers) to analyze the diversity of 22 accessions belonging to 5 species of eleusine. the results revealed 14, 10, and 26% polymorphisms in 17 accessions of e. coracana from africa and asia. they suggested that the issr marker was good as compared to rflp and rapd in terms of the quantity and quality of data output different types of markers are considered for generating reliable diversity assessment. two types of markers in this study have shown low correlation. this may be due to the difference on number of primers used in each marker type. moulin et al. [37] reported the 0.55 correlation value between rapd and issr marker based coefficient matrices. additionally scoring ssr codominant markers as presence and absence scale might have some role on revealing true genetic diversity and low correlation among two coefficient matrices. breeding and conservation work in nepal need to strengthen in order to develop climate resilient high yielding varieties. this crop is poorly studied though many farmers are growing over diverse climate and soil conditions. economic trait based study might have immediate application therefore, further studies are necessary to assess the important traits linked markers through covering large number of landraces collecting from wide areas. this will also help to relate the genetic diversity in association with farming land, and ultimately help to guide conservation works. highly polymorphic markers and distantly related landraces in this study could be of used for breeders, conservationists and landrace owners. finger prints of these landraces will be helpful for identification of landraces and developing ownership certificates for particular landrace. conclusion finger millet landraces collected from different areas have shown genetic diversity that can be valuable resources for variety development and diversity management. estimated genetic distance were ranged from 0.027 to 0.853 with rapd markers and from 0.04 to 0.93 with ssr markers. multivariate analysis generated different groups of these 40 landraces. these landraces were only from two districts, and therefore further analysis need to carry out covering wider areas so that any association between diversity and geographical areas could be estimated. author’s contribution bkj conceptualized the research proposal, monitored lab work and wrote paper. dj finalized the proposal, reviewed papers, and performed the lab works, scoring and data analysis. skg supervised the research activities and supported on data analysis and interpretation. all authors read and approved the final manuscript. competing interests no competing interests were disclosed. nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):1-11 joshi et al. ©njb, bsn 10 funding national agricultural research and development fund (nardf) and nepal agricultural research council funded this research works. acknowledgements authors express sincere thanks to national agricultural research and development fund (nardf) and nepal agricultural research council for financial support. molecular work was carried out in national genebank, khumaltar. we thank madan bhatta, raju chaudhary and indira kafle for their supports. ethical approval and consent not applicable references 1. chandrashekar a. finger millet: eleusine coracana. inadvances in food and nutrition research 2010 jan 1 (vol. 59, pp. 215-262). academic press. https://doi.org/10.1016/s10434526(10) 59006-5 2. upadhyaya hd, gowda cl, pundir rp, reddy vg, singh s. development of core subset of finger millet germplasm using geographical origin and data on 14 quantitative traits. genetic resources and crop evolution. 2006 jun 1;53(4):679-85. https://doi.org/10.1007/s10722-004-3228-3 3. upreti rp. status of millet genetic resources in nepal: wild relatives of cultivated plants in nepal. inwild relatives of cultivated plants in nepal (r. shrestha and b. shrestha, eds.). proceedings of national conference on wild relatives of cultivated plants in nepal 1999 jun (pp. 2-4). 4. upadhyay mp, joshi bk. plant genetic resources in saarc countries: their conservation and management. saarc agriculture and information centre, dhaka. 2003. 5. libird. kodo and fapar ka parikar haru banaune bidhi. nepal: libird; 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botulinum neurotoxin type f (bont/f) domains; cloning; recombinant protein expression; immunoreactivity introduction botulinum neurotoxins (bonts), the most potent of all biological substances known to date, are produced by several species of the genera clostridia (c. botulinum, c. butyricum, and c. baratii) [1-3]. seven immunologically distinct botulinum neurotoxin serotypes bonts/a-g are produced and implicated in botulism poisoning [4]. botulism is a serious neuroparalytic disease [5], which generally occurs through ingestion of preformed toxin or rarely, through infection of wounds. the center for disease control and prevention (cdc) classifies bonts, among the six highest risk threat agents for bioterrorism “class a biological warfare agent” [6, 7]. despite their potential to be used for deleterious purposes, bonts have increasing nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   2 applications in cosmetics [8] and therapeutics for the treatment of numerous dystonias, inflammation, and chronic pain [9-11]. botulinum neurotoxin serotype f (bont/f), is a member of the botulinum neurotoxin family as a single ~150 kda inactive polypeptide chain post-translationally nicked, forming a dichain consisting of a c-terminal ~100 kda heavy chain (hc) and a n-terminal ~50 kda light chain (lc) linked by a disulphide bond [12, 13]. bont/f cleaves its substrate vesicle associated membrane protein (vamp) at position (gln58-lys59) [14, 15], one of three neuronal proteins associated with exocytosis; subsequently inhibiting acetylcholine release, resulting in death by flaccid paralysis [16, 17]. each bont/f partial fragment; light chain catalytic domain (rf-lc), n-terminal half of the heavy chain translocation domain (rfhn), the c-terminal half of the heavy chain receptor binding domain (rf-hc), and cterminal quarter part of the heavy chain receptor binding domain (rf-hcc) plays a specific role in the toxicity mechanism [18, 19]. botulinum neurotoxin lcs operate by zinc dependent proteolysis involved in neurotransmitter exocytosis from presynaptic termini [20]. botulinum neurotoxin hns possesses channel-forming capability in the acidic environment of the endosome, allowing internalization of the toxin, while hcs are involved in specific binding to the presynaptic membrane via gangliosides and a protein coreceptor [21]. lastly, the hcc region of bonts are known to harbor the receptor binding neutralizing epitopes which are targets for antibodies that can specifically bind to the receptor, and show neutralizing activity against bont toxicity [22]. thus, it is imperative to construct each domain for the analysis of their molecular and biochemical activities as well as for the development of potential neutralizing antibodies specific to each. to achieve this goal, bont/f domains from c. botulinum were cloned and expressed using a high expression vector and compatible host e. coli strains to obtain high quality of recombinant proteins suitable for administration into mice. methods chemicals, buffers, and reagents components related to dna manipulation, including ex-taq polymerase, dntp, and restriction enzymes were purchased from takara bio. inc., (shiga, japan). luria bertani (lb) media and cell culture media were purchased from becton dickinson and company (md, usa) and hyclone (ut, usa) respectively. additional chemicals including ampicillin, iptg, freund’s complete and incomplete adjuvant, and buffers were purchased from sigma aldrich (mo, usa). bacterial strains, plasmids, and purification nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   3 systems c. botulinum type f strain langeland [23] and bont/f (wako pure chemicals ind., osaka, japan) was kindly supplied by medy-tox inc. plasmids pgex-4t-1 (amersham pharmacia biotech acquired by ge healthcare, uppsala, sweden) and pet-32a(+) (novagen, emd chemicals inc., affiliate of merck kgaa, darmstadt, germany) were used for the construction of expression vectors. e. coli strains dh5α (takara bio inc.), bl21codonplus-ril and bl21-codonplus(de3)ril (stratagene, ca, usa) were used as host strains for propagation and expression of recombinant proteins, respectively. glutathione-s-transferase (gst) purification modules and thrombin protease were purchased from amersham pharmacia biotech. construction of bont/f domains chromosomal dna was isolated from c. botulinum f str. langeland, which was used as the template for polymerase chain reaction (pcr) for all domains (fig. 1). forward and reverse primers for each domain were designed (table 1) based on the published ncbi sequence of the langeland genome (nc_009699). synthetic genes encoding bont/f lc (light chain), catalytic domain (1308 bp); hn (n-terminal 1/2 of heavy chain), translocation domain (1266 bp); hc (c-terminal 1/2 of heavy chain), receptor binding domain (1263 bp); and hcc (far cterminal quarter part of heavy chain), (501 bp) were designed based on the published bont/f sequence (orf: 883635-887471) langeland str. (ncbi genbank accession: nc_009699) for the plasmid construction of each domain. bp indicates base pair of dna. 1kb marker is provided for scale. table 1. primers used for amplification of bont/f domains. aprimer direction: f, forward; r, reverse. bdirection of each sequence is in 5' to 3' orientation (underlined) including restriction endonuclease sequence (bold) and inserted stop codon sequences (blue) for lc and hn reverse primers. cnucleotide positions based on published c. botulinum f str. langeland genome sequence (ncbi genbank accession: nc_009699). drestriction endonucleases utilized to digest pcr amplified domain and vector for plasmid construction. isolation of genomic dna was done utilizing protocols and chemicals mentioned by wizard genomic dna purification kit (promega, wi, usa). pcr was performed using a bio-rad icycler (ca, usa). reaction mixtures were preheated for 5 min at 94 °c for initial denaturation and then 30 cycles of pcr (denaturation, annealing, and elongation) were performed for amplification of domains as follows: 1 min at 94 °c, 1 min at 57 °c, and 1 min 30 sec at 72 °c for f-lc; 1 min at 94 °c, 1 min at 50 °c, and 1 min 30 sec at 72 °c for f-hn and f-hc; 1 min at 94 °c, 1 min at 54.5 °c, and 40 sec at 72 °c for f-hcc. following completion of cycles, a final extension was lc 1 kb scale s s hn hc 1308 bp 1266 bp 1263 bp hcc 501 bp native bont/f (3837 bp) 883635 887471 _____________________________________________________________________________________________________________________________ primera sequence 5' ! 3'b nucleotide positionsc restriction enzymesd _____________________________________________________________________________________________________________________________ f-lc-f cgtgtcgacatgccagttgtaataaatagtttt 883635-883658 sali f-lc-r ccggcggccgcttattttctaggaataacgctctt 884942-884922 noti f-hn-f cgtggatccggtacaaaggcgccaccgcgacta 884943-884966 bamhi f-hn-r ccgctcgagttaatataaaattagaattttatc 886209-886188 xhoi f-hc-f cgtggatcctttaataaattatataaaaaaatt 886209-886232 bamhi f-hc-r ccgctcgagttagttttcttgccatccatgctc 887471-887448 xhoi f-hcc-f cgtggatccgataagtctattactcagaattca 886971-886994 bamhi f-hcc-r ccgctcgagttagttttcttgccatccatgctc 887471-887448 xhoi _____________________________________________________________________________________________________________________________ fig.1: schematic diagram demonstrating bont/f domains for cloning. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   4 carried out for an additional 7 min at 72 °c. amplified pcr products were purified by agarose gel elution kit (intron biotechnology, the republic of korea), and resultant pcr products were digested with restriction enzymes (table 1) and subcloned into pgex4t-1 vector using t4 ligase kit (promega) for overnight at 16 °c, so that the correct reading frame was incorporated along the thrombin cleavage site under the gst gene (representative constructs made from macvector 10.0.2 (nc, usa) software containing f-lc and f-hn domains are shown in fig. 2). ligated samples were transformed into dh5α by heat-shock (42 °c for 50 sec) method and positive clones with appropriate insert were screened out by use of lb agar plates containing 100 µg/ml ampicillin. positive colonies containing the ligated constructs were transformed into bl21-ril for the expression of type f recombinant domains. expression and purification of recombinant proteins to monitor the induction of recombinant domains in bl21-ril, 2 ml of transformant cultures were inoculated and grown in lb broth and induced at various time intervals using different concentrations of isopropyl β-d-1-thiogalactopyranoside (iptg). in order to purify recombinant proteins, mass production was performed by inoculating 2 ml induced culture in 1 liter followed by centrifugation (12,000 xg) at 4 °c for 20 min. pellet was then resuspended in 10 ml of 50 mm phosphate buffered saline (pbs) and cells were then lysed by sonication, with 10 short bursts of 30 sec followed by intervals of placing samples on ice for 1 min cooling. 2 (a) 2 (b) fig.2: schematic diagram of constructed plasmids generated by macvector 10.0.2 software (symantec corporation). plasmids pgex-4t-1-f-lc (a) and pgex-4t-1-f-hn (b) encoding catalytic and translocation domains. nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   5 after centrifugation at 12,000 xg for 30 min, the pellet was resuspended in 10 ml pbs overnight at 4 °c and supernatant was placed in a gst affinity chromatography column after column was initially washed multiple times with deionized water and pbs, followed by normalization with gst resin. the large scale of gst-fusion proteins was eluted by single step affinity chromatography (containing sepharose 4b beads). fractions containing desired proteins were pooled and dialyzed for 2 h at 4 °c against pbs. for thrombin protease treatment, resin bound gst-fusion proteins were cleaved with 20 units thrombin incubating for 30 min at 37 °c followed by 15 min at room temp (20 25 °c) and final elution with 500 µl pbs. the eluted proteins were further dialyzed to maintain salt concentrations and ph. protein concentrations were measured according to the bradford method using a bio-rad model 550 microtiter plate reader. all proteins were labeled, aliquoted and stored at – 70 °c prior to use. lastly, sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) was carried out on a 12% gel under reducing conditions in order to determine solubility of the eluted proteins. production and analysis of polyclonal antibodies against rf-lc and rf-hn for immunization, three female balb/c mice (four weeks old having around 18-22 gram body weight) were immunized intraperitoneally with 10 µg of antigen mixtures containing recombinant proteins (25 µg gst free f-lc or 25 µg gst free f-hn) in pbs. additionally, an equal volume of freund’s complete adjuvant was separately injected into each mouse. after two weeks lapse, the mice were immunized with the mixture containing 20 µg of recombinant protein and equal volume of freund’s incomplete adjuvant. at the start of the fourth week following the initial immunization, the mice were finally boosted by intravenous injection of the recombinant proteins (30 µg) without adjuvant. three days following final boost, mice were then bled from the tail and tested by indirect elisa. preparation of monoclonal antibodies (hybridoma technology) cell fusion and culture of hybridomas was carried out according to the protocol by kohler et al. [24]. briefly, cultures were incubated at 37 °c in an incubator with 5% co2-in-air and 98% relative humidity. four days following final boost, the mouse showing the highest antibody titer by elisa was sacrificed by cervical dislocation and its spleen was removed aseptically. spleen cells (1x108 viable cells) were mixed with sp2/0ag 14 myeloma cells (1x107viable cells) and nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   6 grown in dulbecco’s modified eagle’s medium (dmem) supplemented with 10% fetal bovine serum (fbs) (hyclone). the addition of 50% polyethylene glycol 4000 (peg) (sigma aldrich) allowed for the fusion of mixed cells; resulting in hybrid cells. hybrid cells were screened by incubation in 96-well plates (nunc) containing hypoxanthine-aminopterin-thymidine (hat) selection medium in combination with mouse feeder cells for one week. antibody production from hybrid cells was monitored through indirect elisa; positive cells were expanded into 24-well plates (nunc) containing hypoxanthine-thymidine (ht) media supplemented with dilute aminopterin according to the standard protocol by harlow and lane [25]. elisa-positive hybridomas were selected and cloned twice via limiting dilution; one cell per well into 96-well plates supplemented with ht media and feeder cells. finally, cloned cell lines were grown in dmem supplemented with 10% fbs and stored in liquid nitrogen. indirect enzyme linked immunosorbent assay (elisa) for the measurement of serum antibody titers, elisa was performed as a standard protocol mentioned by sigma aldrich, with only minor modifications. each well of 96 well plates (nunc, copenhagen, denmark) was coated with antigen (100 µl containing 100 ng of either gst free recombinant protein or 100 ng bont/f) and incubated at 4 °c overnight. each well was washed once with 200 µl of pbs containing 0.05% tween 20 (pbst) and blocking solution (100 µl 1% skim milk) was added and incubated for 30 min at 37 °c. following two more washes, the serum (1: 1000 dilution) in pbs was added as the primary antibody. the plates were incubated at 37 °c for 90 min, and then washed three times as described above. goat anti-mouse igg conjugated with alkaline phosphatase was added as the secondary antibody (diluted 1:2000 with pbs) and incubated for 2 h. after incubation with igg, plates were washed four times prior to substrate treatment and visualization. immobilized antigens were visualized with the p-nitrophenyl phosphate (disodium) in substrate buffer containing 9.7% diethanolamine, and 1 mm mgcl2 and the resultant absorbance was measured at 415 nm with microplate reader (model 550 bio-rad) for 5 10 min at 37 °c. western blot analysis western blot experimentation was adapted from previous methodology [26]. the protein samples were separated by sds-page and electroblotted for ~70 min by using 230 ma current onto a nitrocellulose membrane (schleicher and schuell inc., nh, usa). nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   7 subsequently, the membrane was blocked for nonspecific binding incubating with blocking solution for 1 h and then incubated with primary antibody overnight at room temp. the membrane was washed two times with pbst and incubated with alkaline phosphate conjugated goat anti-mouse igg as a secondary antibody for 2 h. after washing three more times with pbst, the membrane was color developed by using 10 ml alkaline phosphate substrate solution, 50 µl nitro blue tetrazolium chloride, and 50 µl 5-bromo-4chloro-3-indolyl phosphate. results and discussion the cloning of both light and heavy chain domains using synthetic genes has been reported for various bonts [27-33]. however, there exist few studies on the cloning and expression of bont/f domains [34, 35]. construction of recombinant plasmids containing pgex-4t-1-f-lc, -hn, -hc, hcc was made for subsequent gst-tagged expression in e. coli. initially, sequences of bont/f dna domains were pcr amplified from genomic dna of c. botulinum f str. langeland and flanked by restriction sites (table 1). specifically, the amplified products were cloned into pgex-4t-1 after digestion with respective restriction enzymes. ligated constructs (for reference see schematic diagrams of constructed plasmids made by macvector software 10.0.2, fig. 2) were transformed into dh5α for propagation and positive colonies were identified after screening on plates containing ampicillin. subsequently the recombinant plasmids were digested with restriction enzymes (table 1) and appropriate band sizes were identified for both pgex-4t-1 (4.9 kb) and all individual fragment sizes (fig. 3). for confirmation of domain homology incorporated into our vector, sequencing was performed. after sequence analysis, plasmid dna of positive clones had 100% identity with native sequences (data not shown). for protein expression, the four recombinant clones were transformed into e. coli bl21codonplus-ril (strain used for protein expression with vectors driven by non-t7 promoters). the bl21-codonplus-ril was chosen because of its capability to express rare codons which allows for the high-level expression of recombinants [36]. thus, bl21codonplus-ril was a compatible host strain for our pgex-4t-1 plasmid systems (containing a tac promoter). all domains were expressed in the form of fusion proteins with gst localized at the n-terminal of the fusion protein, enabling the nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   8 (i) visible bands of amplified f-lc product (3a), f-hn (3b), f-hc (3c), and f-hcc (3d); (ii) endonuclease restricted bands of purified pgex4t-1-f-lc with sali/noti (3a), pgex-4t-1-f-hn (3b), pgex-4t-1-f-hc (3c), and pgex-4t-1-f-hcc (3d) with bami/xhoi. m denotes dna marker and lane 1, dna sample loaded. ease of purification using gst affinity chromatography. expression of clostridial proteins at 37 °c has been shown to increase protein degradation [37], thus we chose an optimal temperature range of 20 25 °c for our expression studies. conditions for expression of recombinant proteins were as follows: 7 h at 24 °c using 0.5 mm iptg concentration for gst-f-lc, 8 h at 22 °c using 0.4 mm iptg concentration for gst-f-hn, 8 h at 20 °c using 0.25 mm iptg concentration for gst-fhc, and 8 h at 20 °c using 0.10 mm iptg concentration for gst-f-hcc. gst-f-lc and gst-f-hn induction was monitored by sdspage (fig. 4) and were found to be highly over-expressed in soluble form and purified at high concentrations. despite induction at low temperature and iptg concentrations, gst-f-hc and gst-f-hcc were expressed in inclusion bodies. change in expression vectors from pgex-4t-1 to pet32a(+) was then carried out in attempt to obtain soluble forms of gst-f-hc and gst-f-hcc. pet-32a(+) was subsequently transformed into bl21-codonplus(de3)-ril host cells, that utilizes the t7 rna polymerase promoter. m 1 m 1 insert f-lc (1308 bp) vector pgex-4t-1 (4.9 kb) bp 300 400 850 650 1650 3000 2000 4000 5000 500 1000 100 400 500 3000 2000 5000 1000 300 200 650 850 1650 4000 amplified f-lc pcr product (1308 bp) a (i) bp a (ii) amplified f-hn pcr product (1266 bp) 3000 m 1 500 1000 850 650 1650 2000 bp b (i) m 1 insert f-hn (1266 bp) 500 650 1000 850 1650 3000 2000 4000 5000 bp vector pgex-4t-1 (4.9 kb) b (ii) m 1 amplified f-hc pcr product (1263 bp) bp 100 400 500 3000 2000 5000 1000 300 200 650 850 1650 4000 c (i) m 1 insert f-hc (1263 bp) vector pgex-4t-1 (4.9 kb) 300 400 850 650 1650 3000 2000 4000 5000 500 1000 bp c (ii) 2000 1000 1650 4000 m 1 amplified f-hcc pcr product (501 bp) bp 100 400 500 300 200 650 850 d (i) m 1 insert f-hcc (501 bp) vector pgex-4t-1 (4.9 kb) d (ii) 2000 1000 1650 4000 bp 100 400 500 300 200 650 850 fig.3: agarose gel (1%) electrophoresis of pcr product and restriction digested plasmid stained with ethidium bromide and visualized by uv nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   9 iptg induction was carried out in a time dependent manner and sds–page (12 %) analysis of total lysate of e. coli bl21(pgex-4t-1-f-lc (a) and pgex-4t-1-f-hn (b) with or without iptg induction stained with coomassie blue. lane m: protein marker (bio-rad); lanes 1 4: (0, 2, 5, and 7 h for f-lc and 0, 2, 5, and 8 h for f-hn) after iptg induction. lanes 2 4 demonstrate expressed gst-f-lc a) and gst-f-hn (b) with treatment of iptg. kda, protein size expressed in kilodalton. although both vectors contain thrombin cleavage sites and are ideal for production of soluble proteins, neither one was able to alter the formation of inclusion bodies. proteinrefolding procedures were performed using reducing reagents including various molar concentrations of urea or guanidine – hcl to solubilize the inclusion bodies but attempts were unsuccessful. as a result, only lc and hn domains were used for protein analysis and collection of polyclonal serum from immunized mice. gst affinity chromatography was then carried out to collect gst tagged lc and hn. after gst affinity chromatography and cleavage with thrombin protease treatment, recombinant gst free f-lc and gst free f-hn were analyzed on sds-page to monitor expected protein bands. sds-page revealed the appropriate size of both recombinants; gst fused 75 kda and gst free 50 kda (fig. 5). final recombinant protein concentrations were determined to be 1.0 mg/l. in order to analyze specificity of mice polyclonal sera against flc and f-hn, mice were immunized intraperitoneally with antigens; gst free flc and f-hn. to analyze the specificity of the polyclonal serums against f-lc and fhn, mouse polyclonal serum was collected from the tail vain and tested by western blotting and indirect elisa (both of these techniques being highly valuable in the detection of candidate biomarkers). the mouse anti-f-lc polyclonal antibodies were found to specifically recognize f-lc and bont/f (figs. 5a and 6a). similarly, anti-fhn polyclonal antibodies demonstrated a m 1 2 3 4 kda 25 75 50 37 100 150 250 15 10 m 1 2 3 4 kda 25 75 50 37 100 150 250 15 gst-f-lc gst-f-hn sds-page (12%) 4 (a) sds-page (12%) 4 (b) fig.4: induction and expression analysis of recombinant proteins nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   10 strong affinity towards f-hn and native type f toxin (figs. 5b and 6b). however, neither anti-f-lc nor anti-f-hn polyclonal antibodies were able to detect the hc domain (fig. 5), validating the specificity of the polyclonal serums towards their respective region of bont/f. the designated recombinant domains have native structure as holotoxin and it was not unexpected for them to produce anti-sera against native toxin (bont/f). mouse showing highest immunoreactivity was then sacrificed in attempt to isolate a monoclonal antibody specific to f-lc or fhn domains. this could allow further research into the development of a highly effective neutralizing monoclonal antibody for a subunit vaccine and/or passive immunotherapy against bont/f. (i) sds–page of purified gst f-lc and gst free f-lc (a) purified gst f-hn and gst free f-hn (b) by single step gst affinity chromatography separated on a 12% gel and visualized by coomassie blue stain; (ii) western blot analysis performed by raising anti-f-lc (a) and anti-f-hn (b) mouse polyclonal antibody. lane m: protein marker (bio-rad); lane 1, gst-f-lc or hn; lane 2, gst free-f-lc or hn; and lane 3, unpurified gst-f-hc. fig.5: purification and characterization of recombinant bont/f domain proteins sds-page 5b (i) m 1 2 3 m 1 2 3 kda 25 75 50 37 100 150 250 sds-page 5a (i) kda 25 75 50 37 100 150 250 western blot 5a (ii) gst-f-lc gst free-f-lc 25 75 50 37 100 150 250 m 1 2 3 m 1 2 3 kda kda western blot 5b (ii) 25 75 50 37 100 150 250 gst-f-hn gst free-f-hn nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   11 bar graph values represent interaction of anti f-lc (a) and anti-f-hn (b) mouse polyclonal serum antibody with: pc = bont/f as a coating antigen; s. 1 3 = samples of gst free f-lc (a) and gst free f-hn (b) coating antigens respectively & interaction of non-immunized mouse serum with: nc= gst free f-lc (a) and gst free f-hn (b) used as coating antigens respectively. mouse polyclonal serum was harvested from three different mice (from different batches) immunized with gst free f-lc or gst free f-hn. pcpositive control, ncnegative control. n = 3 wells, two-tailed students t-test, s. 1 3 compared to nc, p < 0.05. attempts were unsuccessful in isolating a monoclonal antibody specific towards our constructed bont/f domains (data not shown). however, we have successfully isolated a mouse monoclonal antibody that demonstrates neutralizing activity against native bont/f toxin resulting from bont/f toxiod in vivo immunization and hybridoma technology [38]. conclusion this paper describes molecular and immunological studies on c. botulinum neurotoxin type f domains. synthetic genes encoding bont/f partial fragments: catalytic domain (rf-lc), translocation domain (rfhn), receptor binding domain (rf-hc) and quarter part of hc (rf-hcc) were designed and cloned into e. coli. additionally, expression, purification, and immunoreactivites were analyzed by western blotting and indirect elisa for lc and hn domains. the use of gst tagged recombinant protein technology was chosen in order to optimize detection and acquisition of high purity, stable, and soluble proteins through use of single step affinity chromatography. although host strains, vector types, and induction conditions for expression were optimized to recover recombinant proteins in the soluble fraction, we were unable to purify hc and hcc. immunoreactivity was accessed through gst free f-lc and f-hn through in vivo immunization and polyclonal serum antibody collection, proceeded by western blotting and indirect elisa. the anti-f-lc and anti-f-hn mouse 6 (a) 6 (b) fig.6:immunoreactivity analysis by indirect elisa nepal  journal  of  biotechnology.    jan.  2012,  vol.  2,  no.  1:  1  –  15                                                                                                                    biotechnology  society  of  nepal  (bsn),  all  rights  reserved   12 polyclonal antibodies exhibited a strong affinity towards gst free and gst tagged flc and f-hn respectively, while both recognized the native type f toxin. from this work, we conclude that purified bont/f lc and hn domains are capable of producing highly effective immunogens. moreover, if future attempts prove fruitful in the isolation of monoclonal antibodies (that possess neutralizing activity), towards bont/f fragments, this may lead to more efficient protection against a high neurotoxin dose. implications of this research could span across the development of therapeutic approaches, diagnostic detection systems, and vaccine candidacy for the protection and treatment of botulism as has been elucidated in previous work [34, 37, 39-42]. in summary, we have expressed both recombinant lc and hn domains of bont/f. the recombinant proteins are soluble; elicit an active immune response (polyclonal antibodies) in mice, making them ideal for investigators to process potential subunit vaccines towards bont/f. acknowledgements all authors wish to acknowledge the financial support from medy-tox inc. and for their help in the acquisition of c. botulinum f str. langeland and bont/f (wako pure chemicals ind., osaka, japan). we would also like to express our gratitude to jesus andres cuaron for his contributions on the drafting of this article. references 1. mccroskey lm, hatheway cl, fenicia l, pasolini b, aureli p: characterization of an organism that produces type e botulinal toxin but which resembles c. butyricum from the feces of an infant with type e botulism. j clin microbiol 1986, 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https://www.doi.org/10.54796/njb.v9i2.41909 ©njb, bsn 14 phytochemical analysis and α-amylase inhibitory activity of young and mature leaves of cinnamomum tamala rasna maharjan1 , prakash thapa1 , karan khadayat 1, 2 , surya kant kalauni1 1central department of chemistry, tribhuvan university, kirtipur, kathmandu, nepal 2department of biotechnology, national college, tribhuvan university, naya bazar, kathmandu, nepal received: 5th nov 2020; revised: 22nd sep 2021; accepted: 6th dec 2021; published online: 31st dec 2021 abstract the bioactive chemical components of the plant's origin have been used as primary remedies for a wide array of human diseases including diabetes. the present research deal to evaluate and compare anti-diabetic potential of ethanolic and methanolic, young and mature leaves of medicinally valuable cinnamomum tamala. total phenolic and flavonoid contents of young and mature leaves were determined. in vitro α-amylase inhibition was carried out using 2-chloro-4-nitrophenyl-α-d-maltotrioside (cnpg3) as substrate. phytochemical screening revealed the presence of polyphenols, flavonoids, terpenoids, quinones, carbohydrates, glycosides, diterpenes, tannins, and reducing sugars. the highest total phenolic content and flavonoid content were observed in methanolic extract of mature leaves (13.725 ± 0.54 mg gae/g) and young leaves (12.591 ± 0.71 mg qe/g) respectively. methanolic young leaves extract showed α-amylase inhibition with ic50 value 224.6 ± 2.76 μg/ml as compared to acarbose with ic50 value 5.93 ± 0.14 μg/ml. the result suggests that young leaves of c. tamala had anti-diabetic activity so further work should be carried out. keywords: α-amylase, anti-diabetic, cinnamomum tamala, phytochemicals corresponding author, email: karankhadayat55@gmail.com; skkalauni@gmail.com introduction diabetes mellitus is one of the most common health issues among the serious health concerns around the world. it is a chronic multifactorial endocrine disorder of glucose intolerance. approximately 463 million adults (20-79 years) suffered from diabetes worldwide in 2019 and may escalate to 700 million by 2045. in nepal, about 696,900 adults are living with diabetes [1]. among three type of diabetes, type 1 diabetes occur due to insulin deficiency, type 2 diabetes occur due to inability to utilize insulin and gestational diabetes occur during pregnancy condition [2]. among them, diabetes of type 2 is the most severe and fast-growing in most countries majorly due to rapid urbanization, diet, lifestyles [3,4]. in hyperglycemia, the rise in blood glucose levels generates excessive superoxide anions which produce hydroxyl radicals through haber weiss reaction that can lead to the peroxidation of membrane lipids, oxidative protein damage to cell membranes, and also affects other biomolecules including carbohydrates, proteins, and dna [5]. consequently, long-term diabetes leads to stroke, blindness, heart attack, kidney failure, amputation, and the menace of dying prematurely [2]. in recent years, the therapeutic approach for diabetes patients is diet control, exercise, and hypoglycemic drugs such as sulphonylureas, acarbose, and insulin with undesirable side effects such as liver toxicity, lactic acidosis, and diarrhea [6]. in this regard, inhibition of key carbohydrate digestive enzymes (α-amylase) is considered one of the best therapeutic techniques for the treatment of diabetes and its associated diseases. pancreatic α-amylase is also known as 1, 4-α-d-glucan glucanohydrolase ec (3.2.1.1) plays a vital role in the digestion of starch molecules in the human body. at the first stage, there is partial digestion of starch by salivary amylase enzyme. the enzyme cleaves polymeric substrate (starch) into shorter oligomers. at the second stage, these shorter oligomers are further split into maltose, maltotriose, and small malto-oligosaccharides with the help of pancreatic αamylase in the gut [7]. in this present study, α-amylase catalyzes the endohydrolysis of α-1, 4 glycosidic linkages of 2-chloro-4-nitrophenyl-α-d-maltotrioside (cnpg3) to yield free chromophore, 2-chloro-nitrophenol (cnp) and release 2-chloro-4-nitrophenyl-α-d-maltoside (cnpg2), maltotriose (g3), and glucose (g). the rate of increase in chromophore absorbance is related to the α-amylase activity of the sample [8,9]. in recent years, the scientist has been so much attracted on the exploration of the secondary metabolites of plants as inhibitors for most effective formulation aspect of drugs with few or hardly negative effects. the medicinal plant contains various natural antioxidants such as tocopherols, vitamin c, and nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 https://orcid.org/0000-0003-4460-137x https://orcid.org/0000-0002-3559-9215 https://orcid.org/0000-0002-8125-9756 mailto:karankhadayat55@gmail.com https://orcid.org/0000-0002-4882-4864 mailto:skkalauni@gmail.com nepal j biotechnol. 2021 dec.9 (2):14-20 maharjan et al. ©njb, bsn 15 phenolic compounds which are capable to neutralize oxidative damage and protecting from various diseases including diabetes [10]. plants have been the primary medicinal source for centuries to cure a wide array of human diseases, particularly in developing countries like nepal due to scarce resources, affordability, and insufficient access to conventional treatment. thus, considering the extraordinary biodiversity of nepal, there is a need for scientific investigations of plant species. among the various medicinal plants, c. tamala is the focus of this research. c. tamala (buch.-ham.) t. nees and eberm (luraceace) is one of several traditional remedies used under the ayurvedic system in nepal. its leaves are commonly called tejpat and are used as a common ingredient in cooking. this species is a perennial or evergreen tree up to 7.5 m in height and a girth of about 1.4 cm [11]. young leaves red-brown, smooth and mature leaves sericeous, glabrescent, and rarely glaucous [12]. leaves are natural food preservatives for pineapple juice [13]. besides, tejpat is also a traditional dye-yielding plant [14]. it has been traditionally used as an astringent, stimulant, and carminative [15]. c. tamala leaves exhibited significant biological properties with scientific validation including antioxidant [16,17], antimicrobial [18,19,20], anti-diabetic [21,22,23], anticancer [24], and anti-inflammatory [25]. to the best of the authors’ knowledge, no scientific literature was published regarding the comparison of young and mature leaves extracts of c. tamala to evaluate their bioactivities. the aim of this research was therefore to estimate total phenolic and total flavonoid contents along with the evaluation of in vitro anti-diabetic activity of the extracts of c. tamala by the microplate-based method. materials and methods chemicals and reagent methanol, ethanol, folin-ciocalteu (fc) reagent were purchased from qualigens, sodium carbonate, dimethyl sulphoxide (dmso), potassium acetate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and sodium chloride were obtained from fisher scientific, gallic acid, and quercetin from hi-media, aluminum chloride from merck and acarbose, α-amylase from porcine pancreas, and 2chloro-4-nitrophenyl-α-d-maltotrioside were purchased from sigma. collection and authentication of plant materials fresh young and mature leaves of c. tamala were collected separately from machhegaun, kathmandu in july 2019. a herbarium specimen was deposited at the central department of botany, tribhuvan university, and voucher code rm001 was provided. extraction the collected young and mature leaves were washed properly with water, shade dried, and ground into powder. the extraction was done by cold percolation method. in short, the powder of young and mature leaves was soaked in methanol and ethanol for 72 hrs with occasional shaking at room temperature. after 72 hrs, the solvent was filtered and the filtrate was evaporated using a rotary evaporator under vacuum at 400 c [26]. the percentage yield was calculated by the given formula: % yield = dry weight of extract dry weight of a plant × 100 phytochemical screening the presence of phytochemicals in the young and mature leaves of c. tamala was analyzed through the following standard protocol [27, 28, 29]. estimation of total phenolic content (tpc) crude extract of young and mature leaves of c. tamala was estimated for tpc by using fc reagent [30,31,32]. in brief, 20 μl sample was added with 100 μl folinciocalteu (2 n), accompanied by 80 μl na2co3 (1n). the reaction mixture was incubated in dark at room temperature for 15 minutes until the dark blue color was observed. finally, the absorbance was taken at 765 nm using a spectrophotometer (synergy lx, biotek, instruments, inc., usa). a standard calibration curve for gallic acid (10-100 μg/ml) was prepared and phenolic content was expressed as milligrams of gallic acid equivalent per gram of dry weight of the extract (mg gae/g). all experiments were performed in triplicate and expressed as mean ± standard error of mean. estimation of total flavonoid content (tfc) crude extract of young and mature leaves of c. tamala was estimated for tfc by using the aluminum trichloride (alcl3) method [32,33]. briefly, 20 μl sample with 110 μl distilled water was added with 60 μl ethanol, 5 μl alcl3 (10%), and 5 μl potassium acetate (1 m). the reaction mixture was incubated in dark at room temperature for 30 minutes. after incubation, absorbance was measured at 415 nm using a spectrophotometer (synergy lx, biotek, instruments, inc., usa). a standard calibration curve for quercetin (10 100 µg/ml) was prepared and nepal j biotechnol. 2021 dec.9 (2):14-20 maharjan et al. ©njb, bsn 16 flavonoid content was expressed as milligrams of quercetin equivalent per gram of dry weight of the extract (mg qe/g). all experiments were done in triplicate and expressed as mean ± standard error of mean. in vitro α-amylase inhibition assay the in vitro α-amylase inhibition was performed in a microplate reader as previously described method [34]. firstly, young and mature leaves extract was prepared using 50% dmso, and the reaction was carried out using 50 mm sodium phosphate buffer (ph 7) with 0.9% nacl. concisely, 20 µl of extract of different concentrations (31.25 1000 µg/ml) was mixed with 80 µl of the enzyme at a final concentration of 1.5 u/ml. then, it was preincubated at 37 °c for 10 min and after pre-incubation, 100 µl cnpg3 substrate at a final concentration of 1 mm was added and left for 15 minutes at the same temperature. the absorbance was measured at 405 nm. dmso (not more than 5% was taken as final concentration) was taken as negative control and acarbose as a positive control. all experiments were carried out in triplicate. % inhibition = a(control) − a(sample) a(control) × 100 a (control) = absorbance of enzyme-substrate with 50% dmso a (sample) = absorbance of enzyme-substrate with extract as inhibitor. statistical analysis the data were analyzed by using gen 5 microplate data collection and analysis software of microplate reader and then by ms excel. the ic50 value of crude extract was calculated by using graph pad prism version 8 software and the results were expressed as mean ± standard error mean (sem). results phytochemical screening the percentage yield was found to be higher in methanol as compared to ethanol and are given in table 1. the range of percentage yield of mature and young leaves varies from 7.36 to 18.22%. the young leaf had the highest percentage yield (eey 10.22 % and mey 18.22 %) and the mature leaf had the lowest percentage yield (eem 7.36 % and mem 12.24 %). the different phytochemical tests had shown the presence of polyphenols, flavonoids, tannins, quinones, carbohydrates, glycosides, terpenoids, and reducing sugar whereas basic alkaloids are absent in both extracts of c. tamala (table 2). table 1. percentage yield of different extract of c. tamala s. no. sample percentage yield (%) 1 ethanolic extract of mature leaf (eem) 7.36 2 ethanolic extract of young leaf (eey) 10.22 3 methanolic extract of mature leaf (mem) 12.24 4 methanolic extract of young leaf (mey) 18.22 table 2. preliminary phytochemical screening of different extract of c. tamala s. no. phytochemicals mem mey eem eey 1 polyphenols + + + + 2 flavonoids + + + + 3 tannins + + + + 4 quinones + + + + 5 carbohydrates + + + + 6 glycosides + + + + 7 terpenoids + + + + 8 diterpenes + + + + 9 reducing sugars + + + + 10 basic alkaloids “+” indicates presence, “-” indicates absence total phenolic and flavonoid content the tpc value of ethanolic young and mature leaves extract was 9.83 ± 0.30 mg gae/g and 8.91 ± 0.39 mg gae/g respectively whereas, the tpc value of methanolic young and mature leaves extract was 11.26 ± 0.17 mg gae/g and 13.73 ± 0.55 mg gae/g. the tfc value of ethanolic young and mature leaves extract was 6.82 ± 0.67 mg qe/g and 7.02 ± 0.32 mg qe/g respectively whereas, the tfc value of methanolic young and mature leaves extract was 12.59 ± 0.71 mg qe/g and 11.84 ± 0.66 mg qe/g as shown in table 3. table 3. total phenolic and flavonoid content of different extract of c. tamala s. no. sample tpc mg gae/g tfc mg qe/g 1 mem 13.73 ± 0.55 11.84 ± 0.66 2 mey 11.26 ± 0.17 12.59 ± 0.71 3 eem 8.91 ± 0.39 7.02 ± 0.32 4 eey 9.83 ± 0.30 6.82 ± 0.67 mean ± sem (n = 3) in vitro α-amylase inhibition assay table 4. screening for α-amylase inhibition of different extract s. no. sample concentration % inhibition 1 mem 500 µg/ml < 50 2 mey 500 µg/ml 70.33 ± 0.47 3 eem 500 µg/ml < 50 4 eey 500 µg/ml < 50 5 acarbose 50 µg/ml 96.77± 0.028 mean ± sem (n = 3) the percentage inhibition of young, and mature leaves of c. tamala using methnol and ethanol as solvent were screened for 𝛼-amylase inhibitory activity at 500 µg/ml (table 4). young leaf had the highest inhibition of 𝛼nepal j biotechnol. 2021 dec.9 (2):14-20 maharjan et al. ©njb, bsn 17 amylase enzyme whereas mature leaf showed the lowest inhibition at the same concentration in our study. only methanolic extract of young leaves had shown 70.33% inhibition while screening. so, the methanolic extract of young leaves was further assessed for ic50 value at different concentrations (table 5) and was found to be 224.6 ± 2.76 μg/ml. however, compared to standard drug acarbose, the methanolic extract of young leaves showed moderate 𝛼-amylase inhibitory activity (table 6) and was found to be 5.93± 0.14 as shown in table 7. table 5. α-amylase inhibition of methanolic extract of young leaves (mey) at different concentration concentration inhibition 1 inhibition 2 inhibition 3 mean ± sem 500 µg/ml 70.15 69.62 71.23 70.33 ± 0.47 250 µg/ml 54.80 58.59 58.38 57.26 ± 1.23 125 µg/ml 33.37 35.45 31.89 33.57 ± 1.03 62.50 µg/ml 14.89 9.46 10.04 11.46 ± 1.72 31.25 µg/ml 2.83 3.78 6.78 4.46 ± 1.19 mean ± sem (n = 3) 0 200 400 600 0 20 40 60 80 mey concentration (µg/ml) % i n h ib it io n inhibition 1 inhibition 2 inhibition 3 table 6. α-amylase inhibition of acarbose at different concentration concentration inhibition 1 inhibition 2 inhibition 3 mean± sem 50 µg/ml 96.82 96.75 96.72 96.76± 0.02 25 µg/ml 90.08 89.93 89.80 89.94± 0.08 12.5 µg/ml 77.72 77.07 76.88 77.22± 0.25 6.25 µg/ml 55.44 51.92 53.38 53.58± 1.02 3.125 µg/ml 24.27 23.77 33.52 27.19± 3.16 1.563 µg/ml 5.33 6.64 4.01 5.33± 0.75 mean ± sem (n = 3) 0 20 40 60 0 50 100 150 acarbose concentration (µg/ml) % i n h ib it io n inhibition 1 inhibition 2 inhibition 3 table 7. ic50 values of acarbose and potent sample (mey) s. no. sample ic50 (µg/ml) 1 acarbose 5.93± 0.14 2 mey 224.6 ± 2.76 mean ± sem (n = 3) discussion diabetes has been stated as the primary cause of global morbidity and mortality [35]. retarding the absorption and digestion of carbohydrates in the intestine through inhibition of carbohydrate digestive enzymes such as αamylase is one of the therapeutic strategies to minimizing postprandial hyperglycemia [36, 37, 38]. this study reported the potential in vitro amylase inhibitory properties of c. tamala mature and young leaf by identifying its main phytochemical constituents. extraction of phytochemicals is the preliminary process for recovering and isolating compounds from the pulverization of plant materials [39]. the extraction yield depends upon different parameters such as solvent with varying polarity, ph, temperature, extraction time, and sample composition [40]. in our study methanolic extracts showed (mem, mey) higher yield than that of ethanolic extracts (eey, eem). the difference might be due to the high polarity of methanol that may cause higher solubility of phenolics, flavonoids, alkaloids, and terpenoids compounds in methanol as compared to other solvents including ethanol [40, 41,42]. the phytochemicals obtained from c. tamala are shown in table 2 that was compared with the previously reported study and found similar phytochemical profiling [43]. our findings exhibited that methanolic extracts have the higher tpc and tfc relative to ethanolic extracts as seen in table 3. aryal et al. suggested that numerous phenolic compounds are present in plant extracts that are responsible for enzyme inhibition supported by tpc (mg gae/g) and tfc (mg qe/g) of mem (13.73 ± 0.55, 11.84 ± 0.66), mey (11.26 ± 0.17, 12.59 ± 0.71), eem (8.91 ± 0.39, 7.02 ± 0.32), and eey (9.83 ± 0.30, 6.82 ± 0.67) respectively [44]. phenolics and flavonoids compounds such as quercetin, ferulic acid, nepal j biotechnol. 2021 dec.9 (2):14-20 maharjan et al. ©njb, bsn 18 anthocyanins, catechin, and resveratrol are the reason behind controlling glycemia via α-glucosidase, αamylase, and lipid peroxidation inhibition, insulin secretion, and accelerating glucose uptake [45,46]. the difference in tpc and tfc is due to different nature of extracting solvents [47]. previously, it was revealed that younger leaves are most active in biosynthesis and accumulation of phenolic, flavonoid, and proanthocyanidins than the mature leaves of lantana camara [48]. similarly in our study, among young and mature leaf extracts, only methanolic extract of young leaf showed significant inhibition toward the α-amylase enzyme. the ethanolic extract of young leaf failed to show significant inhibition that might be due to its less polar nature causing lower yield of phenolics and flavonoids compounds. besides that, high flavonoid content of mey might be responsible for high inhibition as compared to other extracts. several potential α-amylase and α-glucosidase inhibitors from medicinal plants belongs to flavonoid class as reported by earlier study [49]. phenolic compounds including flavonoids and tannins have been reported to play a vital role in controlling diabetes [50, 51, 52]. phenolic compounds mainly flavonoids interact strongly with proteins and could inhibit their enzymatic activities by making complexes and changing conformation [53]. the ic50 value of acarbose as positive control showed around 40 times more potency than mey as shown in table 5. this might be due to plant extract contains a jumble of multiple compounds as compared to pure inhibitor compound acarbose [34, 54]. previous studies reported that polyphenol compounds (proanthocyanidins) derived from different plant sources showed potent amylase inhibitors [55, 56]. a-type procyanidin oligomer of cinnamon led to potent antioxidant properties and acts as an insulin sensitizer [57]. besides that, cinnamtannin d-1 (cd1) isolated from c. tamala was reported to shield pancreatic β-cells from palmitic acid-induced apoptosis and oral consumption of cinnamaldehyde had hypoglycemic and hypolipidemic activity in stz-induced diabetic rats respectively [23, 58]. these compounds might be present in the extract of young leaves, so further isolation and molecular characterization are needed. conclusion the present study evaluated phytochemical constituents as well as α-amylase inhibition of methanolic and ethanolic extracts from young and mature leaves of c. tamala. the in vitro results revealed that methanolic extract and young leaves possess higher antidiabetic activity. thus, it was suggested that further investigation for isolation and characterization of a bioactive compound and its kinetic study from mey should be carried out that can act as an anti-diabetic agent. abbreviations gae (gallic acid equivalent), qe (quercetin equivalent), cnpg3 (2-chloro-4-nitrophenyl-α-dmaltotrioside), mey (methanolic extract of young leaf), mem (methanolic extract of mature leaf), eey (ethanolic extract of young leaf), eem (ethanolic extract of mature leaf), tpc (total phenolic content), tfc (total flavonoid content) author’s contributions this research was performed in collaboration with all authors. rm and kk have contributed to the plan of the research work. the first draft was written by rm, responsible for data analysis, who conducted technical work (lab). skk supervised the research project, pt revised the manuscript. the final manuscript was read and approved by all authors. competing interests no competing interests. funding the research was completed by financial support from the university grant commission (sanothimi, bhaktapur, nepal). acknowledgments the authors extend their appreciation to the central department of chemistry, tribhuvan university for providing an opportunity to conduct this research and prof. dr. suresh kumar ghimire of central department of botany, tribhuvan university for plant identification ethical approval and consent not applicable references 1. idf diabetes atlas 9th edition 2019 [internet]. 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broadhurst cl, polansky mm, schmidt wf, khan a, flanagan vp, et al. isolation and characterization of polyphenol type-a polymers from cinnamon with insulin-like biological activity. j agric food chem. 2004;52(1):65-70. https://doi.org/10.1021/jf034916b 57. babu ps, prabuseenivasan s, ignacimuthu s. cinnamaldehyde-a potential antidiabetic agent. phytomedicine. 2007;14(1):15-22. https://doi.org/10.1016/ j.phymed.2006.11.005 2 nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 9‐13  9   biotechnology society of nepal (bsn), all rights reserved   original research article  an evaluation of the fungi isolated from sub‐ epidermal region of post‐harvested stored wheat  grains  shiju mathew1, george thomas2, tufail ahmad3  1 ministry of higher education, aksum university, aksum (ethiopia)  2 department of molecular biology & genetic engineering, allahabad  agricultural institute ‐deemed university, allahabad 211007 (india)  3  department of agriculture process and food engineering, allahabad  agricultural institute – deemed university, allahabad 211007 (india)  abstract  the  criteria  of  wheat  quality  are  as  varied  as  their  different  uses.  wheat,  which  is  suitable  for  a  particular use / product, may have certain characters that make it entirely unsatisfactory for other  purposes.  the  storage  fungi  damage  the  grains  in  several  ways;  they  reduce  the  germinability,  produce undesirable odor and kernel discoloration, decrease the food value and also produce toxins  injurious  to  the  health  of  consumers.  the  sub‐epidermal  mycoflora  of  stored  wheat  grains  predominantly consisted of ubiquitous mould genera aspergillus, alternaria and penicillium possibly  because  of  their  omnipresence,  capacity  to  grow  on  all  possible  substrates  and  a  wide  range  of  temperature  and  humidity.  the  most  frequent  species  observed  in  the  stored  wheat  grains  of  aspergillus  were  a.  niger  and  a.  fumigates,  alternaria  alternata  and  pencillium  citrinum.  among  these the frequency of alternaria alternata was highest which has the capacity to produce mycotoxin  which can contaminate and cause spoilage. the grain losses found in quantity and quality; can be in  the  form  of  depletion  in  seed  viability,  hardness,  color,  size  and  shape,  grain  weight  and  various  biochemical parameters viz., protein, carbohydrate and vitamins under post harvest storages.     key words: wheat, post‐harvest, storage, sub‐epidermal fungi and spoilage.    correspondence author:  e‐mail: shijumathew_biotech@yahoo.com; contact no.: (+251) 920126043  introduction  grain  production  in  any  country  varies  from  year  to  year  and  hence  the  grains  should  be  stored  strategically  from  years  of  overproduction  for  use  in  year of under production. grain quality after harvest is  influenced  by  a  wide  variety  of  biotic  and  abiotic  factors  and  has  been  studied  as  a  stored  grain  ecosystem.  spoilage  of  stored  grain  by  fungi  is  determined  by  a  range  of  factors  which  can  be  classified  into  four  main  groups  including  (a)  intrinsic  nutritional  factors,  (b)  extrinsic  factors  (c)  processing  factors  and  (d)  implicit  microbial  factors.  the  factors  produce  fungal  colonization  within  the  stored  grains  (wallace and sinha, 1981; sinha, 1995). in 1970s, it was  considered for the first time that the stored grain as a  manmade ecosystem which needed to be examined in  a  more  holistic  and  ecological  manner  to  enable  a  proper  understanding  of  the  processes  occurring  and  to  improve  post‐harvest  management  of  stored  food  commodities of all types. the post harvest losses at the  farm  level  have  been  estimated  to  be  3.28  kg/q  in  wheat. the post harvest loss of wheat grain has been  found  to  be  highest  during  storage  (magan  et  al.,  2003). stored grains can have  losses  in both quantity  and quality. losses occur when the grain is attacked by  microorganisms and other organisms including insects,  mites, rodents and birds (neetirajan et al., 2007).  the  wheat  grains  come  in  association  with  the  fungi  from the time of grain maturity and also at the time of  storage.  some  of  these  fungi  are  in  intimate  association  and  are  present  as  dormant  mycelium  under the pericarp or dormant spores on the surface of  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 9‐13  10   biotechnology society of nepal (bsn), all rights reserved   the  kernel.  however,  there  are  a  number  of  fungi  which  are  only  superficially  associated  with  stored  grains.  the  mycelium  was  usually  septate,  thin  and  branching  repeatedly  to  form  a  network  on  the  sub‐ epidermal surface of stored wheat grains. sometimes,  due  to  more  frequent  formation  of  transverse  walls,  beaded cells were seen. the sub‐epidermal mycelium  was observed even in apparently, healthy, undamaged  grains  examined  superficially  with  hand  lens.  the  association of fungi with cereal grains starts from the  field itself. shortly after the grain reaches to maximum  size,  the  lemma  and  palea  protecting  it  are  pushed  apart  exposing  the  grain  to  infection  by  fungi  (machacek  and  greaney,  1938)  and  their  extensive  studies has been carried out in the laboratory on these  aspects  (sankaran  et  al.,  1975;  sankaran,  1976;  sankaran  et  al.,  1976).  an  extensive  microflora  has  been found to be associated with stored wheat grains  (duggeli, 1904; kent‐ jones and amos, 1930; james et  al.,  1946;  christensen,  1956;  poisson  and  guilbot,  1956;  inagaski and  ikeda, 1959; field and king, 1962;  brook  and  white,  1966;  graves  et  al.,  1967;  pelhate,  1968;  hesseltine,  1968;  wallace,  1973).  earlier  in  the  laboratory  a  number  of  cereals  have  been  screened  with respect to microflora associated in storage grains  (basu,  1974;  mehrothra,  1974;  palni,  1975;  jayas,  1995). fungal activity can cause undesirable effects in  grains  including  discolouration,  contribute  to  heating  and  losses  in  nutritional  value,  produce  off‐odours,  losses  in  germinability,  deterioration  in  baking  and  milling  quality,  and  can  result  in  contamination  by  mycotoxins (hocking, 2003; magan et al., 2003).  materials and methods  the  investigation  was  done  at  allahabad  agricultural  institute‐demmed university, allahabad in india. . the  work  was  an  attempt  to  correlate  the  sub‐epidermal  fungal  infestation  and  quality  of  wheat  grain  under  storage. the wheat samples were collected separately  in  3  replicates  for  each  of  the  wheat  (triticium  aestivum l.) varieties viz., u.p. 262 and h.d. 1982 from  f.c.i.  godown,  naini,  allahabad  district,  whole  sale  dealers from naini and muttiganj markets of allahabad  and from the local farmers. the samples were brought  to the  laboratory under aseptic condition where they  were screened (before washing and after washing with  water) for their associated sub‐epidermal fungal flora.  out of these, some samples were heavily infested with  fungi, some were slightly infested and some were not  at  all  infested.  twenty  samples  of  wheat  grains  belonging to two different varieties were screened for  the presence of sub‐epidermal mycelium. five different  sample  collection  sites  were  selected  and  12  month  stored  (moisture  content  8‐16%)  samples  were  screened  within  a  week  of  their  collection.  one  hundered  grains  from  each  samples  were  taken  randomly, they were examined for the presence of sub ‐epidermal mycelium within the grain by the method of  hyde and galleymore (1951) as described below:   1.  the  grain  without  cracks  and  holes  on  their  epidermis were soaked in water for a short period  and  then  the  epidermis  was  peeled  off  with  forceps.  2.  the stored grains were also examined superficially  with a hand lens (10 x).  3.  the  peeled  epidermis  contains  sub‐epidermal  mycelium.  4.  the  epidermis  was  then  placed  in  aniline  blue  (0.2%  in  66%  lactic  acid)  and  warmed  for  5  –  10  minutes which stained the mycelium alone and the  epidermal cells were left unstained.  the  sub‐epidermal  fungi  was  brought  into  culture  by  first  surface  disinfecting  the  soaked  grains  by  immersion  for  two  minutes  in  0.2%  sodium  hypochlorite  and  washing  in  two  changes  of  sterile  water. then the peeling of the epidermis were taken  out  and  transferred  to  five  different  culture  medium  viz.,  czapek’s  solution  agar,  czapek’s  osmophilic  solution  agar,  malt  extract  agar,  wheat  extract  agar  and oat meal agar with the following composition:  1.  czapek’s  solution  agar  media,  oat  meal  agar  media and malt extract agar: the composition of  these media has been mentioned above.  2.  czapek’s  osmophilic  solution  agar:  same  as  czapek’s  solution  agar  media  except  for  sucrose  which is 20% instead of 3%.  3.  wheat extract agar:  wheat extract 20g; agar, 30g;  yeast extract, 0.5g; distilled water, 1000ml.    the culture was incubated at 25± 2 0c for 7 days.  each set of experiment had a control to differentiate  the  laboratory  contaminants  from  the  microflora  actually  associated  with  the  sample.  the  fungal  colonies were isolated and  identified with the help of  authentic  literature.  the  frequency  of  occurrence  of  different  fungi  isolated  from  wheat  samples  was  calculated by the following formula;                                                           nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 9‐13  11   biotechnology society of nepal (bsn), all rights reserved   the  various  media  used  for  the  identification  of  subepidermal  fungi  with  different  composition  of  czepek’s  solution  agar  {nano3  (3.0  g);  k2po4  (1.0  g);  kcl (0.5 g); mgso4 .7h2o (0.5 g); feso4 .7h2o (0.01 g);  sucrose (30.0 g); agar (20.0 g); distilled water (1000.0  ml);  ph  6.5)},  czepek’s  yeast  agar  {  k2hpo4  (1.0  gm);  czepek’s concentrate (10.0 gm); yeast extract (5.0 gm);  sucrose  (30.0  gm);  agar  (20.0  gm);  distilled  water  (1000.0 ml); ph 6.5}, malt extract agar {malt – extract  (20.0 g); peptone (1.0 g); dextrose (20.0 g); agar (20.0  g);  distilled  water  (1000.0 ml)  ph  6.5}, oat  meal  agar  {oat meal (20.0 g); yeast extract  (0.5 g); agar (20.0 g);  distilled  water  (1000.0  ml);  ph  6.5},  potato‐dextrose  agar {potato dextrose (20.0 g); agar (20.0 g); distilled  water  (1000.0  ml);  ph  6.5},  peptone  agar  {peptone  (10.0  g);  dextrose  (20.0  g);  agar  (20.0  g);  distilled  water  (1000.0  ml);  ph  6.5}  and  synthetic  mucor  agar { dextrose (20.0 g); asparagine (2.0 g); kh2po4 (0.5 g);  mgso4  .7h2o  (0.25  g);  thiamine  chloride  (0.5  g);  distilled water (1000.0 ml); ph 6.5}. these media were  sterilized at 1210c and 15 lbs p.s.i. for 20 minutes.  results and discussion  the result of sub‐epidermal fungi in the stored wheat  grain  varieties  u.p.  232  and  h.d.1982  collected  from  different sites of allahabad are given in figure 1. sub‐ epidermal mycelium was seen in all samples screened.  these mycelia were usually septate, thin and branching  repeatedly to form a network. sometimes, due to more  frequent  formation  of  transverse  walls,  beaded  cells  were seen. four species of fungi as shown  in table 1  were  isolated  from  sub‐epidermal  region  of  wheat  grains and the microscopic view  is shown  in figure 2.  the  most  common  storage  fungi  observed  in  the  stored  wheat  grain  samples  was  alternaria  alternata  (14.6%). species of aspergillus were also encountered  with a. fumigatus (10.5%), and a. niger (8.3%) followed  by  penicillium  citrinum  (3.8%).  the  sub‐epidermal  mycelium  was  observed  even  in  apparently,  healthy,  undamaged  grains  examined  superficially  with  hand  lens  prior  to  peeling.  sporophores  were  sometimes  seen emerging from the groove region or from cracks  and holes on the smooth surface of the stored wheat  grain.   the  entry  of  the  fungus  in  stored  grains  could  be  through the epidermis as the sub‐epidermal mycelium  was  observed  even  in  apparently  healthy  grains  examined  superficially  with  a  hand  lens  and  prior  to  peeling. however, sporophores were sometimes seen  emerging in the groove region or from the cracks and  holes  in  the  smooth  surface.  simmonds  (1968)  observed that infection takes place through the cracks  in the epidermis or the opening over the embryo since  abundant fungal fruiting was evident where cracks had  occurred. welling (1968) found that aspergillus species  infected  seeds  independent  of  the  amount  of  injury.  the  mature  stored  wheat  grains  were  found  to  have  more  injury.  hyde  and  galleymore  (1951)  observed  that  mature  grains  contain  more  internal  mycelia.  flannigan  (1974)  also  found  alternaria  alternata  in  78.5%  of  the  wheat  grains  established  fungi  with  alternaria  alternata  was  among  the  ones  which  appeared later.  reports from various countries show that this species  is  found  to  be  the  most  common  post  harvest  fungi.  saponaro and madaluni (1960) reported the presence  of aspergillus  in stored wheat grains  in  italy, wallace  and  sinha  (1962)  in  canada,  kurata  et  al.  (1968),  tsunado (1970) and tsuruta (1970) in japan. however,  james and smith, 1948 from canada reported a. niger  stood second with respect to frequency of occurrence  of fungi in stored wheat grains.  conversely aspergillus  and penicillium are more often considered as ‘storage  fungi’.  they  are  known  to  form  mycotoxins  in  stored  grains and are usually not regarded as fungi that can  produce  mycotoxins  before  harvest  (frisvad,  1995;  wicklow, 1995; hockings, 2003)  acknowledgements  i  gratefully  acknowledge  the  cooperation,  uninterrupted  guidance,  impeccable  and  valuable  suggestions  rendered  to  me  by  prof.  (dr.)  george  thomas during my research work.   s. no.  organisms  frequency of occurrence (%)  1  alternaria alternata  14.6  2  aspergillus fumigatus  10.5  3  aspergillus niger  8.3  4  penicillium citrinum  3.8  table 1: frequency of fungi isolated from sub ‐epidermal region of stored wheat  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1 : 9‐13  12   biotechnology society of nepal (bsn), all rights reserved   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analyzing dna sequence data: an example from  tomato genes  bal k. joshi and dilip r. panthee  north carolina state university, raleigh, north carolina‐ 27695, usa  abstract  dna and amino acid sequences are alphabetic symbols having no underlying metric. use of information theory is  one of the solutions for sequence metric problems. the reflection of dna sequence complexity in phenotype  stability  might  be  useful  for  crop  improvement.  shannon‐weaver  index  (shannon  entropy,  h’)  and  mutual  information  (mi)  index  were  estimated  from  dna  sequences  of  22  genes,  consisted  of  two  gene  families  of  tomato,  namely  disease  resistance  and  fruit  quality.  main  objective  was  use  of  information  theory  and  multivariate  techniques  to  understand  diversity  among  genes  and  relate  the  sequence  complexity  with  phenotypes. the normalized h’ value ranged from 0.429 to 0.461. the highest diversity was observed  in the  gene  crtr‐b  (beta  carotene  hydroxylase).  two  principal  components  which  accounted  for  36.65%  variation  placed these genes into four groups. groupings of these genes by both principal component and cluster analyses  showed  clearly  the  similarity  at  phenotypes  levels  within  cluster.  sequences  similarity  among  genes  was  observed within a family. diversity assessment of genes applying information theory should link to understand  the sequences complexity with respect to gene stability for example stability of resistance gene.     key words: diversity analysis, dna sequences, principal component analysis, tomato genes    correspondence author:  e‐mail: joshibalak@yahoo.com  introduction  sequencing of genomic dna has been started in many  organisms in the world and most of the sequences are  publically available.  international solanaceae genome  project  (sol)  has  started  sequencing  the  genome  of  tomato.  ten  countries  namely  korea,  china,  united  kingdom, india, the netherlands, france, japan, spain,  italy and the united states are involved in the genome  sequencing  project  of  tomato  (mueller  et  al.,  2005).  tomato is the model plant for the study of a number of  economically  important  traits  including  fruit  development  and  plant  defense  (li  et  al.,  2001;  tanksley,  2004).  major  problem  with  these  sequence  data  is  metric  problem  i.e.  difficult  to  analyze  statistically  to  extract  the  biologically  meaningful  information.  use  of  information  theory  is  one  of  the  ways for handling with sequence metric problem data.  information theory which  is based on probability and  statistics quantifies information in the categorical form  of  data  e.g.  alphabetic  sequences  of  dna.  entropy  is  the  key  measure  of  information  which  quantifies  the  uncertainty  involved  when  encountering  a  random  variable.  another  element  of  information  theory  is  mutual  information  which  is  the  amount  of  information in common between two random variables  (schneider, 2003; atchley et al., 2000).  many  resistant  varieties  of  tomato  have  been  developed through the introgression of resistant genes  either from cultivated or from wild species of tomato.  more  than  8  years  is  necessary  to  develop  resistant  variety  through  conventional  breeding  system.  however, due to the high mutation rate  in pathogen,  resistant gene may not be effective for a  longer time  because  of  the  evolution  of  mutant  pathogen.  more  stable  resistant  gene,  if  possible  to  identify  at  early  stage  may  contribute  greatly  in  crop  improvement.  similarly, there is a high variation for fruit quality traits  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 1‐8  2  biotechnology society of nepal (bsn), all rights reserved   of tomato. many of these genes have been sequenced  and  data  are  available  in  the  solanaceae  genomics  network (sgn) (http://sgn.cornell.edu) (mueller et al.,  2005a; mueller et al., 2005b). dna sequences data are  added rapidly to the sgn. however, utilization of these  data  is  restricted  due  to  lack  of  data  analysis  tools.  using  the  information  theory,  various  indices  and  variability  had  been  estimated  from  the  amino  acid  sequences  data  and  subsequently  analyzed  using  multivariate techniques (atchley et al., 1999; atchley et  al., 2000; atchley and zhao, 2007; atchley et al., 2005).  dna sequences are also alphabetic symbols that need  to transform these symbols to biologically meaningful  variables.  approaches  used  by  atchley  et  al.  (1999,  2000,  2005)  can  be  used  to  summarize  the  dna  sequences  data.  multivariate  technique,  for  example  principal  component  analysis  (pca)  and  cluster  analysis  would  help  greatly  to  draw  inference  from  these dna sequences data. the objective of this study  was  to  measure  the  diversity  and  to  study  the  relationship  between  sequence  variation  and  phenotype  among  two  gene  families  namely  disease  resistance and fruit quality.   multivariate analysis allows using all available variable  information  simultaneously  producing  a  single  parameter.  it  has  been  used  for  both  qualitative  and  quantitative  characters  to  measure  genetic  relationships  within  cereal  species  e.g.  barley  (cross,  1992;  hussaini  et  al.,  1977)  and  rice  (kanwal  et  al.,  1983).  the  information  generated  can  be  useful  for  identifying  groups  of  accessions  that  have  desirable  characters for further study and for investigating some  aspects  of  crop  evolution  (brown,  1991;  cowen  and  frey,  1987;  perry  and  mcintosh,  1991).  among  the  different  methods  of  multivariate  analysis,  pca  and  cluster analysis are commonly used. pca is a technique  for analyzing relationships among several quantitative  variables  measured  on a  number  of  objects  (ringnér,  2008).  it  provides  information  about  the  relative  importance  of  each  variable  in  characterizing  the  objects.  cluster  analysis  can  be  used  to  group  units  according to the similarity for certain characteristics.   among  the  different  measures  of  diversity,  shannon‐ weaver index (shannon entropy, h') is being used for  qualitative  traits  (holcomb  et  al.,  1977;  niwranski  et  al., 2002; tolbert et al., 1979) and amino acid sequence  data (atchley et al., 2000, 2005). principally h' includes  both  species  number  and  evenness,  where  a  greater  number of species  increase diversity, as does a more  equitable distribution of individuals among species. as  species  richness  and  evenness  increase,  so  does  diversity.  diversity  index  provides  important  information about rarity and commonness of species in  a  community.  the  ability  to  quantify  diversity  in  this  way  is  an  important  tool  for  biologists.  alphabetic  sequence data can be summarized more precisely with  this  idea  and  multivariate  techniques.  this  paper  describes  the  gene  diversity  in  tomato  using  information theory and multivariate techniques.  materials and methods  tomato  genetic  resource  center  (tgrc)  (http:// tgrc.ucdavis.edu) has  listed a total of 1239 genes and  their  symbols  along  with  their  phenotypes.  among  them, there are 68 resistance genes related to diseases  and 41 genes related to fruit quality of tomato. a total  of 22 genes (table 1) consisting of 8 disease resistance  genes, 12 fruit quality related genes and 2 genes from  potato  genome  were  used  in  this  study.  dna  sequences along with other traits of these genes were  downloaded  from  solanaceae  genomics  network  (http://sgn.cornell.edu).   multiple  alignments  of  dna  sequences  were  done  in  clustelx2  (http://www.clustal.org/).  clustalx2  calculates  the  best  match  for  the  selected  sequences  based on the homology concept, and lines them up so  that the  identities, similarities and differences can be  seen.  shannon  entropy  (h’)  was  estimated  for  each  gene,  not  the  site  of  nucleotides  using  the  fastaentropy  software.    fastaentropy  was  originally  developed for estimating h’ and mi of amino acid sites  (column wise) based on the 20 amino acids ( atchley et  al., 1999,2000; butte and kohane, 2000). we used this  software  in dna sequences data. to verify the use of  fastaentropy in dna sequences data, we estimated h’  of  translated  amino  acids  and  found  the  strong  association  with  dna  sequence‐based  estimates.  normalized  entropy  value  was  used  to  develop  the  entropy  profile  of  each  gene.  normalized  mutual  information (mi) matrix among the genes was used to  generate  the  scatter  biplot  considering  principal  components  i  and  ii  using  ntsyspc  (http:// www.exetersoftware.com/index.html). cluster analysis  was also carried out based on this mi in ntsyspc. pca  permits  reduction  of  the  complexity  or  dimension  of  the  problem  (johnson  and  wichern,  1988;  ringnér,  2008). the technique consists of reducing the structure  of  the  data  matrix  starting  from  a  linear  model  of  getting  new  variables,  referred  to  as  principal  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 1‐8  3  biotechnology society of nepal (bsn), all rights reserved   components.  those  principal  components  with  eigen  values ³ 1.0 were selected. cluster analysis allows one  to  identify  groups  of  objects  or  variables  that  are  similar among themselves (sneath and sokal, 1973).  results and discussion  generally mapping populations in tomato is generated  by crossing cultivated species with wild relatives which  results in maximizing the genetic variation particularly  for disease resistance and fruit quality traits  (frary et  al.,  2000;  fridman  et  al.,  2000).  therefore,  we  used  these  two  gene  families  (disease  resistance  and  fruit  quality)  to  assess  and  characterize  the  sequence  variation. all available genes with dna sequences were  included in our study.  tomato is a diploid with 2n= 24 chromosomes and self  pollinated species. the eight disease resistance genes  are located in chromosomes 1, 3, 4, 5 and 6, mostly in  long arm (table 1). one locus, pseudomonas syringe pv  tomato  resistance  has  4  alleles.  dna  sequences  of  these genes ranged from 1496 to 3987 base pairs. the  number of sequences of alleles of pto were the same.  with regards to 12 fruit quality related genes, mostly  they are located in long arm of chromosomes 3 and 6.  beta carotene gene has 4 alleles with the same number  of  dna  sequences  and  py‐1  gene  has  5  alleles  with  different number of sequences. the allele prov5 of psy‐ 1 has the shortest sequences and never ripe, nr has the  longest dna sequences. two disease resistance genes  from potato genome were of 1508 and 333 number of  base pairs.   shannon entropy profiles along with their index (h’) of  each  gene  are  given  in  figure  1.  the  normalized  h’  value  ranged  from  0.4291  to  0.461.  all  the  alleles  of  beta carotene and pto have the same h’ values. both  genes  of  potato  indicated  more  gene  diversity  and  complexity  as  compared  to  tomato  genes.  within  tomato  genes,  the  highest  diversity  was  observed  in  the gene, crtr‐b (beta carotene hydroxylase) and the  lowest diversity  in phytoene synthase‐1 (prov5). gene  of  white  flower  was  the  most  variable  and  of  yellow  flower  was  the  least  variable.  the  second  highest  complexity  in  dna  sequences  was  observed  in  the  gene  resistance  to  potato  cyst  nematode.  entropy  values  of  all  the  proteins,  generated  after  translating  these  dna  sequences  were  higher  than  their  genes,  but  strong  correlation  between  directly  estimated  h’  values  of  genes  and  h’  of  their  translated  protein  indicates that fastaentropy can be used to estimate h’  from dna sequences (data not shown). on an average,  h’ value of disease resistance gene family was higher  than fruit quality gene family.  the higher the h’ value, the more sequence complexity  in  the  gene.  based  on  this  hypothesis  the  gene  with  high h’ value would be more stable or such gene need  long time to take place the evolutionary changes. this  is very useful particularly to develop disease resistance  variety. breeder can identify the more stable resistance  genes looking on the shannon entropy profile.   mutual information values for genes are given in table  2. the value with 1, for example, among the alleles of  pto (pseudomonas syringae pv tomato resistance) and  b  (beta  carotene)  indicates  that  the  alleles  of  these  genes have the same sequences. the associations   of  psy‐1‐prov5 (yellow color of ripe fruit flesh) with psy‐1‐ 2s  and  psy‐1‐prov4  were  the  highest.  association  of  rangap‐2 was  lower with most of the genes. mutual  information  values  describe  the  extent  of  association   figure 1.  normalized  shannon entropy  profile of tomato  genes  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 1‐8  4  biotechnology society of nepal (bsn), all rights reserved   sn  gene  allele  locus name  chromosome‐arm  phenotype  seq. length  disease resistance genes         1  asc  ‐‐  alternaria stem  canker resistance   3‐long  resistance to alternaria stem canker  1496   2  cf‐9  ‐‐  cladosporium  fulvum resistance   1‐short  resistance to specific races of  cladosporium fulvum  2906   3  hero  ‐‐  heterodera  rostochiensis   4‐short  resistance to potato cyst nematode  (globodera rostochiensis)  4280   4  pto  1  pseudomonas  syringae pv tomato  resistance   5‐long  resistance to pseudomonas syringae  pv tomato, race zero sensitive to  insecticide fenthion  2466   5  pto  2  pseudomonas  syringae pv tomato  resistance   5‐long  resistance to pseudomonas syringae  pv tomato  2466   6  pto  h  pseudomonas  syringae pv tomato  resistance  5‐long  resistance to pseudomonas syringae  without fenthion sensitivity  2466   7  pto  pto‐2  pseudomonas  syringae pv tomato  resistance   5‐long  resistance to pseudomonas syringae  pv tomato  2466   8  mi‐1.2     leucine zipper,  nucleotide binding,  leucine‐rich repeat  6‐short  r gene that confers resistance  against some species of root knot  nematode and specific isolates of  potato aphid and white fly  3987  fruit quality genes            9  b  1  beta‐carotene   6‐long  flesh of ripe fruit orange, due to high  b‐carotene, low lycopene  concentrations  1772   10  b  c  beta‐carotene  6‐long  increased fruit lycopene content,  phenotype similar to b‐og  1772   11  b  m  beta‐carotene  6‐long  high b‐carotene, low lycopene in ripe  fruit  1772   12  b  og  beta‐carotene  6‐long  corolla tawny orange, increased fruit  lycopene  1772   13  crtr‐b  wf  beta carotene  hydroxylase  3‐short  white flower  1340   14  nr  ‐‐  never ripe   9‐long  fruits turn color at normal time, but  develop full pigmentation slowly and  never assume as deep a color as  normal  3240   15  psy 1  (1s)  phytoene synthase 1   3‐short  yellow color of ripe fruit flesh  942   16  psy 1  (2s)  phytoene synthase 1   3‐short  yellow fruit flesh, lighter yellow  flowers  1730   17  psy  1  prov4  phytoene synthase 1  3‐short  yellow color of ripe fruit flesh  1939   18  psy 1  prov5  phytoene synthase 1  3‐short  yellow color of ripe fruit flesh  213   19  psy 1  y  phytoene synthase 1  3‐short  likely allele of r with reddish flesh  tones in ripe fruit  303   20  rin  1  ripening inhibitor   5‐long  fruits green at maturity, later turning  bright yellow, retarded ripening  1192  potato genes            21  star     solanum tubersum  ankyrin repeat     putatively involved in quantitative  resistance to phytophthora infestans  1508   22  ranga p2     gtpase‐activating  protein     a gtpase‐activating protein that  interacts with rx, the potato virus x  resistance gene  333  table 1. list of genes and phenotypes along with their location related to diseases resistance and fruit quality of  tomato (http://tgrc.ucdavis.edu/data/acc/genes.aspx) used in this study  between  gene  pair  and  zero  value  between  them  means  they  are  independent  (atchley  et  al,  1999,  2000).  we  found  all  genes  having  some  degree  of  association with each others.   result of principal component analysis of sequences of  dna  is  given  in  figure  2.  the  first  seven  principal  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 1‐8  5  biotechnology society of nepal (bsn), all rights reserved   components with ≥ 1 eigen value accounted for 70.03%  of  the  total  variance  among  the  genes  based  on  the  dna  sequences.  the  first  principal  component  explained  18.61%  and  second  component  accounted  for 18.03% variance. plotting of these accessions along  first  and  second  principal  components  indicated  that  these  genes  were  placed  in four  groups. most  of the  individual  genes  make  a  separate  cluster  (figure  3).  clustering  these  genes  can  be  helpful  to  identify  the  similarity among the genes.   both  pca  and  cluster  analysis  separated  these  genes  into  four  groups.  these  grouping  were  also  similar  phenotypically.  for  example,  genes  related  to  beta  carotene  make  a  single  cluster  and  genes  related  to  fruit  characters  tended  to  appear  together.  this  indicates that the nature of variation in dna sequences  reflect the similar variation at phenotypic levels.   genes  within  cluster  were  phenotypically  similar.  however,  one  group  consisted  of  both  disease  resistance and fruit quality genes. this might be due to  role  of  secondary  metabolites  in  fruit  quality  and  disease resistance. for example tomato fruit contains  antioxidant, mainly pigment which is also necessary in  defense  mechanism.  potato  gene,  rangap2  made  an  figure 2. plotting of tomato genes considering principal component i (18.61%) and principal component ii  (18.03%) based on mutual information index calculated from the dna sequences   figure 3. upgma cluster analysis of tomato genes generated from the mutual information matrix  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 1‐8  6  biotechnology society of nepal (bsn), all rights reserved   g en e  p to   2  3  4  5  6  7  8  9  10   11   12   13   14   15   16   17   18   19   20   21   p to 2  1  1                                        p to ‐2   1  1  1                                      p to ‐h   1  1  1  1                                    p sy ‐1 ‐2 s  0. 02 7  0. 02 7  0. 02 7  0. 02 7  1                                  p sy ‐1 ‐ p ro v5   0. 08 1  0. 08 1  0. 08 1  0. 08 1  0. 90 8  1                                p sy ‐1 ‐ p ro v4   0. 02 5  0. 02 5  0. 02 5  0. 02 5  0. 26 7  0. 90 8  1                              p sy ‐1 ‐y   0. 05 6  0. 05 6  0. 05 6  0. 05 6  0. 63 7  0. 75 6  0. 65   1                            h er o   0. 00 9  0. 00 9  0. 00 9  0. 00 9  0. 01 3  0. 03 1  0. 01 5  0. 01 6  1                          m i‐ 1. 2  0. 01   0. 01   0. 01   0. 01   0. 01   0. 01 7  0. 01 4  0. 01 3  0. 23   1                        b ‐1   0. 01 1  0. 01 1  0. 01 1  0. 01 1  0. 01 2  0. 03 4  0. 01 1  0. 01 2  0. 07 7  0. 06 4  1                      b ‐c   0. 01 1  0. 01 1  0. 01 1  0. 01 1  0. 01 2  0. 03 4  0. 01 1  0. 01 2  0. 07 7  0. 06 4  1  1                    b ‐m   0. 01 1  0. 01 1  0. 01 1  0. 01 1  0. 01 2  0. 03 4  0. 01 1  0. 01 2  0. 07 7  0. 06 4  1  1  1                  b ‐o g  0. 01 1  0. 01 1  0. 01 1  0. 01 1  0. 01 2  0. 03 4  0. 01 1  0. 01 2  0. 07 7  0. 06 4  1  1  1  1                p sy ‐1 ‐1 s  0. 00 7  0. 00 7  0. 00 7  0. 00 7  0. 02 2  0. 02 1  0. 02 3  0. 02 4  0. 00 9  0. 01   0. 00 5  0. 00 5  0. 00 5  0. 00 5  1              n r‐ 1  0. 00 6  0. 00 6  0. 00 6  0. 00 6  0. 01   0. 03 6  0. 01 2  0. 00 9  0. 00 5  0. 00 3  0. 00 5  0. 00 5  0. 00 5  0. 00 5  0. 01 1  1            ri n ‐1   0. 01 2  0. 01 2  0. 01 2  0. 01 2  0. 01 2  0. 02 9  0. 01 4  0. 01 9  0. 00 6  0. 00 8  0. 00 5  0. 00 5  0. 00 5  0. 00 5  0. 00 7  0. 01   1          st ar   0. 00 6  0. 00 6  0. 00 6  0. 00 6  0. 01 8  0. 02 1  0. 01 8  0. 05 4  0. 00 5  0. 00 5  0. 01 4  0. 01 4  0. 01 4  0. 01 4  0. 02 7  0. 01   0. 01   1        a sc   0. 00 4  0. 00 4  0. 00 4  0. 00 4  0. 00 3  0. 02 2  0. 00 5  0. 02   0. 00 6  0. 00 5  0. 00 8  0. 00 8  0. 00 8  0. 00 8  0. 00 4  0. 00 2  0. 01 1  0. 01 8  1      c rt r ‐w f  0. 00 4  0. 00 4  0. 00 4  0. 00 4  0. 00 8  0. 02 1  0. 01 3  0. 01 2  0. 00 7  0. 00 8  0. 00 8  0. 00 8  0. 00 8  0. 00 8  0. 00 9  0. 00 8  0. 00 5  0. 02   0. 00 4  1    r an g a p ‐2   0. 00 3  0. 00 3  0. 00 3  0. 00 3  0. 00 7  0. 02 8  0. 