microsoft word 11394 nsb saioc 2023.03.27.docx received: 01 dec 2022. received in revised form: 20 mar 2023. accepted: 22 mar 2023. published online: 27 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania saioc a et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11394 doi:10.15835/nsb15111394 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae a shorta shorta shorta short----term survival analysis of severe patients with acute vascular term survival analysis of severe patients with acute vascular term survival analysis of severe patients with acute vascular term survival analysis of severe patients with acute vascular event admitted on event admitted on event admitted on event admitted on intensive care unit departmentintensive care unit departmentintensive care unit departmentintensive care unit department alexandra saioc1,2, vlad-ionuţ nechita3,4*, cosmin i. puia4,5,6 1clinical emergency hospital of oradea, 65 gheorghe doja, 410169 oradea, romania; alexandrangelas@gmail.com 2oradea university, faculty of medicine and pharmacy, department of medical disciplines, doctoral school of biomedical sciences, oradea, romania 3“iuliu hațieganu” university of medicine and pharmacy, department of medical informatics and biostatistics, 6 louis pasteur str., 400349 cluj-napoca, romania; nechita.vlad@umfcluj.ro (*corresponding author) 4third surgery clinic, prof. dr. “octavian fodor” regional institute of gastroenterology hepatology, 19-21 croitorilor street, 400162 cluj-napoca, romania; drpuia@yahoo.fr 5oradea university, faculty of medicine and pharmacy, doctoral school of biomedical sciences, oradea, romania 6“iuliu haţieganu” university of medicine and pharmacy, faculty of medicine, department of surgery, 19-21 croitorilor street, clujnapoca, romania abstractabstractabstractabstract the study evaluated the short-term survival during hospitalization period of the patients admitted on the intensive care unit department with an acute vascular event in comparison with other pathologies. we developed a retrospective cohort study with longitudinal design on 178 subjects admitted in an intensive care unit department. the patients were divided according the presence (33 patients) or absence (145 patients) of an acute vascular event (myocardial infarction or stroke). the survival during the hospitalization period was evaluated also considering the presence of diabetes (58 patients) or diabetes with complications (54 patients). the survival was presented as probability to survive during hospitalization period and median survival time. logrank test was used to compare the groups. patients with myocardial infarction had the average age of 60.57±7.97 years old, with 100% mortality during the hospitalisation period and a median survival time of 16 days. the patients with stroke were 66.34±14.12 years old with 46.15% survival probability and median survival time of 26 days; respectively patients without vascular event were 65.52±15.81 years old, had a survival probability of 46.89% and a median survival time of 28 days. considering all the patients with vascular events, survival probability was 36.36%. diabetes, the presence of arterial hypertension or diabetic complications does not influence significantly the survival in this sample (logrank test, p>0.05). survival of patients with acute vascular event is comparable with that of the patients admitted on intensive care unit for other health condition. a poor prognostic was observed for myocardial infarction. keywords:keywords:keywords:keywords: intensive care unit; myocardial infarction; stroke; survival; vascular event https://www.notulaebiologicae.ro/index.php/nsb/index saioc a et al. (2023). not sci biol 15(1):11394 2 introductionintroductionintroductionintroduction type 2 diabetes is a very important metabolic disorder characterized by chronic hyperglycemia, a burden for the healthcare system worldwide (reed et al., 2021). this disease was mentioned for the first time in a manuscript, about three thousand years ago (hegazi et al., 2015). the highest prevalence in the population have type 2 diabetes (90%), followed by type 1 diabetes (less than 10%) and other rare types. the increase in prevalence for type 2 diabetes is related with bad eating habits and increase body weight (haslam, 2010; maahs et al., 2010; ozougwu et al., 2013). the molecular cause for type 2 diabetes seems to be inappropriate insulin production or secretion in the pancreatic beta cells or the resistance to insulin (thompson and kanamarlapudi, 2013). long-term complications are important in this pathology, considering that is the first cause of blindness, end-stage kidney disease, cardiovascular disease and leg amputation worldwide (thompson and kanamarlapudi, 2013). myocardial infarction is characterized by the death of myocardial cell due to ischemia (thygesesn et al., 2010). mortality decreased in the past years (mozaffarian et al., 2010). dispensarisation on intensive care unit after an acute myocardial infarction is accepted in the standard practice (halpern and pastores, 2013). stroke is another important acute vascular event caused by hemorrhage or ischemia that can require management in the intensive care unit (khassawneh et al., 2012). according to ho et al. (2015) the in-hospital mortality after hemorrhagic and ischemic stroke is 20.4% respectively 15%. for patients with myocardial infarction who developed cardiogenic shock a mortality of at least 40% is described in the literature (saleh and ambrose, 2010). the aim of our study was to evaluate the short-term survival of patients with a major vascular event that needed intensive care management considering associated diabetes in comparison with other cases from intensive care unit department. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods setting and study design a cohort study with longitudinal retrospective design was conducted on patients admitted in the intensive care unit from oradea regional emergency hospital. participants all patients admitted in the intensive care unit department were eligible for the study. patients who were in the direct care of the main author, with a complete medical history and without missing data were included for analysis. the cohort was divided according the presence and the type of vascular event (myocardial infarction or stroke). data source and collection patient’s information was collected from hospital database and patient observation sheets. qualitative data (gender and comorbidities), quantitative (age) and survival information (survival time in days) were collected. statistical analysis the statistics were computed on r commander version 2.8.0 and for survival graphs the kmggplot2 package was used. qualitative data were evaluated as relative frequencies, quantitative data were presented as averages and standard deviations respectively median and interquartile range. the survival time was presented saioc a et al. (2023). not sci biol 15(1):11394 3 as median and 95% confidence intervals. to compare the survival time, we used the logrank test and kaplan meyer curves. the p value lower than 0.05 was considered statistically significant. ethical issues this study received approval no. 33441/07.10.2022 from the ethics committee of the oradea regional emergency hospital. results results results results one hundred seventy-eight patients admitted in the intensive care unit department were evaluated. the groups were built according to the presence of cardiovascular event: twenty-six patients after a stroke (14.61%), seven patients after myocardial infarction (3.93%) and one hundred forty-five subjects without vascular events (81.46%). the main characteristics of the patients and associated comorbidities are presented in table 1. table 1.table 1.table 1.table 1. the main characteristics of the sample (n=178) all subjects all subjects all subjects all subjects (n=178)(n=178)(n=178)(n=178) myocardial myocardial myocardial myocardial infarction (n=7)infarction (n=7)infarction (n=7)infarction (n=7) stroke stroke stroke stroke (n=26)(n=26)(n=26)(n=26) no vascular event no vascular event no vascular event no vascular event (n=145)(n=145)(n=145)(n=145) characteristicscharacteristicscharacteristicscharacteristics age* (years) mean± sd 65.44±15.32 60.57±7.97 66.34±14.12 65.52±15.81 gender, male – no. (%) 101 (56.74%) 5 (7.14%) 16 (61.53%) 80 (55.17%) diabetes no. (%) 58 (32.58) 4 (57.14%) 6 (23.07%) 48 (33.1%) diabetes with hypertension no. (%) 13 (7.3%) 0 1 (3.84%) 12 (8.27%) diabetes with complications no. (%) 54 (30.33%) 4 (57.14) 6 (23.07%) 44 (30.34%) survivalsurvivalsurvivalsurvival death during hospitalization period no. (%) 98 (55.05%) 7 (100%) 14 (53.84%) 77 (53.1%) death on the intensive care unit no. (%) 89 (50%) 7 (100%) 14 68 (46.89%) death after they were transferred to another department no. (%) 9 (5.05%) 0 0 9 (6.2%) median survival time * 27 (95% ci 2433) 16 (95% ci 1na) 26 (95% ci 20na) 28 (95% ci 2435) *presented as median and 95% confidence intervals; na, not applicable. no statistically significant difference in survival (p=0.25 –log-rank test) was observed for diabetic patients that had a survival probability of 48.27% and a median survival time of 27 days (95% ci 19-45) in comparison with the median survival time for those without diabetes with a survival probability of 46.66% and median survival of 27 days (95% ci 24-34). patients with diabetes and associated hypertension have a survival probability of 46.15% with a median survival time of 28 days (95% ci 5-na), and the difference was not statistically significant in comparison to those without hypertension (figure 1). for those with diabetes and specific diabetic complications, the survival probability was 50% and the median survival time on intensive care unit was 28 days (95% ci 18-45). for patients with diabetes and an acute vascular event, the survival probability was 50% with a median survival time of 32.5 days (95% ci 17-na), but the differences with the other patients admitted on intensive care unit were not statistically significant (figure 1). saioc a et al. (2023). not sci biol 15(1):11394 4 (a) (b) (c) (d) figure 1.figure 1.figure 1.figure 1. survival during hospitalization period regarding the presence of diabetes (a), hypertension (b), diabetic complications (c) and vascular events (d) the survival probability for the subjects with stroke admitted in the intensive care unit was 46.15% with a median survival time of 26 days (95% ci 20-na) while none of the subjects admitted with myocardial infarction managed to survive. the median survival time for myocardial infarction was 16 days (95% ci 1na). the overall survival probability for subjects with vascular events admitted in the intensive care unit was 36.36%. for the subjects without vascular event the survival probability was 46.89% and the median survival time of 28 days (95% ci 24-35) (figure 2). the survival probability was significantly inferior for the subjects with myocardial infarction. saioc a et al. (2023). not sci biol 15(1):11394 5 (a) (b) figure 2.figure 2.figure 2.figure 2. survival during hospitalization period regarding the presence (a) and the type (b) of vascular event discussiondiscussiondiscussiondiscussion the myocardial infarction and stroke are considered diseases with golden hour (cho et al., 2019). this study evaluated the short-term survival in case of a major vascular event (myocardial infarction or stroke) in comparison with other subjects admitted in a mixed intensive care unit. myocardial infarction that needed intensive care management proved to have a significantly poor prognostic in comparison with the other subjects, despite a slightly lower average age (table 1 and figure 2). kristoffersen et al. (2012) described a close overall relative frequency of death among the patients after acute myocardial infarction (19.1%) to those that had an acute stroke event (17.6%) during the first 30 days hospitalization period. the rates were higher in our study, considering also the severity of the cases that needed intensive care management. an important factor for survival was also the time and the distance before the patients firstly arrived in the hospital, as berlin et al. (2016) mentioned. driving time to the hospital was less important for stroke patients in comparison with myocardial infarction regarding the prognostic (berlin et al., 2016). myocardial infarction seems to have a poor short –term survival especially stemi in comparison with the stroke where mortality is increased later, after the first approach due to secondary sequelae, hemodynamic instability or pulmonary complications (berlin et al., 2016). a meta-analysis over 141 studies regarding the mortality in the intensive care unit for subjects with diabetes in comparison to those without diabetes, a significant disadvantage for diabetes was observed only for subjects admitted after surgery (or= 1.48 [1.04 to 2.11]), but not for those from the medical or mixed intensive care units (siegelaar et al., 2011). the results were similar with the situation described in our study. the diabetes was not associated with increased mortality (figure 1). the presence of diabetic complications did not make differences on short-term outcome, and this can be explained by a different treatment approach adapted to their needs, and a good care they receive, to have a similar survival chance. also, regarding the chronic pathology and complications, they seem to be managed efficiently (figure 1). krinsley (2003) suggested that hyperglycemia is a common condition for the critically ill patient, being associated with an increased inhospital mortality. patients with high glucose levels due to diabetes seems to be less affected by hyperglycemia in critical situations (rady et al., 2005; egi et al., 2008; falciglia et al., 2009; graham et al., 2010). this can also explain the similarities for the short-term prognostic (figure 1). saioc a et al. (2023). not sci biol 15(1):11394 6 our single-center experience can be valuable to assess the short-term prognostic for severe subjects after an acute vascular event admitted for intensive care. possible drawback for this study is the retrospective design; the small sample size, and the low number of of subjects in the group with vascular event; especially for those with myocardial infarction. a larger sample with prospective follow-up is recommended for further research on this domain. conclusionsconclusionsconclusionsconclusions survival of patients admitted after an acute stroke on a mixed intensive care unit is comparable with that of the patients admitted for other non-vascular conditions. a poor prognostic was observed for myocardial infarction in comparison with stroke or non-vascular subjects. for patients with diabetes or with diabetic complications no differences in survival were observed during short-term prognostic on a mixed intensive care unit department. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions sa was responsible for data collection and manuscript writing. nvi was involved in statistical analysis, interpretation, review and editing of final manuscript. pci provided conceptualization, supervision, study design, final review and correction. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) this study received approval no. 33441/07.10.2022 from the ethics committee of the oradea regional emergency hospital. acknowledgementsacknowledgementsacknowledgementsacknowledgements we thankfully acknowledge the support we received from university of oradea faculty of medicine and pharmacy for this study. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences berlin c, panczak r, hasler r, zwahlen m (2016). do acute myocardial infarction and stroke mortality vary by distance to hospitals in switzerland? results from the swiss national cohort study. british medical journal 6(11):e013090. https://doi.org/10.1136/bmjopen-2016-013090 saioc a et al. (2023). not sci biol 15(1):11394 7 cho kh, nam cm, lee sg, kim th, lee sh, park ec (2019). measuring of quality of care in patients with stroke and acute myocardial infarction: an application of algebra effectiveness model. medicine 98(20):e15353. https://doi.org/10.1097/md.0000000000015353 egi m, bellomo r, stachowski e, french cj, hart gk, hegarty c, bailey m (2008). blood glucose concentration and outcome of critical illness: the impact of diabetes. critical care medicine 36:2249-2255. https://doi.org/10.1097/ccm.0b013e318181039a falciglia m, freyberg rw, almenoff pl, d'alessio da, render ml (2009). hyperglycemia-related mortality in critically ill patients varies with admission diagnosis. critical care medicine 37:3001-3009. https://doi.org/10.1097/ccm.0b013e3181b083f7 graham bb, keniston a, gajic o, trillo alvarez ca, medvedev s, douglas is (2010). diabetes mellitus does not adversely affect outcomes from a critical illness. critical care medicine 38:16-24. https://doi.org/10.1097/ccm.0b013e3181b9eaa5 halpern na, pastores sm (2010). critical care medicine in the united states 2000-2005: an analysis of bed numbers, occupancy rates, payer mix, and costs. critical care medicine 38:65-71. https://doi.org/10.1097/ccm.0b013e3181b090d0 haslam d (2010). obesity and diabetes: the links and common approaches. primary care diabetes 4(2):105-112. https://doi.org/10.1016/j.pcd.2010.04.002 hegazi r, el-gamal m, abdel-hady n, hamdy o (2015). epidemiology of and risk factors for type 2 diabetes in egypt. annals of global health 81(6):814-820. https://doi.org/10.1016/j.aogh.2015.12.011 ho wm, lin jr, wang hh, liou cw, chang kc, lee jd, ... lee th (2016). prediction of in-hospital stroke mortality in critical care unit. springerplus 5(1):1051. https://doi.org/10.1186/s40064-016-2687-2 khassawneh b, ibnian a, yassin a, al-mistarehi a, e’leimat i, ali m, ... el-salem k (2019). the outcome of patients with acute stroke requiring intensive care unit admission. european respiratory journal https://doi.org/10.1183/13993003.congress-2019.pa2283 krinsley js (2003). association between hyperglycemia and increased hospital mortality in a heterogeneous population of critically ill patients. mayo clinic proceedings 78:1471-1478. https://doi.org/10.4065/78.12.1471 kristoffersen dt, helgeland j, clench-aas j, laake p, veierød m (2012). comparing hospital mortality – how to count does matter for patients hospitalized for acute myocardial infarction (ami), stroke and hip fracture. bmc health service research 12:364. https://doi.org/10.1186/1472-6963-12-364 maahs dm, west na, lawrence jm, mayer-davis ej (2010). chapter 1: epidemiology of type 1 diabetes. endocrinology & metabolism clinics of north america 39(3):481-497. https://doi.org/10.1016/j.ecl.2010.05.011 mozaffarian d, benjamin ej, go as, arnett dk, blaha mj, cushman m, ... turner mb (2015). heart disease and stroke statistics – 2015 update: a report from the american heart association. circulation 131:e29-e322 ozougwu jc, obimba kc, belonwu cd, unakalamba cb (2013). the pathogenesis and pathophysiology of type 1 and type 2 diabetes mellitus. journal of physiology and pathophysiology 4:46-57. https://doi.org/10.5897/jpap2013.0001 rady my, johnson dj, patel bm, larson js, helmers ra (2005). influence of individual characteristics on outcome of glycemic control in intensive care unit patients with or without diabetes mellitus. mayo clinic proceedings 80:1558-1567. https://doi.org/10.4065/80.12.1558 reed j, bain s, kanamarlapudi v (2021). a review of current trends with type 2 diabetes epidemiology, aetiology, pathogenesis, treatments and future perspectives. dmso. diabetes, metabolic syndrome and obesity 4:35673602. https://doi.org/10.2147/dmso.s319895 saleh m, ambrose ja (2018). understanding myocardial infarction. f1000 research 7. https://doi.org/10.12688/f1000research.15096.1 siegelaar se, hickmann