00 7  0. 02 2  0. 00 6  0. 00 6  0. 00 3  0. 00 3  0. 00 3  0. 00 3  0. 00 8  0. 00 4  0. 00 3  0. 01 4  0. 00 3  0. 00 7  1  c f‐ 9  0. 00 5  0. 00 5  0. 00 5  0. 00 5  0. 00 7  0. 02 8  0. 00 8  0. 02 3  0. 00 4  0. 00 5  0. 00 8  0. 00 8  0. 00 8  0. 00 8  0. 00 7  0. 00 4  0. 00 4  0. 03 2  0. 00 6  0. 00 7  0. 00 3  ta b le  2 . m u tu al  in fo rm at io n  m at ri x  am o n g  22  g en es  o f  to m at o  a n d  p o ta to  e st im at ed  f ro m  d n a  s eq u en ce s  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 1‐8  7  biotechnology society of nepal (bsn), all rights reserved   individual  cluster.  phytophthora  infestans  resistance  gene from potato clustered with cladosporium fulvum  resistance gene of tomato.   sequences of dna are being deposited in the website  by large amount and such data has a sequence metric  problem. this problem could now be handled following  the method of estimating variability and covariability,  mutual  information  index  and  applying  dimension  reduction  approaches  as  used  in  amino  acids  sequences  (atchley  et  al.,  2000,  2005;  atchley  and  zhao,  2006).  atchley  et  al.  (2000)  summarized  the  amino  acids  sites  of  bhlh  protein  by  estimating  entropy  and  mutual  information  values.  information  theory is useful to look for pattern in dna and protein  sequences  (schneider,  1999).  information  theory  has  been  applied  to  the  analysis  of  dna  and  protein  sequences for analyzing sequence complexity from the  shannon‐weaver  indices  and  comparing  homologous  sites  in  a  set of  aligned  sequences  by  means  of  their  information content.   strong relationship was detected in the present study  between  dna  variation  and  phenotype.  a  large  amount of genetic and molecular information has been  deposited  at  http://sgn.cornell.edu  (jiménez‐gómez  and  maloof,  2009)  that  might  be  useful  to  explore  possible  relationships  using  this  technique.  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i.  morphological  traits.  crop  science 31: 1350‐1355.  ringnér,  m.  2008.  what  is  principal  component  analysis? nature biotechnology 26: 303.  schneider, t.d. 1999. information theory primer. web  document: 1‐9.  sneath,  p.h.a.  and  r.r.  sokal.  1973.  numerical  taxonomy. springer.  tanksley,  s.d.  2004.  the  genetic,  developmental,  and  molecular bases of fruit size and shape variation  in  tomato. the plant cell online 16: s181‐189.  tolbert, d.m., c.o. qualset, s.k. jain and j.c. craddock.  1979.  a  diversity  analysis  of  a  world  collection  of  barley. crop science 19: 789‐794.  nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 doi: https://doi.org/10.3126/njb.v7i1.26945 ©njb, biotechnology society of nepal 8 nepjol.info/index.php/njb. original research article antimicrobial resistance patterns and plasmid profiles of methicillin resistant staphylococcus aureus isolated from clinical samples gaurav agrahari1, amrit koirala1, roshan thapa1, mahesh kumar chaudhary2 and reshma tuladhar1* 1central department of microbiology, tribhuvan university, kirtipur, nepal, 2kist medical college, imadol, kathmandu, nepal, abstract methicillin-resistant staphylococcus aureus (mrsa), showing resistance to several antibiotics is a global health problem associated with considerable mortality and morbidity. antibiotic susceptibility test is a commonly used method to characterize mrsa in epidemiologic studies. additionally, plasmid profile has been reported to be useful in tracing the epidemiology of antibiotic resistance. this research was conducted to determine the antimicrobial resistance patterns and plasmid profiles of mrsa isolated from clinical samples at kist medical college, imadol, kathmandu, nepal. all the clinical specimens sent to the laboratory were processed by standard microbiological techniques and antibiotic susceptibility testing was done by the modified kirby bauer disc diffusion method. further, plasmid profiling was done by alkaline-lysis method. a total of 27 (38.02%) mrsa were isolated from 71 s. aureus positive samples. mrsa showed the highest resistance towards penicillin (92.60%) and ampicillin (92.60%). in contrast, high levels of sensitivity were shown towards vancomycin (85.19%) and tetracycline (85.19%). out of 27 mrsa positive samples, single plasmids were isolated from only 6 (22.22%) mrsa isolates. antibiograms alone are inadequate to accomplish the characterization of mrsa during epidemiological studies. however, plasmid profile analysis in conjunction with the antibiotic susceptibility pattern is valuable in the epidemiological investigation of mrsa, and for reducing mrsa prevalence and treatment cost. keywords: staphylococcus aureus, antibiotic resistance, methicillin resistance s. aureus, plasmid *corresponding author e-mail: resutu@gmail.com introduction methicillin-resistant staphylococcus aureus (mrsa) was first reported in 1961, within a year of methicillin introduction [1]. since then, mrsa strains have spread among hospitals and disseminated worldwide. being recognized as a prominent nosocomial pathogen, in the past few decades mrsa has emerged outside of healthcare settings, spreading in the community [2, 3]. these community-associated mrsa strains have also been shown to be the cause of healthcare-associated infections [4]. due to an increase in mrsa infection, the rate of morbidity and mortality has increased along with the increase in health care costs. in various countries, the prevalence of mrsa has varied from hospital to hospital. indeed, asia accounts for the highest prevalence rates of healthcare-associated methicillin-resistant s. aureus (ha-mrsa) and community-associated methicillin-resistant s. aureus (ca-mrsa) in the world. the majority of hospitals in asia are endemic for multidrug-resistant mrsa (mdrmrsa), with an estimated proportion of 28% (in hong kong and indonesia) to >70% (in korea) among all clinical s. aureus isolates in the early 2010s [5]. in the united states, mrsa infection is the second major cause of death among people after aids per year and is estimated to cause more infections than the other diseases combined [6]. in large united states hospitals (500 or more beds), about 40% of s. aureus infections are methicillin-resistant and are increasing continuously [7]. in the context of nepal, various research and studies showed great variations in the prevalence of mrsa nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 agrahari et al. 2019 ©njb, biotechnology society of nepal 9 nepjol.info/index.php/njb. ranging from 11-50% [8-13]. overuse or misuse of antibiotics may be the common reason for the rapid increase in the frequency of mrsa strains as well as mdr-mrsa [14]. lack of hygienic practices of the medical personnel in hospitals or health care centers and lack of isolation room for the known patients already infected with mrsa strains in health care centers, etc. are the other probable reasons for the increase in mrsa infections [15]. the best understood mobile genetic elements are plasmids, which have been widely studied in epidemiological surveillance of mrsa outbreaks and in tracing antibiotic resistance [16]. it is currently suggested that more than 90% of mrsa strains carry plasmids while numerous studies have supported that the genetic exchange of plasmids containing antibiotic-resistant genes between bacteria plays an important role in the evolution of multidrugresistance [17-19]. in various epidemiological studies, antibiograms and plasmid profiles are commonly used to characterize mrsa. however, to achieve differentiation, antibiograms are frequently inadequate. plasmid profile is more instructive and has been reported to be a useful tool in tracing the epidemiology of antibiotic resistance. this study was therefore, conducted to analyze the discriminatory power of plasmid profile analysis in conjunction with antibiogram in differentiating different strains of mrsa and antibiotic resistance patterns and resistance plasmids of mrsa isolated from clinical samples. materials and methods this cross-sectional study was conducted at kist medical college, imadol, kathmandu, nepal, from may 2014 to november 2014. the study comprises 2044 clinically ill, both indoor and outdoor patients which include children, adult and old of both sexes, aged between one month to ninety years who visited kist medical college, imadol, kathmandu. all the clinical specimens such as pus, urine, blood, sputum and body fluids were collected in strictly sterile, leak-proof, dry containers which were free from all traces of disinfectants. the sample specimens were collected by medical officers or as per the instruction by the medical officers before the use of antibiotics, and then immediately transferred to microbiology laboratory. the samples taken to the laboratory were processed as quickly as possible. staphylococci obtained from different clinical specimens were studied for antimicrobial sensitivity pattern, and the mrsa obtained were subjected to plasmid profiling by the alkaline-lysis method. the identification of the organism was based on standard laboratory criteria (growth in blood agar and mcconkey agar media for 24-48 hours at 37°c, colonial morphology, gram staining, catalase test, oxidase test, and coagulase test) [20]. after differentiation based on coagulase, the isolates were examined for the antimicrobial sensitivity pattern. the organisms were screened for mrsa using cefoxitin disc diffusion test. the plasmid profile of mrsa was done by alkaline lysis method in central department of microbiology, t.u., kirtipur, nepal. isolation of bacterial plasmid dna by alkaline-sds lysis plasmid dna was isolated from mrsa according to sambrook & russel [21]. mrsa isolates were sub-cultured in luria-bertani broth at 37oc and 3 ml of the overnight culture was subjected to plasmid dna extraction by centrifugation at 5000 rpm for 5 min. after washing in tris-ethylene diamine tetraacetic acid (edta) buffer (10mm tris, 1mm edta, ph 8.0, te), the pellet was added to the freshly prepared mixture of naoh and sds (1:1), to which potassium acetate was added. the microfuge tube was inversely mixed and centrifuged at 13,000 rpm for 10 minutes. the supernatant was transferred into a new microfuge tube with adding phenol: chloroform (1:1) and centrifuged at 13,000 rpm for 10 minutes. the upper aqueous phase was collected in a clean microfuge tube to which chloroform was added and centrifuged at 13,000 rpm for 10 minutes. the supernatant was then transferred into a new microfuge tube and cold 95% ethanol was added to precipitate bacterial dna. the pellet was washed with 70% ethanol and dissolved in 50 µl of te buffer. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 agrahari et al. 2019 ©njb, biotechnology society of nepal 10 nepjol.info/index.php/njb. table 1: staphylococcal growth from various clinical specimens. staphylococci were isolated from different clinical samples following standard microbiological techniques. the staphylococcal isolates were screened for s. aureus and cons by the coagulase test. s. aureus, staphylococcus aureus; cons, coagulase-negative s. aureus. type of organism types of clinical sample total no. blood no (%) urine no (%) sputum no (%) pus no (%) body fluids no (%) s. aureus 23 (32.4) 16 (22.5) 5 (7) 22 (31) 5 (7) 71 cons 14 (51.9) 9 (33.3) 1 (3.7) 3 (11.1) 0 27 total 37 (37.8) 25 (25.5) 6 (6.1) 25 (25.5) 5 (5.1) 98 preparation of agarose gel agarose gel of 1 percent was prepared by melting 1 gm agarose in 100 ml of diluted tae buffer using a microwave oven. the molten agarose was allowed to cool to about 50ºc and 50 µl ethidium bromide (1 mg/ml) was added and mixed and was immediately poured into gel tray and the comb was placed. after solidification of the gel, the comb was removed. the gel was placed in a horizontal electrophoresis apparatus filled with tae buffer. loading and electrophoresis of the sample each 5 µl of isolated plasmid dna was mixed with 1 µl of 6x gel loading buffer. the mixture was slowly loaded into the well using disposable micropipette tips. a marker of 1 kb molecular weight was loaded in one well to determine the size of the plasmid dna. electrophoresis was carried out at 100 volts for 1 hour. visualization of the gel the amplified products of the study samples were visualized by using uv trans-illuminator. the gel was photographed by a digital camera and transferred data to the computer for further documentation . results identification and distribution of mrsa in this study different clinical samples were taken from out-patients and various wards of the kist medical college, imadol, kathmandu. for the identification of staphylococci, standard microbiological procedures were followed. this involves the morphological appearance of the isolated colony, staining reactions and biochemical properties. each of the organisms was first isolated in pure form. then colony morphology was noted, which was followed by gram’s staining and biochemical tests. the detailed biochemical, cultural and staining techniques are mentioned in the supplementary section. among all the clinical specimens processed during the study period, 414 specimens showed positive growth among which 98 were isolated and identified as staphylococci by standard microbiological technique. the staphylococcal isolates were screened for s. aureus and coagulase-negative staphylococci by the coagulase test. out of 98 positive staphylococcal growths from different clinical specimens, 71 isolates were found to be coagulase-positive and were defined as s. aureus (table 1). out of 71 isolates of s. aureus, 27(38.02%) isolates were resistant to cefoxitin (<22 mm) and were defined as mrsa. out of 27 mrsa isolates, highest number of mrsa was isolated from pus sample (n=8) followed by urine (n=8) and blood (n=7). however, only 2 mrsa was isolated from sputum specimens and only one from body fluids (figure 1). antibiotic susceptibility pattern of mrsa isolates we next determined the antibiotic susceptibility pattern of mrsa isolates. here, we found that vancomycin and tetracycline (74.07%) were the most effective drugs whereas ampicillin (92.60%) and penicillin (92.60%) were least sensitive to mrsa isolates (figure 2). nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 agrahari et al. 2019 ©njb, biotechnology society of nepal 11 nepjol.info/index.php/njb. figure 1: distribution of mrsa in different clinical specimens. s. aureus were isolated from different clinical samples by standard microbiological techniques and were identified as mrsa by testing with cefoxitin discs having a zone of inhibition less than 22 mm. the number in the pie-chart indicates the number of mrsa isolated from different clinical specimens. s. aureus, staphylococcus aureus; mrsa, methicillin-resistant s. aureus plasmid profiling of mrsa twenty-seven isolates of mrsa isolated from various clinical samples were screened for plasmids. out of these, 21 strains (77.77%) lacked any plasmid and 6 strains (22.22%) were detected with only one plasmid (figure 3). the isolated plasmid and molecular weight of the plasmid can be visualized in the agarose gel shown in figure 3. the plasmid profile of mrsa and plasmid profile with antimicrobial resistance patterns are shown in table 2. figure 3: plasmid profile of mrsa. plasmids were isolated by the alkaline-lysis method and were run in 1% agarose gel. lane 1 and lane 18 were loaded with 1 kb ladder, lane 6, 16, 24, 26, 27 and 28 shows the positive samples with plasmid and are indicated by the red arrow. figure 2: antibiotic sensitivity patterns of mrsa isolates. antibiotics sensitivity of mrsa was determined by the kirby-bauer disc diffusion method. the number in the graph indicates the percentage of the susceptibility of mrsa towards various tested antibiotics. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 agrahari et al. 2019 ©njb, biotechnology society of nepal 12 nepjol.info/index.php/njb. table 2. plasmid profiles and antimicrobial resistance of mrsa. plasmids from mrsa were isolated by alkaline-lysis method and antibiotic susceptibility patterns were determined by modified kirby-bauer disc diffusion method. amp, ampicillin; amc, amoxyclav; cx, cefoxitin; cot, cotrimoxazole; cip, ciprofloxacin; e, erythromycin; ctx, cefotaxime; gen, gentamycin; of, ofloxacin; p, penicillin; va, vancomycin. discussion in this study, cefoxitin disk was used for the detection of methicillin-resistant s. aureus. oxacillin can also be used but the cefoxitin disk diffusion method is more effective in detecting hetero-resistant mrsa. cefoxitin is also an inducer of the meca gene hence suitable for detecting hetero-resistant mrsa. furthermore, it gives a clearer endpoint and is easier to read than tests with oxacillin [22-24]. the present study revealed that the prevalence of mrsa was 38.02% from the total isolated s. aureus. the result of the present study correlates with the findings of the study conducted by rajaduraipandi et al [25] where the prevalence of mrsa was 37.9% and with sanjana et al [26] where 39.9% mrsa was isolated from different clinical samples. in developing countries like nepal, screening of antibiotic susceptibility pattern of mrsa is a great tool for effective treatment and cost minimization. in this study, we determined the antibiotic susceptibility pattern of mrsa through the disc diffusion method. here, we found that 92.60% of mrsa isolates were resistant to conventional drugs such as penicillin and ampicillin. whereas, vancomycin and tetracycline were found to be the most effective drug as 85.19% of mrsa isolates showed sensitivity pattern. thus, tetracycline can be considered as an alternative for vancomycin in the case of vancomycin resistant s. aureus. plasmid profile has been reported as one of the techniques for typing mrsa [27]. here, 27 mrsa isolates were subjected to plasmid isolation by alkaline-lysis method. alkalinelysis in combination with the detergent sds has been used for more than 20 years to isolate plasmid dna from e. coli [28]. exposure of bacterial isolates to strongly anionic detergent at high ph opens the cell wall, denatures chromosomal dna and proteins and releases plasmid dna into the supernatant. in this study, out of 27 mrsa isolates only 6 (22.22%) had plasmids. plasmid profile analysis appears to be of very low discriminatory capacity in the investigation of mrsa epidemiology because of the non-detection of plasmids in the majority of the mrsa strains. this has been reported by terry et al [29] and ugbugo et al [30]. all plasmid-carrying strains harbored a single plasmid. this result was compatible with the study of maithem et al [31] in which all the isolates harbored single plasmid. the acquisition of methicillin resistance is through a highly mobile genetic element, staphylococcal cassette chromosome (sccmec) that carries meca gene and confers methicillin resistance. a study by caddick et al [32] showed that the presence of plasmid dna is involved in the acquisition of multi-drug resistance. however, there exists no significant correlation between plasmid dna and multidrug-mrsa. this could be the possible reason for the low isolation of plasmid in our study. the molecular weights of the isolated plasmids were found to be more than ten thousand base pairs. this result was compatible with the study of badgeremeka et al [33] where all the isolates harbored plasmid with molecular weight ranging from 496912130 bp. all the mrsa strains which harbored the plasmid showed resistance towards β-lactam antibiotics such as cefoxitin, penicillin, and ampicillin. this was also similar to badger emeka et al [33] where plasmid harbored strains had more resistance towards β-lactam antibiotics. thus, it might be concluded that the nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 agrahari et al. 2019 ©njb, biotechnology society of nepal 13 nepjol.info/index.php/njb. resistance of mrsa towards βlactam antibiotics was plasmid-mediated. the detection of meca gene or penicillin-binding protein 2a is considered as a more reliable and confirmatory method for identifying mrsa [34]. furthermore, plasmid profiling along with antibiotic susceptibility patters are considered as a useful method for epidemiological studies and to design antibiotic policies and effective control measures [27, 31]. in this study, we determined the prevalence of mrsa in different clinical specimens along with their antibiotic susceptibility pattern and plasmid profiling. however, the determination of meca gene should be done to confirm mrsa isolates and are limited in this study. the result of this study concluded that there is a great need of regular surveillance in order to monitor resistance patterns and minimize the increase of antibioticresistant micro-organisms. acknowledgements i am really very delighted and want to express my deep thankfulness to all the staff of central department of microbiology (cdm) and kist hospital for their continuous support and help during my entire lab work. last but not the least, i want to deeply thank my family, my roommates and my friends for providing me support, guidance and encouragement. conflict of interest the authors declare no conflict of interest. author contributions gaurav agrahari contributed to the conceptualization, formal analysis, investigation, methodology, visualization, and writing; amrit koirala, roshan thapa, and mahesh kumar chaudhary played a supporting role in the investigation, methodology, and writing; reshma tuladhar contributed to conceptualization, supervision, validation, and writing. references 1. lowy fd: antimicrobial resistance: the example of staphylococcus aureus. j clin invest. 2003 111:1265-1273. 2. chambers hf: the changing epidemiology 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teaching hospital. jiom. 2012 34:13-17. 13. mukhiya rk, shrestha a, rai sk, panta k, singh rn, rai g, et al: prevalence of methicillin-resistant staphylococcus aureus in hospitals of kathmandu valley. njst. 2012 13:185-190. 14. centers for disease control and prevention, office of infectious disease antibiotic resistance threats in the united states, 2013. available at: nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1:8-14 agrahari et al. 2019 ©njb, biotechnology society of nepal 14 nepjol.info/index.php/njb. http://www.cdc.gov/drugresistance/threat -report-2013. accessed january 28, 2015. 15. pittet d, hugonnet s, harbarth s, mourouga p, sauvan v, touveneau s, et al: effectiveness of a hospital-wide programme to improve compliance with hand hygiene. lancet. 2000 356:1307-1312. 16. al-mohana am, al-charrakh ah, nasir fh, al-kudhairy mk: community-acquired methicillin-resistant staphylococcus aureus carrying meca and panton-valentine leukocidin (pvl) genes isolated from the 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22. broekema nm, van tt, monson ta, marshall sa, warshauer dm: comparison of cefoxitin and oxacillin disk diffusion methods for detection of meca-mediated resistance in staphylococcus aureus in a large-scale study. j clinic microbial. 2009 47(1):217-219. 23. felten a, grandry b, lagrange ph, casin i: evaluation of three techniques for detection of low-level methicillin-resistant staphylococcus aureus (mrsa): a disk diffusion method with cefoxitin and moxalactam, the vitek 2 system, and the mrsa-screen latex agglutination test. j clinic microbial. 2002 40(8):2766-2771. 24. mimica mj, berezin en, carvalho rl, mimica im, mimica lm, sáfadi ma, et al: detection of methicillin resistance in staphylococcus aureus isolated from pediatric patients: is the cefoxitin disk diffusion test accurate enough? braz j infect dis. 2007 11(4):415-417. 25. rajaduraipandi k, mani kr, panneerselvam k, mani m, bhaskar m, manikandan p: prevalence and antimicrobial susceptibility pattern of methicillin resistant staphylococcus aureus: a multicentre study. ind j med microbial. 2006 24(1): 34-38. 26. sanjana rk, shah r, chaudhary n, singh yi: prevalence and antimicrobial susceptibility pattern of methicillin-resistant staphylococcus aureus (mrsa) in cmsteaching hospital: a preliminary report. j college medical sci-nepal. 2010 6 (1): 1-6. 27. tayfour ma, eris fn, alanazi ar: comparison of antibiotic susceptibility tests, plasmid profiles and restriction enzymes analysis of plasmid dna of methicillin susceptible and resistant staphylococcus aureus strains isolated from intensive care units. saudi med j. 2005, 26(1): 57-63. 28. birnboim hc, doly j: a rapid alkaline extraction procedure for screening recombinant plasmid dna. nucleic acids res. 1979, 7:1513-1523. 29. terry alli oa, akinloye o, rowley da, butcher pd: a comparative assessment of ribosomal dna polymorphisms in methicillin resistant staphylococcus aureus (mrsa) epidemiology. afr j biomed res. 2002 10: 117 –125. 30. ugbogu oc, akinsade ak, nwokocha ko, ahuama oc: plasmid profile of methicillin resistant staphylococcus aureus isolates from university students. nigerian j microbiol 2011 25: 2363–2368. 31. maithem a, al-hamdani ma, hamad ig: study of plasmid profile, susceptibility patterns of clinical staphylococcus aureus isolated from patients with otitis media in basrah. j basrah res. 2012 38: 79-89. 32. caddick jm, hilton ac, rollason j, lambert pa, worthington t, elliott ts: molecular analysis of methicillin-resistant staphylococcus aureus reveals an absence of plasmid dna in multidrug-resistant isolates. fems immunol med microbiol. 2005 44(3):297-302. 33. badgeremeka li, emeka pm, dibua ume: plasmid profile of multi antibiotic resistant stahylococcus aureus isolated from diabetic wounds from patients at nsukka, south eastern, nigeria. afr j biotech. 2014 13(43): 4148-4152. 34. boyce jm: methicillin-resistant staphylococcus aureus in hospitals and long-term care facilities: microbiology, epidemiology, and preventive measures. infec control hosp epidemiol 1992 13(12):725737 http://www.ncbi.nlm.nih.gov/pubmed/?term=pittet%20d%5bauthor%5d&cauthor=true&cauthor_uid=11073019 http://www.ncbi.nlm.nih.gov/pubmed/?term=harbarth%20s%5bauthor%5d&cauthor=true&cauthor_uid=11073019 http://www.ncbi.nlm.nih.gov/pubmed/?term=mourouga%20p%5bauthor%5d&cauthor=true&cauthor_uid=11073019 http://www.ncbi.nlm.nih.gov/pubmed/?term=mourouga%20p%5bauthor%5d&cauthor=true&cauthor_uid=11073019 http://www.ncbi.nlm.nih.gov/pubmed/?term=sauvan%20v%5bauthor%5d&cauthor=true&cauthor_uid=11073019 http://www.ncbi.nlm.nih.gov/pubmed/?term=touveneau%20s%5bauthor%5d&cauthor=true&cauthor_uid=11073019 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1 :15-20 doi: https://doi.org/10.3126/njb.v7i1.26946 ©njb, biotechnology society of nepal 15 nepjol.info/index.php/njb. original research article detection of pyuria by microscopic urinalysis as a marker of pediatric urinary tract infection dhiraj shrestha1, pratigya thapa2, dinesh bhandari3, balkrishna bhattachan4*, hiramani parajuli5, prakash chaudary5, vijay kumar sharma6, pradeep kumar shah5 1department of microbiology, shi-gan international college of science and technology, kathmandu, nepal. 2department of microbiology, kathmandu upatyakakhanepani limited (kukl), kathmandu, nepal. 3school of public health, university of adelaide, australia. 4biotechnology society nepal, bhaktapur, nepal. 5department of microbiology, tri-chandra multiple college, kathmandu, nepal. 6department of biochemistry, institute of medicine, tuth, kathmandu, nepal. abstract globally, different diagnostic tests of urinary tract infection (uti) are in clinical practices. a reliable test can increase the efficiency of the healthcare system, especially in a developing country like nepal, reducing cost and time. thus, we accessed the possibility of pyuria detected by microscopic urinalysis as a marker of pediatric uti. the prospective study was conducted fromjuly2014 to january 2015 at alka hospital, lalitpur. microscopic urinalysis of 353clean-catch urine samples was done by the wet mount method, followed by urine culture by a semiquantitative method. we confirmed 64 (18.1%) uti cases by culture, the gold standard for uti diagnosis. fever was the most common clinical manifestation in uti cases. the sensitivity, specificity, positive predictive value and negative predictive value of pyuria detected by microscopic urinalysis to identify uti were 50%, 70.9%, 27.6% and 86.5% respectively. in 318 febrile cases, the sensitivity, specificity, positive predictive value and negative predictive value of pyuria detected by microscopic urinalysis to identify uti were 73.2%, 72.6%, 28.3% and 94.8% respectively. the findings suggest pyuria detected by microscopic urinalysis as not a worthwhile marker of pediatric uti. but it is a trust worthy marker in febrile pediatric cases. keywords: febrile, marker, microscopic urinalysis,pediatrics, pyuria, uti *corresponding author email: balkrishnabhattachan@gmail.com introduction urinary tract infection (uti) is a common infection in all age groups [1-3] and affects at least 1% of boys and 3% of girls[4].uti is difficult to diagnose in children, as symptoms are non-specific [5-7].complications of uti in children lead to renal scarring and terminal kidney damage[8].uti management varies with evolving research findings[8].the diagnosis of uti should base clinically and confirmed by urine culture [9]. urine culture is a gold standard for the diagnosis of uti, but it takes up to 24 hrs for final reporting [10]. using microscopic urinalysis allows starting antimicrobial treatment 24 hours sooner than waiting for culture results [11]. microscopic urinalysis can thus be a useful test for the rapid diagnosis of uti in children [10]. but, no single cut-off count of leucocytes exhibits high sensitivity and specificity [5, 12]. at least 5 leucocytes per high power field (hpf) of centrifuged urine is commonly considered as pyuria [13]. pyuria is mostly observed as a result of inflammation, thus it is a common sign of uti [14]. this makes pyuria a suitable marker of uti. this study aimed to access the utility of microscopic urinalysis as a potential marker to diagnose pediatric uti. materials and methods the cross-sectional prospective descriptive study was conducted from july 2014 to january 2015 at alka hospital, lalitpur, nepal. the iso 9001:2008 accredited hospital is a referral hospital at kathmandu valley. a total of 8,692 urine samples were submitted to the microbiology laboratory for culture during the study period. only 353 non-repetitive, clean catch urine samples from infants and children patients, under 13 years of age and with symptoms of uti, were included in the study. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 15-20 shrestha et al. 2019 ©njb, biotechnology society of nepal 16 nepjol.info/index.php/njb. the symptoms were abdomen pain and/or dysuria and/or fever and/or frequency of urine and/or malodorous urine. for infants and younger children, symptoms were fever and parental reporting of malodorous urine. the children who were already on antibiotics therapy were excluded. the clean-catch urine samples were collected in a sterile container. in infants and non-toilet-trained children, a sterile plastic bag was attached to genitalia for clean catch urine collection. in toilet-trained children, voided midstream urine sample was collected. each sample was first subjected to microscopic urinalysis by the wet mount method. in brief, 10ml of urine was centrifuged at 3000rpm for 5min. the supernatant was discarded, and the sediment was re-suspended in 500μlurine. this native urine sediment was dropped on a glass slide and covered by a coverslip. the microscopic examination was performed by the bright-field microscopy (x400).the threshold value of at least 5 pus cells/hpf, which corresponds to at least 25 leukocytes per ml of non-centrifuged urine, was considered as pyuria[13]. in parallel, each sample was subjected to urine culture by a semi-quantitative method. in brief, 1μl urine was streaked on macconkey agar (himedia ltd, india) and blood agar plate (himedia ltd, india) using a calibrated loop of 2mm size. growths were observed after 18-48hrs of aerobic incubation at 37°c. the growth of at least 100colonies on the agar plate, which corresponds to at least 105colony-forming units (cfu) per ml of urine, were considered as culture-positive [15]. data were entered and stored using microsoft excel (version 2010, microsoft corporation, usa). chi-square test of variables was performed whenever applicable and p values below 0.05 were considered significant. results the mean age of patients was 5±3.5 years (ranging from 1 month to 12 years; variance= 12.5). in our study, the male to female ratio of uti suspected cases was 1:1.4. uti was confirmed by culture in 64(18.1%) out of 353 patients. meanwhile male to female ratio of uti confirmed cases was 1:1.2.fever was the most common clinical symptom in uti confirmed cases, 49 (76.6%) followed by malodorous urine, 46 (72%) (table 1). in 18 (62.1%) of 29 males and 14 (40%) of 35 females who were confirmed of uti did not have pyuria (table 2). of 64 uti cases, 32 (50%) casesshowed pyuria and 32 (50%) cases did not show pyuria. this was statistically significant since pyuria was associated with an increased risk of bacteriuria (p<0.05) (table 3). table 1. clinical symptoms in patients symptoms suspected uti cases (% of 353 cases) confirmed uti cases (% of 64 cases) abdomen pain 212 (60.1) 33 (51.6) dysuria 233 (66.0) 32 (50.0) fever 318 (90.1) 49 (76.6) frequency of urine 222 (62.9) 32 (50.0) malodorous urine 71 (20.1) 46 (71.9) table 2: number of pus cells/hpf and bacteriuria in male and female patients pus cells/hpf no. of sample male female culture negative culture positive culture negative culture positive <3 181 70 14 86 11 3-5 56 23 4 26 3 5-8 39 13 3 21 2 8-10 14 2 2 9 1 10-15 20 5 1 12 2 ≥15 43 7 5 15 16 total 353 120 29 169 35 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 15-20 shrestha et al. 2019 ©njb, biotechnology society of nepal 17 nepjol.info/index.php/njb. of 41 febrile uti cases, only 30 (73.2%) cases showed pyuriaand 11(26.8%) did not show pyuria. this was statistically significant since pyuria was associated with an increased risk of bacteriuria in febrile cases (p<0.05) (table 4) discussion uti can only be accurately diagnosed by a combination of clinical and laboratory investigations. wide ranges of practices are seen among physicians [16]. over diagnosis of uti had been a common problem that had led to aggressive antibiotic therapy [1]. we first classified the pool of 353 cases, as uti and non-uti, depending on the culture. thus, 18.1%of cases were confirmed to have uti. the discordance of clinical and laboratory investigations could have resulted in low growth positivity. in our study, male to female ratio of suspected cases was 1:1.4, this was involuntary recruitment bias. meanwhile male to female ratio of uti confirmed cases was 1:1.2. the natural epidemiology pattern of uti shows more prevalence in females[17].the urethra of females are colonized with colonic gramnegative bacteria as they are shorter in length and are in close proximity to the anus, thus females are more frequently affected by uti [18]. in our study, fever was the common symptom, manifested in 76.6% of uti cases. this was similar to reports from other studies [19-21]. there is no unison in the cut-off value of pus cells to consider as pyuria. the cut-off value of ≥5 pus cells/hpf was considered pyuria[13]. out of total 237 samples without pyuria, 13.50%were culture positive; and of 116 samples with pyuria, 27.6%were culture positive. the relationship of pyuria and culture was statistically significant (p<0.05). culture positive without pyuria often occurs in patients with diabetes, enteric fever of bacterial endocarditis whereas pyuria with sterile culture occurs in patients with prior antibiotic use, renal tuberculosis, corticosteroid administration, analgesic nephropathy, or renal calculi [18]. in our study, since no distinction of samples from patients was made on these criteria, both bacteriuria without pyuria and pyuria without bacteriuria may have occurred. the sensitivity, specificity, positive predictive value and negative predictive value of pyuria to diagnose uti were 50%, 70.9%, 27.6% and 86.5% respectively. this was slightly lower but comparable with reports from other studies [19, 22-24]. our study revealed pyuria with less sensitivity and high specificity. this finding indicates that the presence of pyuria may not suggest uti but the absence of pyuria can exclude uti. furthermore, positive predictive table 3: relationship between microscopic urinalysis and culture in all suspected cases pyuria urine culture total (%) culture positive (%) culture negative (%) pyuria 32 (50) 84 (29.1) 116 (32.9) nonpyuria 32 (50) 205 (70.9) 237 (67.1) total 64 (100) 289 (100) 353 (100) sensitivity=50% specificity=70.9% positive predictive value=27.6% negative predictive value=86.5% table 4: relationship between microscopic urinalysis and culture in febrile cases pyuria in febrile cases urine culture total (%) culture positive (%) culture negative (%) pyuria 30 (73.2) 76 (27.4) 106(33.3) nonpyuria 11 (26.8) 201 (72.6) 212(66.7) total 41(100) 277(100) 318(100) sensitivity=73.2% specificity=72.6% positive predictive value=28.3% negative predictive value=94.8% nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 15-20 shrestha et al. 2019 ©njb, biotechnology society of nepal 18 nepjol.info/index.php/njb. value and negative predictive value suggest that using pyuria to diagnose uti in children will result in a significantly larger number of falsepositive and lower false-negative results. therefore our study suggests that pyuria detected by microscopic urinalysis is a less reliable marker for pediatrics uti but can be used to exclude uti as a single test modality. some authors still agree that microscopic urinalysis can identify only a third to half of the patients with positive urine culture [25-27]. we further accessed the reliability of pyuria detected using microscopic urinalysis by dividing the study population based on the presence or absence of symptom fever to improve predictive scores. out of total 318 febrile cases, 9.4%were culture positive along with pyuria and 3.5% samples were culture positive without pyuria. the relationship of pyuria and culture in febrile cases was statistically significant (p<0.05). thus, in febrile cases, the sensitivity, specificity, positive predictive value and negative predictive value of pyuria to diagnose uti increased to 73.2%, 72.6%, 28.3% and 94.8% respectively. our study revealed pyuria with higher sensitivity and specificity in febrile cases. this finding indicates that the presence of pyuria in febrile cases can suggest uti; similarly, the absence of pyuria in febrile cases can exclude uti. furthermore, low positive predictive value and high negative predictive value suggest that using pyuria to diagnose uti in febrile children can result in a higher number of false-positive but lower falsenegative results. this suggests that pyuria detected by microscopic urinalysis is a worthwhile marker for uti in febrile children. furthermore, our study suggests pyuria detected by microscopic urinalysis can serve as a reliable marker of uti in pediatrics in a primary healthcare setting where prevalence is much lower. this can omit unnecessary tests, thus can increase effective diagnosis and cost in the healthcare system. nitrate reduction test and leucocyte esterase (le) test as recommended by the national institute for health and care excellence (nice) can further be used to improve this diagnosis accuracy [5]. nevertheless, our study doesn’t underrate the importance of culture for uti diagnosis in children. but reliable marker can increase the effectiveness of diagnosis excluding unnecessary tests. critical cases need a quick diagnosis for prompt treatment that cannot wait culture result which usually demands 18-48 hours. in an economy lagged country like nepal, this can help to improve and outreach the healthcare facility especially in a primary healthcare system where there is a fundamental lack of enough resources for investigations; and treatment is primarily based on clinical suspicion. thus, pyuria detected by microscopic urinalysis can be a standalone diagnostic test in febrile cases in such settings. conclusion our findings suggest pyuria detected by microscopic urinalysis entertain less sensitivity and specificity, thus pyuriais not the reliable marker of uti in pediatrics. however, the reliability of the pyuria detected by microscopic urinalysis was higher to diagnose uti in febrile pediatric cases, which can be a single test model in low resource settings like the primary healthcare system. conflict of interest none declared acknowledgments none consent to publish not applicable ethical approval and consent to the participant the study was a laboratory-based study and a part of the study was a routine patient care investigation. no patient-related data were collected except the demographic parameters, thus ethical approval was not required. oral informed consent was taken from a guardian on behalf of the patients. availability of data and materials all data generated or analyzed during this study are included in the article. raw data can nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 15-20 shrestha et al. 2019 ©njb, biotechnology society of nepal 19 nepjol.info/index.php/njb. be made available upon request to the corresponding author. funding none authors’ contributions all authors made substantial contributions to the study. ds, vks, and pks conceived and designed the study. ds, pt, bb and hp collected samples, investigated and recorded the laboratory findings at the laboratory. vks and pks supervised and provided methods for the study. ds, bb, hp and pc reviewed works of literature and drafted the manuscript. ds, pt, bb and pccompiled, curated and interpreted data. ds, pt and db critically reviewed and revised the manuscript by compiling, formatting, editing and writing the final version of the manuscript. all authors read and approved the final manuscript. references 1. taneja n, chatterjee s, singh m, singh s, sharma m: pediatric urinary tract infections in a tertiary care center from north india. indian j med res. 2010 131(1):101-105. 2. vasudevan r: urinary tract infection: an overview of the infection and the associated risk factors. j microbiol exp 2014, 1(2):42-54. 3. magliano e, grazioli v, deflorio l, leuci ai, mattina r, romano p, et al: gender and agedependent etiology of communityacquired urinary tract infections. sci world j. 2012 2012:6. 4. watson ar, taylor cm, mcgraw m: forfar and arneil’s textbook of pediatrics, 6th edn. spain: churchill livingstone; 2003. 5. national institute for health and care excellence (nice): urinary tract infection in children diagnosis, treatment and longterm management. nice clinical guidelines, no. 54. in. london: national institute for health and care excellence (nice); 2007. 6. owen d, vidal-alaball j, mansour m, bordeaux k, jones kv, edwards a: parent’s opinions on the diagnosis of children under 2 years of age with urinary tract infection. fam pract. 2003 20(5):531-537. 7. sibi g, devi ap, fouzia k, patil br: prevalence, microbiologic profile of urinary tract infection and its treatment with trimethoprim in diabetic patients. res j microbio. 2011, 6:543-551. 8. haghi-ashteiani m, sadeghifard n, abedini m, taheri-kalani ssm: etiology and antibacterial resistance of bacterial urinary tract infections in children's medical center, tehran, iran. acta med iran. 2007 45(2):153-157. 9. fitzgerald a, mori r, lakhanpaul m, tullus k: antibiotics for treating lower urinary tract infection in children. cochrane database syst rev. 2012(8). 10. maduemem ke, rodriguez yd, fraser b: how sensitive are dipstick urinalysis and microscopy in making diagnosis of urinary tract infection in children? int j prev med. 2019 10:62-62. 11. shaikh n, mattoo tk, keren r, ivanova a, cui g, moxey-mims m, majd m, ziessman ha, hoberman a: early antibiotic treatment for pediatric febrile urinary tract infection and renal scarring. jama pediatr. 2016 170(9):848854. 12. cheng y-w, wong s-n: diagnosing symptomatic urinary tract infections in infants by catheter urine culture. j paediatr child health. 2005 41(8):437-440. 13. subcommittee on urinary tract infection scoqiam: urinary tract infection: clinical practice guideline for the diagnosis and management of the initial uti in febrile infants and children 2 to 24 months. pediatrics 2011, 128(3):595-610. 14. doern cd, richardson se: diagnosis of urinary tract infections in children. j clin microbiol. 2016 54(9):2233-2242. 15. world health organization (who): basic laboratory procedures in clinical bacteriology. 2nd edn. geneva: world health organization; 2003. 16. anígilájé eab, t. t.: prevalence and predictors of urinary tract infections among children with cerebral palsy in makurdi, nigeria. int j nephrol. 2013 2013:7. 17. ghorashi z, ghorashi s, soltani-ahari h, nezami n: demographic features and antibiotic resistance among children hospitalized for urinary tract infection in northwest iran. infect drug resist. 2011 4:171176. 18. forbes b, sham d, weissfeld a: study guide for bailey and scott’s diagnostic microbiology. 12th edn: mosby elsevier 2007. 19. ojha ar, aryal ur: profile of children with urinary tract infection and the utility of urine dipstick as a diagnostic tool. j nep health res counc. 2014 12(28):151-155 20. malla kk, sarma ms, malla t, thapalial a: clinical profile, bacterial isolates and antibiotic susceptibility patterns in urinary tract infection in children – hospital based study. j nepal paediatr soc. 2008 28(2):52-61. 21. brkic s, mustafic s, nuhbegovic s, ljuca f, gavran l: clinical and epidemiology characteristics of urinary tract infections in childhood. med arh. 2010 64(3):135-138. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 15-20 shrestha et al. 2019 ©njb, biotechnology society of nepal 20 nepjol.info/index.php/njb. 22. taneja n, chatterjee ss, singh m, sivapriya s, sharma m, sharma sk: validity of quantitative unspun urine microscopy, dipstick test leucocyte esterase and nitrite tests in rapidly diagnosing urinary tract infections. j assoc physicians india. 2010 58:485487. 23. zorc jj, kiddoo da, shaw kn: diagnosis and management of pediatric urinary tract infections. clin microbiol rev. 2005 18(2):417422. 24. al-daghistani hi, abdel-dayem m: diagnostic value of various urine tests in the jordanian population with urinary tract infection. clin chem lab med. 2005 40(10):10481051. 25. graham jc, galloway a: acp best practice no 167: the laboratory diagnosis of urinary tract infection. j clin pathol. 2001 54(12):911919. 26. hpa: uk standards for microbiolog investigations: investigation of urine. national standard method bsop 41. standard units, microbiology services, public health england issue 7. 2009. 27. lin ds, huang sh, lin cc, tung yc, huang tt, chiu nc, et al: urinary tract infection in febrile infants younger than eight weeks of age. pediatrics 2000 105(2):e20. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 39-45 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 39 nepjol.info/index.php/njb in vitro comparative study of antioxidant and antibacterial activity of selected dietary plants ashok kumar, palvi sharma, anupama mahajan, zarina begum department of biotechnology, shaheed udham singh college of engineering and technology (suscet), punjab abstract ethanolic extracts of garlic (bulb), aloe (leaf), flower bud (buds), turmeric (rhizomes) and ginger (rhizomes) were used for relative analysis of antioxidant and antimicrobial activity. antioxidant activity was determined by dpph [1, 1-diphenyl-2-picryl hydrazyl] assay and expressed with ascorbic acid. it was observed that turmeric and ginger have more antioxidant activity than garlic, aloe and flower bud. these extracts were further studied for antibacterial activity by agar well diffusion and spectrophotometric method against tetracycline as reference. the result showed that flower bud is more effective against escherichia coli, pseudomonas aeuroginosa, bacillus subtilis and staphylococcus aureus compared to other plants extract. however, all the plants extract did show antioxidant and antibacterial activity. keywords: antioxidant, antimicrobial, spectrophotometer and well diffusion *corresponding author email: ashoka.kumar2007@rediffmail.com introduction the medicinal importance of plants consists of some chemical substances that affect physiological action on human body. plants play a specific role in traditional medicine which contain phytochemicals that includes alkaloids, flavonoids, terpenoids, steroids, carotenoids and other phenolic compounds [1,2]. these constituents have antioxidant [3] and antimicrobial activity [4] and serve as defence mechanism against many microorganisms [5]. many of them are used as spices and food supplements [6]. antioxidants are medicaments, having ability to protect human body from oxidative stress induced by free radicals [7]. it has been found that oxidative stress is major causing factor for initiation of many diseases including neuro degenerative, diabetes, cancers and others [8, 9]. the oxidation of lipid, protein and carbohydrates by toxic reactive species, cause dna mutation and lead to damage the cell/tissue and death [10]. free radicals, stimulate oxidative stress and can obviate with antioxidants. antioxidants will be effective in scavenging of free radicals and suppress such disorder [11]. it is found in most of the plants and due to its natural source, have expected to gain comparison with synthetic antioxidants [12]. the natural antioxidants do not induce side effect while synthetic antioxidant induce genotoxicity [13]. these plants are also useful as curative agent against numerous pathogenic infections because of its phytochemicals or secondary metabolites [14, 15]. a number of phytochemicals like anthocyanins, flavonoids and other phenolic compounds have antimicrobial activity. due to drug resistance of pathogens, efforts have been made to find their substitute for treatment of diseases and with knowledge of antimicrobial activity of plant extracts, they may play a significant role in cure of microbial diseases [16]. presently, herbs are used as natural antimicrobial and antioxidant resources [17]. in the present study, five plants viz. garlic (bulb), aloe (leaf), flower bud (buds), turmeric (rhizoids), ginger (rhizoids) are studied which are being used as supplement and herbal medicine world-wide. the aim of present research was to investigate phytochemicals in extracts of different plant as well as to examine antioxidant and antimicrobial activity using in vitro system. there is also comparison of well diffusion method with spectrophotometric method for antimicrobial activity. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 39-45 kumar et al. ©njb, biotechnology society of nepal 40 nepjol.info/index.php/njb table 1: a brief summary of plants. plant family medicinal use reference garlic (alium sativum) amaryllidaceae it has antiseptic, anticoagulant, antibiotic, antioxidant, analgesic, anti-inflammatory and anticancerous properties. [18, 19, 20] ginger (zingiber officinale) zingiberaceae it has antimicrobial, antioxidant, anti-cancerous, anti-inflammatory. [21,22,23,24] turmeric (curcuma longa) zingiberaceae its expressed antimicrobial, antioxidant, anticancerous, anti-inflammatory, anti-diabetic. [25,26,27] clove -flower bud (syzygium aromaticum) myrtaceae it possesses antimicrobial, antioxidant and antiinflammatory activities. [28,29,30] aloe vera (aloe barbadensis miller) asphodelaceae it has antiseptic, antibiotic, antioxidant, analgesic, anti-inflammatory and anti-cancerous properties. [31,32,33] material and methods collection of herbal plants wild species of garlic, ginger, turmeric, flower bud and aloe were taken fresh from the fields. four bacterial strains such as escherichia coli (mtcc 25922), bacillus subtilis (mtcc 3256), pseudomonas aeuroginosa (mtcc 1688) and staphylococcus auerus (mtcc 6810) were used in study. preparation of extract the herbal plants including garlic, ginger, turmeric, flower bud and aloe were washed with distilled water and allow drying in the absence of sunlight at 30 -350c. dried samples were crushed in powdered form for extraction. samples were put in soxhlet apparatus with 90% ethanol and extraction was completed. the extracts were dried under vacuum. the thick paste crude drugs were considered as 100% concentration of extract. each extract were dissolved in 10% dmso (dimethylsulfoxide) for further use. phytochemical screening of plants phytochemical investigation of extracts (10% dmso) were performed as per described earlier [34,35]. antioxidant activity the antioxidant activity of the plant extracts and the standard was assessed on the basis of the radical scavenging effect of the stable 1, 1diphenyl-2-picrylhydrazyl (dpph-0.002%) method [36, 37]. the diluted working solution of the test extracts (10 mg/ml) was prepared using the respective solvents. 10mg/ml of ascorbic acid used as reference for compare results of plant extracts. in 3ml of total reaction solution, 2ml of extract/standard solution and 1.0ml of dpph were mixed and allowed to react at 370c for 30 min. afterward, absorbance value were measured at 520nm and converted into percent antioxidant activity. the percentage antioxidant activity was calculated by following formula. percent (%) inhibition of dpph activity = (a – b)/a *100 where a = absorbance of the blank and b = absorbance of the sample table 2: protocol for phytochemical screening component exit test protocol for test result for confirmation alkaloids 1.0ml extract + 3.0 drops of dragendoff’s reagent orange-red precipitate flavonoids 1.0ml extract + few drops of dil. naoh intense yellow colour total phenol 1.0ml extract + 2.0ml water + few drops of 10% fecl3 blue-green colour tannins 100mg solvent free extract + 1ml 5% fecl3 bluishblack precipitate saponins 1.0ml extract + 20ml water + agitation for 15min. 1cm layer of foam steroids 1.0ml extract + 10ml chcl3 + 10ml conc. h2so4 upper layer – red and lower layer – yellow-green terpenoids 5.0ml extract + 2.0ml chcl3 + 3.0ml conc. h2so4 reddish brown precipitate carbohydrate 2.0ml extract + 2 drops of molish reagent (95% αnapthol in ethanol) + 2.0ml conc. h2so4 purple colour at junction glycosides 5.0ml extract + 2.0ml chcl3 + 2.0ml ch3cooh violet, blue to green colour nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 39-45 kumar et al. ©njb, biotechnology society of nepal 41 nepjol.info/index.php/njb antibacterial activity four pathogenic bacteria namely escherichia coli (mtcc 25922), bacillus subtilis (mtcc 3256), pseudomonas aeuroginosa and staphylococcus auerus (mtcc 6810) were collected from mtcc, chandigarh. the obtained cultures were further sub-cultured for test. for preparation of inoculums, they were sub-cultured in test tube containing 10ml nutrient broth and standardized with saline water. agar well diffusion method agar well diffusion [38] was used with manipulation. 0.5ml (9.0 x 104 cfu/ml) of 48 hrs old cultures of test organisms were inoculated into different sterile petri-plates and near about 20 ml sterile media was poured into each dish. the dishes were gently shaken for proper mixing and allowed to solidify. thereafter, four wells were punched of 5 mm diameter with a sterilized cork borer. for each well 50 µl of different extracts were added. the plates were incubated at 37 0c for 24 hrs and then zone of inhibition in mm was measured. the experiment was carried out in triplicates. spectrophotometric method 0.5ml (9.0 x 104 cfu/ml) strains of 48 hrs old cultures were inoculated in test tubes with 20 ml culture media. to each tube, 50 µl of different extracts were added and incubated for 24 hrs. optical density of grown bacteria was measured at 450 nm wavelength. tetracycline (50 µg/ml) was included as control in this protocol [39, 40]. result the comparative study of antimicrobial and antioxidant activity was conducted with dietary plants like garlic, ginger, turmeric, flower bud and aloe. we have also evaluated the antimicrobial activity by disc diffusion and spectrophotometric method. all the experiment were done in triplicates. phytochemical the phytochemical analysis is preliminary characterization of plant extracts which have bioactive compounds. the phytochemical screening show that all the experimental plants contain phenols and terpenoids. turmeric extract does not contain saponins, tannins, glycosides, alkaloids and carbohydrate. the garlic extract contain all the phytochemicals except tannins as listed below whereas ginger extract don’t have steroids and flower bud extract contain all phytochemical except flavonoids and steroids. aloe contains all the phytochemical such as phenols, flavonoids, saponins, tannins, glycosides, terpenoids, alkaloids and carbohydrates. antioxidant activity among the five extract of plants, ginger showed maximum antioxidant activity and aloe showed the lowest activity and reported as 51.54% for turmeric, 28.17% for garlic, 54.08% for ginger, 10.97% for aloe, 19.41% for flower bud. ascorbic acid standard showed 80.5% activity. further experiments are needed to confirm which phytochemicals show antioxidant activity. there are major articles available that report that these plants extract have antioxidant activity [13, 15, 26, 33]. table 3: phytochemical screening of plants extract phytochemicals herbal plants turmeric garlic ginger aloe flower bud phenols + + + + + flavonoids + + + + saponins + + + + steroids + + + tanins + + + glycosides + + + + terpenoids + + + + + alkaloids + + + carbohydrates + + + + nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 39-45 kumar et al. ©njb, biotechnology society of nepal 42 nepjol.info/index.php/njb figure 1: antioxidant activity of plants extract (10mg/ml) with ascorbic acid (10mg/ml) as standard antimicrobial activity agar well diffusion method table 4 shows the antimicrobial activity of selected plant extracts by agar cup diffusion method and the result is similar to that of spectrophotometric method. the result reveal that garlic extract inhibit maximum growth of e. coli whereas pseudomonas aeruginosa is maximum inhibited by aloe and flower bud extract inhibit maximum growth of bacillus subtilis whereas staphylococcus aureus is maximum inhibited by ginger. spectrophotometric method the antimicrobial activity of ethanolic extract of turmeric, garlic, ginger, aloe and flower bud were tested against bacterial strain such as escherichia coli, pseudomonas aeuroginosa, bacillus b. subtilis and staphylococcus auerus by spectrophotometer. tetracycline was used as standard. escherichia coli showed maximum inhibition against garlic extract whereas minimum inhibition was against aloe. pseudomonas aeuroginosa have maximum inhibition against aloe whereas minimum inhibition was against turmeric extract. bacillus subtilis showed maximum inhibition against flower bud extract whereas turmeric showed minimum inhibition. staphylococcus auerus showed maximum inhibition against ginger whereas turmeric extract showed minimum inhibition. discussion the phytochemical screening result revealed that ethanolic extracts of plants consist phenols, flavonoids, saponins, tannins, glycosides, terpenoids, alkaloids and carbohydrates and this was confirmed by chemical. with antimicrobial result [41], it was observed that all of ethanol extracts of plants demonstrated good antibacterial activity. the result of antimicrobial activity expressed significant result against bacterial strains. from determination of antimicrobial activity it was observed that growth of e. coli and s. auerus is inhibited most by garlic whereas p. aeuroginosa with aloe and flower bud inhibit growth of b. subtilis the most. turmeric and ginger also showed significant antimicrobial activity. in comparing the well diffusion and spectrophotometric method, well table 5: antimicrobial activity (zoi in optical density) of plants extract by spectrophotometric method microorganism standard (tetracycline50μg/ml) turmeric50mg/ml garlic 50mg/ml ginger50mg/ml aloe50mg/ml flower bud50mg/ml positive e.coli (-) 0.093 0.309 0.272 0.344 0.499 0.378 0.842 p. aeruginosa 0.110 0.580 0.430 0.361 0.303 0.521 0.806 b. subtilis 0.092 0.460 0.257 0.236 0.362 0.066 0.741 s. aureus 0.042 0.360 0.197 0.136 0.162 0.259 0.714 table 4: antimicrobial activity (zoi in mm) of plants extract by agar well diffusion method microorganism standard (tetracycline – 50μg/ml) turmeric50mg/ml garlic50mg/ml ginger50mg/ml aloe50mg/ml flower bud50mg/ml e.coli 20.00 9.00 11.00 10.00 9.00 10.00 p. aeuroginosa 22.00 8.00 9.00 8.00 13.00 9.00 b. subtilis 19.00 9.00 13.00 9.00 11.00 15.00 s. auerus 20.00 10.00 13.00 14.00 12.00 13.00 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 39-45 kumar et al. ©njb, biotechnology society of nepal 43 nepjol.info/index.php/njb diffusion method has ability to evaluate the activity of antimicrobial drugs including plant extracts. although, this method has no significant difference from other methods in some report. spectrophotometric method [39], of finding the inhibitory action of drugs and extracts is fast and comfortable to use. statistically, there is no significant difference between result obtained by both method. so, spectrophotometric method can be recommend as suitable and sensitive method for investigation of antimicrobial activity of extracts. the comparative investigation was conducted between selected plants turmeric, garlic, ginger, aloe and flower bud for antioxidant activity. the scavenging of dpph radical is most used protocol to evaluate free radical scavenging ability of plant extracts [42]. from obtained result, it was seen that ginger (54.08%) and turmeric (51.54%) expressed better antioxidant effect although all are good antioxidants. thus, tendency of antioxidant was ascorbic acid > ginger > turmeric > garlic > flower bud > aloe in decreasing order. this observation proved that all the selected plants are free radical inhibitors and possibly act as primary antioxidant in human being. the antioxidant effect on dpph is due to the ability of their hydrogen donation and it has been observed that plant extracts expressed proton donating ability and serve as free radical scavengers and work as possible primary antioxidant. although, percent of antioxidant ability of extracts were not greater than standard. the obtained result might be sufficient for further identification of active phytochemicals and evaluate their antimicrobial and antioxidant activity. the 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volatile constituents from aloe vera (l.) peel. j. agric. food. tech. 2012, 2(5): 79-84. 33. saritha v, anilakumar kr, khanum f: antioxidant and antibacterial activity of aloe vera gel extracts.” international. journal of pharmaceutical & biotech. arch. 2010, 1: 376-384. 34. harborne jb: phytochemical methods london. chapman and hall ltd. 1973 49-188. 35. doss a: preliminary phytochemical screening of some indian medicinal plants. anc sci life. 2009 29(2):12–16. 36. thimmaiah sr: standard methods of biochemical analysis, kalyani publishers, ludhiyana. 1999. isbn 81-7663-067-5. 37. tekao t, watanabe n, yagi i, sakata k: a simple screening method for antioxidant and isolation of several antioxidants produced by marine bacteria from fish and shellfish. biosci. biotechnol. biochem. 1994 58: 1780-1783. 38. olayemi ab and opaleye fi: antibiotic resistance among coliform bacteria isolated from hospital and urban waste waters. world j microbiol biotech. 1999 6:285-2 88 39. meng x, nightingle ch, sweeney kr: quantification of in-vitro post-antibiotic effect nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 39-45 kumar et al. ©njb, biotechnology society of nepal 45 nepjol.info/index.php/njb based on the mean recovery-time. ii: a comparison of colony counting versus photometric methods for determination of bacterial growth. journal of antimicrobial chemotherapy 1991 28, 515-521. 40. dominguez mc, rosa m, borobio mv: application of a spectrophotometric method for the determination of post-antibiotic effect and comparison with viable counts in agar. of antimicrobial chemotherapy 2001 391-398. 41. mahfuzul hoque b, bari c ml, vijay k, juneja d, and kawamoto c s: antimicrobial activity of cloves and cinnamon against food borne pathogens and spoilage bacteria , and activation listeria monocytogenes in ground chicken meat with their essential oils.” international research journal of pharmacy. 2008, 3: 9-21. 42. thu k, yin y, mon, tin a khaing, and ohn m tun., “study on phytochemical properties, antibacterial activity and cytotoxicity of aloe vera l.” world academy of science, engineering and technology, 2013, 4: 77. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 doi: https://doi.org/10.3126/njb.v7i1.26948 ©njb, biotechnology society of nepal 30 nepjol.info/index.php/njb. original research article evaluation of phytochemical, antimicrobial, antioxidant activity and cytotoxic potentials of agave americana binayak raj pandey1, angela shrestha1, nisha sharma1, bhupal govinda shrestha1* 1department of biotechnology, kathmandu university, dhulikhel, nepal abstract ethnomedicinal plants are being used as a source of medicine from ancient time but they lack the proof of modern scientific evidence for their effectiveness. this study focuses on the evaluation of phytochemical, antimicrobial, antioxidant properties of one of the ethnomedicinal plant agave americana from dhulikhel region of nepal. the plant extract was prepared using solvent-based warm soxhlet extraction from the leaves of the plant and antimicrobial activity against six different non-resistant clinical isolates of bacteria (staphylococcus aureus, shigella, klebsiella pneumoniae, escherichia coli, bacillus thuringiensis, and salmonella paratyphi) was evaluated using agar disc diffusion method along with qualitative analysis for presence/absence of phytochemicals. antioxidant activity was measured by dpph assay and the cytotoxicity was evaluated using mcf-7 (human breast adenocarcinoma) cancer cell. presence of phytochemicals like alkaloids, flavonoids, reducing sugars and saponins were detected in the plant extract. the extract was found to show some level of antimicrobial activity against staphylococcus aureus, escherichia coli and bacillus thuringiensis at 50, 100 and 200 mg/ml. the ic50 value of the extract was found to be 7.68 μg/ml. the extracts of agave americana showed 50 % cell-death of mcf-7 in 12 h at 5 µg/ml. although this study provided some scientific evidence for the medicinal value of agave americana, further studies are still needed for the detailed evaluations of every molecule present in this plant along with screening in larger geographical area of nepal. keywords: antimicrobial agents; dpph; phytochemical; cytotoxicity; mcf-7 *corresponding author email: bgs@ku.edu.np introduction medicinal plants have always been a source of treatment of diseases since ancient time [1]. even in the modern era, medicinal plants are still being used as the primary method of disease treatment [2]. in fact, there is the whole system of medicine called ayurveda in the indian subcontinent region which uses medicinal plants as both complementary and alternative medicine and is being popular in other continents too [3]. there are countless literature reviews exhibiting the fact that the large group of compounds in modern medicine are derived from the medicinal plants [4] and some argue that except in the fields of infectious diseases and emergency aids the modern medicine is largely palliative rather than curative [5]. agave americana was selected in this study which had previously shown scientific evidence of efficacy against cancer cell lines. in one of the study, steroidal saponins from agave americana showed cytotoxic activity against hl-60 cell line [6]. breast cancer is one of the major health global health problems worldwide and breast cancer cell lines are being drug-resistant [7, 8, 9]. previous researches done in extract of agave americana reveals its antioxidant potential [10] which hints the possibility of using a. americana as an anticancer drug against breast cancer. this study focuses on the cytotoxic potential of a. americana extracts rather than truly evaluating anticancer properties and the substance that are cytotoxic could be a potential anticancer agent. six different clinical isolates viz., staphylococcus aureus, shigella, klebsiella pneumoniae, e. coli, bacillus thuringiensis, salmonella paratyphi from kathmandu university school of medicinal science (kusms) were chosen for antimicrobial screening, these microbes cause common human diseases like sinusitis, bacillary dysentery, pneumonia, gastroenteritis, paratyphoid fever etc., respectively. bacillus nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 31 nepjol.info/index.php/njb. thuringiensis was also chosen as it shared many common phenotypic and genotypic properties with bacillus anthracis, a causative agent for anthrax which is lethal in humans. all the bacteria were tested for resistant to broadspectrum antibiotics in this study. this research is an effort to test the type of phytochemicals present, antimicrobial activity, antioxidant potential and cytotoxic properties of agave americana found in the dhulikhel region of nepal. cytotoxic property of the extract was evaluated in mcf-7 breast cancer cell line. this research targets to generate supportive evidence for the medicinal values of the agave americana especially against common microbes and breast cancer. materials and methods sample collection 100 gm of agave americana leaves were collected from premises of department of biotechnology, kathmandu university, dhulikhel in a sterile bag. the identification of the plant was done using literature surveys. sample preparation the collected sample was then kept for drying in a well-ventilated dark room having a temperature of 250c for 30 days. leaves were powdered using home grade grinder by grinding for 5 minutes. extract preparation extract of the plant was prepared using the warm soxhlet extraction method [11] in which 6gm of the powdered sample of leaves was taken and packed individually into the manually prepared whatman no 1 filter paper bag and kept into the thimble of the soxhlet apparatus (borosil, gujarat, india). the extraction yield in percentage was calculated from the formula: 𝐸𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑 (%) = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 𝑇𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 ∗ 100 200 ml of absolute methanol was added through the thimble and the apparatus was operated continuously for 24 hours (8 hours at 40oc regularly for 3 days) monitoring the circulation of water into the condenser. then, the soxhlet extracts in absolute methanol were treated with hexane to remove chlorophyll pigment by the help of the separating funnel, the process was repeated 4-5 times until the chlorophyll pigment was completely extracted in hexane and green color stopped appearing in the thimble. petroleum ether was used to remove the fatty acid molecules to prepare the final extract. then, drying was done in a water bath at 50oc and the final extract was resuspended in dimethyl sulphoxide (dmso) for the use in all the assays and screening conducted in this study. phytochemical screening qualitative screening of phytochemicals viz., alkaloids, flavonoids, coumarin, saponins, glycosides, reducing sugar, tannins and polyphenols was done chemically [12, 13, 14]. antimicrobial screening three different concentrations viz. 200 mg/ml, 100 mg/ml and 50 mg/ml of the plant extracts were prepared for assessing the antimicrobial activity against six different common diseasecausing pathogenic non-resistant bacteria (s. aureus, shigella, klebsiella pneumoniae, e. coli, bacillus thuringiensis, salmonella paratyphi) using agar disk diffusion method [15, 16] according to nccls (national committee for clinical laboratory) standard where mha (muellerhinton agar) and mcfarland 0.5 standard were used and confirmation of non-resistance was done by antimicrobial susceptibility testing of microbes against broad-spectrum antibiotics chloramphenicol and tetracycline taking dmso as negative control. antioxidant assay determination of total antioxidant activity through free radical scavenging activity was performed by dpph (2,2-diphenyl-1picrylhydrazyl, obtained from sigma aldrich, usa). free radical scavenging assay [17, 18] where l-ascorbic acid was used as standard and ic50 (ic50 value indicates the concentration needed to inhibit a biological or biochemical nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 32 nepjol.info/index.php/njb. function by half) value was calculated for individual plant extract using uvspectrophotometer at 517 nm. different concentration of 1 μg/ml, 2.5 μg/ml, 5 μg/ml, 7.5 μg/ml, 10 μg/ml, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml of four different plant extracts were prepared using dmso as solvent and antioxidant activities were calculated. for ic50, the capability to scavenge the dpph radical was calculated using the following equation. 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 = 𝐴0 − 𝐴1 𝐴0 × 100 % where, a0 =absorbance of dpph solution a1= absorbance of dpph along with the different concentration of extracts. ic50 was calculated from linear trendline equation obtained by plotting a graph of concentration versus % inhibition. cytotoxicity analysis: mcf-7 cell line was obtained from everest biotech ltd., kathmandu, nepal. the mcf-7 (human breast adenocarcinoma cell line) were seeded in 96 well plate at the density of 104 cells/well in 100 µl of culture medium and allowed for 24 hr incubation at 37°c with 5% (v/v) co2 for attachment and then three different concentration of the plant extracts viz. 5µg/ml, 20 µg/ml and 80 µg/ml prepared in dmso were dissolved completely in the culture medium without disturbing the pellet. readings were taken at 12 hr, 24 hr, 36 hr and 48 hr and the culture plates were kept at 37°c with 5% (v/v) co2 during the time frame. numbers of cells were evaluated from manual observation in hemocytometer using phasecontrast microscope (nikon instruments inc, melville, ny, usa). the negative control for mcf-7 cell line was also used in which the plant extract was absent. results were generated from three independent experiments and each experiment was performed in triplicate. the percentage of cell cell-death for the treated sample was determined by the equation: 𝐶𝑒𝑙𝑙 𝐷𝑒𝑎𝑡ℎ % = 𝑁𝑜 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 𝑇𝑜𝑡𝑎𝑙 𝑛𝑜 𝑜𝑓 𝑣𝑖𝑎𝑏𝑙𝑒 𝑎𝑛𝑑 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 ∗ 100 results extraction yields the extraction yield of agave americana from 6 gm of powder was 16.92 % (table 1) as calculated according to the method followed by zhang et.al. [19]. table 1 extraction yield calculation. the extraction yield was calculated to evaluate the recovery rate from agave americana. phytochemical screening the preliminary phytochemical screening of methanol solvent extract shows the presence of alkaloids, flavonoids, reducing sugars and saponins whereas; it shows the absence of tannins, polyphenols, coumarin and glycosides (table 2). antimicrobial assay the susceptibility test for the antibiotics showed that all the bacteria used in the experiment are susceptible to antibiotics thus are non-resistance strains as shown in table 3 table 2 results of the qualitative phytochemical screening. the extract of agave americana was analyzed for the presence of alkaloids, flavonoids, tannins, polyphenols, reducing sugars, coumarins, saponins and glycosides. phytochemicals alkaloids flavonoids tannins and polyphenols reducing sugars coumarins saponins glycosides present(+) or absent(-) + + _ + _ + _ plant used weight of sample(gm) weight of extract (gm) extraction yield % agave americana 6.0030 1.0155 16.92 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 33 nepjol.info/index.php/njb. as zois (zone of inhibitions) shown by all the microbes were greater than 18 mm in diameter. also, the test was done for dmso which shows that dmso used as a solvent for the extract does not inhibit the growth of the bacteria used. chloramphenicol and tetracycline were used as standard antibiotics to test the nonresistance nature of clinical isolates. values are means (±se) zone of inhibition (zoi) in mm for antibiotics as the positive control and dmso as the negative control are presented in the table 3 (n=3, p <0.05). the extract of agave americana shows antimicrobial activity in a dose-dependent manner against six different non-resistance microorganisms as shown in figure 1. results from figure 1a shows that staphylococcus aureus is mildly susceptible to the extracts of agave americana showing zoi of 9 mm, 8 mm and 7 mm at the concentration of 200 mg/ml, 100 mg/ml and 50 mg/ml, respectively. results from figure 1b, 1c & 1f shows that agave americana did not show any antimicrobial activity against shigella, klebsiella pneumonia and salmonella paratyphi, respectively. results from figure 1d shows that agave americana seem to be mildly inhibitory at 200 mg/ml showing the zoi of 9 mm, 8 mm and 7 mm at the concentration of 200 mg/ml, 100 mg/ml and 50 mg/ml, respectively. results from figure 1e shows that agave americana is mildly inhibitory against bacillus thuringiensis showing zoi of 9 mm, 7 mm and 6 mm at concentration of 200 mg/ml, 100 mg/ml and 50 mg/ml, respectively. dpph free radical scavenging activity absorbance in 517 nm was taken for calculation of % scavenging activity of the a. americana extract using dpph as control and ascorbic acid as an analytical standard. graph (figure 2) was plotted using concentration range against percentage (%) scavenging activity and then linear trendline equations were obtained from the graph (table 4) to calculate ic50 value. table 3 zone size interpretative standards for selected antimicrobial disks and test for resistance. agent zone of inhibition (mm) r e si st a n ta in te rm e d ia te a s u sc e p ti b le a s . a u re u s s h ig el la k . p n eu m o n ia e e . co li b . th u ri n g ie n si s s . p a ra ty p h i chloramphenicola (30 μg disk) (positive control-1) ≤12 13-17 ≥18 25± 0.50 26.3±0 .76 22.5± 0.50 21.3±1 .53 26± 0.50 23± 0.50 tetracyclinea (30 μg disk) (positive control-2) ≤14 15-18 ≥19 25± 1.00 23.8±0 .76 22± 0.50 20.8±0 .76 25.1±1 .04 23.1± 1.04 dmso (negative control) na na na 0± 0.00 0± 0.00 0± 0.00 0± 0.00 0± 0.00 0± 0.00 asource: national committee on clinical laboratory standards (nccls), 1998. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 34 nepjol.info/index.php/njb. figure 1 graphical comparison of antimicrobial activity of a. americana extract against different clinical isolates. figures a, b, c, d, e and f represent zoi (zone of inhibition) of different concentration of the a. americana extracts on the individual clinical non-resistant isolates viz., staphylococcus aureus, shigella, klebsiella pneumoniae, escherichia coli, bacillus thuringiensis and salmonella paratyphi respectively. different concentrations of plant extracts at 200 mg/ml, 100 mg/ml and 50 mg/ml were used for antimicrobial assay and standard antimicrobial discs were used as the positive control to ensure that microbes were not resistant to the antibiotics and false negative result about the extract's efficacy could be prevented. in the figure aa represents agave americana. similarly, c30 and t30 stand for chloramphenicol 30 μg discs and tetracycline 30 μg discs respectively. all the experiments were performed in triplicates and the error bar represents maximum of 5% error in independently performed experiments (n=3). nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 35 nepjol.info/index.php/njb. figure 2 dpph free radical scavenging activity of a. americana. the graph shows antioxidant assay of a. americana extract in comparison to ascorbic acid as the standard. table 4 linear regression trendline of the extract with calculated ic50 value. linear regression trendline equations were obtained from figure 2 and ic50 values were calculated accordingly sample regression trendline equation ic50 value ascorbic acid y = 6.99x+38.87 1.592 μg/ml agave americana y = 9.66x-24.27 7.68 μg/ml the ic50 value of ascorbic acid as standard was 1.592 μg/ml and the ic50 value of agave americana was 7.68 μg/ml (table 4). cytotoxicity analysis: the extract of a. americana showed cytotoxicity against mcf-7 cell line to some extent (figure 3). extract that shows cytotoxicity with less concentration in short time can be considered as having robust cytotoxic properties. our experiments reveals that the mcf-7 cell line as negative control was 100 % viable even after 48 hr thus giving the percent apoptosis a value of zero in figure 3. previously published data from lamichhane et.al. from our laboratory also reveals that count of breast cancer cell lines does not get affected for 72 hr as a negative control [12]. figure 3 comparison of percentage cell-death shown by different concentrations of the extract. the figure represents percent celldeath of mcf-7 shown by extract of agave americana at a varying concentration of 5 µg/ml, 20 µg/ml and 80 µg/ml. all the experiments were performed in triplicates and the error bar represents the standard deviation of independently performed experiments (n=3). extracts of agave americana showed 50 % celldeath of mcf-7 in 12 h at 5 µg/ml concentration and 85 % cell-death in 12 h at 80 µg/ml as shown in figure 3. in 48 h, 80 µg/ml and 5 µg/ml of agave americana extract showed 99 % and 90 % cell-death respectively. discussion there is a good recovery rate from the warm soxhlet extraction method followed as shown in table 1 indicating that the extracts may contain phytochemicals that can be imputed to their biological activities [20]. the phytochemical analysis as shown in table 2 confirms the presence of phytochemicals like alkaloids, flavonoids, reducing sugars, and saponins which may in term be responsible for the antibacterial, antioxidant and anti-cancer activities. the clinical isolates that were used in this study were tested for their non-resistance against broad-spectrum antibiotics chloramphenicol and tetracycline as shown in table 3 to prevent false-negative result and dmso was used as a negative control as it does not exhibit any anti-microbial activities [21]. since compounds from the natural sources are 0 20 40 60 80 100 120 5 20 80 control (-ve) % o f c e ll a p o p to s is concentration of extract (µg/ml) cytotoxicity of agave americana in mcf-7 12 h 24 h 36 h 48 h y = 6.99x + 38.87 y = 9.66x 24.27 -20 0 20 40 60 80 100 120 1 2.5 5 7.5 10 20 40 60 80 100 % s ca v e n g in g o f d p p h concentration μg/ml dpph free radical scavenging activity ascorbic acid a. americana nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 36 nepjol.info/index.php/njb. considered as a safer option than the synthetic ones so these natural compounds can be used and explored for the treatment of the various medical conditions [22]. from figure 1 it can be perceived that the extract of agave americana shows a certain level of antimicrobial activity against s. aureus, e. coli and b. thuringiensis. the low level of antimicrobial activity may be due to the presence of antimicrobial compounds in low concentration. if the pure compound responsible for the antimicrobial activity can be identified and isolated from the extract then the compound can show a higher level of inhibitory action. the potent antioxidant activity in a dosedependent manner was evaluated from the plant extract of agave americana as presented in figure 2 and table 4. from the observation, the antioxidant activity of agave americana cannot be claimed strong. the lower antioxidant activity may be affected by geographical locations as some studies revealed that the same plant from different geographical regions may possess variation in compounds and thus process variation in antioxidant activities [2325]. hence, further screening of a. americana in broader geographical regions should be done to confirm the antioxidant nature. the data in figure 3 shows that agave americana has good cytotoxic activity against mcf-7 breast cancer cell line which can be attributed to the phytochemicals present and antioxidant activity of the plants. 5 µg/ml extract of agave americana showed 50 % cell-death of mcf-7 in 12 h whereas, 20 µg/ml of the extract showed more than 80% cell-death of mcf-7 in 48 hr. the results on cytotoxic activities shown here are first of its kind for a. americana found in nepal. further research is warranted to study the molecular mechanism of their potential anti-cancer activity for the development of anticancer drugs. conclusions various literature reviews in agave americana and the results obtained from this study generates some supportive evidence about its use as an ethnomedicinal plant. this study tries to explain the reason behind the use of a. americana from ancient times in treating diseases. the phytochemical, antimicrobial and antioxidant assays from the extract of a. americana show the presence of promising molecules that may be used in treating diseases. new sources of antibiotics are currently needed in a pursuit to counter the emerging resistant strains of microbes. also,new anticancer drugs are needed to counter the drug resistance shown by cancer cells. the ethnomedicinal plants like a. americana can be sources of these new molecules thus the quest to explore the initial properties of these ethnomedicinal plants should never be put to an end. evaluation of cytotoxic properties in the extract of a. americana is promising which may lay a foundation to research new anti-cancer molecules. although the study can be a piece of scientific evidence for the properties and the use of ethnomedicinal plants like a. americana in terms of its antimicrobial, phytochemical, antioxidant and cytotoxic properties for pharmacological purposes but further detailed studied are still mandatory for us to elucidate a mechanistic way regarding how the individual molecules in these kinds of ethnomedicinal plants behave in-vitro and in-vivo. competing interests the authors declare that they have no competing interests. funding funding for the project was entirely done by the department of biotechnology, kathmandu university, nepal. acknowledgments we are grateful to the taxonomist for the identification of plant species and prof. dr. tika bahadur karki, head of department of biotechnology, kathmandu university, nepal for allowing us to undertake this project. we would like to thank mr. sandeep adhikari, ms by research student for helping us with cytotoxicity analysis and mr. basant lamichhane, ms by research student for giving nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 30-38 pandey et al. 2019 ©njb, biotechnology society of nepal 37 nepjol.info/index.php/njb. their views in selecting medicinal plants. we would also like to appreciate bethanie rammer, managing editor at african journal of laboratory medicine, united states and dr. ulrike olszewski of ludwig boltzmann gesellschaft, austria who gave their valuable time and suggestions in order finalize this research article. references 1. sen s, chakraborty r: revival, modernization and integration of indian traditional herbal medicine in clinical practice: importance, challenges and future. j trad complement med. 2017 7(2):234244. 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naseer r, ashraf m, ashrafuzzaman m: variation in antioxidant attributes at three ripening stages of guava (psidium guajava l.) fruit from different geographical regions of pakistan. molecules. 