m, hoekstra jb, holleman f, devries jh (2011). the effect of diabetes on mortality in critically ill patients: a systematic review and meta-analysis. critical care 15(5):r205. https://doi.org/10.1186/cc10440 thompson a, kanamarlapudi v (2013). type 2 diabetes mellitus and glucagon like peptide-1 receptor signalling. clinical and experimental pharmacology 3:138. https://doi.org/10.4172/2161-1459.1000138 thygesen k, alpert js, jaffe as, simoons ml, chaitman br, white hd (2012). third universal definition of myocardial infarction. european heart journal 33:2551-2567. https://doi.org/10.1093/eurheartj/ehs184 saioc a et al. (2023). not sci biol 15(1):11394 8 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11368 nsb gautam 2023.03.22.docx received: 20 oct 2022. received in revised form: 08 mar 2023. accepted: 15 mar 2023. published online: 22 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania gautam p and pitchaimuthu s (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11368 doi:10.15835/nsb15111368 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae environmental variations on physicochemical properties in environmental variations on physicochemical properties in environmental variations on physicochemical properties in environmental variations on physicochemical properties in vvvvasicine asicine asicine asicine content of content of content of content of adhatoda vasicaadhatoda vasicaadhatoda vasicaadhatoda vasica --- anananan ayurvedic medicinal plantayurvedic medicinal plantayurvedic medicinal plantayurvedic medicinal plant palshikar gautam*, shanmugapandiyan pitchaimuthu prist deemed to be university, department of pharmacy, centre for research and development (crd), thanjavur 613 403, tamil nadu, india; gautampalshikar@rediffmail.com (*corresponding author); shanmugapandiyan@gmail.com abstractabstractabstractabstract adhatoda vasica (acanthaceae) is a medicinal plant used in traditional formulations for the treatment of various disease conditions. the physicochemical properties fluctuate with the season and in response to stress. to obtain the highest medicinal value it is necessary to collect the plant raw material in the correct season. the present study was aim to evaluate the physiochemical fluctuations in the leaves of a. vasica and the vasicine, an antidiabetic constituent in these leaves. leaves were collected in every month of a year at different time and places and authentication and standardization was done. the leaves were evaluated for the macroscopic and microscopic evaluation, quantitative microscopy, proximate phytochemical analysis, extractive values in petroleum ether, chloroform, ethyl acetate, ethanol, water, and determination of the concentration of antidiabetic component vasicine. the leaves showed morphological and microscopical variations in different seasons. the phytochemical composition was the same in all seasons however the levels of extractive values fluctuated in response to seasonal variations. maximum levels of ethanol and water extract were obtained in the winter season also the vasicine concentration was higher in this season. keywords:keywords:keywords:keywords: alkaloids; antidiabetic; herbal; metabolites; variations introductionintroductionintroductionintroduction adhatoda vasica an indian medicinal plant has been used in traditional treatments for numerous human diseases for thousands of years and they continue to be an important therapeutic aid for alleviating the ailments of human kind. in india, it is estimated that 75% of population depends on plants to therapy themselves, of those about 65% populace use medicinal plants habitually to battle certain ailments and almost 45% human use such plants in pharmaceutical industries (dash, 2017). its leaves are extensively used for treating cold, cough, whooping cough and chronic bronchitis and asthma as sedative expectorant, antispasmodic and antiinflammatory drug. there is considerable demand for this plant within the country. the demand is being met from natural sources (shabir et al., 2013). the leaves have been found to be a rich source of alkaloids of which vasicine and vasicinone are bioactive. a non-nitrogenus neutral principle, vasakin, vasicinone, two new quinazoline alkaloids, one of which was named as adhvasinone and two new pyrroloquinazoline alkaloids, desmethoxyaniflorine and 7-methoxyvasicinone were identified from the ethanolic extract of the leaves. anonymous (2004). ethanolic extract of the leaves have hypoglycemic activity (dhar et al., 1968). https://www.notulaebiologicae.ro/index.php/nsb/index gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 2 the world health organization (who) has outlined herbal medicine as culminated labelled medicinal products that incorporate lively ingredients as aerial or underground accessories of plants. of the 3,50,000 higher plant species on earth, more than 75000 species are reported to have at least some medicinal value (shi-lin et al., 2016). since ages, humans have relied on nature for their basic needs for the production of foodstuff, shelters, clothing, means of transportation, fertilizers, flavors, and fragrances, and medicines. plants have formed the basis of sophisticated traditional systems of medicine that have been in existence for thousands of years and continue to provide humankind with new remedies (haidan et al., 2016). the history of herbal medication is equally old as human history. most of these plant-derived drugs were originally identified through the subject of traditional remedies and folk knowledge of indigenous people and some of these could not be substituted despite the tremendous progress in synthetic chemistry. therefore, plants can be depicted as a major source of medicines, not merely as isolated active principles in standardized dosage form but also as crude drugs for the population. modern medicines and herbal medicines are complimentary being used in areas for health care program in various developing countries including india (sen, 2017). in the present scenario, the demand for herbal products is growing exponentially throughout the globe and major pharmaceutical companies are currently carrying on extensive research on plant materials for their potential medicinal value (ekor, 2013). the need of new therapies for glycemic control is the fact that existing treatments have limitations because of their side effects (chaudhary, 2017). the herbal extracts which are effective in lowering blood glucose level with minimal or no side effects are known to be used as antidiabetic remedies (kooti, 2016.). diabetes mellitus is a growing problem worldwide entailing enormous financial burden and medical care policy issues (karachaliou, 2020.). according to international diabetes federation (idf), the number of individuals with diabetes in 2010 crossed 375 million, with an estimated 4.5 million deaths each year, (cho et al., 2018.). according to the world health organization (who), up to 80% of the population in developing countries uses plants and its products as traditional medicine for primary health care (oyebode et al., 2016). the who has listed 26,000 plants, which are used for medicinal purposes around the world. among these, 3500 species are in india (anand et al., 2019). there are about 800 plants which have been reported to show antidiabetic potential. a wide collection of plant derived active principles representing numerous bioactive compounds have established their role for possible use in the treatment of diabetes (tran et al., 2020). hptlc study helps to gain idea about chromatographic pattern of the components. current study involves use of hptlc parameters to study changes in phytoconstituents. macroscopic and microscopic study was performed (thomas et al., 2020). the present study was aim to evaluate the physiochemical fluctuations in the leaves of a. vasica and the vasicine, an antidiabetic constituent in these leaves. different analytical methods are available and reported, such as hptlc for estimation of vasicine extracted from leaves in different seasons. (narayana et al., 1995, soni et al., 2008, das et al., 2005). materials and methodsmaterials and methodsmaterials and methodsmaterials and methods the plant material was collected in every month of the year i.e. in the rainy season (june, july, august, september), winter (october, november, december, january) and summer (february, march, april, may), from places of different altitude i.e. low (560 meters), medium (920 meters) and high (1,312 meters), twice i.e. morning and evening. a voucher specimen (no. bsi/wrc/100-1/tech./2019/02) was deposited in the herbarium of botanical survey of india, pune. this study involves collection of data for changes of active components in alkaloidal and anti-diabetic plant due to season, time and places. the plant was authenticated by botanical survey of india, pune. morphological and microscopic study and phytochemical screening was performed. plant material collected in gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 3 different seasons, time and places was extracted successively and as the percent yield of ethanol meth found to be more as compare to other solvent extracts so ethanolic extract content was used for further analysis. assessment of quality of plant materials the plant materials were assessed as per who guideline. fresh plant parts were subjected to color, odor and taste, determination of shape, size, surface characteristics and appearance. for microscopically examinations, free hand sections of the fresh leaf were cut, cleared with chloral hydrate solution and water, and stained with a drop of hydrochloric acid and phloroglucinol. photomicrographic images were taken by using trino cxr camera. quantitative microscopy leaves were subjected to quantitative microscopy for the following values using reported method stomatal number, stomatal index, palisade ratio, vein islet number, vein termination number. proximate analysis proximate analysis of powdered plant material was carried out using reported methods. following determinations were done; foreign organic matter, loss on drying, total ash, water soluble ash, acid insoluble ash, sulphated ash, water soluble extractives, alcohol soluble extractives, ether soluble extractive value. phytochemical screening the air-dried powder (1 kg) of plant was extracted in soxhlet apparatus with solvents of increasing polarity as follows: petroleum ether; chloroform; ethyl acetate; ethanol. each time before extracting with the next solvent, the material was dried. all the extracts were concentrated by distilling the solvent and the extracts were dried on water bath. then consistency, color, appearance of the extracts and their percentage yield were noted. establishment of qualitative phytoprofile of successive solvent extracts the extracts obtained from successive solvent extraction were then subjected to various qualitative chemical tests to determine the presence of various phytoconstituents like alkaloids, glycosides, carbohydrates, phenolics and tannins, proteins and amino acids, saponins and phytosterols using reported methods (table 1). table 1.table 1.table 1.table 1. hptlc analysishigh performance thin layer chromatography plant name phyto constituent std. area (under curve) mobile phase wavelength (nm) a. vasica vasicin 5053.5 au ethyl acetate: methanol: ammonia (8:2:0.2) 254 results results results results and discussionand discussionand discussionand discussion plant leaf color is green, characteristic odor and taste, shape is oblong simple, petiolate, exstipulate entire margin, a tapering base and acuminate apex, leathery touch, smooth and shining texture. cell wall is single layered epidermis made up of compactly arranged barrel shaped parenchymatous cells. vascular bundle is arc shaped, conjoint, collateral and closed. gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 4 table 2. table 2. table 2. table 2. monthly variation with high altitude at morning (% of alkaloids) n= 3 p< 0.05. table 3.table 3.table 3.table 3. monthly variation with altitude at morning (% yield mg/g) n= 3 p< 0.05. enclosed by a parenchymatous bundle sheath. vessels with pitted thickening, anomocytic or anisocytic stomata, glandular, multicellular uniseriate (50 to 120µ) trichomes, prismatic calcium oxalate crystals and starch grains are present. chemical constituents in leaf shows presence of alkaloids and its percentage varies at different season, time and places (table 2; figure 1). table 4.table 4.table 4.table 4. concentration and area under curve (auc) concentration (µg/ml) area (auc) 0.2 379.9 0.4 550.8 0.6 730 0.8 887 1 1049.9 table 5.table 5.table 5.table 5. hptlc analysis of a. vasica leaf extract rfrfrfrf valuevaluevaluevalue seasonseasonseasonseason areaareaareaarea (au)(au)(au)(au) yield (mg/g)yield (mg/g)yield (mg/g)yield (mg/g) 0.49 summer 1868 1.89 rainyrainyrainyrainy 3149314931493149 3.203.203.203.20 winter 2329 2.36 hptlc chromatogram of vasicine % yield mg/g in leaf extracts show variations at different season, time and places. in months of june-sept. (rainy season) at morning time with high altitude place it shows more yield. the values are expressed as mean sem; p<0.05, (two-way anova followed by tukey’s multiple comparison test) (table 3; figure 2). calibration curve (figure 3) show variations of concentration of vasicine with area under curve. (table 4; figure 3). the presence of vasicin in ethanolic extract was confirmed by hptlc fingerprinting and the yield was 3.20 mg/ gm at rf value 0.49 (table 5; figures 4, 4-a, 4-b and 4-c). altitude month 1 2 3 4 5 6 7 8 9 10 11 12 low 4.30 ±0.57 5.00 ±1.00 5.05 ±1.00 5.50 ±1.00 5.70 ±1.00 5.05 ±1.00 5.33 ±1.52 5.37 ±1.52 5.32 ±1.15 5.50 ±1.00 4.27 ±1.15 4.78 ±1.15 medium 4.34 ±0.57 5.07 ±1.00 5.04 ±1.00 5.02 ±1.00 5.01 ±1.00 5.06 ±1.00 5.03 ±1.52 5.33 ±1.52 5.36 ±1.15 5.04 ±1.00 4.72 ±1.15 4.76 ±1.15 high 4.76 ±0.57 5.34 ±0.57 5.33 ±0.57 5.36 ±0.57 5.39 ±0.57 5.34 ±0.57 5.07 ±1.15 5.75 ±1.15 5.73 ±0.57 5.39 ±0.57 5.05 ±1.00 5.02 ±1.00 altitude month 1 2 3 4 5 6 7 8 9 10 11 12 low 1.00 ±0.20 1.10 ±0.26 1.25 ±0.55 1.43 ±0.11 1.82 ±0.05 2.10 ±0.05 2.27 ±0.05 2.34 ±0.05 2.23 ±0.11 1.42 ±0.05 1.25 ±0.10 1.62 ±0.60 medium 1.624 ±0.20 1.56 ±0.32 1.84 ±0.65 2.03 ±0.10 2.46 ±0.11 2.78 ±0.05 2.84 ±0.05 2.93 ±0.05 2.82 ±0.15 1.97 ±0.10 1.72 ±0.10 2.15 ±0.60 high 2.03 ±0.58 1.84 ±0.05 2.17 ±0.60 2.44 ±0.11 2.83 ±0.05 3.17 ±0.05 3.22 ±0.05 3.36 ±0.05 3.24 ±0.11 2.49 ±0.05 2.23 ±0.10 2.32 ±0.05 gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 5 figure 1.figure 1.figure 1.figure 1. monthly variation with altitude (morning) med.= medium (altitude) figure 2.figure 2.figure 2.figure 2. monthly variation of with altitude (morning) mg/gm= milligram/ gram, med= medium (altitude) figure 3.figure 3.figure 3.figure 3. calibration curve of vasicine gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 6 (a) (b) (c) gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 7 (d) figure 4.figure 4.figure 4.figure 4. densitogram of vasicine (a); densitogram of vasicine from ethanolic extract (summer) (b); densitogram of vasicine from ethanolic extract (rainy) (c); densitogram of vasicine from ethanolic extract (winter) (d) discussiondiscussiondiscussiondiscussion the study of morphological, microscopical and physico-chemical parameters of adhatoda vasica help to differentiate the plant from its other species. the pharmacognostic profile of plants presented here may be useful to supplement information with regard to its identification and will be helpful in establishing standardization criteria. present work is an attempt to compile data regarding variations of chemical constituents due to seasonal changes in selected plants i.e. adhatoda vasica l. the plant belongs to alkaloid category and possessing antidiabetic activity. the plant was authenticated by botanical survey of india, pune. morphological and microscopic study was performed. the powdered drugs were subjected to phytochemical screening. plant material in different seasons was extracted successively and as the percent yield of ethanolic extract found to be more as compare to other solvent extracts and according to solubility of selected phytoconstituents in ethanol, ethanolic extract was selected for further analysis. qualitative chemical examination of extracts revealed presence of alkaloids, and other chemical components. literature study proves that these constituents have antidiabetic activity. the presence of vasicine in ethanolic extract of plant was confirmed by hptlc fingerprinting and the content yield was calculated from au. it was observed that, in different season’s places and time, there is a change in hptlc pattern of the constituents i.e. in rainy season from june to september, at high altitude and at morning 6 am vasicine content was found more. it helps to identify best season and place for collection of plant material from the source so as to gain high yield of active component and to increase the efficacy of the formulation. conclusionsconclusionsconclusionsconclusions seasonal variation is associated with the vegetative and reproductive stages of the plant, it has direct influence with the variation in chemical constituents of the plants. as per ayurveda, there exists a huge gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 8 collection of plants with antidiabetic potential. only few of them have been scientifically proven and a lot more have yet to be explored and proved. a. vasica have shown varying degrees of hptlc chromatogram for vasicine and hence affects hypoglycemic potency in different seasons of collection. future studies may target isolation, purification, and characterization of bioactive compounds present in these plants and formulation of a potent antidiabetic dosage form. the outcome of such studies may provide a starting point for selection of a particular season for collection of raw material to develop potential antidiabetic drugs. future studies may target isolation, purification, and characterization of bioactive compounds present in these plants and formulation of a potent antidiabetic dosage form. the outcome of such studies may provide a starting point for selection of a particular season for collection of raw material to develop potential antidiabetic drugs. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions both authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this work was supported by the botanical survey of india for authentication of plant samples. b.v.d.u.’s poona college of pharmacy for analytical study of extracts, jspm’s jayawantrao sawant college of pharmacy and research, hadapsar, pune for providing laboratory facilities to perform the research work. list of abbreviationslist of abbreviationslist of abbreviationslist of abbreviations a.u.c.area under curve bsibotanical survey of india f.o.m.foreign organic matter hptlchigh performance thin layer chromatography i.d.f.international diabetes federation l.o.d.loss on drying otcover the counter r & dresearch and development t. s.transverse section u.sunited states whoworld health organization conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 9 referencesreferencesreferencesreferences anonymous (2004). review on indian medicinal plants. vol i. indian council of medical research, new delhi, pp 258. arun c, chitharanjan d, vijaya r, dendi k, aditya c, rahul r, … wasique m (2017). clinical review of antidiabetic drugs: implications for type 2 diabetes mellitus management. frontiers in endocrinology 8(6):1-12. https://doi.org/10.3389/fendo.2022.891927 asha t, abhilash k, adinath s, sanjeevani d, lata k (2020). a high-performance thin layer chromatographic method using a design of experiment approach. turkish journal of pharmaceutical sciences 17(2):148-158. https://doi.org/10.4274/tjps.galenos.2018.80958 das c, poi r, chowdhary a (2005). hptlc determination of vasicine and 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naoufal l (2019). a comprehensive review on medicinal plants as antimicrobial therapeutics: potential avenues of biocompatible drug discovery. metabolites 9(11):258. https://doi.org/10.3390/metabo9110258 wesam k, maryam f, zahra a, damoon a, majid a (2016). the role of medicinal plants in the treatment of diabetes. electronic physician 8(1):1832-1842. https://doi.org/10.19082/1832 gautam p and pitchaimuthu s (2023). not sci biol 15(1):11368 10 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions 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received: 02 feb 2022. received in revised form: 14 dec 2022. accepted: 06 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania sharma b and alam a (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11199 doi:10.15835/nsb15111199 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae biogenesis of silver nanoparticles from the shoot extract of biogenesis of silver nanoparticles from the shoot extract of biogenesis of silver nanoparticles from the shoot extract of biogenesis of silver nanoparticles from the shoot extract of delonix delonix delonix delonix regiaregiaregiaregia its characterization (uvits characterization (uvits characterization (uvits characterization (uv––––vis spectroscopy and sem) and vis spectroscopy and sem) and vis spectroscopy and sem) and vis spectroscopy and sem) and evaluation for antimicrobial potentialevaluation for antimicrobial potentialevaluation for antimicrobial potentialevaluation for antimicrobial potential bhawana sharma, afroz alam* banasthali vidyapith, department of bioscience and biotechnology, rajasthan-304022 india; bhawana.sharma1230@gmail.com; afrozalamsafvi@gmail.com (*corresponding author) abstractabstractabstractabstract the current research work explores the production of silver nanoparticles using aqueous extracts of delonix regia (boj. ex hook.) raf. (angiosperms; fabaceae) shoots for the bioreduction of ag metal and its antimicrobial activity. fourier transform infrared spectroscopy (ftir), zeta potential, ultraviolet-visible spectroscopy, scanning electron microscopy (sem), and x-ray diffraction (xrd) have been used to evaluate the produced silver nanoparticles (agnps). both antibacterial and antifungal activity were examined against bacterial and fungal pathogens, viz., escherichia coli and bacillus subtilis, and fungal strains, viz., fusarium oxysporum and aspergillus niger. the presence of silver nanoparticles was observed by the color change, i.e., from pale yellow to dark brown. the zeta potential observed for the produced nanoparticle is -18mv. the sem and xrd revealed the size of synthesized agnps, i.e., 35nm and sem size lies in the range of 40-60 nm. uvvisible absorption spectra were found at wavelength 425 nm. the synthesized nanoparticles are cost-efficient and could be an alternative procedure for the peculiar production of nanoparticles and also act as potential antimicrobial agents. keywkeywkeywkeywords:ords:ords:ords: agnps; antimicrobial activity; delonix regia; ftir; sem; xrd introductionintroductionintroductionintroduction silver nanoparticles (agnps) are gaining popularity in a variety of fields, including medicine, food, industrial applications, and healthcare. they are typically accomplished through a variety of chemical and physical methods such as a specific rod position, laser ablation, and pyrolysis, all of which appear to be extremely dangerous and costly (singh et al., 2010). biogenic silver nanoparticle (agnps) synthesis is efficient, sustainable, cost-effective, and ecofriendly, and has proven to be one of the viable and novel mechanisms for the peculiar production of silver nanoparticles (agnps). in recent years, various metals and materials have been involved in the production of nanoparticles from algae, fungi, bacteria, viruses, and several plants (valli and geetha, 2016). the determination of biological activity of agnps is done under various parameters, which include particle size, shape, and composition, which are used for determining cytotoxicity. due to the attractive physicochemical properties of silver nanoparticles, they have great potential in the fields of medicine and https://www.notulaebiologicae.ro/index.php/nsb/index sharma b and alam a (2023). not sci biol 15(1):11199 2 biology (akter et al., 2018). metallic nanoparticles have exhibited enhanced antioxidant, antimicrobial, and numerous other properties due to their small size and shape, high surface area, and distinct characteristics (mubarak ali et al., 2011; dikshit et al., 2021). delonix regia (boj. ex hook.) raf. (family fabaceae), is a large, medium-sized ornamental tree with fernlike, bipinnately compound leaves. it is commonly known as royal poinciana, flamboyant (english), and gulmohar (hindi). it is mainly found in madagascar, africa, northern australia, and india as a semideciduous tree (latif et al., 2019). distinct parts of the d. regia plant, like leaves, roots, bark, and flowers, are used for the treatment of various ailments, like fever, bronchitis, anemia, pneumonia, diabetes, and inflammation. it exhibits various physiologically active chemical components such as lupeol, coumarin, carotene, lycopene, sterols, gallic acids, quercetin, etc. d. regia is generally used for treating fungal and bacterial infections, inflammation, and liver diseases (siddiquee et al., 2020). therefore, the aim of this study was to analyze the production of environmentally compatible silver nanoparticles through d. regia shoot extract. different techniques were used for the characterization of silver nanoparticles, such as uv, sem, xrd, and their antimicrobial activity. phytoconstituents of delonix regia the plant consists of various pharmacologically active compounds like phenolic compounds that possess many anti-inflammatories, anti-ulcer, anti-microbial, anti-fungal, anti-bacterial, and anti-helmitic properties that are valuable or beneficial in a variety of health conditions (bhorga and kamble, 2019). phytochemical screening results showed the presence of various bioactive constituents, like leaves that contain alkaloids, carbohydrates, and tannins; bark and shoots that contain terpenoids, flavonoids, tannins, alkaloids, and sterols; and flowers that contain saponins, steroids, carotenoids, and tannins. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods hi-media was used to make silver nitrate (agno3). many other reagents, solvents, and materials used are of analytic grade. for the characterization of silver nanoparticles, both milli-q water and double-distilled water were used. collection and identification of plant extract the shoot of d. regia was collected from the campus of banasthali vidyapith (tonk), rajasthan, india. the authentication number of the reference specimen is buri-1407/2022, and it was deposited in the herbarium of banasthali university, rajasthan, india (buri). preparation of d. regia shoot extract the collected shoots were rigorously washed with running tap water afterwards using milli-q water. before the pulverization process, the shoots were properly dried at room temperature for 10 days so that no moisture content was present. to prepare the shoot extract, 10 g of finely powdered shoot was mixed with 250 ml of deionized water and boiled at 60 °c for 30 min. further, the resulting extract was cooled to room temperature before being filtered with whatman no. 1 filter paper. for subsequent research, the extract which was properly filtered is retained at 4 °c (das et al., 2017). preparation of biogenic silver nanoparticles following the bioreduction of silver nanoparticles (agnps), a 1 mm aqueous solution of agnps is produced. a 10 ml d. regia shoot extract was mixed with 90 ml of a 1 mm agno3 solution and incubated at sharma b and alam a (2023). not sci biol 15(1):11199 3 28 °c for 24 hours. this bioreduction of agno3 can be visually apparent by the change in color pattern, i.e., from pale yellow to dark brown, as depicted in figure 1. the confirmation of bioreduction was done by the uv visible spectra analysis pathway (bharathi et al., 2018). figurefigurefigurefigure 1. a1. a1. a1. a. delonix regia shoot extract; b. b. b. b. formation of silver nanoparticles descriptive characterization of silver nanoparticles (agnps) ultravioletvisible spectrophotometer the synthesis of agnps was carried out by using a uv-visible spectrophotometer, noticing a distinctive surface plasmon resonance (spr) peak at a scanning range of 200–800 nm and a speed of 200 nm/min. the dilution of the colored reaction samples was done with deionized water and scanned in ultra-violet visible spectra with a 1 cm quartz cuvette at 25 °c (chouhan and guleria, 2020). x-ray diffraction studies (xrd) the bruker eco d8 advance x-pert pro was used to figure out the x-ray diffraction of silver nanoparticles at 40 kv of voltage and a current strength of 20 ma (mittal et al., 2012). debyescherrer equation was used to calculate x-ray diffraction d= κλ/β cosθ where, d denotes crystal size κ = shape factor (0.94) β = full width in radians at half maximum λ = denotes the x-ray wavelength (1.5418ă) particle size analyzer after 24–48 h of incubation, the particle size was measured using a zetasizer (santhoshkumar et al., 2011). fourier transform infrared spectroscopy (ftir) ftir analysis was used to investigate the identification of functional groups on the surface of silver nanoparticles by a shoot extract of d. regia within an isolation distance of 4 cm (reddy et al., 2019). scanning electron microscopy (sem) the morphology of d. regia stabilized nanoparticles was analyzed by scanning electron microscopy (ahmed et al., 2016). antimicrobial assay the antimicrobial activity of delonix regia shoot extract and its agnps was explained by the disc diffusion method. both potato dextrose agar (pda) and nutrient agar media (na) were used for the sharma b and alam a (2023). not sci biol 15(1):11199 4 preparation of fungal and bacterial species. the agnps, agno3, and antibiotics were loaded into different filter paper discs. the discs were placed on nutrient agar media with bacterial culture and incubated for 12 h at 37 °c, whereas the discs were placed on potato dextrose agar with fungal culture and incubated for 24 hours at 28 °c. after 12 or 24 h, both bacterial and fungal cultures were checked, and the zone of inhibition was recorded (rathi sre et al., 2015). results results results results characterization of agnps ultraviolet visible spectroscopy after 24 h of observation, the ultraviolet-visible absorption spectra of colloidal silver nanoparticles (agnps) were shown. the peak intensity is found near 425 nm, indicating the formation of silver nanoparticles. the agitation of the surface plasmon resonance (spr) band in the u.v. visible region results in the color change from pale yellow to dark brown (aqueous solution), as depicted in figure 2. figure 2figure 2figure 2figure 2. uv visible spectrophotometer of shoot extract of delonix regia (agnps) x-ray diffraction (xrd) the xrd pattern of crystalline agnps is described in figure 3. sharp peaks of agnps were observed at 38.5°, 39.94°, 44.36°, 64.42°, and 77.44°. the calculation of average sizes of crystallized agnps was done using the debye-scherrer equation, and the size of agnps was observed to be 35.5 nm, which confirms the crystalline nature of synthesized silver nanoparticles (agnps). sharma b and alam a (2023). not sci biol 15(1):11199 5 figure 3figure 3figure 3figure 3. x-ray diffraction pattern of agnps using delonix regia shoot extract particle size analyzer the sharp peak of the d. regia shoot extract was found to be 88.94 nm. the obtained results revealed that the diameter of agnps is 129.6 d (nm), as depicted in figures 4 and 5. the zeta potential of d. regia shoot extract was investigated and found to be -18.7 mv. figure 4. figure 4. figure 4. figure 4. particle size of silver nanoparticles (agnps)using delonix regia sharma b and alam a (2023). not sci biol 15(1):11199 6 figure 5figure 5figure 5figure 5. zeta potential of agnps (delonix regia shoot extract) fouriertransform infrared spectroscopy (ftir) for the identification of the chemical bonds that are present in biogenic agnps, the ftir tool is used. the spectra of d. regia shoot extract exhibit various absorption bands at 3367, 2920, 1630, and 1472 cm-1. the band is observed at 3367 cm1 due to ohstretching vibrations. the different bands at 2920, 1630, and 1472 cm-1 indicate the existence of aromatic, carbonyl, and amide groups, as well as alkane groups (-c-h stretching). the minor bands at 787, 733, and 683 cm-1, which are attributes of aromatic phenols, indicate the presence of c-h out of plane bend regions as depicted in figure 6. figure 6figure 6figure 6figure 6. fourier transform infrared spectroscopy of agnps using delonix regia shoot extract scanning electron microscopy (sem) the investigation of the topology and size of the prepared samples was done through sem. the results explained that the crystallized synthesis of silver nanoparticles exists in a rectangular shape, i.e., an average size of 40–60 nm, as depicted in figure 7. sharma b and alam a (2023). not sci biol 15(1):11199 7 figure 7figure 7figure 7figure 7. scanning electron microscopy of synthesized agnps using delonix regia shoot extract antimicrobial activity the antibacterial properties were observed against pathogenic bacterial strains, viz., escherichia coli, bacillus subtilis, while the antifungal activity was done using fungal strains, viz., fusarium oxysporum and aspergillus niger. the disc diffusion method, as shown in figures 8 and 9, was used for both activities. thus, the findings indicate that the bacterial pathogen escherichia coli was detected to have a maximum zone of inhibition in comparison to bacillus subtilis (table 1). in terms of antifungal activity, aspergillus niger had the greater zone of inhibition when compared to fusarium oxysporum (table 2). a. escherichia coli b. bacillus subtilis figure 8.figure 8.figure 8.figure 8. antibacterial activity of silver nanoparticles using delonix regia shoot extract sharma b and alam a (2023). not sci biol 15(1):11199 8 a. fusarium oxysporum b. aspergillus niger figure 9figure 9figure 9figure 9. antifungal activity of shoot extract of delonix regia table 1. table 1. table 1. table 1. antibacterial activity of synthesized silver nanoparticles (agnps) s. no. bacterial species zone of inhibition (mm) agnps (pe) antibiotics (chloramphenicol) control (agno3) 1. escherichia coli 13±0.21b 22±0.35c 12±0.08a 2. bacillus subtilis 12 ±0.10b 21±0.29c 10±0.09a table 2table 2table 2table 2. antifungal activity of synthesized agnps s. no. fungal species zone of inhibition(mm) agnps (np) antibiotics (fluconazole) control (agno3) 1. aspergillus niger 14±0.25b 25±0.46c 12± 0.09a 2. fusarium oxysporum 10 ±0.08b 20± 0.32c 11 ±0.08a statistical analysis the data are shown as averages of three replicates (n = 3). all obtained data were evaluated by ibm spss statistics 20 software. three-way interactions were executed between the chosen variables. for each output variable, multiple-comparison a tukey's p<0.05 post hoc test was performed to compare the variance of the data. all data are presented as means standard deviation. sharma b and alam a (2023). not sci biol 15(1):11199 9 discussiondiscussiondiscussiondiscussion scientists are very interested in nanoparticles' significant antimicrobial impact because of the increasing microbial resistance to antibiotics and the development of antimicrobial resistant strains. one of the most encouraging approaches for avoiding microbial resistance is the use of nanoparticles, as they have the capability to fight drug resistance via multiple approaches. silver nanoparticles (agnps) are highly useful in this context as they are effective antibacterial entities due to their enhanced reactivity, resulting from their high surface-tovolume ratio (almatroudi, 2020). hence, biosynthesized agnps emerge as one of the most viable alternative methods for the curing of microbial infections. in present study, we have investigated the uv-visible absorption spectra found near the wavelength of 425 nm. the particle size indicated its diameter, i.e., 129.6 nm, and the zeta potential of synthesized silver nanoparticles was investigated to be -18mv. the x-ray diffraction of synthesized silver nanoparticles revealed the intense peak at 38.50°, 39.94°, 44.36°, 64.42° and 77.44° and the average size of crystallized agnps was 35 nm. sem study of silver nanoparticles disclosed that they are rectangular in shape and their size ranges from 40-60 nm. the fourier transform-infrared spectrum of d. regia shoot extract revealed different peaks at 3363, 2920,1630 and 1472 cm-1 which indicates the presence of bonded oh-group, alkanes, carbonyl and amide bands. the antimicrobial activity showed the maximum zone of inhibition found in the bacterial activity, viz., e. coli and in fungal activity, the maximum zone of inhibition was found in a. niger. the aqueous shoot extract of d. regia was known to be an effective reducing and capping agent promoting efficient reduction of agnps and production of crystalline, spherical and stable at much lower concentration of plant extracts. similar observations were noticed by other workers (abu-dief et al., 2021; bala et al., 2017; anitha and sakthivel, 2016). conclusionsconclusionsconclusionsconclusions the synthesis of silver nanoparticles has been found to be environmentally friendly and cost-effective, with numerous applications in fields such as biosensors, cosmetics, preservatives, antimicrobials, and so on. the aqueous shoot extract of delonix regia was known to be an effective bio reducing and capping agent, promoting efficient reduction of silver nanoparticles and the production of crystalline, spherical, and stable plant extracts and silver salts at much lower concentrations. various techniques, such as xrd, ftir, and sem, are used to characterize the biogenic synthesis of agnps. according to the findings of this study, plant-mediated synthesis of biogenic agnps has a variety of antimicrobial properties that are useful in pharmaceuticals and medical applications. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions both authors came up with the idea and designed the piece. sharma b carried out the experiment under alam a's direct supervision. sharma b performed all statistical analysis and drafted the manuscript. both authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. sharma b and alam a (2023). not sci biol 15(1):11199 10 acknowledgementsacknowledgementsacknowledgementsacknowledgements the authors would like to thank professor ina shastri, vice-chancellor, banasthali vidyapith, rajasthan, for her support and encouragement. we would also like to thank dst for providing networking assistance through the fist programme at the department of bioscience and biotechnology, banasthali vidyapith, as well as the dbt-funded bioinformatics center. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences abu-dief am, abdel-rahman lh, abd-ei sayed ma, zikry mm (2021). green synthesis of silver nanoparticles using delonix regia extract, characterization and its application as adsorbent for removal of cu(ll) ions from aqueous solution. asian journal of applied chemistry research 9(1):1-15. https://doi.org/10.9734/ajacr/2021/v9i130202 ahmed s, saifullah, ahmad m, swami bl, ikram s (2016). green synthesis of silver nanoparticles using azadirachta indica aqueous leaf extract. journal of radiation research and applied sciences 9(1):1-7. http://doi.org/10.1016/j.jrras.2015.06.006 akter m, sikder tm, rahman mm, ullah aa, hossain kfb, banik s, hosokawa t, saito t, kurasaki m (2018). a systematic review on silver nanoparticlesinduced cytotoxicity: physicochemical properties and perspectives. journal of advanced research 9:1-16. https://doi.org/10.1016/j.jare.2017.10.008 almatroudi a (2020). silver nanoparticles: synthesis, characterization and biomedical applications. open life sciences 15(1):819-839. https://doi.org/10.1515/biol-2020-0094 anitha p, sakthivel p (2016). synthesis and characterization of silver nanoparticles using delonix elata leaf extract and its anti-inflammatory activity against human blood cells. international journal of engineering, science and technology 4(1):330-336. bala sd, sathiyaseelan mf, dons t (2017). green synthesis of silver nanoparticles using delonix regia leaf extract and evaluation of their antimicrobial efficacy. world journal of pharmaceutical sciences 5(3):209-215. http://www.wjpsonline.org/ bharathi d, josebin md, vasantharaj s, bhuvaneshwari v (2018). biosynthesis of silver nanoparticles using stem bark extracts of diospyros montana and their antioxidant and antibacterial activities. journal of nanostructure in chemistry 8(1):83-92. http://doi.org/10.1007/s40097-018-0256-7 bhorga ph, kamle s (2019). comparative phytochemical investigation and determination of total phenols and flavonoid concentration in leaves and flowers extract of delonix regia (boj. ex. hook). journal of drug delivery and therapeutics 9(4-s):1034-1037. https://doi.org/10.22270/jddt.v9i4-s.3761 chouhan s, guleria s (2020). green synthesis of agnps using cannabis sativa leaf extract: characterization, antibacterial, anti-yeast and α-amylase inhibitory activity. materials science for energy technologies 3:536-544. https://doi.org/10.1016/j.mset.2020.05.004 das rk, pachapur vl, lonappan l, naghdi m, pulicharla r, maiti s, brar sk (2017). biological synthesis of metallic nanoparticles: plants, animals and microbial aspects. nanotechnology for environmental engineering 2(1):1-21. http://doi.org/10.1007/s41204-017-0029-4 dikshit pk, kumar j, das ak, sadhu s, sharma s, singh s, gupta pk, kim bs (2021). green synthesis of metallic nanoparticles: applications and limitations. catalysts 11(8):902. https://doi.org/10.3390/catal11080902 latif ms, abbas s, kormin f, mustafa mk (2019). green synthesis of plant-mediated metal nanoparticles: the role of polyphenols. asian journal of pharmaceutical and clinical research 12(7):75-84. http://doi.10.22159/ajpcr.2019.v12i7.33211 sharma b and alam a (2023). not sci biol 15(1):11199 11 mittal ak, kaler a, banerjee uc (2012). free radical scavenging and antioxidant activity of silver nanoparticles synthesized from flower extract of rhododendron dauricum. nano biomedicine and engineering 4(3):118-124. mubarakali d, thajuddin n, jeganathan k, gunasekaran m (2011). plant extract mediated synthesis of silver and gold nanoparticles and its antibacterial activity against clinically isolated pathogens. colloids and surfaces b: biointerfaces 85(2):360-365. http://doi.10.1016/j.colsurfb.2011.03.009 reddy p, ruj s, fathima n (2019). ecofriendly synthesis of silver nanoparticles using delonix regia flower extract and its characterization by uv and sem and in vitro study on its toxicity and antioxidant potential. international journal of pharmacy and biological sciences 9(1):21-33. santhoshkumar t, rahuman aa, rajakumar g, marimuthu s, bagavan a, jayaseelan c, kamaraj c (2011). synthesis of silver nanoparticles using nelumbo nucifera leaf extract and its larvicidal activity against malaria and filariasis vectors. parasitology research 108(3):693-702. http://doi.10.1007/s00436-010-2115-4 siddiquee ma, ud din parray m, mehdi sh, alzahrani ka, alshehri aa, malik ma, patel r (2020). green synthesis of silver nanoparticles from delonix regia leaf extracts: in-vitro cytotoxicity and interaction studies with bovine serum albumin. materials chemistry and physics 242:122493. http://doi.10.1016/j.matchemphys.2019.122493 singh a, jain d, upadhyay mk, khandelwal n, verma hn (2010). green synthesis of silver nanoparticles using argemone mexicana leaf extract and evaluation of their antimicrobial activities. digest journal of nanomaterials and biostructures 5(2):483-489. rathi sre pr, reka m, poovazhagi r, arul kumar m, murugesan k. (2015). antibacterial and cytotoxic effect of biologically synthesized silver nanoparticles using aqueous root extract of erythrina indica lam. spectrochimica acta part a: molecular and biomolecular spectroscopy 135:1137-1144. http://doi.10.1016/j.saa.2014.08.019 valli g, geetha s (2016). green synthesis of copper nanoparticles using cassia auriculata leaves extract. international journal of chem tech research 2:5-10. the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11451 nsb el boukhari 2023.03.16.docx received: 23 jan 2023. received in revised form: 03 mar 2023. accepted: 10 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania el boukhari a et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11451 doi:10.15835/nsb15111451 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae flow cytometry and chromosome numbers variation in argan flow cytometry and chromosome numbers variation in argan flow cytometry and chromosome numbers variation in argan flow cytometry and chromosome numbers variation in argan tree tree tree tree argania spinosaargania spinosaargania spinosaargania spinosa (l.) skeels(l.) skeels(l.) skeels(l.) skeels ali el boukhari 1,2, salma tabi1,4, abdelhamid el mousadik2, rachida el boullani2, abdelghani tahiri1, meriyem koufan1, hamid benyahia3, rachid bouharroud1*, naima ait aabd1* 1regional center of agricultural research of agadir, national institute of agricultural research (inra), avenue ennasr, b.p. 415 rabat principale, rabat 10090, morocco; abdelghani.tahiri@inra.ma; meriyem.koufan@inra.ma; rachid.bouharroud@inra.ma (*corresponding author); naima.aitaabd@inra.ma (*corresponding author) 2ibn zohr university, faculty of sciences, laboratory of biotechnology and valorization of natural resources, pob 8106, agadir, morocco; ali.elboukhari@edu.uiz.ac.ma; a.elmousadik@uiz.ac.ma; r.elboullani@uiz.ac.ma 3regional center of agronomic research of kenitra, national institute of agronomic research (inra), avenue ennasr, b.p. 415 rabat principale, rabat 10090, morocco; hamid.benyahia@inra.ma 4sultan my sliman university, faculty of sciences and technics, laboratory of natural resources, environment and health, b.p 523, beni mellal, morocco; salma.tabi@usms.ma abstractabstractabstractabstract argania spinosa l. skeels is an endemic species of west-central morocco, which is characterized by a high diversity of morphological and genetic traits. it constitutes a natural resource for oleo-agro-sylvo-pastoral uses. all conservation and genetic breeding strategies aimed to domesticate argan require a good knowledge of the plant material. however, several studies focused on agronomical, morphological, phytochemical, and molecular characterization, while the cytogenetic aspects were less investigated. the objective of this work is to identify the chromosome number and ploidy level on the national argan collection at the agadir regional agronomic research center, morocco. the determination of the chromosome number was carried out on root tips of germinated seeds collected from five trees genotypes selected on various morphological aspects. as a result, chromosome count on active root tip cells showed variation in the number (2n = 20; 2n = 22; 2n = 24) with a stable ploidy level (2n = 2x) that is confirmed by flow cytometry. these results combine two previous findings (2n=20, 2n=24) and reveal a third existence of twenty-two chromosome. as a conclusion, a. spinosa has three chromosomal numbers which represent the genetic diversity of the chromosomal number that this species exhibits. more studies are required to explain this variation on chromosome numbers for future breeding programs and to avoid incompatibilities. keywords:keywords:keywords:keywords: argane tree; chromosome number; cytogenetic; flow cytometry; genetic diversity https://www.notulaebiologicae.ro/index.php/nsb/index el boukhari a et al. (2023). not sci biol 15(1):11451 2 introductionintroductionintroductionintroduction argania spinosa (l.) skeels (sapotaceae), the only representative of the family in morocco, is an allogamous monoecious tree with entomophilic pollination (nerd et al., 1998; ajerrar et al., 2020). it is an endemic species of west-central morocco; its forest area is estimated to 800,000 hectares (msanda et al., 2021). the arganereae offers several ecosystem services, namely the storage and sequestration of carbon, habitats of several endemic species, contributes to the conservation of biodiversity, improves soil fertility, prevents soil erosion, regulates and creates microclimates (karmaoui, 2016). the biosphere reserve of argania covers an area of 2,560,000 ha (msanda et al., 2021), in which the local population (3 million people, including 2.2 million in rural areas) exploits this natural resource for energy purposes (wood and shell) as well as a fodder resource (leaves, and fruit pulp). therefore, they are dependent on the exploitation of argan forests (laaribya et al., 2017). the economic value of the argan tree is based mainly on its almonds, from which highly marketable oil is extracted (m'hirit, 1987). argan oil is very known for its quality, with an interesting saponifiable composition, excellent effects, and several benefits (berrougui et al., 2003; drissi et al., 2006; koufan et al., 2020a). argane’s shells are used in combustion for heating and even as a bio-composite base. biochar from argan shells enriches the soil by improving nutrient and water retention (bouqbis et al., 2016). in addition, the pulp, bark, leaf, root, and even press cake have interesting medicinal uses (moukal, 2004). because of its importance and for its protection, unesco declared the arganereae on december 8th, 1998, as the first biosphere reserve in morocco. in 2021, unesco declared may 10th the international day of the argane tree. well known for its high genetic diversity, efforts of scientific research achieved detection and characterization of specie’s genetic diversity on the morphological, agronomical, biochemical and molecular level and marked its exploitable genetic potential (el mousadik et al., 1996; ait aabd et al., 2012; ait aabd et al., 2013; ait aabd et al., 2015; mouhaddab et al., 2017; koufan et al., 2020b). unlike the cytogenetic approach, that is less studied. the breeding program from wild types seems to be the straighter method for genetic improvement and before conception of argane orchards. genetic diversity can be found in genome sequence and the number of chromosomes (de vicente et al., 2004). polyploidy means increasing the number of chromosomes in an animal or plant cell (multiple of the number of haploids). it can be an auto-polyploidy (duplication without cytokinesis) or an allopolyploidy (fusion of two unreduced gametes). also, the genetic variation can be at the origin of the anomalies of the chromosomal number like aneuploidy. it is defined as the presence of an abnormal number of chromosomes. in general, chromosome number variation can be a speciation factor. it plays a fundamental role in plants evolution, diversification, and ecological adaptation. karyotype assessments by counting the number of metaphase chromosomes is time consuming and laborious. highly skilled manipulators are needed to accomplish it, in addition, tissues containing few numbers of dividing cells, which may not be readily available. however, flow cytometry has arisen as a far more reliable methodology for the determination of genome size and ploidy level. it permits measurement of the fluorescence of large numbers of stained nuclei within seconds, more samples within minutes (seker et al., 2003). in fact, with the enormous biodiversity at the biometric, phenological, agro-morphological, and molecular level, the allogamous pollination system, and pollen incompatibility (ait aabd et al., 2022), the existence of chromosomal variation remains to be checked in the argan tree species. indeed, this study aims to investigate the chromosome number on a collection of genotypes morphologically and phenologically differentiated. el boukhari a et al. (2023). not sci biol 15(1):11451 3 materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant material the study was undertaken on five genotypes selected previously based on their morpho-biometry and phenology (tree shape, height, number of trunks, flowering period, fruit shape and fruit ripening duration). the trees were planted at melk-zhar experimental farm, regional agronomic research center, agadir, morocco. chromosome counting the protocol of majourhat et al. (2007) was adopted with minor modification. mature fruits were collected from each tree and kept until drying. after pulping and crushing seeds, they were disinfected using sodium hypochlorite, rinsed with sterile water, and then germinated on petri dishes with moist filter paper at 26 °c. root tips were cut out when the radicles were about 2-3 cm long. they were pre-cooled four hours in ice water. the samples are then fixed in glacial ethanol-acetic acid (3: 1) for 16 h at 4 °c, then transferred to 70% ethanol solution and kept at 4 °c. the root tips were hydrolysed in 5 n hcl for 10 min. finally, the samples were stained in orcein acetic solution for two hours then squashed under coverslips. slides were observed under different objectives to find well spread cells and show the best meta-phasic plates. mitotic chromosomes were photographed under 1000x magnification using an optical microscope. each photographed cell was drawn to count it accurately. to prevent experiment artefacts, more than 3 counts were made per each preparation and plant. flow cytometry cells were isolated from specimens collected directly from each genotype. leaf samples (5 mm2) were chopped with razor blade in presence of 400 µl nuclei extraction buffer in petri dishes. the samples were filtered directly into a sample tube of 50 µm and stained with 1,5 ml of 4’,6-diamidino-2-phenylindole buffer stain. following 5 min incubation at room temperature, stained samples were run in flow cytometer. analysis was repeated twice for each genotype. results results results results chromosome numbers chromosomal counting is carried out on well spread, drawn, and photographed plates. it was difficult to count the chromosomes given their small size (0.59 to 1.69 µm) (majourhat et al., 2007). despite, three chromosome numbers were counted: 20, 22 and 24 (figure 1), which means three base numbers (n=10, n=11, n=12). the number of chromosomes varied between mother trees. indeed, three genotypes with 22, one with 24, and one with 20 were observed. el boukhari a et al. (2023). not sci biol 15(1):11451 4 figure 1. figure 1. figure 1. figure 1. chromosomes count on a. spinosa: microscopic photography (a, b, c) and draw (a, b, c) of meta-phasic plate showing 22 chromosomes, 20 chromosomes and 24 chromosomes. scale bars represent 5 µm. flow cytometry profiles figures 2, 3 and 4 show different raw flow cytometer profiles. first peak (around 50) is background noise and the second peak is cells fluorescence intensity emission. profile 1 represent the maximal fluorescence intensity measured (around 230), profile 2 with intermediate intensity (around 200) while profile 3 represent the minimal fluorescence intensity detected (around 150). it was observed that fluorescence intensity of dapi stain varied according to genotype. cells intensity detected is correlated to relative nuclei dna content. figure 2. figure 2. figure 2. figure 2. argania spinosa flow cytometry profile 1, cells count represented on the y axis while fluorescence intensity is represented on the x axis el boukhari a et al. (2023). not sci biol 15(1):11451 5 figure 3. figure 3. figure 3. figure 3. argania spinosa flow cytometry profile 2, cells count represented on the y axis while fluorescence intensity is represented on the x axis figure 4. figure 4. figure 4. figure 4. argania spinosa flow cytometry profile 3, cells count represented on the y axis while fluorescence intensity is represented on the x axis discussiondiscussiondiscussiondiscussion miège (1954) reported 20 chromosomes on a. spinosa somatic cells, while humphries et al. (1978) discovered 24 chromosomes on pollen mother cells. the current study on a. spinosa showed a new finding as the counting of 2n = 22 chromosomes which has never been reported. the highest diversity observed on argan tree forest could give an explanation of this variability of chromosome numbers. based on flow cytometry analysis, the 3 chromosomes numbers (20, 22 and 24) were confirmed and showed none variation in ploidy level. in fact, the intensity dissimilarity observed between genotypes cannot be a score as a variation in ploidy level. nevertheless, this slight variation may be due to the genome size or genome composition el boukhari a et al. (2023). not sci biol 15(1):11451 6 variations (dapi binds specifically to at nucleotides). understanding the genetic diversity of the argan tree is an essential basis that requires more scientific effort to achieve the essential objective of the genetic improvement of this species. the basic data are chromosome numbers, but chromosome size, morphology, and staining characteristics may also be of important value (stace et al., 2000). systematic investigations are mainly based on molecular methods, while chromosome data provide essential and basic information, which may for example help in interpreting results from molecular studies (baltisberger and widmer, 2006). it is worth noting that the high genetic diversity revealed by molecular tools can be explained at chromosome’s scale. possession of more or fewer chromosome numbers influences the presence/absence of alleles on the whole genome. there is no report of intraspecific cytological variation in sapotaceae until now. johnson (1991) evaluated the number of chromosomes of 95 sapotaceae species. all specimens showed n = 10, 11, 12, 13, or 14 and almost at the diploid level. the highest numbers (n = 13, 14) predominate in the tribes chrysophylleae and omphalocarpeae, whereas the numbers in mimusopeae, isonandreae and sideroxyleae are almost on n = 10 or 11. intraspecific cytological variation is frequent in plants, especially polyploidy. however, intraspecific variation in chromosome number is rare (severns et al., 2008). among species, genome size variation can occur as different ploidy levels (terlević et al., 2022) or chromosome number variation. it can vary or not at the same ploidy level (bagheri et al., 2022). moreover, genome size variation can be correlated with morphological traits (hoang et al., 2019). in general, descending dysploidy and polyploidy played crucial roles in chromosome number evolution in angiosperm (carta et al., 2020) and may be the origin of this observed variation. in addition, abnormal meiocytes may be observed as a variation on chromosome numbers such as cytomixis, which is defined as the migration of chromatin between adjacent cells through cytoplasmic connection channels (bellucci et al., 2003). this phenomenon detected in the meiocytes of several plants can cause variation in the chromosome number. however, this variation can be prevailing in populations of wild-type (kaur and singhal, 2015), having the form of different types of aneuploidy (trisomy, tetrasomy, double trisomy) or polyploidy (diploid, triploid, tetraploid, octaploid). tetrasomy can be the origin that explains this variability (2n=20: no tetrasomy, 2n=22: one chromosome tetrasomy, 2n=24: two tetrasomy chromosomes). the union of two n+1 gametes can increase the number of chromosomes and restore euploidy in the offspring (2n+2) (mayrose and lysak, 2021). in addition, chromosome drive may be responsible for this diversity in chromosomal numbers (camacho et al., 2000). organisms can use a greater number of genes with an increase in the number of allelic variants. this is considered as interesting to plants in terms of synthesis rate and or the variability of the metabolic compounds produced (pan et al., 2009). duplicated genes can be maintained in copies (duplication divergence) which often undergo neo-functionalization or under-functionalization (comai, 2005). a cytomorphologic study by counting chromosomes from the somatic cells (buds) on more genotypes is desirable to confirm these findings and achieve a genotype characterization associated with phenotypes. in addition, deepening in the study of the nature of chromosomes that makes the difference, their segregation, and their sequences is crucial. furthermore, an analysis of genome size by a non-specific staining (like iodide propidium) to set standard genome size is important to facilitate the detection of cytotypes for rapid breeding program then avoid incompatibility during cross pollination process. conclusionsconclusionsconclusionsconclusions a. spinosa has three possible chromosome numbers representing the highest genetic diversity observed. this diversity reflects another side of the adaptability of this species to mitigate extreme climate conditions. these findings will make possible determination of high diversity and ecotypes identification belonging to this species based on chromosomal numbers. el boukhari a et al. (2023). not sci biol 15(1):11451 7 authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions naa conceptualization; aeb, naa, st, hb, at, rb, methodology; naa, aem, reb, mk, validation; aeb, naa, hb, mk investigation; aeb, naa, aem, reb, rb, writing—original draft preparation; aeb, naa, at, rb writing—review and editing; naa, aem, reb, mk supervision; naa, mk, rb funding acquisition. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this work was supported and funded by national institute of agronomic research of agadir, morocco (grant id. prmt 2020-24). conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences ait aabd n, el asbahani a, el alem y, el finti a, msanda f, el mousadik a (2013). variation in oil content and fatty acid composition in preselected argan trees with morphological characters and geographical localization. mediterranean journal of nutrition and metabolism 6(3):217-225. https://doi.org/10.3233/s12349-013-0134-2 ait aabd n, msanda f, el mousadik a (2012). univariate and multivariate analysis of agronomical traits of preselected argan trees. notulae botanicae horti agrobotanici cluj-napoca 40:308-316. https://doi.org/10.15835/nbha4028209 ait aabd n, msanda f, el mousadik a (2015). genetic diversity of the endangered argan tree (argania spinosa l.) 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(2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11549 doi:10.15835/nsb15211549 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae evaluation of crossevaluation of crossevaluation of crossevaluation of cross----genus transferability of ssr markers from other genus transferability of ssr markers from other genus transferability of ssr markers from other genus transferability of ssr markers from other legumes to two closely related legumes to two closely related legumes to two closely related legumes to two closely related onobrychisonobrychisonobrychisonobrychis (fabaceae) taxa(fabaceae) taxa(fabaceae) taxa(fabaceae) taxa ioan băcilă1, dana şuteu1,4*, ana coste1, zoltán r. balázs2,3,4, gheorghe coldea1 1national institute of research and development for biological sciences, institute of biological research, department of experimental biology, 48 republicii st., 400015 cluj-napoca, romania; ioan.bacila@icbcluj.ro; dana.suteu@icbcluj.ro; ana.coste@icbcluj.ro; gheorghe.coldea@icbcluj.ro 2babeş-bolyai university, faculty of biology and geology, department of molecular biology and biotechnology, 1 kogălniceanu st., 400084 cluj-napoca, romania; zoltan.balazs@ubbcluj.ro 3babeș-bolyai university, faculty of biology and geology, center for systematic biology, biodiversity and bioresources 3b, 1 kogălniceanu st., 400084, cluj-napoca, romania 4babeș-bolyai university, doctoral school of integrative biology, 1 kogălniceanu st., 400084 cluj-napoca, romania; dana.suteu@icbcluj.ro (*corresponding author) abstractabstractabstractabstract microsatellite markers previously developed for other leguminous species were tested for cross-genus transferability and evaluated for their potential usefulness in providing an improved assessment of the genetic relationships between two closely related taxa belonging to onobrychis genus (fabaceae). candidate microsatellite markers were tested for polymorphism and replicability in sixteen populations of o. montana dc. subsp. transsilvanica (simonk.) jáv. and o. montana. out of the 23 ssrs, there were identified seven polymorphic loci. in total 32 alleles were detected and the number of alleles per locus varied from two to six. pic values ranged from 0.375 to 0.6454, and four ssrs displayed a pic > 0.5. relative uniform rates of genetic diversity were obtained. in case of o. montana dc. subsp. transsilvanica (simonk.) jáv. the observed and expected heterozygosity ranged from 0.100 to 0.952 and from 0.219 to 0.525, respectively, while for o. montana ranged from 0.166 to 0.750 and from 0.083 to 0.375, respectively. seven polymorphic ssrs with clear and reproducible amplification were identified. these markers proved to be very efficient for unambiguous population discrimination based on both geographic and taxonomic criteria. hereafter, these ssr markers can be used as tools for evolutionary studies in onobrychis genus, as well in providing knowledge on patterns of the species phylogeography. keywords:keywords:keywords:keywords: cross-genus transferability; leguminous; microsatellite; onobrychis; polymorphism https://www.notulaebiologicae.ro/index.php/nsb/index băcilă i et al. (2023). not sci biol 15(2):11549 2 introductionintroductionintroductionintroduction the onobrychis genus includes about 206 species (powo, 2023), cross-pollinated, diploid (2n = 14, 16) or tetraploid (2n = 28) (mohsen and nasab, 2010), perennial or annual herbs or shrubs. the genus extends throughout the europe (excepting scandinavia and the british isles), central and eastern asia, and north africa (powo, 2023). within the genus, onobrychis montana dc. subsp. transsilvanica (simk.) jáv. (ciocârlan, 2009; sârbu et al., 2013) (≡ onobrychis transsilvanica (nyárády and nyárády, 1957); ≡ onobrychis montana dc. var. transsilvanica (simk.) beck (borza, 1949) is an endemic taxon in the romanian carpathian chain. it shares close, yet controversial, taxonomic relationships and a strong morphological resemblance with the allopatric species onobrychis montana dc. our previous study (băcilă et al., 2015) represented the first attempt to provide some molecular insights for this carpathian endemic taxon with the use of aflp and cpdna markers. because these markers failed to clearly resolve the distinction between o. montana and o. montana dc. subsp. transsilvanica (simonk.) jáv., other molecular markers, more informative ought to be identified. microsatellites (or single sequence repeats ssrs) are codominant markers characterized by high levels of polymorphism, thus being widely recognized as very powerful and informative in both animal and plant species (ellegren, 2004). the hypervariable nature of ssrs produces allelic variations even among very closely related varieties. therefore, they are considered the markers of choice for the characterization of core collections and for the management of germplasm collections (kumar et al., 2023). one of the characteristics that make these markers particularly interesting in genetic diversity studies is their high rate of transferability to closely related species (gupta et al., 2003; simko, 2009). nevertheless, significantly low values of crosstransferability have been observed for genomic ssrs, which are known to be more polymorphic and located in less conserved regions of the genome (peakall et al., 1998; sourdille et al., 2001). we selected and tested for transferability and polymorphism 23 expressed sequence tag -est-ssrs originated from other leguminous species: glycine max, medicago sativa, medicago trunculata, and phaseolus vulgaris (peakall et al., 1998; yu et al., 2000; gaitán-solís et al., 2002; julier et al., 2003; gutierrez et al., 2005; zhang et al., 2007). previously, demdoum et al. (2012) successfully cross-amplified 14 of these markers in o. pyrenaica sennen, o. argentea boiss. and o. viciifolia scop., while the remaining nine markers were noted by avcı et al. (2014) as polymorphic in 58 onobrychis species from turkish flora. the main purpose of this study was to test the cross-genus transferability of several ssr markers into o. montana dc. subsp. transsilvanica (simonk.) jáv. and o. montana and provide a preliminary evaluation of their usefulness for assessing the genetic relationships between the two taxa. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods sampling and dna extraction ten populations belonging to o. montana dc. subsp. transsilvanica (simonk.) jáv. and six populations of o. montana were sampled from the alps and the carpathians mountains (table 1). more details on the sampling strategy, on the populations and on the dna extraction can be found in băcilă et al. (2015). băcilă i et al. (2023). not sci biol 15(2):11549 3 tabletabletabletable 1111. sampled populations of o. montana and o. montana dc. subsp. transsilvanica (simonk.) jáv.: taxon, numbering, population code, country of origin (ro – romania; fr – france; po – poland; sk – slovakia; mne – montenegro), mountain range, sampling locality, geographic coordinates (partially reproduced from băcilă et al., 2015) taxontaxontaxontaxon nononono population population population population codecodecodecode countrycountrycountrycountry rangerangerangerange locality/massiflocality/massiflocality/massiflocality/massif coordinates coordinates coordinates coordinates (longitude °e/ (longitude °e/ (longitude °e/ (longitude °e/ latitude °n)latitude °n)latitude °n)latitude °n) o. montana dc. subsp. transsilvanica (simonk.) jáv. 1 otrm ro sw carpathians piatra iorgovanului peak, retezat mts. 45°16’55.96” 22°50’45.09” 2 otr ro sw carpathians piule peak, retezat mts. 45°18’25.7” 22°54’31.4” 3 otm ro se carpathians cearcănu peak, maramureşului mts. 47°38’57.96” 24°49’54” 4 otch ro se carpathians toaca peak, ceahlău mts. 46°59’35.3” 25°57’57.3” 5 otgh ro se carpathians ocsem peak, giurgeuhăşmaş mts. 46°40’41” 25°50’11” 6 otc ro se carpathians zăganu peak, ciucaş mts. 45°29’22” 25°58’39.1” 7 otpc ro se carpathians piatra craiului mică peak, piatra craiului mts. 45°33’10.3” 25°15’47.6” 8 otb ro se carpathians caraiman peak, bucegi mts. 45°24’56.7” 25°29’51.71” 9 otbv ro se carpathians postăvaru peak, bârsei mts. 45°33’58.88” 25°33’02.22” 10 otf ro se carpathians jgeabul văros peak, făgăraş mts. 45°36’20.82” 24°35’37.68” o. montana 11 omac fr alps col d’izoard, cottian alps 44°49’36” 6°43’48” 12 oma fr alps col du lautaret, dauphiné alps 45°04’09.13” 6°24’05.23” 13 omj fr alps colomby de gex, jura mts. 46°19’38.75” 6°0’4.35” 14 omad mne dinaric alps durmitor, dinaric alps 43°06’26.75” 19°0.1’10.38” 15 omtw po w carpathians wawoz krakow, high tatras 49°10’27.24” 20°08’11.97” 16 ombt sk w carpathians saddle between mt. muran and mt. novy, belianske tatras 49°14’55” 20°11’00” ssr fingerprinting 23 microsatellites (original code names: mtba01b04r2, mtba27d09f1, mtbb36f05f1, mtba04c08r1, mtbb22g10f1, mtbc47b06f1, mtbb44f02r1, ag81, bi74, al79, bg178, al46, aw265, aw567861, pv-at001, bm141, mtic326, mtic272, mtic230, mtic21, bm175, bm152, bm137) developed by peakall et al. (1998), yu et al. (2000), gaitán-solís et al. (2002), julier et al. (2003), gutierrez et al. (2005), and zhang et al. (2007) for other leguminous species were tested for transferability in o. montana dc. subsp. transsilvanica (simonk.) jáv. and o. montana. băcilă i et al. (2023). not sci biol 15(2):11549 4 each primer pair had to be optimized, as poor amplification or unspecific bands were otherwise present. following amplification and analysis of gel patterns, only seven ssr primer pairs were selected, fluorescently dyed (6-fam) and used in subsequent reactions. for the amplification of these seven microsatellites, four different pcr programs were used in order to obtain a clear and reproductible amplification (table 2). table 2table 2table 2table 2. pcr programs used for ssr amplifications. pvat001, mtbb22g10, mtba27d09, ag81, pvat001, mtbb22g10, mtba27d09, ag81, pvat001, mtbb22g10, mtba27d09, ag81, pvat001, mtbb22g10, mtba27d09, ag81, bg178, bm141, and mtic272bg178, bm141, and mtic272bg178, bm141, and mtic272bg178, bm141, and mtic272 represent the original names of the markers (see also table 3 for references) pcr pcr pcr pcr stepsstepsstepssteps pvat001pvat001pvat001pvat001 mtbb22g10mtbb22g10mtbb22g10mtbb22g10 mtba27d09, mtba27d09, mtba27d09, mtba27d09, ag81ag81ag81ag81 bg178, bm141, bg178, bm141, bg178, bm141, bg178, bm141, mtic272mtic272mtic272mtic272 initial denaturation 94 °c, 2 min 94 °c, 3 min 94 °c, 3 min 95 °c, 5 min denaturation 94 °c, 45 sec 94 °c, 45 sec 94 °c, 45 sec 94 °c, 30 sec annealing temperature 50 °c, 45 sec 50 °c, 1 min 51 °c, 1 min 50 °c, 30 sec elongation 72 °c, 1 min 72 °c, 1.5 min 72 °c, 1.5 min 72 °c, 1 min repet steps 2-4 35x 35x 40x 35x final elongation 72 °c, 5 min 72 °c, 10 min 72 °c, 10 min 72 °c, 10 min the pcr products were purified with sephadex sephacryl (1:1) (ge healthcare bio-sciences ab, usa) and then diluted 50 times. 1.5 μl of dilution were added to 10 μl mix of hidi formamide and genescan 500 rox size standard (applied biosystems, thermo fisher scientific, usa) and subjected to capillary electrophoresis on an abi prism 3130 genetic analyzer (applied biosystems, thermo fisher scientific, usa). the characteristics of the seven primer pairs are presented in table 3. table 3table 3table 3table 3. characteristics of seven microsatellite loci used for cross-transferability in onobrychis sp. locuslocuslocuslocus primer sequence (5’primer sequence (5’primer sequence (5’primer sequence (5’----3’)3’)3’)3’) allele size allele size allele size allele size range (bp)range (bp)range (bp)range (bp) referencereferencereferencereference mtba27d09 f:gaagaagaaaaagagatagatctgtgg r: ggcaggaacagatccttgaa 100-326 gutierrez et al., 2005 mtbb22g10 f: ccagtggcagctacggtacta r: gagacggaggagaagttgctt 149-161 gutierrez et al., 2005 ag81 f: attttccaactcgaattgacc r: tcatcaatctcgacaaagaatg 134-184 peakall et al., 1998 bg178 f: acccactcaactcaacacacac r: ttctccttgaccaaccttgatt 184-187 zhang et al., 2007 pv-at001 f: gggagggtagggaagcagtg r: gcgaaccacgttcatgaatga 157-266 yu et al., 2000 bm141 f: tgaggaggaacaatggtggc r: ctcacaaaccacaacgcacc 103-487 gaitán-solís et al., 2000 mtic272 f: aggtggatggagagagtca r: tcatgaatagtggcactcaa 132-210 julier et al., 2003 data analysis alleles scoring was performed with genemapper v.4.0 software (applied biosystems, thermo fisher scientific, usa). powermarker v.3.25 (liu and muse, 2005) was used to calculate the total number of alleles, gene diversity and polymorphism information content (pic). descriptive statistics as: number of alleles and observed [ho] and expected heterozygosities [he], were estimated per population using genalex 6.5 (peakall and smouse, 2006). a frequency matrix was generated and subsequently used within splitstree v.4.10 (huson and bryant, 2006) to compute unweighted pair group method with arithmetic mean (upgma) băcilă i et al. (2023). not sci biol 15(2):11549 5 phylogenetic tree based on the shared allele distance and the neighbor-net method. bootstrap values were calculated from 1000 replicates. results results results results 23 ssrs were tested in o. montana dc. subsp. transsilvanica (simonk.) jáv. and o. montana and consistent amplification was obtained for 18 of them (71.26%), while the rest provided multiple nonspecific bands. however, due to lack of polymorphism and low reproductibility, only seven ssr (table 3) were selected for the subsequent characterization of the onobrychis sp. populations. a total number of 32 alleles were detected, each ssr amplified 2–6 alleles, and the average number of alleles per ssr was 4.571. pic values ranged from 0.375 to 0.6454, with an average of 0.5089 (table 4). only four ssrs (mtba27d09, mtbb22g10, pv-at001, and mtic272) displayed a pic > 0.5, and therefore were considered informative. relative uniform rates of genetic diversity were obtained, ranging from the lowest value of 0.5 (ag81, bg178, and bm141) to the highest value of 0.7 (mtic272). the gene diversity and pic values pointed out that mtic272 represented the most informative locus in the two onobrychis species analysed (table 4). tabletabletabletable 4444. number of alleles, pic, and gene diversity values for seven ssr loci analysed in onobrychis sp. ssr locusssr locusssr locusssr locus no. of allelesno. of allelesno. of allelesno. of alleles gene diversitygene diversitygene diversitygene diversity picpicpicpic mtba27d09 6 0.6500 0.5957 mtbb22g10 4 0.6618 0.6033 ag81 4 0.5000 0.3750 bg178 2 0.5000 0.3750 pv-at001 6 0.6486 0.5931 bm141 6 0.5000 0.3750 mtic272 4 0.7000 0.6454 meanmeanmeanmean 4.5714.5714.5714.571 0.59430.59430.59430.5943 0.50890.50890.50890.5089 ho and he ranged in case of o. montana dc. subsp. transsilvanica (simonk.) jáv. from 0.100 to 0.952, and from 0.219 to 0.525, respectively, while for o. montana, they ranged from 0.166 to 0.750 and from 0.083 to 0.375 (table 5). table 5table 5table 5table 5. genetic characterization of seven polymorphic microsatellite loci tested across sixteen populations of onobrychis sp. ho = observed heterozygosity; he = expected heterozygosity. locuslocuslocuslocus o. montana o. montana o. montana o. montana dc. subspdc. subspdc. subspdc. subsp. transsilvanica . transsilvanica . transsilvanica . transsilvanica (simonk.) (simonk.) (simonk.) (simonk.) jáv.jáv.jáv.jáv. onobrychis montanaonobrychis montanaonobrychis montanaonobrychis montana hhhhoooo hhhheeee hhhhoooo hhhheeee mtba27d09 0.366 0.183 0.611 0.305 mtbb22g10 0.550 0.275 0.166 0.083 ag81 0.100 0.05 0.333 0.167 bg178 0.952 0.525 0.667 0.333 pv-at001 0.066 0.033 0.222 0.111 bm141 0.433 0.216 0.277 0.139 mtic272 0.450 0.225 0.750 0.375 the upgma analysis (data not shown, manuscript in preparation) managed to clearly differentiate all the 16 populations of onobrychis, exhibiting taxonomic and geographic delineation. băcilă i et al. (2023). not sci biol 15(2):11549 6 discussiondiscussiondiscussiondiscussion the rate of ssr cross-genera transferability was 18 out of 23 tested markers (71.26%). this value was lower than 81%, as previously reported by demdoum et al. (2012), but higher than other related data (eujayl et al., 2004). the intra-genus amplification rate was considered to be around 50% (peakall et al., 1998), but this value quickly declined inter-genera. zhang et al. (2007) found 18-22% transferability from medicago to trifolium, while peakall et al. (1998) reported only 1-3% transferability of glycine’s ssr to other leguminous genera. however, a narrow proportion of microsatellites was found to be polymorphic in onobrychis (38.8% out of the 18 transferable ssrs). several markers showed multiple bands that could not be eliminated by calibrating the pcr conditions. the generation of multiple products during cross-species amplification may occur by mutation, rearrangements and duplications in the flanking region and/or changes in the number of repeats (peakall et al., 1998), similar results being reported by gutierrez et al. (2005) in their study of est-ssr in leguminous. eventually, only seven ssr loci were selected on the base of polymorphism and reproducibility and they were subsequently used for characterization and genetic diversity evaluation of 16 populations of o. montana dc. subsp. transsilvanica (simonk.) jáv. and o. montana. these seven markers showed medium pic values (average 0.5089) (table 3). the number of alleles per locus ranged from 2 to 6 (table 3), lower than previously reported by other studies (4-14) (falahati-anbaran et al., 2007). since the studied onobrychis species are diploid or tetraploid species (o. montana dc. subsp. transsilvanica (simonk.) jáv. 2n=14, löve, 1975; and respectively o. montana 2n=28; löve, 1984), the number of detected alleles seemed to be low. a possible explanation is the pcr amplification bias, which could cause the loss of the less frequent alleles and predominant detection of the most common alleles, therefore leading to an under estimation of the number of alleles per loci in each population (peakall et al., 1998). although the level of polymorphism exhibited by the seven employed microsatellites was relatively low and only four of them (mtba27d09, mtbb22g10, pvat001, and mtic272) were informative (pic > 0.5), it was possible to differentiate all the analysed populations by taxonomic and even geographic criteria. conclusionsconclusionsconclusionsconclusions within the studied group represented by 16 populations of o. montana dc. subsp. transsilvanica (simonk.) jáv. and o. montana the rate of ssr cross-genera transferability was 18 out of 23 tested markers (71.26%). subsequently, only seven ssr loci were selected on the base of polymorphism and reproducibility. a total number of 32 alleles were detected, the average number of alleles per ssr being 4.571. relative uniform rates for pic and genetic diversity were obtained, pointing out that mtic272 represented the most informative microsatellite. although the level of polymorphism of the seven analysed microsatellites was relatively low, they managed to clearly differentiate all the analysed populations based on taxonomic and geographic criteria. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions the contributions of authors to the manuscript are as follows: conceptualization: ib, gc; field work: gc; data curation: ib, ac, zrb, dș; formal analysis: ib and dș; funding acquisition: ib; investigation: ib; methodology: ib; project administration: ib; writing original draft: ib; writing review and editing: ib, ac, zrb, gc and dș. all authors read and approved the final manuscript. băcilă i et al. (2023). not sci biol 15(2):11549 7 ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements we are grateful to andreas tribsch, nadir alvarez, rolland douzet, zbigniew mirek, liviu filipaș, mihai puşcaş, adrian ilie stoica, and tudor ursu for their valuable help in collecting the plant material. this work was financially supported by a grant from the romanian national authority for scientific research, cndi–uefiscdi, project number pn–ii–ru–pd–2012–3–0005; 15/26.04.2013, as well as by the core project bioresgreen, subproject bioclimpact no. 7n/03.01.2023, code 23020401. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences avcı s, ilhan e, erayman m, sancak c (2014). analysis of onobrychis genetic diversity using ssr markers from related legume species. journal of animal and plant sciences 24(2):556-566. băcilă i, şuteu d, coldea g (2015). genetic divergence and phylogeography of the alpine plant taxon onobrychis transsilvanica (fabaceae). botany 93:257-266. https://doi.org/10.1139/cjb-2014-0175 borza a (1949). conspectus florae romaniae regionumque affinium, vol. ii. cartea românească press, cluj. ciocârlan v (2009). flora ilustrată a româniei – pteridophyta et spermatophyta. ceres press, bucharest. demdoum s, muñoz f, delgado i, valderrábano j, wünsch a (2012). est-ssr cross-amplification and genetic similarity in onobrychis genus. genetic resources and crop evolution 59:253-260. https://doi.org/10.1007/s10722-0119681-x ellegren h (2004). microsatellites: simple sequences with complex evolution. nature reviews genetics 5:435-445. https://doi.org/10.1038/nrg1348 eujayl i, sledge mk, wang l, may gd, chekhovskiy k, zwonitzer jc, mian mar (2004) medicago truncatula estssrs reveal cross-species genetic markers for medicago spp. theoretical and applied genetics 108:414-422. https://doi.org/10.1007/s00122-003-1450-6 falahati-anbaran m, habashi aa, esfahany m, mohammadi sa, ghareyazie b (2007). population genetic structure based on ssr markers in alfalfa (medicago sativa l.) from various regions contiguous to the centres of origin of the species. journal of genetics 86(1):59-63. https://doi.org/10.1007/s12041-007-0008-9 gaitán-solís e, duque mc, edwards kj, tohme j (2002). microsatellite repeats in common bean (phaseolus vulgaris) isolation, characterization, and cross-species amplification in phaseolus ssp. crop science 42(6):2128-2136. https://doi.org/10.2135/cropsci2002.2128 gupta pk, rustgi s, sharma s, singh r, kumar n, balyan hs (2003). transferable est-ssr markers for the study of polymorphism and genetic diversity in bread wheat. molecular genetics and genomics 270:315-323. https://doi.org/10.1007/s00438-003-0921-4 gutierrez mv, vaz patto mc, huguet t, cubero ji, moreno mt, torres am (2005). cross-species amplification of medicago truncatula microsatellites across three major pulse crops. theoretical and applied genetics 110:12101217. https://doi.org/10.1007/s00122-005-1951-6 huson dh, bryant d (2006). application of phylogenetic networks in evolutionary studies. molecular biology and evolution 23(2):254-267. https://doi.org/10.1093/molbev/msj030 băcilă i et al. 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(2023). not sci biol 15(2):11549 9  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11377 nsb sevindik 2023.03.22.docx received: 09 nov 2022. received in revised form: 10 feb 2023. accepted: 07 mar 2023. published online: 22 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania sevindik e et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11377 doi:10.15835/nsb15111377 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae chemical composition of chemical composition of chemical composition of chemical composition of vitex agnusvitex agnusvitex agnusvitex agnus----castuscastuscastuscastus l. flowers collected from l. flowers collected from l. flowers collected from l. flowers collected from populations distributed in aydpopulations distributed in aydpopulations distributed in aydpopulations distributed in aydiiiinnnn, , , , türkiyetürkiyetürkiyetürkiye emre sevi̇ndi̇k1*, deniz karakoyun1, i̇brahim m. güldere1, fatih borhan1, elif apaydin2, emre kabi̇l3 1aydın adnan menderes university, faculty agriculture, department of agricultural biotechnology, south campus, cakmar, aydin, türkiye; ph.d-emre@hotmail.com (*corresponding author); denizkrkyn98@gmail.com; mguldere00@gmail.com; fatihborhan986@gmail.com 2giresun university, center research laboratory application and research center, giresun, türkiye; eliferenapaydin@gmail.com 3yalova university, armutlu vocational school, food processing division, 77500 yalova, türkiye; emre.kabil@yalova.edu.tr abstractabstractabstractabstract essential oils (eos) are compounds that are biosynthesized autonomously by aromatic plants as their secondary metabolites. vitex agnus-castus plant leaves, flowers and fruits are of pharmacological importance. in this study, the chemical composition determination of the essential oils of the vitex agnus-castus flowers with different colors (white, pink, purple) growing in aydin ecological conditions was carried out with the extraction using clevenger hydro-distillation apparatus and then the essential oil composition was determined by gas chromatography-mass spectrometer (gc-ms). as a result of the study, ‘çakmar’ pink-flowered population had the main component (-)-allospathulenol (14.59%), ‘çakmar’ purple-flowered and ‘koçarli’ purple-flowered populations had the main component pyrrolo (3,2,1-jk) carbazole (14.42% and 14.68%, respectively), while ‘çakmar’ white flowered, ‘koçarli’ white flowered and ‘koçarli’ pink flowered populations had the main component caryophyllene (15.54%, 12.64%, and 19.05 respectively). keywords:keywords:keywords:keywords: gc-ms; essential oils; vitex agnus-castus; türkiye introductionintroductionintroductionintroduction all over the world, various plants have been used for various reasons. especially, medicinal and aromatic plants are grown for use in preventing diseases, as flavoring agents in incense, cosmetics, preserving food and improving the taste of many types of food (toplan et al., 2022; yilmaz et al., 2022). in addition, many studies reporting medicinal plants with important biological activities such as antioxidant, antimicrobial, antiviral, and antiproliferative activities have been revealed (zhang et al., 2014; suleman et al., 2021; gecer, 2022). vitex is one of the largest genera of the lamiaceae family (formerly belonging to the verbenaceae family), and consists of 217 species. some of the known and studied vitex species are vitex negundo vitex agnus-castus, vitex trifolia, vitex rotundifolia, vitex cymosa and vitex peduncularis (souto et al., 2020; kamal et al., 2022). the vitex genus is represented by two species (v. agnus-castus and v. pseudo-negundo (hausskn. ex bornm.) hand.-mazz) in https://www.notulaebiologicae.ro/index.php/nsb/index sevindik e et al. (2023). not sci biol 15(1):11377 2 the flora of türkiye (eryiğit et al., 2015). vitex agnus-castus is defined as vitex, agnos and lygos by ancient literature (kolancı, 2017). in turkish, is known as 'hayıt, ayd, hayed, beşparmak grass, and bitter ayit' (kavaz et al., 2022). it spreads especially in the eastern black sea, marmara, aegean, mediterranean, and partially in the southeastern anatolia region of türkiye (fakir et al., 2014). when this plant is looked at from an aesthetic point of view; the length of its spike-shaped flowers, its plump flower structure, its rich colour range from whitelight pink to purple, its sustained blooming, and the striking appearance of its large seeds on the spike formed after its flowers, increase the variety of usage areas in herbal landscaping (girmen and karagüzel, 2005). vitex agnus-castus is rich in numerous bioactive substances it is a well-known herbal plant (zhelev et al., 2022). v. agnus castus fruits, flowers and leaves contain phenolic acids and derivatives, flavonoids, tannins, iridoids and diterpenoids (sarikurkcu et al., 2009; săvulescu et al., 2019; bakr et al., 2020). since ancient times, local flowers and fruits have been applied in various cultures (özderin, 2021). traditionally, chasteberry extract has been used to treat many gynecological conditions, including menstrual disorders, premenstrual syndrome (pms), corpus luteum insufficiency, infertility, acne, menopause, impaired lactation, and various gynecological problems (stojković et al., 2011; ogaly et al., 2021). in this study, essential oil content of different colored (white, pink, purple) v. agnus-castus flowers grown in aydın ecological conditions was determined. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods collection of plant samples and extraction of essential oils for the essential oil extraction process, approximately 500 grams of v. agnus-castus flower samples were collected from different regions of aydın, türkiye, six populations with white, purple, and pink flowers in three different colors. the collected flower samples were kept with their moisture evaporated in an oven at 35 °c for 24 hours, then distilled for about 4 hours by hydro-distillation method in the clevenger apparatus to extract the essential oil, and kept at +4 °c for chromatographic analysis. chromatographic analysis by gc-ms the qualitative analysis of the essential oil obtained from the plant sample was dedected by gas chromatography-mass spectrometry (gc-ms) analysis system. helium was used as the carrier gas and separation was performed with an hp-5ms capillary column coated with 5% phenyl-methylpolysiloxane (30.0 m x 250µm x 0.25 µm). gc-ms temperature program was applied from 50 °c to 80 °c ramped with 5 °c/min waiting for 2 minutes, 80 °c to 100 °c ramped with 2 °c/min waiting for 1 minute, 100 °c to 150 °c ramped with 2 °c/min waiting for 1 minute, 150 °c to 240 °c ramped with 2 °c/min waiting for 1 minute and finally from 240 °c to 270 °c ramped with 2 °c/min waiting for 7 minutes (rambla et al., 2015; apaydın, 2018). the volatile compounds which dedected were identified with wiley and nist mass spectrometry libraries. results results results results and discussionand discussionand discussionand discussion essential oils (eos) which are biosynthesized by aromatic plants as their secondary metabolites and represented by a strong odor are natural, volatile and complex mixtures of lipophilic compounds, often including terpenes, phenol-derived aromatic compounds and aliphatic compounds (bakkali et al., 2008; santos et al., 2016; marić et al., 2021). there have been numerous studies to determine the chemical composition of essential oils obtained from chasteberry (moudachirou et al., 1998; senatore et al., 2003; sarikurkcu et al., 2009; stojković et al., 2011; duymuş et al., 2014; fakir et al., 2014; gulsoy toplan et al., 2015; ulukanlı et al., 2015; eryiğit et al., 2015; tin et al., 2017; zhelev et al., 2022 etc.). in our study, essential oil composition of v. sevindik e et al. (2023). not sci biol 15(1):11377 3 agnus-castus flowers analyzed (components with a total ratio of ≥2%) is shown in table 1, while gc-ms chromatogram images are shown in figures 1, 2, 3, 4, 5 and 6. as a result of the study, ‘çakmar’ pink-flowered population had the main component (-)-allospathulenol (14.59%), ‘çakmar’ purple-flowered and ‘koçarlı’ purple-flowered populations had pyrrolo (3,2,1-jk)carbazole (14.42% and 14.68% respectively), while ‘çakmar’ white flowered, ‘koçarlı’ white flowered and ‘koçarlı’ pink flowered populations had the main component of caryophyllene (15.54%, 12.64%, and 19.05 respectively). pca (principal component analysis) (simca 14.1) was used to determine the effect of flower varieties on volatile compounds. table 1. table 1. table 1. table 1. composition of the essential oil from vitex agnus-castus flowers of different populations çakmar pink flowersçakmar pink flowersçakmar pink flowersçakmar pink flowers çakmar purple flowersçakmar purple flowersçakmar purple flowersçakmar purple flowers çakmar white flowersçakmar white flowersçakmar white flowersçakmar white flowers rtrtrtrt componentcomponentcomponentcomponent %%%% rtrtrtrt componentcomponentcomponentcomponent %%%% rtrtrtrt componentcomponentcomponentcomponent %%%% 7.423 α-pinene 2.45 21.516 cyclohexene 3.24 10.530 1.8-cineole 2.22 8.539 sabinene 2.99 24.446 trans β-farnesene 4.63 21.499 α-terpinyl propionate 4.90 15.108 cyclopentasiloxane 2.05 25.556 bicyclogermacrene 6.08 23.490 caryophyllenecaryophyllenecaryophyllenecaryophyllene 15.54 21.591 1-p-menthen-8-yl acetate 6.19 27.839 spathulenol 6.11 24.423 trans-β-farnesene 4.66 23.554 caryophyllene 5.53 27.965 caryophyllene oxide 4.28 27.782 spathulenol 6.36 24.480 trans β-farnesene 4.35 28.480 ledol 2.37 27.925 caryophyllene oxide 5.85 28.051 ((((----))))----allospathulenolallospathulenolallospathulenolallospathulenol 14.59 29.418 delta.-cadinene 4.08 29.390 delta.-cadinene 5.57 28.120 caryophyllene oxide 3.71 34.866 guaia-3,9-diene 2.07 36.187 n-(m-fluorophenyl)maleımıde 4.68 28.675 ledol 2.56 36.250 n-(mfluorophenyl)maleımıde 13.70 36.977 pyrrolo(3,2,1-jk) carbazole 11.46 29.447 . α-cadinol 2.57 36.685 1-(3-hexyl)-2,5dimethylbenzene 3.57 38.688 α-selinene 4.29 36.279 4(1h)-pteridinone 6.54 37.040 pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1----jk)jk)jk)jk) carbazolecarbazolecarbazolecarbazole 14.42 39.346 sigmosceptrellin-b 2.34 37.103 pyrrolo(3,2,1-jk) carbazole 10.77 38.728 bakkenolıde-a-dıol 6.28 38.763 cycloheptane 4.67 39.386 kolavelool 2.29 koçarlı purple flowerskoçarlı purple flowerskoçarlı purple flowerskoçarlı purple flowers koçarlı white flowerskoçarlı white flowerskoçarlı white flowerskoçarlı white flowers koçarlı pink flowerskoçarlı pink flowerskoçarlı pink flowerskoçarlı pink flowers rtrtrtrt componentcomponentcomponentcomponent %%%% rtrtrtrt componentcomponentcomponentcomponent %%%% rtrtrtrt componentcomponentcomponentcomponent %%%% 8.493 sabinene 3.49 9.987 β-phellandrene 2.12 6.811 α-pinene 3.00 10.513 1.8-cineole 3.79 25.179 (+)-2-carene 6.10 8.058 sabinene 4.24 21.505 4-terpinenyl ester of isobutanoicacid 5.63 28.915 caryophyllenecaryophyllenecaryophyllenecaryophyllene 19.05 9.998 β-phellandrene 2.19 23.485 caryophyllene 7.47 32.468 bicyclogermacrene 6.19 15.360 cyclopentasiloxane 5.32 24.423 trans-.β.