2012 17(3), 3165-3180. doi: 10.3390/molecules17033165 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1 :103-112 doi: https://doi.org/10.3126/njb.v7i1.26955 ©njb, biotechnology society of nepal 103 nepjol.info/index.php/njb. review article polyphenols: nature's gift as dietary phytoconstituents against different human ailments rojina thapa1, rambika thapa1, sunita mahat1 and rupak thapa2* 1 himalayan college of agricultural sciences & technology 2nist college, banepa, abstract recently, researches and scientists are showing a lot of interests in bioactive compounds of plants and its products as these constituents are of high valued. polyphenols are phytochemical constituents which are the integral components in plants and their products that are associated with defensive mechanism against infections and various oxidative stress by free radicals. green tea, fruits, vegetables, cereals and red wines are rich sources of polyphenolic constituents which attributes prevention from infections and diseases because of its antioxidant properties, anti-diabetic properties, and radical scavenging properties. oxidative stress which is the primary reason for different ailments in humans is due to the free radicals that are present even during the normal health condition. polyphenolic acids like cinnamic acid, romarinic acid, stilbenes like resveratrol, flavonoids like catechin, taxifolin, quercetin, and lignans like sesamin, pinoresinol, podophyllotoxin etc. are found to be effective against wide range of human diseases like oxidative stress, cardiovascular disorder, neurodegenerative diseases, aging, and cancer. these phytoconstituents prevent the diseases and provide relieving sensation via different mechanisms.here, the study shows the importance of polyphenols with respect to the relevance of human health. as there are the promising applications of various bioactive constituents in a wide range of disease, further research should be encouraged on the mechanism of action and bioavailability of polyphenols. keywords polyphenols; antioxidants; bioavailability; cardiovascular diseases; diabetes; aging; cancerintroduction corresponding author emailrup_thapa123@hotmail.com introduction plants produce different secondary metabolites among which polyphenols are of great importance, which is engaged in protection from various pathogens and radiation (1]. polyphenols occur abundantly in fruits and vegetables that we consume in daily lives [1-3]. food consists of polyphenols which attribute to the astringency, taste, coloration, and oxidative stability [3-4]. past research and study strongly provide evidence that the constant dietary intake of food with exuberant polyphenols provide defence against various diseases like diabetes [5], cancer [6], cardiovascular diseases [7] and neurodegenerative diseases [8] (figure 1) as the food with phenols and its derivatives are propitious to health, polyphenols and its derivatives demands are augmenting. classification of polyphenols around 10,000 polyphenolic compounds have been discovered and identified [9]. almost all polyphenols originate from a basic precursor, phenylalanine or shikimic acid. phenylalanine is a substitute for methyl group of alanine whereas shikimic acid is a cyclohexane, an anionic form of shikimate [9-10]. normally, these precursors are associated with sugar residues in association with hydroxyl groups, carboxylic acids, organic acids, lipids, amines, etc. on the basis of phenol ring possessed by phenol and on the basis of structural components that they incorporate, phenols can be classified into different groups [11]. most important classification of polyphenols includes phenolic acids, stilbenes, flavonoids, and lignans [9-11]. illustration of different classes of polyphenols is provided by table 1. phenolic acids these are aromatic acid compounds which comprises a phenolic ring and an organic carboxylic acid framework. these are found ©njb, biotechnology society of nepal 104 nepjol.info/index.php/njb. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 103-112 thapa et al. 2019 figure 1: polyphenols found abundantly as plant secondary metabolites occurring naturally in vegetables, fruits, cereals, green tea and red wines. research and studies have shown that plant extracts are rich in polyphenolic constituents which are potent radical scavengers and impart great antioxidant activities providing significant protection against wide range of diseases like cardiovascular diseases, cancer, neurodegenerative diseases, aging, and diabetes. copiously in fruits and vegetables and are further divided into two naturally occurring broad classes of hydroxybenzoic acids (figure 2) and hydroxycinnamic acids (figure 3) that are synthesized from the non-phenolic component of benzoic and cinnamic acid respectively [21]. the study has shown that the content of hydroxybenzoic acid in the case of an edible plant is usually low, except in the case of red fruits and onions. clearly, hydroxycinnamic acids are found more commonly than hydroxybenzoic acids [22]. phenolic acids are found in greater amount in dried fruits, beans, mushrooms, chicory, coffee etc. (21-22]. stilbenes stilbenes consists of two phenyl components united via two carbon methylene bridge. table 1. types of polyphenols: sources and functions polyphenols food sources examples functions references phenolic acids straberries, coffee, nut, vinegar, turmeric, rosemary, cinnamon cinnamic acid, ellagic acid, rosmarinic acid antioxidant, radical scavengers and anticarcinogenic activities 12-14 stilbenes grapes, red wine resveratrol reduce oxidative stress, anti-aging, antioxidants. 15-16 flavonoids green tea, onion, berries, citrus fruits. apple catechin and epicatechin, taxifolin, quercetin antimicrobial, antioxidant and antidiabetic 17-18 lignans wide variety of plant species podophyllotoxin and its derivatives anticancer therapy 19-20 ©njb, biotechnology society of nepal 105 nepjol.info/index.php/njb. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 103-112 thapa et al. 2019 stilbene may be either e-stilbene (trans-isomer] (figure 4) or z-stilbene (cis-isomer) (figure 5) (23]. e-stilbene consists a central ethane double bond replaced with phenyl groups on each carbon atoms of double bond whereas in zstilbene it consists of cis ethane double bond substituted by a phenyl group on both carbon atoms of the double bond. human diet contains a low amount of stilbene [24]. the most widely researched stilbene is resveratrol, which occurs naturally. red wines and grapes comprise a higher number of stilbenes. flavonoids flavonoids (figure 6) are bioactive polyphenols which have low molecular weight with the 15-carbon skeleton, which comprises two phenyl rings and a heterocyclic ring. flavonoids are the most indispensable group which is studied widely and they can again be classified into thirteen different subclasses [25]. over 4,000 flavonoids have been discovered and studied, most of which gives pigmentation to flour, fruits, and leaves. flavonoids which are of eminent importance include flavonols, flavanones, isoflavones, flavones, and anthocyanins (figure 7) [25-26]. lignans lignans contain a structure which is derived from dimerization of two cinnamic acid residues [figure 8). lignans are derived from 2 phenyl propane moiety and sound plant source is vegetables, cereals, lentils and linseed [27]. among the listed sources of lignans, linseed is the richest one. major constituents of lignan include seco iso lariciresinol and minor being matai retinol [27-28]. recent findings polyphenol consumption regarding the dietary consumption polyphenols, little research has been done, though it is of immense importance. on a study carried out in the usa, flavonoid consumption was found out to be approximately 1gm/d [29]. among various groups of polyphenols, the flavonoid is studied widely. daily intake of flavonoids has been estimated to be approximately 20 mg/d in european countries [30]. in asian countries, there is significant consumption of soya which amounts to approximately 30 mg/d of isoflavones [30]. dietary intake of polyphenols increases when vegetables and fruits like apple, pears, grapes etc. are consumed in the significant amount [2930]. the worldwide consumption of tea and coffee aids in consumption of hydroxycinnamic acids. people who drink several cups of tea or coffee might intake approximately 700 mg hydroxycinnamic acids /d, whereas people who don’t drink tea or coffee do not intake even ©njb, biotechnology society of nepal 106 nepjol.info/index.php/njb. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 103-112 thapa et al. 2019 25 mg/d [31]. there is a variation on polyphenol intake and this is due to the individual preferences on different food items. in conclusion, polyphenol intake normally reaches 1g/d if people are subjected to adequate fruits and vegetables on daily basis [29-31]. bioavailability of polyphenols the term “bioavailability” is used to describe the perception of the speed and degree to which a metabolite approaches its spot action. in simple words, it is that portion of an ingested component which barges into the systemic circulation and distinct areas where it can employ its action [32-34]. the ultimate aim of bioavailability is to find out better-ingested components, active metabolites, and polyphenols that lead to the formation of the active metabolite. table 2. important factors affecting bioavailability of polyphenols factors examples references intestinal factors enzyme activity, intestine and colon microflora 33, 35 systemic factors age, physiological condition, and diseases 33, 36 food factors fat, fiber and food matrix 33, 37 processing factors heat, homogenization and cooling methods 33 other components bond with polyph-enols or proteins 33, 38 in order to demonstrate the biological properties polyphenols essentially rely on bioavailability. the structural framework plays an important role in the rate at which these polyphenols are ingested in intestines [39]. although some polyphenols are absorbed in small intestine, a great number of polyphenols that we intake cannot be readily absorbed in its original nature. in order to be absorbed, polyphenols have to be modified via intestinal enzyme and microflora present in it [40]. polyphenols are hydrolysed in intestinal cells and also the modification continues at liver via sulfation, glucuronidation, and methylation [41]. it is not the quantity of polyphenol that we consume determine the speed of absorption, but the chemical structure. it is a detoxification process that limits their toxic effects [39-41]. in addition to that, it facilitates biliary and urinary excretion by augmenting their hydrophilicity. some of factors that affect bioavailability of polyphenols are listed on table 2. polyphenol as the natural antioxidants a molecule which has the capacity to inhibit the oxidation of other molecules is called antioxidants. during the oxidation process, there is the transference of electrons or protons from reference substance [42]. oxidation process elicits free radicals which have the capability to start the deleterious reaction in the cell which ultimately deteriorates cell and in worse scenarios may lead to cell death [42]. antioxidants, on the other hand, has the ability to terminate this deleterious reaction by scavenging the free radicals and by inhibiting the oxidation process [43]. polyphenols are robust antioxidants and are depicted as freeradical scavengers [44]. this property is due to their hydrogen donating ability. polyphenols provide h+ atoms readily. this free radical scavenging property is mainly due to its immense reactivity of hydroxyl components [45]. polyphenols and cardiovascular diseases cardiovascular diseases are mostly seen as a causative factor for mortality in developed countries [46]. different factors are responsible for the coronary heart diseases out of which environmental and hereditary are the most prominent ones [47]. besides genetic factors, lifestyle and the way of living and food consumption play important role in case of cardiovascular diseases [48]. lack of physical activities, unhealthy food habits, the intoxication of the body with cigarettes &, alcohol and stress full life are the main ©njb, biotechnology society of nepal 107 nepjol.info/index.php/njb. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 103-112 thapa et al. 2019 contributing factors for the prevalence of cardiovascular diseases [49]. different research and studies have shown that diet rich in polyphenols and flavonoids like beans, fruits and vegetables have curative and inhibitory effects against cardiovascular diseases [50-52). it was observed from the study that, there was 11% reduction from the adverse effect of cardiovascular diseases when three cups of black tea are taken daily [53-54). cardiovascular diseases are prevented by the polyphenols because it has the ability to ameliorate the activity of an enzyme and increase the bioavailability of nitric oxide for endothelium [47-49). consumption of polyphenol-rich food products has a relaxing effect on the endothelium. furthermore, food products rich in polyphenols like, flavonol has the effect of reducing hypertension and also lowers the blood pressure with improved function of endothelial tissues which prevents aggregation of platelet and lowers inflammation [55-56]. as black tea and cocoa contain high polyphenolic content, high drinking of these products lowers the risk of high blood pressure. [57-58] polyphenols and cancer cancer is a multi-step process. this includes initiation, development, and metastasis. tumour may or may not cause deteriorating effects to the cell. once the initiation starts, it inevitably progresses to malignancy imparting negative effects. afterward, these cancerous cells transfer from one part to another by the process known as metastasis [59]. polyphenols have the protective function and elicit the decrease in initial stage tumors. different studies have shown that polyphenolic constituents like phenolic acids [60], stilbenes [61], flavonoids [62], phenol [63] and lignans [64] have proved to be effective against different human cancer cell lines. polyphenol is to know for their inhibiting properties to cancerous cells via different mechanisms, which can be estrogen activity, antiproliferative activity, programmed cell death (apoptosis], limiting oxygen to the cancerous cell, production and synthesis of detoxifying enzymes, alteration in cell signaling and regulating immunity-inducing factors in the host [65-66]. cancerous cells are the results of carcinogens which are activated by the assertion of cytochrome p450, but the polyphenols have the tendency to affect this activity and have been identified as inhibiting factor which restricts this promotion of p450 expression [67-68]. polyphenols induce phase ii proteins which detoxify the toxicity and engender enhanced immunity against harmful xenobiotics [66]. a study in green tea has revealed that its components like catechins, theaflavins, and cherubins have promising potential against cancer [69]. they have found to restrict the formation of cancerous cells by the programmed cell death of prostate cancer cells. similarly, quercetin and resveratrol have found to inhibit all stages of cancer development and have found to be advantageous against skin, lung, colon, and breast cancer [70]. polyphenols and diabetes diabetes is a condition which leads to increase in blood sugar level in the blood. according to nlh, two main types of diabetes [type 1 and type 2) are common, type 2 being common [https://medlineplus.gov/diabetes.html]. polyphenolic compounds work against diabetes by inhibiting the intestinal glycosidases and glucose transporter [72]. different types of polyphenols like phenolic acids, stilbenes, flavonoids, phenol, and lignans are found to inhibit the s-glut-1 transfer of glucose in the small intestine [72-73]. resveratrol and saponins also have been found to detain the transport of glucose in the alimentary canal. different studies have shown that quercetin, resveratrol, catechin, epicatechin, and epigallocatechin have antidiabetic properties [74]. stilbene too has potent antidiabetic property and controls diabetes by modulating sirt1 which is responsible for homeostasis of insulin and glucose [75]. report on llc-pk1 cells shows that anthocyanins and grape juice have inhibiting effect towards ©njb, biotechnology society of nepal 108 nepjol.info/index.php/njb. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 103-112 thapa et al. 2019 glucose mediated oxidative stress and cytotoxicity [76]. polyphenols and aging different theories have been proposed regarding aging among which oxidative stress due to free radicals is one of the most approved ones [77]. oxidative stress and detrimental effect due to it is present even under good health condition, but the damage rate elevates at the age increases due to weak repair mechanism at old age [78]. aging is the consequence of the wide range of deleterious alteration in the cells as the age progresses ultimately leading to weakness and death [79]. antioxidant and anti-inflammatory compounds have properties of reducing oxidative stress by scavenging the free radicals which work as effective anti-aging compounds [80]. studies show that anthocyanins and grape polyphenol resveratrol show high scavenging potential with high antioxidant as well as antiinflammatory activities [84]. they are known to restricts the peroxidation of lipids and also inhibit cyclo-oxygenase which is inflammatory mediators [80-83]. dietary intake rich in flavonoids and polyphenolic compounds like fruits and vegetables shows high antioxidant activity. berries, beans, spinach, apple and green tea have been reported with high antioxidant contents and found advantageous against agerelated senescence and age-related problems [85-86]. adequate consumption of polyphenols ameliorate the negative effects of aging and also have been found to delay the onset of aging [87]. polyphenols and neurodegenerative diseases neurodegenerative diseases are the consequences of oxidative stress and free radicals which damage brain cells [88]. medicine plus list 7 diseases under degenerative nerve diseases which include: friedreich's ataxia, alzheimer's disease, parkinson's disease, lateral sclerosis, huntington's disease, amyotrophic, lewy body disease and spinal muscular atrophy. neurodegenerative diseases can be fatal even leading to the demise of patients. polyphenolic compounds are seen to have positive effects on nerve degenerative diseases as it has the power to scavenge the free radicals which impart detrimental effects in the body. resveratrol, the integral component in grapes and wine, have been reported to scavenge free peroxide, hydroxide and other free radicals [90]. people who drink 2-3 glasses of red wine daily have been shown to 80% decrease in the evidence in alzheimer’s disease [91]. drinking green and polyphenolic-rich diet act potent agent for neuroprotection via cell signaling, programmed cell death [apoptosis] proliferation and differentiation [92]. green tea has been found effective against parkinson’s diseases by inhibiting mptp (n-methyl-4phenyl-1, 2, 3, 6-tetrahydropyridine), which is the integral factor for parkinson’s diseases [9193]. conclusion this review tries to outline the current understanding of polyphenols and its biological beneficial effects in human health. dietary intake of polyphenolic compounds daily imparts important protection against wide range of detrimental diseases like cardiovascular disorders, diabetes, cancer, aging and nerve-degenerative diseases. this positive advantageous aspect of polyphenols on human health depends on their intake and their bioavailability. it is well documented through studies and researches all around the world that polyphenols have the wide range of applications in human health but still the mechanism of action of various polyphenolic constituents are yet to be understood. this positive advantageous aspect of polyphenols on human health depends on their intake and their bioavailability. the role of polyphenols in human health is a promising area of the research study. this review outlines the promising wide range application of polyphenolic compounds and prominent hope for chronic detrimental human diseases. ©njb, biotechnology society of nepal 109 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baral1, ram raja gurung3, balkrishna bhattachan4 1pokhara bigyan tatha prabidhi campus, pokhara nepal 2central blood transfusion service, nepal red cross society, pokhara, nepal 3deurali-janata pharmaceutical pvt. ltd., kathmandu, nepal 4siddhi memorial hospital, bhaktapur, nepal abstract hepatitis b virus (hbv) and hepatitis c virus (hcv) infections lead to chronic diseases and are the most common causes of liver cirrhosis and cancer in developing countries like nepal. the study is carried out to determine the seroprevalence of hbv and hcv by using a rapid kit method and elisa method to find out its risk factors. the cross-sectional study was done among blood donating people from 16th august 2016 to 19th november 2016. blood donors in pokhara valley were screened for anti-hcv antibodies, anti-hbv antibodies using third generation elisa kits and automated elisa processor in serology laboratory at central blood transfusion service (cbts) of nepal red cross society (nrcs) in pokhara, nepal. 1777 (87.2%) units were male blood donors and 260 (12.6%) units were female donors out of 2037 participants. gender wise, the ratio between male and female was 1:0.1. hbv and hcv infection rate in blood donors were detected at 0.7% (15/2037) and 0.5% (8/2037) respectively. hbv infection rate in volunteer blood donor people was 0.7% (14/1881) which was higher than the replacement donors i.e. 0.6% (1/156). similarly, in hcv infection rate in volunteer donor were 0.4% (8/1881). hbv infected people are detected higher than the hcv infected people among the blood donors. in addition, there was no significant relationship between positive results of hbv and hcv tests with the gender, age, tattoo, donor type. keywords: blood transfusion, sera, hcv, hbv, elisa, nepal *corresponding author email: balkrishna_bhattachan@hotmail.com introduction hepatitis b virus (hbv) is an enveloped, hepadnaviridae with partially double-stranded dna [1]. it is one of the cause of potentially lifethreatening liver infection and is a major global health problem. it can cause chronic liver disease and puts people at high risk of death from cirrhosis of the liver and liver cancer [2]. worldwide, an estimated two billion people have been infected with the hepatitis b virus and more than 240 million have chronic (long-term) liver infections. in developing countries like nepal, the highest prevalence of hbv was found in the 6-15 year age group [3]. common modes of transmission include perinatal, unsafe injection practices, unsafe blood transfusions, and unprotected sexual contact [4]. hepatitis c virus (hcv) is single-stranded rna, enveloped flavivirus that causes liver disease [5]. the disease can range in severity from a mild illness lasting for few weeks to a serious, lifethreatening condition that can lead from cirrhosis of the liver to liver cancer. about 170 million people are infected worldwide [6]. hcv is most commonly transmitted through exposure to infectious blood and it may be transmitted through gender with an infected person or sharing of personal items contaminated with infectious blood, but these are less common [7]. this study is conducted among blood donors in pokhara valley in order to study the hbv and hcv seroprevalence, among different groups and subgroups of blood donors. the main aim of this study is to determine the seroprevalence of hbv and hcv by rapid kit and elisa method and find out its associated risk factors. open land route facilitates the biological and virological spread of diseases [8]. so, in the case of hcv and hbv, millions of migrant workers act as a biological transporter. however, documentation of hcv and hbv with past infection is frequent nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 33-38 shah et al. ©njb, biotechnology society of nepal 34 nepjol.info/index.php/njb in person with routine parenteral exposures which indicate that the transmission by this line is prominent. accidental parenteral exposures such as needle-stick injuries are quite common in patients. another route of transmission is having tattoos, drug addicts, heterosexual and homosexual gender, liver diseases, and blood transfusion. perception and awareness level of people has been changed after the jaundice outbreak occurred in biratnagar during april-july 2014 [9]. methods and materials study design and setting cross-sectional and descriptive type of study was done. the study sample comprised of 2037 blood donors. healthy donors included in this study were donating blood in blood transfusion center, pokhara at mobile health camp organized by red cross society. this study was conducted from 16th august to 19th november 2016. study area and population blood donors were selected according to the criteria of blood transfusion service (bts) based on the national guidelines for bts. the categories of donors that were present in this study were volunteers and replacement who may be the first time or repeated donor. individuals selected by donor screening criteria as per nrcs’ standard operating procedure (sop) of 2006 ad. participants were requested to fill a questionnaire form along with written consent were collected. each donor was given an id number for later trace out if needed. inclusion criteria: healthy donors were included for blood donation after taking an interview. the detailed inclusion criteria for blood donors are: donor should be in the age group between 18 to 60 years, body weight more than 45 kg for male and 35 kg for female, within normal range blood pressure 120/80 mm without medication and hemoglobin should not be less than 12.6 g/ml. exclusion criteria: those who are found unhealthy on the interview were excluded for donation. the exclusion criteria for blood donors are:in the case of a female, menstruating, pregnant and breastfeeding mother are not allowed for donation and patients taking antibiotics. sample collection and processing the blood bags were transported from blood donation campaign site to blood bank in igloo box containing ice bars to maintain the temperature near 40c. a few drops of blood is used for blood grouping and 5ml of blood was allowed to clot or centrifuged at 3000 rpm for 2 minutes in a dry screw-capped test tube to separate serum samples to perform the test for hbv and hcv. detection of hbv and hcv by using rapid kit test collected sera were subjected for routine mandatory screening for hbsag and anti-hcv antibodies by enzyme immunoassay-based rapid tests according to the standard protocol described by respective company (hepacard, j. mitra and co., new delhi, india, and hcv tridot, j. mitra and co., new delhi, india) [10]. detection of hbv and hcv by using elisa method sera from blood donors for hcv were tested against (anti-hcvs) antigen by third generation enzyme-linked immunosorbent assay (elisa) and for detection of hbv by hbsag elisa (hcv tri-dot test, j. mitra and co., new delhi, india; hepacard, j. mitra; genedia hcv elisa.3.0, green cross, kyunggido, korea; and enzygnost hbsag 5.0, dade behring, marburg, germany) [11]. statistical analysis pearson's chi-square test was used to determine the significant association of dependable variables. winpepi software (version 11.65) was used for quantitative data analysis. a p-value < 0.05 was considered to be statistically significant at 95 % ci. results a total of 2037 blood samples were collected from donors. the blood was screened by rapid test and elisa test to detect hbv and hcv infection. gender wise, the ratio between male and female was at 1:0.1. the infection rate of hbv and hcv in blood donors were detected to be 0.7 % (15/2037) and 0.5% (8/2037) respectively (figure 1). nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 33-38 shah et al. ©njb, biotechnology society of nepal 35 nepjol.info/index.php/njb figure 1: infection of hepatitis b virus and hepatitis c virus in blood donors (confirm by elisa method) the infection rate in a male for hbv is 0.7% (13/1777) which was higher than hcv 0.4% (8/1777). similarly, the infection rate in the female for hbv is 0.7% (2/260) which was also higher than hcv (0.0%) but no statistically significant, (p=0.280) result was obtained (table 1). in hbv, the highest rate of infection was detected 0.9% (7/764) in 31-45 year old of blood donors which was followed by 0.7 % (1/138) in 46-60 year old, and 0.6% (7/1135) in 18-30-year-old people. similarly, the highest rate of hcv infection were detected 0.7% (1/138) in 46-60 years old people followed by 0.6% (5/764) in 3145-year-old and 0.2% (2/1135) in 18-30 years old people, p=0.588, (figure 2) figure 2. age wise distribution with hbv and hcv among blood donors hbv infection rate of volunteer donor people was 0.7% (14/1881), which is higher than the replacement donor 0.6% (1/156). similarly, hcv infection rate of volunteer donor was 0.4% (8/1881) with p=0.455 (table 2). table 2: donor type-wise distribution with hepatitis infections among blood donors donor type disease total no. pvalue hbv no. (%) hcv no. (%) replacement donor 1 (0.6) 0 (0.0) 156 0.455 volunteer donor 14(0.7) 8 (0.4) 1881 total 15 (0.7) 8 (0.4) 2037 hbv infection rate in people without tattoo was 0.7% (14/1847) higher than the people with tattoo 0.5% (1/190). similarly, hcv infection rate in people without tattoo was 0.3% (6/1847) higher than 0.1% (2/190) in people with a tattoo with p=0.214 (table 3). hbv infection rate of married blood donors was 0.9% (12/1262) whereas 0.3% (3/775) was found in unmarried blood donors. similarly, hcv infection rate of married blood donors was 0.7% (7/1262) whereas 0.1% (1/775) was found in unmarried blood donor with p=0.65 (table 4). table 4. marital status wise distribution with hepatitis infection among blood donors disease total p-value marital status hbv no. (%) hcv no. (%) no 3 (0.3) 1 (0.1) 775 0.651 yes 12 (0.9) 7 (0.7) 1262 total 15 (0.7) 8 (0.4) 2037 discussion the group of viruses (hepatitis a, b, c, d, and e) that cause acute and/or chronic infection and inflammation of the liver give rise to a major public health problem globally [12]. hepatitis b and c viruses are major agents of severe illness and death. the burden of acute hepatitis, liver 0.7 0.4 0 0.2 0.4 0.6 0.8 hbv hcv p e rc e n ta g e hepatitis types positive (%) table 1: sex-wise distribution with hepatitis diseases among blood donors gender hepatitis total no. pvalue hbv no. (%) hcv no. (%) male 13 (0.7) 8 (0.4) 1777 0.280 female 2 (0.7) 0 (0.0) 260 total 15 (0.7) 8 (0.4) 2037 table 3. tattoo wise distribution with hepatitis infection among blood donors disease total p-value tattoo used hbv no. (%) hcv no. (%) no 14 (0.7) 6 (0.3) 1847 0.214 yes 1 (0.5) 2 (0.1) 190 total 15 (0.7) 8 (0.4) 2037 0.6 0.9 0.7 0.2 0.6 0.7 0 0.2 0.4 0.6 0.8 1 18-30 year 31-45 year 46-60 year p e rc e n ta g e age group hbv (%) hcv(%) nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 33-38 shah et al. ©njb, biotechnology society of nepal 36 nepjol.info/index.php/njb cirrhosis and cancer due to this hepatitis b and c viruses is high worldwide (about 2.7% of all deaths) and is forecasted to become a higher ranked cause of death over the next two decades. it is estimated that 57.0% of cases of liver cirrhosis and 78.0% of cases of primary liver cancer have resulted from hepatitis b or c virus infection [13]. the infection rate of hbv and hcv in blood donors were detected on 0.7% and 0.5% respectively. in the previous study reported that the hepatitis b and hepatitis c infection rate was detected by 2.6% and 0.3% respectively out of 307 tested patients [14]. the high percentage of hbv and hcv infection was obtained may be due to less no. of public awareness of hepatitis known infected person did not participated in the blood donation. the unsafe behavioral practices such as body pierce, tattoo, use of injectable drug, unprotected sex etc. also led to increase the transmission of infection. the positive rate of hepatitis infection was found to be higher in a male patient than female but there is no statistical significant relationship found between gender and hepatitis [14]. in hbv infection, most of the infection rate (0.9%) was detected in age group ranging 31-45 years old whereas most of the infection rate in 46-60 years old people in hcv was detected at 0.7%. the highest positive rate was found in the patients of age group 31-60 years old [14]. the infection rate of hbv and hcv in volunteer donor people were found to be 0.7%, and 0.4% respectively. it may be, due to blood transfusion, hepatitis can be transmitted in another body. study also revealed that the eastern region of nepal had the highest percentage of hcv infection (48.0%) [15]. seroprevalence of hbv among mothers were found 17.9% in 151 mothers of the total study population in the selected village development committees (vdcs) [16]. the highest rates of hcv and hbv co-infection was found to be 58% and 70% respectively 30-39 years age group. hbv infection rate was 0.7% higher in people without tattoo whereas the hcv infection rate in people without tattoo was 0.3% higher. because in our case, the number of participants without tattoo was higher than the number of blood donors with tattoo. although, it is widely known fact that cosmetic procedure, tattooing on a body can result in a hbv and hcv infection if the needles and equipment used are not properly sanitized. besides, the poor tattooing procedure may have chances of microbial contamination. hbv and hcv infection rate in married blood donors were 0.9% and 0.7% respectively. researchers concluded the marital status is a significant risk factor [18]. they detected hbv and hcv were found 1.7% and 1.0% respectively in a married participant. hbv and hcv can be transferred from unprotected sexual contact if their partner is infected with the hepatitis virus. people with unsafe sexual relationships and addiction to injectable drug users should always be careful as the chances of the virus getting transmitted are high among them. this severity of cases may be due to the lack of vaccination information in the population. the replication of hbv and hcv in hiv patients should be actively monitored while receiving antiviral therapy and this monitoring system should be made a part of clinical care [17]. in the context of nepal, people have has less knowledge of vaccination and those who knows avoid it because of hepatitis vaccination cost and no. of clinic visits to complete vaccination doses. the implications of screening tests for hbv or hcv co-infection in hiv patients are of great need in nepal [18]. limitations of the study: this study was carried out in a limited population and only involved the donors attending on mobile health camp organized by bts-nrs. so, the results obtained from this study might not be enough to indicate the actual burden of the disease in the large population. still, the study found the existence of endemic viral hepatitis among the people and need health policies to reduce the burden of viral hepatitis in society. pcr based detection method was not incorporated due to feasibility issues regarding time and resources constrain. conclusions hbv and hcv infections can be transfer by piercing needles, general contact, blood nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 33-38 shah et al. ©njb, biotechnology society of nepal 37 nepjol.info/index.php/njb transfusion, drug users. therefore, policymaker should provide awareness and knowledge for people about it. general people must take a hepatitis vaccine. government should provide hepatitis vaccine at an affordable price. government and its allied body should provide infection control support. finally, people should be aware of its associated risk factors. the infection rate in prior blood transfusion people, hbv and hcv infections found in the same ratio. moreover, due to its risk factors, like age, gender, tattoo, marital status, donor type can be transmitted the vital role in the mode of transmission of hepatitis. conflict of interest the authors declare that they have no competing interests. acknowledgments we would like to convey our gratitude to mr. dhrubamadi lamichhane, coordinator of central blood transfusion service and all the members of nrcs (cbts) who were helpful and made my lab work possible. and, thankfulness to mr. arjun gyawali and allied staffs. without their support, we could not able to conduct this study. consent for publication not applicable ethical approval and consent to the participant no patient-related data were collected. ethical approval was therefore not required. the study was laboratory-based basic science study. written informed consent was taken from all participating patients or from guardian on the behalf of their children. availability of data and materials all supplementary files, data generated and analyzed during this study will be made available as per reasonable request to the corresponding author. source of support no funding author’s contributions gbs and kg designed the study. gbs collected sample at nrcs-cbts. gbs and bpb performed investigation and recorded the laboratory findings with the validation. kg supervised and provided a methodology for the study. bpb, bb & rrg administered the project, reviewed literature, and wrote original manuscript bb and rrg curated data to perform statistical data analysis and data interpretation. rrg & bb also reviewed, proofread, and revision of the draft by compiling, formatting, editing and writing the final version of the article. thus, all authors made a substantial contribution to the study. all of them read and approved the final manuscript. references 1. brooks g, carroll kc, butel j, and morse s: jawetz melnick & adelberg’s medical microbiology (26th ed.) 2012 usa: mcgrawhill. 2. jindal a, kumar m, and sarin sk: management of acute hepatitis b and reactivation of hepatitis b. liver international. official journal of the international association for the study of the liver 2013 33 (suppl 1):164-175. 3. shrestha sm, and shrestha s: chronic hepatitis b in nepal: an asian country with a low prevalence of hbv infection. trop gastroenterol. 2012 33:95–101. 4. who (2013). hepatitis, (access on www.who.int/topics/hepatitis/factsheets/en /). 5. younossi zm, stepanova m, mishra a, venkatesan c, henry l, hunt s: the impact of chronic hepatitis c on resource utilization and inpatient mortality for medicare beneficiaries between 2005 and 2010. alimentary pharmacology & therapeutics, 2013 38(9): 1065-1075. 6. mullis ce, laeyendecker o, reynolds sj, ocama p, quinn j, boaz i, et al: high frequency of false positive hcv elisa in rakai, uganda. clinical infectious diseases, an official publication of the infectious diseases society of america, 2013 57(12):1747-1750). 7. rudra s, chakrabarty p, hossain ma, akhter h, and bhuiyan mr: seroprevalence of hepatitis b, hepatitis c, hiv infections in blood donors of khulna, bangladesh. mymensingh medical journal 2010 19(4) :515-519. 8. nepal a. and kunwar b: evidence of hepatitis c virus infection and associated treatment in nepal. j mol biomark diag, 2016 7 (270):1-4. doi: 10.4172/2155-9929.1000270 9. subba nr: managing hepatitis outbreak in biratnagar nepal. science journal of public http://www.who.int/ nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 33-38 shah et al. ©njb, biotechnology society of nepal 38 nepjol.info/index.php/njb health, 2015 3(6): 808-814. doi: 10.11648/j.sjph. 20150306.12 10. tiwari br., ghimire p, kandel sr, and rajkarnikar m: seroprevalence of hbv and hcv in blood donors: a study from regional blood transfusion services of nepal. asian j transfus sci. 2010 4(2): 91–93. doi: 10.4103/0973-6247.67026 11. karki s, ghimire p, tiwari br, & rajkarnikar m: hbsag serosurveillance among nepalese blood donors. ann trop med public health. 2008 11(1):15–18. 12. gaze r. de, carvalho dm, santoro-lopes g, tura lf: from hepatic diseases and jaundice to viral hepatitis: the configuration of a kaleidoscope. revista de saude publica, 2013 47(1): 116-122. 13. who: (2013). immunization, vaccines, and biologicals, https://www.who.int/immunization/en/ 14. pokheral n, bhandari d, and jha b: the pattern of hepatitis b and c infections among patients attending a tertiary care hospital in kathmandu, nepal. j inst med, 2016 38(2):2-3. 15. ionita g, malviya a, rajbhandari r, william schluter w sharma g, kakchapati rs, and dixit s: seroprevalence of hepatitis b virus and hepatitis c virus co-infection among people living with hiv/aids visiting antiretroviral therapy centers in nepal: a first nationally representative study. intl j infect dis. 2017 60: 64–69. 16. shedain pr, devkota md, banjara mr, ling h, dhital s: prevalence and risk factors of hepatitis b infection among mothers and children with hepatitis b infected mother in upper dolpa, nepal. bmc infectious diseases 2017 17(1): 667. doi:10.1186/s12879-017-2763-4. 17. chuang wl, dai cy, chang wy, lee lp, lin zy, chen sc: viral interaction and responses in chronic hepatitis c and b co-infected patients with interferon-alpha plus ribavirin combination therapy. antivir ther, 2005 10, 125-133. pmid: 15751770 18. supramn hs, gokhale s, sathian, b. et al ( 2015). hepatitis b virus (hbv) and hepatitis c virus (hcv) co-infection among hiv infected individuals at tertiary care hospital in western nepal. nep j epidemol, 5(2), 488-49 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2937303/?report=classic https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2937303/?report=classic https://dx.doi.org/10.4103%2f0973-6247.67026 nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 27-31 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 27 nepjol.info/index.php/njb in vitro cultivation of newly reported wild edible mushroom volvariella bomybycina from nepal pradip kumar chaudhary1, 2*, mitesh shrestha1, 2, bal hari poudel1, mahesh kumar adhikari1, 3 1central department of biotechnology, tribhuvan university, nepal 2nepal academy of science and technology, khumaltar, nepal 3national herbarium & plant laboratories, department of plant resources, ministry of forests and soil conservation, nepal abstract wild edible mushrooms are becoming endangered all over the world. very few wild edible mushrooms are found in natural habitat. volvariella bombycina is an edible and medicinal mushroom. the mushroom was collected in natural habitat growing on populus tree. mycelium of the mushroom was developed in pda slant tubes by tissue culture method, incubated at 25oc for 1-2 weeks. spawn was developed in wheat grains after incubation at 25oc for 2-3 weeks. substrates were formulated for the development of fruiting bodies by combination of paddy straw, saw dust and rice husk. fruiting bodies of v. bombycina was cultivated in these substrates after incubation at 28 ± 2oc for 2-4 weeks. the work describes the optimized process for in vitro culture of wild edible mushroom volvariella bomybycina. key words: mushroom; wild; edible; cultivation; volvariella bombycina *corresponding author email: pradipkchaudhary@biotechtu.edu.np introduction wild edible mushrooms possessing medicinal and nutritional properties have been collected and consumed by people since time immemorial [1]. nepal is rich in biodiversity due to its complex variation in geomorphology and phytogeography [topology, climate and altitudinal]. it is known for being rich in mushroom diversity [2]. due to this, the exploration for novel mushrooms is of paramount importance. among such, one important edible and medicinal mushroom, volvariella bombycina has just been recorded in 2016 by adhikari m.k on populus tree from tribhuvan university, kirtipur [6]. volvariella bombycina [schaeff.:fr] singer, commonly known as silky agaric, silky sheath, silky rosegill, silver-silk straw mushroom, tree mushroom grows on populus tree in nepal and is distributed in china [3], north america [4], india [5], nepal [6], pakistan [7] and korea [8]. v. bombycina is a tropical and subtropical species and belongs to the family pluteaceae [6]. v. bombycina usually grows in a shady place on the rotting wood, leaf debris, rich agricultural soil especially in coffee and palm plantation [9]. it is an edible [9, 10] tasty, with a modest and pleasant flavor [11] with potential for commercial cultivation [13, 14]. the morphology of fruiting bodies of v. bombycina are highly variable, suggesting a strategy for adaptation against environmental stresses caused by a range of extrinsic factors [3]. the life cycle, hymenial cell type, cystidia and basidia of v. bombycina are similar to that of v. volvacea. however, on contrary to v. volvacea, it requires slightly lower temperature for mycelial growth and fruiting starts from 26oc to 30oc [14]. v. bombycina produces various bioactive secondary metabolites; ergosta-4,6,8[14],22-tetraene-3-one, ergosterol peroxide, indole-3-carboxaldehyde, indazole [15],and isodeoxyhelicobasidin in liquid culture [16]. the mushroom possesses several biological properties such as antioxidative, antitumor, hypocholesterolemic [17] and antimicrobial properties [18]. volvariella bombycina has a great commercial value all over the globe since it can be consumed as a substitute for meat because of its high protein content and for its enormous medicinal value [19] . attempts for its' cultivation have not been reported from nepal till date with few reported attempted from china [3] and india [19]. this difficulty in cultivation could be attributed to variability in nature of fruiting bodies [3]. similarly, previous methods were not suitable for commercial cultivation. this research utilized the easily available local substrates such as paddy straw and others to nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 27-31 chaudhary et al. ©njb, biotechnology society of nepal 28 nepjol.info/index.php/njb induce development of fruiting bodies from wild edible mushroom v. bombycina. materials and methods collection, identification and handling of fruiting bodies v. bombycina was collected from the trunk of populus tree found at tribhuvan university, kirtipur in paper tissue and cultured on the same day. the collected mushroom was identified by morphological and spore print characteristics. some specimens were dried under natural light at room temperature for several days. herbarium was prepared and deposited at national herbarium & plant laboratories, godawari, nepal under department of plant resources [dpr]. isolation of pure culture of mycelium isolation of pure culture of mycelium was performed by tissue culture method [20]. sterilized inner part of tissue [mycelia] from pileus and stipes were inoculated in sterilized potato dextrose medium [pda] slant tubes under sterile condition and incubated [bod incubator, optics technology] at 25oc for 1-2 weeks. pure culture of mycelium was obtained by repeated sub culturing process. grain spawn the protocol was followed as developed by chang and miles with slight modifications. 5 kg of wheat grains was washed thoroughly in clean water, soaked for one day and mixed with 7.5g of caco3. the grain mixture was filled in heat resistant polypropylene bags of cover size 28 x 10 cm2 and their mouths were closed using rubber bands. the bags were sterilized in autoclave at 15 lb/in2 pressure at 121o c for 3045 minutes. pure culture of mycelium was transferred aseptically to sterilized bags filled with substrate. the bags were finally incubated [bod incubator, optics technology] at 25o c for 23 weeks [20]. bed preparation and spawn run good paddy straws were selected and chopped into 3-4 inches; washed thoroughly and soaked in boiling water for 60-90 minutes and allowed to cool after draining excess water. sterilized rice husk and saw dust was added at concentration of 3-5% by weight of paddy straws. 60 x 30 cm2 sterilized polypropylene bags were used for packaging of substrate and spawn. spawns were inoculated at every 5 cm layers during packaging of substrate. thus, prepared bags were incubated at 25oc in incubator [bod incubator, optics technology]. the spawn run period was 8-10 days and humidity was maintained at 80-90%. mouth of the polypropylene bags were opened for 1-2 hours once the spawn run period was over for proper air circulation under sterile condition. the incubation was done till the primordial formation. development of fruiting bodies during fruiting bodies development, the mouths of the bags were opened up to 6 hours a day under sterile condition for air circulation. humidity and temperature were maintained up to 90% and 28 ± 2oc respectively. the fruiting bodies were harvested at egg stage and then in mature stage. results morphology of the mushroom figure 1: v. bombycina a. hymenial surface b. upper surface c. spore print d. spores [40x]. v. bombycina was observed as large pileus creamy white, dry, and covered with silky hairs measuring ~10 cm in diameter. stipe is 13 cm long and 1-2 cm thick. the stipe was tapering upwards, cylindrical often curved, dry, white, smooth without a ring. the volva is cup-shaped with irregular margin. it measures 4 cm long, 2 cm wide, thick, white to yellowish or brownish, and mouth open sac like. lamellae are free, at first whitish, later becoming pink, crowded, margin entire figure 1 a&b]. spore print was observed as rosy or pink in color [figure 1c]. v. bombycina spores were elliptical, smooth, cystidia long; variously shaped. pileipell is nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 27-31 chaudhary et al. ©njb, biotechnology society of nepal 29 nepjol.info/index.php/njb without gelatinized hyphae. clamp connection was not seen (figure 1d) pure culture of mycelium and spawn preparation figure 2: v. bombycina a. pure culture of mycelium b. microscopic view [100x] and c. mother spawn. white mycelia were isolated on the pda tubes. septate mycelium was observed under microscopic view (figure 2 a & b]. the mother culture of the mycelium of v. bombycina was initiated after 4-5 days of incubation. the pure culture of the mycelium was isolated by repeated sub culturing on pda tubes. incubation period varies between 15-20 days depending upon density of mycelium. the spawn was developed in wheat grains. the mycelium was fully colonized in wheat grains after incubation of 2-3 weeks (figure 2 c). fruiting bodies development figure 3: cultivated fruiting bodies a. early stage b. mature stage. mycelium colonization was observed in substrates after one week of incubation. primordial was initiated during 10-15 days of incubation. button stage and mature fruiting bodies were observed during 15-20 days and 2030 days respectively (figure 3) discussion the genus volvariella, is easily recognized with pink lamellae and rosy or pink in color spores in figure 1 d. figure 4: showing growth curve of mycelium of v. volvacea and v. bombycina at 25oc the stipe of the fruiting bodies doesn’t have an annulus. it has volva at the base of the stipe. the lamellae of volvariellla species are whitish at first, which later becomes pink [6]. growth rate of mycelium of v. bombycina showed slower in contrast to v. volcacea (figure 4). according to jonathan & awotona in 2011, mycelia of v. bombycina showed best growth at 28o c and ph 6.8 [21]. it has been reported that straw alone is not sufficient as a composting material as it contains a little quantity nutrients and has a slow rate of decomposition [25]. the cultivation of wild edible mushroom v. bombycina was grown by using local agricultural waste substrate, paddy straw supplemented with saw dust and rice husk. formulation of mushroom substrates presents a very essential factor for the multiplication of mycelium, as it allows the penetration of the mycelium, which influences the fruiting of mushroom. mushroom produces various enzymes which makes it capable of utilizing the complex lignocellulosic organic compounds [22]. it took 1-2 weeks for complete mycelial colonization in the substrates. incubation allows the colonization of mycelium in the substrate of the paddy straw mushroom, mycelium of v. bombycina grows at slightly low temperature [25oc-30o c] compared to v. volvacea and requires relative humidity 80-90%. a variety of waste materials have been used for cultivation of volvariella mushroom that include paddy straw, water hyacinth, oil palm bunch and oil palm pericarp waste, banana waste, sawdust, cotton waste, sugar cane bagasse, composted mixtures of tropical wood wastes and pineapple skin waste. these substrates provide essential macro elements for crop nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 27-31 chaudhary et al. ©njb, biotechnology society of nepal 30 nepjol.info/index.php/njb production [potassium, calcium, phosphorous, magnesium, nitrogen and sodium] [20]. the formation of fruiting primordial or initials requires minimal ventilation and light penetration to stimulate the synchronized fruiting. formation of fruiting primordial is one of the most critical stage which depend upon physical factors viz. temperature, ventilation and light [23]. the transition from the vegetative hyphae to the formation of primordial and the subsequent development of fruiting bodies depends on many factors, genetic as well as environmental. environmental factors can affect the yield the yield, timing of fruition and other characteristics of the crop depending upon its genetic potential [20]. consequences between environmental factor and mushroom growth substrates have been reported to play an important role in inducing the formation of fruiting bodies [24]. conclusion hence, in vitro cultivation procedure for volvariella bombycina under laboratory conditions was optimized. the optimized protocol can be utilized for commercial scale cultivation with further processing and optimization. author’s contribution pkc, bhp and mka conceived and designed the experiments; pkc performed the experiments; pkc and ms analyzed the data; pkc wrote the paper; ms revised and proof read the manuscript. all authors have read and approved the final version of the manuscript. conflict of interests the authors declare no competing interests for publication of this paper. ethical issues as the research involved no human or animal specimens, ethical approval for conducting this research work was not necessitated. acknowledgement the authors are grateful to central department of biotechnology, tribhuvan university for supplementing the required laboratory services, materials and reagents. the authors would also like to thank prof. rajani malla, and prof. krishna das manandhar for their helpful insights. references 1. boa er: wild edible fungi: a global overview of their use and importance to people. food & agriculture org; 2004. 2. adhikari mk: mushrooms of nepal. second edition. k.s. adhikari; 2014. 3. chiu sw, moore d, chang st: basidiome polymorphism in volvariella bombycina. mycol res british mycological society. 1989 92[1]:69–77. 4. shaffer rl: volvariella in north america. mycologia. 1957 49[4]:545. 5. lakhanpal tn, kumar a, kaisth k: fleshy fungi of north-western himalaya-i. a temperate white from of volvariella bombycina. indian j mushrooms. 1986 12:1-4. 6. adhikari mk: volvariella bombycina: a mycofloral species from nepal. j plant resour. 2017, 15[1]:1–3. 7. sabir sm, hayat i, hussain i, gardezi sra: proximate analysis of mushrooms of azad kashmir. plant pathol j 2003 2[2]:97–101. 8. seok sj, kim ys, weon hy, lee kh, park km, min kh: taxonomic study on volvariella in korea. mycobiology 2002 dec 1, 30[4]:183. 9. zoberi mh. introduction. in: tropical macrofungi: some common species. london: palgrave macmillan uk, 1972 10. dickinson ch, lucas ja: the encyclopedia of mushrooms. orbis, 1979. 11. fischer d, bessette a: edible wild mushrooms of north america: a field-to-kitchen guide. university of texas press, 2010. 12. huang n, wu s: a preliminary study on the silver-silk straw mushroom, volvariella bombycina [pers. ex fr.] sing. mushroom newsl trop. 1982 3:6–9. 13. elliott t, challen m: the breeding system of the silver-silk straw mushroom, volvariella bombycina. mushroom newsl trop. 1985 6:3–8. 14. chiu sw, chen m, chang s-t: differentiating homothallic volvariella mushrooms by rflps and ap-pcr. mycol res. 1995 99[3]:333–6, doi: 10.1016/s0953-7562[09]80909-x, . 15. xu g-h, choo s-j, kim y-h, ryoo i-j, seok s-j, ahn j-s: secondary metabolites of volvariella bombycina and their inhibitory effects on melanogenesis. j microbiol biotechnol. 2010 20[1]:78–81. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 27-31 chaudhary et al. ©njb, biotechnology society of nepal 31 nepjol.info/index.php/njb 16. xu g-h, kim y-h, choo s-j, ryoo i-j, zheng cj, seok s-j: isodeoxyhelicobasidin, a novel human neutrophil elastase inhibitor from the culture broth of volvariella bombycina. j antibiot. 2009 62[6] :333–4, doi: /10.1038/ja. 2009.24 17. badalyan s: edible and medicinal higher basidiomycetes mushrooms as a source of natural antioxidants. int j med mushrooms. 2003 5[2], doi: 10.1615/interjmedicmush.v5.i2.40. 18. schillaci d, arizza v, gargano ml, venturella g: antibacterial activity of mediterranean oyster mushrooms, species of genus pleurotus [higher basidiomycetes]. int j med mushrooms. 2013 15[6]:591–4, doi:10.1615/intjmedmushr.v15.i6.70. 19. muthusamykaran, tamilkani p, senthilkumar g, vijayalakshmi s, panneerselvam a: volvariella bombycina of tamil nadu. int j inf res rev. 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volvacea. 1983, 56-63. . nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 doi: https://doi.org/10.3126/njb.v7i1.26954 ©njb, biotechnology society of nepal 74 nepjol.info/index.php/njb. original research article phenotypic characterization of beta-lactamases producing gram-negative bacteria in a tertiary hospital, nepal elina maharjan1,2, pooja shakya1, balkrishna bhattachan3, bharat prasad baral4, dhiraj shrestha5,6* 1department of microbiology, kathmandu college of science & technology (kcst), nepal. 2research center for applied science and technology (recast), tribhuvan university, nepal. 3siddhi memorial hospital, bhaktapur, nepal. 4annapurna child and women hospital, pokhara, nepal. 5department of microbiology, tri-chandra multiple campus, kathmandu, nepal. 6department of microbiology, shi-gan international college of science and technology (sicost), kathmandu, nepal. abstract infections caused by beta-lactamases producing gram-negative bacteria are increasing, thus posing a challenge to the management of such infections. the surveillance data of such bacteria is limited in nepal so this study aimed to detect the beta-lactamase producing gram-negative bacteria in a tertiary setting. a total of 604 clinical samples, including urine, blood, sputum and body fluids, were cultured and identified by the routine standard laboratory protocols. antibiotic susceptibility testing was done by kirby bauer disc diffusion method following clinical and laboratory standard institute guidelines (2014). extended-spectrum betalactamases (esbl) producers were identified by combined disk method and metallo-betalactamases (mbl) producers were identified by imipenemedta combined disk method. out of 604 samples, 282 (46.7%) samples showed significant growth, of which 229 (81.2%) were gramnegative bacteria. of 229 gram-negative bacteria, 200 (87.3%) were multidrug resistant, 67 (29.3%) were esbl producers and 16 (7.0%) were mbl producers. klebsiella pneumoniae were among higher esbl producers and pseudomonas aeruginosa were among higher mbl producers. the findings suggest higher antibacterial resistance among gram-negative bacteria with the added burden of beta-lactamase production. imipenem was effective against 125 of 229 gramnegative bacteria tested. thus, imipenem can be the drug of choice for empirical management. the higher multidrug resistance and higher beta-lactamases production among gram-negative bacteria warrant the continuous monitoring, surveillance, early detection, and infection control practices of such bacteria. keywords: antibacterial resistance, extended-spectrum beta-lactamases, esbl, gram-negative, metallo-beta-lactamase, mbl *corresponding author email: hiraj.diamond@gmail.com introduction antibacterial resistance (abr) is an increasingly serious threat to human health, challenging the effective management of infections. abr increase the health care cost as a result of prolonged illness, additional tests and pricier drugs [1]. annually, more than 750,000 deaths are caused by resistant bacteria. the median overall increased cost to treat a resistant bacterial infection is around 700 usd [2]. abr is a natural genetic change, thus newer mechanisms are emerging and spreading, raising multidrug-resistant (mdr) bacteria [1]. one of the most worrisome resistance mechanisms is the production of betalactamases, of which extended-spectrum betalactamases (esbls) and metallo-betalactamases (mbls) are the most impacting ones. esbls can hydrolyze all beta-lactam antibiotics including cephalosporins except cephamycins or carbapenems [3]. esbls are often plasmid-mediated. since it was first reported from germany in 1983, it has then spread worldwide [4]. in nepal, esbls were first reported in 2006 [5]. similarly, mbls can hydrolyze all broad-spectrum beta-lactams including cephalosporins, and carbapenems, except monobactams [6]. since its first report from japan in 1991, it has also been reported nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 maharjan et al. 2019 ©njb, biotechnology society of nepal 75 nepjol.info/index.php/njb. worldwide [7]. mbls were first reported in nepal in 2009 [8]. the acquisition, expression, and dissemination of beta-lactamase producing genes in pathogens have posed the major public health concern today [9]. limited data are available on surveilling beta-lactamase producing clinical isolates in nepal. abr surveillance data can guide the physician in choosing appropriate therapy for effective management of infectious disease without extensive testing. thus, this study aimed to produce updated data on surveilling beta-lactamase producing clinical isolates in a tertiary healthcare setting. this would help to formulate antimicrobial stewardship policy to circumvent the rising threat of abr. materials and methods study setting, design and study population a prospective hospital-based study was conducted in the department of microbiology at annapurna neurological institute and applied science, kathmandu, nepal from march to november 2014. a total of 604 clinical samples from patients of all ages and both sexes, visiting the hospital and requesting a routine investigation, was included in the study. the samples include 263 urines (clean catch urine and catheter tip), 140 sputum samples, 73 blood samples and 128 body fluids (pus, pus swab, csf, bile fluid, pleural fluid, peritoneal fluid, synovial fluid, and tracheal secretion). contaminated samples and repeated samples from the same patient were excluded to avoid selection bias. laboratory processing of the samples the samples were subjected to the standard microbiological procedures for isolation and identification of bacteria. in short, the samples were inoculated onto macconkey agar (himedia, india) and blood agar (himedia, india) plate by streaking, followed by incubation at 37℃. growths of bacteria were observed after 18-48hrs. the identification of gram-negative bacteria was done by gram’s stain morphology, cultural characteristics, and conventional biochemical tests. biochemical tests employed were catalase test, oxidase test, indole test, citrate utilization test, methyl red test, voges-proskauer test, christensen’s urease test, triple sugar iron test, decarboxylase test, and phenylalanine deaminase test. antimicrobial susceptibility testing the antimicrobial susceptibility test of all identified bacteria against antibiotics of various classes was done in vitro by kirby-bauer disc diffusion method [10]. a zone of inhibitions (zoi) for each disc was measured and the results were interpreted as per clsi guideline m100-s24 [11]. control strains escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used for the quality control of the test. detection of extended-spectrum betalactamase producing strain esbl producing isolates were identified by a phenotypic method as described in clsi guideline m100-s24 [11]. screening test for esbl: isolates exhibiting resistance against ceftriaxone (30µg) (zoi ≤25mm) and/or ceftazidime (30µg) (zoi ≤22mm) and/or cefpodoxime (10µg) (zoi ≤17mm) and/or cefotaxime (30µg) (zoi ≤27mm) were screened for esbl production. combined disc (cd) method as a confirmatory test for esbl: zois of isolates against ceftazidime disc (30µg) and cefotaxime (30µg) were compared against zois of isolates against ceftazidime disc (30µg) containing clavulanic acid (10µg) and cefotaxime (30µg) containing clavulanic acid (10µg) when placed 25mm apart (center to center). the isolates showing the difference of 5mm or more between either of the two zois of the disc and clavulanate added disc was confirmed positive for esbl production. klebsiella pneumoniae atcc 700603 (esbl positive) was used as control strains. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 maharjan et al. 2019 ©njb, biotechnology society of nepal 76 nepjol.info/index.php/njb. table 1. sample and significant growth pattern total samples culture positive gram-negative bacteria gram-positive bacteria gender (m: f ratio=1.45:1) male 357 (59.1%) 174 (61.7%) 143 (62.4%) 31 (58.5%) female 247 (40.9%) 108 (38.3%) 86 (37.6%) 22 (41.5%) departments of patients inpatients 471 (78%) 180 (63.8%) 157 (68.6%) 23 (76.7%) outpatients 133 (22%) 102 (36.2%) 72 (31.4%) 30 (56.6%) age groups (years): infants and children, adolescents, adults, elders ≤9 19 (3.2%) 3 (1.1%) 2 (0.9%) 1 (1.9%) 10-19 56 (9.3%) 29 (10.3%) 26 (11.4%) 3 (5.7%) 20-59 313 (51.8%) 129 (45.7%) 99 (43.2%) 30 (56.6%) ≥60 216 (35.8%) 121 (42.9%) 102 (44.5%) 19 (35.8%) total 604 (100%) 282 (100%) 229 (100%) 53 (100%) detection of metallo-beta-lactamase producing strain since during the study no standard protocol was available for the detection of mbl, mbl producing isolates were identified by the commonly used phenotypic method. screening test for mbl: isolates exhibiting resistance against ceftazidime (30µg) (zoi<18mm) were screened for mbl production. the resistance against imipenem (10μg) and/or meropenem (10μg) was not used as a screening tool so as to avoid missing the detection of hidden mbl in bacteria. bacterial suspension equivalent to 1:10 dilution of 0.5 mcfarland was used for lawn culture in mueller-hinton agar before incorporating antibiotic discs [12]. combined disc (cd) method as a confirmatory test for mbl: zoi of isolate against imipenem disc (10 µg) was compared against zoi of isolates against imipenem disc (10µg) containing 292µg (10µl of 0.1m) ethylenediamine-tetraacetic acid (edta) when placed 25mm apart (center to center). the isolates showing difference of 4mm or more between two zois was confirmed positive for mbl production [12, 13]. p. aeruginosa atcc 27853 (mbl negative) and p. aeruginosa pa 105663 (mbl positive) was used as control strains. results distribution of samples and isolates of 604 samples, only 282 (46.7%) were culture positive. higher number of samples from males were culture positive 174 (61.7%). similarly, a higher number of samples from the inpatient department were culture positive, 180 (63.8%). culture positivity increased with the age of patients. among 282 culture positive, 229 (81.2%) were gram-negative bacteria (table 1). eight different species of gram-negative bacteria were identified. among these, e. coli were the predominant, 78 (34.1%). e. coli was also predominant in urine samples, 61(55.0%) (table 2). nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 maharjan et al. 2019 ©njb, biotechnology society of nepal 77 nepjol.info/index.php/njb. antibiotic susceptibility of gramnegative bacteria of 229 gram-negative bacteria, e. coli showed higher resistance against ampicillin, cefpodoxime, ceftriaxone, and cefixime, while imipenem and chloramphenicol were found effective. p. aeruginosa showed higher resistance against cefixime and cefpodoxime, while imipenem was found effective. similarly, other gram-negative bacteria showed higher resistance against ampicillin, cefpodoxime, and ceftriaxone, while imipenem was found fairly effective (table 3). distribution of beta-lactamase producers of 229 gram-negative bacteria, 200 (87.3%) were found mdr. of these bacteria, 67 (29.3%) were esbl producers and 16 (7.0%) were mbl producers. esbl production was higher among sputum isolates i.e. 35 (52.2%) and mbl production was higher among urine isolates i.e. 8 (50%) (table 4).distribution of betalactamase production among gram-negative bacteria of 229 gram-negative bacteria, mdr was found higher among klebsiella spp. isolates. esbl production was higher among k. pneumoniae 25 (37.3%), e. coli 16 (23.9%) and p. aeruginosa 16 (23.9%). similarly, mbl production was higher among p. aeruginosa 9 (56.3%) and e. coli 3 (18.8%) (table 5). discussion rise of beta-lactamases among pathogens has now been a prime threat to global health. betalactamase production has been reported in p. aeruginosa, acinetobacter spp. and enterobacteriaceae [14]. the evidence has shown that abr has a significant adverse impact on clinical outcomes and increases costs due to the consumption of health care resources [15]. of 229 gram-negative bacteria, 29.3% were found esbl producers. similar results were reported by previous studies as 25% in 2013 [16], 25.8% in 2014 [17], 24% in 2015 [18], 26.9% in 2015 [19] and 34.5% in 2017 [20]. esbl producers are increasingly disseminating in nepal, 0.6% in 2006 to 40% in 2017 [5, 16-22]. esbl production was higher among k. pneumoniae (37.3%), e. coli (23.9%) and p. aeruginosa (23.9%). table 2: distribution of gram-negative bacteria in different clinical samples bacteria sample total urine blood sputum body fluid acb* 2 (1.8%) 0 (0%) 4 (5.5%) 2 (4.9%) 8 (3.5%) citrobacter spp. 1 (0.9%) 0 (0%) 0 (0.0%) 0 (0.0%) 1 (0.4%) e. coli 61 (55.0%) 0 (0%) 9 (12.3%) 8 (19.5%) 78 (34.1%) k. pneumoniae 16 (14.4%) 0 (0%) 33 (45.2%) 22 (53.7%) 71 (31.0%) k. oxytoca 15 (13.5%) 0 (0%) 6 (8.2%) 1 (2.4%) 22 (9.6%) p. aeruginosa 12 (10.8%) 2 (50%) 20 (27.4%) 7 (17.1%) 41 (17.9%) proteus spp. 4 (3.6%) 0 (0%) 1 (1.4%) 1 (2.4%) 6 (2.6%) salmonella spp. 0 (0.0%) 2 (50%) 0 (0.0%) 0 (0.0%) 2 (0.9%) total 111 (100%) 4 (100%) 73 (100%) 41 (100%) 229(100%) *acinetobacter calcoaceticus-baumannii complex nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 maharjan et al. 2019 ©njb, biotechnology society of nepal 78 nepjol.info/index.php/njb. similar results were reported in e. coli as 25.8% by pokhrel et al in 2014 [17], 22.4% by raut et al in 2015 [23], 26.9% yadav et al in 2015 [19]. similarly, comparable results were reported in k. pneumoniae as 22.4% by raut et al in 2015 [23] and 43.3% nepal et al in 2017 [22]. esbl production was higher among sputum isolates i.e. 35 (52.2%). of 229 gram-negative bacteria, 7% were found mbl producers. similar results were reported by previous studies as 7.1% khanal et al in 2013 [16], 3.2% pokhrel et al in 2014 [17] and 4% nepal et al in 2017 [20]. but a lower rate of 1.3% was reported by mishra et al in 2012 [12] and a higher rate of 15% was reported ansari et al in 2015 [18]. mbl producers are increasingly disseminating in nepal [12, 16-18, 20]. mbl production was higher among p. aeruginosa isolates, 56.3%. besides p. aeruginosa and acinetobacter spp., mbl production was also found in e. coli and k. pneumoniae as well. mbl in enterobacteriaceae was reported by similar studies reported [16-18, 20]. however, this contrast with the result of similar previous studies [12] which reported mbl production only in p. aeruginosa and acinetobacter spp. mbl production was higher among urine isolates i.e. 50% of total mbl producers. genotypic methods are considered superior tools for surveillance of such esbl and mbl producers. but the evolution of newer betatable 3: antibiotic susceptibility of gram-negative bacteria antibiotics e. coli (n=78) (%) p. aeruginosa (n=41) (%) other gnb (n=110) (%) s i r s i r s i r amikacin 30.8 14.1 55.1 58.5 17.1 24.4 39.1 11.8 49.1 ampicillin 5.1 0.0 94.9 12.2 0.0 87.8 4.5 0.9 94.5 aztreonam 10.3 15.4 74.4 34.1 19.5 46.3 20.0 10.0 70.0 ceftriaxone 5.1 2.6 92.3 19.5 0.0 80.5 5.5 0.9 93.6 cefixime 2.6 5.1 92.3 2.4 0.0 97.6 8.2 0.0 91.8 cefotaxime 3.8 7.7 88.5 17.1 2.4 80.5 10.0 0.9 89.1 cefpodoxime 5.1 0.0 94.9 12.2 0.0 87.8 5.5 0.0 94.5 ceftazidime 9.0 2.6 88.5 17.1 4.9 78.0 10.0 0.9 89.1 ciprofloxacin 14.1 1.3 84.6 19.5 2.4 78.0 11.8 0.0 88.2 chloramphenicol 38.5 9.0 52.6 58.5 17.1 24.4 30.9 18.2 50.9 co-trimoxazole 11.5 2.6 85.9 14.6 0.0 85.4 11.8 2.7 85.5 gentamicin 21.8 5.1 73.1 41.5 4.9 53.7 20.9 2.7 76.4 imipenem 39.7 15.4 44.9 78.0 0.0 22.0 56.4 4.5 39.1 nitrofurantoin 18.0 4.9 77.1 nt nt nt nt nt nt norfloxacin 12.8 1.3 85.9 9.8 4.9 85.4 13.6 0.0 86.4 piperacillin/ tazobactam nt nt nt 63.4 9.8 26.8 nt nt nt *gnb=gram-negative bacteria, s=sensitive, i=intermediate, r=resistance, nt=not tested nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 maharjan et al. 2019 ©njb, biotechnology society of nepal 79 nepjol.info/index.php/njb. lactamase genes would make use of such tools tough, owing to the lack of primers. moreover, using such expensive tools in resource lacking settings, like nepal, would be a fairy tale scenario. a combined disc phenotypic method using imipenem and edta for mbl, ceftazidime/clavulanate or cefotaxime /clavulanate for esbl offers a cheaper alternative. also, phenotypic method proves not only the presence of beta-lactamases producing genes but also their expression. this method has been reported to have a sensitivity of 100% and specificity of 98%, thus reliable [12, 24]. of 229 gram-negative bacteria, e. coli showed higher resistance against ampicillin, cefpodoxime, ceftriaxone, and cefixime while imipenem and chloramphenicol were found effective. p. aeruginosa showed higher resistance against cefixime and cefpodoxime while imipenem was found effective. similarly, other gram-negative bacteria showed higher resistance against ampicillin, cefpodoxime, and ceftriaxone while imipenem was found fairly effective. imipenem was found as an effective drug against most of the tested gram-negative isolates. this concords with similar reports which reported imipenem as the most sensitive drug [25, 26]. as per the expert consensus, isolates resistant to two or more classes of antibiotics are considered mdr strains [27]. of 229 gram-negative bacteria, 87.3% of the isolates were found mdr strains. mdr was found higher among klebsiella spp. isolates (93.5%). similarly, 87.8% of p. aeruginosa and 50% of acinetobacter spp. isolates were found mdr strains. some studies reported higher mdr in these bacteria [16, 18, 20]. in our study, nearly all esbl and mbl producers were mdr strains. this limits physicians with therapeutics for the management of infections. such esbl producing strains can be inhibited by the use of beta-lacatamase inhibitors like clavulanate, but mbl producers are resistance to these inhibitors and mbl inhibitors are yet to be trialed in human. the reserve drug for mdr gramnegative bacteria is emptied with the evolution of newer beta-lactamases. colistin has always been the ultimate weapon in the fight against mdr superbugs in case all other therapeutic options fail. however, resistance against colistin has been reported in recent times, rendering our drug arsenal completely empty for such superbugs [28-30]. abr is a crisis that must be managed with the utmost urgency to contain it. such surveillance that generates updated data is required for the implementation of sound strategies and public health actions to contain abr. abr requires concerted cross-sectional action by governments table 5. distribution of gram-negative isolates producing esbl and mbl bacteria total isolates mdr bacteria esbl producers mbl producers acb* 8 4 4 (6.0%) 2 (12.5%) citrobacter spp. 1 1 0 (0.0%) 0 (0.0%) e. coli 78 65 16 (23.9%) 3 (18.8%) k. oxytoca 22 21 5 (7.5%) 0 (0.0%) k. pneumoniae 71 66 25 (37.3%) 2 (12.5%) p. aeruginosa 41 36 16 (23.9%) 9 (56.3%) proteus spp. 6 6 0 (0.0%) 0 (0.0%) salmonella spp. 2 1 1 (1.5%) 0 (0.0%) total 229 200 (100%) 67 (100%) 16 (100%) *acinetobacter calcoaceticus-baumannii complex nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 74-81 maharjan et al. 2019 ©njb, biotechnology society of nepal 80 nepjol.info/index.php/njb. and society as a whole. currently, ‘the global action plan on antimicrobial resistance-2015’, as formulated by who [31], must be implemented as envisioned to tackle the abr. conclusion the findings suggest higher mdr, esbl, and mbl not only among p. aeruginosa and acinetobacter spp. but also among enterobacteriaceae family, including e. coli and k. pneumoniae. imipenem showed promising sensitivity against most of the gram-negative isolates, thus it can be the antibiotic of choice for management of such infections. evolving betalactamases against newer generation betalactams have posed a serious threat to public health. only the continuous monitoring, surveillance, early detection, and infection control practices can ensure the effective management of such resistant strains. conflict of interest the authors declare no conflict of interest. acknowledgments we acknowledge asst. prof. archana katuwal and dr. ranga bahadur basnet for supervising this study and providing the methodology for the study. consent to publish not applicable ethical approval and consent to the participant no patient-related data were collected and thus ethical approval was not required. the study was a laboratory-based study and a part of the study was a routine patient care investigation. oral informed consent was taken from all patients or from a guardian on behalf of the patients. availability of data and materials all data generated or analyzed during this study are included in the article. raw data can be made available upon request to the corresponding author. funding none authors’ contributions all authors made substantial contributions to the study. em, ds and ps conceived and designed the study. em and ps collected sample, investigated and recorded the laboratory findings at the hospital. ds, bb and bpb curated the data and analyzed the data. em and ds administered the project, reviewed literature, and drafted the manuscript. ds helped with critical review and revision of the manuscript by compiling, formatting, editing and writing the final version of the manuscript. all authors read and approved the final manuscript. references 1. antimicrobial resistance [https://www. who.int/en/news-room/fact-sheets/detail /antimicrobial-resistance ] 2. the global threat of antibiotic resistance [https://www.reactgroup.org/antibioticresistance/the-threat/] 3. philippon a, labia r, jacoby g: extendedspectrum beta-lactamases. antimicrob agents chemother.1989 33(8):1131-1136. 4. knothe h, shah p, krcmery v, antal m, mitsuhashi s: transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical 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7, no. 1 : 63-73 doi: https://doi.org/10.3126/njb.v7i1.26952 ©njb, biotechnology society of nepal 63 nepjol.info/index.php/njb. original research article pharmacological in vivo test to evaluate the antidepressant activity of polyherbal formulation rinki kumari1, k. ilango2, g.p.i. singh3, g.p. dubey1* 1institute of medical sciences, banaras hindu university, varanasi221 005 2interdisciplinary school of indian system of medicine, srm university, katankulathur, chennai 3adesh university, barnala road, bathinda, punjab abstract the antidepressant effects of the polyherbal formulation (pf) (contain four extracts of medicinal plants namely: nyctanthes arbortristis, hippophae salcifolia, ocimum tenuiflorum and withania somnifera ) was examined by evaluating the extent of reduction of behavioural alterations and neurotransmitter in the rats stressed by forced swim test (fst). in the present study, compared with the model control group (fst), the altered behavioural parameters were attenuated significantly (p < 0.05) in the group treated with the pf (100,200 and 400 mg kg−1), comparable with the standard drug treated group, sertraline (10mg kg−1). the pf and sertraline significantly (p < 0.05) increased the level of the neurotransmitter such as serotonin, dopamine, acetylcholine and noradrenalin whereas decreased the level of monoamine oxidase along with oxidant in the brain of the stressed rats. pf and sertraline were also involved in the reduced oxidant and generated antioxidant in the stressed rats. the results indicated that polyherbal formulation exhibited significant antidepressant activity, as indicated by its ability to decrease force swim stress, induced immobility time in rats as well as restoring the biogenic amines to normal level that were altered by the swim induced stress in whole rat brain. therefore, pf can be a potential candidate for treatment of depression as well as a potent antidepressant. however, further studies are required to substantiate the same. keywords: depression, antidepressant, nyctanthes arbortristis, hippophae salcifolia, ocimum tenuiflorum, withania somnifera, forced swim test. *corresponding author e-mail: gpdubey13@gmail.com introduction depression is an etiologically heterogeneous group of chronic psychiatric illness with a high prevalence rate (21%) and the second leading cause of the loss of human disability-adjusted life year [1-4]. the prevalence rate of depression is 6-8 % in female and 3-5% in male [5]. clinically characterized by a wide range of symptoms that reflect alternation in cognitive, psychomotor, biological, motivational, behavioral, emotional process and refers to either negative effect or absence of positive effects. the studies have suggested, depression also affects the quality of life of many people and has become a major cause of suicidal death [6-9]. although, treatment of depression entirely depends on clinically available synthetic antidepressants (that based on serendipitous, act via monoamine neurotransmitters, such as serotonin and noradrenaline). only certain portion of the patients show full remission in response to these antidepressants and other hand, associated with more side-effects and the chronic toxicity that affect almost every organ system. these available drugs have potential for adverse effects on cognition and behavior [1011]. to obtain better therapeutic benefits and minor adverse reactions, there is a pressing need for alternative antidepressant from natural source i.e., herbal remedies, used traditionally, now documented with safe profile [12-13]. recently, it has been reported the use of polyherbal formulation exhibiting synergistic activity achieving maximum beneficial potency as compared to single herb [14-15]. the use of pf finds the way of curing depression. ayurveda declared, more than a nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 64 nepjol.info/index.php/njb. few plants of this formulation, the so-called “medhya” plants, possess such antioxidant, antidepressant and anxiolytic properties. nyctanthe sarbor-tristis, (n. arbor-tristis,) (family oleaceae) is an herb that has been used in india for centuries as an anti-inflammatory [16], analgesic, antipyretic, ulcerogenic activities [1720], immunostimulant activity and anxiolytic activity. hippophae salcifolia (h. salcifolia) (seabuckthorn) are deciduous shrubs in the family elaeagnaceae that traditionally used as anti-stress and improve, enhance physical endurance, mental function alone but there are limited published data in related to centrals nervous system disorders [19-21]. it has also shown anti-oxidant, immuno-modulatory, antiinflammatory and homocysteine lowering effects because it is a rich source of flavonoids, vitamins, proteins, amino acids, folic acid, phytosterol, alpha-tocopherol and phenolic compounds [21]. ocimum tenuiflorum (o.tenuiflorum) (family lamiaceae), has wild growth and available throughout the eastern world tropics and contains eugenol, oleanolic acid, ursolic acid, rosmarinic acid, etc. it has a vast number of therapeutic applications such as cardiopathy, haemopathy, leucoderma, asthma, bronchitis, catarrhal fever, otalgia, hepatopathy, vomiting, lumbago, hiccups, ophthalmia, gastropathy, genitourinary disorders, ringworm, verminosis and skin diseases etc due to presence of abundance of naturally occurring polyphenols in ayurveda, greek, roman, siddha and unani. these polyphenols possess potential effects of neuroprotection. due to high concentration of eugenol act as cox-2 inhibitor and analgesic effect [22-28]. withania somnifera (w. somnifera) is known as ashwagandha, an ayurvedic formulation mentioned in traditional medicine system, used as a mild sedative, aphrodisiac rejuvenative and possess a very good effect in all psychological disorders. the roots of w. somnifera consist very active compounds known as withanolides, is a strong potent psycho neuro pharmacologically active molecules and is subjected for antidepressant activity. various studies have shown it is effective in the treatment of chronic fatigue, nervous exhaustion, and memory loss and neurodegenerative disorders and there are limited published data [29-30]. even though pf has antidepressant potential, there is no sufficient evidence for its effects in animal models of depression. according to ayurveda“sarangdhar samhita” dated centuries ago in 1300 ad has highlighted the concept of polyherbalism in this ancient medicinal system to achieve additional therapeutic effectiveness [31]. recent studies have shown that different herbal active bio molecules of pf, possess potential effects of neuroprotection [32-35]. so far, no study has appeared about the protective effects of this polyherbal formulation on depression induced by forced swim stress. considering substantial neuroprotective properties of pf, we sought to investigate whether pf could improve level of neurotransmitter induced by forced swim stress in rats; and to explore the underlying mechanisms for the same. materials and methods animals albino wistar rats (with 2 months of age and both genders), weighing 150 to 200g were taken from the central animal house of adesh university, bathinda, punjab. all animals were kept in polypropylene cages with paddy husk as bedding. the rats were housed in groups of 6 animals per cage and acclimatized to a colony room with a controlled ambient temperature 23 ± 2ºc, relative humidity of 50–70% and a 12hour natural light/dark cycle. the animals had free access to pelleted feed of standard composition containing all macro and micro nutrients and purified water ad libitum and were acclimated for 7 days prior to the consequent studies. the animals were examined at regular intervals by a trained personal for any behavioural abnormalities and behavioural studies were carried out during the light phase (10.00 am–1.00 pm). the animals were used only once for each experiment. the experiments were performed after approval of the protocol by the ethics committee of the adesh university, bathinda punjab with reference number; nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 65 nepjol.info/index.php/njb. aimsr/mc/estt/07/2009/718 & dated 16-072009. collection of polyherbal hydroalcoholic extract the hydro-alcoholic extracts of four plantsnyctanthe sarbortristis (75mg), occimum tenniflorum (50mg), hippophe salcifolia (40mg) and withania somnifera (35mg) in the combination as a polyherbal formulation (pf). the source of hydro-alcoholic extract of four plants from bajinath pharmaceutical pvt. ltd., paprola himachal pradesh. pf was freshly suspended in distilled water and administered per oral (p.o.) in a constant volume of 10 ml/kg. the dose of extracts per rat was calculated with the values obtained in gravimetric assay and estimated at 2000g/kg/day. different doses of test drug (100,200,400 mg/kg/day) were administered orally by using oral gavage in rats, 40-45 min before the behavioural tests. sertraline hcl was used as the reference standard drug for evaluating antidepressant activity. sertraline hcl suspension was prepared using saline. drug and analytical chemical sertraline hydrochloride, (1s,4s)-4-(3,4dichlorophenyl)-n-methyl-1,2,3,4-tetrahydronaphthalen-1-amine selective reuptake inhibitor antidepressant, was gifted from sun pharmaceutical company baddi, himachal pradesh and used as positive control for antidepressant action. all other reagents and solvents were of analytical grade. polyherbal formulation was dissolved in 0.5% carboxyl methyl cellulose (cmc) with 1% tween 80 (solubility enhancer). the dissolution of the extract wasfreshly done from the powder immediately before its administration. a control group received equal volume of distilled water. experimental design there were six groups in this present study: group i: 0.5% carboxyl methyl cellulose (cmc) plus unstressed, group ii: water plus stressed, groups iii: sertraline hydrochloride (10 mg·kg−1, i.p.) plus stressed and the at 30 min before exposure to stress), group vi and iv: pf (polyherbal formulation) plus stressed (100, 200 and 400 mg·kg−1 p.o., respectively) (treatment at 60 min before exposure to stress). pf and sertraline hydrochloride were each given orally by gavage, 60min before each stressor once every day for 7th, 14th and 28 days. measurement of immobility period by forced swim test (fst) the forced swim test was performed according to the method described by porsolt [36] with slight modifications [37].this test consisted of two parts, an initial training period of 15 min and an actual test for 5 min after 24 h. the rats were individually forced to swim inside vertical plastic jar (26 cm × 12 cm × 26 cm) containing water to a height of 15 cm at 25 ± 2 °c for a 5 min session every day for 7 days. when they were placed in the glass jar for the first time the rats were initially highly active, vigorously swimming in circles, and trying to climb the wall or diving to the bottom. after 3–4 min, their activity began to fall down and immobility increased. after 5–6 min, immobility reached a raised ground where the rat remained immobile more than 70% of the time. after 15 min in the water, the rats were removed, wiped with dry cloth and allowed to dry before being returned to their cages. the plastic jar was emptied and washed thoroughly after testing for each rat. the rats were again placed in the jar 24 h later after initial administration of the drug and extracts and their activity was recorded within 5 min. the duration of immobility was measured using a stop watch. an animal was judged to be immobile whenever it remained floating passively in the water in a slightly hooked but vertical position and sometime horizontal position, with no additional activity other than that necessary to keep its head above water [3637]. behavioral assessment numerous other behavioral parameters were assessed in the rats on 7th, 14th and 28th days after forced swim stress test. all the animals were subjected for other behavioral analysis such as anxiety and memory. the mirror chamber test was used as a measure of anxiety [38] while for cognitive behavior the elevated nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 66 nepjol.info/index.php/njb. plus maze test was used, developed by kulkarni [39]. biochemical assay biochemical tests for neurotransmitter and other biomarker in all rats on day 7th, 14th and 28th, after mentioned behavioral analyses, the rats were immediately sacrificed by cervical dislocation. the brains were immediately removed and 10 % (w/v) tissue homogenate was prepared in 0.1 mol·l−1 phosphate buffer (ph 7.4). the homogenates were centrifuged for 15-20 min at 10000 g and the supernatants were used for analyses of serotonin, melatonin, monoamine oxidase, noradrenalin, acetylcholine, lipid peroxidation and glutathione levels. the serotonin and melatonin were estimated according to the methods described by snyder et al [40] and ozaki et al [41] respectively in the brain tissue. the level of monoamine oxidase in the rat brain tissue was measured by using the procedure of xu et al. [42] with a minor modification and noradrenalin and acetylcholine, estimated by high performance liquid chromatography with fluorescence detection (hpltc) method [28].lipid peroxidation was assayed byof the presence malondialdehyde, a biomarker in form of thiobarbituric acid-reactive substances by the method of wills et al [43] whereas glutathione in brain was analyzed according to the method described by ellman et al [44]. data analysis behavior assay was analyzed statistically using graph pad prism a one-way analysis of variance (anova), followed by turkey’s test. the results were expressed as the mean ± standard error of the mean (sem.). differences with p values < 0.05 were considered statistically significant. for biochemical assays, one-way anova was applied on the percentage values of each group calculated with respect to positive and negative control group. the values were expressed as mean ± sem. results body weights on 7th, 14th, and 28th days at the beginning of the experiment, there were no significant differences in body weight among the six groups (p> 0.05), with the values (mean ± sd) being 104± 2.45, 101.71 ± 6.2, 103.52 ± 1.15, 105.59 ± 12.45, 107.98 ± 15.6, 104.9±7.78 and for groups 1–6 respectively. the body weights at the 7th, 14th and 28th days of the treatments were also not significantly different among the groups (p > 0.05); the values of 7th days were 106± 3.2, 105.51 ± 7.45, 104.92 ± 2.15, 108.47 ± 13.1, 109.91 ±6.12,105.9±5.78 and 14 days were 133.3 ±15.57, 129.35 ±28.62, 123.95 ± 15.43, 131.09 ±28.78, 135.64 ±24.16 and 125 ±31.22 whereas on the 28days the value of body weight were 135.80 ±12.6, 131.33 ±17.98, 133.19 ±12.2, 135.9 ±13.04, 139.19 ±44, and 133.33± 5.2, 1–6 respectively. therefore, the results suggested that polyherbal formulation or sertraline didn’t show any toxicity effect in treatment group. effects of polyherbal formulation (pf) on the immobility time in the forced swim (fst) test and its anti-anxiety after treatment with the pf for 7,14 and 28 consecutive days, there was a significant reduction in immobility time in the forced swim test, in a dose-dependent manner, compared with the experimental control (stressed) rats (p < 0.05, figure 1). the reduction in immobility after the treatment with the pf demonstrated its antidepressant-like potentials, since decreased swimming performance would increase the immobility time in the forced swim test. sertraline was used as the standard drug for antidepressant activity on the forced swim test. as presented in figure 1, sertraline caused a significant reduction in immobility time. effect of test drug on biochemical parameters. pf were also exerted anti-anxiety like effect because it decreased time latency to enter in mirror chamber and also increased number of entries in stressed rat. figure 2 a to c showed the standard drug, sertraline, was more effective than the pf (p < 0.05, figure. 