-farnesene 4.08 39.129 (+)-epibicyclosesquiphelland rene 2.54 25.110 (+)-2-carene 3.34 25.522 bicyclogermacrene 5.23 45.486 2-methyl-6propylpyridine-3carboxylic acid 3.02 28.829 caryophyllenecaryophyllenecaryophyllenecaryophyllene 12.64 27.811 spathulenol 7.25 48.810 (z)-β-caryophyllene 3.32 30.563 trans-. β-farnesene 5.38 27.942 caryophyllene oxide 4.23 50.407 1,2,4-trıazolo(3,4c)(1,2,4)benzotrıazın-1(5h)one 2.98 32.463 bicyclogermacrene 4.50 29.396 α-cadinol 3.53 51.437 β-isomethylıonone 4.96 45.520 2h-1,4-benzothiazine 3.04 36.222 n-(m-fluorophenyl)maleımıde 13.15 53.685 levopimaradiene 2.38 48.822 (z)-β-caryophyllene 3.08 36.674 1-(3-hexyl)-2,5dimethylbenzene 3.54 55.682 cycloheptane 2.60 50.464 1,2,4-trıazolo(3,4c)(1,2,4)-benzotrıazın1(5h)-one 3.49 37.006 pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1----jk)jk)jk)jk) carbazolecarbazolecarbazolecarbazole 14.68 51.431 β-ısomethylıonone 3.74 38.711 bakkenolıde-a-diol 6.72 52.879 3-fluoro-5,7dimethylquinol-2(1h) one 3.67 sevindik e et al. (2023). not sci biol 15(1):11377 4 39.358 sigmosceptrellin-b 2.62 55.654 methylpropyleyne 7.73 56.604 kolavelool 3.57 68.706 4,4'-dimethyl-2,2',5,5'tetramethoxy-1,1'biphenyl 2.02 figure 1. figure 1. figure 1. figure 1. gas chromatogram of the essential oil of ‘çakmar’ pink flowers figure 2. figure 2. figure 2. figure 2. gas chromatogram of the essential oil of ‘koçarlı’ purple flowers sevindik e et al. (2023). not sci biol 15(1):11377 5 figure 3. figure 3. figure 3. figure 3. gas chromatogram of the essential oil of ‘çakmar’ purple flowers figure 4. figure 4. figure 4. figure 4. gas chromatogram of the essential oil of ‘çakmar’ white flowers sevindik e et al. (2023). not sci biol 15(1):11377 6 figure 5. figure 5. figure 5. figure 5. gas chromatogram of the essential oil of ‘koçarlı’ white flowers figure 6. figure 6. figure 6. figure 6. gas chromatogram of the essential oil of ‘koçarlı’ pink flowers the biplot diagram for the first and second components shows the correlation of the studied flowers based on the volatile compounds detected. according to the data obtained, the first and second components (pc1 and pc2) explained 59.7% of the total variance. except for ‘çakmar’ purple flower, ‘çakmar’ and ‘koçarlı’ flower populations were collected in two different groups according to pc1 (figure 7). sevindik e et al. (2023). not sci biol 15(1):11377 7 figure 7.figure 7.figure 7.figure 7. biplot of the first two principal components in chasteberry fruits collected from aydın,türkiye, tin et al. (2017) determined 1.8 cineole (8.24%) as the main component. gulsoy toplan et al. (2015) examined the essential oils of v. agnus-castus fruits collected from zonguldak, edirne, balıkesir, muğla, antalya and the main components were determined as βcaryophyllene (11.7%), bicyclogermacrene (22.1%), 1.8-cineole (17.3%), 1.8-cineole (13.2%), sabinene and bicyclogermacrene (12.1%), respectively. in the essential oils of the fruits of the chasteberry plant collected from izmir/turkey, eryiğit et al. (2015) determined trans-caryophyllene (19.17%) as the main component. duymuş et al. (2014) determined sabinene (22.7%) and 1.8-cineole (22.4%) as the main components of fruits and the fruitless herbs, respectively, of v. agnus-castus collected from i̇zmir, türkiye. fakir et al. (2014) determined essential oil content of v. agnus castus collected from isparta, türkiye during the flowering period and the main component was α-pinene (26.99), while the main component was 1.8 cineole (28.34%) during the fruit ripening period. ulukan et al. (2015) determined the main component as 1.8-cineole (24.38%) from essential oils of chasteberry leaves collected from osmaniye, türkiye. sarikurkcu et al. (2009) identified 1.8cineole (24.98%) as the main component of chasteberry fruits from manisa, türkiye. senatore et al. (2003) determined 1.8-cineol (21.6%) as the most abundant in the essential oil obtained from the white flowered fruit of vitex agnus-castus collected from i̇çel, türkiye, and caryophyllene (30.9%) as the most in essential oil obtained from the fruits of the purple flowered ones. in montenegro, stojkovic et al. (2011) found the main component as sabinene (17.8%) in immature chasteberry fruits, 1.8-cineole (16.3%) in mature fruits, and 1.8cineol (22.0%) in leaves. bakr et al. (2020) have determined 1.8 cineole (44.98 %) as the most abundant component in leaves of v. agnus-castus collected from the city of giza, egypt. zhelev et al. (2022) identified 1.8-cineole (16.9–18.8%) as the main component in chasteberry fruits collected from south-central bulgaria and north-east bulgaria. moudachirou et al. (1998) determined 1.8-cineole (22.6%) as the main component in the essential oil content of the leaves of vitex agnus-castus collected from benin. in the past, in some studies of essential oils on the fruits, leaves and flowers of the vitex agnus-castus, 1.8 cineole, α-pinene, caryophyllene, trans-caryophyllene, β-caryophyllene, sabinene and bicyclogermacrene were determined as main components. sevindik e et al. (2023). not sci biol 15(1):11377 8 when we compare our study with previous ones, the results are not consistent. the reason for this could be that the geographical and environmental conditions in which the samples distributed were different, confirming that the essential oil composition and content of the plants could be different (sevindik et al., 2016). in our study, for ‘çakmar’ white flower, ‘koçar’ white flower and ‘koçarlı’ pink flower populations, the main component was determined as caryophyllene. sesquiterpenoids are an extremely large group of secondary metabolites found in plants. among them, the caryophyllene or β-caryophyllene group is the most common in nature. in the last few years, β-caryophyllene has represented an important topic of study (francomano et al., 2019; gyrdymova and rubtsova, 2021). in the past, the antimicrobial effect of β-caryophyllene has been tested against some human pathogens (dahham et al., 2015). conclusionsconclusionsconclusionsconclusions in this study, we evaluated the chemical composition of essential oils obtained from white, pink and purple chasteberry flowers. as a result, we found (-)-allospathulenol, pyrrolo(3,2,1-jk)carbazole and caryophyllene as main components. these findings may be a source for further antimicrobial, antiinflammatory, anti-cancer, and antioxidant studies. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions e.s, d.k i̇.m.g and f.b collected flowers samples. the experiments were performed and analyzed by e.s. e.a and e.k. e.s, e.a and e.k wrote the paper. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research was supported by the tubitak 2019-2/2209-a (project no: 1919b011904175) conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. sevindik e et al. 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(2023). not sci biol 15(1):11377 11 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11379 nsb biswas 2023.03.16.docx received: 11 nov 2022. received in revised form: 03 mar 2023. accepted: 10 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania biswas i and das mohapatra pk (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11379 doi:10.15835/nsb15111379 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae paraburkholderia tropicaparaburkholderia tropicaparaburkholderia tropicaparaburkholderia tropica pki7 and pki7 and pki7 and pki7 and kosakonia arachidis kosakonia arachidis kosakonia arachidis kosakonia arachidis pki8: two pki8: two pki8: two pki8: two newly reported tannase newly reported tannase newly reported tannase newly reported tannase producing bacteria isolated from forest soil and producing bacteria isolated from forest soil and producing bacteria isolated from forest soil and producing bacteria isolated from forest soil and study of their tannase producing potentialitystudy of their tannase producing potentialitystudy of their tannase producing potentialitystudy of their tannase producing potentiality ishita biswas 1,2, pradeep k. das mohapatra1,3* 1raiganj university, department of microbiology, raiganj 733134 uttar dinajpur, west bengal, india; ishitabiswas1307@gmail.com 2s.b.s. government college, department of botany, hili 733126 dakshin dinajpur, west bengal, india 3raiganj university, professor a. k. bothra environment conservation centre, raiganj 733134, uttar dinajpur, west bengal, india; pkdmvu@gmail.com (*corresponding author) abstractabstractabstractabstract tannins are the polyphenolic secondary metabolites of plants and having antimicrobial properties due to their protein precipitation abilities and other toxic effects towards microbes. however, with the help of an inducible enzyme called tannase, a variety of microbes including bacteria can resist tannin toxicity. tannase catalyses the dissociation of ester bonds in gallotannins like tannic acid, resulting in release of gallic acid and glucose. because of its vast industrial applications and ease of manipulation, interest in identifying and applying bacteria as tannase producers has grown in recent years. the bacterial strains reported in the present work were isolated from soil and identified as paraburkholderia tropica pki7 and kosakonia arachidis pki8 through morphological, biochemical, and 16s rdna molecular approach. simultaneous tannase and gallic acid production by these two bacterial strains were observed through submerged fermentation. this is the first report of paraburkholderia tropica pki7 and kosakonia arachidis pki8 strains as tannase producing bacteria. the initial tannase production were 0.75 u/ml and 1.49 u/ml respectively for p. tropica pki7 and k. arachidis pki8 after 24 h of submerged fermentation while gallic acid release was 8.4 µg/ml and 10.74 µg/ml respectively. there is a potential scope in higher tannase and gallic acid biosynthesis by the two strains. keywords:keywords:keywords:keywords: 16s rdna; bacterial strains; gallic acid; submerged fermentation; tannase; tannin introductionintroductionintroductionintroduction tannins are water-soluble plant secondary metabolites that belong to the polyphenolic family. after cellulose, hemicelluloses, and lignin, they are estimated to be the fourth most prevalent element in plants. based on their structures, they are divided into two groups: hydrolysable and condensed. tannins are important in plant defence mechanisms because they protect plants from microbial attack by rendering viruses and bacteria inactive (jana et al., 2013). despite their benefits, tannins are considered as antinutritional and antimicrobial compounds because they can precipitate proteins, block enzymatic reactions, and interfere with other critical microbial processes (jiménez et al., 2014). in nature, however, a variety of bacteria can withstand tannin toxicity https://www.notulaebiologicae.ro/index.php/nsb/index biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 2 by digesting tannin with the help of an enzyme called tannase (kumar et al., 1999; govindarajan et al., 2019; lekshmi et al., 2020). tannase (tannin acyl hydrolase, ec 3.1.1.20) is an industrially important intraor extracellular enzyme that belongs to the "hydrolase" enzyme class and is mostly found in bacteria, fungi, and yeast (biswas et al., 2022). they are induced by tannin and hydrolyse the ester and depside linkages of a variety of compounds, including gallotannins, ellagitannins, gallic acid esters, epicatechin gallate, and generate gallic acid and glucose. tannase has wide applications in a variety of industrial disciplines, including food, feed, drinks, pharmaceuticals, leather, and dye production (biswas et al., 2020). it is predominantly utilised in the production of gallic acid, acron wine, instant tea, and fruit juice and beer clarification. it demonstrates its practical applicability in the treatment of tannin-containing waste water and leather industry effluent treatment (das mohapatra et al., 2020). the by-product gallic acid of tannin breakdown is a potential antioxidant and having anticancerous properties (mitra et al., 2021). in comparison to the entire bacterial population, there are few reports in the literature on the bacterial origin of tannase (biswas et al., 2020). as a result, looking for new and potential tannase producers is a never-ending effort, because large-scale enzyme synthesis necessitates maintaining a bacterial strain in a continuous culture. furthermore, because tannase is an inducible enzyme, optimising culture conditions for maximum enzyme output is quite difficult. paraburkholderia tropica is a plant growth promoting bacteria (pgpb) and persists in nonleguminous plants and has been found in the rhizosphere, rhizoplane, internal tissues of sugarcane stem from brazil and south africa, as well as maize plants from mexico (garcía et al., 2020). p. tropica demonstrates a variety of in vitro skills that are engaged in promoting plant growth, including the fixation of nitrogen, the formation of organic acids that solubilize phosphate, and the generation of antifungal compounds. p. tropica sp. has boosted maize productivity in terms of in vivo plant-growth promotion; in addition, wheat inoculation with p. tropica mto-293 led to an increase in production under field conditions, however there was no discernible difference (garcía et al., 2020). kosakonia arachidis is a plant-growth-promoting bacterium with activities such as 1aminocyclopropane-1-carboxylate (acc) deaminase, iaa production, and nitrogenase. groundnut (arachis hypogaea l. 'alr-20) rhizospheric soil was previously isolated from the tamil nadu agricultural university experimental plot in coimbatore, india, containing k. arachidis. the root length of indian mustard, tomato, and rice plants was considerably increased by seeds inoculated with k. arachidis (cyg seed germination pouch, mega international). methanol dehydrogenase is produced by k. arachidis (madhaiyan et al., 2010). these above two bacteria were mainly documented as plant-growth-promoting bacterium in literature (madhaiyan et al., 2010; garcía et al., 2020). although there are reports on other species of paraburkholderia as tannase producers (biswas et al., 2020) however no report was found for p. tropica pki7 and k. arachidis pki8 strains. in the present study this is the first report of these two newly isolated bacteria paraburkholderia tropica pki7 and kosakonia arachidis pki8 as potential tannase producers. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods isolation and screening of potent tannase producer soil sample was taken from the kulik forest floor in raiganj, uttar dinajpur, west bengal, india (figure 1). using a selective tannic acid agar media, a number of tannase-producing bacterial strains were identified using a serial dilution approach (mondal and pati, 2000). the serially diluted soil samples were then inoculated in the specific tannin medium and incubated for 72 hours at 35 °c. tannase producers were chosen based on the appearance of a distinct halo zone encircling the bacterial colony. two bacterial strains with the highest biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 3 clear zone to colony size ratio (cz/cs) were selected and designated as pki7 and pki8. the strains were kept at 4 ℃ and subcultured on tannic acid agar slant at regular intervals. figure 1. figure 1. figure 1. figure 1. study area map of kulik forest, raiganj, uttar dinajpur, west bengal, india identification of selected bacterial strain the selected bacterial strains pki7 and pki8 were identified based on their morphological, biochemical, and 16s rdna sequencing properties (biswas et al., 2021). gram staining, colony morphology, and pigmentation were used to characterise bacteria morphologically, whereas biochemical characterization for the isolates included carbohydrate utilisation, catalase production, oxidase production, hydrolysis of starch and casein, citrate utilisation, nitrate reduction, and gelatine liquefaction. carbohydrate fermentation studies were carried out using phenol red as an indicator and a basal medium supplied with appropriate carbohydrate such as glucose, fructose, lactose, sucrose, mannose, and mannitol. the colony morphology such as form/shape, colour, margin, elevation, size, appearance, and optical quality of the bacterial isolates were also studied. using a dna isolation kit (zymotm, usa), the genomic dna of each isolate was extracted. about 10 ml of a culture that had been cultivated for 24 hours (nutrient broth) was centrifuged at 8000 rpm for 15 minutes, and dna was extracted from the cell pellet in accordance with the kit's instructions. using a uv spectrophotometer (nanodrop, thermo scientific), the quality and amount of dna were assessed. gene segment of 16s rdna of the bacteria was amplified by pcr up to 30 cycles using 27f (5′agagtttgatcctggctcag-3′) and 1492r (5′-ggttaccttgttacgactt-3′) primers biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 4 (suzuki and giovannoni, 1996) and purified samples of pcr were sequenced through sanger sequencing. the obtained forward and reverse reading sequences were assembled into contigs using the cap3 assembly programme (huang and madan, 1999), and decipher v2.0 (wright, 2016) was used to screen for the presence of chimaeras. the sequencing was then submitted to the ncbi (https:/ submit.ncbi.nlm.gov/subs/genbank/) and received the accession number. blast was used to match the 16s rdna sequences of the bacterial isolates to the nucleotide database of ncbi genbank. ten sequences were chosen based on their maximum identity score, and the sequences were aligned using the multiple sequence alignment software application clustalw. megax software was used to create a phylogenetic tree based on the tamura-nei model, which included 1000 bootstrap replications and a standard pattern of partially deleted nucleotide changes (tamura and nei, 1993; kumar et al., 2016). preparation of inoculum and production of tannase through submerged fermentation inoculum was prepared by growing a loop full amount of stock culture of the bacterium in 50 ml sterile tannic acid medium (ph 5.0) as described above at 35 oc for 20h. the inoculation medium and the fermentation medium were identical. the bacterial isolates pki7 and pki8 were grown for 24 hours on a rotary shaker (120 rpm) at 35 °c with 50 ml of sterilised media in a 250 ml erlenmeyer flask. the culture broth was centrifuged at 5000 g for 15 min to check for the production of tannase and gallic acid in the supernatant. study of bacterial growth the cell concentration was evaluated using turbidimetry at 620 nm to measure the growth by uv-vis spectrophotometer (das mohapatra et al., 2020). enzyme assay tannase activity in the fermented medium was determined according to the colorimetric method of mondal et al. (2001). the specific extinction coefficient of tannic acid at 530 nm was 0.577. one unit (u) of tannase was defined as the amount of enzyme, which can hydrolyse 1 μmole of the ester linkage of tannic acid per minute. estimation of gallic acid gallic acid content of the culture filtrate was estimated by the method of bajpai and patil (1996). the following equation was used to determine the concentration of gallic acid using a specified extinction coefficient: concentration of gallic acid (mg/ml) = 21.77 (a254.6) 17.17 (a 293.8) results results results results and discussionand discussionand discussionand discussion isolation and screening of potent tannase producer on tannic acid agar plates, microorganisms isolated from soil samples were screened. after 72 hours of incubation on tannic acid – agar plates, a diverse population of bacteria and fungi was discovered. pki7 and pki8 were chosen as a powerful tanninolytic bacteria based on higher clear zone to colony size (cz/cs) ratio of 1.36 and 1.78 respectively with clear zone around the colonies of 15 mm and 16 mm respectively on tannic acid agar plate (figure 2a and 2c). after 72 hours of incubation, the strain pki8 changes pigmentation and appears brownish in colour in tannic acid agar medium (figure 3). the bacterial isolates were cultured in liquid medium with tannic acid (1% w/v) as the sole carbon source, and after 24 hours of incubation, the tannin degradation potentiality of pki7 and pki8 were found to be 7.6% and 15.67% respectively. biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 5 figure 2. figure 2. figure 2. figure 2. growth of pki7 (a) and pki8 (c) on tannic acid agar medium showing zone of tannin hydrolysis after 24 h of incubation; while b showing tannin tolerant bacteria figure 3. figure 3. figure 3. figure 3. growth of pki8 on tannic acid agar medium showing pigmentation after 72 h of incubation identification of selected bacterial strain the selected bacterial isolates pki7 and pki8 were characterized and identified based on the morphological, biochemical, and molecular characteristics. colonies of pki7 were tiny, circular, off white in colour, opaque, smooth in texture, flat with entire margin while pki8 colonies had a diameter of 3-4 mm, were off white in colour, smooth, erose, opaque, and had an entire margin. table 1 shows the colony features of pki7 and pki8 in further detail. the cells of both the strains were aerobic, gram-negative rods (figure 4a and 4b). strain pki7 was found to produce spore but pki8 did not sporulate. strain pki7 were found to be aerobic, gram-negative rods and were positive for catalase and nitrate reduction while negative to gelatin utilization which indicates that it belongs to the family burkholderiaceae while the main characteristics of pki8 were acid and gas production from d-glucose and other carbohydrates, catalase positivity while oxidase negativity, and inability to convert nitrate to nitrite. this strain has also been discovered to grow on mac conkey's medium, with red colonies. these traits led to the conclusion that the isolate pki8 belongs to the enterobacteriaceae family. table 1.table 1.table 1.table 1. colony characteristics of the bacterial isolates isolates characteristics size (mm) shape colour margin elevation opacity texture /surface appearance paraburkholderia tropica pki7 tiny circular off white entire flat opaque smooth dull kosakonia arachidis pki8 moderate (34) circular off white (24h)/ brown (72 h) lobate raised opaque smooth dull biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 6 figure 4. figure 4. figure 4. figure 4. gram staining view of pki7 (a) and pki8 (b) table 2 summarises the morphological and biochemical properties of the isolates. table 2table 2table 2table 2. morphological and biochemical characterization of bacterial isolates characteristics studied result (paraburkholderia tropica pki7) (kosakonia arachidis pki8) gram’s staining shape rod rod colour off white off white motility + + oxygen tolerance + + spore + citrate utilization + + starch hydrolysis casein hydrolysis + gelatin hydrolysis indole production + + mr/vp test + catalase + + oxidase + nitrate reduction + carbohydrate fermentation test: (acid production) glucose + fructose + sucrose + lactose mannitol + + mannose + + maltose + (+) positive, (-) negative biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 7 the voges-proskauer test revealed positive result for the strain pki8 but negative for pki7 while for gelatinase both are negative. nitrate cannot be converted to nitrite by pki8 but pki7 is potent in this task. fermentation of glucose, fructose, sucrose, mannitol, mannose, and maltose by pki8 produces acid and gas but not through pki7, however lactose fermentation produces neither by them (table 2). citrate utilisation was positive for both, whereas casein and starch hydrolysis were negative for pki8 but pki7 showed positive response for casein hydrolysis. these characteristics revealed that the bacterial strain pki7 belongs to the genus paraburkholderia and pki8 to the genus kosakonia. the growth profile of the bacterial isolates in nutrient broth medium has been shown in figure 5. figure 5. figure 5. figure 5. figure 5. growth curve of pki7 (a) and pki8 (b) 16s rdna profiling and a blast study were used to confirm the identity of isolate. the 16s rdna nucleotide sequence of strain pki7 was found to be 99.03 identical to that of paraburkholderia tropica strain 202 (genbank accession as mk389460.1) while pki8 was 98.95 percent identical to that of kosakonia arachidis strain ah-143 (genbank accession as nr 116403.1). strain pki7 was found to be clustered with paraburkholderia tropica strain 202 on the phylogenetic tree and therefore the bacterial strain was designated as paraburkholderia tropica strain pki7 (genbank accession number mw784720.1) (figure 6), on the other hand, pki8 was found to be clustered with kosakonia arachidis strain ah-143 on the phylogenetic tree, therefore, the bacterial strain was named as kosakonia arachidis strain pki8 (genbank accession number mz127794.1) (figure 7). this is the first report on tannin fermentation and tannase production by paraburkholderia tropica pki7 and kosakonia arachidis pki8, two newly isolated tannase producers. 