2 a, b and c). nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 67 nepjol.info/index.php/njb. figure. 1: effect of pf on the immobility time in the stressed rats. sertraline was used as a positive control. figure.2: effect of the pf on anxiety (a) latency of entries (s) (b) time spent and (c) number of entries effect of polyherbal formulation (pf) treatment on biochemical parameter: brain serotonin levels serotonin levels were estimated in the whole brain content of rats. fst model exhibited significantly lower value of serotonin levels, expressed as percentage values with respect to the control group. more than a 40% decrease in serotonin levels was observed due to depression induced model group. pf treatments showed significantly increased brain serotonin levels in model group (figure 3). also, sertraline was increased the level of serotoninin the brain of stressed rats as compared to model group. figure. 3: effect of the pf on level of serotonin in stressed rat’s brain mao enzyme activity fst model rats resulted in a significant increase in total mao enzymatic activity in the brain as compared to the sertraline and test drug treatment group. the effects of pf and sertraline on the mao activities in the rat brain are shown in fig 4. test drug at doses of 100, 200 and 400 mg/kg suppressed the brain monoamine oxidase activities. the data represented 25. 2% increases in total mao activities in the whole brain content of fst rats with respect to sertraline treated rats respectively. pf treatment significantly reduced the level of total mao enzyme activities in the brain within 28 days at 400mg/kg. similar results were observed for the sertraline treatment. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 68 nepjol.info/index.php/njb. figure. 4: effect of the pf on level of mao in stressed rat’s brain noradrenalin figure. 5: effect of the pf on level of noradernalin in stressed rat’s brain noradrenalin levels were estimated in the whole brain content of rats. model group exhibited significantly lower value of noradrenalin levels compared to the control group. pf treatments showed significantly increased brain noradrenalin levels in treated group (figure 6). figure. 6: effect of the pf on level of acetylcholine in stressed rat’s brain also, sertraline showed recovered brain noradrenalin levels increase as compared to model group. at the dose, 400mg/kg significantly increased the level of noradrenalin in the brain of depressive rats and this concentration is effective doses for recovery of noradrenalin in the brain. acetylcholine pf gradually increased the level of acetylcholine in the brain of depressive rats while 200mg/kg concentration significantly increased level of acetylcholine. these level are lower as compared to standard drug figure 6. lipid peroxidation and glutathione pf gradually increased the level of malondialdehyde in the brain of depressive rats while these level are lower as compared to standard drug figure 7. simultaneously; we observed the glutathione level in brain of stressed rats decreases. figure. 7: effect of the pf on level of reduced glutathione in stressed rat’s brain figure. 8: effect of the pf on level of lipid peroxidation biomarker in stressed rat’s brain discussion after screening of plants having role on central nervous system four plants namely nyctanthes nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 69 nepjol.info/index.php/njb. arbortristis (parijata), hippophae salicifolia (amlavetas), occimum tennuiflorum (van tulsi) and withania somnifera were selected on the basis of their biological activity. hydro-alcohol extract of four medicinal plants(nyctanthe sarbortristis; hippophae salcifolia ;ocimum tenuiflorum ; withania somnifera) has been reported to possess potent different activityanti-inflammatory,anti-oxidant,cox-2 inhibitor and analgesic effect and chronic fatigue, nervous exhaustion, memory loss, neurodegenerative disorders etc, respectively due to presence of major effective constituents such as nyctanthic, lupeol, ascorbic acid, flavonoids, folic acid, oleanolic acid, ursolic acid, rosmarinic eugenol and 35 withanolides with 12 alkaloids and several sitoindosides [1630]. therefore, all the four plants have shown their action on various targets involved with depression like monoamine content, proinflammatory markers, behavioral pattern etc and the selected four plants were taken and after determination of bio-activity the plants were taken for standardization and quality control studies and to develop a polyherbal formulation beneficial in the management of depression of varying etiology. we hypothesized pf could be effective in the management of diseases caused by imbalance of monoamines or neurotransmitter, inflammatory, injury fatigue and oxidative stress. to test the hypothesis, we evaluated the effects of the nyctanthic, oleanolic, eugenol, withanolides, flavonoids and folic acid rich hydro-alcoholic extract on behavioral alterations in stressed rats. the present study can be summarized by following finding: 1. the force swim stress procedure caused depressive-like behavior in treatment rats, as observed by different assays such as mirror chamber test and elevated plus maze test; 2. significant reduction in depressivelike behaviors was evident in the stressed rats treated with pf and sertraline (positive control); 3. forced swim test procedures imbalance the neurotransmitter and induced inflammatory injury along with oxidative damage by increasing lipid peroxidation in the treated rats and 4. pf and sertraline treatments exerted protective effects against forced swim stress and increase the level of neurotransmitter and decrease the glutathione in the rat brain. numerous studies have been indicated that stressful life events and chronic stresses are risk factors for several neurodegenerative diseases including depression because it is capable to altering physiological homeostasis of body and imbalance the neurotransmitter in brain [45-47]. depression is an incapacitating psychiatric ailment which is characterized by a pervasive low mood, loss of interest in usual activities, diminished ability to experience pleasure (anhedonia), withdrawal of interest, feelings of worthlessness, and suicidal tendencies [1-9]. most commonly, fst is employed behavioral model of depression and used to evaluate the antidepressants drug [48]. the poly herbal formulation as found to be safe in as no mortality was observed following treatment. the immobility exhibited by test animals in these models is an indicative of a behavioral despairness which reflects a state depressive state. group iv to vi showed a significant decrease in their immobility time showed a moderately significant decrease in immobility time compared to control and model group. sertraline showed extremely significant reduction in the immobility time. on the other hand, anxiety is a part of which is psychological and behavioral state induced in animals and humans by stress-like conditions and characterized by fear and annoyance. serotonin level is decreases in the anxiety condition [38]. in our present study, anxiety evaluated by mirror chamber test [39] and we found the pf and sertraline both significantly increase the time spent, number of entries and decreases latency entries (figure 2)and increase the serotonin in the stressed rats. the elevated plus maze test is used to evaluate cognitive behavior and memory performance [49]. according to the report [49,50], the rodents are like to live in enclosed and dark area. our data of this behavior assays showed that pf, changes in both open arm entries and time spent in open arms significantly (p < 0.05) and sertraline is also increased the open arm entries and time spent in open arm (p < 0.05) whereas model group showed significant reduction in nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 70 nepjol.info/index.php/njb. open arm entries and the time spent in open arm (p < 0.05). therefore, this study showed that pf and sertraline, both promote glucocorticoid production and release in the adrenal cortex [50]. as we know sertraline acts by the selective serotonin reuptake inhibitor (ssri) pathway and has been used as a standard drug in majority studies. the beneficial effect of sertraline in the fst model seems to be due to increased availability of these neurotransmitters nor epinephrine (ne), acetylcholine, melatonin and serotonin (5ht) at the post synaptic site following reuptake inhibition due presence of potential phytocontituents and so forth produces anti-depressive actions. on the other hand, depression is a multifaceted mental illness resulting from alterations in central serotonergic, melatonin, noradrenergic, and system along with mao activity. the monoaminergic hypothesis of depression postulates that the major neurochemicals process in depression, is the impairment of monoaminergic neurotransmission and the concomitant decrease in extracellular concentration of noradrenaline and serotonin [36,44,51]. most of the prescribed antidepressants inhibit serotonin or noradrenaline reuptake and mono amineoxidase inhibitors act by increasing the synaptic availability of these neurotransmitters [36,44]. the dopaminergic system is also an important target implicated in the regulation of mood disorders, as preclinical and clinical studies have indicated a diminished dopamine turnover in depression [36]. as a result, depressive disorders are commonly due to imbalance in neurotransmission and it bring this imbalance back to balance consists in inhibiting the uptake of neurotransmitters into the neurons, thus neurotransmitters levels increasing in the brain and shown to improve the clinical symptoms of depression. among them are chemical entities like tricyclic antidepressants or herbal extracts as demonstrated here and by many others [34-38]. consequently, it was thought to be worthwhile to estimate all the five neurotransmitters in brain of rats depressed by forced swimming and tail suspension test. model group have shown decreased levels of 5-ht, melatonin, na, and acetylcholine with increases the activity of mao, indicating a state of depression while treated animals exhibited increased levels of these biogenic amines and low level of mao activity. in that test drug 400 mg/kg produced a significant increase in 5-ht, melatonin, na, and acetylcholine levels at 28th days. this effect was comparable to the standards used. various studied have been shown the oxidative stress has been associated in the pathophysiology of many neurological disorders and caused anxiety and stress lead to depression [52-53] due to reactive oxidative stress (ros) as like hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide. these are involved in the oxidative damage to lipids, neuronal inflammation and reduced intracellular antioxidant altered the membrane function and signaling process in the brain [54-55]. in the present study, forced swim test caused the stress, lead to oxidative damage in stressed rats and decreases the glutathione, represents a main cellular non-protein and antioxidant that redox regulator in protecting oxygen species. it also regulate lipid peroxidation and also decrease in catalase activity. studies had been reported that catalase and lipid peroxidation, are lined to depressant-like behavior. in the present studies we found that malondialdehyde (biomarker of lipid peroxidation) is higher in the fst model whereas its level was reduced in pf and sertraline treated rats because the both drugs induced the more synthesis of reduced glutathione, involved in diminished catalase activity [56-57]. studies have been shown these catalase activity increases lipid peroxidation through metals transition reaction resulting generate the radical hydroxyl, which cause oxidative injury, inflammation and neuronal damage. therefore, pf and standard drug increased the level of glutathione in the stressed rats and protecting the neuron from oxidative stress and damage. as a result, all of these plants are classified in ayurveda as rasayanas which are apparent to promote mental health, improve nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 63-73 kumari et al. 2019 ©njb, biotechnology society of nepal 71 nepjol.info/index.php/njb. immunity and enhance long life of individuals [58-59]. further studies will reveal the mechanisms by which test drug synergistically enhances the antidepressant activity especially on the serotonergic system. taken together, our data proved that the anti-depressive therapeutic effects of test drug are possible with 400mg/kg doses, when it compared with standard. this might be due to different mechanisms by which four extracts act on the neurotransmitter system. conclusion in the present study, forced swim stress (fst) was used to developed depressive like behavior in rats for 7, 14 and 28 days. it was an increased immobility time, anxiety and impaired cognitive behavior in the experiment rats (stressed). it was also imbalanced biochemical in fst model (stressed rats) through the reduction of different neurotransmitter that altered behavioral parameters and it were attenuated significantly by administration of the polyherbal formulation (pf) comparable to sertraline. both drugs were capable to reduce the oxidative stress and decreases the level of reactive oxidative species through increased the level of antioxidant in the brain. it was protected the neuron from the neuronal damage and improved the cognitive behavior. however, pf, centrally exerts an antidepressant-like effect in the fst by a mechanism involving inhibition of serotonin reuptake, inhibition of mao activity and evaluated the level of acetylcholine, melatonin and noradrenaline in the brain. given that these targets have been increasingly reported to be involved in the pathophysiology of depression and on the antidepressant efficacy. thus, poly herbal extract possesses antidepressant action demonstrated by modulation of serotonergic pathway, mao receptors, acetylcholine, noradrenalin and melatonin in rats. declarations conflict interests the authors declare that they have no conflict interests. acknowledgments the authors would like to thank the adesh university punjab for their assistance and support. the authors wish to express their gratitude to dr arvind shah, dranand shah and all staff member of our laboratory. references 1. nemeroff cb, owens mj:treatment of mood disorders. nature neurosci. 2002 5:1068–1070. 2. shreevathsa m, ravishankar b, dwivedi r: antidepressant activity of mamsyadi kwatha: an ayurvedic compound formulation. ayu. 2013 34(1): 113–117. 3. belmaker rh, 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bach aw, lan nc, johnson dl, abell cw, bembenek me, kwan sw: cdna cloning of human liver monoamine oxidase a and b:molecular basis of differences in enzymatic properties. proc natl acadsci. 1988 85:4934. 54. iversen l: neurotransmitter transporters and their impact on themdevelopment of psychopharmacology. br j pharmacol. 2006 147. 55. millan mj: the role of monoamines in the actions of established and “novel” antidepressant agents: a critical review. eur j pharmacol. 2004 1–3. 56. l’opez-mu˜noz f, alamo c, juckel g, and assion hj: half a century of antidepressant drugs—on the clinical introduction of monoamine oxidase inhibitors, tricyclics, and tetracyclics part i: monoamine oxidase inhibitors. j clin psychopharmacol. 2007 555– 559. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 46 nepjol.info/index.php/njb isolation and screening of antibiotics producing streptomyces spp from the soil collected around the root of alnus nepalensis from godawari amina baniya 1,2, sushma singh 1,2, minu singh 1,2, pragya nepal 1, mahesh adhikari 1, sagar aryal2, anurag adhikari 2* 1 asian institute of management and technology, lalitpur, nepal 2 kathmandu research institute for biological sciences, lalitpur, nepal abstract actinomycetes are considered as the most invaluable prokaryotes whose genome mining show a great number of putative secondary metabolite biosynthesis pathways as well as gene clusters ranging from 20 to 50 per genome. the genus streptomyces has been explored for its ability to produce 60% antibiotics worldwide. alnus nepalensis (alder) has been found to harbor diverse eubacteria in its rhizosphere. to evaluate the antibiotic production potential from actinomycetes, we collected soil samples from rhizosphere (5-7 cm deep) of alder tree. primary screening was done by cross-streak method against multidrug resistant (mdr) such as methicillin resistant staphylococcus auereus (mrsa), vancomycin resistant enterococcus feacalis (vre), imepenem resistant acinetobacterbaumannii, vancomycin resistant klebsiella pneumonia and imepenem resistant e. coli as well as non-mdrs (e. coli, bacillus subtilis, klebsiella pneumoniae, s. aeureu and enterococcus feacalis). extraction of antibiotics was done using rota-vapour from extract obtained by solid-substrate fermentation technique followed by solvent extraction. secondary screening was done using well diffusion assay against mdrs. among total of 40 isolates of actinomycetes recovered, 14 showed remarkable zone of inhibition (zoi) to various mdrs. nasa 303 showed 26 mm of zoi against vre, nasa 101 had zoi of 34 mm against mrsa, nasa 319 had 33.7 mm zoi against imepenem resistant e. coli, nasa 306 had 36 mm of zoi against vancomycin resistant klebsiella pneumoniae, and nasa 108 showed zoi of 29.6 mm against imepenem resistant e. faecalis. this investigation revealed that the actinomycetes found in rhizosphere of alder tree had mdr killing potent antibiotics, which needs to be further explored. keywords: actinomycetes, alnus nepalensis, antibiotics *corresponding author email: adhikari.a@kribs.org.np introduction nepal is a narrow rectangular country in the himalayas. it is bounded by cold, arid tibetan plateau in north and hot humid planes to the south. being a transition zone between the two extremes of north and south, the vegetation and the microbes in this goldilocks zone has thrived spectacularly. the alder (alnus nepalensis) of nepal [1] is one of the examples for the diversified ecosystem, which houses amazing diversity of microbes within its rhizosphere region. alder is semi-deciduous tree; present throughout the himalaya at 500-3000m elevation from pakistan through nepal and bhutan to yunnan in southwest china. moreover, the alder houses microbes capable of producing antimicrobial metabolites that prevents it from being infected by different pathogenic fungi and bacteria. its rhizosphere can be screened insearch of novel antibiotic producing microbes [2]. actinomycetes are filamentous bacteria that belong to the phyla actinobacteria and the order actinomycetales. actinomycetes are considered as the most invaluable prokaryotes in medicinal and biotechnology industries because of their ability to produce number of bioactive molecules, particularly of the antibiotic compounds. streptomyces that belong to the genus actinomycetes has been considered for the production of 60% of the antibiotics [3-5]. the rapidly decreasing costs of genome sequencing has made genome mining, an invaluable resource for drug discovery: a great number of putative secondary metabolite biosynthesis pathways have been discovered using these genome data [6, 7]. in actinomycetes species, about 20-50 gene clusters per genome encoding pathways for secondary metabolite biosynthesis are present [6, 8]. intriguingly, a nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 47 nepjol.info/index.php/njb large number of these pathways are cryptic: they are not expressed under standard laboratory conditions, and their products are therefore unknown. exciting proof-of-principle successes have already been achieved in awakening cryptic secondary metabolites [10]. it has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media (in-vitro) and remain unexplored [9]. the rrna and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species. accessing this “missing” microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today [10]. in recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species [11]. however, by and large, the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives [12]. one of the oldest unresolved microbiological phenomena is why only a small fraction of the microbiological population grows on artificial media. the “uncultivable” microbial majority arguably represents our planet’s largest unexplored pool of biological and chemical novelty. here we utilize this approach and develop a novel platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. we followed the simple method of isolation of actinomycetes by using the selective media starch casein agar. microbial derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of existing ones are formidable challenges. bacteria are known producers of metabolites used in pharmaceuticals and industries respectively. however the emergence of multidrug resistant pathogens has rekindled the need to discover new antimicrobials from the environment. among many metabolites, antibiotics are one produced by streptomyces spp. which is known to be found around the rhizosphere of alnus nepalensis. the findings of this study will therefore shed light on isolating and screening of streptomyces spp. that produces antibiotic from the soil around the root of alnus nepalensis. the world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era. there is an urgent need to investigate alternative treatment options while there are still a few antibiotics left. new resistance mechanisms emerge and spread globally threatening our ability to treat common infectious diseases, resulting in death and disability of individuals who, until recently, could continue a normal course of life. without effective anti-infective treatment, many standard medical treatments will fail or turn into very high risk procedures. infections caused by resistant microorganisms often fail to respond to the standard treatment, resulting in prolonged illness, higher health care expenditures, and a greater risk of death. when infections become resistant to first-line drugs, more expensive therapies must be used. a longer duration of illness and treatment, often in hospitals, increases health care costs as well as the economic burden on families and societies. the achievements of modern medicine are put at risk by antimicrobial resistance. without effective antimicrobials for prevention and treatment of infections, the success of organ transplantation, cancer chemotherapy and major surgery would be compromised. thus, world urgently needs new and more than ever effective antimicrobials in order to treat the ever-increasing threat of antibiotics resistance. material and methods sample collection and growth of actinomycetes one gram soil sample was collected from random rhizosphere region of alder tree, situated at an altitude of 1580 msl and brought to laboratory for optimization by dry heat (100° c for 1 hour) and wet heat (80° c for 40 minutes) methods. starch casein agar (sca) media was taken as selective media and 10-fold serial dilution technique was followed using nacl. spread plate technique was performed and nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 48 nepjol.info/index.php/njb isolated colonies of actinomycetes were selected and revived for pure culture. isolation and characterization of actinomycetes identification of actinomycetes: it was done by morphological method. the morphological method consists of macroscopic examination and characterization. microscopically the actinomycetes isolates were differentiated by their colony characters, e.g. size, shape, color, consistency etc. colonies showing white powdery characteristics was identified. isolated colonies were sub-cultured to obtain pure culture. actinomycetes were named as nasa 101 to nasa 115, nasa 201 to nasa 203, nasa 301 to nasa 324, nasa 401 to nasa 404 respectively. primary screening: perpendicular streak method was used to screen actinomycetes from library against all collected mdr [methicillin resistant staphylococcus aureus (mrsa), vancomycin resistant enterococcus feacalis (vre), imepenem resistant actinobacter baumannii, imepenem resistant e. coli and vancomycin resistant klebsiella pneumonia] as well non-mdr (klebsiella pneumonia, s. aureus, bacillus subtilis, e. coli, e. facealis). extraction of antibiotics solid substrate fermentation: 100 gm of rice grain was selected for fermentation by pouring in jam bottle and mixed with 100 ml buffer solution and autoclaved. 40 such jam bottles were made. for each nasa culture inoculation of culture was done in each bottle and left for 10 days at 28°c in environment chamber. this was smashed and dried again in environmental chamber and then smashed rice was dissolved into 200 ml of methanol and ethylacetate on 1:1 ratio. then this was kept on water bath shaker at 28°c for 24 hrs at 120 rpm. after 24 hrs the solution was filtered from whatman paper. extraction using rota vapor: filtrate was taken for extraction and crude antibiotic compounds were obtained after use of rota vapor at 60° temperature in 100 rpm. this crude compound was in the form of liquid and obtained after color phase change. secondary screening: well diffusion method was performed against similar mdr and non-mdr freshly grown in trypton soya broth for 3 hours. mha media plates were made and 1-4 holes were punched; one hole contained solvent (methanol and ethylacetate) in ratio of 1:1. other 3 holes contained different extract from rota vapor method. all the plates with solvent and numbers of extract were left for nearly 1-2 hrs for diffusion. then these plates were swabbed with mdr and non-mdr microorganisms according to the requirements. spread plate technique was used for it. one control plate with antibiotic disc placed on mha media was also prepared. these spread plates with microorganisms were incubated at 37° c for 24 hrs. this plating was done for all the extract along with solvent. then zone of inhibition was observed around extract holes. results figure 1: nasa 101 showing zoi against mrsa we obtain actinomycetes isolates after primary screening from soil of rhizosphere of alder tree found in different sites around godawari (table 1). these actinomycetes showed antibacterial activities when cross-streaked with non-mdr microorganisms and mdr microorganisms. some actinomycetes showed antibacterial activities after primary screening but not after secondary screening. nasa 103, nasa 104, nasa 105, nasa 106, nasa 107, nasa 109, nasa 110, nasa 111, nasa 114, nasa 115, nasa 201, nasa 301, nasa 302, nasa 305, nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 49 nepjol.info/index.php/njb figure 2: nasa 108, 113 showing zoi against mdr enterecoccus feacelis figure 3: nasa 303, 304, 306, 307, 308, 318 showing zoi against mdr klebsiella pneumoniae nasa 310, nasa 311, nasa 314, nasa 315, nasa 316, nasa 317, nasa 320, nasa 324 (table 2 and table 3). among the 40 isolates of actinomycetes from rhizosphere of alder tree, about 14 isolates showed antibacterial activity against different mdr bacteria. nasa 101 (zone of inhibition zoi-34mm) was found to inhibit growth of mrsa (figure 1). nasa 108(zoi-29.66mm) and nasa 113(zoi-26.4mm) showed antibacterial activity against imepenem resistant e. faecalis (figure 2). nasa 303 inhibited both vre and mdr klebsiella pneumoniae. mdr klebsiella pneumoniae (figure 3) was inhibited by nasa 303 (zoi-34mm), 304 (zoi-29.7mm), 306 (zoi36mm), 307 (zoi-32mm), 308 (zoi-31mm) and table 1: description of samples collected from different sites of godawari and number of obtained actinomycetes s.n sample sites districts specific soil sample area soil depth (cm) numbers of isolates and their code 1. j1 lalitpur leech prominent area 5 nasa 306, 321 2. e1 lalitpur entrance to phulchwoki 5 nasa 307, 308, 309 3. e2 lalitpur entrance to phulchowki 5 nasa 108 4. o1 lalitpur slope area of phulchowki 5 nasa 113, 304, 313, 312 5. o11 lalitpur slope area of phulchwoki 5 nasa 303 6. o3 lalitpur slope area of phulchwoki 5 nasa 318, 319 7. pp lalitpur phulchwoki 5 nasa 101 nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 50 nepjol.info/index.php/njb figure 4: nasa 307, 318, 319 showing zoi against imipenem resistant e. coli 318 (zoi-28.5mm). nasa 307 (zoi-22.7mm) also showed antibacterial activity against mdr e. coli (figure 4) so did nasa 318 (zoi-31.7mm) and 319 (zoi-33.7mm). also, nasa 303 (zoi-26mm), 309 (zoi-25.6mm), 312 (zoi-20mm), 313 (zoi22mm) and 321 (zoi-24.6mm) showed antibacterial activity against vre (figure 5) morphological features for example aerial mycelium, substrate mycelium, soluble pigment, reverse side and growth of selected 14 strains were studied (table 4). nasa 101 showed ash gray aerial mycelium and yellow substrate mycelium; soluble pigment was absent and the reverse side was whitish gray showing good growth (figure 6). table 2: primary screening of actinomycetes for antibacterial activity by perpendicular streak method isolates non-mdr microorganisms mdr microorganisms b. subtilius e. coli k. pneumoniae e. faecalis s. aureus mrsa vre ipr e. coli k. pneumonia nasa 101 + + + + + + nasa 103 + + nasa 104 + nasa 105 + + nasa 106 + nasa 107 + + nasa 108 + + + + nasa 109 + + nasa 110 + + + + nasa 111 + nasa 113 + + nasa 114 nasa 115 + + + + + + nasa 201 + + + + + + + + nasa 301 + + + nasa 302 + + + nasa 303 + + nasa 304 + + + nasa 305 + + + nasa 306 nasa 307 + + + + nasa 308 + + + + nasa 309 + + nasa 311 nasa 312 + + + nasa 313 + + nasa 314 + + + nasa 315 + + nasa 316 + + nasa 317 nasa 318 + + + nasa 319 + + + nasa 320 + + + nasa 321 + + + nasa 324 + + nasa 401 + + + nasa 403 + nasa 404 + + = zone of inhibition absent; + = zone of inhibition present nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 51 nepjol.info/index.php/njb figure 5: nasa 303, 309, 312, 313, 321 showing zoi against vre figure 6: growth of isolated actinomycetes in sca media discussion the rising problem in the treatment of bacterial infections can be accounted to the presence of pathogens that are resistant to the currently available antibiotics and hence these pathogens are termed multidrug resistant (mdr) pathogens. mdr pathogens reported from nepal includes escherichia coli [13], salmonella and shigella species, escherichia coli, salmonella spp., klebsiella pneumoniae, pseudomonas aeruginosa, citrobacter freundii , proteus spp [14] , methicillin resistant staphylococcus aureus (mrsa)[15], vibrio cholera [16]. mdr pathogens reported from south asia includes acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumonia [17], shigellasonnei [18] , escherichia coli [19], salmonella enteric [20]. there is certain resemblance in mdr pathogens found in context of nepal and south asia yet there are differences in resistant levels of certain bacteria to different drugs. it shows mdr pathogens can be specific to its geographical location. actinomycetes producing vancomycin producing is streptomyces orientalis [21], methicillin is produced by penicillium sps. [22] and imepenem by streptomyces cattleya [23]. thus, actinomycetes are found to produce drugs such as vancomycin, imepenem, etc., due to which we selected actinomycetes in our project. isolation of actinomycetes from soil was done using a different methodpretreatment of soil for 1 week by air drying followed by isolation of actinomycetes using selective media composed of starch yeast extract agar (sye), yeast extractmalt extract agar (isp2), oatmeal agar (isp3), inorganic salt-starch agar (isp4), starch-casein agar (sca), glucose asparagine agar (gaa), actinomycete isolation agar (aia) and marine agar (ma)[24], whereas other authors used crowded plate method for isolation of actinomycetes [25]. microbially derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 52 nepjol.info/index.php/njb table 3: secondary screening of antibacterial activity of the actinomycetes isolated isolate no. name of the test organism (inhibition zone diameter in mm) vre mrsa imepenem resistant escherichia coli vre klebsiella pneumonia imepenem resistant enterococcus feacelis nasa 101 34 nasa 108 29.66 nasa 113 26.4 nasa 303 26 34 nasa 304 29.7 nasa 306 36 nasa 307 22.7 32 nasa 308 31 nasa 309 25.6 nasa 312 20 nasa 313 22 nasa 318 31.7 28.5 nasa 319 33.7 nasa 321 24.6 table 4: morphological feature of obtained actinomycetes symbol of strain aerial mycelium substrate mycelium soluble pigment reverse side growth nasa 101 ash gray yellow whitish gray +++ nasa 108 whitish gray slimey yellow whitish gray +++ nasa 113 white gray yellow gray +++ nasa 303 ash gray slimy yellow dark gray +++ nasa 304 white white dark gray ++ nasa 306 ash gray gray dark gray +++ nasa 307 whitish gray yellow slimey yellow +++ nasa 308 white yellow slimey yellow ++ nasa 309 whitish gray slimey yellow slimey yellow +++ nasa 312 ash gray slimey yellow slimey yellow +++ nasa 313 dark gray white white ++ nasa 318 whitish gray white white +++ nasa 319 dark gray white slimey yellow +++ nasa 321 dark gray white slimey yellow +++ = absent; ++ = moderate growth; +++ = good growth nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 53 nepjol.info/index.php/njb existing ones are formidable challenges. a total of 40 isolates of actinomycetes were obtained, among which secondary screening nasa 101 was found to inhibit mrsa, and nasa 303, 309, 312, 313, 321 were found to inhibit vre. mrsa is resistant to methicillin antibiotic and nasa 101 must produce antibiotic of different structure than that of methicillin [26]. similarly, the structure of antibiotics produced by nasa isolates (table 3) inhibiting vre and vancomycin resistant klebsiella pneumoniae, should be of different structure than that of vancomycin [26]. likewise, the structure of antibiotics produced by nasa isolates (table 3) showing zone of inhibition to imipenem resistant e. coli and enterococcus feacalis should be different than that of imipenem [26]. from this we can say that the class of antibiotics produced by nasa isolates may be unique one in its structural and functional basis. the results of primary and secondary screening did not match quite well in our project. some of the strains showing zone of inhibition in primary screening did not show inhibition during secondary screening (well diffusion method). example: nasa 101, nasa 105, nasa 115, nasa 201, nasa 401, nasa 404, nasa 301, nasa 313, nasa 321, nasa 324. this may be due to the protocol followed for extraction of antibiotics, effect of solvent used in extraction process. extraction of secondary metabolites, such as antibiotics, are generally done by maintaining the compositions of the media containing yeast extract, dextrose, starch, casein hydrolysates, ammonium sulphate, calcium carbonate. this kind of composition leads to increased production of antibiotics [27]. antibiotics are also extracted by using nutrient broth and the method of chromatography which helps in efficient extraction of minute amount of antibiotics that may be present in the broth [28]. but we extracted antibiotics using fermentation followed by rota vapor technique using solvents such as methanol and ethylacetate (1:1). since we used sca media for the primary screening, the antibiotics could have expressed quite well. however, we used solid substrate fermentation method for extraction of antibiotics using rice as the media with other chemical supplements which may not allow antibiotics to be expressed on those isolates [29]. this might be due to the differences in morphology of actinomycetes when grown in solid and liquid media as filamentous mycelia and fragmenting mycelia respectively [30]. in the secondary screening zone of inhibitions were observed in nasa 108, nasa 303, nasa 307, nasa 308, nasa 309, nasa 318, nasa 319 and nasa 321 strains but not in the primary screening. this might be due to the solid fermentation method of the nasa strains on rice medium, favoring more to these antibiotics expressing nasa strains on the secondary screening than that on sca medium, not favoring them to be expressed during the primary screening. many previous studies have reported a high degree of concordance in the zone of inhibition between the primary and the secondary screening extracts [30]. it was found that root soil of the analyzed trees such as pine, alder, birch contained more microorganisms than soil distal to the roots. thus, we selected the rhizosphere of alder tree, the indigenous tree of nepal. the presented analysis of the diversity of actinomycetes in the isolation (different soils and rhizosphere of different trees) indicated a lack of significant differences between the microorganisms. the only sources of isolation which were relatively diverse were the root-free soil of birch and the birch rhizosphere. the lowest diversity of actinomycetes was found in bulk soil and the action rhizal rhizoplane of alder [31]. multidrug resistant bacteria are found in context of nepal that are entirely native to this geographical location and thus differs from those found in other parts of the world. hence, we need to screen and isolate actinomycetes that are native to this environmental condition as well as novel in order combat those mdrs. conclusion in this study, we isolated actinomycetes from soil around the roots (rhizosphere) of alder tree. we demonstrated that the rhizosphere of alder tree houses clinically important microorganisms. further study of these isolated strains can be done to determine exact molecules responsible nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 54 nepjol.info/index.php/njb for anti-mdr property. whereas the macroscopic morphology of the isolates could merely provide presumptive genus identification, 16s rrna assay and subsequent sequencing analysis, in the future, may possibly resolve their species level identity. since, mdrs are unique to certain geographical locations, actinomycetes capable of combating such mdrs must be prevailed in that particular environmental condition. thus, intensive research to isolate such clinically significant actinomycetes must be encouraged using indigenous natural resources. references 1. jackson jk: manual of afforestation in nepal. second edition. in. kathmandu, nepal: forest research and survey centre.; 1994. 2. golinska p, dahm h: occurence of actinomycetes in forest soil. dendrobiology 2011:5-9. 3. fguira lf, fotso s, ameur-mehdi rb, mellouli l, la: purification and structure elucidation of antifungal and antibacterial activities of newly isolated streptomyces sp. strain us80. research in microbiology 2005. 4. mellouli l, ameur-mehdi rb, mansour s, sa: isolation, purification and partial characterization of antibacterial activities produced by a newly isolated streptomyces sp. us24 strain. research in microbiology 2003. 5. singh ls, baruah i, bora tc: actinomycetes of loktak habit: isolation and screening for antimicrobial activities. biotechnology 2006. 6. medema, trefzer a, kovalchuk a, van den berg m, muller u, h m: the sequence of a 1.8-mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways. genome biol evol 2010. 7. m. g, kol s, gomez-escribano j, bibb m, takano e: deletion of a regulatory gene within the cpk gene cluster reveals novel antibacterial activity in streptomyces coelicolor a3(2). microbiology 2010. 8. xj w, yan y, zhang b, an j, wang j, tian j: genome sequence of the milbemycinproducing bacterium streptomyces bingchenggensis. j bacteriol 2010. 9. ts b, widmaier d, temme k, mirsky e, santi d, voigt c: synthesis of methyl halides from biomass using engineered microbes. j am chem soc 2009. 10. jf m, liras p: engineering of regulatory cascades and networks controlling antibiotic biosynthesis in streptomyces. curr opin microbiol 2010. 11. mh m, breitling r, bovenberg r, takano e: exploiting plug-and-play synthetic biology for drug discovery and production in microorganisms. nat rev microbiol 2011. 12. hm s, mirsky e, voigt c: automated design of synthetic ribosome binding sites to control protein expression. nature biotechnology 2009. 13. baral p, neupane s, marasini bp, ghimire kr, lekhak b, shrestha b: high prevalence of multidrug resistance in bacterial uropathogens from kathmandu, nepal. bmc research notes 2012. 14. pratikshya poudela* pp, nabaraj adhikaric, pradeep kumar shahd: multi-drug resistant bacterial isolates associated with blood stream infection american scientific research journal for engineering, technology, and sciences (asrjets) 2015. 15. bhatta dr, cavaco lm, nath g, gokhale s: threat of multidrug resistant staphylococcus aureus in western nepal. asian pacific journal of tropical disease 2015. 16. gupta pk, pant nd, bhandari r, shrestha p: cholera outbreak caused by drug resistant vibrio cholerae serogroup o1 biotype eltor serotype ogawa in nepal; a crosssectional study. antimicrobial resistance & infection control 2016. 17. bialvaei az, kafil hs: colistin, mechanisms and prevalence of resistance. current medical research and opinion 2015. 18. the hc, rabaa ma, duy thanh dp, lappe, niall de south asia as a reservoir for the global spread of ciprofloxacin-resistant shigella sonnei: a cross-sectional study. plosmedicine 2016. 19. ram s, vajpayee p, singh rl, shanker r: surface water of a perennial river exhibits multi-antimicrobial resistant shiga toxin and enterotoxin producing escherichia coli. ecotoxicology and environmental safety 2009. 20. kupferschmidt k: drug-resistant typhoid fever becoming an epidemic in africa and asia. science 2015 21. levine dp: vacomycin: a history. clinical infectious diseases 2006. 22. dutfield g. in.; 2009. 23. bodner mj, li r, phelan rm: definition of the common and divergent steps in carbapenem β-lactam antibiotic biosynthesis. chembiochem 2011. 24. malek na, khan chowdhury aj, zai zz: selective isolation of actinomycetes from mangrove forest of pahang, malaysia. in: international conference on agriculture, biology and environmental sciences (icabes'14): 2014; indonesia. 25. nanjwade basavaraj k, chandrashekhara s, shamarez am, goudanavar ps, manvi fv: isolation and morphological characterization of antibiotic producing actinomycetes. tropical journal of pharmaceutical research 2010. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 46-56 baniya et al. ©njb, biotechnology society of nepal 55 nepjol.info/index.php/njb 26. national center for biotechnology information. pubchem compound database. in.; 2017. 27. shetty pr, buddana sk, tatipamula vb, naga yvv, ahmed j: production of polypeptide antibiotic from streptomyces parvulus and its antibacterial activity. brazilian journal of microbiology 2014. 28. charyulu em, g s, g rs, arumugam g: antimicrobial activity of secondary metabolite from marine isolate, pseudomonas sp. against gram positive and negative bacteria including mrsa. indian journal of experimental biology 2009. 29. gebreyohannes g, feleke m, samuel s, nagappan r: isolation and characterization of potential antibiotic producing actinomycetes from water and sediments of lake tana, ethiopia. asian pac j trop biomed 2013. 30. mohan ysyvj, sirisha b, prathyusha k, r p: isolation, screening and characterization of actinomycetes from marine sediments for their potential to produce antifungal agents. global journal of biology, agriculture and health sciences 2014. 31. golińska p, dahm h: occurrence of actinomycetes in forest soil. dendrobiology 2011. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1:20-24 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 20 nepjol.info/index.php/njb development of cost optimized horizontal gel electrophoresis running unit for developing countries (nepal) sajesan aryal, aroj hada, sanjay hamal, abhishek prajapati, paras mani timilsina, sandeep adhikari * department of biotechnology, kathmandu university abstract the use of expensive lab techniques has left many high schools and even university students unacquainted with the basic experimental procedures and protocols in developing country including nepal. horizontal gel electrophoresis is one of the expensive protocols, which every student in the laboratory may not get an equal chance to access individually. however, this technique, being indispensable and inevitable in molecular biology principles, is of abounding importance for students to be familiar with. thus, realizing its importance, we present an extremely simple and inexpensive design of gel-electrophoresis unit, which emulates electrophoresis analysis with the use of nichrome and aluminum wires as a substitute for platinum wires, together with daily used plastic materials. because of these factors, the approximate cost of unit design is significantly reduced to an amount of 10 usd. the efficiency of the substitute wires was confirmed and it resulted in satisfactory data characterized by good resolution of the dna fragments. the inexpensive nature, good results and simplicity of the device make it an ideal unit for teaching and learning in developing countries. keywords: horizontal gel electrophoresis, molecular biology, nepal, nichrome. *corresponding author email: acsandeep2014@gmail.com introduction in graduate courses and research environment, the use of gel electrophoresis seems to be an expensive technique. the main concern lies on the fact that many developing countries limit their courses on theoretical concepts unaccompanied by experimental practices due to cost ineffectiveness. learning theoretical science concepts and lack of opportunity to apply the very concept on the real world only provides an abstract vignette of knowledge for the students. “the core reason that may underlie upon the fact that many developing countries emphasizes teaching theory in secondary, higher secondary and sometimes even in undergraduate courses, is the absence of expensive laboratory equipment. ”( ens et al, 2012) [1]. gel electrophoresis is expensive equipment which many colleges and even some universities of developing countries cannot afford in adequate numbers so that all the students can equally access them. thus, this article proposes an inexpensive and result oriented simpler design alternative for macromolecule separation replacing the expensive equipment. gel electrophoresis is one of the most widely used molecular biology technique for the separation and analysis of the macromolecule (dna, rna, and proteins) fragments based on their size and charge. the technique is based on the use of electric field to separate different sized macromolecule fragments in a porous gel matrix. the electric field is applied in such a way that one end of the gel has a positive charge and another end has a negative charge. as macromolecules like dna and rna have a negative charge due to the presence of phosphate groups, they tend to move from the negative pole to the positive pole of the gel under the application of electric field. [2] finally, after the samples have been separated using gel electrophoresis technique, bands representing molecules of different sizes can be detected. hereby, we designed a similar electrophoresis unit which emulates the original laboratory gel electrophoresis unit, constructed with readily available inexpensive materials and showed its efficacy with the electrophoresis of dna samples as described below. the test and analysis of the protocol were performed at molecular biology laboratory at kathmandu university (ku), dhulikhel, nepal. the major focus during the project was given on the key element of construction of electrophoresis unit and nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 20-24 aryal et al. ©njb, biotechnology society of nepal 21 nepjol.info/index.php/njb preparation of gels and buffers, which generally remains invisible to the students during the teaching process. along with these parts, diagnostic tests were repeatedly performed to check the durability of the protocol and the quality of result obtained by visualizing the dna bands under uv radiation. materials and methods two micro-wave safe plastic boxes of (16.8 x 11.2) cm and (10.75 x 11) cm dimension were bought from a local plastic store. also, a plexi-glass of dimension (11 x 11) cm was bought from a glassware store. a 30 cm measuring scale was bought from a local stationary. nichrome, aluminum and two multimeter wire one black and another red in color was bought from a local electric store. instrumentation a micro-wave safe plastic box of (16.8 x 11.2) cm dimension was used as the electrophoresis tank. a plexiglass of dimension (11x11) cm was attached to the bottom of this tank leaving some spaces on the both side of the plastic box so that it mimics the platform where gel-box is supposed to sit as in laboratory equipment. two kinds of electrode arrangement were tested. first, double wounded nichrome wire as both cathode and anode. second, double wounded nichrome as cathode and triple wounded aluminium as anode. variation of electrodes in anode was brought to test the durability and effectiveness of the two different wires; nichrome and aluminium. this was done so, because the electrode at anode corroded at higher rate in comparision with the electrode at cathode. for the gel holding tray, a suitable plastic box of dimension (10.75 x 11) cm was fitted in the buffer tray. leaving 1 cm from the bottom of the gel holding tray, the entire tray was cut horizontally. again, the two smaller opposite ends of the gel holding tray were cut vertically so that it mimics the shape and form of the standard gel box used in the laboratory. for the gel comb, a ruler of considerable thickness was chosen. each groove of 0.5 cm with 0.5 cm gap in between was made. ruler was cut on either side to make it exactly fit and enter inside the buffer tray. it was cut in such a way that the teeth of the ruler do not touch the bottom of the gel holding tray. two multimeter wires were fitted in the lid of electrophoresis unit, by drilling the extreme corners but of the same side of the lid and the wires were fitted into the drilled holes. the tip of both the wires was cut and joined with two alligator leads which fit in the available voltage regulator. figure 1: overall instrumentation of the electrophoresis unit gel and running buffer 1% agarose gel was used in all of the processes. before the agarose solution was polymerized into gel, 0.5 µg/ml of ethidium bromide (etbr) was added after gel reached 65º c and stirred. ethidium bromide acts as an intercalating agent between the two strands so that upon visualization of the dna bands in the uv it can be properly viewed. [3, 4] the running buffer was prepared according to standard laboratory practices (either 0.5x tbe or tae). the dna samples were loaded in the wells of the gel loading tray and the tray was immersed into the buffer solution completely. then, the lid of the electrophoresis tray was closed so as to complete the circuit and the voltage was applied. care was taken that the wells on the gel were on cathode (negative terminal) so that on applying electric field dna could move towards the anode (positive terminal). a constant voltage was set up and a time of 120 min for all test runs. the current was applied. after the designated time, the gel was removed and then visualized under the uv radiation and the dna bands were observed. [5] nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 20-24 aryal et al. ©njb, biotechnology society of nepal 22 nepjol.info/index.php/njb result and discussion efficiency of the electrophoresis unit: for nichrome as both cathode and anode figure 2a: dna bands from an agarose (1%) gel with tbe buffer run for 55 v for 2 hours using nichrome as both the electrodes. i. figure: 2b: gel image of (i) 1 kb dna ladder and (ii) lambda dna/hindiii obtained from neb (gel run on 1% agarose concentration). firstly, our unit was run using standard dna samples of λ dna hind iii digest, λ dna, λ dna hind iii digest and 1 kb dna ladder, in the order from left to right, as shown in figure 2a. all the samples were standards obtained from neb (new england biolabs). the dna ladder in lane 4 was separated into 10 fragments identical to the standard gel image obtained from neb [6]. the lambda dna digested by hindiii in lane 3 separated into 5 fragments which contradicted with the standard neb gel image. this may have happened because the time of the run was not enough for the separation to complete. the 1 kb dna ladder in lane 4 separated into 10 fragments as shown in figure: 2a. comparing the two images (figure 2a and figure 2b), it is clear that the band separation of each sample in both the runs were similar (the standard obtained from neb and the one obtained from our unit). from these results, we are able to say the efficiency of migration of dna samples is quite satisfactory while using nichrome electrodes in place of platinum electrodes. figure 2c: dna ladder standard calibration curve (log10fragment size (kb) vs. migration (mm)) obtained from the data interpreted from fig (1a). distances were measured from image processing software: image [8] for nichrome cathode and aluminum anode secondly, the unit was run using nichrome at cathode and aluminum at anode. samples of lambda dna and lambda dna hind iii digest (from left to right) were used and the following gel image was obtained (figure 3a). the lamda dna digested by hindiii in lane 2 separated into 5 distinct bands as shown in the gel image below (figure 3a). a fragment of the lambda dna/hindiii digest was not clearly observed. this may have happened due to not enough run time or low intensity of the band. all the samples were standards obtained from neb (new england biolabs). figure 3a: λdna/hindiii bands from an agarose (1%) gel with tbe buffer run for 55 v for 2 hour in apparatus using nichrome as cathode and aluminum as anode. comparing the two images (figure 3a and figure 2b (ii)), it is clear that the band separation of each y = -0.0144x + 1.9363 r² = 0.9792 -0.5 0 0.5 1 1.5 0 50 100 150 200 lo g 1 0 (k b ) migration (mm) dna ladder standard curve ii nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 20-24 aryal et al. ©njb, biotechnology society of nepal 23 nepjol.info/index.php/njb sample in both the runs were similar and differ by only one band (the standard obtained from neb and the one obtained from our unit). from these results, we are able to say the efficiency of migration of bands is quite satisfactory while using nichrome at cathode and aluminum at anode in place of platinum electrodes. figure 3b: lambda dna/hind iii standard curve (fragment size (kb) vs. migration (mm)) obtained from the data interpreted from figure 2b. from these observations, we verified that use of nichrome or aluminum wires as electrodes provide similar resolution to that of commercially available platinum wired units. analysis of instrument's durability: electrode materials, aluminum and nichrome could be used for a total runtime of 6-8 hours. the use of nichrome wires as both the electrodes allowed a run of 6-8 hours after which significant deterioration of the nichrome wires at anode was observed while the metal wire at cathode remained intact. degradation of anode was visible as decrement of the wire diameter. therefore, efficiency of another metal (aluminum) at anode was checked. although corrosion and pitting of aluminum at anode as white flocculent material was observed during electrophoresis, electrodes remained intact for normal runs of about 3 times (6 hours). aluminum wire at anode reacted with components of the buffer electrolyte during the run and formed a protective layer in the form of white flocculent material. the material did not interfere with the run as seen by the gel image (figure 2a) [6]. then another thrice wounded aluminum or chromium wire can be fitted in an l-shaped manner easily as described in instrumentation protocol. in contrast, nichrome at cathode seemed very effective as it did not show any signs of corrosion even for a long run over 8 times (16 hours). these data of wire durability were obtained after triplicate experimental runs. variation of electrodes in anode was brought to test the durability and effectiveness of the two different wires; nichrome and aluminium. this was done so because the electrode at anode corroded at higher rate in comparision with the electrode at cathode. this occurs due to the oxidation of anode. electons travel from anode to cathode. hence metal of anode is continuously converted to ion and anode is pitted and consumed continuously (corrosion). this is the reason for using platinum as electodes. platinum is a transition metal in group 10 (viii b) of the periodic metal. they are noble metals and hence are relatively non-reactive. platinum does not react with air , water etc and does not corrode at any temperature. hence platinum is very suitable to use as electrodes. however its price is very high i.e. 930 usd/ozt. the cost of nichrome wire is 2.29 usd/kg and that of aluminium is 0.0582 usd/ozt [7]. micro-wave safe plastic boxes were chosen over glass to ensure that the heat produced from the electrodes would not damage the interiors of the box at a low price. the interior layer of the plastic box was not damaged even after the overall experiment run. thus, it was concluded the use of micro-wave safe plastic box, which are easily available at relatively low cost can effectively be used in the protocol. plexiglass at the bottom of the gel box also didn't show any signs of damage during the overall period of run. also, the wires and alligator clips resulted as quite good alternatives in the design of the unit. cost estimate and comparison: the electrophoresis unit was made with a goal to reduce the cost of the overall unit so that universities and colleges in developing countries are able to provide quality practical education to y = -55.628x + 95.186 r² = 0.9252 0 50 100 0 0.5 1 1.5 lo g 1 0 (k b ) migration (mm) dna/hindiii standard curve table 1: cost estimation of the units apparatus cost (usd) microwave safe box 5 plexi-glass 2 multimeter wires 2 nichrome & aluminum wires 1 total 10 (approx.) nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 20-24 aryal et al. ©njb, biotechnology society of nepal 24 nepjol.info/index.php/njb the students with less expense. the detailed cost estimation of the unit is table 1. the cost of a commercial gel electrophoresis apparatus including the gel-holding tray and the combs usually range from 500 usd to 2000 usd depending on the various models from different manufacturers (sigmaaldrich, thermofisher, fisher scientific,etc ) . table 1: price obtained from related manufacturer’s website. name/type price (in usd) thermo scientific™ owl™ easycast™ b2 mini gel $688.32 $837.80 fisherbiotech™ horizontal electrophoresis systems $312.64 $1275.83 thermo scientific™ owl™ d4 $1,155.29 invitrogen™ novex™ $570.00 conclusion we came to a solid conclusion based on the above observations that the unit with chromium electrodes was quite effective for the normal use, with quality result and resolution of the bands at a lower cost compared to the commercially available units. this protocol upon optimization can be effectively embraced by the high schools, colleges and even universities to demonstrate the basic principle of gel electrophoresis and provide each student with an equal opportunity to get acquainted with the physical as well as working mechanisms of the gel electrophoresis equipment at a low cost. from these observations, we verified that our unit is well suited for the demonstration of gelelectrophoresis procedures and applications in high school biology labs and even universities where each student may not have an equal access to the laboratory apparatus when mentored by their tutor due to its expensive nature, in a developing country like nepal. references 1. ens s, olson a, dudley c, ross n, siddiqi a, umoh k, schneegurt m: inexpensive and safe dna gel electrophoresis using household materials. biochem mol biol educ. 40(3), 198-203. doi:10.1002/bmb.20596 2. lee p, costumbrado j, hsu c, kim y: agarose gel electrophoresis for the separation of dna fragments. j visual exp 2012. 3. britos l, goyenola g, oroño s: simple protocol for secondary school hands-on activity: electrophoresis of pre-stained nucleic acids on agar-agar borate gels. biochem mol biol educ. 32(5), 341-347. 4. green m, and sambrook j: molecular cloning: a laboratory manual 4th ed. cold spring harbor: cold spring harbor laboratory press. 2012 5. sigmon j and larcom l: the effect of ethidium bromide on mobility of dna fragments in agarose gel electrophoresis. electrophoresis 1996, 17:1524-1527. 6. infomine: investment mine. http://www. infomine.com/investment/ (2017). accessed 10 june 2017 7. new england biolabs: product catalog. https://www.neb.com/products (2017). accessed 10 june 2017. 8. bancroft and wilder d: electrolytic theory of corrosion. j. phys. chem. 1924; doi: 10.1021/j150242a001 9. imagej: image processing and analysis in java. https://imagej.nih.gov/ij/ (2004). accessed 15 may 2016. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1 :113-123 doi: https://doi.org/10.3126/njb.v7i1.26956 ©njb, biotechnology society of nepal 113 nepjol.info/index.php/njb. review article potential of ayurvedic drugs in differentiating neuronal stem cells from human breast milk: a review rinki kumari1* 1advanced centre for traditional and genomic medicine, institute of medical science, banaras hindu university, up, india abstract recently, stem cell therapy has revolutionized excellent clinical therapy with the potential of stem cells to differentiate into various cell types and it may help to replace different cell lines of an organism. frequent, clinical trials are carried out to merge the new scientific stem cell information and techniques with traditional knowledge and plant extracts that may result in less toxic, affordable, and highly available natural alternative therapeutics. ayurveda, the traditional indian system of medicine has given great emphasis to the promotion of health. ayurveda therapies are based on the restoration of body balance and nourishment of dhatus. rasayana concept of ayurveda explains tissue regeneration and cell renewal. rasayana drugs and therapies provide research opportunities for the biology of regeneration. specific medhya rasayana stimulates and nourishes respective medha (dhi, driti, and smriti) dhatus. interpretation of this description offers clues for specific differentiation of neuronal stem cells from human breast milk (hbm) in the presence of some herbal extracts. the previous studies suggest that neuronal stem cells differentiate from human breast milk (human mesenchymal stem cell) more effectively with madhya rasayana drugs. the present review highlights the potential of ayurveda and its possible contributions in regenerative medicine. keywords: human breast milk, stem cell, mammary gland, nervous system, nestin-positive, non-invasive *corresponding author email: rinkiv3@gmail.com introduction in recent times, stem cell therapy (sct) is on top position in the clinical area of regenerative therapy and these cells are used with the enormous goal with clinical application in the medical field. these cells also prevent and treat various clinical issues with a unique aspect including regenerate (self-renewal) &repair injured tissues and organs in the organism. the various study declared that these cells have the ability to distinguish into a specific adult cell. globally, clinical sct is growing from a strong root and have promising clinical therapeutic setup in an organized form. definite generated cells and tissues from pluripotent stem cells are grafted on injured tissue to repair along with to regenerate specific tissue or organ [1-2].these cells have the power to make each tissue and organ within the specific frame. stem cells, able to construct every type of human tissues or organ and due to unlimited self-renewal to produce progeny exactly the same as the originating cell with the extremely regulated mode. stem cells are also differentiate into different adult specific cell lineages under appropriate clinical conditions [3].therefore, scs are geared up to generate a specific cell type that becomes a part of the healthy living system or living organism. recently, mcginley et al., (2016), defined stem cells are most applicable in clinical field and involved to generate whole organs for organ replacement [4]. rapidly, these are also involved in treating untreatable neurodegenerative diseases such as parkinson's disease [5] alzheimer's disease [4], and diabetes mellitus [6]. the multipotent human stromal cells are categorized as adult stem cells and isolated from adult tissues including bone marrow and various non-marrow tissues like adipose tissue, adult muscle, corneal stroma or the dental pulp of deciduous baby teeth these sources are knows as nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 114 nepjol.info/index.php/njb. invasive and painful. other some study have reported that human breast milk is a rich source of multipotent mesenchymal stem cells. breast milk could be an alternative source of stem cells for autologous stem cell therapy. in addition some clinical waste products such as placenta, umbilical cord blood as well as body or tissue fluids such as synovial fluid amniotic fluid and menstrual blood useful for stem cell isolation. these sources are completely non-invasive and non-pain able to have less ethical issues [2]. recently, several studies have committed that these cells have the potential to differentiate into a number of cell lineages types including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), neurons, glia, tendon-ligament and adipocytes (fat cells which give rise to marrow adipose tissue) etc. with much promise for use in clinical stem cell therapy [7-8]. human embryonic stem cells (hescs) are the foremost versatile, consequent from blastocysts (three to five-day recent embryo) that grow from embryo to fetus (ectoderm, mesoderm, and hypoblast are the 3 germ layers).these cells can form any type of cell and tissue within the body; once stem cells are directed to a particular lineage, they can regenerate cells and tissues as part of normal growth and repair. although, at this stage, usually, they cannot move away from their dispersed cell type. adult cells are typically only able to replicate into the precise cell lineage for examplethe cells are from skin cells can only replicate into other skin cells. therefore, researchers have found ways to induce pluripotency ability to replicate into other cell lines or tissue lineage among some adult cells for possible use in regenerative medicine [9]. nowadays, hmscs are being studied more extensively worldwide due to having scientifically verified essential clinical potentials. joshi and bonde (2014) have compared studied thath mscs have some more advantages over embryonic stem cells (hescs) such as it can simply isolate from different sources and can easily to in vitro culture along with negligible ethical issues allied with their application. although it has most required alternative significant advantage never recognized as foreign cells once utilized in stem cell therapy in any clinical cases, due to its hladr (human white corpuscle antigen-antigen d related)-negative characteristic unique feature [10]. currently, various reports have committed that the research tasks in the field of hmscs are being excellent experiment and studies; due to its scientifically proven abilities and enormous clinical potential. in addition, hmscs have some important advantages over embryonic stem cells (hescs) like easily isolate from different sources; easy to in vitro culture; negligible ethical issues allied with their application. although several studies have evidence to provide these cells never recognized as foreign cells when used in stem cell therapy in various clinical cases because of their hla-dr (human leukocyte antigenantigen d related)-negative characteristic [10]. in 2017, witkowskazimny et al., have attempted to provide that human breast milk (hbm) is good for health with high-quality of nutritional and immunological properties. it is recognized as the ideal breastfeeding to infant to improve their health in societies. hbm contains a heterogonous component of immunological, biochemical and cellular properties that have a potential role at the different stage of lactation, the degree of breast fullness, infant feeding and the health of the breastfeeding dyad [11]. numerous studies have suggested that these components have the potential to significantly enhance the immunity of infants because of the presence of the different components of micro and macronutrients and these components are involved to protect infants from infections (due to containing increased numbers of infectionfighting white blood cells). additionally, provides nourishment, fortification, and organic process programming to an infant, with short and semipermanent effects [11-13].moreover, the presence of maternal cells or heterogeneous cells in hbms (figure 1), including leukocytes, epithelial cells, lactocytes (milk-secretory cells), and myoepithelial cells (from the ducts and alveoli of nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 115 nepjol.info/index.php/njb. the mammary gland) are involved in various developmental stages of infants. figure 1. schismatic presentation of heterogeneous cell in human breast milk although, these cells have numerous biomedical application due to heterogeneous nature [11]. recently, evidence has declared that human breast milk showed positive nestin, which marked the presence of important cellular components i.e., progenitor/stem cells. various studies were able to explain the commensal and beneficial bacteria likelactic acid bacteria and bifidobacteria's presence with good spring, in human breast milk, which is important for the proper development of infants. it is also full of a hierarchy of regenerative and progenitor cells. therefore, hbm composed dynamic cellular components and is the good proportion of singular cell types which can be altered by various factors [11]. based on the various evidence, human breast milk has been recognized as a mainly applicable clinical therapeutic agent, and it is a most suited applicable agent like other drug therapy or regenerative medicine [14]. cregan et al., and his team explained the presence of a protein marker known as neuroectoderm marker (multipotent stem cell marker) such as nestin-positive putative mammary stem cells in hbm. however, it is also a presence in the heterogeneous subpopulation of milk with low frequency. scientists have reported that nest in showed similar properties of other stem cells. frequently, expressed in follicle stem cells and also in their differentiated progeny [15]. it has most useful clinical application, to differentiate into different cells and tissues like neural, bone marrow, pancreatic and epithelial cells like hescs [14-15]. in addition, the presence of cell surface markers like cytokeratin (ck) 5, hoechst 33342 and nesting are not able to explain the differentiation potential of these mammary cells isolated from human breast milk [14]. even though, in the mammary gland, stem cells have the capability to regulate program and immediately, alters into the full secretary state during pregnancy and in the postpartum period [16]. some other researchers have also supported the findings of cregan et al., that hbm derived stem cells had the capability to differentiate into neural cell lineages as like both embryonic and mesenchymal stem cells. on the basis of other studied that the exposure of the milk cells into neurogenic medium, leads to differentiation into all three essential neural lineages 1) ß-tubulin as a neuron marker expressed by neurons, 2) o4 marker expressed by oligodendrocytes, 3) the gfap marker expressed by astrocytes [17]. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 116 nepjol.info/index.php/njb. hosseini et al., showed that both mammary glands and nervous system, are originated from an embryo. as a result milk cells are a good source of neural cells and involved in the development of the enteric nervous system. it consisting of a mesh-like network of neurons that regulate the function of the gastrointestinal system [17]. literature search reported that pluripotent stem cells have the ability to generate self-renewing stem cells and presence in hbm. these cells have a specific feature of multi lineage segregation latent for all three germ layers i.e., ectoderm, mesoderm, and endoderm [17]. various studies confirmed that hbm stem cells are also express classic essential embryonic stem cell surface markers including octamer-binding transcription factor 4 (oct4), sex determining region y-box (sox2), seea4, tra 1–60/81 and homeobox (nanog). cell surface markers confirmed that hbm stem cells possibly will perform similar to both embryonic and mesenchymal stem cells [17-18]. ancient traditional medicinal systems like ayurveda, siddha, unani and also revealed in the ancient vedas and originated in india. ayurveda means "science of life" and longevity" that developed between 2500 and 500 bc [19]. from the ancient era, ayurveda always recommends the entire system to be in this world with a long healthy life. several pieces of literature expressed that ayurveda has knowledge and source to the regeneration of cell to treat and cure clinical diseases via uses of the different herbal product. these herbal extract (a single and polyherbal formulation) significantly decrease various clinical disease burden. although, still, the mechanisms of action of single and polyherbal extracts are mostly unknown [19]. ayurveda consent with a dynamic exchange in terms of continuous regeneration of dhatus with the physiological process of doshas i.e. vata, pitta and kapha. as per modern concept, the tissues undergo constant destruction and regeneration in the body. ayurveda suggested that several healthy dietary, lifestyle and herbomineral interventions for dosha balance and dhatu nourishment resulting in healthy long life through tradition rehabilitation. traditionally rehabilitation such as panchakarma and rasayana are used in ayurveda for rejuvenation of cells. in addition, natural drugs can assist in the management of neurodegenerative disease burden by making the interaction between tridosha and triguna in a well-balanced circumstance. several scientific evidence showed that natural medhya rasayana drugs providing medhya (intellect promoting) effect to improve the memory of the patients [19]. on the basis of current knowledge about human breast milk stem cells and ayurvedic concepts of regeneration, both together have a major contribution to the development of regenerative herbal medicine with an integrative approach in the modern clinical area [19]. rasayana is ayurvedic drug branches which refer to rejuvenating therapy and immune modulation. according to ayurveda, rasayana therapy or jarachikitsa are not involved only covers health management but also delay aging or vayasthapan, by offering treatment of aging through the rejuvenation process. the enhancement of rasa (essence) is the quit essential quality that all rasayana medicines possess, ultimately looking to promote health and vigor of the tissues. rasahasa characteristic tendency to improve the nutritional status of the human body and reduces stress through three basic mechanisms -1.rasa (nutrient effect) 2.agni (digestion and metabolism), 3.srotas (microcirculation and tissue perfusion). however, rasayana drug is concerned to promote physical and mental health, improve defense mechanisms of the body and enhance life longevity [19]. recently, several lines of evidence have reported that more than 200 rasayana drugs are used for a reduced different type of clinical neurodegenerative burden. due to the presence of various antioxidants active molecules in these herbal drugs. in ayurveda, medhya rasayana is used for the brain and mood disorder treatment and improving the status of medhya issues [1920]. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 117 nepjol.info/index.php/njb. previous evidence has supported that human mesenchymal stromal cells (hmscs) contain a multi-potent cluster of cells. frequently, it can be easily isolated from developed tissue like bone marrow, adipose tissue, including medical waste material (umbilical cord, placenta, and human breast milk). joshi et al., (2014) have shown thath mscs capable to differentiate into a wide range of cell lineages. ita has promising potential role to stem cell therapy and this clinical role of mscs, increasing the demand of msc technique [2,10]. different literature search reported regarding presented stem cell therapy and commonly, use of various chemical stimulant compounds including recombinant cytokine, growth factors, stimulate proliferators and involved in the differentiation process for the normal development of cells. such compounds have two sites of their application in one site necessitate for the regulation of process and another way, associated with various undesirable effects or disadvantages together with toxicity and excessive costs. therefore, these negative characteristics of chemical agents forced to scientists for search alternative natural product to complete the requirement for normal process and stimulants to be used as growth factors for stem cell proliferation in stem cell therapy [2].however, study exploring the potential and benefits of human breast milk stem cells in the feeding of infants including stem therapy and regenerative medicine. in the present study, we review the effects of alternative medicines either single or mixture, able to promote cell proliferation and differentiation of hmscs in neuronal lineage, with their underlying mechanisms. various literature search regarding alternative medicines or plant-based remedies have been used in traditional medicine practices from many yearsago for reducing a wide range of diseases. herbal medicines act as a potential promising alternative way of offering substantial improvement of patient conditions via significantly decreased disease symptoms. although the mechanisms of action of a single and polyherbal form of extracts mostly remain undetermined and under process. the study tries to define the effect of different herbal extracts on stem cells proliferation and differentiation, and along with the effect of cytotoxic, may provide indepth insights into their disease-curing mechanisms [21-22]. recently, the use of medicinal herbs or herbal preparation has supported new complementary and alternatives source of growth or stimulant factors for the proliferation of stem cell and these herbal medicines are potentially beneficial for treating various clinical challenges. ayurveda declared, more than a few plants of this formulation, the so-called ‘media' plants, possess different properties. these plant extracts have the neuro-protective ability and in vitro studies was proven that decreased induced neurotoxicity, so that can be able to treat various neurodegenerative disorders like parkinson's disease, and alzheimer's disease[23-25]. scientifically, proved that media' plants act as stimulants to proliferate stem cells and fulfill the required number of cell for patient transplantations. for example, shorearobusta resin, and yashada bhasma have shown a protective effect in brain and deoxyribonucleic acid (dna) damage [26] another important indian common herbal medicine, phyllanthus emblica, its fruit has effectively involved in reducing dna damage in brain cells and also improved genomic stability in neurons and astrocytes [27]. in other in vitro study, phyllanthus emblica inhibiting the activities of hyaluronidase and collagenase type 2 [28]. effects of medicinal plants as a stimulant on human stem cells proliferation in this part of the article, discuss the regarding medicinal plants and their active phytoconstituent show to involve an increasing number of stem cell and differentiation in a specific lineage. numerous studies have reported that herbal extracts either single orpolyherbal formulation have a positive effect in the increasing number of stem cell, due to containing a plethora of active phytochemicals such as polyphenols and flavonoids. polyherbalism, in "sarangdharsamhita" has been highlighted as the concept of polyherbal and supported the active nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 118 nepjol.info/index.php/njb. phytochemicals components of single plants have been well established. usually, bioactive molecules are present inan individual plant with less amount and insufficient to achieve the desired therapeutic effects. therefore, a few scientific studies evidence have exposed that combined various plant may produce a more positive effect, due to herb-herb interaction or synergism, as compared to individual use of the plant extract[29-31]. the promising effect of the various parts of medicinal plants including roots, leaves, stem, and fruits, are used in wide range of herbal drug preparations for the treatment of different clinical diseases. in traditional medicine, these plantderived bio-active chemicals are synergistically very useful in treating and curing clinical diseases and also decreasing their symptoms. different clinical studies elaborate different activity of bio-active chemicals on the endothelial or vascular genesis, angiogenesis, antiadipogenic, osteogenic, neurogenic, and also showed proliferative effects on human mesenchymal stem cells. these studies were confirmed by rna expression analysis [2]. curcumin(1e,6e)-1,7-bis(4-hydroxy-3-methoxy phenyl) hepta-1,6-diene-3,5-dione or diaryl heptanoid,)(figure-2) is a bright yellow phytopolyphenol compound or bioactive molecule and found in flower of curcuma longa (turmeric), belonging to the family and zingiberaceae (a member of the ginger) and one of the oldest indian medicine. it possesses a wide range of pharmacologic properties –anticancer, antioxidant and anti-inflammatory properties. it is also used as an herbal supplement and also in cosmetics ingredient, food coloring and food flavoring. curcumin is an active biochemical which can be stimulate developmental and adult hippocampal neurogenesis it may enhance neural plasticity along with its repair [32]. figure 2. showing curcumin, a linear diarylheptanoid (consist of two aromatic rings (aryl groups) joined by a seven carbons chain (heptane) and having various substituents). wang et.al., (2014) showed the neuroprotective effects of diarylheptanoid in animal models through the inhibition of aβ generation and subsequently, induced autophagy by downregulatingpi3k/akt/mtor signaling pathway [33]. some other studies showed these yellow bioactive molecules exhibited the biphasic effects on the propagation and discrimination of stem cells and also on spinal cord neural progenitor cells and embryonic neural progenitor cells [32,34-35]. ginkgo biloba is one of the oldest chinese plant species and possess different tradition medicinal properties like-dementia, eye problems, intermittent claudication, tinnitus, and other health problems including asthma, bronchitis, and kidney and bladder disorders and act as a dietary supplement. it was proven that ginkgo nuts are members of the royal court for senility. zhuang et al., showed that ginkgo extract contains salvianolic acid b (sal b induces the proliferation of nspcs) and involved in the proliferation of endogenous neural stem cells in vascular dementia rats. leaves of ginkgo (figure3) contains many active molecules like 22–24 % flavonoids and 5–7 % terpene trilactones (gingolides and bilobalide) and also egb 761(ginkgolid a, b, c) and bilobalide (egb 761) and showed neural modulatory. it acts as antitumor, anti-aging, hepato-protective and cardioprotectiveagents [36]. figure 3 leave of ginkgo biloba. some recent studies have evidence that this extract involved in the regulation of different neurotransmitter level and capable to strongly inhibit monoamine oxidase a and synaptosomal nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 119 nepjol.info/index.php/njb. uptake of da, 5-ht and norepinephrin. a few of other in vitro studies showed this extract can significantly enhance the proliferation of mouse cochlear nscs and differentiation into functional neurons [37]. another chinese medicinal extract, scutellaria baicalensis georgi, known as chinese skullcap (figure-4), is a species of flowering herbs and belonging to the lamiaceae family, it is also known as a golden herb from the garden of chinese medicinal plants. it found in many european, east asian countries, and the russian federation. it is frequently used in chine and officially include in chinese pharmacopoeia. its dried root is known as huang-qin and used in the preparation of several chinese herbal medicines. root has been applied in the treatment of various clinical issues such as diarrhea, dysentery, hypertension, hemorrhaging, insomnia, inflammation and respiratory infections. scutellaria baicalensi contains active compound baicalin (flavonoid) and promotes neural differentiation but inhibits glial formation by regulating expression of stat3 and bhlh in e15–16 embryonic neural precursor cells (npcs)[38]. figure4 plant of chinese skullcap source https://www.flickr.com/photos/salvias/56665 69724. carica papaya (figure-5) belong to family caricaceae and its origin is in the tropics of the americas, perhaps from southern mexico and neighboring central america. the leaves, seeds, roots and unripe pulp of papaya showed different medicinal activity like antidiabetic, antiobesity, antitumor, antimicrobial agents and healing properties. also involved in the treatment of dengue fever and ulceretic [39]. other in vitro study the leaf extract up regulated the synthesis of thrombopoiesis related cytokines interleukin-6 and also upregulated stem cell factor by hmscs, isolated from exfoliated figure 5 carica papaya source https://www.flickr.com/photos/plsc100/31730 9400/ deciduous teeth. it has an effect on improving thrombocyte counts in both human and murine models, therefore, c. papaya leaf extract as a precious potential therapeutic agent for clinical diseases [39]. korean mistletoe, known as viscum album (european mistletoe), has been traditionally used as a medicinal plant. the extract of the plant contain lectins act as therapeutically active biomolecules and shown to be cytotoxic against tumor cells. the various report as shown proliferative activities on placenta-derived hmscs. the extract can regulate cytotoxicity on hepg2 cancer cells at low concentrations of 1–5 pg/ml but proliferates naive placenta-derived hmscs by way of autophagy mechanisms [40].in some studies, it can inhibit telomerase activity and result in dna fragmentation and tumor cell apoptosis[41]. dhanvantar kashaya (a decoction of herbs having regeneration property) prepared on the base of the concept of polyherbal formulation (pf). in vitro study, this formulation has regeneration propertyon wharton jelly mesenchymal stem cells (wjmscs). pf has used as a stimulant growth enhancer increased the proliferation rate; also decreased the turnover time, and also delayed senescence. it is able to improve the yield and quality of stem cells in vitro nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 120 nepjol.info/index.php/njb. and act as a non-toxic supplement for the culturing of a cell [42]. dan-qi-tongmai-pian, is chinese herbs extract namely prepared by combination of astragalus and salvia, has shown negative apoptosis of bmderived hmscs through stimulating c-iap-1/2 expression and restricting caspase-3 activation [43]. ayurveda literature search regarding medicinal plant, in ayurveda pharmacopeia, out of 200 rasayanadrugs, some herbal extracts such as mucuna gigantean, angelica sinensis and salvia miltiorrhiza are neurogenic and has a specific tissue affinity with specific target action (gamitva in ayurveda-‘reaching the target'.) in the brain. these remedies have shown much promise in the proliferation and differentiation of hmscs in neuronal cell differentiation [2]. the effect of these neurogenic medicine on hmscs are shown to differentiate into neural cells in vitro, confirmed by rna expression of some neurogenic markers like epidermal growth factor (egf) or brain-derived neurotrophic factor (bdnf), some of protein and nestin, a marker of neural precursors [44]. angelica sinensis (danggui) is commonly known as dong quai or "female ginseng" and its family apiaceae. its originates in china and grows in cool high altitude mountains like japan, and korea. numerous studies have shown its dried root identified as radix angelica sinensis (ras) are frequently used in china. it is more effective to treat various clinical disorders blood deficiency, menstrual disorders such as dysmenorrhea and irregular menstrual cycle and promote blood circulation [45]. radix angelica sinensis (ras) contain many active chemicals like ferulic acid, in vitro it can reduce β-amyloid peptide (induced neurotoxicity& tau phosphorylation). other study showed ferulic acid, able to inhibit neurotoxic β-amyloid peptide aggregation in animal models. recently, some report showed that higher percentages of neurallike cell differentiation from adhmscs compared with hmscs treated with butylated hydroxyl anisole. it is a commonly used neuronal inducer, act as a stimulant. these finding supported thatferulic acid significantly treat neurodegenerative disorders [45-46]. mucunagigantea (family leguminosae) extracts contain active constituent l-dopa, (act as a precursor for dopamine neurotransmitter). the extract of mucunagigantea, involved in stem cell therapy specifically nerve-related treatment and also enhances stem cell related treatment capacity.in 2012, kongos reported that human breast milk-derived hmscs, when treated with 1 % acetic acid extract of mucunagigantea, showed high proliferative properties in neural differentiation. it can also promote up-regulation of mrna expression of nestin (neural marker) and also β-iii tubulin (an immature neuron marker) [2]. salvia miltiorrhiza (chinese or traditional chinese or pinyin: dānshēn), also known as red sage, chinese sage, tan shen, or danshen, perennial plant. it belongs to lamiaceaefamily. frequently, used in traditional chinese medicine and native place in china and japan. its root extracts differentiating wharton jelly-derived hmscs into neural cells with high expression of mrna nestin, β-tubulin, neurofilament, and glial fibrillary acidic protein, and showed significant morphological changes. the study was also confirmed by high expression of a neural cell marker i.e., neurite outgrowthpromoting protein [47]. moringaoleifera (family moringaceae) is an indian fast-growing, deciduous plant and widely cultivated for its young seed pods. its seeds contain many bioactive molecules morning [4-(αl-rhamnosyloxy) benzyl isothiocyanate; gmgitc], is an isothiocyanate. in vitro studies showed isothiocyanate induced pdlscs toward neural progenitor differentiation. it can increase the expression of neuronal genes, involved in neuron cortical development. furthermore, moringin are also up regulated genes involved in osteogenesis and adipogenesis [48-49]. gardenia is a flowering plant (figure-6), belongs to coffee family, rubiaceae, and its native place are tropical and subtropical regions of africa, asia, madagascar, and pacific islands. its crude extract elevated the numbers of neun positive neurons in the hippocampal dg after 8 weeks of nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 113-123 kumari 2019 ©njb, biotechnology society of nepal 121 nepjol.info/index.php/njb. treatment and increased the surface density of brdu-positive cells. this extracts act as an antidepressant and promoted neurogenesis in the hippocampus [50]. figure6 flower of gardenia source https://en.wikipedia.org/wiki/gardenia conclusion stem cell therapy has an enormous positive prospective view in various scientific applications among the presence of ayurvedic stimulating mediator like herbal extract. as known from ancient time herbal drug are trusted medicine for the treatment of disease and reduced their symptoms because of the presence of different active molecules either single compounds or complex combined compounds which act synergetic effect for better therapeutic value. these extracts showed in vitro study the unbelievable positive effect on stem cell differentiation and proliferation still the mechanism of herbal medicine is unknown and modes of action should widely study. according to the literature reviewed, stem cell therapy are targeting various neurogenesis and it works for brain repair in different neurodegenerative treatment. as well known, in combination of stem cell with an herbal extract which acts as stimulants have shown amazing finding and provide excellent treatment of clinical issues. stem cell differentiation into a different specific part like chondrogenic, osteogenic, vasculogenic, neurogenic potential might bevery important for future clinical therapeuticsandgenerated tissue use for replacements in various cases like osteoporosis, heart disease, parkinson's disease. however, various studies on hmscs derived from clinical waste and human breast milk, are need explored, because these are allied with lessermoral issues rather than other source and associated with the easiest sampling method. various literature searches about stimulant, most of the artificial stimulants are associated with major adverse effect and also very expensive rather than herbal extracts. herbal stimulants showed a great effect on proliferation and differentiation of stem cell in neuron cell growthand cost-effective, highly available, safe alternative source for stem therapeutic application globally. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate not applicable. abdollahpour, g: a review on leptospirosis. proceedings of leptospirosis research conference, japan, matsoyamam ;1990: 34‐ 37 references 1. strauer be, kornowski r : stem cell therapy in perspective. circulation. 2003 107:929–34. 2. udalamaththa vindya lankika, chanika dilumi jayasinghe and preethi vidya dagama :potential role of herbal remedies in 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8:9153. 50. yu y, he j, zhang y, luo h, zhu s, yang y, et al.: increased hippocampal neurogenesis in the progressive stage of alzheimer's disease phenotype in an app/ps1 double transgenic mouse model. hippocampus.2009 19(12):1247-53. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 doi: https://doi.org/10.3126/njb.v7i1.26947 ©njb, biotechnology society of nepal 21 nepjol.info/index.php/njb. original research article ethanol extract of choerospondias axillaris fruit pulp enhances haematological parameters in oncorhynchus mykiss cultured in nepal shubha ratna shakya12* and shyam narayan labh2 1central department of zoology, tribhuvan university 2amrit campus, tribhuvan university, nepal abstract the present outdoor experiment was carried out to evaluate the effect of dietary supplementation of lapsi choerospondias axillaris (roxburgh, 1832) on haematological parameters in rainbow trout oncorhynchus mykiss in nepal. the lapsi fruits were obtained from local market of kathmandu. the feeding trail was conducted for 90 days. about 270 trout with similar body weight (5±1g) were distributed randomly at the rate of 15 fishes per cage (1m3) into 18 cages placed in raceway pond. six practical diets containing 40% protein were prepared as t1 (0.0 g kg-1) t2 (0.1 g kg-1), t3 (0.2 g kg-1), t4 (0.4 g kg-1), t5 (0.8 g kg-1) and t6 (1.6 g kg-1) supplemented by the ethanol extract of lapsi fruits along with other usual ingredients viz. fish meal, wheat flour and cod liver oil etc. at the end of the experiment the haematological parameters were measured. a significant difference (p< 0.05) in haematological parameters was observed between the treated diets fed groups to that of control diet fed group. total erythrocytes count (rbc), haematocrit (hct), haemoglobin concentration (hb), and erythrocyte indexes (mcv, mch and mchc) were found significantly higher in t4 (0.4 %) diet fed trout as compared to the control. a minimum of 0.4 % (0.4g kg-1) lapsi fruit extract in fish feeds gave more increase in haematological parameters of oncorhynchus mykiss. inclusion of lapsi fruit extract at 0.4 % concentration is therefore could be used effectively in aquaculture. keywords: oncorhynchus mykiss, growth, lapsi, haematocrit, haemoglobin *corresponding author email: shubharatnashakya@gmail.com introduction aquaculture is one of the important sectors contributing significantly in the nepalese economy. aqua farmers are encouraged towards intensification of culture system to increase production and profit in the world [1]. in intensive aquaculture stress level in fish impairing immune responses against pathogens leads to disease outbreaks in fishes [2, 3]. disease is one of the most important constraints of fish production both in culture system, as well as wild condition. fish production is decreased due to the occurrence of disease caused by different pathogens in aquaculture. to overcome this problem and to achieve the sustainable development of aquaculture, control of infectious diseases, parasites and maintenance of good health of cultured fish concern the most. various chemical agents, herbal extracts, and nutritional factors which stimulate the nonspecific defence mechanisms are used for growth performance and control of infectious diseases in fish [4] caused by various pathogens. despite the use of a large number of antibiotics, drugs, pesticides, and chemotherapeutics to control diseases, they are not so effective and ecologically unsafe. several studies have confirmed the presence of active ingredients responsible for various biological activities [5] in plants and many of them have already been tested against various diseases to test its immunostimulant efficacy [6] in aquaculture. there are several medicinal plants used in fish aquaculture are cassia alata, calophyllum inophyllum, clinacanthus nutans, clinacanthus sp., glinus oppositifolius, hura crepitan, momordica charantia, ocimum sanctum (red), ocimum sanctum (white), ochrocarpus siamensis, phyllanthus acidus, phyllanthus amarus, phyllanthus debelis, phyllanthus reticulatus, phyllanthus urinaria, psidium guajava, tinospora crispa, tinaspora cordifolia [7]. additionaly, many herbal extracts have been tested or its efficacy nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 shakya and labh 2019 ©njb, biotechnology society of nepal 22 nepjol.info/index.php/njb. against bacterial challenge in fish [8]. dietary supplementation of anthra quinone extract, zingiber officinale and curcuma longa stimulated immunity and enhanced resistance against pathogen aggravated stress in fish. similarly, feed incorporated with natural herb enhanced the non specific immune responses in c. mrigala against the pathogen aggravated stress of pseudomonas aeruginosa [9, 10]. nowadays, the use of natural plants as growth promoter and immune-stimulator along with their compounds such as essential oils and herbal extracts has improved the non-specific immune system in fishes. use of medicinal plants is a suitable alternative to synthetic antibiotics, chemical growth agents and synthetic immune-stimulants. herbal drugs improve the immune system and increase the host’s resistance to disease by increasing the number of white blood cells and production of antibodies [11]. citarasu et al., (2006) reported that immune-stimulants are substances, which increase the non-specific defense mechanism and provide resistance against pathogenic organisms [12]. thus the use of cheaper and effective natural herbal extracts in the diet of fishes is gaining momentum which is less toxic and reduces the residual load to the aquatic environment [13]. these herbal extracts in aqua feed are less toxic and reduce residual load in the aquatic environment. dietary natural herbs incorporation not only enhance growth but also lead to an augmentation in non-specific immunity, antioxidation, enzyme activity, and disease resistance in fish due to presence of active phytochemicals [14, 8,15]. improving immune system in valuable fish species such as rainbow trout is the most important in cold water aquaculture. food additives, growth stimulants and immunestimulants, including chemical agents, nutritional factors, bacterial and probiotic components, and animal or plant extracts affect the immune system and body defenses against disease agents [16]. increased use of antibiotics, growth stimulants and chemical compounds leads to increased resistance of microorganisms against antibiotics and drugs. antibiotic resistance is one of the most fundamental problems in aquaculture industry [17]. drug resistance, decreased meat quality and high cost lead to the use of natural stimulants in fish farming, especially herbal and traditional plants [18]. nowadays, the use of natural plants as growth and immune stimulant factors along with their compounds such as essential oils and herbal extracts has improved the non-specific immune system in fish farming. using traditional and medicinal plants as safety stimulants is a suitable alternative to synthetic antibiotics, chemical growth agents and immune stimulants. immune stimulants, especially herbal medicine improve the immune system and increase the host’s resistance to disease via increasing the number of white blood cells and production of antibodies [11]. lapsi, choerospondias axillaris (roxburg, 1832) of family anacardiaceae is grown in 301 village development committees of 29 hill districts of nepal [19]. its fruit is rich in vitamin c content [20] and enhance the immune system [21] of the organisms. phenolic and flavonoid compounds [22] present in lapsi fruit pulp are antioxidant agents which can inhibit free radicals, so they can be effective in preventing many oxidative diseases such as cancer. these compounds also have antibacterial and antifungal effects [23]. rainbow trout oncorhynchus mykiss (walbaum, 1792),) is the most preferred cold water fish species in aquaculture industry of nepal it is one of the commercially cultured cold water fish in nepal. it is an exotic fish species introduced in 1968 and 1971 from india and in 1988 from japan to substitute fish import in star hotels catering for tourists, use of cold-water resources for aquaculture and to promote of fishing tourism in hill and mountains of nepal for uplifting the living standard of people [24]. it is an important commercial fish in nepal with maximum market demand and acceptability as food by the consumers due to their taste and flesh. the “one village one product (ovop)” program has selected rainbow trout as one of the products in nepal from 2063/64. this program nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 shakya and labh 2019 ©njb, biotechnology society of nepal 23 nepjol.info/index.php/njb. was implemented in nuwakot district in the same fy 2063/64 in order to promote and product cold water fish farming. haematological parameters are gradually becoming a routine practice for monitoring health status in fish [25, 26, 27] to interpret physiological responses to environmental stress [28, 29] and changes in the proportion of blood cells may be indicative of a disease or an exposure to chemicals [30]. blood indices must be analyzed when animals are exposed to pollutants [31], stress [32], infections [33, 34], parasitism [35] and seasonality [30]. blood parameters changes during diseases or any changes occurring in the organism as a result of injuries to organs or tissues related to infectious diseases [36, 37]. it has been observed that blood parameters such as haematocrit, haemoglobin concentration and rbc count are related to environmental factors such as water temperature and salinity [38]. the knowledge of the haematological parameters can be used as an effective and sensitive index to monitor physiological and pathological changes in fishes [39]. normal ranges for various blood parameters in fish have been established by different researchers in fish physiology and pathology [40,41].these parameters are also closely related to the response of the fish to the environment, an indication that the environment where fish lives could exert some influence on the blood characteristics [42, 43]. haematological parameters of rainbow trout fingerlings fed with lapsi fruit extract supplemented diet have not studied. thus, the present study was conducted to evaluate the efficacy of lapsi extract in different amount in diets on some haematological parameters of rainbow trout in intensive aquaculture. table 1. ingredients of experimental diets (%) ingredients experimental diets (% inclusion) g kg-1 t1 t2 t3 t4 t5 t6 fish meal† 29.31 29.31 29.31 29.31 29.31 29.31 soya meal‡ 14.52 14.52 14.52 14.52 14.52 14.52 groundnut oil cake† 9.17 9.17 9.17 9.17 9.17 9.17 rice powder† 14.16 14.16 14.16 14.16 14.16 14.16 wheat flour† 14.43 14.43 14.43 14.43 14.43 14.43 corn flour† 11.37 11.37 11.37 11.37 11.37 11.37 sunflower oil† 3 3 3 3 3 3 cod liver oil† 2 2 2 2 2 2 vitamin & mineral premix§ 1 1 1 1 1 1 c. axillaris extract† 0 0.01 0.02 0.04 0.08 0.16 betain hydrochloride†† 0.02 0.02 0.02 0.02 0.02 0.02 bht(butylated hydroxytoluene)†† 0.02 0.02 0.02 0.02 0.02 0.02 cmc (carboxymethyl cellulose) †† 1 0.99 0.98 0.96 0.92 0.84 total 100 100 100 100 100 100 †ingredients like fish meal, soya meal, groundnut oil cake, rice powder, wheat flour, corn flour, sunflower oil and cod liver oil were procured from local market of kathmandu valley. ‡ruchi soya industries, raigad, india. §composition of vitamin mineral mix (emix plus) (quantity 2.5kg -1) vitamin a 55,00,000 iu; vitamin d3 11,00,000 iu; vitamin b2 2,000 mg; vitamin e 750 mg; vitamin k 1,000 mg; vitamin b6 1,000 mg; vitamin b12 6 µg; calcium pantothenate 2,500 mg; nicotinamide 10 g; choline chloride 150 g; mn 27,000 mg; i 1,000 mg; fe 7,500 mg; zn 5,000 mg; cu 2,000 mg; co 450 mg; ca 500 g; p 300g; llysine 10 g; dl-methionine 10 g; selenium 50 mgl-1; selenium 50 mgl-1; satwari 250 mgl-1; (lactobacillus 120 million units and yeast culture 3000 crore units). †fruits of c. axillaris were obtained locally and then extracts were prepared from the pulp of lapsi fruits. ††himedia laboratories, mumbai, india. nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 shakya and labh 2019 ©njb, biotechnology society of nepal 24 nepjol.info/index.php/njb. the use of lapsi in diet in aquaculture is thus anticipated to be an excellent strategy for the prevention of infectious microbial diseases and to replace antibiotics and chemotherapeutic. materials and methods study area and period the experiment was carried out in soodo rainbow trout culture farm (nepal) for 90 days. rainbow trout were obtained from hatchery of soodo rainbow trout farm, private fish farm in ranipauwa, nuwakot district in nepal. experimental design the trout were acclimated for seven days feeding on control diet. a total of two hundred seventy acclimated trout of similar size (average weight 92.37±0.039 g) were randomly allocated to 18 wooden bordered plastic cages placed in race way pond, 15 fish in each cage. there were three replicates for each treatment. t2, t3, t4, t5, and t6 were treatment groups and t1 was control. temperature ranged from 12 °c to 19 °c, dissolved oxygen ranged from 4.13 to 6.12. mg-1 and ph ranged from 7.33 to 7.67 throughout the study period. feed formulation and preparation of experimental diets the crude extract of the pulp of lapsi fruits was prepared by using ethanol (70%) as described by [43]. six experimental diets (40% protein) were prepared containing similar ingredient composition. diet one was control diet (t1) without extract of lapsi fruits. other five diets were supplements with lapsi fruit’s extract t2 (0.1 g kg-1), t3 (0.2 g kg-1), t4 (0.4 g kg-1), t5 (0.8g kg-1) and t6 (1.6 g kg-1). table 1 shows the ingredients of the feeds. proximate analysis of feeds the proximate composition of the experimental diets (table 1) was analysed following the standard methods of the association of official analytical chemists [44]. the moisture content was determined by drying at 105 oc to a constant weight. nitrogen content was estimated by automated kjeldahl apparatus (2200 kjeltec auto distillation, foss tecator, sweden) and crude protein was estimated by multiplying nitrogen percentage by 6.25. ether extract (ee) was measured using a soxtec system (1045 soxtec extraction unit, tecator, sweden) using diethyl ether (boiling point, 4060 oc) as a solvent and ash content was determined by incinerating the samples in a muffle furnace at 600 oc for 6 hours. nitrogen free extract (nfe) was calculated by difference i.e., nfe = 100-(cp+ee+cf+ash). nitrogen free extract (nfe) = 100(cp+ee+cf+ash) maintenance and feeding each cage was covered by mosquito net to prevent the fish from jumping out. all cages were kept in the same raceway pond under adjust the feeding status of trout. the fish were hand fed at 3 % of body weight two times a day at a randomly 5 trout were weighed from each cage biweekly interval to balance the diet. blood collection three fish were randomly sampled from each cage and were anaesthetized with buffered tricaine methane suffocate (ms-222; 5 mg l−1). blood was drawn from the caudal vein of treated and control fish using 2 ml heparinized plastic syringes and gauge hypodermic needles. the syringe is flushed with ethylenediamine tetraacetic acid (edta) as the anticoagulant. table 2. proximate composition of experimental diets (%) ingredients experimental diets t1 t2 t3 t4 t5 t6 dry matter (dm) 97.15 97.43 97.59 97.71 96.93 97.014 moisture 2.85 2.57 2.41 2.29 3.07 2.986 crude protein (cp) 31.16 31.07 31.32 31.14 31.22 31.239 ether extract (ee) 6.56 6.37 6.11 6.98 6.755 6.855 crude fiber 8.32 8.32 8.43 8.79 8.845 8.997 ash 9.23 8.73 9.53 7.69 7.84 7.458 nfe 44.73 45.51 44.61 45.4 45.34 45.451 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 shakya and labh 2019 ©njb, biotechnology society of nepal 25 nepjol.info/index.php/njb. the collected blood was transferred to eppendorfs of 1.5 ml capacity and stored in refrigerator for further analysis. these blood samples were used for determining erythrocyte count [45] and hemoglobin concentration [46]. heamatocrit values (hct) were calculated according to the formulae mentioned by britton [47). haematological examination total red blood cells were determined in a 1:100 dilution of blood sample in shaw solution [48] with an improved neubauer haemocytometer according to the procedure described by [49].the total numbers were reported as 106 per mm3 for rbc and 103 per mm3 for wbc [50]. haemoglobin concentration was estimated by standard cyanmethemoglobin method [51]. heamatocrit value (hct) was determined after blood centrifugation in micro-haematocrit capillary tube, using a micro-haematocrit centrifuge and micro-haematocrit reader [52]. the mean cell volume (mcv), mean cell haemoglobin (mch), and mean cell haemoglobin concentration (mchc) were calculated [45] using the formulae: mcv (fl) = (hct / rbc) x 10 mch (pg) = (hb / rbc) x 10 mchc (g/dl) = (hb / hct) x 100 statistical analysis the results are presented as means ± se, difference between haematological parameters among control and treatment groups were analyzed by one way analysis of variance (anova) followed by duncan’s multiple range test. results the haematological parameter of rainbow trout (oncorhynchus mykiss) fed with different doses of lapsi fruit extract was shown in table 4. the red blood cells count was significantly higher in treated groups compared to the control group (t1). overall, a higher rbc count was found in the group fed with t4 diet (5.86±0.737) compared with group fed with t1 diet (4.15±0.396). haemoglobin increased significantly in trout fed with treated diets compared with control (p < 0.05). the maximum concentration of haemoglobin was recorded in the group fed with t4 diet (15.24±3.204) followed by t5, t6, t3, t2 and the t1 (table 3). haemocrit (hct) was increased significantly in treated groups compared with control (p < 0.05). the maximum hct value was recorded in t4 (36.83±12.42) group and minimum in control group (30.57±1.06). the minimum mcv value was observed in t4 group (130.94±4.00) and maximum values was recorded in control group (184.57±11.29) and mch maximum value was recorded in control group (38.50±11.85) and minimum in t4 (30.77±5.84). mchc increased significantly in treated groups compared with control (p< 0.05). mchc maximum value was recorded in t4 (29.99 ±0.07) and minimum in control group (21.27±6.62). rbcred blood cell count, hbhaemoglobin, hcthaematocrit value, mcvmean corpuscular volume, mch-mean corpuscular haemoglobin, mchc-mean corpuscular haemoglobin concentration. t1= control (without lapsi extract), t2= 100 mg lapsi extract/kg, t3= 200 mg lapsi extract/kg, table 3. haematological parameters of rainbow trout (oncorhynchus mykiss) fed with diets of different levels (mean ± sd) of lapsi fruit’ pulp extract for 90 days of the experiment treatments haematological parameters rbc (x106 mm-3) hb (g/dl) hct (%) mcv (fl) mch (pg) mchc (g/dl) t1 4.15±0.396 9.52±1.313 30.57±1.06 184.57±11.29 38.50±11.85 21.27±6.62 t2 4.59±1.523 12.07±3.444 31.73±7.62 177.87±4.00 36.23±2.15 22.05±6.34 t3 4.61±0.912 13.94±2.327 32.65±0.53 142.16±8.03 35.56±7.25 24.94±22.83 t4 5.86±0.737 15.24±3.204 36.83±12.42 130.94±4.00 30.77±5.84 29.99 ±0.07 t5 5.81±0.36 14.57±2.082 35.58±9.94 157.58±13.65 32.96±3.46 28.49±6.80 t6 4.88±0.66 14.22±1.151 33.75±9.23 165.51±26.03 34.97±6.17 25.41±9.54 nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 shakya and labh 2019 ©njb, biotechnology society of nepal 26 nepjol.info/index.php/njb. t4= 400 mg lapsi extract/kg, t5 = 400 mg lapsi extract/kg and t6 = 1600 mg lapsi extract/kg. discussion in fishes, blood is a patho-physiological reflector of the entire body and the haematological parameters such as leucocyte count, erythrocyte count, hematocrit and hemoglobin are routinely tested to monitor the health status of fish and any deviation from the normal ranges may indicate a disturbance in the physiological process. [53,54]. the changes in the blood parameters of c. gariepinus caused by stress due to exposure to environmental pollutant, diseases or by pathogens have been studied by many researchers especially in capture fisheries [55,56]. hematological parameters may provide an index of the physiology status of fish which are directly influenced by sex, size, seasons, slow or fast movement and age of fishes [57] and indirectly influenced by quality of water, temperature, food availability physiological status of fish [58], microbial infection of fish and toxicants [59]. ologhobo (1992) [60] reported that the most common blood variables consistently influenced by diet are the haematocrit (hct) and haemoglobin (hb) levels. the changes in rbc count, hb concentration, hct, mcv, mch and mchc show health status of fish [61]. haemoglobin, rbc, hct, mcv, mch and mchc values obtained in the experiment almost agreed with earlier workers. according to the results, lapsi extract supplemented diets could increase hemoglobin content and rbc numbers in treated groups compared to control group. this agreed with the work of [62] who also reported similar increase in haematological parameters in nile tilapia. in agreement with the present findings, sahu et al., (2007) [63] found higher rbc counts in labeo rohita fingerlings fed with magnifera indica kernel when compared to control. shalaby et al., (2006) [64] reported significant increases in rbc, hb and htc of nile tilapia fed with garlic that support the present study. faisal (2003) [65] reported significant decrease of mch and mcv in clarias garepins fed with garlic that is also similar to the findings of the present study. c. gariepinus fed a. barbadensis leaves paste showed significant (p < 0.05) increase in haematocrit (hct), haemoglobin, rbc, wbc in comparison to the control group. this was in agreement with the work of haghigi and rohani (2013) [66] who observed similar increase in the haematological parameters in rainbow trout fed ginger powder. these were also similar findings of farahi et al. (2012) [67] who reported significant enhancement (higher values) of wbc and pcv in diet supplemented with m. officinalis and aloe vera. concerning the effect of the lapsi supplemented diet on haematological parameters of rainbow trout (oncorhynchus mykiss) indicated increased haematocrit, haemo-globbin, erythrocyte, mch, and mchc in comparison to the control group (p<0.05) (table. 3). these could be attributed to the fact that, the lapsi in the diet has increased the haematological parameter values as a result of hematopoietic stimulation [68, 69, 70]. conclusion number of chemicals and drugs used in aquaculture cause severe environmental problems. most of the antibiotics and drugs are banned because of their negative impact and effluent remittance in the fish muscle which may cause side effect to the consumer. herbal drugs, safer to fish and the environment are the viable alternative to antibiotics and other banned drugs. most of the herbs and herbal extracts are given orally. the farmer does not know about the importance of herbal drugs. so it is necessary make aware on the use of biological and ecofriendly approach of herbs and their products in aquaculture to the farmers. it will reduce the production risk, negative impact and production cost and also increasing fish production with sustainable way. in conclusion, the present study revealed that lapsi, choerospondias axillaris incorporated diet fed to rainbow trout, oncorhynchus mykiss improved haematological parameters. hence, 0.4 mg kg-1 lapsi extract could be used as a supplement in the diet of fish for better health. however, more research should be carried out on the uses of nepal journal of biotechnology. d e c . 2 0 1 9 vol. 7, no. 1: 21-29 shakya and labh 2019 ©njb, biotechnology society of nepal 27 nepjol.info/index.php/njb. choerospondias axillaris in order to obtain optimal health. acknowledgment i would like thankful to former head dr. ranjana gupta for providing necessary facilities. help 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amentoflavone and kaempferol glycosides from the aerial parts of cissampelos pareira hari prasad devkota*, sena miyazaki, shoji yahara school of pharmacy, kumamoto university, kumamoto 862-0973, japan abstract aerial parts of cissampelos pareira l. (family: menispermaceae), commonly known as “batulpate” in nepal, are used for the treatment of fever, indigestion and to stop bleeding after child birth. in this study, a biflavonoid, amentoflavone; and two kaempferol glycosides, kaempferol 3-o--d-glucopyranoside and kaempferol 3-o-d-glucuronopyranoside were isolated from 70% meoh extract of the aerial parts. structures of these compounds were elucidated on the basis of 1hand 13c-nmr spectral data. to the best of our knowledge, it is the first report on the isolation of these compounds from a plant belonging to family menispermaceae. keywords: cissampelos pareira; menispermaceae; batulpate; flavonoids; amentoflavone *corresponding author email: devkotah@kumamoto-u.ac.jp introduction cissampelos pareira l. (family: menispermaceae), commonly known as “batulpate” in nepal, is a climbing herb with perennial root-stock. it is distributed throughout nepal to about 3000 m altitude. the aerial parts and roots are widely used as traditional medicines in various ailments. the juice of the plant is administered orally to stop bleeding after child birth and to counteract the loss of blood. it is also used as tonic, diuretic and for the treatment of fever and indigestion. the juice is also applied locally to treat skin diseases and the decoction is used to relieve the pain of dislocated bones. the roots are used for the treatment of indigestion, constipation, cough and cold and applied locally in snake bite [1]. previous studies have reported antiviral [2], antiprotozoal [3] and antifertility [4] activities of the extracts of aerial parts of c. pareira. regarding chemical constituents, alkaloids are reported as major constituents in leaves [5] and roots [6-10]. ramirez et al. [3] reported a chalcone-flavone dimer, cissampeloflavone as an antiprotozoal agent from the aerial parts and ganguly et al. [4] also reported the presence of flavonoids in the leaves of c. pareira. however, there are no other reports about individual flavonoids present in c. pareira aerial parts. thus, in present study, we focused on the phenolic constituents in the aerial parts and isolated one biflavonoid [1] and two flavonoid glycosides [2, 3], which are reported in this paper. materials and methods general experimental procedures optical rotations were measured with a jasco dip-1000kuy polarimeter. 1h-, 13c-nuclear nuclear magnetic resonance (nmr) spectra were measured on a jeol -500 spectrometer. chemical shifts are given in ppm with reference to tetramethyl silane (tms). column chromatography was carried out with mci gel chp20p (75 ~ 150 m, mitsubishi chemical industries co. ltd., tokyo, japan), sephadex lh-20 (amersham pharmacia biotech, tokyo, japan) and silica gel 60 (0.040-0.063 mm, merck kgaa, darmstadt, germany). thin layer chromatography (tlc) was performed on a precoated silica gel 60 f254 (aluminum sheet, merck kgaa, darmstadt, germany) and 10% sulfuric acid spray followed by heating was used for derivatization. plant material fresh aerial parts of c. pareira were collected on june 2010, from lumle, kaski district, nepal and shade dried for two weeks. a voucher specimen has been deposited at graduate school of pharmaceutical sciences, kumamoto university, kumamoto, japan. extraction and isolation the shade dried aerial parts (58.0 g) were extracted with 70% methanol (meoh) (1.5 l x 2 times) at room temperature and the combined extracts were evaporated under reduced pressure to obtain dried nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 1-4 devkota et al. ©njb, biotechnology society of nepal 2 nepjol.info/index.php/njb figure 1: structures of the isolated compounds table 1: 1hand 13c-nmr data of compound 1 in dmsod6 c no . c h, mult. (j in hz) c no. c h, mult. (j in hz) 2 163.9* 2” 163.7 * 3 102.5 6.82, s 3” 102.4 6.77, s 4 181.6 4” 182.0 5 161.3 5” 160.4 6 98.8 6.18, d (2.1) 6” 98.7 6.36, s 7 163.5* 7” 161.4 8 93.8 6.44, d (2.1) 8” 103.9 9 157.2 9” 154.6 10 103.9 10” 103.9 1' 121.2 1”’ 121.2 2' 127.6 8.02, d (2.4) 2”’ 128.0 7.58, d (8.8) 3’ 121.2 3”’ 115.7 6.70, d (8.8) 4' 159.5 4”’ 160.4 5' 116.1 7.12, d (8.5) 5”’ 115.7 6.70, d (8.8) 6’ 131.2 7.99, dd (2.4, 8.5) 6”’ 128.0 7.58, d (8.8) extract (10.0 g). the extract was then suspended in water and separated into water soluble and insoluble fractions. the water soluble fraction (7.9 g) was subjected to mci gel chp20p column chromatography (cc) and eluted successively with water, 40%, 70% and 100% meoh to obtain total 8 fractions (fr. 1~8). a flavonoid rich fraction, fr. 6 (200 mg, 70% meoh eluate) was subjected to sephadex lh-20 cc (meoh) to obtain 5 subtractions (subfr. 6-1~6-5). subfractions 6-3 and 6-4 were combined (30 mg) and subjected to silica gel cc (chcl3:meoh:h2o = 8:2:0.1) to obtain kaempferol 3-o--d-glucopyranoside; compound 2 (5.0 mg) and kaempferol 3-o--d glucuronopyranoside; compound 3 (2.0 mg). the water insoluble fraction (2.1 g) was subjected to sephadex lh-20 cc (meoh) to obtain 8 fractions (fr. i1~i8). among them, fr. i7 (74 mg) was subjected to silica gel cc (chcl3:meoh = 20:1) to obtain amentoflavone; compound 1 (2.4 mg). amentoflavone (1) a pale yellow amorphous powder; 1hand 13cnmr (in dmso-d6), table 1. kaempferol 3-o--d-glucopyranoside (2) a pale yellow amorphous powder; []d20 -35.8º (c 0.75, pyridine); 1hand 13c-nmr (in cd3od+d2o) table 2. kaempferol 3-o--d glucuronopyranoside (3) a pale yellow amorphous powder; []d20 -51.2º (c nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 1-4 devkota et al. ©njb, biotechnology society of nepal 3 nepjol.info/index.php/njb 0.52, pyridine:water = 1:1); 1hand 13c-nmr (in dmso-d6), table 2. table 2: 1hand 13c-nmr data of compounds 2 and 3 c no. 2 (in cd3od+d2o) 3 (in dmso-d6) c h, mult. (j in hz) c h, mult. (j in hz) 1 2 157.0 156.2 3 135.1 133.0 4 179.0 177.3 5 161.6 161.0 6 100.0 6.26, d (1.8) 98.6 6.02, d (1.8) 7 164.6 164.7 8 95.3 6.40, d (1.8) 93.8 6.22, d (1.8) 9 159.2 156.3 10 105.6 103.5 1' 122.6 121.0 2', 6' 132.0 7.97, d (8.5) 130.9 8.00, d (8.5) 3', 5' 116.2 6.96, d (8.5) 115.0 6.83, d (8.5) 4' 160.2 160.0 1” 103.7 5.02, d (6.7) 100.8 5.53, d (7.6) 2” 74.9 3.17―3.90 74.1 3.30―3.80 3” 77.4 3.17―3.90 75.4 3.30―3.80 4” 70.5 3.17―3.90 72.1 3.30―3.80 5” 77.0 3.17―3.90 76.4 3.30―3.80 6” 61.8 3.17―3.90 172.2 results and discussion in this study, the aerial parts of c. pareira, traditionally used for the treatment of various disorders in folk medicine were investigated for their chemical constituents. three compounds (1-3) were isolated from the 70% meoh extract by repeated column chromatography on mci gel chp20p, sephadex lh20 and silica gel. the structures of these compounds were identified as amentoflavone [11], kaempferol 3-o--d glucopyranoside [12] and kaempferol 3-o--d glucuronopyranoside [13] (figure 1) on the basis of nmr spectral data and comparison with reported values in literatures. to the best of our knowledge, it is the first report on the isolation of these compounds from a plant belonging to family menispermaceae. flavonoids and biflavonoid are one of the most widely studied chemical classes of natural products for their distribution in plant families, chemical diversity and various heath beneficial effects. amentoflavone has been isolated and identified from more than 120 plant species and reported to have several pharmacological effects including potent antioxidant and anti-inflammatory activities [14]. similarly, kaempferol and/or its glycosides were also reported from more than 400 plant species and showed potent antioxidant, anti-inflammatory, antitumor and antimicrobial activities [15]. in conclusion, three compounds, amentoflavone [1], kaempferol 3-o--d-glucopyranoside [2] and kaempferol 3-o--d-glucuronopyranoside [3] were isolated 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and pharmacokinetics of amentoflavone, a naturally-occurring biflavonoid. molecules. 2017 22:299. 15. calderón-montaño jm, burgos-morón e, pérez-guerrero c, lópez-lázaro m: a review on the dietary flavonoid kaempferol. mini rev med chem. 2011 11:298-344. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 25-32 issn 2091-1130 (print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 25 nepjol.info/index.php/njb fecal carriage of extended spectrum β-lactamases (esbl) producing escherichia coli and klebsiella spp. among school children in pokhara, nepal bharat prasad baral1,2, binita koirala sharma2, krishna gurung1, ram raja gurung4, hosuru subramanava supram2, balkrishna bhattachan3* 1pokhara bigyan thatha prabidi campus, pokhara , nepal 2manipal teaching hospital, pokhara, nepal 3siddhi memorial hospital, bhaktapur, nepal 4deurali-janta pharmaceuticals pvt. ltd., kathmandu, nepal abstract extended-spectrum β-lactamases (esbl) producing microbes in recent years have been a major problem in developing countries like nepal, with limited treatment options. this study aimed to determine the prevalence of esbl producing e. coli and klebsiella spp. in school children in pokhara, nepal. the study was conducted from june to october, 2015 at the microbiology laboratory of manipal teaching hospital, pokhara, nepal. antibiotic susceptibility test (ast) was done after isolation and identification of bacterial isolates. then, presence of esbl enzymes in e. coli and klebsiella spp. were tested by combination disc diffusion test using cefotaxime and ceftazidime alone and with clavulanic acid. out of total 309 school children, 211 (68%) bacterial isolates were detected from stool samples. among them, e. coli and klebsiella spp. were detected in 97 (46%) and 39 (19%) stool samples respectively. bacteria isolated from 14 (5%) stool samples were multi-drug resistant (mdr) positive. after applying combined disk method, 88 (29%) isolates were found to be esbl producer. emerging prevalence rate of esbl producing e. coli and klebsiella spp. are major problem in medical history. therefore, rapid need of surveillance for effective management of such mdr-strain is required. keywords: extended-spectrum β-lactamase (esbl), e. coli, klebsiella spp., children, nepal *corresponding author email: balkrishna_bhattachan@hotmail.com introduction extended-spectrum β-lactamases (esbl) producing organism was first isolated in 1983, germany, since it has been increasingly reported worldwide [1, 2]. the prevalence of esblpositive isolates depends upon wide range of environmental, genetic, geographical, age group, and type of infections [3]. emerging and increasing incidence of esbl related infections has been observed throughout the world [4]. esbls are class a β-lactamases carbapenems and are often plasmid-mediated enzymes with various genotypes [1]. the most common genotypes are the shv, tem, and ctx-m types [5]. other clinically important types include veb, per, bel-1, bes-1, sfo-1, tla, and ibc [6]. enterobacteriaceae family of gram-negative organisms; in particular, k. pneumoniae, k. oxytoca and escherichia coli produce esbls [7]. they are also produced by other gram-negative organisms, such as acinetobacter baumannii, proteus spp., pseudomonas aeruginosa, and salmonella spp. [8]. patients suffering from esbl producers have high mortality, longer hospital stay, high health care cost, and longer antibiotic therapy as compared to those patients suffering from nonesbl producers [9]. furthermore, they pose significant therapeutic challenges in the daily management of infectious diseases due to their resistance to additional classes of antibiotics reducing the effectiveness of alternative antimicrobial regimens [10]. worldwide, several studies [1, 5, 6, 8] have been conducted but only a few data are available concerning the genetic characterization of clinical isolates from nepal [7, 11]. the acquisition and expression of esbl enzymes among e. coli and klebsiella spp. have posed a serious public health problem. this study was conducted to determine the esbl producing bacteria in school going children without any clinical symptoms to observe the fecal carriage. study designing and setting the study was conducted in pokhara lekhanath municipality-26, where samples were collected nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 25-32 baral et al. ©njb, biotechnology society of nepal 27 nepjol.info/index.php/njb from three government (n=147) and three private schools (n=176). children from 4 to 15 years were enrolled who were apparently healthy and not taking any antibiotics 1 week prior to our study. after taking written informed consent and questionnaire from their parents, stool samples were collected in a sterile screw-capped container. demographic data like age, sex, antibiotic intake within 15 days, were included in questionnaire form. a total of 323 children were enrolled, where all children provided stool samples, except 14 who provided insufficient stool samples without any labeling. sample collection and processing around one gram of fresh stool sample was collected in a clean, well labeled, and sterile screw-capped plastic container. collected samples were kept in an icebox, transported within 1 hour, and processed immediately following standard operating procedures (sops) of microbiology to the laboratory of manipal teaching hospital (mth) in pokhara, nepal. if there was a delay in processing the specimens were kept in a freezer at 2–8°c. the general procedure of sample processing were explained in the flowchart (figure 1). isolation and identification of e. coli and klebsiella spp.by culture method stool samples were inoculated onto macconkey agar plates, which were incubated at 37°c for 24 hours. after proper incubation, plates were observed for the growth of the bacteria. isolated colonies were sub-cultured in nutrient agar and then plates were incubated at 37°c for 24 hours. gram-positive and negative bacteria were initially confirmed by gram staining and thereafter, identified by conventional biochemical tests such as (mrvp, indole, motility, protease, citrate, & o/f). antimicrobial susceptibility testing (ast) ast of all isolates was performed by kirby bauer's disc diffusion method and interpretation of the results was done as described in clinical laboratory standard institute (clsi) guideline 2016 [18]. for antibiotic susceptibility testing, antibiotic discs (himedia laboratories pvt. ltd., india) cefotaxime (30μg), ceftazidime (30μg), gentamycin (10μg), imipenem (10μg), nitrofurantoin (300μg), norfloxacin (10μg), piperacillin-tazobactam (100μg/10μg), and amikacin (30μg) were used. control strains of e. coli atcc 25922 were used in parallel as a part of quality control. organisms showing resistance towards two or more classes of antimicrobial agents were considered to be multidrug-resistant (mdr). screening and confirmation of esbl producing strains all the e. coli isolates were subjected to the screening test for esbl detection. screening test for esbl detection was done according to the clsi guidelines. isolates showing inhibition zone size of ≤ 22 mm with ceftazidime (30 μg), ≤ 25 mm with ceftriaxone (30 μg), and ≤ 27 mm with cefotaxime (30 μg) were interpreted as screening test positive for esbl production [18]. for the confirmatory test for esbl, two or three colonies of organisms were suspended in 0.5 ml of sterile broth and the turbidity matched to 0.5 mcfarland. using a sterile cotton swab the broth culture was uniformly swabbed on mha. all the e. coli isolates which were resistant to at least ceftazidime, ceftriaxone and/or cefotaxime were subjected to the esbl confirmatory test using ceftazidime (30 μg) and ceftazidime-clavulanic acid (30 μg & 10 μg) and the cefotaxime (30 μg) and cefotaxime-clavulanic acid (30 μg & 10 μg) combination disks. the tests were interpreted and a difference of 5 mm between zone of inhibition of a single disk and in combination with clavulanic acid (inhibitor) was confirmed to be produced by an esbl positive isolate [18]. klebsiella pneumoniae atcc 700603 was used as a control strain. statistical analysis pearson's chi-square test was used to determine the significant association of dependable variables. winpepi software (version 11.65) was used for quantitative data analysis. a p-value < 0.05 was considered to be statistically significant at 95 % ci. nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 25-32 baral et al. ©njb, biotechnology society of nepal 28 nepjol.info/index.php/njb figure 1: flowchart of isolation and identification of e. coli and klebsiella spp. from collected stool samples results as summarized in table 1, in 143 females, e. coli and klebsiella spp. were isolated from 43 (30%) and 18 (16%) respectively. in 166 male children, e. coli and klebsiella spp. were isolated from 54 (33%) and 21 (13%) respectively. e. coli and klebsiella spp. were most frequently found in children aged 6-10 years both in females (31% and 32%) and males (14% and 46%), respectively. of the total samples tested, 211 (68.3%) bacterial isolates were detected. among them, e. coli and klebsiella spp. were detected in 97 (46%) and 39 (19%) stool samples respectively. of the total tested samples, 14 (16%) isolates of bacteria were figure 2. antibiotic resistance pattern of klebsiella spp. 0 40 17 20 10 67 44 50 norfloxacin ceftazidime gentamicin amikacin imipenam piperacillin-… ceftaxime nitrofurantoin table 1.age and sex wise distributions in e. coli and klebsiella spp. in school children gender age (year) female male total e. coli klebsiella spp. total e. coli klebsiella spp. 4-5 7 2 (29) 0 (0) 6 3 (50) 0 (0) 6-10 104 32 (31) 15 (14) 127 41 (32) 19 (46) 11-15 32 9 (28) 3 (9) 33 10 (30) 2 (20) total 143 43 (30) 18 (16) 166 54 (33) 21 (13) table 2. distribution of e. coli and klebsiella spp. on the basis of esbl producer and mdr bacterial isolates total no. isolates (%) (n =211) multidrug resistance (mdr) isolates number (%) (n =14) esbl screening positive (%) (n =116) esbl producer confirm (%) (n =88) e. coli 97 (46) 9 (64) 84 (72) 74 (84) klebsiella spp. 39 (19) 5 (36) 32 (10) 14 (16) others gramnegative bacteria 27 (13) nt nt nt gram-positive bacteria 48 (23) nt nt nt nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 25-32 baral et al. ©njb, biotechnology society of nepal 29 nepjol.info/index.php/njb figure 3: antibiotic resistance pattern of e. coli. figure 2 summarizes that klebsiella spp. was resistance to piperacillin-tazobactam (67%); nitrofurantoin (50%) and cefotaxime (44%). as depicted in figure 3, the resistance pattern of e. coli was also found highest in piperacillintazobactam (62%) followed by norfloxacin (60%) and cefotaxime (50%) etc. found mdr strains, while 116 isolates were screened as esbl positive. by combined disk method, 88 isolates were confirmed to be esbl producer. among the total mdr bacteria, 9 (64%) were e. coli and 5 (36%) were klebsiella spp., 74 (84%) isolates of e. coli and 14 (16%) of klebsiella spp. were confirmed as esbl producer (table 2). discussion esbl producing gram-negative bacilli, particularly k. pneumoniae and e.coli, recently arose worldwide in a hospital and community-acquired infections (cais) as serious pathogens, and has been progressively rising. this upsurge introduces additional difficulties in treating patients affected by ebsls since it puts at risk the activity of numerous wide-spectrum antibiotics [7]. the present study exhibits that out of the 211 clinical isolates, 88 (42%) were found to be esbl producers, and these results were in accordance with a previously conducted study [11, 18]. additionally, the ratio of positive cases for the male to female children in this study was 1.2:1, which contrasted another study where the ratio for male to female patients was 1:2.2 [7]. however, this difference in the ratio may be due to a gender bias of male and female samples initially in both the experiments. thus, gender may not play a role in esbl susceptibility of a person. furthermore, e. coli positive cases were found in 97 (31%) clinical isolates, which was the predominant organism in this case. conversely, another study found e. coli to be positive in 81% of their samples, which is remarkably higher than that of our study [7]. the high occurrence of positive result may suggest that there is a possibility of normal flora of human e. coli. anti-microbial resistance (amr) is a major public health problem in patient care in both developed and developing countries. therefore, in this study, we analyzed eight different antibiotics. among eight antibiotics, piperacillin-tazobactam resistant was predominant in both e. coli (62%) and k. pneumoniae (67%), which differed from previous studies [13–15]. piperacillintazobactam is commonly used to treat intraabdominal, lower respiratory, urinary tract, and gynecological and skin/soft tissue infections, as well as for fever in patients with neutropenia [16]. the predominance of the resistant bacteria for these antibiotics could imply that illnesses associated to e. coli & k. spp. are fairly prevalent in the pokhara and have been treated just as normally. in addition, the rates of resistance to cefotaxime, ceftazidime, gentamycin, and amikacin for e. coli presented in this study are 50%, 60%, 10%, and 11% respectively. however, this contrasts another study, where the resistance of e. coli were found 99%, 78%, 29% and 4% to cefotaxime, ceftazidime, gentamycin, and amikacin respectively [17]. this may suggest that the e. coli collected from nepal has not been exposed to the four antibiotics quite as much as in other more developed places, which could probably mean that it is still possible to more easily manage the esbls in nepal effectively 24 60 10 11 9 62 50 5 0 10 20 30 40 50 60 70 80 90 100 e. coli (%) nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 25-32 baral et al. ©njb, biotechnology society of nepal 30 nepjol.info/index.php/njb compared to places with higher antibiotic exposure and resistance. from the eight antibiotics that were analyzed, imipenem was the most effective drug; however, it should not be administered as the empirical drug unless the infection is life-threatening, since carbapenems are considered the drug of last resort. if this situation is not taken seriously and misuse the carbapenem drugs then carbapenem resistant bacteria may evolve. in this study, 4.5% mdr strains were detected, which was very low compared with other studies 64.0%, and 64.9% [19]. this may be the case due to the overexposure of the antibiotics to the bacteria in external environments and animals, whereas in the related district it may not be the case. furthermore, 84% e. coli was confirmed as esbl producer followed by k. pneumoniae, (9%) and k. oxytoca (7%). a similar result was found in the study where the researchers reported 91% e. coli and 8% k. pneumonia [7]. conversely, the findings of another study [20] showed the variable result with e. coli isolates, 33(9%) were esbl producers. the identification of esbl producing organisms is a major challenge in the clinical world and, due to the selective pressure caused by heavy use of expanded-spectrum cephalosporins, lapses in effective infection control measures and affinity of these enzymes for different substrates, outbreaks are increasing [21]. an antibacterial choice is often complicated by mdr leading to over-prescription and misuse of antibiotics. as indicated by the present and previous findings, it appears to be mandatory to include esbl detection in routine laboratory practice so as to limit the rapid spread of esbl producing organisms. amr can be tackled by following continuously the 5 steps viz. awareness; surveillance; stewardship; research & innovation; infection prevention & control accordance with the “one health” concept in agriculture, veterinary, environment and human. limitation of study: we couldn’t perform genotyping test and mbl test in bacterial isolates due to a limited resource and time limitation. conclusions: the prevalence of esbl producing e. coli and klebsiella spp. was more. the preponderance of esbl producing e. coli and klebsiella spp. were resistant to the in-use antibiotics used from stool samples. imipenem was the most potent antibiotic and could be the drug of choice for treatment of infections caused by esbl strains. this clinical threat of raised up esbl prevalence is creating significant therapeutic problems prompting an immediate need to formulate strategic policy initiatives to reduce their prevalence. conflict of interest the authors declare that they have no competing interests. acknowledgments: we are indebted to volunteers, teacher and school principal. in addition, we are thankful to hospital staff and doctors. without their support, we couldn't able to complete this research. consent for publication not applicable consent to participant written informed consent was taken from all participating patients or from guardian on the behalf of their children. ethical approval and consent to the participant no patient related data were collected. ethical approval was therefore not required. the study was laboratory-based basic science study. written informed consent was taken from all participants or from guardian on the behalf of their children. availability of data and materials all supplementary files, data generated and analyzed during this study will be made nepal journal of biotechnology. d e c . 2 0 1 8 vol. 6, no. 1: 25-32 baral et al. ©njb, biotechnology society of nepal 31 nepjol.info/index.php/njb available as per reasonable request to the corresponding author. source of support no funding was obtained. author’s contributions bpb and bks designed the study and collected sample at manipal teaching hospital. drb and bpb performed investigation and recorded the laboratory findings with the validation. hss supervised and provided a methodology for the study. bpb, bb and rrg administered the project, reviewed literature, and wrote the original manuscript. bb and rrg curated data to performed statistical data analysis and data interpretation. rrg & bb also reviewed, proofread, and revision of the draft by compiling, formatting, editing and writing the final version of the article. thus, all authors made a substantial contribution to the study. all of them read and approved the final manuscript. references 1. thenmozhi s, moorthy k, sureshkumar bt, suresh m: antibiotic resistance mechanism of esbl producing enterobacteriaceae in clinical field: a review. int j pure appl biosci. 2014, 2:207– 226. doi:10.5935/1678-9741.20110056. 2. rupp me, fey pd: extended spectrum beta-lactamase (esbl)-producing enterobacteriaceae: considerations for diagnosis, prevention and drug treatment. drugs. 2003, 63:353–365. 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leptospirosis is a bacterial zoonotic disease caused by spirochetes of the genus leptospira that affects human  and a wide range of animals. the direct method of diagnosis of leptospirosis, has been so far by culture isolation  but it is time consuming and potentially biohazardous. another traditional method is the detection of antibodies  (serological  tests)  which  is  also  a  time  consuming  method  and  fails  to  identify  the  infecting  serovar.  to  overcome  these  limitations  associated  with  the  cultivation  and  serology,  we  developed  pcr  assay  targeting  partial lipl21 gene and lipl41 gene of leptospires using in‐house designed p28/29 and p30/31 primers, with a  product size of 385bp and 427bp. the amplicons were subjected to restriction enzyme digestion using rsai, pvu  ii and hindiii for product of p28/29 and clai, taqi and rsai were used for product of p30/31. the protocols were  standardized  and  the  assay  targeting  the  partial  lipl21  and  lipl41  gene  was  found  to  be  specific  for  eight  pathogenic leptospires out of nine leptospires tested. the products were then cloned in pgemt easy vector and  sequenced to facilitate further studies.  pcr could detect the target bacterial gene without any ambiguity and  showed  good  efficiency  in  detection  of  targeted  species  in  the  sample.  this  simple,  rapid  and  cost‐effective  method can be applicable in a prediction system to prevent disease outbreak by these leptospira species and  can be considered as an effective tool for disease diagnosis of leptospira species.    key words: pcr, lipl21, lipl41, molecular diagnostics, transformation.     correspondence author:   sharanaiah umesha, dos in biotechnology, university of mysore, karnataka, india.  e‐mail: umeshgroup@yahoo.co.in  introduction  the  economic  importance  of  bovine  leptospirosis  includes  loss due to abortion,  loss of milk production  and  elevated  veterinary  costs  and  predominantly  human  infection  (levett  et  al.,  2005).    leptospirosis  also known as weil's disease, canicola fever, canefield  fever, nanukayami fever, 7‐day fever and many more.   leptospirosis recognized as one of the most common  zoonoses.  leptospirosis  is  commonly  transmitted  to  humans through contact of animal urine with unhealed  breaks  in  the  skin,  eyes  or  with  the  mucous  membranes. outside tropical areas, leptospirosis cases  have a relatively distinct seasonality with most of them  occurring during august‐september/february‐march in   year (zhang et al., 1992).   the spectrum of human disease caused by leptospires  is extremely wide, ranging from subclinical infection to  a  severe  syndrome  of  multi  organ  infection  resulting  high mortality. this syndrome, icteric leptospirosis with  renal  failure,  was  first  reported  more  than  100  years  ago  by  adolf  weil  in  heidelberg.  however,  an  apparently  identical  syndrome  occurring  in  sewer  workers  was  described  several  years  earlier.  earlier  descriptions  of  diseases  that  were  probably  leptospirosis  were  reviewed  recently  (serres  et  al.,  1995).  leptospira (from the greek word leptos means fine or  thin and latin word spira means saprophytic species).  leptospira was first observed  in 1907  in kidney tissue  slices of a leptospirosis victim who was reported to die  of “yellow fever” (abdollahpour, 1990).      the direct  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  23  biotechnology society of nepal (bsn), all rights reserved   in  artificial  medium,  with  leptospira  present  in  an  infected  animal.  several  leptospiral  outer  membrane  proteins  have  been  shown  to  attach  to  the  host  extracellular matrix  and  to  factor h,  suggesting  these  proteins may be important for adhesion of leptospira  to  host  tissues  and  in  resisting  complement,  respectively.  the  outer  membrane  of  leptospira,  like  those of most other gram‐negative bacteria, contains  lipopolysaccharide  (lps).  differences  in  the  highly  immunogenic lps structure account for the numerous  serovars  of  leptospira.  consequently,  immunity  is  serovar  specific;  current  leptospiral  vaccines,  which  consist  of  one  or  several  serovars  of  leptospira  endemic  in  the  population  to  be  immunized,  protect  only  against  the  serovars  contained  in  the  vaccine  preparation. leptospiral lps has low endotoxin activity.   an unusual feature of leptospiral lps is that it activates  host  cells  via  tlr2  rather  than  tlr4.    the  unique  structure  of  the  lipid  a  portion  of  the  lps  molecule  may  account  for  this  observation.  finally,  the  lps  o  antigen content of l.  interrogans differs  in an acutely  infected versus a chronically infected animal.  the role  of  o  antigen  changes  in  the  establishment  or  maintenance  of  acute  or  chronic  infection,  if  any,  is  unknown  (zhang  et  al,.  1992;  bharti  et  al.,  2003).  leptospires  enter  into  the  body  of  a  susceptible  host  through mucous membrane or abraded skin.   after 4  to 10 days, the host becomes bacteraemic, this period  lasting from hours to 7 days, and may be characterised  by pyrexia and anorexia (abdollahpour, 1990).   materials and methods  leptospira  serovars.  a  total  of  9  leptospira  serovars  namely,  l.  icterohaemorrhagiae,  l.  canicola,  l.  pamona,  l.  autumnalis,  l.  javanica,  l.  pyrogenes,  l.  australis,  l.  hardjo  and  l.  inadai  were  procured  from  the  repository  of  pd_admas(project  directorate  on  animal  disease  monitoring  and  surveillance),  uas,  bangalore, india.   cultivation  of  leptospira.  leptospira  serovars  mentioned above were grown in luria‐bertani (lb) agar  (difco detroit, mi, usa) supplemented with 1‐3% nacl  at 370c overnight.  dna  preparation.  template  genomic  dna  was  extracted/isolated from pure culture of corresponding  leptospires,  using  qiaamp  dna  minikit  (qiagen,  germany) and other alternative method described by     hoshino  et  al.,  1998.  for  it    freshly  grown  bacterial  method for diagnosis of leptospirosis, has been so far  by  culture  isolation  but  it  is  time  consuming  and  potentially  hazardous.    another  traditional  method  is  the detection of antibodies (serological tests) which is  also a time consuming method and fails to identify the  infecting  serovar.    classification  and  identification  of  leptospires  using  serology  is  a  difficult  and  tedious  procedure.    recently  molecular  biology  techniques  have been introduced for detection.  these techniques  are predominantly used for facilitating the study of the  epidemiology  of  leptospirosis.    genetic  taxonomy  involves  dna/dna  hybridisation  and  gc  content  the  guanine‐plus‐cytosine  mol  percentages  (g+c  mol  %)  content of dna. the genotypic classification based on  dna  hybridization  defined  21  genome  species  of  leptospira  that  include  29  serogroups  and  269  serovars.  yasuda  et  al.,  1987  proposed  a  new  classification  of  leptospira  based  on    dna  homology  study on 46 pathogenic and non pathogenic serovars.  woodward  and  redstone,  1994  developed  a  polymerase  chain  reaction  (pcr)  combined  with  restriction  fragment  length  polymorphism  which  can  be used for differentiation of leptospira serovars.  it is  suggested  that  the  pcr  combined  with  restriction  fragment length polymorphism is useful tool for rapid  detection  and  preliminary  differentiation  of  leptospires (levett et al., 2001).  although  over  269  serovars  of  leptospira  have  been  described,  all  members  of  the  genus  have  similar  morphology. leptospira are spiral‐shaped bacteria that  are 6‐20 μm long and 0.1 μm in diameter.  one or both  ends  of  the  spirochete  are  usually  hooked.  because  they are so thin,  live leptospira are best observed by  dark field microscopy. the bacteria have a number of  freedom degrees; when ready to proliferate via binary  fission, the bacterium noticeably bends in the place of  the future split (levett et al., 2005). leptospira have a  gram‐negative‐like  cell  envelope  consisting  of  a  cytoplasmic  and  outer  membrane.  however,  the  peptidoglycan layer is associated with the cytoplasmic  rather than the outer membrane, an arrangement that  is unique to spirochetes. the two flagella of leptospira  extend from the cytoplasmic membrane at the ends of  the  bacteria  into  the  periplasmic  space  and  are  necessary  for  the  motility  of  leptospira.  the  outer  membrane  contains  a  variety  of  lipoproteins  and  transmembrane  outer  membrane  proteins.  as  expected,  the  protein  composition  of  the  outer  membrane differs when comparing leptospira growing  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  24  biotechnology society of nepal (bsn), all rights reserved   step at 720c for 1 min and a final elongation at 720c for  5 min. finally annealing temperature was standardized  to 600c for 1 min (fig. 1a).   the  pcr  standardization  using  primers  p30/31  was  initiated in a 25 ml reaction volume containing 10x taq  buffer  with  kcl  (100mm  tris  hcl,  50mm  kcl,  0.8% nonidet p40), 1.5mm mgcl2, 2.5mm of each dntps, 2u  of  taq  polymerase,  25pmoles  of  each  primer  and  0.25ml of dna (40ng/ml) extract from l. javanica. the  test  was  done  for  several  combinations  annealing  temperatures from 550c to 650c. the reaction started  with an initial denaturation temperature of 950c for 5  min followed 35 cycles of denaturing at 950c for 1 min,  annealing at 600c for 1 min at g ± 50c, elongation step  at 720c for 45 sec and a final elongation at 720c for 5  min.  finally  annealing  temperature  was  standardized  to  600c  for  1  min  (fig.  1b).    optimized  annealing  temperature details shown in table (table 2).  agarose  gel  electrophoresis.  a  15  ml  aliquot  of  each  pcr  product  was  mixed  with  3µl  of  6x  loading  dye  (fermentas) and loaded on to the wells of 2% agarose  gel in tae buffer. it was run at 80v for about 45 min.  dna bands were visualized under uv‐trans illuminator  and documented using gel documentation system with  quantity  one  software  (biorad,  u.s.a)  and  the  pcr  product  was  eluted  using  eppendorf  qiaquick  gel  extraction kit (qiagen, germany).  colonies were suspended in 500 µl te buffer or 50µl of  mid‐log phase bacterial culture was added to 450µl of  te buffer, then the cell suspension was boiled for 10  min  followed  by  quick  cooling  on  ice  for  5  min,  and  centrifuged  at  12,800  g  for  5  min.    the  supernatant  was collected and stored at ‐300c until use.  primer  design.  all  available  sequences  of  the  target  genes  among  leptospira  spp.  were  downloaded  from  the  genbank  (http://www.ncbi.nlm.nih.gov/genbank/ genbanksearch.html). perlprimer software by marshall  et al., (2004) was used to design the primers. sequence  for  lipl21  holding  accession  number  ay688426  and  lipl41  sequence  of  leptospira  borgpetersenii  serovar  hardjo‐bovis  holding  accession  number  cp000348.1  was retrieved from ncbi‐genbank.  it was then used to  get  conserved  sequences.  the  conserved  part  of  the  nucleotide sequence was retrieved and used for primer  designing. the criteria that was set for primer design  was primer length is 20±2 target temperature is 55±1° c.    the  overall  theoretical  specificities  of  the  newly  designed  primers  were  checked  using  blast  search.   details  of  the  primers’  sequences,  their  expected  amplicons sizes after pcr, etc. are summarized in table  (table 1).  standardization  of  polymerase  chain  reaction  (pcr)  protocol. in order to achieve the best sensitivity for the  pcr  using  primer  p28/29  several  combinations  of  annealing  temperatures  from  580c  to  680c  were  tested. the following conditions were chosen: pcr was  performed  in  25  ml  reaction  volume  containing  10x  taq buffer with kcl (100mm tris hcl, 50mm kcl, 0.8%  nonidet p40), 1.5mm mgcl2, 2.5mm of each dntps, 2u  of  taq  polymerase,  25pmoles  of  each  primer  and  0.25ml  of  dna  (40ng/ml)  extract  from  l.  icterohaemorrhagiae.  pcr  chemicals  were  procured  from fermantas. the reactions were carried out in an  eppendorf thermocycler. initially, pcr conditions were  optimized  by gradient  pcr.  the  reaction  started  with  an  initial  polymerase  activating  temperature  of  950c  for 5 min followed 35 cycles of denaturing at 950c for 1  min, annealing at 630c for 1 min at g ± 50c, elongation  primer  target species/gene  sequence(5’‐3’)  amplicon length  reference (accession  no.)  p28/f  leptospira/lipl21  ccagcactgacaccggacaaa  385bp  ay688426  p29/r  leptospira/lipl21  ccggaaccaaccgctttacat  385bp  ay688426  p30/f  leptospira/lipl41  gacctcagtaaacgcgccgatat  427bp  cp000348.1  p31/r  leptospira/lipl41  cagcggcttcgtccaatcct  427bp  cp000348.1  table 1. details of the newly designed primers for specific identification     pcr reaction mixture(25ml)     p28 /p29     p30/p31     10x buffer                         2.50ml  mgcl2                                                  1.50ml  dntps                                                  2.00ml  primer‐forward                        0.50ml  primer‐reverse                         0.50ml  template                                          0.25ml  taq pol                                    2.00ml  nuclease   free water     15.75µl  table 2. details of standardized thermal cycling  condition   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  25  biotechnology society of nepal (bsn), all rights reserved   min. immediately mixture  was kept over ice for 2min.   0.8ml of lb broth was then added to it and incubated  at  37°c  for  90  min  in  an  orbital  shaker.    it  was  then  centrifuged at 4000 rpm for 10 min and the pellet was  plated on lb agar plate containing ampicillin (50mg/ml)  and  40ml  of  2%  x‐gal  (5‐bromo‐4‐chloro‐3‐indolyl‐b‐ galactopyranoside) and 8ml of 2% iptg (isopropyl‐b‐d– thiogalactopyranoside).  the  plates  were  then  incubated over night at 37°c.  well‐isolated  white  colonies  (fig.  4)  were  picked  and  transferred  to  5ml  of  lb  broth  containing  5ml  of  ampicillin (50mg/ml).  the tubes were then incubated  at  37°c  in  the  shaker  for  overnight.  plasmid  dna  isolation and purification was done using qiaprep spin  miniprep kit (qiagen, germany) and microcentrifuge.  characterization  of  recombinants.  the  plasmids  isolated  from  the  white  colonies  were  further  characterized by the insert release after digestion with  ecori  (g  ˉaattc)  restriction  enzyme.  the  enzyme  ecori was used to cleave and release the insert in the  plasmid  (fig.  5a  and  5b).  the  reaction  mixtures  were  prepared as per the table below and were subjected to  digestion at 37°c in a water bath for 1h.  details of the  reaction mixture are summarized in table (table 5).  pcr  for  confirmation.  the  pcr  were  carried  out  for  confirmation of the recombinants using primer p28/29  and  p30/31  under  the  standardized  conditions  as  described  previously  using  the  plasmid  dna  as  template. it was performed in a 25 ml reaction volume  containing  10x  buffer  (100mm  tris  hcl,  50mm  kcl,  0.8%nonidet  p40),  1.5mm  mgcl2,  2.5mm  of  each  dntps, 2u of taq polymerase, 25pmoles each primer  and 1ml of template (1:10 diluted).     5ml  aliquot  of  pcr  products  mixed  with  1µl  of  6x  loading dye (fermentas) and loaded on to the wells of  2.5% agarose gel. it was run at 80v for about 45 min.  dna bands were visualized under uv‐transilluminator  and  documented  using  biorad  gel  documentation  system with quantity one software (fig. 6a and 6b).   characterization  of  amplicons.  the  pcr  product  of  primer  p28/29  and  primer  p30/31  were  subjected  to  re  digestion.  the  enzymes  rsai(gtˉac)  and  hindiii (aˉagctt)  were  used  to  characterize  pcr  product  of  p28/29  (fig.  2a)  and  enzymes  clai(atˉcgat),  taqi (tˉcga)  and  rsai(gtˉac)  were  used  to  characterize  pcr product of p30/31 (fig. 2b).   the reactions were  set as under and incubated at 37°c in a water bath for  4h.  restriction  enzymes  and  its  corresponding  buffer  were  procured  from  fermentas.    clai  and  its  corresponding  buffer  were  procured  from  promega.   details  of  the  reaction  mixture  are  summarized  in  table 3.   a 15 ml aliquot of each digested products were mixed  with 3µl of 6x loading dye (fermentas) and loaded on  to the wells of 2% agarose gel in tae buffer. it was run  at  80v  for  about  45  min.  dna  bands  were  visualized  under uv‐transi lluminator and documented using gel  documentation  system  with  quantity  one  software (biorad, u.s.a) (fig. 2a and 2b).  specificity  the  pcr  assay.  the  specificity  of  both  the  assay developed was tested by taking 10‐20ng of each  template dna isolated from the different serovars and  the  pcr  was  carried  out  under  the  standardized  conditions as shown in table (table 4).  a 10ml aliquot of each pcr product mixed with 2ml of  6x loading dye (fermentas) was run on a 2.5% agarose  electrophoretic gel in tae buffer and dna bands were  visualized under uv‐transilluminator and documented  using  gel  documentation  system  with  quantity  one  software (biorad, u.s.a) (fig. 3a and 3b).  transformation.  ligation  mixture  was  prepared  by  adding 1ml of pgemt easy (50ng) vector at the rate of  and 1ml of t4 dna ligase (3u/ml) to 5ml of 2x buffer.  5ml  of  dna  (50ng/ml)  was  added  to  this  mixture  for  ligation. ligation was done at 4°c for overnight.  ligation  mixture  was  added  to  ice  cold  200ml  of  competent cells and tapped gently, it was incubated on  ice for 30 min., then heat shock was given at 42°c for 1  reaction  mixture  lipl21  lipl41  rsai  hindiii  clai  taqi  rsai  buffer  enzyme  dna  water  bsa  2.0 ml  1.0 ml  8.0  ml  11.0 ml  ‐  2.0 ml  1.0 ml  8.0 ml  11.0 ml  ‐‐‐‐‐‐  2.0ml  1.0ml  5.0ml  10.0ml  2.0ml  2.0ml  1.0ml  5.0ml  12.0ml  ‐  2.0ml  1.0ml  5.0ml  12.0ml  ‐  table 3. details of the restriction enzyme  digestion reaction of lipl21 and lipl41   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  26  biotechnology society of nepal (bsn), all rights reserved   out  reactions  at  various  annealing  temperatures  ranging from 58°c to 68°c on l.  icterohaemorrhagiae  serovar. this showed non‐specific amplification at 58° c,  58.7°c,  60.8°c  and  63.5°c.  so  pcr  reaction  using  p30/31 was carried out on l.javanica template at two  different mgcl2 concentration i.e. (0.5, 1.0µl). no bands  could be detected for pcr amplification at 0.5 µl mgcl2  but  specific  band  of  430bp  was  observed  at  1.0  µl  mgcl2.    standardization  was  done  by  carrying  out  reactions at various annealing temperatures from 55°  to  65°c  for  1  min.  good  amplification  bands  were  observed  for  pcr  reactions  carried  out  at  annealing  temperatures  60.5°c,  61.8°c,  63.1°c,  64.2°c,  65.0°c  and 65.5°c (fig. 1b).  the 427bp amplicons thus amplified was subjected to  re.  the  restriction  enzyme  pattern  of  this  amplicon  confirms  it  to  be  a  lipl41  partial  gene  of  leptospira  genus (fig. 2b).  the  specificity  of  this  primer  was  tested  on  nine  different serovars under optimized conditions. the pcr  assay showed nonspecific amplification along with the  specific  band.  primer  degradation  was  suspected  and  the  pcr  reactions  at  the  optimized  conditions  were  repeated with fresh primer set p30/31. pcr reactions  were  again  carried  out  on  nine  serovars  at  mgcl2  results and discussion  specificity  of  leptospira  spp.  primers.  initially,  pcr  using  p28/29  was  standardized  by  carrying  out  reactions  at  various  annealing  temperatures  ranging  from 58°c to 68°c on l.  icterohaemorrhagiae serovar  which showed amplification at 58°c, 58.7°c and 60.8°c  (fig.1a).  a  single  band  of  approximately  385bp  was  observed at 60°c annealing temperature. a set of pcr  reactions  were  done  by  varying  the  template  concentration from 5ng to 60ngand could amplify the  specific  band  at  all  the  template  concentrations.  the  pcr  products  were  pooled  and  characterized  by  subjecting  to  re  digestion  using  rsai  and  hindiii  restriction  enzymes,  both  of  which  confirmed  the  product  size  of  p28/29  to  be  385bp  (fig.2a).  the  specificity of this primer was tested on nine different  serovars namely l. icterohaemorrhagiae, l. canicola, l.  pamona,  l.  autumnalis,  l.  javanica,  l.  pyrogenes,  l.  australis,  l.  hardjo  and  l.  inadai  under  standardized  condition. the assay targeting the partial sequence of  lipl21  was  found  to  be  specific  for  eight  pathogenic  leptospires out of nine  leptospires tested (fig. 3a)  for the pcr assay targeted to the partial sequence of  lipl41,  standardization  was  done  initially  by  carrying  figure  1.  (a)  specificity  and  detection  of  annealing  temperature of lipl21 gene. annealing temperature  was  standardized  to  600c  for  1  min.    lane1‐6,  pcr  products  using  dna  template  of  l.  icterohaemorrhagiae.    lane  1‐6  annealing  temperature  of  each  lane  corresponding  to  58oc,  58.7oc,  60.8oc,  63.5oc,  66.1oc,  68oc,  respectively.   lane  7,  100bp  dna  ladder  (fermentas).    gel  run  performed using 2% agarose.   figure  1.  (b)  specificity and detection of annealing  temperature of lipl41 gene. annealing temperature  was standardized to 600c for 1 min.   lane2‐7, pcr  products using dna template of l. javanica.  lane 2‐ 7 annealing temperature of each lane corresponding  to  60.5oc,  61.8oc,  63.1oc,  64.2oc,  65.0oc,  65.6oc,  respectively.    lane  1  and  8,  100bp  dna  ladder  (fermentas).  gel run performed using 2% agarose.   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  27  biotechnology society of nepal (bsn), all rights reserved      organism  pcr reaction mixture (25ml)        p28 /29     p30/31     l. javanica  l. autumnalis  l. hardjo  l. pyrogenes  l. pamona  l. australis  l. icterohaemorrhagiae  l. inadai  l. canicola  10x buffer                      2.50ml  mgcl2                                              1.50ml  dntp mix                                     2.00ml  forward primer                    0.50ml  reverse primer                     0.50ml  template                                      0.25ml  taq pol                                2.00ml  nuclease free water     15.75ml     lipl21  lipl41  buffer  (10x)  enzyme  dna  water       2.0 ml       1.0 ml       8.0 ml     11.0 ml       2.0 ml       2.0 ml       5.0 ml       10.0 ml         total     20.0 ml      20.0 ml  table 4. details of the standardized conditions for pcr   table 5. details of the restriction  enzyme digestion reaction for insert  release   figure  2.  (a)  characterization  of  amplicons  using  restriction enzymes rsai in lane 2 and hindiii in lane  3 and lane dna template without re enzyme in lane  1,  lane  4,  100bp  gene  ruler.      gel  run  performed  using 2% agarose.   figure  2.  (b)  characterization  of  amplicons  using  restriction enzymes clai  in lane 3, taqi in lane 3 and  rsai in lane 5, dna template with out re enzyme in  lane  2,  lane  1  and  6,  100bp  gene  ruler.    gel  run   performed using 2% agarose.   concentration  of  1mm.  this  assay  proved  to  be  only  specific  to  l.  javanica  serovar  out  of  nine  serovars  tested (fig. 3b).  the  pcr  product  of  both  p28/29  and  p30/31  of  size  385bp  and  427bp  respectively  were  then  individually  cloned  into  pgemt  easy  ta  cloning  vector  and  transformed  to  jm109  e.  coli  competent  cells  and  plated on lb ampicillin plates with iptg and x‐gal (fig.  4). the discrete five white colonies and a blue colony  were then picked, cultured and plasmid was  isolated.  the  recombinants  were  then  characterized  by  releasing  the  insert  (fig.  5a  and  5b)  using  ecori  restriction  enzyme  followed  by  pcr  reaction  (fig.  6a  and 6b) for confirmation. the cloned sequences were  then  sent  for  sequence  analysis  (mwg  biotech  pvt.  ltd., bangalore) in order to facilitate further studies.  one of the main advantages of using this technique on  various  biological  samples  is    possibility  to  detect  pathogenic  leptospires  first  by  pcr  assay  and  subsequently  confirming  it  through  re  digestion  thus  improving the specificity of the test. further sequence  analysis may reveal the associated serovar causing the  disease.  the  direct  method  of  diagnosis  of  leptospirosis  is  by  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  28  biotechnology society of nepal (bsn), all rights reserved   culture  isolation,  but  it  is  time  consuming  and  potentially  biohazardous.  another  traditional  method  is the detection of antibodies (serological tests) but it is  also time consuming and fails to identify the infecting  serovar. to overcome the limitations of cultivation and  serology, we have used pcr amplification of leptospiral  dna for the diagnosis of leptospirosis at an early stage  of illness.  in  a  previous  study  by  (merien  et  al.,  1992  and  gravekamp  et  al,.  1993),  the  16s  rrna  gene  target  could not differentiate pathogenic and nonpathogenic  leptospira.    the  development  of  pcr  assay  targeting  partial  sequence  of  lipl21  and  lipl41  gene  of  pathogenic leptospires using in‐house designed p28/29  and p30/31 primers, with a product size of 385bp and  427bp,  respectively.  these  primers  were  designed  by  using  gene  tool  lite  software.  the  gene  sequence  coding for the respective surface protein was subjected  to  nucleotide  blast  to  get  conserved  sequences.  the  most  conserved  sequence  was  then  chosen  and  primers were designed.  the  newly  developed  pcr  using  in‐house  designed  primers produces expected   amplicons sizes i.e. 385bp  for lipl21 and 427bp for lipl41.  afterwards when this  pcr  protocol  was  applied  to  examine  all  the  test  samples  of  this  species  also  produces  specific  amplicons.  no amplicons of specific size was observed  in  case  of  non‐target  leptospira,  or  other  bacterial  figure 3. (a) the specificity of this primer tested on  nine different serovars under optimized conditions  for lipl21.  lane 3‐11 different serovars, lane 2, nega‐ tive control and lane 1, 100bp gene ruler. gel run  performed using 2.5% agarose.   figure 3. (b) the specificity of this primer tested on  nine  different  serovars  under  optimized  conditions  for lipl41.  lane 2‐5 different serovars and lane 1and  6, 100bp gene ruler.  gel run performed using 2.5%  agarose.   figure 4.    plate showing  recombinant  colonies   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  29  biotechnology society of nepal (bsn), all rights reserved   species.  besides non‐specific amplicon of different size  did not appear in any case.  in conclusion, the assay targeting partial sequence of  lipl41 gene showed high sensitivity and specificity for  l. javanica out of 9 leptospires tested. the pcr assay  targeting  partial  sequence  of  lipl21  gene,  which  showed  good  sensitivity  and  specificity  with  all  the  nine serovars tested and may also likely to detect other  pathogenic  serovars  as  the  lipl21  sequence  targeted  under the assay is conserved among all the pathogenic  leptospires  hence  may  be  valuable  tool  for  early  diagnosis  of  pathogenic  leptospires  directly  from  biological  samples  with  clinical  suspicion  of  leptospirosis.  figure 5. (a) insert release of lipl21from recombinant  pgem‐te  vector.    gel  run  performed  using  2.5%  agarose.   figure 5. (b) insert release of lipl41 from recombi‐ nant pgem‐te vector.  gel run performed using 2.5%  agarose.   figure  6.  (a)  pcr  for  lipl21  gene  for  confirmation.   gel run performed using 2.5% agarose.   figure  6.  (b)  pcr  for  lipl41  gene  for  confirmation.   gel run performed using 2.5% agarose.   references  abdollahpour,  g:  a  review  on  leptospirosis.   proceedings of leptospirosis research     conference,  japan, matsoyamam ;1990: 34‐37.  bharti, ar, nally je, ricaldi jn, matthias ma, diaz mm:  leptospirosis:  a  zoonotic  disease  of  global  importance. lancet infect dis 2003, 3:757–771.   gravekamph c, kemp vad, franzend m, carrington d,    schoone gj,  eys gjjmv,  everardr c0r,  hartskeeraln  ra,  terpstra  wj:    detection  of  seven  species  of  pathogenic  leptospires  by  pcr  using  two  sets  of  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 22‐30  30  biotechnology society of nepal (bsn), all rights reserved   primers.  j of gen microbiol  1993, 139: 1691‐1700.  hoshino  k,  yamasaki  s,  mukhopadhyaym,  ak,  chakraborty  s,  basu  a,  bhattacharya  sk,  nair,  gb,  shimada  t:  development  and  evaluation  of  a  multiplex pcr assay for rapid detection of toxigenic  vibrio cholerae o1 and o139.   fems  immunol med  microbiol 1998, 20: 201–207.   levett,  pn,  songee  l,  branch,  carol  u,  whittington,  charles  n,  edwards,    paxton  h:    two  methods  for  rapid  serological  diagnosis  of  acute  leptospirosis.  clin and diag. lab immunology 2001, 8(2):349‐351.  levett  pn,  morey  re,  galloway  rl,  turner  de,  steigerwalt ag, mayer lw: detection of pathogenic  leptospires  by  real‐time  quantitative  pcr.    j  med  microbiol 2005 54(1):45‐49.  merien  f,  amouriaux  p,  perolat  p,  baranton  g,  saint  girons i:  polymerase chain reaction for detection of  leptospira  spp.  in  clinical  samples.  j  clin  microbiol  1992, 30(9):2219‐2224.  marshall    oj:  perl  primer:  cross‐platform,  graphical  primer design for standard, bisulphite and real‐time  pcr. bio inf applications note 2004, 20 (15): 2471– 2472.  serres gd, levesque b, higgins r, major m, laliberre d,  bouliance n, duval b: need for vaccination of sewer  workers against leptospirosis and hepatitis a.  occup  and envt. med 1995,52: 505‐507.  woodward    m  j,  redstone  js  :  differentiation  of   leptospira serovars by polymerase chain reaction and  restriction  fragment  length  polymorphism.  vet  rec  1993, 132: 325‐326.  yasuda ph, arnold g, steigerwalt, katherine r, sulzer,  arnold f, kaufmann, faye rogers     don, j, brenner:  deoxyribonucleic  acid  relatedness  between  serogroups  and  serovars  in  the  family  leptospiraceae  with  proposals  for  seven  new  leptospira species.  int  j  systc bact 1987, 37(4): 407 ‐415.  zhang, y., dai, b.  (1992) detection of leptospiral dna  in the serum of 175 patients with early leptospirosis  by polymerase chain reaction.  hua xi yi ke da xue  xue bao 23(3):256‐60.   nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:21-26 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 21 nepjol.info/index.php/njb assessment of quality of underground drinking water: very near (≤20 meters) and far (>50 meters) from the river munal subedi1*, mamata gharti magar1, gita shrestha rajbhandari2 1department of microbiology, amrit campus, tu, nepal. 2department of botany, amrit campus, tribhuvan university, nepal. abstract water quality information is needed to assess the state of water contamination in a variety of community, including those that rely primarily on unimproved underground sources of drinking water. the study was carried out with an aim to assess the quality of ground water in particular sites of the kathmandu valley. the ground water samples were collected from shallow well, tube well and deep tube wells located at specific places of the valley. the research was focused on physiochemical and bacteriological analysis of underground water from sites near to bagmati river (≤20 meters) and from sites far from bagmati river (>50 meters). the sampling sites were scattered from sinamangal to minbhavan. total sample size was 100, with 50 in each stratum. study processing was done during the period from february 2013 to may 2013. six physiochemical parameters namely ph, conductivity, ammonia level, chloride level, nitrite level, nitrate level and biological parameters (coliform and fecal coliform) of each sample was tested. based on the research work, it was recorded that the underground water close to river (≤20 meters) has comparatively high physiochemical and biological parameter (fecal coliform) than underground water that were farther from the river (>50 meters). fecal coliform was predominant 58% (29/50) in water nearer to river rather than in water farther from the river 20% i.e. (10/50). similarly, the values of physiochemical and biological parameter increased comparatively with more distance i.e. ≤10 meters from river. the finding indicated that the underground water near to river is more polluted than far from the river. key words: bacteriological quality, biological, drinking water, fecal coliform, ground water, physiochemical. *corresponding author email: munalsubedi@yahoo.com introduction the unplanned urbanization and industrialization have resulted in excess use of environment, in particular water resources [1]. groundwater is the only alternative source for safe and reliable drinking water. groundwater is characterized by low temperature, low redox potential, high carbon dioxide and mineral content, less amount of suspended solids, and lack of microbial contaminants. water from underground sources such as hand pump, shallow and deep well is often of better quality than surface water or other open water sources if the soil is fine-grained and its bedrocks do not have cracks, crevices, and bedding plants, which permit the free passage of polluted water especially within the metropolitan zones. the most common and widespread risk associated with drinking water is its contamination either directly or indirectly by sewage or other wastes of human and animal origin. bacterial as well as chemical pollution of water sources may occur and is mostly derived from watershed corrosion and drainage from sewage, swamps, or soil with high humus content or seepage through river. prolonged discharge of industrial effluents, domestic sewage and solid waste dump cause the ground water to become polluted and create health problems [2]. due to inadequate amount of distributed drinking water (pipe water) by kathmandu upatayka khane pani limited (kukl), many citizens in kathmandu valley are seeking the alternative way to meet their demand. about 45% people of the valley depend on underground water for drinking and other domestic purposes [3]. underground water is one of the alternative sources for drinking water in the valley. however, in many places within the valley, it is suspected that water is non-potable because it is often contaminated with various pathogenic as well as opportunistic microflora and toxic chemical compounds by different means, such as improper disposal of garbage, unmanaged sewer system and polluted river. the ground water pollution in urban areas is mostly due to infiltration of urban storm water, leakage of waste water and septic reservoirs, and improper industrial activities [4]. thus, ground water very near (≤20 meters) to polluted river might be polluted due to seepage of polluted river into the underground water resulting in contamination with different chemicals as well as pathogens. thus households near (≤20 meters) to nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 21-26 subedi et al. ©njb, biotechnology society of nepal 22 nepjol.info/index.php/njb river may be more polluted than the households which are far (>50 meters) from the river side. as a result, underground water very near to river serves as the commonest vehicle of transmission of a number of infectious waterborne diseases as well as factor for leaching heavy metals from the river. changes in water quality are reflected in its physical, chemical, and biological conditions; and these in turn are influenced by physical and anthropogenic activities [5]. if we are to provide safe water and prevent possible waterborne diseases, assessment of water quality and water use patterns based on environmental, social, economic and cultural characteristics of a given area is essential [6]. here, we have studied the quality of underground water of kathmandu valley near the river which is ≤20 meters and far from the river which is >50 meters. further, we assessed the physiochemical parameters like, ph values, conductivity, turbidity, and chloride content, nitrite content, nitrate content, ammonia content, of underground water along with the microbial quality (especially coliforms and fecal coliforms) of underground water obtained from the two strata [7-9]. methodology figure 1: map of kathmandu valley showing the study area from sinamangal to new baneshwor. the study was continuously carried out for four months. locations from sinamangal to minbhavan site that included both banks of bagmati river (kathmandu) were used. 300 ml of underground water samples were collected from 100 different households located near site (≤20 meters) and far site (>50 meters) from bagmati river. in order to accomplish the objectives, sample sites have been divided into two strata (near and far). stratum 1: 50 samples were taken from the households that were very near the bagmati river (<20 meters). the sites near the river were chosen assuming that contamination of underground water correlates with proximity from the polluted river that indicates possible seepage of polluted water into the underground water through cracks and crevices. stratum 2: in this stratum, 50 samples were selected from those households, which are at least 50 meters far from bagmati river. it was assumed that a distance of at least 50 meters will avoid intrusion by polluted river water. one hundred samples i.e. 50 from areas close to river and 50 from areas far from river were tested. during sampling, interview was conducted to know the depth of hand pipe and well, and also to find out the exact number of people who have been using groundwater for drinking as well as other purposes. samples were collected from sinamangal to min-bhavan from both sides of river. the samples were then transported to a laboratory for analysis. processing was done on the same day, within 6 hours of collection or preservation at 40c was carried out when an immediate analysis was not possible. analysis was performed for the determination of different physicochemical parameters and biological parameters of underground water. the results were compared with standard values recommended by world health organization (who) for drinking purposes [7,8]. the laboratory analysis of samples was done using standard methods in the laboratory of the department of microbiology, amrit campus [9]. to determine coliforms, most probable number was carried out and was confirmed by using differential media i.e. m-endo agar, and biochemical media, triple sugar iron agar [8,10]. further confirmation was carried by indole, methyl red voges proskeur and citrate utilizations test. physiochemical parameters of water were tested according to standard protocol (table 1). precautions were taken to prevent any cross contamination during the experiment. the experiments for the biological parameters were performed under aseptic conditions using sterile equipment for sample collection as well as nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 21-26 subedi et al. ©njb, biotechnology society of nepal 23 nepjol.info/index.php/njb processing such as use of sterile bottle for water bottle for collection, disinfection of work table, use pre-purity and postpurity plates for the biochemical test. however, for the physiochemical parameters, fresh, carefully prepared chemicals were used in appropriate amounts. table 1: physiochemical parameters s. no test parameter methods 1 ph ph meter 2 conductivity conductivity meter 3 chloride (mg/l) iodometric method 5 ammonia (mg/l) phenate method 6 nitrate (mg/l) brucine method 7 nitrite (mg/l) uv visible spectrophotometer method source (apha, 1998) result and discussion number of households that used underground water for sole drinking purpose was comparatively lower (20%) in area ≤20 meters distance from river than those in area >50 meters distance from river i.e. 60%. however, use of water for other purposes such as cooking and bathing were equal in both distances (figure 2). figure 2: no. of household that use underground water for different purpose 76% (38) of households in areas ≤20 meter distance from the river used conventional sand filter method for the treatment of water while the rest of the households used water without treatment. however, 100% of the households in area >50 meter distance used conventional sand filter method for the treatment of water (figure 3). any alteration in water ph is accompanied by the change in other physiochemical parameters [11]. our study showed that water was alkaline in most of the samples in areas ≤20 meters and >10 meter distance and ph ranged from 7.2-8.4. however, in areas ≤10 meter distance, ph ranged from 7.3-8.9, whereas in water samples from areas >50 meters distance ph ranged from 6.9-7.9. ph value of different studied samples in different distances were within the range prescribed by who (6.5-8.5) except in ≤10-meter distance where the upper range was exceeded. high value of ph may be due to waste discharge and microbial decomposition of organic matter in the water body [12]. because the distance between the river and the underground water in some cases is less than 10 meters and depth is around 30 feet there might be a possible seepage of river water to the underground drinking water. figure 3: no. of houses using sand filter as treatment for underground water electrical conductivity is one of the tools to assess the purity of water. often, high electrical conductivity correlates with contamination by anthropogenic sources [13,14]. electrical conductivity was found in the range 764-946 mho/cm in the samples from areas >10 to ≤20 meter distance. in ≤10 meter distance, it was found in the range of 790-1202 mho/cm, whereas in water sample from areas >50 meters distance it was found to be in the range of 658-982 mho/cm. electrical conductivity was comparatively higher range in ≤10 meters. it is correlated with the presence of salinity of water. one of the reasons for high salinity is the high concentration of cation such as sodium, calcium and magnesium along with chloride, phosphate and nitrate anions [15]. in water sample distance ≤10 meters, the higher range of electrical conductivity may be due to possible leaching of river water in underground drinking water. the high concentrations of chloride in combination with nitrate or ammonium show that the water is contaminated with domestic sources [16]. increase in chloride concentration in discharge of municipal 30 50 50 10 45 50 0 10 20 30 40 50 60 water for drinking purpose water for cooking purpose bathing and washing purpose n o . o f h o u se h o ld s >50m <20m 0 10 20 30 40 50 60 water treatment no water tretment ≥50meter ≤20meter nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 21-26 subedi et al. ©njb, biotechnology society of nepal 24 nepjol.info/index.php/njb and industrial waste has been reported [17]. in river ganga at vanarasi, chaudhary and ojha (1985) found that chloride value ranged from 5.9 to 7.9 mg/l. chloride was found in the range 14.5-68.2 mg/l in samples from areas >10 to ≤ 20 meters [18,19]. in ≤10 meter distance, it was found in the range of 39.7-70.2 mg/l whereas in sample >50 meters distance it was found to be 23.9-48.2 mg/l. it is within the desirable limit prescribed by who, which is 250 mg/l. according to versari et al. (2002) chloride concentration higher than 200 mg/l is considered to be a risk for human health and may cause unpleasant taste of water [19]. ammonia content in water may be harmful to health since it can be converted to nitrate. if only ammonia is present in the water then, pollution by sewage must be very recent [20]. the occurrence of no2 with ammonia indicates that sometime has lapsed since the pollution has occurred. if all the nitrogen is present in nitrate form, a long time has been passed after pollution because water has purified itself and all nitrogenous matter has been oxidized [20]. the presence of ammonia in ground water is quite generally a result of natural degradation processes [20]. ammonia in higher concentration is toxic to man. the toxicity of ammonia increases with ph because at higher ph most of the ammonia remains in the gaseous form [21]. ammonia was found in the range of 0.75-5.5 mg/l in samples from areas >10 meters to ≤20 meters distance. in water from areas ≤10 meters distance, ammonia concentration was higher i.e. in the range 4.2-21.4 mg/l, whereas in water samples from area >50 meters distance ammonia concentration was found to be 0.5-10 mg/l. all samples exceeded the who guideline for ammonia which is 0.1 mg/l [6,7]. although, ammonia pollution is not always due to domestic pollution, high ammonia content in deep well can be due to the underlying intercalated layers of peat and lignite. ammonia of mineral origin is rare in natural water but its presence is quite generally a result of natural degradation processes most inevitably due to ammonification of organic matter [22]. in present study, nitrate and nitrite were found to be in the range of 0.3-3.5mg/l and 0.1-2 mg/l, respectively in water samples from areas >10 meters and ≤20 meters distance. in samples from ≤10 meters distance, nitrate and nitrite concentrations were found to be in the range of 1.46.2 mg/l and 1.6-8.4 mg/l, respectively, whereas in water samples taken from areas >50 meters distance these were found to be 0.1-0.8 mg/l and 0.1-2.1 mg/l. although these parameters are comparatively more in water sample ≤10 meter distance, all the values are within the range of who. according to who, the maximum contaminant levels for nitrates at 50 mg/l no3and maximum contaminant level for nitrite is 50 mg/l no3-. also, there have been recorded cases of “blue-baby” syndrome caused by nitrate concentrations only slightly higher than 10 mg/l no3[23]. there is a positive correlation of high nitrate drinking water concentrations to elevated gastric cancer occurrences in chile and england [24]. the microbiological analysis of water was performed by most probable number. mpn index of analyzed water samples showed wide variation and ranged from 4 to ≥2400 coliforms/100 ml. similarly, mpn index was found in the range from 9 to ≥2400 coliforms/100ml in samples from ≤20 meters and >10 meters distance. in ≤10 meters distance, it was found in the range from 21 to ≥2400, whereas in samples from >50 meters distance, it was found to be 4 to ≥2400 coliforms/100ml. this shows that 80% (20/25) of water samples taken from distance ≤20 meters and ≥10 meters is contaminated with coliforms. similarly, 80% (20/25) of water samples taken from distance ≤10 meters is contaminated with coliforms and 46% (23/50) of water samples from >50 meters distance is polluted with coliforms. on analysis of these data, water nearer to river has more coliforms than water farther from the river. the result showed that most of the samples ice. 80% (40/50) water sample nearer from the river (<20 meters) has exceeded the who standard. similarly, samples 46% (23/50) water sample far from the river (>50 meters) has exceeded the who standard. several sources of contamination could be suggested and could include the possibility contamination from improper management of sewer system [6, 25]. e. coli was found to the predominant organism in total coliforms and in most of contaminated drinking water [25-28]. likewise, in our study, e. coli was the predominant organism among all nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 21-26 subedi et al. ©njb, biotechnology society of nepal 25 nepjol.info/index.php/njb isolated coliforms. similarly, fecal coliform was predominant 58% (29/50) in water nearer to river than in water farther from the river 20% i.e. (10/50). presence of fecal coliform indicates that water is polluted with sewage or from improper management of sewer system [29]. comparison of coliforms and fecal coliforms in two distances from the river is shown figure 4. figure 4: comparative analysis of coliform and fecal coliform present in underground water >50 meters, ≤20 meters and <10 meters from the river side. limitations of the study the study could have been extended further for the isolation and identification of other pathogenic bacteria such as salmonella typhi, vibrio cholerae, campylobacter jejuni, helicobacter pylori etc, the presence of which could strengthen the study and confirm the actual presence of the infectious agents in the underground water due to the seepage of the polluted river. conclusion present finding indicates that the underground water nearer to river (≤20 meters) showed comparatively high values of physiochemical and biological parameters than underground water farther (>50 meters) from the river. however, the values of physiochemical and biological parameter increased comparatively if water is taken from even nearer distance i.e. ≤10 meters from river. some households are using underground water as drinking purpose without or with treatment (sand filter). this might be one of the factors causing water borne diseases as this filter does not assure the reduction of chemical and biological parameter to meet standard value. thus, water from the underground needs to be treated to reduce the physiochemical parameter and should be disinfected or boiled before consumption to avoid water-borne diseases. it is important to make people who are using underground water i.e. very near to river that the polluted river has consequences to their drinking water sources aware. the government, local agency should raise awareness to the people about the quality of water who are using underground water. finally, all approaches should be made to make the river water free from chemical and organic pollution. conflict of interest none authors contribution ms was involved in the study conception and design, data analysis and interpretation. ms and mgm were involved in sample collection, drafting manuscript and correction. gsr was involved in reviewing of the manuscript. acknowledgement authors thanks university grants commission (ugc), nepal for providing financial assistance to conduct this research. references 1. singh sp, pathak d & singh r: hydrobiological studies of two ponds of satna (m.p.) india, eco.env.and cons. 2002 8(3): 289-292. 2. raja re, lydia s, princy m & chritopher g: physico-chemical analysis of some groundwater samples of kotputli town jaipur, rajasthan. indian j environ prot. 2002 22(2):137-40. 3. moppw: optimizing water use in kathmandu valley; hmg/nepal, 2003. 4. robertson wd, cherry ja, and sudicky ea: ground water contamination from two small septic systems on sand aquifiers 1991 ground water, 29(1): 82-92. 5. asian development bank and international centre for integrated mountain development, kathmandu (adb/icimod). 2010 6. united nations: the millennium development goals report. united nations department of public information. new york, 2005. 7. world health organization (who): guidelines for drinking water quality. 3rd edition, geneva. 2008. 8. world health organization (who): assessing microbial safety of drinking water improving approaches and methods. 1st edition, geneva. 2003. 9. american public health association (apha): standard methods for washington, d.c.1998 10. aneja kr: experiments in microbiology, plant pathology and biotechnology; new age international, india, 2003. 27 5 6 13 6 4 10 14 15 0 10 20 30 >50m ≤20 >10 <10 absence of coliform total coliform nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 21-26 subedi et al. ©njb, biotechnology society of nepal 26 nepjol.info/index.php/njb 11. wetzel rg: limology; saunders co. philadelphia, usa, 743;1975. 12. patil sg, chonde sg, jadhav as & raut pd: impact of physico-chemical characteristics of shivaji university lakes on phytoplankton communities, kolhapur, india; research journal of recent sciences. 2012 1(2): 56-60. 13. pant br: ground water in kathamandu valley of nepal; environ monit assess. 2010 178:147-185 14. de sousa dn, mozeto aa, carneiro rl and fadini ps: electrical conductivity and emerging contaminant as markers of surface fresh water contamination by waste water; sci total environ. 2014 484:19-26. 15. chan cl zalifah mk & norrakiah as: microbiological and physicochemical quality of drinking water the malaysian j analyt sci. 2007 11(2): 414-420. 16. bhatt lr lacoul hd lekhak hd & jha pk: physicochemical characteristics and phytoplankton of taudaha lake, kathamandu poll res. 1999 18(4): 353-358. 17. ownby cr & kee da: chloride in lake. eric proc cong grat lake. 1967: 382-389. 18. chaudhary uk & ojha csp: environmental impact of the river ganga at varanasi proc, env impact assessment of water resources; project unive, roorkee, 1985: 760-771. 19. versari a, parpinello gp & galassi s: chemometric survey of italian bottled mineral waters by means of their labeled physicochemical composition; j food compos anal. 2002 15: 251-264. 20. manandhar s & sharma s: practical approach to microbiology, 3rd edition; national book centre, botahity, kathmandu, 2003. 21. goel pk: water pollutions: causes, effects and control new delhi-110002, india. h.s. poplai; new age international limited,india, 1997. 22. pandey vp, chapagain sk & kazarna: evaluation of ground water environment of kathmandu valley. environ earth sci. 2010 60:1329-1342 23. washington state department of health: nitrates in drinking water. 1999. retrieved from <http://www.doh.wa.gov/ ehp/dw/npp.htm>, 11/3/99 24. canter w: nitrates in groundwater; lewis publishers. 1997. 25. subedi m & aryal m: public perception about drinking jar water and its bacteriological analysis: nepal med coll j. 2010 12(2):110-114 26. rai sk, ono k, yanagida ji, kurokawa m and rai ck: status of drinking water contamination in mountain region in nepal. nepal med coll j. 2009 11: 281-283. 27. pant br: ground water in kathamandu valley of nepal; environ monit assess. 2010 178:147-185 28. prasai t, joshi dr, lekhak b, & baral mp: microbiological analysis of drinking water of kathmandu valley. scientific world. 2007 5:112114. 29. subedi m: bacteriological quality of drinking water supplied in different schools of kathmandu valley; nepalese journal of microbiology. 2011 2:50-55. nepal j biotechnol. 2 0 2 0 j u l y ; 8(1):17-28 doi: https://doi.org/10.3126/njb.v8i1.30206 ©njb, bsn 17 research article phytochemical evaluation, antioxidant and antimicrobial activities of various extracts from leaves and stems of bryophyllum pinnatum imaobong e. daniel , ekemini i. akpan, edidiong c. utam department of chemistry, university of uyo, pmb 1017, uyo, akwa ibom state, nigeria article history:received: 5 mar 2020; revised: 7 jul 2020; accepted: 20 jul 2020; published online: 31 jul 2020 abstract antioxidant and antimicrobial activities of different extracts (methanol and ethyl acetate) of leaf and stem of bryophyllum pinnatum were studied. the screening for the secondary metabolites was carried out using the standard methods. the antioxidant capacities of the different extracts were assessed using dpph (2,2-diphenyl1-picrylhydrazyl) radicals and ferric reducing antioxidant power (frap) while the antimicrobial activity of the extracts obtained were screened against gram-positive, gram-negative bacteria and fungi (staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, salmonella spp., vibrio cholerae, candida albicans and aspergillus niger) using agar well diffusion method. both extracts obtained from leaf and stem of b. pinnatum contained most of the phytochemical compounds tested for. however, anthocyanins and anthraquinone were not detected in leaf extracts while coumarin was absent in stem extracts. quantification of bioactive compounds showed that both extracts contained the highest concentration of polyphenols (34.49 ±0.47 mg gae/g and 32.32 ±1.2 mg gae/g for methanol leaf and stem extracts respectively) while the least concentration was recorded for alkaloids (0.03±0.02 mg/g for methanol stem extract). results revealed that the extracts showed dosedependent scavenging of dpph as well as the ability of the extracts to reduce fecl3 solution, with methanol extracts exhibiting the highest scavenging and reducing capacity. however the leaves of b. pinnatum had greater antioxidant activity than the stem by dpph and ferric reducing assays, with ic50 values ranging from 3.147µg/ml to 3.80µg/ml for dpph and 331.9 451 µg/ml for frap assays. the antimicrobial activity of various solvent extracts of leaf and stem reveal that microorganisms exhibited different sensitivities towards these extracts in a dose-dependent manner. methanol leaf extract showed no activity against e. coli while p. aeruginosa was insensitive to ethyl acetate leaf extract. for stem extracts, a. niger, v. cholerae and p. aeruginosa were resistant to methanol extract while a. niger, salmonella spp. and p. aeruginosa was resistant towards ethyl acetate stem extract. the results obtained in this study showed that b. pinnatum is a reservoir of bioactive compounds and both extracts exhibited significant antimicrobial and antioxidant activity. keywords: bryophyllum pinnatum, antioxidant activity, agar well diffusion, aspergillus niger, polyphenols, ferric reducing power assay. corresponding author, email: imaudoekwere@gmail.com introduction a medicinal plant is any plant in which one or more of its organs contain substances that can be used for the synthesis of useful drugs, and also serve as a major lead for modern drug design [1, 2]. numerous researchers on medicinal plants and herbal drug production reported that bioactive components of medicinal plant occur in the leaves, flowers, roots, stem, bark or wood. these bioactive compounds commonly known as phytochemicals include terpenes, alkaloids, flavonoid, bioflavonoid, benzophonones, xanthenes as well as some metabolites such as tannins, saponins, cyanates, oxalate and anthraquinones [3, 4, 5, 6]. several plants containing secondary metabolites possess antioxidant, antimicrobial and other biological potentials [7]. natural antioxidants, such as phenolic compounds are gaining importance due to their benefits for human health, decreasing the risk of degenerative diseases by reduction of oxidative stress and inhibition of macromolecular oxidation [8, 9]. several studies indicate that medicinal plants which are rich reservoir of bioactive compounds contain compounds that are significant in therapeutic application against human and animal pathogen, including bacteria, fungi and viruses [10, 11]. nepal journal of biotechnology publisher: biotechnology society of nepal issn (online): 2467-9313 journal homepage: www.nepjol.info/index.php/njb issn (print): 2091-1130 mailto:imaudoekwere@gmail.com https://orcid.org/0000-0001-6097-901x nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 18 studies show that nearly 80% of the world’s population still relies on traditional medicines for primary health care, most of which is the use of plant extracts [12]. in the past few years, uncertainties concerning the safety of synthetic antimicrobial drugs have led to an increase in the demand for natural compounds such as plants rich in antimicrobials [13]. this is because herbal medicines have been reported to be safe, affordable, acceptable, available and without any adverse side effects especially when compared with synthetic drugs [14, 15]. in developing countries, there is a gradual revival of interest in the use of medicinal plants especially herbal preparations in the local healthcare systems because of the increasing problems of multidrug resistance (mdr) to human pathogenic bacteria [16, 17]. one of such plants used in the treatment of a wide range of ailments is bryophyllum pinnatum (b. pinnatum). b. pinnatum belongs to the family crassulaceae and the common names include life plant, love plant, miracle leaf, and canterbury bells. it is widely distributed in tropical africa, america, hawaii, india, china, australia, and madagascar [18]. b. pinnatum is a succulent plant, 50 – 200 cm tall and about 3.2 cm wide, and reproduces via seeds and also vegetatively from leaf bulbils [19, 20]. they are medium green above blotched with purple underneath. it has flashy, dark green leaves. its flower is in paniculate cymes 20 – 80 cm long. it has fruit whose follicles are 10 -14 mm long enclosed in the persistent papery calyx. the seeds are numerous in each fruit. the leaves and leaf juice have been used traditionally as anti-inflammatory, antipyretic, antimicrobial, antioxidant, antitumor, antidiabetic, anti-ulcer, antiseptic, hypocholesterolemic, and cough suppressant [21]. in nigeria, the plant is particularly known for its effective wound healing properties and detachment of the umbilicus of infants, for the treatment of earache, burns, abscesses, ulcer, insect’s bites, whitlow, diarrhea and lithiasis [19, 20]. the lightly roasted leaves are used externally for skin fungus and inflammations and the leaf infusion is an internal remedy for fevers [22]. b. pinnatum leaves are used to expel worms, cure acute and chronic bronchitis, pneumonia and other forms of respiratory tract infections [23]. it is used for all sorts of respiratory conditions such as asthma, cough and bronchitis. it is employed for the treatment of kidney stones, gastric ulcers and oedema of the leg [24]. materials and methods collection of plant materials fresh leaves and stem of b. pinnatum were obtained from etinan and ikot ekpene local government areas in akwa ibom state, nigeria, and authenticated by the department of botany, university of uyo, nigeria, and a voucher specimen [voucher no: uuph27(a)] was kept in the herbarium for future reference. extraction procedure the leaves and stem were thoroughly washed with distilled water and air-dried for 2 weeks. the dried parts were pulverized and the powdered plant parts were separately divided into portions. 750g of the different plant parts were macerated with 1.7 l each of methanol and ethyl acetate at room temperature for 72 hrs. after 72 hrs, the different extracts were filtered separately off through a cotton plug and finally with a whatman no. 1 filter paper. the liquid filtrates were concentrated and evaporated to dryness using a rotary evaporator (wgtv311-v, wilmad-labglass, usa) at 40 °c, and each extract was transferred into well-labeled sterile glass vials and stored at 4 °c before use [25]. phytochemical screening of plant extracts the leaf and stem extracts (methanol and ethyl acetate) were screened for the presence of various bioactive components (phytochemicals) using standard procedures [6, 26, 27]. test for anthraquinones to 6g of the different plant parts in this study, 10 ml of benzene was added. after 10 minutes, the solution was filtered and 10 ml of 10% ammonia was added to the filtrates and shaken. the presence of pink, violet, or red color signified the presence of anthraquinones in the ammonia phase [26]. determination of tannins 10 ml each of bromine water was added to the 0.5 g of leaf and stem extracts of b. pinnatum. the discoloration of bromine water indicated the presence of tannins. test for saponins 5.0 ml of distilled water was added to the different plant extracts in a test tube. the froth formed was nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 19 mixed with few drops of olive oil. the formation of foams showed the presence of saponins. tests for flavonoids shinodatest. magnesium strip and hcl were mixed with plant extracts. the development of pink colour confirmed the presence of flavonoid. tests for glycosides liebermann’s test. 2.0 ml of acetic acid and 2 ml of chloroform were added to the different extracts. the mixtures were allowed to cool after which concentrated h2so4 was added. the appearance of green color signified the presence of aglycone, steroidal part of glycosides. keller-kiliani test. a solution of glacial acetic acid (4.0 ml) with 1 drop of 2.0% fecl3 mixture as well as 1 ml h2so4 was added to 10 ml of the different plant extracts. a brown ring formed between the layers indicated the presence of cardiac steroidal glycosides. salkowski’s test. 2 ml of conc. h2so4 was added to the plant crude extract. a reddish-brown color formed indicated the presence of the steroidal aglycone part of the glycoside. test for terpenoid 2.0 ml of chloroform was added to 5 ml plant extracts and evaporated on the water path. the mixture was boiled with 3 ml of h2so4. a grey color formed confirmed the presence of terpenoids. test for steroids 2 ml of chloroform and concentrated h2so4 were added with the 5 ml plant crude extract. in the lower chloroform layer, red color appeared that indicated the presence of steroids. test for coumarins 1ml of 10% sodium hydroxide solution was added to 1ml of the different plant extracts. formation of yellow colour indicated the presence of coumarins test for reducing sugar (deoxy sugars) 0.5g of each extract was macerated with 20 ml of distilled water and filtered. to 1 ml of the filtrates, 1 ml of alkaline copper reagent was added. the mixture was boiled for 5 min and allowed to cool. then 1 ml of phosphomolybdic acid reagent and 2 ml of distilled water was added and the absorbance read at 420 nm test for phenols the extract (500 mg) was dissolved in 5ml of distilled water. to this, few drops of neutral 5% ferric chloride solution were added. a dark green color indicated the presence of phenolic compounds. test for quinone 1ml of each of the various extracts was treated separately with alcoholic potassium hydroxide solution. quinones give coloration ranging from red to blue. test for amino acids and proteins biuret test: to 0.5 mg of extract equal volume of 40% naoh solution and two drops of 1% copper sulphate solution was added. the appearance of violet colour indicated the presence of protein. ninhydrin test: about 0.5 mg of extract was taken and 2 drops of freshly prepared 0.2% ninhydrin reagent was added and heated. the appearance of pink or purple colour indicated the presence of proteins, peptides or amino acids. anthocyanins 2 ml of 2 n hcl and ammonia was added to 2 ml of the different extracts. the appearance of pink-red which later turned blue-violet indicated the presence of anthocyanins. test for alkaloids extracts were dissolved individually in dilute hydrochloric acid and filtered. the filtrates were used to test the presence of alkaloids. dragendorff’s test: 1ml of the filtrate was treated with few drops dragendorff’s reagent. formation of orange-brown precipitate indicated the presence of alkaloids. mayer’s test: filtrates were treated with mayer’s reagent. the formation of a yellow cream precipitate indicated the presence of alkaloids. quantitative estimation of phytoconstituents the phytochemicals which are present in the methanol and ethyl acetate stem and leaf extracts of b. pinnatum was quantitatively determined using standard protocols. determination of polyphenols the total phenolic content in the extracts were determined by the modified folin-ciocalteu method as described by singleton and rossi [28] and modified by ayoola et al. [29]. sample extract was dissolved in methanol (1 mg/ml). an aliquot of 0.5 nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 20 ml of each plant extract (1 mg/ml) was mixed with 5 ml of folinciocalteu reagent which was previously diluted with distilled water (1:10 v/v). the mixture was shaken slightly and allowed to stand at 22 °c for 5mins. after, 4 ml (75 g/l) of sodium carbonate (na2co3) was added, and the tubes containing the mixtures were allowed to stand for 30 min at 40 °c to develop colour. absorbance was then read at 765 nm using the uv spectrophotometer (shimadzu, japan). results were expressed as gallic acid equivalent in (mg/g) of extracts (10–100 mg/ml). all samples were analyzed in triplicate. determination of total flavonoids total flavonoid contents were determined using aluminium chloride colorimetric method [30]. a volume of 0.5 ml of 2% alcl3 ethanol solution was added to 0.5 ml of sample solution. after one hour at room temperature, the absorbance was measured at 420 nm. using uv spectrophotometer (shimadzu, japan). yellow color indicated the presence of flavonoids. total flavonoid content was calculated as quercetin equivalent (mg/g). the calibration curve ranged from 10 – 100 mg/ml. determination of total alkaloids total alkaloids were determined according to the standard method as described by harbone [27]. 200 ml of 10% acetic acid in ethanol was added to 5 g of different plant extracts. the mixtures were covered and allowed to stand for 4 h. the solutions were decanted and filtered and the extracts were further concentrated in a water bath until one-quarter of the original volume was obtained. concentrated ammonium hydroxide was added dropwise to the concentrated extract until the precipitation was complete. the precipitate was collected and washed with dilute ammonium hydroxide and then filtered. the residue was dried and weighed. determination of total tannins 50 ml of distilled water was added to 500 mg of the plant extracts and was shaken for 1 h in a mechanical shaker. the solution obtained was filtered into a 50 ml volumetric flask and made up to the mark. 5 ml of the filtrate was pipetted out into a test tube and mixed with 2 ml of 0.1 m fecl3 in 0.1 n hcl and 0.008 m potassium ferrocyanide. the absorbance was measured at 120 nm within 10 min [31]. determination of total saponins to 20 g of each plant extracts, 100 cm3 of 20% aqueous ethanol were added. the samples were heated at 55°c for 4 h with continuous stirring. the mixture was filtered and the residue re-extracted with 200 ml 20% ethanol. the combined extracts were reduced to 40 ml over water bath at about 90°c. 20 ml of diethyl ether was added to the concentrates and shaken vigorously. the aqueous layer recovered was further purified by adding 60 ml of n-butanol. the combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride and heated in a water bath. after evaporation, the samples were dried in the oven to a constant weight and the saponin content was calculated [32]. in vitro antioxidant assay antioxidant activity by dpph assay the free radical scavenging activity of the different extracts was measured in vitro by 2,2’-diphenyl-1picrylhydrazyl (dpph) method as described by brand-williams et al. [33]. 0.5 mm solution of dpph was added to sample solutions at different concentrations (20 – 100 μg/ml). a control (abs control) containing methanol and dpph solution was also prepared. all solutions obtained were then incubated for 1 hour at room temperature. absorbance was measured at 517 nm. vitamin c was used as standard and the same concentrations of it were prepared as the test solutions. the percentage of inhibition of samples was calculated from obtained absorbance by the equation: 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑜𝑓 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠 𝑡𝑒𝑠𝑡/𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐴𝑏𝑠 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 a percent inhibition versus concentration curve was plotted and the concentration of sample required for 50% inhibition was determined and represented as ic50 value for each of the test solutions. ferric reducing/antioxidant power (frap) assay the reducing property of the extract was determined by assessing the ability of the extracts to reduce fecl3 solution [34]. briefly appropriate concentrations of the extracts were mixed with 2.5 ml of 200 mm of sodium phosphate buffer (ph 6.6) and 2.5 ml of 1% potassium ferrocyanide. the mixture was incubated at 50°c for 20 min after which 2.5 ml of 10% trichloroacetic acid was added. the mixture was then centrifuged at 650 rpm for 10 min. supernatant nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 21 (the upper layer) (5 ml) was mixed with equal volume of deionized water and 1 ml of 0.1% ferric chloride, and the absorbance was measured at 700 nm. the ferric-reducing-power capacities of the plant extracts and standard antioxidants were expressed graphically by plotting absorbance against concentration [34]. ascorbic acid was used as a positive reference. the experiment was done in triplicate. antimicrobial activity collection of test organisms microorganisms used were obtained from the microbial stock collection unit of the department of microbiology, university of uyo, akwa ibom state. the test organisms used were 1 gram-positive bacterium (staphylococcus aureus), 4 gram-negative bacteria (escherichia coli, pseudomonas aeruginosa, salmonella spp., vibrio cholerae) and two fungi (candida albicans, aspergillus niger). these organisms were sub cultured to obtain pure and fresh isolates. the pure bacterial cultures were maintained on nutrient agar medium and fungal culture on potato dextrose agar (pda) medium. isolates were identified using standard microbiological procedures by carrying out gram’s reaction and biochemical tests to confirm the species. preparation of test organisms before inoculation mcfarland standard was used as a reference to adjust the turbidity of bacterial suspensions. the bacterial suspensions were standardized following the clsi guidelines for aerobic bacteria. all of the test microorganisms were grown in mueller hinton broth for 18–24 h, followed by the matching of bacterial suspension to the turbidity equivalent to 0.5 mcfarland solutions (1-2×108 cfu/ml). different concentrations (10, 20, 40, 60 and 80 mg/ml) of the extracts were prepared and kept in corked test tubes. seeding of muller – hinton agar plates 0.1 ml of each diluted isolates was aseptically transferred into mueller–hinton agar (oxoid, uk) plates and aseptically spread evenly using sterile hockey stick. the seeded plates were left for 30 mins for the isolates to diffuse into the medium. sterile cork borer of 5 mm was used to bore holes on the agar plates. 0.1 ml of each of the extracts was then dropped in the holes and labeled accordingly. the diameters of inhibition zones were measured [35]. results phytochemical screening the results of the qualitative phytochemical screening of methanol and ethyl acetate of leaf and stem extracts of b. pinnatum as presented in table 1 revealed the presence of amino acids, quinone, phenol, reducing sugar, coumarin, steroids, terpenoids, glycosides, flavonoids, saponins, tannins and alkaloids in both plant parts. however, anthocyanins and anthraquinones were not detected in the methanol and ethyl acetate leaf extracts. the results of the quantitative phytochemical screening of methanol and ethyl acetate of leaf and stem extracts of b. pinnatum in table 2 shows that the concentration of alkaloids ranged from 0.03±0.02 to 0.90±0.13 mg/g. alkaloids were not detected in the ethyl acetate stem extract. the concentration of saponins varied from 0.45±0.43 to 1.12±0.21 mg/g, with methanol leaf extracts having the highest concentration (1.12±0.21 mg/g). the results of flavonoids (y=0.142x-0.0.177: r2=0.983) in both plant part extracts showed that the stem extracts contained the highest concentration of flavonoids (0.53±0.13 and 0.31±0.01 mg for methanol and ethyl acetate extracts respectively) while the table 2: quantification of the phytochemicals in the extracts of bryophyllum pinnatum phytochemica ls (mg/g) leaf extracts stem extracts methanol ethyl acetate methanol ethyl acetate alkaloids 0.16±0.01 0.90±0.13 0.03±0.02 nd saponins 1.12±0.21 1.02±0.22 1.05±0.11 0.45±0.43 flavonoids 0.21±0.14 0.11±0.14 0.53±0.13 0.31±0.01 tannins 4.98±1.31 2.8±±0.21 0.64±0.24 0.25±0.12 poly phenols 34.49±0.47 21.2±2.2 32.32±1.2 17.9±0.62 nd = not detected table 1: qualitative analysis of different parts of bryophyllum pinnatum plant parts leaf extract stem extract solvents methanol ethyl acetate methanol ethyl acetate anthocyanins absent absent present present amino acid present present present present quinone present present present present phenol present present present present reducing sugar present present present present coumarin present present present present steroids present present present present terpenoids present present present present glycosides present present present present flavonoids present present present present saponins present present present present tannins present present present present anthraquinone absent absent present present alkaloids present present present absent nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 22 least concentration was present in the leaf extracts with (0.21±0.14 and 0.11±0.14 mg/g for methanol and ethyl acetate extracts respectively). tannin content (y=0.168x -0.094: r2=0.975) was higher in methanol leaf extract (4.98±1.31 mg/g) while the least concentration was recorded for ethyl acetate stem extract (0.25±0.12 mg/g). methanol leaf extract recorded the highest concentration of polyphenols (34.49±0.47 mg/g) while ethyl acetate stem extract contained the least concentration (17.9±0.62 mg/g). (y=0.149–0.121 x: r2 = 0.967). antimicrobial activity tables 3 and table 4 show the result of the antibacterial activity of leaf and stem extracts of b. pinnatum respectively tested against five bacterial and two fungi strains at different concentrations (10, 20, 40, 60 and 80 mg/ml). all the extracts showed strong antimicrobial activity against test organisms in a dose-dependent manner. results from table 3 showed that s. aureus, c. albican, v. cholerae, salmonella spp. and p. aeruginosa were susceptible to the methanol leaf extract of b. pinnatum at all concentrations. however, antibacterial activity was not observed against e. coli. for ethyl acetate leaf extracts, the tested microorganisms showed varying degree of susceptibility at various concentrations except v. cholerae and p. aeruginosa, which were resistant to ethyl acetate extract at all concentrations. table 4 showed that a. niger, v. cholera and p. aeruginosa were resistant to methanol stem extract at all concentrations while salmonella spp. was resistant at 10 mg/ml. the zone of inhibition of s. aureus ranged from 7±0.11 to 15±0.01 mg/ml while that of table 3: antimicrobial activity of methanol and ethyl acetate leaf extracts of bryophyllum pinnatum against the human pathogenic bacteria by disc diffusion method. crude extracts isolates zone of inhibition (mm) 10 mg/ml 20 mg/ml 40 mg/ml 60 mg/ml 80 mg/ml methanol s. aureus 8.00±0.01 10.00±0.3 12.0±1.2 14±0.2 20±0.5 a. niger na na na 10±0.05 14±0.3 candida albican 16±0.34 20±0.21 23±1.2 30±1.2 34±1.41 vibrio cholera 8±0.03 10±0.25 11±0.23 15±0.11 22±1.21 escherichia coli na na na na na salmonella spp. 10±0.12 13±0.22 16±1.3 20±1.31 23±1.10 p. aeruginosa 14±0.01 20±1.22 23±1.2 27±1.22 30±0.9 ethylacetate s. aureus 8±0.01 9±0.01 10±0.13 13±0.03 14±0.12 a. niger 8±0.1 9±0.11 10±0.6 12±0.11 13±0.21 candida albican 13±0.3 17±0.33 21±0.02 26±0.22 30±0.12 vibrio cholera na na na na na escherichia coli 13±0.01 15±0.01 17±0.03 20±0.41 25±0.61 salmonella spp. na 9±0.01 10±0.05 12±0.04 14±0.12 p. aeruginosa na na na na na key: na= no activity, s. aureus= staphylococcus aureus, a. niger=aspergillus niger, p. aeruginosa= pseudomonas aeruginosa table 4: antimicrobial activity of methanol and ethyl acetate stem extracts of bryophyllum pinnatum against the human pathogenic bacteria by disc diffusion method. crude extracts isolates zone of inhibition (mm) 10mg/ml 20mg/ml 40mg/ml 60mg/ml 80mg/ml methanol s. aureus 7±0.11 10±0.02 11±0.12 13±0.11 15±0.01 a. niger na na na na na candida albican 9±0.02 10±0.01 13±0.2 15±0.13 17±0.11 vibrio cholera na na na na na escherichia coli 8±0.22 11±0.03 14±0.15 17±0.21 19±1.2 salmonella spp. na 6±0.01 9±0.01 11±0.12 13±0.11 p. aeruginosa na na na na na ethyl acetate s. aureus na 9±0.01 10±0.13 11±0.01 12±0.12 a. niger na na na na na candida albican 10±0.3 11±0.22 13±0.14 15±0.23 22±1.02 vibrio cholera 8±0.01 11±0.5 14±0.22 16±0.13 18±0.54 escherichia coli 13±0.11 16±0.13 20±0.01 24±0.62 28±0.43 salmonella spp. na na na na na p. aeruginosa na na na na na key: na = no activity, s. aureus = staphylococcus aureus, a. niger =aspergillus niger, p. aeruginosa= pseudomonas aeruginosa nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 23 c. albican varied from 9±0.02 to 17±0.11 mg/ml. at 10 mg/ml, the zone of inhibition of e. coli was 8±0.22 mg/ml while at 80mg/ml, the zone of inhibition was 19±1.2 mg/ml. ethyl acetate stem extract did not show any antimicrobial activity against a. niger, salmonella spp. and p. aeruginosa. higher antimicrobial activity was observed against c. albican (10±0.3 -22±1.02 mg/ml) and e. coli (13±0.11 28±0.43 mg/ml). antioxidant activity dpph free radical scavenging activity figure 1: dpph scavenging activity of leaf extract of bryophyllum pinnatum methanol : y = 11.26x + 14.56: r2 = 0.612 vit c: y =13.16x +18.48: r2 = 0.590 ethyl acetate: y = 9.231x+ 14.84: r2 = 0.550 figure 2: dpph scavenging activity of stem extract of bryophyllum pinnatum methanol: y = 10.36x + 10.28; r2 =0.677 ethyl acetate y = 9.365x +8.62; r2 = 0.693 figures 1 and figure 2 show the results of dpph assay of methanol and ethyl acetate of leaf and stem extracts of b. pinnatum respectively. vitamin c was used as the standard. the extracts have shown dosedependent scavenging of dpph radicals. the result also showed that at 100 μg/ml dose, the leaf extracts (methanol and ethyl acetate) inhibited dpph radical by 72.5% and 60.2% respectively while at the same concentration, methanol and ethyl acetate stem extracts inhibited dpph radical at 63. 8% and 57.6% respectively compared to the standard drug, vitamin c (85.4%). a graph of % inhibition against various concentrations was plotted and the ic50 was calculated from the different regression graphs and the results are presented in table 5. the ic50 in dpph assay ranged from 3.147 to 4.41 μg/ml. table 5: ic 50 in μg/ml for antioxidant activity of bryophyllum pinnatum extracts. ic 50 for vitamin c in dpph is 2.39, while that for frap is 220.24 antioxidant activity leaf extracts stem extracts methanol ethyl acetate methanol ethyl acetate dpph 3.147 3.80 3.83 4.41 frap 331.9 451 428.30 618.38 frap assay the ferric-reducing-power capacities of the plant extracts and standard antioxidants were expressed graphically by plotting the absorbance against concentration and results presented in figures 3 and. all the plant extracts showed concentrationdependent reducing power. the ic50 of the ranged from 331.9 μg/ml. to 618.38 μg/ml. figure 3: ferric reducing power activity of leaf extract of bryophyllum pinnatum vit c: y = 0.227x + 0.004; r2 = 0.886 methanol: y= 0.151x – 0.123; r2 = 0.991 ethyl acetate y = 0.0.110; r2 = 0.984 figure 4: ferric reducing power activity of stem extract of bryophyllum pinnatum 0 20 40 60 80 100 0 20 40 60 80 100 % in h ib it io n concentration in ug/ml methanol ethyl acetate vit c 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 10 20 30 40 50 a b s o rb a n c e concentration in ug/ml methanol ethyl acetate vit c 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 10 20 30 40 50 a b so rb a n ce concentration in ug/ml methanol ethyl acetate 0 10 20 30 40 50 60 70 80 90 0 20 40 60 80 100 % i n h ib it io n concentration in ug/ml methanol ethyl acetate vit c nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 24 methanol y = 0.117x – 0.112, r2 = 0.919 ethyl acetate y = 0.081x – 0.089; r2 = 0.929 discussion the preliminary phytochemical screening of the different extracts of b. pinnatum obtained from leaves and stem was done to assess the presence of bioactive compounds and the results were presented in table 1. the results showed that the leaf and stem extracts contained moderate to high concentrations of most of the phytochemical compounds screened for. however, anthocyanins and anthraquinones were not detected in the leaf extracts while coumarins were not found in the stem extracts. the various phytochemical compounds detected in the different plant extracts (table 1) are known to have health benefits, physiological activities and medicinal importance [36]. quantitative analysis was done to check the concentration of major bioactive compounds such as polyphenols, flavonoids, alkaloids, saponins, and tannins in the different extracts of b. pinnatum and the results is presented in table 2. from the results obtained, the leaf extracts contained the highest concentration of phytochemicals quantified. however, the stem extract contained the highest concentration of flavonoids. this may be attributed to the fact that the stem may be a reservoir of flavonoids. both extracts followed a similar trend as follows; polyphenols> tannins> saponins> flavonoids > alkaloids. the polyphenolic content of the different extracts ranged from 17.9±0.62 to 34.49±0.47 mg gae/g. many studies have corroborated the anticarcinogenic, anti-bacterial, anti-viral or antiinflammatory activities of phenolic compounds [37, 38, 39]. they are also known as a powerful chainbreaking antioxidants. these groups of compounds have received much attention as potential natural antioxidants in terms of their ability to act as efficient radical scavengers and metal chelators. it has been reported that the antioxidant activity of phenol is mainly due to their redox properties, hydrogen donors and singlet oxygen quenchers [40]. the presence of phenolic compounds in b. pinnatum indicates that these plants may be used as an antioxidant and anti-microbial agent. the result of the quantification of tannins indicated that the different extracts of b. pinnatum contained varying concentrations of tannin, with methanol leaf extract having the highest concentration (4.98±1.31mg/g). plant tannins extracted from various sources have been also shown to possess antitumor-promoting effects, analgesic, antioxidant, anti-inflammatory and antimicrobial activities [19, 41-44]. tannin exerts its antioxidant activity by scavenging free radicals, inhibiting lipid peroxidation and chelating of metal [45]. the concentration of flavonoids in this study ranged from 0.53±0.13 mg/g to 0.11±0.14 mg/g. interestingly, the methanol stem extract had the highest concentration of flavonoids while the least was recorded for ethyl acetate leaf extract. studies have shown that the intake of flavonoid-rich diets lowers heart diseases [46]. this is because flavonoids lower high blood pressures, as well as cholesterol in animal studies and, have strong antiinflammatory properties [47]. they also inhibit low density lipoprotein (ldl) oxidation by free radicals flavonoids exert their antioxidant activity by scavenging free radicals, chelating metals and inhibiting lipid peroxidation. the –oh at c3 of the flavonoid structure plays a role in chelating and scavenging activity [48]. their antimicrobial properties of flavonoids are probably because they form complexes with both extracellular and soluble proteins, as well as the bacterial cell wall. they could also disrupt cell membranes if lipophilic enough [49]. the presence of flavonoids in extracts of b. pinnatum studied enhances its antimicrobial and antioxidant capacity. saponins are widely distributed in plants and research has shown that they have many pharmacological actions and biological activities like anti-inflammatory, molluscicidal, antimicrobial, antispasmodic, anti-diabetic and anticancer, hypocholesterolemic, antioxidant, anticonvulsant and analgesic, anthelmintic and cytotoxic activities [21, 50, 51]. the result obtained in this study showed that b. pinnatum contained moderate levels of saponins. the result obtained for the quantification of alkaloids indicated that the extracts contained moderate levels of alkaloids. ethyl acetate leaf extract contained the highest concentration of alkaloids (0.90±0.13 mg/g) while the lowest concentration was recorded for methanol stem extract (0.03±0.02 mg/g). this may be as a result of the nature of the extractable alkaloids present in the nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 25 extract. naturally occurring alkaloids and their synthetic derivatives have analgesic, antispasmodic and bactericidal activities [44]. the phytochemical compounds present in the different extracts of b. pinnatum may be responsible for its antimicrobial and antioxidant properties. evaluation of antimicrobial activity of the different extracts of the studied plants parts was determined against different microorganisms. extent of sensitivity of the test organisms to the plant extracts was assessed by measuring the zone of inhibition after 24 hrs incubation. table 3 and 4 showed the antimicrobial activity of b. pinnatum leaf and stem extracts respectively using different extracting solvents. all the tested extracts revealed antimicrobial activity showing different selectivity. the result revealed varying degree of inhibition on the different test isolates, with more significant inhibition seen with a higher extract concentration. the results revealed that the methanol leaf extract of b. pinnatum was most effective against the test organisms. p. aeruginosa showed the highest susceptibility (30±0.9 mm at 80 mg/ml) to b. pinnatum methanol leaf extract, while s. aureus showed the least susceptibility (20±0.5 mm). however, for ethyl acetate leaf extract, the most susceptible bacteria was e. coli (25±0.61mm at 80 mg/ml) while the least was salmonella spp. (14 ±0.12 mm). the results also revealed that all the test fungi were susceptible to the leaf extracts. methanol and ethyl acetate leaf extracts showed maximum inhibition at 34±1.41 mm and 30±0.12 mm against c. albican respectively. the result of the antimicrobial activity of the different stem extracts revealed that e. coli showed the highest sensitivity to methanol and ethyl acetate extracts at 19±1.2 mm and 28± 0.43 mm, respectively. the least susceptibility was recorded for salmonella spp. and s. aureus at 13±0.11 mm and 12± 0.12 mm for methanol and ethyl acetate stem extracts respectively. the results also revealed that all the test fungi were resistant to the different stem extracts except c. albicans. it should be noted that the effects of the stem and leaves extract against the bacteria also differed; the variation observed in the phytochemical compounds detected in the extracts may possibly account for their varied bioactivity [52, 53]. the antimicrobial activity could be attributed to some of the detected compounds in these plant extracts such as tannins, saponins, alkaloids, flavonoids and terpenoids [54, 49, 55]. the antioxidant capacity of the different extracts of b. pinnatum was evaluated by two different assays: free radical scavenging action on dpph radicals and ferric reducing power. this is because no single assay can represent the total antioxidant capacity, and for this reason different and complementary assays were used to evaluate the extract antioxidant activities: the scavenging ability of b. pinnatum extracts (leaves and stem) on dpph free radical is shown in figures 1 and figure 2. the result indicated that all of the assessed extracts of b. pinnatum were able to reduce the stable, purple-coloured radical dpph to the yellow coloured dpph-h form. the results showed a dose-dependent scavenging power, where activity increased as the concentration increased for both extracts. the result also showed that at 100 μg/ml dose, the leaf extracts (methanol and ethyl acetate) inhibited dpph radical by 72.5% and 60.2% respectively while at the same concentration, methanol and ethyl acetate stem extracts inhibited dpph radical at 63.8% and 57.6% respectively compared to the standard drug, vitamin c (85.4%). the ic50 for dpph and frap assays were calculated from a linear regression analysis of the observed inhibition percentage against concentration. the results showed that the ic50 value for dpph assay ranged from 3.142 – 4.441 µg/ml the dpph radical scavenging ability of the extracts showed the following trend vitamin c<methanol leaf extract<ethyl acetate leaf extract<methanol stem extract<ethyl acetate stem extract (table 5). it is interesting to note that the lower the ic50 value, the higher the scavenging activity of the plant extract. the leaf extracts recorded the lowest ic50 values hence its higher antioxidant properties. it was evident that the extracts showed hydrogen donating ability and therefore the extracts could serve as free radical scavengers, acting possibly as primary antioxidants [56]. ferric reducing antioxidant power (frap) assay is used to evaluate the capacity of natural antioxidant to donate an electron or hydrogen [57]. in the reducing power assay, the presence of antioxidants in the samples would result in the reduction of fe3+ to fe2+ by donating an electron [58]. the ferric reducing activity of the leaf and stem extracts of b. nepal j biotechnol. j u l y 2 0 2 0 ; 8(1):17-28 daniel et al. ©njb, bsn 26 pinnatum are presented in figures 3 and figure 4. all extracts exhibited a similar concentration-dependent activity pattern as given in the dpph analysis. the reducing power of the extracts followed the order of methanol leaf extract>methanol stem extract>ethyl acetate leaf extract>ethyl acetate stem extract. the reducing properties are generally associated with the presence of reductones which have been shown to exert antioxidant action by breaking the free radical chain and donating a hydrogen atom. reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation [59-61]. from the results obtained, it can be inferred that the extracts studied possess reducing power and therefore, could serve as electron donors, terminating the radical chain reactions. the ic50 value was used as a significant indicator of antioxidant ability. the ic50 (table 5) for ferric reducing activity ranged from 331.9 to 618.38 μg/ml. the antioxidant activity of these plants could be due to the ability to scavenge specific free radicals and/or due to the interaction with redox chemistry of iron ions. conclusion methanol and ethyl acetate extracts of leaf and stem of b. pinnatum were obtained in this study. phytochemical screening revealed the presence of important bioactive compounds such as polyphenols, tannins, saponins, flavonoids and alkaloids. it is worthy of note that all the extracts in this study exhibited broad-spectrum antimicrobial activity on the test microorganisms with highest activity recorded for methanol extracts. the extracts exhibit significant antioxidant activity-dpph radical scavenging and ferric reducing when compared with standard compounds. methanol extracts of different plant parts demonstrated the most significant antioxidant activity and also highest polyphenolic content which could be responsible for the activity. the results obtained in this study showed broadspectrum antibacterial activity as well as antioxidant potential of b. pinnatum; hence further isolation, purification, identification and structure elucidation of the phenolic phytochemical constituents should be carried out. author’s contributions this work was carried out in collaboration with all authors. author ied designed the study, wrote the procedures and the first draft of the manuscript. author ecu and ecu managed the literature searches and the experimental process. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. funding the authors declared that no grants were involved in supporting this work. acknowledgments the authors wish to thank the department of biochemistry and the department of microbiology, university of uyo for their assistance during this research work. ethical approval and consent not applicable. references 1. world health organization (who). resolution-promotion and development of training and research in traditional medicine. who document. 1977;30:49-50. 2. vijayalakshmi r, ravindhran r. preliminary comparative 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1department of microbiology of tri-chandra campus, kathmandu, nepal 2western regional hospital, pokhara, nepal 3 siddhi memorial hospital, bhaktapur, nepal 4deurali-janta pharmaceuticals pvt. ltd., kathmandu, nepal abstract shigellosis, a disease caused by shigella species. it is a major public health problem in developing nations like nepal, where communities having poverty; poor sanitation, personal hygiene, and water supplies. the main aim of our study is to isolate and identify shigella spp. from gastroenteritis patients and to find out its drug resistance pattern. a cross-sectional study was carried out based on routinely attending outpatients and inpatients. a total of 225 stool samples collected from gastroenteritis patients were processed from 20 april to 24 september 2014 in western regional hospital, pokhara, nepal. standard microbiological procedures were followed for the isolation of shigella spp. after that slide agglutination kit method was used for identification of shigella spp. finally, kirby-bauer disc diffusion method was done for an antimicrobial resistance test. of the total 225 gastroenteritis patients, 133 were detected as bacterial positive cases. among positive cases, shigella spp. was identified in 10.5%. age wise, an infection rate of shigella in patients <15-years old was found higher i.e. 7.3% than in patients ≥ 15 years old i.e. 4.5% with the (p = 0.432) at 95% ci. the infection rate of s. dysenteriae, s. flexneri, and s. sonnei was detected in 28.6%, 57.1%, and 14.3% respectively. for the antimicrobial test, eight types of antibiotics were used. the most resistance pattern of isolated shigella spp. was found in nalidixic acid, and co-trimoxazole 92.8% followed by ampicillin 64.3% and ciprofloxacin 42.8% etc. our study reported that endemicity of shigellosis with s. flexneri is the predominant group in gastroenteritis patients. this finding suggests that co-trimoxazole, nalidixic acid, ciprofloxacin and ampicillin should not be used experimentally as first-line drugs for shigellosis treatment. keywords: shigella, antibiotic resistance pattern, shigellosis, gastroenteritis, tertiary care hospital, nepal *corresponding author email: balkrishabhattachan@gmail.com introduction shigellosis is caused by members of the bacterial genus shigella. it is a severe and occasionally life-threatening gastrointestinal infection. worldwide, shigella spp. is the most common cause for acute and bloody diarrhea (dysentery). they are also responsible for a significant proportion of the burden of morbidity and mortality associated with diarrheal disease [1, 2]. the incidence of shigellosis in developing countries is nearly 20 times more than in developed countries [3]. annually, it is estimated that there are 125 million infections and 14,000 deaths due to shigellosis in asia. [4]. the first emerging antimicrobial studies conducted in the 1950s, reported multiple drug resistance transmitted by plasmids among shigella spp. from many countries [5, 6]. as a result of the considerable global burden, low infectious dose, clinical severity, and frequent reports of emerging antimicrobial resistance against first-line and, more recently, secondline therapies [7-9]. over 70% of shigella isolates were resistant to two or more drugs including ampicillin and co-trimoxazole during 2002 to 2007 in india [10]. reports from indonesia, bangladesh, malaysia and nepal, showed an increasing prevalence of shigella isolates that are resistant to multiple drugs like trimethoprim-sulphamethoxazole, ampicillin, nalidixic acid and tetracycline [11 16]. shigellosis is associated with significant morbidity and mortality among the pre-school children. it is caused by any one of the four species of shigella, namely serotype a s. dysenteria, serotype b s. flexneri, serotype c s. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 14-20 thapa et al. ©njb, biotechnology society of nepal 15 nepjol.info/index.php/njb sonnei, and serotype a s. boydii, and the classification is based upon the serological antigen [17]. the frequencies of s. flexneri, s. sonnei, s. boydii, and s. dysenteriae are 16.0%, 7.0%, 2.0%, and 1.0% in developed countries and 60.0%, 15.0%, 6.0%, and 6.0% in developing countries, respectively [18]. in nepal, shigella spp. was detected in 13.6% of stool samples examined at nepalgunj medical college and teaching hospital, banke, between september 2011 and april 2013 [19]. the current study was carried out in stool specimens collected from gastroenteritis patients at a tertiary care hospital in pokhara, nepal. the main aim of this study is to isolate and identify shigella spp. from gastroenteritis patients and find out its antimicrobial resistant pattern by modified kirby-bauer disk diffusion method. materials and methods study design and setting a cross-sectional study was conducted in 225 stool samples collected during 20 april to 14 september 2014. clinical specimens of gastroenteritis patients were examined which is referred by a physician. the patient’s symptoms like diarrhea, abdominal pain, vomiting, reddish & watery stool; age, gender etc. were also recorded. both outpatient and in patients with acute gastroenteritis but not undertaking any antibiotics were taken as subject from western regional hospital. sample collection one gram of fresh stool sample was collected in a clean and sterile screw-capped plastic container. the collected samples were labeled properly with the patient’s name, age, sex, address, and date. these samples were processed immediately following standard operating procedures (sops) of microbiology in the laboratory of western regional hospital, pokhara, nepal. if there was a delay in processing freshly passed stool sample within 2 hours, the specimens were kept in a refrigerator at 2-8oc. macroscopic examination microscopic physical examination of stool sample was done for the color, consistency (formed, semi-formed, unformed), presence of blood, mucus or pus. isolation and identification of shigella on culture method: 0.1 ml of stool sample were inoculated in macconkey (ma), xylose lysine deoxycholate (xld) and salmonella-shigella (ss) agar (himedia lab. pvt. ltd. india) and incubated at 37oc for 24 hours. the suspected colonies were grouped as gram positive and gram negative by gram’s staining reaction. biochemically, shigella are found indole negative, citrate negative, urea negative and hydrogen sulphide negative in tsi agar’s slant and nonmotile [14]. identification of shigella spp. in slide agglutination test shigella spp. was further tested by type-specific antisera of group a, b, c and d shigella antisera in a serological kit (denka seiken co. ltd. 3-42, nihonbashi kayabacho, chuo-ku, tokyo, japan). agglutination appeared on (group a) monovalent, (group b) polyvalent, (group c) polyvalent and (group d) polyvalent in kit, were identified for s. dysenteriae, s. flexneri, s. boydii and s. sonnei respectively. antibiotic susceptibility test antibiotic susceptibility tests of different clinical isolates against various antibiotics were performed by modified kirby-bauer disk diffusion method in mueller hinton agar (himedia laboratories, india) according to clinical laboratory standards institute (clsi) guidelines [20]. the antibiotics used for analysis were ampicillin, azithromycin, ciprofloxacin, co-trimoxazole, ceftriaxone, gentamycin, and nalidixic acid. the diameters of inhibition of zone for shigella were compared with strains escherichia coli atcc 25922 (national committee for clinical laboratory sciences, document m100-56: sixth informational supplement). statistical analysis pearson’s chi-square test was used to determine the significant association of dependent variable by using win-pepi software (copyright j. h. abrmson, 23 aug nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 14-20 thapa et al. ©njb, biotechnology society of nepal 16 nepjol.info/index.php/njb 2016 version 11.65) with the p-value > 0.05 is considered as significant at 95% ci. results of the total 225 gastroenteritis patients, 133 were as detected bacterial positive cases (grampositive = 54 and gram-negative = 79). among bacterial positive cases, shigella spp. was detected in 10.5% (14/133). the infection rate of s. flexneri, s. dysenteriae, and s. sonnei were detected in 57.1% (8/14), 28.6% (4/14) and 14.3% (2/14) respectively (figure 1). sex wise distribution of all positive cases in different species of shigella in gastroenteritis patients: among bacterial positive cases, male patients were 129 and female patients were 96 in the ratio of 1.3:1. in male patients, infection rate of s. flexneri was the highest i.e. 42.8% (6/14), followed by s. dysenteriae 7.1% (1/14) and s. sonnei 7.1% (1/14). whereas in female patients, the highest rate of infection was observed for s. dysenteriae, i.e. 21.1% (3/14), followed by s. flexneri 14.3% (2/14) and s. sonnei 7.1% (1/14) were depicted in figure 2. figure 2: sex wise distribution of all positive cases in different species of shigella in gastroenteritis patients. age-wise distribution of gastroenteritis patients with shigella and its infection: infection rate for shigella was 7.3% (10/137) and 4.5% (4/88) in patient <15 years old and >15 years old respectively, confirming that infection rate for shigella spp. is higher in children patients than in patients elder than 15 years old with the p-value (p = 0.432) at 95% ci (table 2). antibiotic resistance wise distribution of gastroenteritis patients with shigella and its species infection: in antibiotic resistance test, the most resistance pattern of isolated shigella were found in nalidixic acid and co-trimoxazole 92.8% (13/14) followed by ampicillin 64.3% (9/14) and ciprofloxacin 42.8% (6/14), detail description of shigella spp. were shown in table 3. discussion worldwide, acute gastrointestinal infections including diarrhea are among the leading causes of morbidity and mortality among children, particularly in underdeveloped countries [21]. poor access to safe water, inadequate sanitary conditions, lower literacy rate, and unavailability of healthcare facilities in the remote area are the major factors 0 10 20 30 40 50 42.8% 7.1% 7.1% 0 14.3% 21.1% 7.1% 0 male female total stool specimen (n=225) stool culture (n= 225) no isolated bacteria (n=92) isolated bacteria (n= 133) gram-positive bacteria (n = 54) gram-negative bacteria (n = 79) lf (lactose fermenting) (n= 45) nlf (non-lactose fermenting) (n = 34) shigella spp. (n= 14) s. dysenteriae (n= 4) non lactose fermenter s. flexneri (n= 8) non lactose fermenter s. sonnei (n=2) late-lactose fermenter s. boydii (n=0) non lactose fermenter figure 1. flow chart for the isolation and identification of shigelle isolates. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 14-20 thapa et al. ©njb, biotechnology society of nepal 17 nepjol.info/index.php/njb predisposing diarrheal illness among developing countries [22]. shigella spp. was detected in 10.5%, among which, infection rate of s. flexneri, s. dysenteriae, and s. sonnei was detected in 57.1%, 28.6%, and 14.3%, respectively in gastroenteritis patients. s. boydii could not identify, this might be due to small sample size or error in the kit method in diarrheal patients, or might be used by kit method rather than molecular methods. in the previous study, it was reported that s. flexneri (43.2%) is the predominant of the four species followed by s. dysenteriae (41.5%), s. boydii (7.6%) and s. sonnei (7.6%) in nepal [19]. similarity, the prevalence of s. flexneri is identified in 42.0% of isolates, while s. dysenteriae in 27.5%, s. boydii in 21.7% and s. sonnei in 8.7% in eastern nepal [18]. in western nepal, s. flexneri, s. dysenteriae, s. boydii, and s. sonnei were accounted respectively for 43.1%, 27.7%, 21.5%, and 7.7% of the total number of shigella isolated [14]. infection rate for shigella spp. was higher (7.3%) in gastroenteritis patients of <15 years old than in patients ≥15 years old (4.5%) which is not statistically significant (p=0.432). this finding is consistent with the study done by khan et al and shakya et al found shigella 42.0% and 38.4% and 30.1% respectively [14, 18, 32]. the reported high prevalence of shigella spp. from children aged 1-10 years, compared to the other age groups. they also reported high prevalence of shigella spp. from male patient compared to the female [14, 32]. shigellosis or severe bacillary dysentery is a disease of public health importance because it is associated with increased mortality and morbidity especially among the children of developing countries [23]. the seasonal tendency of shigellosis was summer-monsoon [24]. our study was conducted in summermonsoon (april to september); when school going children may get dysentery and diarrhea due to unsafe water like rainfall, flood and drinking contaminated water. on the other hand the no. of male cases with the pathogen exposer were high may be due to they go out from home more frequently for playing, eating purpose that compares to female. for antibiotic resistance pattern, eight types of antibiotics were used. nalidixic acid and cotrimoxazole (92.8%) was found to be the most resistant antibiotics in isolated shigella spp. followed by ampicillin (64.3%) and ciprofloxacin (42.8%). among isolated shigella spp., almost all of the s. flexneri is most resistant to co-trimoxazole (100.0%) while s. dysenteriae is resistant to nalidixic acid (100.0%). in addition, most of the s. sonnei was found 100.0 % resistant in ampicillin, nalidixic acid, and cotrimoxazole. in a study, resistance rate of table 2: age with isolated shigella spp.; s. flexnari, s. dysenteriae, and s. sonnei in patients of gastroenteritis characteristics total stool sample (n=225) shigella (n=14) p value s. flexnari (n=8) s. dysenteriae (n=4) s. sonnei (n=2) p-value no. (%) no. (%) no. (%) no. (%) age (year) <15 137 10 (7.3) >0.05 5 (3.6) 3 (2.1) 2 (1.4) >0.05 ≥15 88 4 (4.5) 3 (3.4) 1(1.3) 0 (0.0) table 3 antibiotic resistance pattern of shigella and its species antibiotics s. flexnari (n=8) s. dysenteriae (n=4) s. sonnei (n=2) total (n=14) no. (%) no. (%) no. (%) no. % ampicillin (10µm) 4 (50.0) 3 (75.0) 2 (100.0) 9 (64.3) azithromycin (10µm) 1 (12.5) 0 (0.0) 0 (0.0) 1 (7.1) ciprofloxacin (5µm) 2 (25.0) 3 (75.0) 1 (50.0) 6 (42.8) ceftriaxone (30µm) 2 (25.0) 2 (50.0) 0 (0.0) 4 (28.6) co-trimoxazole (25µm) 8 (100.0) 3 (75.0) 2(100.0) 13 (92.8) gentamycin (10µm) 2 (25.0) 0 (0.0) 0 (0.0) 2 (14.3) nalidixic acid (30µm) 7 (87.5) 4 (100.0) 2 (100.0) 13 (92.8) nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 14-20 thapa et al. ©njb, biotechnology society of nepal 18 nepjol.info/index.php/njb shigella spp. for nalidixic acid was 95.6%, ampicillin 85.5%, co-trimoxazole 82.6%, gentamicin 24.6%, ofloxacin 21.7% etc. [19]. 33% of the total shigella studied was found multi drug resistant (mdr) i.e. they showed resistance to 3 or more antibiotics. however, none of the shigella spp. was resistant to azithromycin and ceftriaxone. ciprofloxacin resistance was seen only among shigella dysenteriae strains [14]. shigella spp. resistant to nalidixic acid (95.4%); ampicillin (84.6%) cotrimoxazole (81.5%) and ciprofloxacin (46.2%) were detected in nepal [16]. over the past decades, it has been reported that a significant number of shigella spp. isolates resistant to normally prescribed drugs [25]. in the early 1990s, many isolates of shigella spp. were susceptible to norfloxacin, nalidixic acid, gentamicin and furazolidone [26, 27]. however, in the late 1990s, most shigella isolates showed an increased resistance to these antibiotics [28, 29] but most were susceptible to ciprofloxacin [30]. nowadays, alternative drugs such as the third generation cephalosporins are being commonly used. although, the present study shows that shigella strains are rapidly acquiring resistance to these substituted drugs as well. the emergence of plasmid-borne resistance to cephalosporins is another reason which further reduces the therapeutic option for the treatment of shigellosis [19]. shigella spp. is highly necessary to start a prompt and rational antibiotic regimen to minimize the clinical effects of severe dysentery and its complications [31]. we could not able to perform confirmatory tests like other biochemical tests, molecular level test like pcr, gel electrophoresis due to lack of resources and additional clinical patients issues like clinical complication and informed consents. moreover, the study was limited to the single hospital so the results do not represent a broad population. conclusions our findings also revealed that endemicity of shigellosis with s. flexneri is the predominant strain in gastroenteritis patients. to prevent school going children from shigellosis they should keep away from untreated water. as shown by antibiotic resistant pattern, cotrimoxazole, nalidixic acid, ciprofloxacin,and ampicillin should not use experimentally as first-line drugs for the treatment of shigellosis. frequent analysis of resistance pattern and periodic reporting of shigella spp. is necessary for shigellosis therapy. in addition, continuous cultural surveillance of multidrug-resistant test of shigella spp. is necessary to know changing the time-to-time resistant pattern of its serogroup. isolation and sensitivity testing for shigella spp. should be done regularly. monitoring of emergence of resistance is highly recommended. ethical approval and consent to participant all shigella strains were routinely collected in the microbiology laboratory. no patient-related data were collected. ethical approval was therefore not required. the study was laboratory-based basic science study. written informed consent was taken from all participating patients or from guardian on the behalf of their children. consent for publication not applicable availability of data and materials all supplementary files, data generated and analyzed during this study will be made available as per request to co-author. source of support: none conflict of interest: none declared author’s contributions kt and ak designed the study. kt collected sample at western regional hospital. kt and ak performed an investigation and recorded the laboratory findings with the validation. jbk supervised and provide methodology for the study. rrg and bb administered the project, reviewed literature, and written original manuscript; curated data to perform statistical nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 14-20 thapa et al. ©njb, biotechnology society of nepal 19 nepjol.info/index.php/njb data analysis and data interpretation. rrg helped in review and revision of the draft by compiling, formatting, editing and writing the final version of the article. thus, all authors made a substantial contribution to the study. all of them read and approved the final manuscript. acknowledgements we are indebted to staff and volunteer at western regional hospital and tri-chandra college’s friends who help us continuously. without their supports, we could not conduct this research. references 1. thapar n, sanderson ir. 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shigella-shigellosis. centers for disease control and prevention, atlanta, ga, usa 2016. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 14-20 thapa et al. ©njb, biotechnology society of nepal 20 nepjol.info/index.php/njb http://www.cdc.gov/shigella/generalinformation.html. 22. sangeetha av, parija sc, mandal j, and krishnamurthy s: clinical and microbiological profiles of shigellosis in children. j of hlth, pop and nutr. 2014 32 (4): 580–586. 23. gupta s, mishra b, muralidharan s, srinivasa h: ceftriaxone resistant shigella flexneri, an emerging problem. indian j of med sci. 2010 64 (12): 553–556. 24. bennish ml, salam ma, hossain ma, myaux j, khan eh, chakraborty j, et al: antimicrobial resistance of shigella isolates in bangladesh, 1983-1990: increasing frequency of strains multiply resistant to ampicillin, trimethoprim-sulfamethoxazole, and nalidixic acid. clin infect dis. 1922 14: 1055-1060. 25. jesudason mv: shigella isolation in vellore, south india (1997-2001). indian j med res. 2002 115: 11-13. 26. thapa br, ventkateswarlu k, malik ak, panigrahi d. shigellosis in children from north india: a clinic pathological study. j trop pediatr. 1995 41: 303-307. 27. niyogi sk, mitra u, dutta p: changing patterns of serotypes and antimicrobial susceptibilities of shigella species isolated from children in calcutta, india. j infect dis. 2001 54: 121-122. 28. sack br, rahman m, yunus m, khan he: antimicrobial resistance in organisms causing diarrheal disease. clin infect dis. 1997 24: 102-105. 29. khan ai, huq s, malek ma, hossain mi, talukder ka, faruque asg et al: shigella serotypes among hospitalized patients in urban bangladesh and their antimicrobial resistance. epidemiol infect. 2004 132: 773-777. 30. khan wa, seas c, dhar u, salam ma, bennish ml: treatment of shigellosis: v. comparison of azithromycin and ciprofloxacin. a double-blind,randomized, controlled trial. ann intern med. 1997 126: 697-703. 31. aggarwal p, uppal b, ghosh r, et al: multi drug resistance and extended spectrum beta lactamases in clinical isolates of shigella: a study from new delhi, india. tra med infect dis. 2015 14 (4): 407–413. 32. shakya g, acharya j, adhikari s, rijal n: "shigellosis in nepal: 13 years review of nationwide surveillance", bmc health popul nutr. 2016 35:36 http://www.cdc.gov/shigella/general-information.html http://www.cdc.gov/shigella/general-information.html nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 58-63 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 58 nepjol.info/index.php/njb effect of herbs and herbal products feed supplements on growth in fishes: a review shubha ratna shakya department of zoology, tribhuvan university, amrit campus, kathmandu, nepal abstract the herbs and herbal products added to the feed cure many diseases, promote growth, reduce stress, improve immunity and prevent infections in fish under culture. the addition of herbs and herbal products in fish diet is cheaper and environmental friendly with low side effect to the fish and consumers. hence, their use as drugs in disease management in aquaculture is gaining popular. they are better than various antibiotics and vaccines used in the treatment of diseases. the present review highlights the importance of herbs and herbal products supplementation in fish feed for better fish production. keywords: aquaculture, growth promoter, herb, fishes *corresponding author email: shubharatnashakya@gmail.com introduction fish of commercial importance are farmed in captivity under controlled conditions to fulfill the demand of white meat for human consumption. in commercial fish farming, the production is maximized by increasing the weight of individual fish [1, 2]. an artificial feed used in the aquaculture improves fish growth with maximum weight in short time [3]. new substances are added in fish feed to improve feed conversion efficiency that result in fish growth [4]. many studies show that inclusion of herbs in fish diet has positive effect on growth and disease free fishes. excess use of various antibiotics, hormones and other synthetic drugs to control diseases and improve fish growth in aquaculture is the reason behind the emergence of drug resistant bacteria and production of toxic substances harmful to the environment and human health [5] and suppress immunity in the host [6]. thus, their use has been criticized all over the world [7]. the herbs being cheaper, eco-friendly with minimum side effects are used as alternative to antibiotics in fish health management. world health organization (who) encourages supplemented diets incorporated with medicinal herbs or plants which minimizes the use of chemicals in fish diet [8]. in this context herbs and herbal products can be used in fish diet to increase feed consumption in fish under culture [9]. thus, this review is an informative collection in relation to fish growth through herbal feed supplements which may be useful for aqua farmers. bioactive compounds present in various plants are used in animal nutrition to stimulate feed intake, improve secretion of digestive enzyme and activate immune responses. these plants are also known to possess antibacterial, antiviral and antioxidant properties [10]. in aquaculture practices many herbs and herbal products are included in the fish diet to cure diseases, promote growth, reduce stress, stimulate appetite, boost immunity and prevent infections in producing healthy fishes [1115]. the flavor imparted by herbs and herbal products added in fish diet changed the eating patterns, increased feed consumption and stimulated digestion by increasing the secretion of saliva, various digestive enzymes, bile, pancreatic enzymes activity and mucus in fishes [16,17]. some herbal feed supplements used in aquaculture various herbs such as hygrophila spinosa, withania somnifera, zingiber officinalis, solanum trilobatum, andrographis paniculata, psoralea corylifolia, eclipta erecta, ocimum sacnctum, picrorhiza kurooa, phyllanthus niruri and tinospora cordifoliaare used to reduce stress, increase immunity and control bacterial activities. penaeus monodon in culture fed nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 58-63 shakya ©njb, biotechnology society of nepal 59 nepjol.info/index.php/njb with diet containing these herbs improved growth [12]. similarly, garlic, onion, marjoram, caraway, basil, anise, fennel, licorice, black seed and fenugreek are known to promote growth [13], feed conversion [15], improve protein digestibility and retain energy [18, 19] in aquatic animals. promising results were achieved when harada (1990) used garlic as stimulatory effect on olfaction instead of chemotherapeutics on oriental weather loach (misgurnus anguillicaudatus) and japanese amberjack (seriolaquin queradiata) [20]. this is similar with what was reported by lee and gao (2012) on pelodiscus sinensis, ctenopharyngodon idellus, cyprinus carpio, carassius auratus and oreochromis niloticus [16]. allicin is an active compound of garlic which induces increase in feed intake. zeng (1996) also reported that adding 50 mg kg-1of synthesized allicin to tilapia diet increased more than 2-3% of its weight gain after 45 days of culture [21]. the use of other culinary herbs such as red clover (trifolium pratense), caraway (carum carvi) and basil (ocimum basilium) have shown positive results as growth promoting agents in oreochromis niloticus [22]. methanol extract of green tea (camellia sinensis) enhanced the growth, survival rate, feed utilization and protein content in black rockfish (sebastess chlegeli) [23]. garlic supplemented diet improved weight gain (wg) and specific growth rate (sgr) in orechromis niloticus [24]. feed containing 3% garlic powder improved wg, feed efficiency (fe), and sgr in oreochromis niloticus [25]. in the same species high growth rate was observed in feeding diet with 2.5% garlic [26]. garlic supplemented diet increased weight and sgr in tilapia [27]. diet containing 3.2% garlic powder showed best growth in oreochromis niloticus [28]. rainbow trout fed with 1.0% garlic diet increased growth and improved feed utilization [29]. similarly, oreochromis niloticus fed with garlic supplemented diets showed significant improvement in weight gain, feed conversion and protein efficiency [30]. labeo rohita fed with herbal supplemented diet improved feed consumption resulting in better growth due to high protein synthesis [31]. leaves of sesbania grandiflora, moringa oleifera, coleus aromaticus, ocimum basilium and solanum verbascifolium have been found to promote growth in oreochromis mossambicus [32]. o. mossambicus fed with diet containing moringa oliefera showed maximum increased weight and specific growth rate. the maximum increase in length was observed in the fishes fed with ocimum basilicum supplemented diet. thus, plant ingredients are included in fish diet for their better growth. red clover (trifolium pretense) mixed with diet promoted growth in oreochromis aureus [33]. juvenile pike perch (sander lucioperca) fed on diets supplemented with medicinal plants grew faster than those fed with the control diet [34]. in common carp cyprinus carpio, guppy poecilia reticulata, cichlid cryptoheros nigrofasciatus, and red sea bream pagrus major diet supplemented with medicinal plants improved growth. the use of ginseng herb (ginsana g115) in diet enhanced the growth in oreochromis niloticus fingerlings [35-39]. the use of antibiotics can be replaced by optimized dose of garlic to enhance growth performance and meat quality [40]. metwally (2009) recommended supplementation of garlic in fish feed to promote growth and increase survival rate [29]. the use of phyllanthus emblica in any doses to feed of fish results in maximum growth [41]. john et al., (2007) used four different plants such as eichinacea purpurea, allium sativum, nigella sativa, and origanum marjoranaas feed additives which enhanced growth and improved survival of oreochromis niloticus [42]. various commercial herbal additives have been introduced in aquaculture for fish growth. sangrovit® (commercial product containing isoquinoline alkaloid sanguinarine) at low levels (25–100 mg kg-1) in diet promoted growth in tilapia [43]. tilapia fed with sangrovit® supplemented diets consumed more feed as compared to control and showed improved growth. in catla catla, 5% inclusion of cynodon dactylonin diet improved the growth, feed efficiency, body composition, digestive enzyme and anti-protease activity [44]. the significant nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 58-63 shakya ©njb, biotechnology society of nepal 60 nepjol.info/index.php/njb increase in sgr, feed conversion ratio (fcr) and 100% survival rate in the experimental groups were due to the presence of essential amino acids in cynodon dactylon [45]. catla catla fed with cynodon dactylon incorporated diet showed the increased activity of digestive enzymes such as amylase and protease which enhanced digestion and absorption of nutrients essential for fish growth [44]. inclusion of 5% ulva in the diet of nile tilapia improved the growth, feed efficiency, nutrient utilization and body composition [46]. oreochromis niloticus fingerlings fed with dietary herbal powder (superliv®) improved weight gain, fcr, protein efficiency ratio (per) and sgr [47]. bioflavonoids, plant chemicals with estrogenic activity, present in superliv® powder stimulated growth in common carp [48]. sambhu and jayaprakash (2000) recommended the use of 1% of livol (ihf-1000) to enhance maximum growth and improve nutrient digestibility in prawn [49]. livol (ihf-1000) is a purely herbal product containing different plant ingredients which improves digestion thereby leading to better growth in cultivable fishes [50, 51, 52]. the fish fed with different doses of immuplus improved growth and inflammatory response (increase protease and amylase activity) [53]. the higher growth in treated fish is due to better utilization of feed through improved secretion of digestive enzymes and higher deposition of fats and protein in carcass [53]. dietary administration of small quantities of vitamin c is known to improve fish growth [54, 55, 56]. chitosan and levamisole supplementation in diet of common carp enhanced its growth [57]. ji et al., (2007) observed that the herbs promoted cellular lipid and fatty acid utilization and protein accumulation resulting in better growth performance in pagrus major [38]. the dietary superliv® powder at different concentrations promoted growth of oreochromis niloticus fingerlings [58]. the bioflavonoid compounds are present in superliv® powder which stimulated growth in common carp [48]. the use of fenugreek in labeo rohita and oreochromis mossambicus [59. 60], rosemary in oreochromis niloticus [61, 62], thyme, rosemary and fenugreek in oreochromis mossambicus [63] improved growth performance, disease resistance and immunity. the thyme diet improved growth and nutrient utilization in the european sea bass (dicentrarchus labrax) [64]. it has been shown that herbs stimulated the secretion of pancreatic enzymes, important factors in nutrient digestion and assimilation [65]. the dietary addition of vitamin c at the rate of 400 mg kg-1 increased average weight and specific growth rate of hybrid carp [66]. vitamin c fed fishes showed higher growth rate [67]. pandey et al., (2012) showed use of vitamin c and e as herbal drugs act as growth promoter and cure diseases of fishes and other aquatic animals [14]. labh and shakya (2016) studied the effect of lapsi (choerospondias axillaries, roxb.) fruit pulp in diet of nile tilapia and common carp in the laboratory of central department of zoology, kathmandu. in the study 0.4 g ethanol lapsi fruit extract per kg is sufficient to enhance growth, survival, feed utilization and protein content of the body [68]. importance of herbal feed supplements in aquaculture many factors such as density, water quality, feed quality and various environmental factors such as temperature, salinity and dissolved oxygen content of water play important role in increasing the meat quality and production in aquaculture. besides the above mentioned factors disease outbreak, environmental conditions like rain, excessive temperature, floods, landslides, entry of predatory fish in the culture ponds or reservoirs, algal bloom etc. are also the cause of decreased aquaculture product. these factors cause stress and underutilization of normal feed leading to low feed conversion ratio. the antibiotics and synthetic drugs used to control diseases could damage the organs like liver and affect growth of fishes under culture leading to decreased productivity. to increase the economically viable production by solving the above problems, a nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 58-63 shakya ©njb, biotechnology society of nepal 61 nepjol.info/index.php/njb proper herbal feed supplementation is required to take care of overall health of fishes and withstand the environmental challenges without affecting the growth survivability. conclusion many studies are being carried to study the effectiveness of herbal supplementation in fish feed to manage fish diseases and produce healthy fish. the outcomes of the studies suggest the use of herbs and herbal products feed supplements for healthy fishes in culture. conclusively, the herbal feed supplements promote growth, minimizes stress, improves immunity and prevents various infections in fishes that will help to produce healthy fishes for human consumption. references 1. schuchardt d, vergara jm, palaciso hf, kalinowski ct, cruz cmh, izquierdo ms & robaina l: effects of different dietary protein and lipid levels on growth, feed utilization and body composition of red porgy (pagrus pagrus) fingerlings. aqua nutr. 2008 14(1): 1-9. 2. 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rezar v: use of herbs and spices and their extracts in animal nutrition. acta agriculturae slovenica. 2009 94(2): 95-102. 66. labh sn & chakrabarti r: effects of different dietary supplements of vitamin c l-ascorbate-2triphosphate calcium (latp-ca) on growth, tissue vitamin c and liver ultrastructure of indian major carp mrigal cirrhinus mrigala (h) during intensive aquaculture. j theor exp biol. 2011 7: 195-201. 67. lee kj & dabrowski k: interaction between vitamins c and e affects their tissue concentrations growth lipid oxidation and deficiency symptoms in yellow perch (perca flavescens). br j nutr. 2003 89: 589-596. 68. labh sn & shakya sr: effects of dietary lapsi (choerospondias axillaris roxb.) on survival, growth and protein profile of common carp (cyprinus carpio l) fingerlings. int j zool stud. 2016 1(5): 36-415. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 5-13 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 5 nepjol.info/index.php/njb analysis of phytoconstituents and biological activities of different parts of mahonia nepalensis and berberis aristata rojeena thusa*, sushika mulmi central department of chemistry, tribhuvan university, kirtipur, nepal abstract the phytochemicals and biological activities of extracts from leaves and stem of mahonia nepalensis and berberis aristata were carried out. phytochemical screening showed the presence of alkaloids, steroids, polyphenols, quinones, glycoside, flavonoid, terpenoid and cardiac glycoside in the hexane, ethyl acetate and methanol extracts of leaf and stem of these two plants. the column chromatography of methanol extract of stem of mahonia nepalensis resulted in isolation of four pure compounds mn1, mn2, mn3 and mn4. out of four isolated compounds, two were identified as mn1:sitosterol and mn2: berberine by comparison of melting point, co-tlc, ir and uv spectra of authentic sample. potent pharmacological activity of mahonia nepalensis and berberis aristata were revealed from antimicrobial activity and brine shrimp bioassay. methanol extracts of stem of mahonia nepalensis and berberis aristata showed significant zone of inhibition of 18 mm and 21 mm respectively against the staphylococcus aureus. methanol extract of berberis aristata were comparatively little stronger against staphylococcus aureus than methanol extract of mahonia nepalensis. lc50 values (μg/ml) of methanol extracts of stem of berberis aristata and mahonia nepalensis were found to be 8.058x10-4 and 8.3 whereas methanol extracts of leaf of mahonia nepalensis and berberis aristata were 389.04 and 1303.166 respectively. key words: mahonia nepalensis, phytochemical, berberis aristata, lc50 values, *corresponding author email: rojeenathusa@gmail.com introduction nepal is well known for superb collection of medicinal and aromatic plant resources that grow luxuriously in tropical forests to alpine meadow [1]. since past, parts of these medicinal plants or their extracts have been used as traditional medicine. traditional medicines are safe, easily accessible and affordable form of health care for much of the rural population in nepal. plants used in traditional medicine are important sources of novel biomolecules with application for the manufacture of pharmaceuticals and cosmeceuticals [2]. berberidaceace is a family of shrubs and herbs, mainly of the northern temperate zone, with simple, pinnate, or peltate leaves with spines. there are 14 genera in this family, and mahonia and berberis are among them. mahonia is readily distinguished from berberis by its compound leaves, spineless stems and inflorescence of several dense spikes. berberis is distinguished by its undivided spiny toothed leaves and its spiny stems with yellow wood [3]. mahonia nepalensis belongs to the family berberidaceae and is known vernacularly as “jamanemandro” in nepali and “michiki swan” in newari. it is medium sized fully hardy perennial evergreen shrub with yellow flowers in winter. this shrub has an ultimate height of 6m /19.7ft. its origin is in nepal. it is widely distributed in the high mountainous areas at altitude of about 1000m 2000m in nepal, sikkim, bhutan, china, vietnam, etc. it is useful in architectural and security barrier in garden, traditional essential flower for conducting bel bibaha and bratabanda in newar community. the stem and wood of this plant have antiinflammatory, anti-bacterial, anti-fungal activity. it is particularly used for the treatment of skin diseases like eczema, psoriasis, etc. this plant contains alkaloids as the major compounds which belong to class protoberberines and bisbenzylisoquinolines [4] berberine [5], jattrrorzhine, o-methyl puljabine [6], isotetradine, homoaromaline etc. were isolated from the stem of this plant [7]. berberis aristata also belongs to berberidaceace family and is commonly known as “chutro” in nepali, “daruhaldi” in hindi and “indian berberry” in english. it is spinous herb native to northern himalaya region, widely distributed from himalayas to sri lanka, bhutan and hilly areas of nepal. it is used in ayurvedic medicine from very long time. the plant is used traditionally in inflammation, diarrhea, wound healing, skin disease, menohrrhagia, jaundice, and affection of eyes. a very valuable ayurvedic nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 6 nepjol.info/index.php/njb preparation" rashut" is prepared from this plant [5]. it is useful in treatment of jaundice, diabetes, cancer, malaria etc. and has good anti-oxidant property, anti-pyretic activity, analgesic activity, anti-fungal activity and anti-microbial property, anti-inflammatory, anti-platelet activating factor [8]. berberine, berbamine [9], oxycanthine, epiberberine, palmatine, dehydrocaroline, jattrorhizine, columbamine, dihydrokarachine, karachine [10], taximaline [11], oxyberberine, aromaline [12], pakistanine, 1-o-methyl pakistanine [13], pseudo palmatine chloride, pseudo berberine chloride, lanost-5-en-β-ol [14] were isolated from its stem while citric acid, malic acid from fruit and e-caffeic acid, quercetin, chlorogenic acid, meratin and rutin from flower have also been isolated from this plant [15]. due to their important pharmaceutical importance, we have tried to isolate the plant metabolites and determine the photoconstituents of different parts of these two valuable plants and studied their biological properties, isolated some of the important compounds and tried to characterize and identify the isolated compound as well. materials and method sample collection and extraction the stem and leaves of mahonia nepalensis and berberis aristata were collected from bhaktapur and palpa district respectively in april, 2013 and thoroughly dried in shade. about 50 g of stem and leaves of the two plants were ground to fine powder. the grinded parts were then successively extracted with hexane, ethyl acetate and methanol on the basis of their increasing polarity by using soxhlet apparatus. the extracts were concentrated using rotatory evaporator and left for removal of solvent. after the solvents were completely removed, they were used for different purposes like phytochemical screening, biological activities study and isolation of chemical constituents. phytochemical screening a small amount of dry extracts of plant materials were subjected to phytochemical screening. the method employed was based on the standardized procedure with slight modification [16-18]. test for tannin/polyphenols to a portion of extract diluted with water, 3-4 drops of 10% fecl3 is added, a blue color was observed for gallic, tannins and green color for catecholic tannin. test for reducing sugar to 0.5ml of plant extract, 1ml of water and 5-8 drops of fehling’s solution was added and heated over water bath. brick red precipitate indicated the presence of reducing sugar. test for quinines to the extract, freshly prepared feso4 solution (1ml) and ammonium thiocyanate (few crystals) were added and treated with conc. h2so4 drop by drop. the deep red color was persistent indicating the presence of quinines. test for glycosides for testing the glycosides, the protocol was slightly modified. to the extract, 5ml molisch’s reagent was added and conc.h2so4 was added drop wise without disturbing the solution. a violet ring at the junction of two liquids was observed and on shaking the solution turned violet completely indicating the presence of glycosides. test for flavonoid i. shinoda test: 4ml of extract solution was treated with 1.5ml of 50% methanol solution. the solution was warmed and metal magnesium was added. to this solution, 5-6 drops of conc. hcl was added and red color was observed for flavonoid and orange color for flavones. ii. 5ml of dilute nh3 solution was added to the aqueous filtered solution of each fraction followed by the addition of conc.h2so4. the appearance of yellow color indicated the presence of flavonoid. the yellow color disappeared after some time. test for terpenoid about 0.2g of each sample was mixed with 2ml of chloroform first and 3ml of conc.h2so4 was nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 7 nepjol.info/index.php/njb added to each mixture. the formation of a reddish brown coloration at the interface indicates the presence of terpenoid. test for alkaloids i. meyer’s test: to 2ml of extract, 1ml of meyer’s reagent (picric acid solution) was added. white precipitate and pale yellow precipitate indicates presence of alkaloids. ii. dragendroff’s reagent test: 2ml of extract and each fraction were warmed with 2%. h2so4 for 2min. after filtration of the reaction mixture a few drops of dragendroff's reagent were added to each filtrate. orange red precipitate indicates presence of alkaloids. test for saponins about 2g of powdered sample was boiled in 20ml of distilled water in a water bath and filtered.10ml of filtrate was mixed with 5ml of distilled water and shaken vigorously. the appearance of frothing indicated the presence of saponins. test for volatile oils 2ml extract was shaken with0.1ml of naoh and a small quantity of dil. hcl. a white precipitate was formed which indicated presence of volatile oils. test for cardiac glycosides 5ml of plant extract was treated with 2ml of glacial acetic acid containing one drop of fecl3 solution. a brown ring at the interface indicated a deoxy sugar characteristic of cardenolides. a violet ring was appeared below the brown ring, while in acetic acid; a greenish ring was formed just gradually throughout thin layer which showed the presence of cardiac glycosides. test for steroids 1gm of plant extract was dissolved in few drops of acetic acid. it was gently warmed and cooled under tap and a drop of conc.h2so4 was added alongside of tube. appearance of green color indicated presence of steroids. separation of compound by using column chromatography 23.2g methanol extract of stem of mahonia nepalensis was adsorbed in 20.07g silica gel (60120) mesh and loaded in the column having diameter 3cm and length 60cm packed with150 mg activated silica gel (60-120)mesh. the 40.5cm long column was eluted with gradient of hexane, ethyl acetate, and methanol to obtain no. of fractions which resulted in isolation of compound mn1, compound mn2, compound mn3 and compound mn4. compound mn1was obtained on concentration of fraction (25-27) in rotator evaporator while compound mn2 from fraction (190-191), compound mn3 and mn4 from fraction (200-206), compound mn1: needle shaped white crystals, melting point (mp) 135° c, showed single spot on tlc with r.f value 0.43 (20% ethyl acetate in hexane). compound mn2: needle shaped yellow crystals, mp 2000c-205oc. (decomposed), showed single spot on tlc with r.f value 0.45 (20% methanol in chloroform). compound mn3: needle shaped brown colored crystals, mp 140˚c, showed single spot on tlc with r.f value 0.81 (30% methanol in chloroform). compound mn4: needle shaped dark orange brown colored crystalline, mp 140˚c and decomposed at 160˚c, showed single spot on tlc with r.f value 0.86 (30% methanol in chloroform). antimicrobial tests the extracts of mahonia nepalensis and berberis aristata were screened for their antimicrobial activity i.e. determination of zone of inhibition against tested organism by agar well diffusion method [19]. four strains of bacteria namely staphylococcus aureus, kleibsella pneumonia (atcc 700603), salmonella typhimurium (atcc 242), and salmonella typhimurium (atcc 14028) were used for antibacterial assay. staphylococcus aureus and kleibsella pneumonia were obtained from national public health laboratory, teku, nepal and salomonella typhimurium and salmonella typhimurium were obtained from institute of medicine, maharajgunj, nepal. these organisms were placed in muller-hinton agar (mha) in the refrigerator at 4°c prior to subculture. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 8 nepjol.info/index.php/njb agar well diffusion method was used to test the anti-bacterial properties of the crude extract and mha was used as medium. the bacterial inoculums were sub-cultured in nutrient broth for 12-18 hours at 37°c. sterile petri plates were taken and mha was poured, allowed to set and maintained the thickness of media at 4mm in each plate. seven wells were bored in the medium and extracts of five plant material, a standard antibiotic disc, nalidixic acid 30μg, as positive control and dmso as solvent control was added into them. the inoculated plates were incubated at 37°c for 18-24hrs and the diameters of the zone of inhibition of microbial growth were measured in millimeters. brine shrimp bioassay brine shrimp bioassay was performed using standard procedure. brine shrimp eggs were hatched in artificial sea water. the brine shrimp larvae were cultured under prescribed laboratory condition [20] and used against methanol extracts. the number of survived shrimps was counted and lc50 value was calculated. result and discussion isolation of plant metabolites the different parts of plants specially leaf and stem selected on the basis of their medicinal use in ethno medicine were successively extracted on the basis of increasing polarity. the yield of these extract and their physical characteristic were shown in (table 1). the highest yield % was observed for the methanolic extract of stem of b.aristata and was found to be 22.6 while the yield % for methanolic extract of stem of mahonia nepalensis table 1: percentage yield of different plant extracts and their physical characteristics. plant part extract % yield color consistency m. nepalensis stem hexane 0.53 yellow sticky m. nepalensis stem ethyl acetate 4.2 yellow sticky m. nepalensis stem methanol 12.6 dark orange powdered m. nepalensis leaf hexane 4.6 green sticky m. nepalensis leaf ethyl acetate 6.8 dark green sticky m. nepalensis leaf methanol 22.4 dark green powdered b. aristata stem hexane 0.88 yellow sticky b. aristata stem ethyl acetate 5.8 yellow sticky b. aristata stem methanol 22.6 dark orange powdered b. aristata leaf hexane 1.2 green sticky b. aristata leaf ethyl acetate 1.8 dark green sticky b. aristata leaf methanol 3.8 dark green powdered table 2: phytochemical screening of different plant extracts test tannin reducing sugar quinone glycoside flavonoid terpenoid alkaloid saponin volatile compd cardiac glycoside steroid hsm ++ + +++ + + +++ esm + + +++ + + + +++ msm +++ + +++ +++ +++ + + +++ hlm ++ + +++ + +++ elm + + +++ + + +++ mlm +++ + +++ +++ +++ + + +++ hsb ++ + +++ + + +++ esb + + +++ + + +++ msb +++ + +++ +++ +++ + +++ hlb ++ + +++ + +++ elb + + +++ + +++ mlb + + +++ + +++ +++ +++ + +++ (+ sign indicate presence, sign indicate absence) nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 9 nepjol.info/index.php/njb was only 12.6%.similarly the highest yield % of methanolic leaf extract of mahonia nepalensis was 22.4 whereas the yield % of methanolic leaf extract of b.aristata was only 3.8%. hexane extract were generally sticky and has comparatively low yield while ethylacetate extract yield was medium. phytochemical screening the phytochemical screening of all plant materials were done on the basis of the procedure given by alamzeb k (2013), talukdar and chaudhary (2010), and prof. i. culie (1990) [16-18]. the results obtained are given below in table 2. isolation and identification from stem of m. nepalensis compound mn1: the compound mn1 was white crystalline having mp 135ºc and showed single spot on tlc in 20% ethylacetate in hexane solvent system with r.f value 0.43. it gave greenish red test in liberman burchard test which indicated that the compound was sterol. it was identified as β-sitosterol with the help of co-tlc and melting point. the structure of β-sitosterol (figure 1). figure 1: chemical structure of β-sitosterol compound mn2: the compound mn2 is needle shaped yellow colored crystalline compound having mp. 2000c-205oc (decomposed). it showed a clear single spot on tlc in 20% methanol in chcl3 solvent system with r.f value 0.4. it gave pale yellow precipitate in meyer’s test and orange red precipitate in dragendroff's test which indicated that the compound was alkaloid. the ir spectra showed peaks of 3549cm-1, 3402 cm-1,3317 cm-1, 3224 cm-1 (n-h stretch ), 3055 cm-1 (c-h stretch in aromatic functional group) ,2908 cm-1 , 2846 cm1(c-h stretch of alkenes), 2121 cm-1(c triple bond n stretch), 1605 cm-1 (c-c stretch in ring, aromatic), 1396 cm-1, 1365.60 cm-1(c-h bending)), 1293.02 cm-1, 1204.44 cm-1 (c-o stretch c-n stretch in aromatic amines), 1111 cm-1, 1041 cm-1(c-o-c bending), 970 cm-1(o-h bending), 887 cm-1, 825 cm-1, 640 cm-1,524 cm-1, 424 cm-1, 393 cm-1(c-h out of plane bending) which were identical with that of the authentic sample of berberine (figure 3 and 4).the uv spectrum of the isolated compound showed that max wavelength of 353.8 nm at 0.861a, min wavelength of 307nm at 0.310a and max wavelength 271.6nm at 2.267a and min wavelength 251.8nm at 0.335a. these uv spectral data of isolated compound were also found to be identical with that of authentic sample of berberine (figure 5 and 6) respectively. comparing the spectral data of ir spectra and uv spectra and melting point with reference to the authentic sample, it was identified that the compound mn2 was berberine. the structure of berberine (figure 2). figure 2: chemical structure of berberine figure 3: ir spectrum of mn2 nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 10 nepjol.info/index.php/njb figure 4: ir spectrum authenticated berberine figure 5: uv spectrum of mn2 figure 6: uv spectrum of authenticated berberine compound mn3: the compound mn3 is needle shaped orange red colored crystalline compound having mp-1400c. it showed a clear spot was observed in 30% methanol in chcl3 with r.f value 0.81.it gave pale yellow precipitate in meyer’s test and orange red precipitate in dragendroff's test which indicated that the compound was alkaloid. the ir peaks were 4420cm-1, 3996.51 cm-1, 3340.71 cm-1,3240.41 cm-1(n-h stretch), 3062.96 cm-1 , 3016.67 cm-1, 2947.23 cm-1, 2885.52 cm-1, 2831.50 cm-1 (c-h stretch in aromatic functional group), 2762.06 cm-1, 2654.05 cm-1, 2623.19 cm-1, 2376.30 cm-1, 2345.44 cm-1 (n-h stretch), 2121.70 cm-1, 2052.26 cm-1 (c triple bond n stretch) ,1928.82 cm,1604.77cm-1,1527.62 cm-1 (c-c stretch in aromatic),1442.75 cm-1 (c-h bending), 1018.41 cm-1 (c-o-c stretch) ,972.12 cm-1(o-h bending),900.40 cm-1, 879.54 cm-1, 807.82 cm-1, 600.8 cm-1, 509.21 cm-1,362.62 cm-1 (c-h out of plane bending). the ir peaks of authentic sample of 7,8 dihydro methoxy berberine were 1605cm-1, 1510 cm-1, 1050 cm-1, 975 cm-1, and 850 cm-1 [21]. the fingerprint region peaks of isolated compound were very much identical with that of the authentic sample 7,8-dihydro-8methoxy berberine for ir spectrum of mn3 (figure 7) figure 7: ir spectrum of mn3 figure 8: uv spectrum of mn3 the uv spectral data λmax/nm (etoh) of the authentic sample were 285 and 365. the uv spectral data of 7,8-dihydro-8-methoxy berberine were very much identical with that of the authentic sample 7,8-dihydro-8-methoxy berberine (figure 9). comparing these spectral data of ir spectra and uv spectra and melting point with reference to the authentic sample, compound mn3 may be 7,8-dihydro-8-methoxy berberine.the confirmation of compound is in progress. the structure of 7, 8-dihydro-8methoxy berberine (error! reference source not found.9). nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 11 nepjol.info/index.php/njb figure 9: chemical structure of 7, 8-dihydro-8methoxy berberine compound mn4: a needle shaped dark orange brown colored crystalline compound mn4 was obtained in pure and dry state. a sharp melting table 3: antimicrobial susceptibility test crude extract vol. of extract staphylococcus aureus kleibsella pneumonia control control hexane stem extract of m.nepalensis 30μl resistant 21mm resistant 30mm ethyl acetate stem extract of m.nepalensis 30μl resistant 21mm resistant 30mm methanol stem extract of m.nepalensis 30μl 18mm 21mm resistant 30mm hexane stem extract of b. aristata 30μl 7mm 21mm resistant 30mm ethyl acetate stem extract of b. aristata 30μl 14mm 21mm resistant 30mm methanol stem extract of b. aristata 30μl 21mm 21mm resistant 30mm hexane leaf extract of b. aristata 30μl resistant 21mm resistant 30mm ethyl acetate leaf extract of b. aristata 30μl resistant 21mm resistant 30mm methanol leaf extract of b. aristata 30μl resistant 21mm resistant 30mm hexane leaf extract of m.nepalensis 30μl resistant 21mm resistant 30mm ethyl acetate leaf extract of m.nepalensis 30μl resistant 21mm resistant 30mm methanol leaf extract of m.nepalensis 30μl resistant 21mm resistant 30mm point was found to be 140˚c and decomposed at160˚c. a clear spot was observed in 30% methanol in chcl3 with r.f value 0.86. further more, it gave pale yellow precipitate in meyer’s test and orange red precipitate in dragendroff's test which indicated that the compound was alkaloid. the exact structure and name of the compound will be confirmed after analysis of nmr, mass and uv spectra. biological activities of plant extract the antimicrobial susceptibility test the study involved the antimicrobial activity tests of the different extracts. different bacteria were used for the test of the activity of the extracts. the results of the qualitative antimicrobial screening of different extracts were as shown in (table 3). comparative study of antimicrobial activity the results of the antimicrobial susceptibility test of the different extracts showed that the crude hexane extracts and ethyl acetate extract of stem and leaf of m. nepalensis were resistant to all of the bacteria species tested while ethyl acetate extract of b. aristata was found to be moderately active against staphylococcus aureus while hexane extract of stem of b. aristata was slightly active against s. aureus. but the methanol extracts of stem of both plant species were found to be strongly active towards the gram positive bacteria staphylococcus aureus. the methanol extract of b. aristata showed same pharmacological effect of nalidixic acid control while methanol extract of m. nepalensis showed little lower pharmacological effect than that of control. hence both of these methanol extract of m. nepalensis and b. aristata were pharmacologically active against staphylococcus aureus while they were resistant to bacteria kleibsella pneumonia. the methanol extract of leaf of both plant species were also found to be resistant to above bacteria species. dmso was used as solvent control and it showed no effect. it was also found that salmonella typhimurim and salmonella typhimurium strains of bacteria were resistant to nalidixic acid (data not shown) brine shrimp bioassay for the study of biological activity of plant material, the newly hatched brine shrimp nauplii were exposed to the plant extracts. the biological activities were evaluated on the basis of their toxicities towards these nauplii. in this nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 12 nepjol.info/index.php/njb method, lc50 values (μg/ml) for different fractions were determined and those having less than 1000 are considered as pharmacologically active. the results obtained during this study are given in (table 4). table 4: cytotoxicity value of different plant extracts methanol extract lc50 (μg/ml) stem of m.nepalensis 8.3 leaf of m.nepalensis 389.04 stem of b. aristata 8.058*10-4 leaf of b. aristata 1303.166 comparative study of cytotoxicity against brine shrimps the results of brine shrimp bioassay showed that stem and leaf of mahonia nepalensis have 8.3 (μg/ml) and 389.04 (μg/ml) lc50 values while stem of berberis aristata have 8.058*10-4 (μg/ml) lc50 values which showed their bio-activity. stem of berberis aristata and mahonia nepalensis were comparatively highly cytotoxicity while leaf of mahonia nepalensis was moderately cytotoxic and leaf of berberis aristata was found to pharmacologically inactive against brine shrimp. conclusion phytochemical screening of hexane extract, ethyl acetate extracts and methanol extracts of the two plants revealed the presence of quinones, glycoside, terpenoid, cardiac glycoside, and steroid. however, alkaloids was found in all type of stem extracts. hence, distribution of different group of compounds was somewhat phytochemical equivalent in both species. the column chromatography of methanol extract of stem of mahonia nepalensis resulted in isolation of four different compounds mn1, mn2, mn3 and mn4 in which compound mn1 was identified as -sitosterol for the first time and mn2 as berberine. metabolic stem extract of mahonia nepalensis and berberis aristata both were strongly pharmacologically active as that of nalidixic acid against staphylococcus aureus. the hexane and ethyl acetate extract of stem and leaf of mahonia nepalensis were resistant to all of the bacterial species whereas hexane extract ethyl acetate extract of berberis aristata were pharmacologically active against stappylococcus aureus. in addition, all the other extracts were found to be inactive against gram negative bacteria like kleibsella pneumoniae, salmonella typhimurim, salmonella typhimurim. methanol stem extract of mahonia nepalensis and methanol stem extract of berberis aristata were highly cytotoxic against brine shrimp while leaf of mahonia nepalensis was moderately cytotoxic and leaf of berberis aristata was pharmacologically inactive against brine shrimp nauplii. conflict of interest the authors declare no conflict of interest. author contribution r.t was responsible for performing research under the supervision of s.m. r.t performed bibliographic researches and participated in discussion. the manuscript was designed, organized and written and edited by r.t. all authors have read and approved the final manuscript. ethical clearance no ethical rules had been violated during sample collection and experimentation. sample of mahonia nepalensis and berberis aristata had been collected from bhaktapur and palpa. fund source the research had been conducted from personal fund. central department of chemistry, tribhuwan university, nepal had allowed conducting the research in the laboratory of chemistry department. acknowledgement authors are sincerely grateful to the head of department, prof. dr. megh raj pokhrel and former head of department, dr. kedar nath ghimire of central department of chemistry, tribhuvan university for supporting and providing laboratory access in central department of chemistry. we are also grateful to dr. amar prasad yadav, dr. surya kant kalauni and dr. bimala subba for their kind cooperation and all the members of central department of chemistry. references 1. shrestha kk, tiwari nn, ghimire sk: mapdon-medicinal and aromatic plant database of nepal. in proceedings of nepal nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1:4-13 thusa & mulmi ©njb, biotechnology society of nepal 13 nepjol.info/index.php/njb japan joint symposiumon conservation and utilization of himalayan medicinal resources, (eds)watanabe t.a.; takano; bista, m.s. and sainju, h.k. nonprofit organization (npo)/society for conservation and development of himalayan medicinal resources(scdhmr)japan, 2002: 53-74 2. phooboo s, devkota a, jha pk: medicinal plants in nepal. an anthology of contemporary research. 2008: 1-24 3. polium o, stainton a: flowers of himalaya. oxford university press, oxford india paperbacks: 20-22 4. plant database facts sheet (http://www.plantdatabase.net/mahonia nepalensis) (accessed 27 april, 2013) 5. govindachari tr, pai br, rajadurai, s, rao ur: alkaloids of mahonia nepalensis d.c. j ind chem soc. 1957: 41-48 6. nguyen tm, tran at, hoang th, chau vm, ninh kb, phan vk : secobisbenzylisoquinoline alkaloid from mahonia nepalensis d.c. j chem. 2007 46 (5), 63-68 7. nguyen tm, tran at, hoang th, chau vm, nin kb, phan vk: bisbenzylisoquinoline alkaloid from mahonia nepalensis, j chem. 2009 47(3): 365-370 8. sharma k, bairwa r, chauhan n, shrivastava b, saini nk: berberis aristata. indian j re ayu pharm. 2011 2(2): 383-388 9. chatterjee rp: j indian chem soc. 1951, 28: 225 10. blasko g: karachine: an unusual protoberine alkaoid. j amer chem soc. 1982 104(7): 20392041 11. blasko g, sharma, m, taxilamine: a pseudobenzylpyroquinoline alkaoid. heterocycle 1982 1982 19(2): 257-259 12. rahmann a, ansari aa: alkaloids of berberis aristata –isolation of aromaline and oxyberberine. j chem soc pak. 1983 5(4): 283 13. bhakuni ds, shoheb a, poppali sp: medicinal plants: constituents of berberis aristata. indian j chem. 1968 6(2): 123 14. katiyar d, singh rk, singh s, singh v: isolation and characterization of lanost-5en-3-ol from the heart wood of berberis aristata dc, ijpi’s j pharmacog herb form. 2011 1(3): 8-13 15. saied s, batool s, naz s: phytochemical study of b. aristata. j of basic & applied science, 2007 3(1): 1-4 16. alamzeb m, khan, mr, ali s, shah, sq, mamoon, and ur: antimicrobial properties of extracts and compounds isolated from berberis jaeschkeana. bangladesh j pharmacol. 2013 8: 107-109 17. talukdar a, chaudhary b: phytochemical screening of ethanolic extracts of rubiacordiofolia, pharma & bio. sci., 2010, 1(4): 530-536 18. culei i: comparative antioxidant activity of individual herbal components used in ayurvedic medicine. phytochemistry 2003 63(1): 97-104 19. perez c, bazevquo pm: an antibiotic assay by the agar well diffusion method. acta biologicae et medicine experimentalis. 1990 15: 113-115 20. meyer bn, ferrigni nr, putnam je, jacobsen lb, nichols de, mclaughlin jl: brine shrimp: a convenient general bioassay for active plant constituents. j med plan res. 1982 45: 3134 21. tian jh, yang cw: chemical constituents from the stem of mahonia japonica. j chi chem soc. 2004 51: 443-446 nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 39-45 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 39 nepjol.info/index.php/njb trichoderma species as biocontrol agent against soil borne fungal pathogens srijana bastakoti1*, shiva belbase1, shrinkhala manandhar2, charu arjyal1 1department of microbiology, tri-chandra multiple campus, ghantaghar, kathmandu, nepal 2plant pathology laboratory, nepal agricultural research council (narc), khumaltar, lalitpur, nepal abstract soil borne pathogenic fungi are of major concern in agriculture which significantly decreases the plant yield. chemically controlled plant imposes environmental threats potentially dangerous to humans as well as other animals. thus, application of biological methods in plant disease control is more effective alternative technique. this study was carried out to isolate trichoderma species from soil sample and to assess its in vitro biocontrol efficacy against fungal pathogens viz. sclerotium rolfsii, sclerotionia sclerotiorum, fusarium solani and rhizoctonia solani. biocontrol efficacy testing of isolates against different fungal pathogens was performed by dual culture technique. in this study, five different trichoderma species were isolated from 26 various soil samples and were tested against four fungal soil-borne pathogens. inhibition percentage of radial growth of sclerotium rolfsii by three of the trichoderma isolates was found to be 100%; about 62% and 68% of maximum inhibition was observed against rhizoctonia solani and fusarium solani respectively whereas sclerotionia sclerotiorum was inhibited maximum up to 23%. this in vitro study revealed that although trichoderma species plays an important role in controlling all type of soil borne fungal plant pathogens, however, isolates as biocontrol agent against sclerotium rolfsii was found to be more efficient in comparison to other pathogens. keywords: biocontrol, trichoderma species, inhibition percentage, soil borne pathogens and dual culture technique *correspondence author email: srijanabastakoti33@gmail.com introduction soil borne plant pathogenic fungi cause heavy crop losses all over the world. agriculture has been facing the destructive activities of numerous pests and pathogens from an early time, which leads not only to the reduction of yield of the crops but also the aesthetic value. chemical control of such plant pathogens disturbs the environment, subverts ecology, degrades soil productivity, and mismanages water resources [1, 2]. in addition to this, due to the growing cost of pesticides, particularly in the less affluent region of the world and consumer demands for pesticide-free food has led to the search for the substitutes for these products. biological control of plant diseases, especially those caused by soil borne plant pathogens and nematodes, by microorganisms has been considered a more natural and environmentally acceptable alternative to the existing chemical treatment methods [3]. the renewed interest in biocontrol among agriculture biologists is due to its eco-friendly protection against weeds, insects, and plant diseases, a long lasting effect, and safety features. some of the bacterial antagonists, however, also have been found to show direct growth promoting effects on crop plant inoculants [4, 5]. biocontrol agent (bcas) can inhibit the growth of soil borne pathogens through various biocontrol mechanisms such as ability to grow much faster than them for space and nutrients, producing many powerful plant degrading enzymes such as lytic enzymes, proteolytic enzymes and more than 200 types of antibiotics which are highly toxic to any macroand microorganism [6]. the ability to produce multiple antibiotics probably helps to suppress diverse microbial competitors, some of which are likely to be plant pathogens and thus enhance biological control. the antibiotics have been shown to be particularly effective at suppressing growth of the target pathogen in vitro and/or in situ. some examples of antibiotics reported to be involved in plant pathogen suppression are 2, 4-diacetylphloroglucinol produced by pseudomonas fluorescens f113 against pythium species which causes the damping off disease, gliotoxin produced by trichoderma virens against rhizoctonia solani which causes root rot in plants [7]. moreover, enzymes produced by bcas are able to hydrolyze chitin, proteins, cellulose, and hemicellulose. thus contributing to direct suppression of plant pathogens. there are selective examples of bcas able to produce enzymes effective against certain plant pathogens [8]. plant disease is a complex phenomenon and, is an interaction among the host, the parasite and the environment. it can be defined as the disturbance in the mailto:srijanabastakoti33@gmail.com nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 39-45 bastakoti et al. ©njb, biotechnology society of nepal 40 nepjol.info/index.php/njb rhythmical equilibrium of a host in respect of structure or physiology or both, and which may lead to the death of a part or entire host or reduce value of its products. the mycelium of sclerotium rolfsii survives best in sandy soils, whereas, the sclerotia survive best in moist, aerobic conditions found at the soil surface [9]. similarly, fusarium solani and rhizoctonia solani are the most important fungal pathogens, which develop in both cultured and non-cultured soils, causing the symptoms of damping off and root rot diseases to the wide range of vegetable and crop plants including tomato [10]. for the effective biocontrol of soil borne pathogens a major consideration is the antagonist’s proliferation after introduction into the soil. trichoderma species is considered as promising biological control agents against numerous phytopathogenic fungi since it is able to inhibit the phytopathogenic fungi either by including resistance and plant defense reaction or by direct confrontation through mycoparasitism and competition or by producing antibiotics [11]. trichoderma species not only produce potential antibiotics, for example the peptaibols, but also mycotoxins and more than 100 metabolites with antibiotic activities including polyketides, pyrones, terpenes, metabolites derived from amino acids and polypeptides [12]. trichoderma species being a biocontrol agent have shown efficacy against diseases caused by pathogens such as fusarium oxysporum, rhizoctonia solani, pythium aphanidermatium, fusarium culmorum, gaeumannomyces graminis var. tritici, sclerotium rolfsii, phytophthora cactorum, botrytis cinerea and alternaria species [13]. thus, trichoderma species has been considered a viable alternative method to manage plant diseases [14]. the role of trichoderma species is not only to control the growth of pathogenic microbes, but there are various other uses for trichoderma species such as, enhance plant defense responses, stimulate colonization of rhizosphere and stimulates plant growth, root growth [15,16]. materials and methodology collection of soil sample twenty-six soil samples were collected from different geographical region of nepal: himalayan region, hilly region and terai region. both fresh and decaying soil samples from old and new heaps were collected into clean polythene bags cultivated with crops like cauliflower, potato, orange, wheat, pea, mustard, coffee, carrot, garlic, banana and tomato.some of the trichoderma isolate were obtained from the soil of hilly region (kathmandu, lalitpur, nuwakot) cultivated with oranges, pea wheat, cauliflower, potato. similarly, soil sample collected from terai region (sarlahi, siraha, bardiya) having warm and humid climate was cultivated with mustard, broad bean, eggplant representing growth of some trichoderma isolates. jumla, having warm and temperate climate was also one of the spot for the collection of soil sample. primarily, soil samples were collected from the zone which has used only organic fertilizer and has not used any pesticides, fungicides or any other chemical agent, to know natural availability of biocontrol agent. it is because, biocontrol agents are not much available in such agricultural land with excessive use of chemical fertilizers since it leads to the inhibition of biocontrol agents present in that soil. isolation of biocontrol agent, trichoderma species the collected soil sample from different geographical regions of nepal was used for isolation of trichoderma species. from the serial dilution, soil samples of 0.5 ml each was poured in selective media potato dextrose agar (pda) for isolation of trichoderma species and soil plating were performed. the plate was then allowed to incubate for 3-4 days at 25-26°c. incubation results the fuzzy growth of fungus on the pda plate was observed which was also seen after seven days of cultivation. identification of trichoderma isolates after the isolation of all isolates, it was examined under a microscope (olympus ch20, at plant pathology laboratory, narc, khumaltar, lalitpur) for the identification and confirmatory of trichoderma species. macroscopic visualization showed rapid growth rate and colonies are wooly becoming compact in time; and the surface colony color was white and scattered greenish patches become visible as the conidia are formed and may form concentric rings at times while on the reverse, the color is pale, tan, or yellowish. after then, growth observed in those plates was taken for microscopic study, colony characteristics and morphology of each trichoderma isolate was observed. microscopic observation of specimens was done by the sticky tape method [17]. examination of the shape, size, arrangement and development of conidiophores (repeatedly branched conidiophores) or phialides (irregularly verticillate, bearing clusters of divergent, often irregularly bent, flask shaped phialides) or conidia (unicellular, round or ellipsoidal, green in color, smooth walled or rough conidia) provided a tentative identification of trichoderma species. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 39-45 bastakoti et al. ©njb, biotechnology society of nepal 41 nepjol.info/index.php/njb figure 1: an overview of overall methodology dual culture of isolates with fungal pathogens all the isolates obtained from the soil sample were evaluated in vitro for their biocontrol efficacy against fungal pathogens by dual culture methods. the soil borne fungal pathogens sclerotium rolfsii, rhizoctonia solani, fusarium solani and sclerotionia sclerotiorum were provided from the plant pathology lab of nepal agricultural research council (narc) at khumaltar, lalitpur, nepal. these fungal pathogens were isolated from naturally infected tomato, egg-plant, mustard plant and brassicas respectively causing wilt and rot disease and obtained in pure culture form. first of all, 20 ml of prepared pda was poured in petri plates and allowed to solidify. after solidification of pda media, 5 mm diameter mycelia disc from the margin of 7 day-old culture of trichoderma species and the soil-borne pathogens were placed on the opposite ends of the plate at equal distance from the periphery on the same day. there were 3 replications for each treatment. the experimental design used was set up with eight petri dishes for each isolate. in control plates, a sterile disc, whatman no.1 filter paper of 5 mm diameter, was placed at opposite side of targeted fungal pathogens. the experiment was performed in complete aseptic condition and both the control plates and test plates were incubated at 25 ± 1°c for 7 days. the colony diameter of radial growth of targeted fungal pathogens was measured after each 1st, 2nd, 3rd and 4th days of incubation period at two locations from center of the test placed and average diameter were calculated. finally, percent inhibition of average radial growth was also calculated in relation to the growth of the controls [18] and the flowchart for methodology for this experiment is shown in figure 1. l = [(c – t)/c] x 100 where, l = inhibition percentage; c = radial growth measurement of the pathogen in control t = radial growth of pathogen in the presence of trichoderma isolates. results isolation of biocontrol agent and microscopic view the growth of trichoderma species in pda plates were observed after 7 days of inoculation and incubation of soil samples. isolated trichoderma species from soil samples used as biocontrol agents is represented in figure 2. figure: 2 isolation of trichoderma species from collected soil samples figure 3: microscopic view of trichoderma isolate ts 206 with repeatedly branched conidiophore, flask shaped phialides and rough conidia 19% 81% presence of biocontrol agent(trichoderma ) nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 39-45 bastakoti et al. ©njb, biotechnology society of nepal 42 nepjol.info/index.php/njb out of 26 soil samples tested, the trichoderma species were isolated in 5 soil samples. the green or white colored fuzzy form growth indicated the trichoderma species and was confirmed by the microscopic examination as shown in figure 3. inhibition percentage shown by trichoderma isolates against soil borne pathogens inhibition percentage of the test soil-borne pathogens in the presence of trichoderma species was performed by dual culture technique which requires both the control plates and test plates. one of the control plates of sclerotium rolfsii is shown in figure 4 indicating three replications. figure 4: colony growth in control plates of sclerotium rolfsii after 96 hrs of incubation shows the whole area of a petri plate is rapidly covered with mycelium, including aerial hyphae which may cover the lid of the plate among the test pathogens, trichoderma isolates named as ts 209, ts 206 and ts 181 showed almost 100% inhibitions against sclerotium rolfsii by covering the growth of whole pathogen in the plate. also, the isolates ts 214 after five day of incubation was found to work more actively against sclerotium rolfsii as shown in figure 5. figure 5: dual culture plate of sclerotium rolfsii with trichoderma species after 5th day of incubation shows a very well inhibition of white spores like mycelium of s. rolfsii by ts 214 isolates representing effective biological action over pathogens. but the sclerotionia sclerotiorum was not much controlled by trichoderma isolate ts 215 indicating inhibition percentage of 22.01%. dual culture plate of trichoderma isolates against sclerotionia sclerotiorum is as shown in figure 6. in the case of dual culture with fusarium solani, one of the isolates of trichoderma species showed the maximum inhibition percentage of 63.3% whereas, rest of the four isolates showed inhibition below 45%. similarly, isolates against rhizoctonia solani also revealed growth inhibition of pathogens. figure 6: dual culture plate of 5 different trichoderma isolates against sclerotionia sclerotiorum (s.s. 1 indicating first replication) after 96 hours of incubation shows competitive effect with almost equal mode of action over each other. the maximum inhibition percentage observed was 66.08% which indicated effective reduction of the colony growth of rhizoctonia solani after 96 hours cultivation. other three isolates were also able to inhibit growth of rhizoctonia solani pathogen up to 55.7%, 47.2%, 43.77% and lowest inhibition percentage of 29.7% was shown by one of trichoderma species. therefore, among these test pathogens, trichoderma species showed strong and effective biocontrol activity against sclerotium rolfsii. the in vitro study also showed that trichoderma species effectively reduced the growth of both fusarium solani and rhizoctonia solani with almost equal inhibition percentage. inhibition percentage of the test soil-borne pathogens in the presence of trichoderma species by dual culture technique is shown in figure 7. discussion in this study, the results of dual culture revealed the rapid and effective growth of trichoderma isolates used against sclerotium rolfsii in comparison to other pathogens (sclerotionia sclerotiorum, fusarium solani and rhizoctonia solani). nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 39-45 bastakoti et al. ©njb, biotechnology society of nepal 43 nepjol.info/index.php/njb figure 7: inhibition percentage of all four soil borne fungal pathogens by five trichoderma isolates shows individual biocontrol action of isolates over placed pathogens. in vitro efficacy testing of biocontrol agent against soil borne pathogen was studied by dual culture technique. three replication of trichoderma species were used during this technique for the calculation of average diameter growth of respective pathogens for a reliable result. the colony growth of sclerotium rolfsii on the 4th day incubation was found to be covered by the growth of trichoderma species. due to overgrowth of trichoderma species in the plate, the growth of test fungal pathogen was found to be highly inhibited. antagonistic interactions of trichoderma species isolate ts 215 showed excellent activity against sclerotium rolfsii, which is known to cause wilt and rot diseases in plants. the damping off disease caused by sclerotium rolfsiiis an important disease of tomato (lycopersicon esculentum) and other vegetable crops when treated with biocontrol agent trichoderma harzianum, about 5262% disease reduction was achieved in another study [19]. whereas, in this study, sclerotium rolfsii isolated from infected tomato was found to be inhibited nearly up to 100% by three isolates of the trichoderma species and two isolates showed inhibition of 84.5% and 72.8% respectively. similarly, this study showed the mycelium growth inhibition of the rhizoctonia solani of 66.08-29.07% by isolates of trichoderma whereas in another study, three different isolates of trichoderma against soil borne pathogen rhizoctonia solani was observed to be 74.4-67.8% [20]. however, some of the antagonists tested were not found to be very effective against test pathogens under the in vitro observations but they may show better result under their natural field condition as their activities depend on the physicoof the environment [21]. on the dual culture plate of trichoderma species with sclerotioni asclerotiorum, competitive effect was observed. here pathogen continued its radial growth and trichoderma species placed in the same plate also showed radial growth. when colony growth of pathogen and biocontrol agent came in contact with each other, both showed its action over each other, however, trichoderma isolates was able to shown less efficacy over growth of pathogens. in this study, biocontrol activity of trichoderma isolate ts 215 also showed active action against soil borne pathogen fusarium solani showing inhibition percentage more than 60%. similarly, from other experiment, under in vitro conditions, the result revealed that trichoderma harzianum, isolates n-8, was found to inhibit the radial mycelial growth of the pathogen effectively by 68.22% [22]. the inhibition shown by the antagonists may be due to release of antibiotic or antibiotic like substances or hyphal parasitism which results in direct inhibition of growth of the pathogen by disintegrating the hyphal wall resulting in the penetration, absorption and lysis of the mycelium. biological control is a promising tool to maintain current level of agricultural production by reducing the release of polluting chemical pesticides to the environment as well as making the plants free from infecting soil borne pathogens, leading to high yield of crops. it is a complex process made up from several successive steps but primarily contact is made between fungal antagonist and host (pathogen) surface. plant pathogenic fungi and nematodes is a widespread problem and the use of chemicals is hardly successful. according to this study, biological control is seen as a better alternative and may be helpful, especially against soil borne fungal pathogens sclerotium rolfsii, fusarium solani, rhizoctonia solani and sclerotioni asclerotiorum. one of the main advantages of the biocontrol agents is that it is cost effective and leads no harm to the crop. most frequently species of bacillus, pseudomonas and trichoderma are used for biological control of fungal pathogens. among them, one of the fungal biocontrol agents used in this study is trichoderma species. they are common saprophytic fungi found in almost any soil and rhizospheric microflora. the reason for choosing trichoderma species as potential biocontrol agents is because of their ability to reduce the incidence of disease caused by plant pathogenic fungi [23]. possible mechanisms of antagonism employed by trichoderma species includes nutrient and niche competitions, antibiosis by producing volatile components and non-volatile antibiotics that are inhibitory against a nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 39-45 bastakoti et al. ©njb, biotechnology society of nepal 44 nepjol.info/index.php/njb range of soil borne fungi, as well as parasitism. antagonistic microorganisms like trichoderma reduce growth, survival or infections caused by the pathogens by different mechanisms like competition, antibiosis, mycoparasitism, hyphal interactions, and enzyme secretion [24]. it is widely known that environmental parameters such as abiotic (soil type, soil temperature, soil ph and water potential) and biotic (plant species and variety, microbial activity of the soil) factors as well as other factors such as method and timing of applications may have influence on the biological control efficacy of trichoderma isolates [25].the soil samples were collected from different districts that include hot region as well as cold region. the soil samples of cold region comparatively showed slow growth rate than that of hot region. initially, in the pda plates trichoderma species is found to grow slowly, it may be due to the presence of other pathogens which have capacity to dominate or slow down the growth of trichoderma species. it may also be due to dispersal of spores in the environment. this can be the reason why some trichoderma species took almost one month for isolation. the in vitro screening of the antagonistic potential used in this work allowed a systematic investigation of several trichoderma isolates. the applied trichoderma species in this study revealed the most effective inhibition of growth of fungal pathogen sclerotium rolfsii and biocontrol action over rest of the test pathogens was limited. however, positive results obtained from this in vitro studies are only indicative, as experimental conditions do not take all ecological and endemic factors into account. for this reason, much more work needs to be done to explore the possibility of the use of this biocontrol agent in field condition to control the diseases caused by sclerotium rolfsii and several other soil borne fungal pathogens by adopting appropriate techniques for field trial. further actions need to be carried out to prepare cost-effective, easy–toproduce and easy-to-apply formulations of different species of trichoderma isolates. conclusion it can be concluded that the isolated trichoderma species reduced the growth of all the four soil borne pathogens: sclerotium rolfsii, fusarium solani, rhizoctonia solani and sclerotionia sclerotiorum significantly in different level and, therefore, can be incorporated for integrated disease management of soil borne plant pathogens. hence, trichoderma species can be used as a potential biocontrol agent against these pathogens. however, its efficacy against sclerotium rolfsii was found to be more in comparison to others. therefore, this research can have promising potential in agricultural field to protect plants affected with various fungal pathogens, especially sclerotium rolfsii. acknowledgement we would like to extend our gratitude to dr. baidya nath mahato, chief and principal scientist of plant pathology division, nepal agricultural research council (narc) for providing us an opportunity to conduct this study, puspa shrestha for providing guidance during the experiments at plant pathology laboratory and all the staffs for their support. we are also grateful to the teachers and staffs of department of microbiology, tri-chandra multiple campus for their support in completing this task. conflict of interest none 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dubey sc, suresh m and singh b: evaluation of trichoderma species against fusarium oxysporum fsp. ciceris for integrated management of chickpea wilt. biol contr. 2007 40: 118-127. 24. ponnusamykonar p, venkitasamy k and varatharajan p: in vitro study of antagonistic effect of trichoderma sp., on tea plant pathogen, phomopsistheae. archives of applied science research 2011 3(4):352-358. 25. behzad h, mousa t, mohammad rm, mahdi d: biological potential of some iranian trichoderma isolates in the control of soil borne plant pathogenic fungi. afr j biotechnol. 2008 7: 967-972. https://www.researchgate.net/researcher/2112895117_a_foroutan https://www.researchgate.net/publication/305423547_biocontrol_of_soybean_charcoal_root_rot_disease_by_using_trichoderma_spp?ev=auth_pub https://www.researchgate.net/publication/305423547_biocontrol_of_soybean_charcoal_root_rot_disease_by_using_trichoderma_spp?ev=auth_pub https://www.researchgate.net/publication/305423547_biocontrol_of_soybean_charcoal_root_rot_disease_by_using_trichoderma_spp?ev=auth_pub nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 32-38 issn 2091-1130(print)/issn 2467-9319 (online) original research article ©njb, biotechnology society of nepal 32 nepjol.info/index.php/njb prevalence of extended spectrum beta-lactamase producing escherichia coli and klebsiella species from urinary specimens of children attending friendship international children’s hospital ujjwal rimal1, shovana thapa2, roshani maharjan1* 1department of microbiology, trichandra multiple college, ghantaghar, kathmandu, nepal. 2department of microbiology, international friendship children’s hospital, maharajgunj, kathmandu, nepal abstract extended-spectrum β-lactamase producing e. coli and k. pneumoniae is a serious threat to the patients. these organisms are major extended spectrum beta lactamase (esbl) producers. the objective of this study was to determine the prevalence of extended spectrum βlactamase producing strains of escherichia coli and klebsiella spp isolates from the urine sample of children visiting international friendship children hospital. during the seven months, between june 2016 to december 2016, 1018 midstream urine samples(msu) were collected from patients suspected of having uti. the samples were investigated by conventional semi-quantitative culture technique and identification of e. coli and klebsiella spp. was done by microscopy and biochemical test. antibiotic susceptibility test of isolates was performed by modified kirby bauer disc diffusion test. esbl screening test was done by using 3rd generation cephalosporin and confirmation done by combination disc diffusion method. out of total 1018 msu samples investigated, 200(19.64%) isolates of e. coli and 28(2.7%) isolates of klebsiella spp. making a total of 228(22.39%) were found to cause significant bacteriuria. 76(33.33%) isolates, from those causing significant bacteriuria, were multi-drug resistant organisms. out of 228 isolates, 54(23.68%) were esbl producers, that includes 51(25.5%) escherichia coli and 3(12.5%) klebsiella pneumoniae. esbl producers were more common in in-patient (36.17%) than out-patient (20.44%). most of the esbl producers were resistance to amoxicillin, followed by cotrimoxazole and ciprofloxacin respectively. they were highly sensitive to imipenem, tigecycline, amikacin, piperacillin-tazobactam, and nitrofurantoin. high prevalence of esbl producing e. coli and klebsiella pneumoniae was found among children. regular and routine monitoring of esbl producing isolates is essential. key words: urinary tract infection(uti), extended spectrum beta lactamase(esbl), e. coli, klebsiella *corresponding author e mail: roshani_mh@hotmail.com introduction urinary tract infection (uti) is a term applied to a variety of clinical conditions ranging in severity from asymptomatic which is carrier status in the urine to symptomatic acute infection of the kidney with resultant sepsis [1]. the clinical symptoms of uti usually include frequency, dysuria, pyuria, suprapubic tenderness, back pain, fever and urgency [2]. the most common uropathogenic gram negative bacteria are escherichia coli and klebsiella pneumoniae [3]. extended-spectrum β lactamases (esbls) are a group of enzymes with the ability to hydrolyze and cause resistance to the oxyiminocephalosporins (i.e.cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefepime) monobactams (i.e. aztreonam) [4]. beta-lactamases are among the most heterogeneous group of resistance enzymes. despite a significant amount of amino acid sequence variability, betalactamases share a common overall topology [5]. there are several esbls genotypes. these are shv, tem, and ctx-m [6]. other clinically important genotypes include oxa, veb, per, bes-1, bel-1, sfo-1, tla, and ibc [7]. “classical” esbls are derived from tem and shv enzymes whereas “non-classical” esbls are derived from enzymes other than tem or shv. 90% of ampicillin resistance in e. coli is due to the production of tem-1 [8]. based on different combinations of changes, currently, 195 tem-type enzymes have been described. tem-2, the first variant described, differed from tem-1 through the substitution of a lysine for a glutamine at position 39 [6]. a shift in the distribution of different esbls has recently occurred in europe, with a dramatic increase of ctx-m enzymes over nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 32-38 rimal et al. ©njb, biotechnology society of nepal 33 nepjol.info/index.php/njb tem and shv variants [9]. since 2000, e. coli producing ctx-m β-lactamases have emerged worldwide as an important cause of communityonset urinary tract infections (utis) and this has been called the ctx-m pandemic [10]. these enzymes were named for their greater activity against cefotaxime than other oxyimino-betalactam substrates (e.g. ceftazidime, ceftriaxone, or cefepime) [7]. rather than arising from mutation, they represent examples of plasmid acquisition of beta-lactamase genes normally found on the chromosome of kluyvera species, a group of rarely pathogenic commensal organisms [9]. in france, a novel derivative of oxa-10 (numbered oxa-28) was found in a p. aeruginosa isolate [11]. the rapid increasing rate of esbl production among enterobacteriaceae, mainly e. coli and klebsiella pneumonia, have created a serious global public health problem [12]. availability of limited treatment options for the infections caused esbl producing bacteria makes the treatment very difficult and often results into treatment failure [13]. scant number of studies had been reported from nepal assessing the esbl producers among e. coli and klebsiella spp in children. hence the study was carried out to determine the prevalence of esbl producing escherichia coli and klebsiella spp isolates from the urine samples of children. materials and methodology sample collection and identification of bacteria the cross-sectional study was carried out in pathology department of international friendship children hospital, maharajgunj, kathmandu from june 2016 to december 2016. patients under the age of 13 years or their guardians visiting the pathology department were directly interviewed for his/her clinical history during the sample collection. during the study period, 1018 midstream urine samples were collected and processed according to the standard laboratory methods by forbes et al [14]. semi-quantitative culture technique was used to detect the presence of significant bacteriuria. the bacterial culture was done on macconkey agar (ma) and blood agar (ba) and incubated overnight at 37oc for isolation of pure culture. diagnosis of uti was made when there were colony count exceeding 105 cfu/ml of urine specimen. the isolates were identified based on morphology, gram’s staining, motility, and standard biochemical tests as described in forbes et al [14]. modified kirby-bauer’s disc diffusion method was employed for the antibiotic susceptibility test of potential pathogenic isolates as per standard technique on muller hilton agar [15]. nitrofurantoin(300mcg), amoxicillin (30mcg), cotrimoxazole (25 mcg), ciprofloxacin (5mcg), ceftriaxone (30 mcg), ceftazidime (30 mcg), gentamicin (10 mcg), imipenem (10 mcg) and meropenem (10 mcg) were used for antimicrobial susceptibility testing. screening of esbl producing strains of e. coli and k.pneumoniae the screening test for the production of esbl was performed using both ceftazidime (caz) (30μg) and ceftriaxone (ctr) (30μg) disks. if the zone of inhibition was less than or equal to 22mm for caz and/or less than or equal to 25mm for ctr, the isolate was considered a potential esbl-producer as recommended by clsi [16]. phenotypic confirmation of esbl production susceptible screened esbl producers were subjected to combined disk test as recommended by the clsi [16]. combination disk method used for the confirmation of esbl-producing strains in which caz (30μg), alone and in combination with clavulanic acid (ca) (10μg) were used. after incubating overnight at 37°c, ≥ 5 mm increase in the zone diameter for either antimicrobial agent which was tested in combination with clavulanic acid (cac) versus its zone when tested alone, was interpreted as positive for esbl production. results table 1: microbiological profile of bacterial isolates s.n bacterial isolates number percent 1 escherichia coli 200 82.30% 2 klebsiella pneumoniae 24 10% 3 klebsiella oxytoca 4 1.64% 4 citrobacter spp 3 1.23% 5 pseudomonas aeruginosa 2 0.82% 6 acinetobacter 2 0.82% 7 staphylococcus aureus 8 3.29% total 243 100% out of the total 1018 mid-stream urine samples, 243(23.87%) samples were found to have a significant growth. out of 243 culture positive cases, escherichia coli (200) (82%) was found to be nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 32-38 rimal et al. ©njb, biotechnology society of nepal 34 nepjol.info/index.php/njb the most common isolates followed by klebsiella pneumoniae (24) (10%), klebsiella oxytoca (4) (1.65%), citrobacter species (3) (1.15%), pseudomonas aeruginosa (2) (0.85%), acinetobacter species (2) (0.85%) and the gram positive isolates, staphylococcus aureus (8) constitutes (3.30%) (table 1). most of the patient were of age group 1-5 (60.96%) followed by age <1 (18.42%) (table 2). table 2: demographic distribution of e. coli and klebsiella spp age group sex of patient total male (%) female (%) <1 year 12 (28%) 30 (72%) 42(18.42%) 1-5 49 (35%) 90 (65%) 139(60.96%) 6-10 11 (34.3%) 21 (65.6%) 32(14.03%) 11-13 3 (20%) 12 (80%) 15(6.57%) total 75 153 228 out of 200 e. coli isolates, 169(84.5%) isolates were sensitive to nitrofurantoin followed by gentamycin 146(73%) and cotrimoxazole 110(55%). more than 30% isolates were resistant to third generation cephalosporins i.e ceftriaxone and ceftazidime (table 3). table 3: antibiotic susceptibility pattern of escherichia coli antibiotics susceptibility pattern (n=200) sensitive (%) intermediate (%) resistance (%) amoxicillin 14 (7) 23 (11.5) 163 (81.5) ciprofloxacin 91 (45.5) 31 (15.5) 78 (39) cotri-moxazole 110 (55) 22 (11) 68 (34) nitrofurantoin 169 (84.5) 20 (10) 11 (5.5) gentamycin 146 (73) 21 (10.5) 33 (16.5) ceftriaxone 108 (54) 30 (15) 62 (31) ceftazidime 98 (49) 34 (17) 68 (34) out of total 24k. pneumoniae isolates, 19(79.2%) were sensitive towards gentamycin, followed by nitrofurantoin 17 (71%), ciprofloxacin 16 (67%) and ceftriaxone 16 (67%) (table 4). table 4: antibiotic susceptibility pattern of k. pneumoniae isolates. antibiotics susceptibility pattern(n=24) sensitive (%) intermediate (%) resistance (%) amoxicillin 0 (0) 0 (0) 24 (100) ciprofloxacin 16 (67) 4 (17) 4 (17) cotrimoxazole 14 (58) 3 (13) 7 (29) nitrofurantoin 17 (71) 1 (4) 6 (25) gentamycin 19 (79.2) 3 (12.5) 2 (8.3) ceftriaxone 16 (67) 2 (8) 6 (25) ceftazidime 14 (58) 4 (17) 6 (25) among total 4 k. oxytoca isolates, all 4 were resistant to amoxicillin, howeversensitive to nitrofurantoin., 3 (75%) of them were sensitive to to cotrimoxazole, followed by ciprofloxacin. 68 (34%) of e. coli isolates, 6(25%) of klebsiella pneumoniae isolates and 2 (50%) of klebsiella oxytoca isolates, making a total of 76(33.33%) isolates were multi-drug resistant (table 5). table 5: distribution of mdr isolates bacterial isolates total mdr (%) escherichia coli 200 68 (34%) klebsiella pneumoniae 24 6 (25%) klebsiella oxytoca 4 2 (50%) total 228 76(33.33%) out of 228 isolates, 61 isolates gave esbl screening test positive. 51 isolates of e. coli and 3 isolates of klebsiella pneumoniae making a total of 54(23.68%) were confirmed as esbl producers. none of k. oxytoca was esbl producer (table 6). table 6: detection of esbl by combination disk method. organism significant growth screening test positive confirmatory test(combination disk method) increase in diameter ≥5mm escherichia coli 200 57 51(25.5%) klebsiella pneumonia 24 4 3(12.5%) klebsiella oxytoca 4 total 228 61 54(23.68%) out of 75 isolates from male, 12(16%) were esbl producer and out of 153 isolates from female 42(27.54%) were esbl producer. more female was infected as compared to male, also chi-square test suggests a significant association in between sex of a patient and esbl producers(p-value<0.05) (table 7). table 7: gender wise distribution of esbl producing isolates. sex esbl producer esbl nonproducer total p-value male 12(16%) 63(84%) 75 <0.05 female 42(27.45%) 111(72.54%) 153 total 54(23.68%) 174(76.31%) 228 among culture positive cases, 47 were from inpatient and remaining 181 were from out-patient. among 47 in-patient, 17(36.17%) was found to be esbl positives. similarly, among 181 out-patient, 37(20.44%) was found to be esbl positives (table 8). all 51 esbl producing isolates of e. coli were sensitive to imipenem, piperacillin-tazobactam, tigecycline and amikacin. 94.2%and 92.2% of e. coli were resistant to ceftriaxone and ceftazidime, respectively. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 32-38 rimal et al. ©njb, biotechnology society of nepal 35 nepjol.info/index.php/njb furthermore, 88.2% and 31.4% of e. coli were resistant to cefepime and meropenem, respectively (table 9). all of the 3 esbl producing k. pneumoniae isolates were sensitive to imipenem, meropenem, piperacillin-tazobactam, tigecycline, and amikacin. all of the isolates were resistant to cefepime and amoxicillin. table 9: antibioitic susceptibility pattern of esbl producing strain of e. coli antibiotics e. coli (n=51) sensitive sensitive % resistance resistance % amoxicillin 51 100 ciprofloxacin 13 25.5 38 74.5 cotrimoxazole 13 25.5 38 74.5 nitrofurantoin 48 94.1 3 5.9 gentamicin 32 66.7 19 37.3 ceftriaxone 3 5.9 48 94.1 ceftazidime 4 7.8 47 92.2 imipenem 51 100 0 0 meropenem 35 68.6 16 31.4 cefepime 6 11.8 45 88.2 piperacillin 51 100 tigecycline 51 100 amikacin 51 100 discussion out of 1018 mid-stream urine sample, 243 (23.87%) showed significant growth. similar result was obtained by bhandari (2013) where growth positivity was found to be 23.36% [17]. similarly, the study carried out in india by niranjan et al (2014) yielded 18.5% significant growth [18]. however, our result is low as compared to that reported from south africa (51%) by habte et al (2009) [19]. a study conducted in nepal by bhatta et al (2013), demonstrated similar result with 27.3% significant growth [20]. the rate of infection found in female patients was 163/684 (23.83%) and in male, the rate of infections was found to be 80/354 (22.59%). there wasn’t any huge difference between rate of infection in male and female children patient. this result is in contrast to the earlier studies by thakur et al (2013) where 56.64% female and 43.36% male patient were infected [21]. the growth positivity with 33.5% among female patients and 23.7% in male patients was observed in a similar study by baral et al (2012)[22]. the predominant isolate was e. coli (82.30%) followed by klebsiella species (11.53%) and staphylococcus aureus (3.29%). e. coli is a predominant isolate, because e. coli can bind to the glycol-conjugate receptor of the uroepithelial cells of human urinary tract so it can initiate infection itself. e. coli is isolated in 90% of infection and strains are characterized by presence of unique virulence determinant the pilus (gal-gal) receptor [23]. similar result was obtained by other studies [24, 25]. e. coli was found to be resistant towards amoxicillin (81.5%), co-trimoxazole (35%), and ciprofloxacin (38%). in our study, 25% of k. pneumoniae were resistant to both ceftriaxone and ceftazidime. other studies from nepal reported that the resistance rates of k. pneumoniae to thirdgeneration cephalosporin were between 20% to 75% [26,27]. likewise, 25% and 8.3% of k. pneumoniae were found resistant to nitrofurantoin and gentamicin, respectively. all k. pneumoniae strains were resistant (100%) to amoxicillin, while resistance rate for e. coli was 81.5%. similar result was observed in a study carried out at madagascar, where 80% of the e. coli isolates were resistant to amoxicillin [28]. in our study, e. coli showed a high resistance rate 68% to co-trimoxazole whereas the k. pneumoniae showed resistance rate 30% to co-trimoxazole. a comparable resistance rate of 80% and 45% to cotrimoxazole was shown among esbl producing e. coli and k. pneumoniae isolates in a study conducted in iran [29]. bazzaz et al (2009) reported esbl-producing isolates of k. pneumoniae and e. coli as 59.2% [30]. in this study, 17% isolates of k. pneumoniae were resistant to ciprofloxacin. resistant to fluoroquinolones is due to the result of alterations in target enzyme (dna gyrase and topoisomerase iv) and because of change in drug entry and efflux [31]. klebsiella pneumoniae sensitivity towards gentamicin was found in 79.2% which was significantly higher than that was reported in karanchi, pakistan where a resistance rate for gentamicin was 46.7% [32]. however, the rate was only slightly higher than to the findings from previous study done in nepal where the sensitivity was 72.9% [22]. these significant variations may be attributed to selective pressures table 8: department wise distribution of esbl producers. in-patients out-patients total p-value esbl producer 17(36.17%) 37(20.44%) 54 <0.05 esbl nonproducer 30(63.82%) 144(79.55%) 174 total 47 181 228 nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 32-38 rimal et al. ©njb, biotechnology society of nepal 36 nepjol.info/index.php/njb by drugs in different regions. resistance of aminoglycosides is done by the enzymes that cause modification of drug by phosphorylation, acetylation or adenylation and less or more by other methods [33]. in our study, 31.4% of esbl positive e. coli was resistant to meropenem, whereas all isolates were sensitive to imipenem which is similar to the study done in peshawar, pakistan [34]. the emergence of carbapenem resistance in k. pneumoniae is typically attributed to the production of klebsiella pneumoniae carbapenemase (kpc) [35]. we found the 76 (33.33%) isolates of e. coli and klebsiella spp were multidrug resistant. similar findings were observed in the study done by tuladhar et al (2003), in a hospital in kathmandu, where 35.21% of bacterial strain were mdr [36]. but the result was in contrast to the study by upadhaya et al (2013) where 48% were mdr [37]. the prevalence of mdr varied among different studies and outcome of the prevalence may depend on various factors such as mdr criterion, how the antibiotics are used, and organism encoding multiple resistance gene which is becoming more prevalent. out of 228 isolates, 54 (23.68%) were esbl producer. our finding was lower in comparison to the study conducted by dahal et al (2016), in a community hospital of kathmandu that reported 47.75% as esbl producer [25]. a study done in india reported nearly 40% of urinary isolates of e. coli and k. pneumoniae were esbl positive [38]. our result was similar to the study done by poudyal (2010) where 25.7% esbl producer were isolated from urine sample [39]. however, our result showed higher prevalence as compared to the study by logan et al (2014) in the united states with esbls representing only 0.28% of all e. coli, k. pneumoniae, and p. mirabilis in children from 1999 to 2001 and later increasing to 0.92% of all isolates in 2010–2011 [40]. the occurrence of esbls among clinical isolates varies greatly from country to country, among the hospitals, within the country. in our study, 25.5% of e. coli isolates and 12.5% of klebsiella pneumoniae isolates were esbl producer. these findings were comparable to the study done by chander and shrestha (2013) in nepal tertiary hospital in which 13.51% of e. coli and 16.55% of klebsiella pneumoniae were esbl producers [41]. esbl occurrence among e. coli and klebsiella is of great concern since infections caused by these bacteria are very common and resistance of the organism may be due to the presence of capsule that gives some level of protection to the cells, presence of multidrug resistance efflux pump, they also spread easily, are pathogenic and efficient at acquiring and disseminating resistance plasmid [42]. more female (27.45%) were infected as compared to male (16%). similar result was obtained by bhandari (2013) [17]. though some of our results were very much contrast with other studies, the prevalence rate of esbl producing isolates in our study was considered significant and high. the higher number of esbl producers might be due to more reliance on third generation cephalosporins to treat gram negative infections and unscrupulous hospital antibiotic policy. moreover, high prevalence of esbl isolates among children might be due to immaturity of the immune system in these infection in these age group. further studies are required to know the current burden in these population. conclusion high prevalence of esbl producing e. coli and k. pneumoniae was observed in our study. e. coli is predominant esbl producers than k. pneumoniae. children under age five were found to be highly infected with urinary tract infection. escherichia coli and klebsiella spp are emerging highly as a multi-drug resistant. imipenem, tigecycline, amikacin, piperacillin-tazobactam were found to be the most effective drug against the esbl producing isolates. nitrofurantoin and gentamycin can be used as a drug of choice against non-esbl producing isolates. declaration ethics approval and consent to participate ethical approval was taken from the ethical review committee of international friendship children’s hospital and tri-chandra multiple campus. informed consent form was obtained from parents of the participants before their participation. competing interests we have read nepal journal of biotechnology policy on declaration of competing interest and declare that we have no competing interests. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 32-38 rimal et al. ©njb, biotechnology society of nepal 37 nepjol.info/index.php/njb authors’ contribution mr. ujjwal rimal, mrs. roshani maharjan, and dr. shovana thapa jointly performed the study. acknowledgement the authors would like to convey special thanks to staffs of international friendship children hospital, participant children, their parents and to all the people who directly or indirectly helped us in completing this work. references 1. tanagho ea, mcaninch jw: smith's general urology: bacterial infections of the genitourinary tract. united states of america: mcgraw-hill companies inc, 2004, 203-227. 2. gupta k, hooton tm, naber kg: international clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by the infectious diseases society of america and the european society for microbiology and infectious 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ansari s, ghimire p et al: comparative evaluation of urine isolates among kidney transplanted and other uti suspected patients visiting national public health laboratory, (nphl) teku, nepal. int j biomed adv res. 2013 4(6):369-75. 38. babypadmini s, appalaraju b: extended spectrum lactamase in urinary isolates of e. coli and klebsiella pneumoniae prevalence and susceptibility patterns in a tertiary care hospital. indian j medical microbiol. 2004 22:172– 174. 39. poudyal s: prevalance of b lactamase producing multidrug resistant bacterial pathogens isolated from different clinical samples at national public health laboratory, nepal. msc thesis. tribhuvan university, submitted to central department of microbiology. 2010 40. logan lk, braykov np, weinstein ra, laxminarayan r. extended-spectrum betalactamase-producing and third-generation cephalosporin-resistant enterobacteriaceae in children: trends in the united states, 1999–2011. j pediatr infect dis soc. 2014 3:320–8. 41. chander a, shrestha cd: prevalence of extended spectrum beta-lactamase producing escherichia coli and klebsiella pneumoniae urinary isolates in tertiary care hospital in kathmandu, nepal. bmc research notes. 2013 6(1):487 42. chudhary u and aggarwal r: extended spectrum b lactamase (esbl)an emerging threat to clinical therapeutics. indian j med microbiol rev. 2004 22: 75-80 nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 4657 issn 2091-1130(print)/issn 2467-9319 (online) revie w article ©njb, biotechnology society of nepal 46 nepjol.info/index.php/njb apoptosis: implications in viral and mycobacterium tuberculosis infections gorakh raj giriǂ, uddhav timilsinaǂ* faculty of life sciences and biotechnology, south asian university, new delhi, india abstract apoptosis is a form of programmed cell death leading to genetically controlled self-destruction of cells. it is essential in the development, maintenance, and regulation of cells during physiological as well as pathological conditions. deregulation of apoptotic mechanisms is associated with various pathological diseases including cancer, autoimmune disorders, viral and bacterial infections. virus and mycobacterium tuberculosis elicit host cell apoptosis as a part of host immune defense or pathogen dissemination. they inhibit both extrinsic and intrinsic pathways of apoptotic mechanisms facilitating pathogen survival and escape from host immune defense. keywords: apoptosis, virus, mycobacterium tuberculosis, immune response *corresponding author email: timilsinau@gmail.com introduction apoptosis is a form of programmed cell death which is the most common form of physiological cell death in eukaryotes, evolutionarily conserved from yeast to humans. it leads to the genetically controlled sequence of events that eventually give rise to spatially and temporally regulated selfdestruction of cells [1,2]. apoptotic mode of cell death is an active process, critical in the development of multicellular organisms and the maintenance and regulation of cell populations during physiological and pathological conditions [3,4]. deregulation of apoptosis leads to various pathological conditions including cancer, autoimmune disorders, and spreading of viral infections while aids, neurodegenerative disorders, and ischemic diseases are caused or enhanced by accelerated apoptosis [3,5–8]. both viral and mycobacterium tuberculosis (mtb) infections modulate host cell apoptosis for their benefits [6,9–11]. this review briefly summarizes the mechanisms of apoptotic deaths and their regulation and significances in viral and mycobacterial infections. apoptosis various extracellular and intracellular stimuli trigger apoptosis. ligation of cell surface receptors, dna damage (because of defects in dna repair mechanism, cytotoxic drugs, or irradiation), lack of survival signals, contradictory cell cycle signaling or developmental death signals are some of the signals evoking apoptosis [1]. apoptosis depends on the activation of a proteolytic cascade of pro-caspases into active caspases. these caspases are synthesized in cells as inactive zymogens called as pro-caspases. procaspases are cleaved by pre active caspases at one or two specific aspartic acids splitting them into two subunits, one small and another large. the assembly of two heterodimers of small and large subunits results in the formation of active caspases. the pro-caspases fall into two classes initiator and executioner [12,13]. apoptotic stimuli trigger activation of initiator caspases (caspases 2, 8, 9, 10) which in turn cleave and activate the executioner caspases (caspases 3, 6, 7) [14]. the executioner caspases cleave thousands of substrates responsible for the characteristic morphological and biochemical features of apoptotic cells [14]. the three main established routes of apoptosis in mammals are extrinsic, intrinsic and perforin/granzyme pathways [2,15]. irrespective of the death stimuli or apoptotic paths, all the three routes lead to the activation of executioner caspases 3, 6 and 7 (figure 1). nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 46-57 giri and timilsina ©njb, biotechnology society of nepal 47 nepjol.info/index.php/njb figure 1. diagram showing general apoptosis process through three main pathways: extrinsic (death receptormediated viz. fasl, fas, trail-r1, and r2) pathway, intrinsic (mitochondria-dependent) pathway and perforin (granzyme)-mediated pathway. the extrinsic pathway starts with the binding of death receptor ligand (dr ligand) to the cell surface death receptors including tissue necrosis factor (tnf) receptor superfamily include cd95 and tnfrelated apoptosis-inducing ligand (trail)-r1/-r2, with the rapid activation of the initiator caspase 8. in the intrinsic pathway, stress (reactive oxygen species, ros, uv, genotoxic stress, etc.) results in the perturbation of mitochondria membrane permeability, release of the proteins such as cytochrome c from the inner mitochondrial membrane space. the release is regulated in part by bcl2 family members, with anti-apoptotic (bcl2/bcl—xl/mcl1) and pro-apoptotic (bax, bak, and tbid). once released, cytochrome c binds to apoptotic protease-activating factor 1 (apaf1), which results in the formation of the apaf1-caspase 9 apoptosome complex and activation of the initiator caspase 9. the activated initiator caspases 8 and 9 then activate the effector caspase 3, 6 and7 with normal cell apoptosis or another t-cell effector mechanism. cytotoxic t lymphocytes (ctl) or natural killer (nk) cells secrete the transmembrane poreforming molecule perforin and release cytoplasmic granules (granzyme a/b) into tumor cells or virus-infected cells. granzyme a activates dna degradation by dnase nm23-h1while granzyme b cleaves pro-caspase 8, pro-caspase 3 or bid. extrinsic pathway of apoptosis external apoptotic signaling mediates the activation of transmembrane death receptors that transmit apoptotic signals after binding to extracellular death ligands such as fasl or tumor necrosis factor-α (tnfα) [16]. death receptors belong to tumor necrosis factor receptor (tnfr) superfamily including tnfr-1, fas/cd95 and tnf receptor-related apoptosis-inducing ligand (trail) receptors dr-4 and dr-5 [17]. proteins of tnfr family result in trimerization and activation of intracellular death domain after ligand binding. adaptor proteins like fadd or tradd get recruited through their death domains to the death domains of activated death receptors forming death inducing silencing complex (disc). death effector domains of fadd or tradd recruit pro-caspase 8 leading to their autocatalytic activation and release of active caspase 8. activated caspase 8 then cleaves and activates downstream executioner caspases 3 and 7. in some cases, the extrinsic death signals can crosstalk with an intrinsic pathway through caspase 8-mediated proteolysis of the bh3-only protein bid. truncated bid can translocate to mitochondria and induce the release of cytochrome c and assembly of apoptosome triggering activation of pro-caspase 9 [18–20] (figure 1). nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 46-57 giri and timilsina ©njb, biotechnology society of nepal 48 nepjol.info/index.php/njb intrinsic pathway of apoptosis intracellular death signals such as dna damage, oxidative stress, starvation and others trigger intracellular apoptotic pathway. all of these stimuli cause changes in inner mitochondrial membrane resulting in the opening of the mitochondrial permeability transition (mpt) pore, loss of the mitochondrial transmembrane potential (δψm) and release of two groups of pro-apoptotic proteins from the intermembrane space into the cytosol [21]. the first group of released proteins constitutes cytochrome c, smac/diablo, and the serine protease htra2/omi that promotes caspase-dependent mitochondrial pathway [22– 24]. cytochrome c binds and activates apaf-1 (apoptosis protease activating factor 1) which hydrolyzes bound datp to dadp. replacement of dadp with datp/atp leads to apaf-1cytochrome c complex to oligomerize into a wheel like a heptamer called apoptosome. pro-caspase 9 gets recruited in the apoptosome through its caspase recruitment domain (card) [25] and gets activated and cleaved which then triggers activation of downstream executioner caspases (figure 1) [25]. smac/diablo and the serine protease htra2/omi, on the other hand promote apoptosis by inhibiting iap (inhibitors of apoptosis) proteins [23,24]. the second group of released proteins includes aif, endonuclease g, and cad which translocate to the nucleus and cause dna fragmentation and condensation of peripheral nuclear chromatin [26–28]. in addition to the release of mitochondrial factors, the loss of the δψm leads to regulation of biochemical homeostasis of the cell viz. atp synthesis gets stopped, redox molecules like nadh, nadph and glutathione are oxidized, and reactive oxygen species are enhanced [29–32]. perforin/ granzyme pathway of apoptosis cytotoxic t lymphocytes (ctl) or natural killer (nk) cells can exert their cytotoxic effects on tumor cells and virus-infected cells by secretion of the transmembrane pore-forming molecule perforin with the subsequent release of cytoplasmic granules through the pore into the target cell [33]. these granules constitute the serine proteases granzyme a and b. granzyme b can cleave proteins at aspartate residues and thus activate pro-caspase 8 and bid. direct activation of pro-caspase 3 and cleavage of icad could also be the results of granzyme b. thus granzyme b dependent routes of apoptosis may be mitochondrial or direct [26]. granzyme a activates caspase-independent apoptosis [34] (figure 1). inside the cell, it enables dna degradation by dnase nm23-h1. granzyme a cleaves set complex (nucleosome assembly protein that usually inhibits dnase nm23-h1 gene) thereby releasing the inhibition of dnase nm23-h1 leading to dna degradation [34]. regulation of apoptosis the components of apoptotic pathways are genetically encoded and ready for action. most cells are just waiting for the death stimuli to trigger these pathways. thus a tight regulation of apoptosis is mandatory. b-cell lymphoma-2 (bcl-2) family proteins play a crucial role in the regulation of apoptosis through their ability to control mitochondrial permeability [35]. bcl-2 family comprises three subfamilies that contain between one and four bcl-2 homology (bh) domains. antiapoptotic bcl-2 subfamily includes four bh domains, and most of them are membraneassociated proteins. the pro-apoptotic bax-like subfamily comprises membrane-associated proteins that lack bh4 domains, while the bh3only subfamily includes a diverse group of proteins containing only bh3 domains [36]. the mammalian bh3-only protein family currently consists of eight members (bid, bad, bim, bak, bik, noxa, puma, and hrk). among eight members, noxa, puma, and bid are transcriptionally upregulated by p53. bid is activated by caspase 8-dependent proteolysis. phosphorylated bad is trapped by 14-3-3 protein and sequestered in the cytoplasm. once bad is unphosphorylated, it gets freed and is translocated to mitochondria. bim and bmf are microtubules, and actin microfilaments tethered proteins and disruption of cytoskeleton liberates them [37,38]. the anti-apoptotic bcl-2 family of proteins (bcl-2, bcl-xl, bcl-w, mcl1, bcl2a1 and bcl-b) blocks apoptosis by preventing bh3-only nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 46-57 giri and timilsina ©njb, biotechnology society of nepal 49 nepjol.info/index.php/njb table 1: list of viral and mtb proteins involved in apoptosis deregulation viruses/mtb (proteins) modulation in apoptotic process references adenovirus proteins (e3-10.4k and e3-14.5k) reduce fas presentation, inhibit tnf-mediated apoptosis [42, 43] epstein-barr virus lmp-1 protein acts like constitutively activated tnf receptor [44] myxoma virus protein m-t2 viral mimic protein of tnf receptor [45] cowpox virus crm protein prevents tnf-mediated apoptosis [46] vaccinia virus protein a53r prevents tnf-mediated apoptosis [47] hiv-1 tat decreases susceptibility to trail, tnfα, and fas. upregulates fasl, bax, caspase 8 and rcas-1 expression, upregulates bcl2 and c-flip expression, downregulates caspase 10 expression [50,68-72] herpesviruses: flice inhibits disc formation [51] human cytomegalovirus: vica inhibits caspase 8 [52] sv40 virus large t antigen binds to and sequesters p53 [53] human papillomavirus e6 protein p53 ubiquitination and degradation [55] adenovirus e1b-55k protein p53 ubiquitination and degradation [56] adenovirus e1b-19k binds to bak preventing bax-bak oligomerization [56] human herpesviruses: bcl-2 ortholog blocks the mitochondrial release of cytochrome c [59] epstein-barr virus: bcl-2 ortholog blocks the mitochondrial release of cytochrome c [60] kaposi’s sarcoma-associated γ-herpes virus: bcl-2 ortholog blocks the mitochondrial release of cytochrome c [59] human cmv protein: vmia inhibits fas-mediated apoptosis [61,62] poxviruses serpin crma suppresses caspase 1 and 8, inhibits tnf and fas-mediated apoptosis [63] baculovirus protein p35 inhibits caspases 1, 3, 6, 7, 8 and 10 [64,65] african swine flu virus: viap inhibits caspase 3 [66] hiv-1 gp120 syncytia formation, upregulates fas, fasl, and tnfα expression, upregulates trail receptors: dr4 and dr5, acts as a molecular mimic of fas, reduces expression of bcl2, phosphorylates mtor and p53, increases expression of puma and activates p38 [48,67] hiv-1 nef increases the membrane expression of tnf [49] hepatitis b virus px protein inactivates p53 [57] west nile capsid protein binds to and sequesters p53 [54] arenaviruses matrix protein z activation of bh3-only proteins?, an indirect interaction with p53 and pi3k/akt with the help of pml? [74-78] enterovirus 71 2b protein direct interaction with and activation of bax [80,81] mtb proteins (mcl-1, a1?) upregulates tnf, fas, and caspase 8 expression, stimulates ros-dependent activation of apoptosis signal-regulating kinase, phosphorylates and degrades flip, mompmediated apoptosis, upregulates flip expression, secretes more stnfr2, increases expression of anti-apoptotic protein bcl-w, inhibition of the pro-apoptotic protein bad [101,104106, 113115] ? refers to mechanism not yet verified. nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 46-57 giri and timilsina ©njb, biotechnology society of nepal 50 nepjol.info/index.php/njb protein induced oligomerization of the proapoptotic bcl-2 proteins bax and bak in the mitochondrial outer membrane. some bh3-only proteins (bid and bim) interact with almost all anti-apoptotic bcl-2 proteins whereas others (noxa) interact only with specific bcl-2 members [35,37,38]. in conclusion, under distinct apoptotic stress signals, bh3-only proteins interfere the fine-tuned balance of homo or hetero-oligomerization between pro-apoptotic members bax/bak and anti-apoptotic members bcl-2/bcl-xl and release the intermembrane space proteins like cytochrome c to trigger apoptosis.iaps (inhibitors of apoptosis proteins) are a family of proteins having antiapoptotic activity [32]. including naip, c-iap1, ciap2, xiap, and survivin, there are eight human iap homologs. xiap, c-iap1, and c-iap2 directly inhibit caspases 3, 7, and 9. smac/diablo, when released from mitochondria, binds to xiap and releases caspases from xiap-caspase complex thereby enabling their activation [39,40]. the cellular flice-like inhibitory proteins (c-flips) inhibit activation of caspase 8 and thus prevent apoptosis [34]. virus-mediated modulation of apoptosis in most cases of viral infections, immune and inflammatory responses, as well as apoptosis of the infected host cell, are triggered. meanwhile, some viruses utilize apoptosis as a mechanism of killing cells and spreading virus by targeting a variety of crucial steps in the pathways that block or delay apoptosis. thus viral infection elicits host cell apoptosis as a part of host immune defense or viral survival component [41]. virus modulates the extrinsic pathway of apoptosis many viruses can efficiently modulate the extrinsic pathway of apoptosis. adenovirus proteins e3-10.4k and e3-14.5k reduce the presentation of fas molecules on the surface of the cells that results in resistance to fas-mediated cell death [42]. these proteins also resist tnfmediated apoptosis [43]. epstein-barr virus lmp-1 (latent membrane-1) protein acts like constitutively activated tnf receptor which interacts with tnf receptor-associated death domain (tradd) protein [44]. the myxoma virus protein m-t2, a viral mimic protein of tnf receptor, cowpox virus cytokine response modifying (crm) proteins and vaccinia virus protein a53r inhibits tnf-mediated apoptosis [45, 46, 47]. membrane-bound hiv-1 gp120 induces apoptosis through syncytia formation while it triggers apoptosis by various mechanisms like upregulation of fas, fasl, and tnfα expression, upregulation of trail receptors dr4 and dr5, and acting as a molecular mimic of fas [48]. hiv-1 nef protein downregulates the expression of cd4 and mhc i molecules but heightens the membrane expression of tnf and related cytokines [49]. hiv-1 tat mediates apoptotic resistance in the infected cells by decreasing susceptibility to trail, tnfα, and fas, but it reconciles apoptosis in uninfected bystander cells by upregulation of fasl [50]. various herpes viruses encode viral flice-like inhibitory proteins (flips), which contain death effector domain but lack caspase activity, inhibit extrinsic apoptotic pathway at the point of disc formation [51]. the human cytomegalovirus encodes vica, which associates with caspase 8 and blocks its activation [52] (table 1). virus modulates the intrinsic pathway of apoptosis many viruses alter apoptosis utilizing the tumor suppressor p53. sv40 virus large t antigen and west nile capsid protein binds to p53 and sequesters it in an inactive complex [53,54]. moreover, human papillomavirus e6 protein and adenovirus e1b-55k protein promote ubiquitin mediated degradation of p53 [55,56] and hepatitis b virus px protein binds and inactivates p53 [57]. virus-encoded orthologs of anti-apoptotic bcl2 proteins are also crucial players in the modulation of apoptosis. adenovirus e1b-19k is similar to bcl2 which binds to bak preventing bax-bak oligomerization [58]. human herpes viruses, epstein-barr virus and kaposi’s sarcomanepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 46-57 giri and timilsina ©njb, biotechnology society of nepal 51 nepjol.info/index.php/njb associated γ-herpes virus use bcl-2 orthologs to block the mitochondrial release of cytochrome c [59,60]. although human cytomegalovirus (cmv) protein vmia shares no sequence homology to bcl2, it is functionally similar to bcl-2 and inhibits fas-mediated apoptosis [61,62]. some viruses use iap orthologs that can inhibit caspases. for example, poxviruses serpin crma suppresses caspase 1 and 8 and inhibits tnf and fasmediated apoptosis [63]. likewise, african swine flu virus produces viap that inhibits caspase 3 and baculovirus protein p35 is another viap with a potential to inhibit caspases 1, 3, 6, 7, 8 and 10 [64,65,66]. hiv-1 gp120 triggers apoptosis by reduced expression of bcl2, phosphorylation of mtor and p53, increased expression of proapoptotic protein puma and activation of p38 [67]. hiv-1 tat inhibits apoptosis in infected cells by upregulation of bcl2 and c-flip expression [68,69] and downregulation of caspase 10 expression [70]. the same protein triggers apoptosis in bystander cells by upregulation of bax, caspase 8 and rcas-1 expression [71,72], and bim-mediated intrinsic apoptosis [73]. matrix protein z of some arenaviruses (new world arenavirus, tacarible virus (tcrv), and the attenuated vaccine strain of junín virus (junv) candid #1) activates caspase 9 thereby triggering the intrinsic apoptotic pathway [74,75]. though the exact molecular mechanism of viral protein zmediated apoptosis is still not clear, in vitro experiments suggest a direct activation of bh3only proteins and an indirect interaction with proteins like p53 and pi3k/akt through cellular oncoprotein promyelocyte leukemia protein (pml) [74–76]. the old world arenaviruses, the lymphocytic choriomeningitis virus (lcmb) and lassa virus (lasv) do not cause apoptosis of infected cells [77,78]. caspase-mediated cleavage of nucleoproteins (nps) of old world areanviruses generates multiple truncated isoforms of nps [74,79]. a decoy function of nps has been proposed in which the cleavage of highly expressed nps within the cell suppresses the cellular targets of caspases thereby inhibiting the apoptosis of the infected cell [74]. enterovirus 71 2b protein directly interacts with and activates the proapoptotic protein bax leading to the activation of mitochondrial pathway of apoptosis [80,81] (table 1). mycobacterium-mediated modulation of apoptosis bacterial pathogens are known to have antiapoptotic mechanisms. mycobacterium tuberculosis (mtb) causes persistent infection indicating that it employs effective mechanisms to inhibit host cell death [82]. published studies highlight both proapoptotic as well as anti-apoptotic capabilities of virulent mtb [83,84], however the underlining molecular mechanisms are still not well understood. though there is a lack of published data favoring mtb-mediated apoptosis of host cells, increased apoptosis of primary human macrophages or human macrophage-like cell lines (u937 and thp1) were reported upon infection with virulent mtb in vitro [85–87]. human alveolar macrophage-derived from bronchoalveolar lavage of tuberculosis patients also showed increased apoptotic death compared to healthy subjects [88,89]. apoptosis of mtb infected cells accompanied by the recruitment of uninfected macrophages through upregulation of mmp9 on epithelial cells surrounding the granuloma helps in the dissemination of the bacteria [90]. in the studies involving the zebrafish and mouse lung models, the pro-apoptotic nuog mtb mutant induced enhance innate response, longer survival and rapid dissemination of the bacteria [91,92]. thus, evidence suggests that host cell apoptosis is crucial for host resistance to mtb infection. considerable less apoptosis of human alveolar macrophage or macrophage-like cell lines when infected with virulent mtb compared to infection with less virulent strains was reported [93–96]. furthermore, fact that inhibition of apoptosis of human and murine macrophages by apoptosisinducing species m. kansaii after over-expression of mtb-nuog/seca2/pkne [97–99] and resistance to fasl and tnfα-mediated apoptosis of mtb nepal journal of biotechnology. d e c . 2 0 1 7 vol. 5, no. 1: 46-57 giri and timilsina ©njb, biotechnology society of nepal 52 nepjol.info/index.php/njb infected cells provide the evidence that mtb inhibits host cell apoptosis [100]. mtb modulates the extrinsic pathway of apoptosis gene expression profiling study suggests that numerous apoptosis-related genes are downregulated in active tuberculosis patients compared to latently infected subjects. though expressions of tnf, fas, and caspase 8 upregulate in active tuberculosis patients, simultaneous marked expression of flip, inhibits host cell apoptosis [101]. mtb infected macrophages are known to secrete more soluble tnf receptor 2 (stnfr2) which binds to tnfα thereby inhibiting its binding with the tnfr1 [100,102–104]. upon infection with mtb, tnf production in the mouse cell line raw264 stimulates ros-dependent activation of apoptosis signal-regulating kinase thereby phosphorylating flip [105]. ubiquitinproteasome-mediated degradation of phosphorylated flip activates caspase 8 leading to apoptosis [105] (table 1). mtb modulates the intrinsic pathway of apoptosis mtb infection upregulates the expression of antiapoptotic genes like mcl-1 and a1, both of which encodes for anti-apoptotic bcl-2-like proteins [106– 110]. alternative splicing gives rise to two isoforms of myloid cell leukemia-1 (mcl-1) protein. one is the anti-apoptotic full length mcl1l that possesses bh domains 1, 2 and 3 and a transmembrane domain. another is the proapoptotic short variant mcl-1s that lacks bh1, bh2 and the transmembrane domain. mcl-1s dimerizes with and antagonizes the function of mcl-1l thereby regulates the mitochondrial permeability [109,111]. furthermore, chemical inhibition of mcl1 in mouse peritoneal macrophages infected with mtb significantly triggered apoptosis [112]. it will be interesting to dissect the role of both isoforms of mcl-1 in mtb-mediated apoptosis evasion. also, the expression of anti-apoptotic protein bcl-w gets upregulated [113], while the inactivation of the pro-apoptotic bad protein occurs upon mtbh37rv infection [114]. infection of macrophages with attenuated mtb leads to momp-mediated apoptosis without mpt induction [10,115] (table 1). in contrast, macrophages infected with virulent mtb induce both momp and mpt causing irreversible mitochondrial swelling leading to necrosis [115]. conclusion programmed cell death via apoptosis is crucial in maintaining cells in health and pathological conditions. both viral and mtb infections modulate the apoptotic pathways of infected as well as neighboring bystander cells. though the majority of virally infected cells undergo apoptosis favoring viral dissemination, viral proteins help specific host cells to evade apoptosis thereby preferring viral persistence. mtb infection prominently evades host cell apoptosis leading to the persistent survival of the pathogen. understanding the molecular mechanisms of deregulation of apoptosis in viral and mtb infection may provide insights into revealing new targets for curing these pathological conditions. conflict of 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af‐10 20101228 opensource, new update from 28. december 2010  correspondence author:   klaus.ammann@ips.unibe.ch      peer reviewed contribution available on the following websites:  public research initiative: www.pubresreg.org   european federation of biotechnology: http://www.efb‐central.org/  s. no.  title  page no.  1.0.  issue  31  2.0.  summary  31  3.0.  differences between gm‐ and non‐gm‐crops overestimated  32  3.1.  early phase of risk assessment: discovery of the dynamics of dna processes.  32  3.2.  molecular processes similar in natural mutation and transgenesis  32  3.3.  more recent publications about genomic  comparisons of gm‐ and non‐gm crops  33  4.0.  natural genetically modified plants, dna as a highly dynamic system  36  4.1.  dramatic rearrangement of r gene loci: this class of genes diversifies more rapidly than  other genes in the crops studies  36  4.2.  jumping genes: their dynamics falsify the erroneous picture of regulators that dna is a  stable string of genes  38  4.3.  helitrons contribute to the lack of gene colinearity observed in modern maize inbreds  39  4.4.  polyploids, alloploids in flowering plants  39  4.5.  horizontal geneflow between pro‐caryotes and eu‐caryotes  40  5.0.  some conventional breeding causes lots of genomic alteration  40  6.0.  regulatory dissent over molecular differences causes transatlantic divide  41  6.1.  perspectives for a dissolution of this divide  41  8.0.  cited literature  42  7.0.  conclusion  42  1.0. issue  the  difference  between  gm‐  and  non‐gm‐crops  has  been  overestimated,  as  soon  as  genetic  engineering  has  been  applied  to  crop  breeding.  the  uncontested  understanding among scientists and in particular in risk  assessment community was that gm crops pose some  novel  risks,  unprecedented  in  conventionally  bred  crops. this has then condensed  in the united nations  cartagena  protocol  on  biosafety1,  which  needs  to  be  questioned in certain basic aspects.  2.0. summary  after an early phase of risk assessment,  including the  results  of  the  asilomar  conference  on  biosafety,  an  early  divide  in  risk  assessment  basic  concepts  developed  between  canada,  the  usa  and  europe  including  a  majority  of  un  signatory  countries.  researchers  like  werner  arber,  based  on  earlier  molecular  insights  and  on  his  own  experience  in  genetic  engineering  claim  that  related  to  molecular  processes  there  is  no  difference  between  genetically  engineering  and  natural  mutation.  this  transatlantic  1 cartagena protocol on biosafety: http://www.cbd.int/biosafety/   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  32  biotechnology society of nepal (bsn), all rights reserved   divide  can  be  solved  with  some  more  innovative  regulatory proceedings.   3.0. differences between gm‐ and  non‐gm‐crops overestimated  3.1. early phase of risk assessment:  discovery of the dynamics of dna processes.  in  the  wake  of  molecular  breeding,  in  particular  with  the  first  successes  of  “gene  splicing”,  the  safety  debates  started  soon  after  the  discovery  of  the  dna  structure by watson & crick (watson & crick, 1953a, b;  wilkins  et  al.,  1953),  followed  by  the  asilomar  conference  (berg  et  al.,  1975;  berg  &  singer,  1995)  ‐  see  also  some  historical  accounts  (chassy,  2007;  friedberg, 2007; klug, 2004). the fascination about the  novelty  of  transgenesis  was  justified,  but  also  overwhelming,  and  the  many  unforeseen  scientific  breakthroughs  following  were  unprecedented  in  the  history  of  molecular  biology.  unfortunately,  the  enthusiasm  also  lashed  back  in  an  overacting  in  risk  assessment,  when  the  first  gm  crops  went  into  production.  the  debate  on  how  gm  crops  should  be  regulated, started very early with an emerging divide  between  regulation  in  the  us  and  great  britain,  including  later  the  whole  of  europe  (bennett  et  al.,  1986; national‐research‐council, 1989).  the seemingly absolute novelty of genetic engineering  on the molecular  level has been contested already  in  the  early  days  of  molecular  biology  in  the  1930s  and  1950s  with  the  discovery  of  cellular  systems  for  genome  restructuring  discovered  with  the  classic  papers  of  mcclintock  (mcclintock,  1930,  1953),  more  details about jumping genes see chapter 4.2.  3.2.  molecular  processes  similar  in  natural  mutation and transgenesis  the  concept  of  violated  intrinsic  naturalness  of  the  genomes is still erroneously maintained by proponents  of  organic  farmers  (van  bueren  et  al.,  2008;  van  bueren & struik, 2004, 2005; van bueren et al., 2003).  this  concept of  singling  out  transgenity  is  falsified by  the publications of arber (nobel laureate 1978).   genetic  engineering  has  been  brought  into  evolutionary  perspective  of  natural  mutation  by  authorities  such  as  werner  arber:  his  view  remains  scientifically  uncontested  that  molecular  processes  in  transgenesis and natural mutation are basically similar  (arber,  1994,  2000,  2002,  2003,  2004;  arber,  2010).  the same claim is made with a more organismal view  by hackett (hackett, 2002).  arber  compared  designed  genetic  alterations  (including  genetic  engineering)  with  the  spontaneous  genetic  variation  known  to  form  the  substrate  for  biological evolution (arber, 2002):  “site‐directed  mutagenesis  usually  affects  only  a  few  nucleotides.  still  another  genetic  variation  sometimes  produced by genetic engineering is the reshuffling of genomic  sequences,  e.g.  if  a  given  open  reading  frame  is  brought  under a different signal for expression control or if a gene is  knocked out. all such changes have little chance to change in  fundamental  ways,  the  properties  of  the  organism.  in  addition,  it  should  be  remembered  that  the  methods  of  molecular  genetics  themselves  enable  the  researchers  anytime  to  verify  whether  the effective  genomic  alterations  correspond to their intentions, and to explore the phenotypic  changes  due  to  the  alterations.  this  forms  part  of  the  experimental  procedures  of  any  research  seriously  carried  out. interestingly, naturally occurring molecular evolution, i.e.  the  spontaneous  generation  of  genetic  variants  has  been  seen to follow exactly the same three strategies as those used  in genetic engineering. these three strategies are:   (a)  small local changes in the nucleotide sequences,   (b)  internal reshuffling of genomic dna segments, and  (c)  acquisition of usually rather small segments of dna from  another type of organism by horizontal gene transfer.  however,  there  is  a  principal  difference  between  the  procedures of genetic engineering and those serving in nature  for  biological  evolution.  while  the  genetic  engineer  pre‐ reflects his alteration and verifies its results, nature places its  genetic variations more randomly and largely independent of  an identified goal. under natural conditions, it is the pressure  of  natural  selection  which  eventually  determines,  together  with the available diversity of genetic variants, the direction  taken  by  evolution.    it  is  interesting  to  note  that  natural  selection  also  plays  its  decisive  role  in  genetic  engineering,  since  indeed  not  all  pre‐reflected  sequence  alterations  withstand the power of natural selection. many investigators  have experienced the effect of this natural force which does  not allow functional disharmony in a mutated organism.”  arbers  numerous  writings  (arber,  2000,  2003,  2004)  confirm  this  important  comparison  on  the  genomic  level  of  evolutionary  and  modern  plant  breeding  processes.  but  there  is  of  course,  despite  all  the  similarities,  one  major  difference:  whereas  natural  mutation acts completely in a natural time scale, that  is,  the  mutants  will  need  hundreds  to  hundred  of  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  33  biotechnology society of nepal (bsn), all rights reserved   thousands of years to overcome selective processes in  nature until they really succeed and take over against  their natural competitors, this  is totally different with  the transgenic crop products: they run through a r&d  phase, and a regulatory process of an average of 15 to  20  years  until  being  completely  deregulated.  but  somewhere along this process they will be propagated  to the millions  in the field, covering  in a evolutionary  extremely short time span millions of hectares.  in  a  recent  paper,  (coll  et  al.,  2009)  come  to  the  conclusion, that gene expression profiles of mon810  and  comparable  non‐gm  maize  varieties  cultured  in  the  field  are  more  similar  than  are  those  of  conventional  lines.  their  bibliography  supports  this  view with numerous peer reviewed publications.    it  is  therefore  no  surprise  that  a  natural  transgene  species  has  been  discovered  in  a  widespread  grass  genus (ghatnekar et al., 2006).  (miller  &  conko,  2004)  provide  important  arguments  supporting this view:   the authors raise also in a justified way doubts about  the  commonly  used  concept  of  transgenesis.  in  the  light of pre‐recombinant dna produced in great variety  by  conventional  breeding  with  thousands  of  foreign  genes.  “in  these  examples  of  prerecombinant‐dna  genetic  improvement,  breeders  and  food  producers  possess  little  knowledge  of  the  exact  genetic  changes  that  produced  the  useful  trait,  information  about  what  other  changes  have  occurred concomitantly in the plant or data on the transfer of  newly  incorporated  genes  into  animals,  humans  or  microorganisms.  consider,  for  example,  the  relatively  new  man‐made wheat  'species' triticum agropyrotriticum, which  resulted from the wide‐cross combination of the genomes of  bread wheat and a wild grass sometimes called quackgrass or  couchgrass    (banks  et  al.,  1993;  sinigovets,  1987)  t.  agropyrotriticum,  which  possesses  all  the  chromosomes  of  wheat as well as the entire genome of the quackgrass, was  independently  produced  for  both  animal  feed  and  human  food  in the former soviet union, canada, the united states,  france,  germany  and  china.”  see  also  the  ask‐force  contributions on the web by k. ammann 20092 on the same  subject.    3.3. more recent publications about genomic   comparisons of gm­ and non­gm crops  recent  publications  demonstrate,  that  transgenesis  e.g. has less impact on the transcriptome of the wheat  grain  than  traditional  breeding  (batista  et  al.,  2008;  baudo et al., 2006; shewry et al., 2007), (more details:  (ammann, 2008, 2009)).  two  figures  may  to  visualize  the  lower  impact  on  transcriptome  expression  of  transgenic  crops  compared to conventional ones:  volcano plots from (batista et al., 2008): in all observed  cases of the comparison between transgenic and non‐ transgenic  crops  the  observed  alteration  was  more  extensive  in  the  mutagenized  than  in  the  transgenic  plants:  “controversy regarding genetically modified (gm) plants and  their  potential  impact  on  human  health  contrasts  with  the  tacit acceptance of other plants that were also modified, but  not considered as gm products (e.g., varieties raised through  conventional breeding such as mutagenesis). what is beyond  the phenotype of these improved plants? should mutagenized  plants  be  treated  differently  from  transgenics?  we  have  evaluated the extent of transcriptome modification occurring  during  rice  improvement  through  transgenesis  versus  mutation  breeding.  we  used  oligonucleotide  microarrays  to  analyze gene expression in four different pools of four types  of rice plants and respective controls: (i) a gamma‐irradiated  stable  mutant,  (ii)  the  m1  generation  of  a  100‐gy  gamma‐ irradiated  plant,  (iii)  a  stable  transgenic  plant  obtained  for  production  of  an  anticancer  antibody,  and  (iv)  the  t1  generation of a transgenic plant produced aiming for abiotic  stress  improvement,  and  all  of  the  unmodified  original  genotypes as controls. we found that the improvement of a  plant variety through the acquisition of a new desired trait,  using  either  mutagenesis  or  transgenesis,  may  cause  stress  and thus lead to an altered expression of untargeted genes.  in all of the cases studied, the observed alteration was more  extensive  in  mutagenized  than  in  transgenic  plants.  we  propose  that  the  safety  assessment  of  improved  plant  varieties  should  be  carried  out  on  a  case‐by‐case  basis  and  not  simply  restricted  to  foods  obtained  through  genetic  engineering.” (batista et al., 2008)  plots  from  (baudo  et  al.,  2006)  are  also  clearly  demonstrating,  that  transcriptome  comparisons  between  transgenic  and  non‐transgenic  comparable  traits show substantial equivalence.  “detailed global gene expression profiles have been obtained  for a series of transgenic and conventionally bred wheat lines  expressing additional genes encoding hmw (high molecular  weight) subunits of glutenin, a  group of endosperm‐specific  seed  storage  proteins  known  to  determine  dough  strength  and  therefore  bread‐making  quality.    differences  in  endosperm  and  leaf  transcriptome  profiles  between  2ask‐force contribution k. ammann 2009: regulation: misconcepts  cause high costs and huge delays in regulation of gm crops: http:// www.efb‐central.org/index.php/forums/viewthread/59/   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  34  biotechnology society of nepal (bsn), all rights reserved   untransformed and derived transgenic lines were consistently  extremely  small,  when  analysing  plants  containing  either  transgenes only, or also marker genes. differences observed  in gene expression in the endosperm between conventionally  bred material were much larger in comparison to differences  between  transgenic  and  untransformed  lines  exhibiting  the  same complements of gluten subunits. these results suggest  that the presence of the transgenes did not significantly alter  gene  expression  and  that,  at  this  level  of  investigation,  transgenic  plants  could  be  considered  substantially  equivalent  to  untransformed  parental  lines.”  (baudo  et  al.,  2006)  in another recent paper on transcriptomic comparison,  (kogel  et  al.,  2010)  come  to  the  following  similar  conclusions (see also the figures):  “in  summary,  our  results  substantially  extend  observations  that  cultivar‐specific  differences  in  transcriptome  and  metabolome  greatly  exceed  effects  caused  by  transgene  expression.  furthermore,  we  provide  evidence  that,  (i)  the  impact of a low number of alleles on the global transcript and  metabolite profile is stronger than transgene expression and  that, more specifically, (ii) breeding for better adaptation and  higher  yields  has  coordinately  selected  for  improved  resistance to background levels of root and leaf diseases, and  this  selection  appears  to  have  an  extensive  effect  on  substantial  equivalence  in  the  field  during  latent  pathogen  challenge.” (kogel et al., 2010)  in  another  recent  paper,  dealing  with  biosynthetic  comparison between tubers and leaves of potato traits  (ferreira  et  al.,  2010),  the  authors  come  again  to  similar  conclusions,  as  expressed  in  an  interview  of  gmo safety of the senior author3   in http://www.gmo‐ safety.eu/en/news/741.docu.html  :  “the  impact  of  transgenes  is  basically  limited  to  their  immediate  function” . and further on:   impact  of  transgenes  is  basically  limited  to  their  immediate function” . and further on:   “gmo  safety:  the  following  statement  was  deduced  from  your findings: conventional breeding causes more changes in  plants than the  introduction of a single transgene. can you  make  such  a  generalization?  after  all,  you  only  looked  at  barley.  have  comparable  studies  been  carried  out  on  other  genetically modified crops?   uwe  sonnewald:  as  far  as  i  know,  this  was  the  first  time  that  both  methods had been used in a simultaneous investigation. researchers have  studied either gene expression or plant substances in wheat, potatoes and  maize and have come to very similar conclusions. the impact of transgenes  is basically  limited to their  immediate function. for example,  if  i  insert a  gene for fructan biosynthesis in potatoes, it is hardly surprising that these  potatoes then produce fructan and so differ in this way from their parent  fig. 1  volcano plots for differentially expressed genes. differentially expressed genes appear above the thick horizontal lines. genes induced _2‐ fold are on the right of the right vertical lines, and the ones repressed _2‐fold are on the left of the left vertical line. the numbers corresponding  to the differentially expressed genes induced _2‐fold for each experiment (red‐shadowed area) are red, and those corresponding to the genes  repressed _2‐fold (blue‐shadowed area) are blue. the green‐shadowed area corresponds to differentially expressed genes that were up‐ or down‐ regulated _2‐fold (green‐colored numbers). blue‐colored genes are those with p between 0 and 0.5, and red‐colored genes are those with p  between 0.5 and 1. from (batista et al., 2008)  3see http://www.gmo‐safety.eu/en/news/741.docu.html   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  35  biotechnology society of nepal (bsn), all rights reserved   lines. but only negligible additional differences were found.  i know of no  instance  where  a  more  significant  change  in  gene  expression  has  been  caused  by  a  single  transgene.  however,  great  variability  exists  between  individual varieties of all the crops mentioned and the obvious explanation  for  this  is  that  often  the  breeding  objective  is  to  create  resistance  to  external stress factors, and this involves a large number of genes.”   again  the  same  conclusions  are  drawn  by  another  comprehensive paper of a large international collective  of authors (barros et al., 2010):   “the aim of this study was to evaluate the use of four non‐ targeted  analytical  methodologies  in  the  detection  of  unintended  effects  that  could  be  derived  during  genetic  manipulation of crops. three profiling technologies were used  to compare the transcriptome, proteome and metabolome of  two transgenic maize lines with the respective control line. by  comparing the profiles of the two transgenic  lines grown  in  the  same  location  over  three  growing  seasons,  we  could  determine  the  extent  of  environmental  variation,  while  the  comparison  with  the  control  maize  line  allowed  the  investigation  of  effects  caused  by  a  difference  in  genotype.  the  effect  of  growing  conditions  as  an  additional  environmental effect was also evaluated by comparing the bt ‐maize  line with the control  line from plants grown  in three  different  locations  in one growing season.  the  environment  was shown to play an  important effect  in the protein, gene  expression and metabolite levels of the maize samples tested  where 5 proteins, 65 genes and 15 metabolites were found to  be differentially expressed. a distinct separation between the  three  growing  seasons  was  also  found  for  all  the  samples  grown in one location. together, these environmental factors  caused  more  variation  in  the  different  transcript  ⁄ protein  ⁄ metabolite profiles than the different genotypes.” (barros et  al., 2010).  figure  2b  demonstrates  no  evident  differences  between gm – and non‐gm maize:  interestingly  enough, the  parallel  short  report  on  the  website of usda (www.isb.vt.edu) was first published  fig. 2 scatter plot representation of transcriptome comparisons of: (a) transgenic b102‐1‐1 line vs. control l88‐31 line in endosperm at 14 dpa  (left), 28 dpa (middle) or leaf at 8 dpg (right); (b) conventionally bred l88‐18 vs. l88‐31 line in endosperm at 14 dpa (left), 28 dpa (middle), or leaf  at 8 dpg (right); (c) transgenic b102‐1‐1 line vs. conventionally bred l88‐18 line in endosperm at 14 dpa (left), 28 dpa (middle), or leaf at 8 dpg  (right). dots represent the normalized relative expression level of each arrayed gene for the transcriptome comparisons described. dots in black  represent statistically significant, differentially expressed genes (deg) at an arbitrary cut off > 1.5. the inner line on each graph represents no  change in expression. the offset dashed lines are set at a relative expression cut‐off of twofold. in the adjacent colored bar (rectangle on the far  right of the figure), the vertical axis represents relative gene expression  levels: reds  indicate overexpression, yellows average expression, and  greens under‐expression. values are expressed as n‐fold changes. the horizontal axis of this bar represents the degree to which data can be  trusted: dark or unsaturated color represents low trust and bright or saturated color represents high trust. from (baudo et al., 2006).  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  36  biotechnology society of nepal (bsn), all rights reserved   (without  notifying  the  authors)  under  a  clearly  misleading  headline    “molecular  profiling  techniques  detect  unintended  effects  in  genetically  engineered  maize”, it was subsequently corrected on intervention  by  the  authors  to  the  original  headline  given  in  the  manuscript:  “molecular  profiling  techniques  as  tools  to  detect  potential  unintended  effects  in  genetically  engineered maize” (barros, 2010).   based on the extensive review of (wilson et al., 2006),  transgenesis results into deletions and insertions in the  genome of considerable size, just as radiation mutation  breeding  can  cause:  (meza  et  al.,  2002)  show  in  genetically transformed plants:   “transgene  silencing  has  been  correlated  with  multiple  and  complex  insertions  of  foreign  dna,  e.g.  t‐dna  and  vector  backbone  sequences.  no  striking  differences  were  seen  between the ts and c lines. the majority of the deletions are  <75 bp, with an average of 36 bp. the smallest deletion was 1  bp. in four cases, deletions of >100 bp were found, the largest  of 1537 bp.  normally, the deletion represented a continuous  stretch of genomic dna (fig. 2a and table 2). a somewhat  more complex pattern was observed  in only one  line (ex2±4  line 8), where a deletion of 35 bp at the integration site was  followed  by  60  bp  of  genomic  dna  preceding  a  second  deletion of 825 bp.” (meza et al., 2002).  it is one of the most frequent misunderstandings, that  transgenesis  causes  more  genomic  disturbance  than  conventional  breeding.  it  is  a  very  frequently  encountered  fundamental  mistakes  of  many  risk  assessment  papers  related  to  gmos:  they  lack  the  baseline  comparison  –  which  in  the  case  of  environmental  risk  assessment  should  also  comprise  the  important elements of agricultural practice. here,  in  chapter  3.2.  and  3.3.  we  demand  a  scientifically  founded  baseline  comparison  between  the  various  breeding methods.  4.0.  natural  genetically  modified  plants,  dna  as  a  highly  dynamic  system  as  a  preface  to  this  chapter,  one  should  realize  the  fantastic  variability  of  cultivars,  here  demonstrated  with  an  illustration  from  (parrott,  2010)  about  the  already ancient colorful maize landraces (fig. 4).  it is also ironic and a clear confirmation of green myths,  that  one  of  the  genetically  most  altered  plants,  the  sunflower,  to find  it  as  a  symbol  of  naturalness  for  a  major political party in germany (fig. 5).  it is also worthwhile to visit the site of david tribe with  gmo  pundit,  he  offers  an  extensive  site  on  genomic  comparison  between  gmos  and  non‐gmos,  with  an  impressive  collection  of  “natural  transgenic  plants”4.  see  in  particular  the  series  of  links  under  natural  gmos, parts 1 to 12 and 13 to 26.  some of the arguments used by david tribe are taken  up  here  and  enriched  with  more  arguments  and  references:  4.1. dramatic rearrangement of r gene loci:  this class of genes diversifies more rapidly  than other genes in the crops studies  one of the major sources of genetic variability (clearly  an  evolutionary  necessity)  is  described  by  (leister,  2005)  on  the  origin,  evolution  and  genetic  effects  of  nuclear  insertions of organelle dna,  illustrated  in the  fig. 5.   in box 1, (leister, 2005) describes in detail the various  possibilities  of  gene  flow  and  reasons  for  genomic  change:  “box  1.  dna  flow  between  different  genetic  compartments  six types of dna transfer are conceivable between the three  fig. 3  pca score plots of maize grown at petit over three consecutive  years.  separation  between  the  non‐gm  and  gm  varieties  for  (a)  microarray  data,  (b)  proteomics  data,  (c)  1h‐nmr  spectra,  (d)  gas  chromatographic ⁄ mass spectrometric (gc ⁄ ms) metabolite profiles.  from (barros et al., 2010).  4david tribe’s blogspot on natural gmos: http:// gmopundit2.blogspot.com/2005/12/collected‐links‐to‐scientific.html   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  37  biotechnology society of nepal (bsn), all rights reserved   dna‐containing  organelles:  nucleus,  plastid  and  mitochondrion.    in  ptdnas,  no  sequence  of  nuclear  or  mitochondrial  origin  has  yet  been  detected,  indicating  that  nucleus‐to‐plastid or mitochondrion‐to‐plastid transfer occurs  extremely  rarely  or  not  at  all.  during  the  early  phase  of  organelle  evolution,  organelle‐to‐nucleus  dna  transfer  (designated in figure i as ‘a’) resulted in a massive relocation  of functional genes to the nucleus: in yeast, as many as 75%  of  all  nuclear  genes  could  derive  from  proto‐mitochondria  [62], whereas w4500 genes in the nucleus of arabidopsis are  of  plastid  descent  [63].  cases  of  present‐day  organelle‐to‐ nucleus  dna  transfer,  revealed  by  the  presence  of  numts  and nupts, are known in most species studied so far.  among  the few eukaryotic organisms in which norgdna has not been  detected are the malaria mosquito (anopheles gambiae) and  the  honeybee  (apis  mellifera).  mitochondrial  chromosomes  contain  segments  homologous  to  chloroplast  sequences,  as  well  as  sequences  of  nuclear  origin,  providing  indirect  evidence  for  plastid‐to‐mitochondrion  and  nucleus‐to‐ mitochondrion transfer of dna (figure i: ‘b’ and ‘c’). thus, a  few  percent  of the  mtdna  of  flowering  plants  derives  from  ptdna,  whereas  retrotransposons  seem  to  be  the  major  source  of  nucleus‐derived  mtdna.    interestingly,  although  plastid‐to‐mitochondrion  and  nucleus‐tomitochondrion  dna  transfer have been detected in almost all plant mitochondrial  chromosomes sequenced so far [64,65], there is no evidence  for the incorporation of ndna into the mitochondrial genome  of maize [66].”  conclusions of an earlier paper of (leister et al., 1998):  “our data suggest a dramatic rearrangement of r gene loci  between  related  species  and  implies  a  different  mechanism  for  nucleotide  binding  site  plus  leucine‐rich  repeat  gene  evolution compared with the rest of the monocot genome”  and further on in the same paper:  “here we describe the isolation and characterization of nbs‐ fig. 4   maize from the guatemalan highlands, showing that cross pollination takes place naturally between the  landraces. photos courtesy of  eduardo roesch, from (parrott, 2010).  fig.  5  sunflowers,  helianthus  annuus  cultivar,  one  of  the  most  artificial horticultural plants as a symbol for the political party of the  greens  from  germany:  bündnis  90,  die  grünen.  http://gruene‐ senden.de/schlagzeilen/archiv.html  fig. 6  schematic overview of known types of intercompartment dna  transfer. (a) organelle‐to‐nucleus; (b) chloroplast‐to‐mitochondrion;  (c) nucleus‐to‐mitochondrion. from (leister, 2005)  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  38  biotechnology society of nepal (bsn), all rights reserved   when one such unit is incorporated at the locus of a gene, it  may affect genic action. the altered action  is detected as a  mutation. subsequent changes at the  locus,  initiated by the  extragenic unit, again can result  in change  in genic action  ; consequently,  a  new  mutation  may  be  recognized.  the  extragenic units undergo transposition from one  location to  another in the chromosome complement. it is this mechanism  that is responsible for the origin of instability at the locus of a  known  gene;  insertion  of  an  extragenic  unit  adjacent  to  it  initiates  the  instability.  the  extragenic  units  represent  systems in the nucleus that are responsible for controlling the  action  of  genes.  they  have  specificity  in  that  the  mode  of  control of genic action in any one case is a reflection of the  particular system in operation at the locus of the gene.  one  extragenic system controlling genic expression is composed of  two interacting units. it is the so‐called dissociation‐activator  (ds‐ac) system.   both ds and ac undergo transposition. the  ds  component,  when  inserted  at  the  locus  of  a  gene,  is  responsible for modification of genic expression. subsequent  changes  at  the  locus,  initiated  by  ds,  result  in  further  modification  of  genic  expression.  the  ac  component  in  this  two‐unit system controls when the changes at ds will occur.  from  the  conclusions  stated  above,  it  was  anticipated  that  the ds‐ac system could operate at any locus of known genic  action.  this  is  because  the  ds  unit  may  be  transposed  to  various locations in the chromosome complement. to obtain  this  type  of  instability  at  any  one  locus  of  kn,own  genic  action, it is only necessary to provide adrquate means for its  detection. the methods used to obtain and detect this type of  instability  at  the  ai  locus  in  chromosome  3  and  at  the  a2 locus in chromosome 5 are described.  a detailed analysis of  one such case is presented in this report.”  later commentaries of fedoroff were summarizing the  scientific  achievements  of  mcclintock,  acknowledging  her scientific merits: (fedoroff, 1992, 1994; fedoroff et  al., 1995; fedoroff, 1984; fedoroff, 1991). especially in  the  review  published  in  the  scientific  american,  transposons  are  well  summarized  as  a  generally  occurring  phenomenon,  having  changed  considerably  the concept of genomics, this is well illustrated in the  fact of the multicolored maize kernels (fig. 7).  see  a  photo  from  a  landrace  preserved  as  a  cultivar  from  thusis,  switzerland,  visualizing  the  dynamics  of  transposition (fig. 8).  more  comments  on  mcclintocks  scientific  breakthrough  in  (lewin,  1983;  shapiro,  1997),  the  latter  probably  the  first  to  coin  the  term  ‘natural  genetic  engineering’.  unfortunately,  the  dynamics  of  life dna processes was not taken properly into account  when  the  cartagena  protocol  on  biosafety  was  conceived.  lrr homologues via pcr from two monocot species, rice and  barley, based on structurally conserved motifs  in dicot nbs‐ lrr r genes. we have analyzed their sequence diversity and  their linkage to genetically characterized r genes. the results  from a comparative mapping in rice, barley, and foxtail millet  indicates  a  rapid  evolution  of  r  genes  in  each  species  and  suggests  possible  mechanisms  to  generate  diversity  in  resistance loci.” and:  “at  present,  rapid  sequence  divergence  and  ectopic  recombination  are  equally  possible  mechanisms  to  explain  the  lack of  intraspecific syntenic relationships detected with  our  set  of  r‐like  gene  probes.  regardless  of  whether  the  former or latter (or both) mechanism drives the evolution of  monocot nbslrr genes, the data shown here provides strong  evidence that this class of genes diversifies more rapidly than  the  rest  of  the  tested  monocot  genomes.”    (leister  et  al.,  1998)  4.2.  jumping  genes:  their  dynamics  falsify  the erroneous picture of regulators that dna  is a stable string of genes  the seemingly absolute novelty of genetic engineering  on the molecular  level has been contested already  in  the  early  days  of  molecular  biology  in  the  1930s  and  1950s  with  the  discovery  of  cellular  systems  for  genome  restructuring  discovered  with  the  classic  papers of mcclintock (mcclintock, 1930, 1953).   1.  a  case  of  semi‐sterility  in  zea  mays  was  found  to  be  associated  with  a  reciprocal  translocation  (segmental  interchange)  between  the  second  and  third  smallest  chromosomes.  2.  through  observations  of  chromosome  synapsis  in  early  meiotic  prophases  of  plants  heterozygous  for  the  interchange it has been possible to locate approximately  the  point  of  interchange  in  both  chromosomes.  the  interchange was found to be unequal.  3.  an  analysis  of  the  chromosome  complements  in  the  microspores of plants heterozygous for the  interchange  indicated  that  of  the  four  chromosomes  constituting  a  ring,  those  with  homologous  spindle  fiber  attachment  regions can pass to the same pole in anaphase i and do  so in a considerable number of the sporocytes.  and  in the paper of 1953, usually cited as the classic  publication,  leading  decades  later,  including  her  relentless fight for the “jumping genes concept”: here  the full summary of her paper:  “previous  studies  of  the  origin  and  mode  of  expression  of  genic instability at a number of known loci in maize led to the  following  conclusions.  extragenic  units,  carried  in  the  chromosomes, are responsible for altering genic expression.  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  39  biotechnology society of nepal (bsn), all rights reserved   repeats  and  cause  no  duplication  of  host  genome  sequence upon insertion.  4.4.  polyploids,  alloploids  in  flowering  plants  in  his  blog  series  part  9,  david  tribe  sums  up  polyploidisation dynamics of higher plants6 :    “during  only  the  past  decade  [i.e  post  1985]  molecular  approaches  have  provided  a  wealth  of  data  that  have  dramatically reshaped views of polyploid evolution, providing  a much more dynamic picture than traditionally espoused. in  particular, molecular data:  (i)  demonstrate  that  both  autopolyploids  and  allopolyploids  exhibit  a  high  frequency  of  recurrent  formation (multiple origin),  (ii)  reveal  that  multiple  polyploidization  events  within  species  have  significant  genetic  and  evolutionary  implications, and  (iii)  contradict  the  traditional  view  of  autoploidy  as  being  rare and maladaptive (soltis & soltis, 1993).    perhaps one of the most important contributions of molecular  data to the study of polyploid evolution is the documentation  that  a  single  polyploid  species  may  have  separate,  independent  origins  from  the  same  diploid  progenitor  species.    multiple origins of polyploids have now been documented in  bryophytes  (wyatt  et  al.,  1988)  and  in  >40  species  of  ferns  (e.g.,  (werth  et  al.,  1985)  and  (ranker  et  al.,  1989)  and  angiosperms (e.g., refs. (brochmann et al., 1992; doyle et al.,  1990;  soltis  et  al.,  1995;  song  &  osborn,  1992).  in  fact,  molecular data  indicate that multiple origins of polyploids  are the rule and not the exception (soltis & soltis, 1993). in  4.3. helitrons contribute to the lack of gene  colinearity  observed  in  modern  maize  inbreds  it  was  david  tribe  in  his  blogspot  on  natural  transgenics part 7 linking helitrons to natural gmo’s 5   “until  recently,  it  was  assumed  that  the  order  of  gene  sequences within modern maize would be virtually invariant.  recent  discoveries  have  shown  that  gene  co‐linearity  is  not  always the case. several  laboratories (1‐3) have found dna  regions rich in gene sequences that are present in some maize  inbred  lines  but  absent  at  homologous  sites  in  other  lines.  this  variation,  termed  "intraspecific  violation  of  genetic  co‐ linearity" or "plus/minus genetic polymorphism," was shown  by  (lal  &  hannah,  2005)  in  a  recent  issue  of  pnas  to  be  caused  by  a  newly  described  transposable  element  family  termed helitrons.”  in  a  recent  review,  (lal  et  al.,  2009)  summarize  the  importance  of  a  recently  discovered  superfamily  of  transposable  elements.  the  authors  critically  analyze  the  proposed  mechanisms  of  helitron  transposition,  their impact on genome evolution and the process by  which these enigmatic elements capture and multiply  host genes. intriguingly, maize helitrons share striking  structural similarity  to  bacterial  integrons.  these  elements  capture  gene  sequences via site‐specific recombination and generate  circular  intermediates  (hall  &  collis,  1995).  both  helitrons  and  integrons  are  mobile,  lack  terminal  fig.  7  development  time  and  frequency  of  transposition  differ  in  mutations  caused  by  the  insertion  of  different  defective  spm  elements.  if  transposition  takes  place  late  in  the  development,  the  clones of revertent cells are small and therefore so are the pigmented  spots (a) . if transposition takes place at about the same time but at a  lower frequency, there are fewer such clones and fewer spots (b).  if  the  transposition  that  resores  gene  function  takes  place  earlier,  the  revertant clones and the spots of the pigmented tissue are larger (c).  from (fedoroff, 1984).  fig.  8    landrace  preserved  as  a  cultivar  from  thusis,  switzerland,  visualizing  the  colorful  dynamics  of  transposition:  photo  klaus  ammann   5david tribes blogspot no. 7: http:// gmopundit.blogspot.com/2006/01/natural‐gmos‐part‐7‐nanobot‐ genetic.html  6david tribes blogspot no. 9: http:// gmopundit.blogspot.com/2006/01/natural‐gmos‐part‐9‐ different.html   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  40  biotechnology society of nepal (bsn), all rights reserved   duplicative  horizontal  gene  transfer  and  differential  gene  conversion is proposed as a hitherto unrecognized source of  genetic diversity.”  the conclusion from this chapter 4.5. is again that gene  exchange in the course of evolution has been proven,  and thus “evolutionary transgenes” are part of nature.  the  conclusion  again:  transgenesis  belongs  to  nature  and  it  is  scientifically  not  justified  to  make  a  fundamental  distinction  between  natural  organisms  (strictly  without  transgenes)  and  artificial  organisms  containing trangenes with methods of targeted genetic  engineering.     5.0. some conventional breeding  causes lots of genomic alteration  one  should  also  take  into  account,  that  many  of  the  conventional breeding methods such as colchicination  (awoleye  et  al.,  1994;  barnabás  et  al.,  1999)  and  radiation  mutation  breeding  (reynolds  et  al.,  2000;  shirley  et  al.,  1992)  are  obviously  more  damaging  to  the genome (schouten & jacobsen, 2007), and  it  is  in  addition not possible to clearly define what impact the  un‐targeted process could have caused. (molnar et al.,  2009) reported  in detail about radiation treatment of  the  chromosome  morphology  of  wheat  hybrids:  dicentric  chromosomes,  fragments,  and  terminal  translocations  were  most  frequently  induced  by  gamma‐radiation,  but  centric  fusions  and  internal  exchanges  were  also  more  abundant  in  the  treated  plants than  in the control amphiploids. the  irradiated  amphiploids  formed  fewer  seeds  than  untreated  plants, but on the other hand normal levels of fertility  were recovered in their offspring. on the positive side  the  authors  are  confident  that  intergenomic  translocations  will  facilitate  the  successful  introgression  of  drought  resistance  and  other  alien  traits  in  bred  wheat.  but  it  has  to  be  admitted  that  repair  mechanisms  on  the  dna  level  are  powerful  (baarends et al., 2001; dong et al., 2002; morikawa &  shirakawa,  2001).  it  is  only  logical  that  opposition  within organic farming towards genetic engineering  is  now  expanding  also  to  some  of  those  conventional  breeding  methods,  some  go  even  so  far  as  to  reject  marker  assisted  breeding  –  typically  for  the  organic  agriculture  scene,  this  trend  is  based  on  the  myth  of  “intrinsic integrity of the genome”, for which term it is  not possible in the literature to find a proper scientific  definition based on comparisons (ammann, 2008). the  addition  of  rejected  breeding  methods  would  ultimately  lead to an absurd situation, where most of  several  species  studied  in  detail  with  molecular  markers,  recurrent  polyploidization  was  shown  to  occur  with  great  frequency  during  short  time  spans  and  in  small  geographic  areas  ((brochmann  et  al.,  1992;  soltis  et  al.,  1995).  for  example,  tragopogon  mirus  and  tragopogon  miscellus  may  have  formed  as  many  as  9  and  21  times,  respectively,  in  a  small  region  of  eastern  washington  and  adjacent  idaho  during  just  the  past  50  years  (soltis  et  al.,  1995).  the  frequent  recurrence  of  polyploidization  also  has  major  evolutionary  implications,  suggesting  that  polyploids  are  much more genetically dynamic than formerly envisioned.”    polyploidy  is  one  of  the  most  distinctive  and  widespread modes of speciation in higher plants. thirty  to 70%  of angiosperms, including many important crop  plants,  are  estimated  to  have  polyploidy  in  their  lineages  (song  et  al.,  1995),  again  a  strong  argument  for the high dynamics of the genome of higher plants.    4.5.  horizontal  geneflow  between  pro­ caryotes and eu­caryotes  there  is  a  rich  literature  documenting  –  on  an  evolutionary  scale  –  that  horizontal  transfer  of  genes  (hgt)  between  pro‐caryotes  and  eu‐caryotes  are  not  uncommon: however, according to (keeling & palmer,  2008) many records of hgt (consortium, 2001) are not  confirmed  by  phylogenetic  analysis  proving  incongruent  sequences  (stanhope  et  al.,  2001).  this  means  that  potentially,  molecular  processes  can  transfer  foreign  genes,  so  –  actually,  all  living  organisms  are  in  that  sense  “transgenic  organisms”,  but  only  considering  evolutionary  time  scales  of  millions of years time span for the transfer event. to  be  clear,  there  is  no  evidence  of  horizontal  gene  transfer  coming  from  the  relatively  new  practice  in  modern  breeding  methods  of  genetic  engineering  (smalla & sobecky, 2002; smalla & vogel, 2007). even  the much publicized case of hgt with a transgene  in  the  human  guts  is  based  on  clearly  wrong  interpretation  and  false  claims  (ammann,  2002).  however, for mitochondrial dna things are different:  according  to  (archibald  &  richards,  2010),  mitrochondrial  dna  can  be  exchanges  rather  frequently:   “parasitic  plants  and  their  hosts  have  proven  remarkably  adept  at  exchanging  fragments  of  mitochondrial  dna.  two  recent  studies  (mower  et  al.,  2010;  richardson  &  palmer,  2007)    provide  important  mechanistic  insights  into  the  pattern,  process  and  consequences  of  horizontal  gene  transfer,  demonstrating  that  genes  can  be  transferred  in  large chunks and that gene conversion between foreign and  native genes leads to intragenic mosaicism. a model involving  nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  41  biotechnology society of nepal (bsn), all rights reserved   in  a  letter7  to  the  executives  of  the  convention  on  biological  diversity  (cbd),  the  public  research  and  regulation  initiative  (prri)  is  asking  for  a  scientific  discussion in order to exempt a list of gm crops from  the  expensive  regulatory  process  for  approval,  here  only the final statement:  “bearing in mind that the method of transformation itself is  neutral,  i.e.  that  there  are  no  risks  related  to  process  of  transformation, prri believes that there are several types of  lmos and traits for which ‐ on the basis of the characteristics  of the host plant, the functioning of the  inserted genes and  experience with the resulting gmo ‐ it can be concluded that  they are as safe as its conventional counterpart with respect  to  potential  effects  on  the  environment,  taking  also  into  account human health. “  in  a  recent  paper,  an  indiscriminate  continuation  of  food biosafety research  is questioned on the basis of  all the above arguments by herman et al. (herman et  al., 2009) with good reason:  “compositional studies comparing transgenic crops with non‐ transgenic  crops  are  almost  universally  required  by  governmental  regulatory  bodies  to  support  the  safety  assessment  of  new  transgenic  crops.  here  we  discuss  the  assumptions  that  led  to  this  requirement  and  lay  out  the  theoretical  and  empirical  evidence  suggesting  that  such  studies are no more necessary for evaluating the safety of  transgenic crops than they are for traditionally bred crops.”  6.1.  perspectives  for  a  dissolution  of  this  divide  these new perspectives create hope, that solutions can  be found:  in  a  first  phase  some  of  the  widespread  transgenic  crops like transgenic maize with the cry1ab endotoxin  should  be  exempt  from  regulation,  which  is  indeed  possible  according  to  art.  7.4  in  the  cartagena  protocol.  in  cop‐mop58  2010  in  japan  it  should  be  possible, to amend the protocol with the introduction  of  a  dynamics  which  allows  to  start  the  regulatory  process with an initial phase focusing on the process of  transgenesis,  first  following  procedures  proposed  for  non‐target  insects  by  (raybould,  2010;  romeis  et  al.,  2008), but  in due time shifting  later the focus on the  product,  making  it  possible  to  abbreviate  the  regulatory process wherever possible and feasible.   the modern time traits would have to be rejected and  breeding would be forced to start from scratch.  basically, many of the first generation gm crops should  be  today  subject  to  a  professional  debate  on  deregulation, and there  is good and sturdy reason to  state  that  many  of  these  gm  crops  should  not  have  been  treated  in  such  a  special  way  in  the  first  place,  they can be compared  in their risk potential to many  crops created with traditional methods.  this should not be misunderstood as a plea for general  deregulation  of  gm  crops,  rather  for  a  strict  and  science  based  risk  based  regulation  focusing  also  on  products, not on processes alone.  somatic  hybridization  also  deserves  a  short  mention  here,  the  method  enabled  the  artificial  hybridization of crops which have no genomic natural  compatibility,  see  the  review  of  (waara  &  glimelius,  1995),  progeny  analysis  of  some  hybrid  combinations  also  reveals  inter‐genomic  translocations  which  may  lead  to  the  introgression  of  the  alien  genes  .  furthermore, fusion techniques enable the resynthesis  of  allopolyploid  crops  to  increase  their  genetic  variability  and  to  restore  ploidy  level  and  heterozygosity after breeding at reduced ploidy level in  polyploid crops.  6.0.  regulatory  dissent  over  molecular  differences  causes  transatlantic divide  this actually includes a critical questioning about some  basic  rules  of  the  united  nations  convention  on  biological diversity (cbd): transgenic crops of the first  generation  should  not  have  been  generally  subjected  to  regulation  purely  based  on  the  process  of  transgenesis alone; rather it would have been wiser to  have a close look at the products in each case, as john  maddox  already  proposed  in  1992  in  an  editorial  in  nature  (anonymous,  1992).  this  is  also  the  view  of  canadian  regulators  (andree,  2002;  berwald  et  al.,  2006; macdonald & yarrow, 2002), where the novelty  of  the  crop  is  the  primary  trigger  for  regulation.  this  transatlantic  (and  transoceanic)  contrast  has  been  commented by many (aerni et al., 2009; bennett et al.,  1986; kalaitzandonakes et al., 2005; ramjoue, 2007a,  b;  snyder  et  al.,  2008;  thro,  2004),  and  although  for  many years a solution and mediation seemed to be too  difficult, contrasts can be overcome:  7prri  letter  :  http://www.pubresreg.org/index.php? option=com_docman&task=doc_download&gid=490  8fifth meeting of the conference of the parties serving as the meet‐ ing of the parties to the cartagena protocol on biosafety (cop‐mop  5),    11  –  15.  10.  2010  nagoya,  japan  http://bch.cbd.int/protocol/ meetings/   nepal journal of biotechnology. jan. 2011, vol. 1, no. 1: 31‐48  42  biotechnology society of nepal (bsn), all rights reserved   the difficulties start there, where a clear definition of  pnts  is  needed  to  come  to  a  decision:  it  means  that  plants  produced  using  recombinant  dna  techniques,  chemical  mutagenesis,  cell  fusion,  cis‐genics  or  any  other  in‐vitro technique  leading to a novel trait, need  to undergo risk assessment in the canadian system. no  wonder the canadian definition of novel traits is rather  wordy, but remains broad minded:  “a  plant  variety/genotype  possessing  characteristics  that  demonstrate  neither  familiarity  nor  substantial  equivalence  to those present in a distinct, stable population of a cultivated  seed  in  canada  and  that  have  been  intentionally  selected,  created  or  introduced  into  a  population  of  that  species  through a specific genetic change.”  7.0. conclusions  there can be no doubt that product‐based regulatory  approaches  are  truest  to  the  scientific  principle  that  biotechnology  is not  inherently more risky than other  technologies that have a long and accepted history of  application in agriculture and food production, it is also  less  prescriptive  than  process‐based  systems,  see  mclean et al. (mclean et al., 2002).  a conceptual framework is proposed by ifpri/isnar  in  2002,  the  international  service  for  national  agricultural  research  (mclean  et  al.,  2002),  a  careful  evaluation  of  process‐based  versus  product‐based  triggers in regulatory action can also lead to a merger  of  both  seemingly  so  contrasting  concepts  into  a  legalized  decision  making  process  on  which  trigger  should be chosen in a case by case strategy:  “process‐based  triggers  are  the  rule  in  almost  all  countries  that  have  developed  national  biosafety  regulatory  systems;  there are exceptions, however, where the novelty of the trait  determines  the  extent  of  regulatory  oversight  and  not  the  process  by  which  the  trait  was  introduced.  while  such  a  product‐based approach to defining the object of regulation  is  truest  to  the  scientific  principle  that  biotechnology  is  not  inherently  more  risky  than  other  technologies  that  have  a  long  and  accepted  history  of  application  in  agriculture  and  food  production,  it  is  less  prescriptive  than  process‐based  regulatory systems.”  many  of  the  debates  on  those  two  concepts  suffer  from a lack of clear‐cut definitions, it will be important  to have a close look at the canadian regulatory system  and the definition of pnts (plants with novel traits). in  canada, the trigger for risk‐assessment is the novelty of  the plant rather than the methods used to produce it.  8.0. cited literature  aerni, p., rae, a., & lehmann, b. (2009) nostalgia versus  pragmatism? how attitudes and interests shape the  term sustainable agriculture in switzerland and new  zealand. food policy, 34, 2, pp  227‐235 http:// www.sciencedirect.com/science/article/b6vcb‐ 4v1mfkr‐1/2/b72610f6397bc5572a076cbe0ae3e599  and http://www.botanischergarten.ch/sustainability/ aerni‐nostalgia‐versus‐pragmatism‐2009.pdf    ammann, k. (2002) university of newcastel report  summaries: no significant horizontal transgene transfer  detected in human guts. in berne debates blog,  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