0 0,5 1 1,5 2 0 4 8 12 16 20 24 o .d . a t 6 2 0 n m incubation time (h) pki7 pki8 biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 8 figure 6. figure 6. figure 6. figure 6. phylogenetic tree (neighbor-joining consensus tree) on the basis of 16s rrna sequence of gene showing the distance between strain pki7 with other strain of paraburkholderia species. the numbers over branches represent bootstrap confidence values (%) based on 1000 replicates. the genbank accessions are shown in parenthesis figure 7. figure 7. figure 7. figure 7. phylogenetic tree (neighbor-joining consensus tree) on the basis of 16s rrna sequence of gene showing the distance between strain pki8 with other strain of kosakonia species. the numbers over branches represent bootstrap confidence values (%) based on 1000 replicates. the genbank accessions are shown in parenthesis biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 9 estimation of tannase activity and gallic acid the estimated initial enzyme yield by p. tropica pki7 and k. arachidis pki8 was found to be 0.75 and 1.49 u/ml respectively and gallic acid production was 8.4 and 10.74 µg/ml respectively at 35 ℃ with medium ph 5.0 (table 3). table 3table 3table 3table 3. initial enzyme activity and gallic acid production by the bacterial isolates bacterial strains ph temperature tannase activity (u/ml) gallic acid production (µg/ml) paraburkholderia tropica pki7 5.0 35 ℃ 0.75 8.4 kosakonia arachidis pki8 1.49 10.74 raghuwanshi et al. (2011) identified a potent tannase-producing bacteria bacillus sphaericus from soil and found that it had the greatest enzyme activity (11.2 iu/ml) under optimal conditions. similarly, jana et al. (2013) found that under optimal conditions bacillus subtilis pab2 produced more tannase (10.69 u/ml) and detected the maximum quantity of gallic acid accumulation (6.45 mg/ml) after 36 hours of fermentation. another report observed that for b. subtilis am1 and l. plantarum cir1, the maximum tannase production levels were 1400 and 1239 u/l after 32 and 36 hours of fermentation, respectively while highest gallic acid release for b. subtilis am1 was 24.16 g/l and for l. plantarum cir was 23.73 g/l (aguilar-zárate et al., 2015). borah et al. (2023) documented highest tannase activity and gallic acid yield of 0.0497 u/ml and 225 µg/ml respectively by bacillus gottheilii m2s2. conclusionsconclusionsconclusionsconclusions the bacterial strains paraburkholderia tropica pki7 and kosakonia arachidis pki8 were tested for tannase activity and gallic acid production. under submerged fermentation conditions, both the isolates can produce extracellular tannase. further, the strains were able to produce gallic acid. more research is being done to improve the culture conditions for maximal enzyme production. this is the first report of paraburkholderia tropica pki7 and kosakonia arachidis pki8 as potential tannase producers. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization and design of experiments (ib and pkdm); methodology (ib); data collection, analysis, and interpretation (ib); supervision (pkdm); writing original draft (ib); writing – review, and editing (pkdm). all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. biswas i and das mohapatra pk (2023). not sci biol 15(1):11379 10 conflict of conflict of conflict of conflict of interestsinterestsinterestsinterests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences aguilar-zarate p, cruz ma, montañez j, rodriguez-herrera r, wong-paz je, belmares re, aguilar cn (2015). gallic acid production under anaerobic submerged fermentation by two bacilli strains. microbial cell factories 14(1):17. https://doi.org/10.1186/s12934-015-0386-2 bajpai b, patil s (1996). tannin acyl hydrolase (ec 3.1. 1.20) activity of aspergillus, penicillium, 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opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11352 nsb panfilova 2023.06.26.docx received: 19 sep 2022. received in revised form: 20 apr 2023. accepted: 13 jun 2023. published online: 26 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania panfilova a et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11352 doi:10.15835/nsb15211352 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae iiiinfluencenfluencenfluencenfluence of biologics on the productivity of winter wheat varieties of biologics on the productivity of winter wheat varieties of biologics on the productivity of winter wheat varieties of biologics on the productivity of winter wheat varieties under irrigation conditionsunder irrigation conditionsunder irrigation conditionsunder irrigation conditions antonina panfilova*, margaryta korkhova, nataliia markova mykolayiv national agrarian university, faculty of agricultural technologies, 9 georgiya gongadze str., mykolayiv, 54020, ukraine; panfilovaantonina@ukr.net (*corresponding author); korhovamm@mnau.edu.ua; 7markovanataliya@ukr.net abstractabstractabstractabstract the aim of the research was to determine the influence of varietal characteristics, pre-sowing seed treatment and irrigation on the yield of winter wheat (triticum aestivum l.). experimental studies were carried out in the conditions of mykolayiv national agrarian university (ukraine) during 2019-2022 yrs. it was found that among the studied variety of winter wheat, the highest grain yield was distinguished by the variety ‘ozerna’ and ‘duma odes’ka’. on average, for pre – sowing seed treatment and irrigation options, these variety formed yields at the level of 6.78 and 6.95 tha–1, which was higher than the grain yield of ‘anatoliya’ variety by 4.1 up to 6.5%, and then the yield of ‘ovidii’ variety by 3.9 up to 6.3%. it was proved that preparations for pre-sowing seed treatment contributed to the formation of more productive stems and grain mass from one ear in winter wheat plants. so, on average, over the years of research, variety and irrigation options, for the use of azotofitr, the number of productive stems increased by 34-74 pcs./ m2 compared to other seed treatment options. at the same time, the yield of winter wheat grain was also slightly higher with the use of azotofit-r biologics. it was found that the use of irrigation provided an increase in grain yield by 1.66-2.19 tha–1 or 23.1-27.2%, depending on the studied winter wheat variety. at the same time, the ‘duma odes’ka’ variety had a higher grain yield under irrigation (8.06 tha–1), while in conditions without irrigation, a slightly higher grain yield was formed by plants of the ‘ozerna’ variety (5.87 tha–1). on average, over the years of research, the highest yield of winter wheat grain was provided by the cultivation of the ‘duma odes’ka’ variety for pre-sowing seed treatment with azotofit r biologics and irrigation–8.38 tha–1. keywords:keywords:keywords:keywords: biologics; irrigation; variety; winter wheat; yield introductionintroductionintroductionintroduction agrarian science has faced the problem of is the humanity provision with food, the major part of which is provided by grain crops. today, grain production has a special place in the structure of the agro-industrial complex. it is the grain and products of its processing which are vital products that ensure the food security of the state, play an important role in the socio-economic development of the national economy, form the basis of agricultural exports and determine the degree of its participation in international cooperation (panfilova and mohylnytska, 2019). https://www.notulaebiologicae.ro/index.php/nsb/index panfilova a et al. (2023). not sci biol 15(2):11352 2 wheat production and utilization accounts for 28% of the global cereal crops. wheat consumption has been steadily increasing due to population expansion and urbanization. consequently, wheat supplies approximately one-fifth of human calories in a variety of forms. wheat will remain a crucial component of human nutrition, and increasing its production is therefore an important requirement for food security (hongjie et al., 2019; nazarenko et al., 2020). winter wheat makes the fullest use of the existing bioclimatic potential of the regions of ukraine. the highest productivity of this crop can be obtained if modern adapted variety with a high genetic potential of productivity are used, taking into account the soil and climatic conditions of the zone, improving and developing highly effective, scientifically based nutrition and protection systems against harmful organisms (markovska and hrechyshkina, 2020). due to the increase in the world's population, the decrease in acreage and gross crop yields due to military operations, the question arises of finding innovative methods to increase the yield of these crops and, as a result, increase the world's food reserves. wheat yield varies from year to year under the influence of moisture availability of crops during the growing season, the variety grown and agricultural techniques for growing the crop, in particular the introduced nutrients (suciu et al., 2018; panfilova et al., 2020; ma et al., 2022; zhang et al., 2022). wheat production in europe countries is particularly dependent on synthetic fertilizers because the use of animal manure is very limited, many of the soils are naturally low in levels of soil organic matter and there are only a few legumes present in main crop rotations that could supply symbiotically fixe nitrogen (biel et al., 2016). research of many foreign and domestic scientists (ying, 2019; hashem et al., 2019; gamayunova and panfilova, 2020; panfilova et al., 2020; müller and behrendt, 2021; nemea et al., 2021; holtappels et al., 2021; panfilova, 2021; panfilova et al., 2021) the most effect of biologics and growth-regulating drugs on the yield and quality of agricultural crops has been proven. to increase the yield of agricultural products, modern nanotechnologies based on growth-regulating preparations containing carbon and cuprum are widely used in scientific research, which protect plants from pathogenic organisms and negative environmental factors (jacob et al., 2020; singh et al., 2021; afreena et al., 2022). research by reda (2021) the positive effect of biostimulants and bioprotectors based on seaweed extracts, humic substances, protein hydrolysates, amino acids, and plant extracts on increasing crop productivity has been proven. no less important in increasing the productivity of agricultural crops is the choice of variety (visioli et al., 2018). replacing old variety with new, more productive, competitive ones with wide agroecological plasticity and increased adaptive properties to adverse environmental conditions, better adapted to the soil and climatic conditions of a certain area and an increased level of agricultural technology is one of the most rational and economic means of increasing the grain yield of winter grain crops with high quality indicators (krivenko and pochkolina, 2019; hudzenko et al., 2021). research by bazalii et al. (2019) it is established that when identifying variety and forms of various types of high-yielding wheat, which has an increased adaptive potential, it is necessary to conduct a step-by-step assessment under different growing conditions. nowadays in the state register of plant varieties, there are more than 460 cultivars of winter wheat. for effective breeding work, the initial material must be studied in detail to meet specific parameters and requirements (wu and zhatova, 2022). obtaining a comparative assessment of new variety and selecting promising ones for further study and introduction into production is impossible without ecological variety testing, so the study of new promising variety of winter wheat in production conditions using modern biologics and irrigation is relevant. panfilova a et al. (2023). not sci biol 15(2):11352 3 materials and methodsmaterials and methodsmaterials and methodsmaterials and methods experimental researches were carried out during 2019-2022 yrs on the experimental field of the mykolayiv national agrarian university, ukraine. the object of research was triticum aestivum l. – variety ‘ovidii’, ‘duma odes’ka’, ‘ozerna’ and ‘anatoliya’. the technology of its cultivation, except for the studied factors, was generally accepted to the existing zonal recommendations for the southern steppe of ukraine. the soil of the experimental field was a typical southern chernozem of residual-slightly saline heavy loess on loess with a humus content (0-30 cm) from 3.1 up to 3.3% and a neutral reaction of the soil solution (ph – 6.8-7.2). the arable soil layer contained an average of 15-25 mobile forms of nitrates (by grandval lyagu), 41-46 mobile phosphorus (by machigin) and 389-425 mg/kg of exchange potassium (on a semi-lamp photometer). agricultural techniques for growing winter wheat of various species were generally accepted for the zone, with the exception of variants that were studied according to the experimental scheme. its predecessor was sown peas. the scheme of experience included the following variants: factor a variety: 1. ‘ovidii’; 2. ‘duma odes’ka’; 3. ‘ozerna’; 4. ‘anatoliya’. factor b – seed treatment: 1. water treatment (control); 2. azotofit–r (0.3 l/t); 3. phytocide (1.5 l/t); 4. mycophrend-r (1.0 l/t); 5. organic-balance monophosphorus (0.5 l/t). factor c humidification conditions: 1. without irrigation; 2. irrigation (1 water-charging irrigation before sowing 800-1000 m3 ha–1, 2 vegetation irrigation of 400-500 m3 ha–1). the total area of the experimental plot is 50 m2, the accounting plot is 26 m2, the repetition rate is four times. records and observations, the number of productive stems, the mass of grain from one ear and grain yield were determined by the method of state variety testing (tkachyk et al., 2017). mathematical and statistical processing of research results was performed using variance analysis using the statistica 10.0 program (ushkarenko et al., 2020). results results results results and discussionand discussionand discussionand discussion the productivity of winter wheat varies from year to year under the influence of moisture availability of crops during the growing season, the variety grown and agricultural techniques for growing the crop, in particular the use of biologics for pre-sowing seed treatment. it is possible to get a guaranteed high yield of winter wheat grain in the southern steppe zone of ukraine, regardless of climatic conditions, only on irrigated land. the productivity of winter wheat plants mainly depends on the density of the productive stem and the mass of grain from one ear. the largest number of productive stems is 917 pcs./m2 under irrigation conditions was formed in the variety ‘duma odes’ka’ with the use of pre-sowing seed treatment with azotofit-r. slightly lower indicators were observed in all the studied variety in conditions without irrigation, but the trend regarding the use of drugs remained the same (table 1). panfilova a et al. (2023). not sci biol 15(2):11352 4 table 1.table 1.table 1.table 1. number of productive stems of winter wheat variety depending on pre-sowing seed treatment and irrigation, pcs./m2 (average for 2020-2022 yrs) variety (factor а) seed treatment (factor в) water treatment (control) azotofit-r phytocyde mycophrend-r organic-balance monophosphorus without irrigation (factor с) ‘ovidii’ 566 619 570 619 630 ‘duma odes’ka’ 609 651 638 657 649 ‘ozerna’ 598 656 639 654 649 ‘anatoliya’ 581 647 640 735 625 irrigation (factor с) ‘ovidii’ 714 826 758 816 776 ‘duma odes’ka’ 801 917 877 889 842 ‘ozerna’ 758 844 787 816 805 ‘anatoliya’ 708 764 745 755 757 lsd0.5: factor a: 17-36; factor b: 43-48; factor c: 22 30 the most productive stems of winter wheat plants were noted for growing the crop on irrigation and using azotofit-r biologics for pre-sowing seed treatment. thus, on average over the years of research, in this version of the experiment, 764 pcs./m2 of productive stems were formed in plants of the anatolia variety, and 826 and 844 pcs./m2 in the ‘ovidii’ and ‘ozerna’ variety, respectively. best of all, the ‘duma odes’ka’ variety responded to seed treatment before sowing with azotofit-r, which had 917 pcs./m2 of productive stems. it should be noted that the variety react differently to irrigation conditions. so, on average, over the years of research and according to the factor of pre sowing seed treatment, plants of the ‘ozerna’ and ‘duma odes’ka’ variety formed the most productive stems under irrigation conditions 802 and 865 pcs./m2 respectively. while in conditions without irrigation, the most productive stems were found in the variety ‘duma odes’ka’ and ‘anatoliya’ 641 and 646 pcs./m2 respectively. the factors studied by us also affected the mass of grain from one ear of winter wheat, which, depending on the experiment option, fluctuated on average over the years of research from 0.90 to 0.98 g. at the same time, it should be noted that this indicator was highest for growing the studied variety of winter wheat under irrigation conditions and conducting pre-sowing seed treatment with azotofit–r biologics, which exceeded the indicators of other experiment options by 1.0-8.2%. in the conditions of the southern steppe of ukraine, precipitation in the summer period falls extremely unevenly, mainly has a storm character, is lost to runoff and evaporation, especially with a low content of organic substances in the soil, so irrigation is the only way to provide plants with moisture during this period (drobitko et al., 2022). on average, over the years of our research and according to the options for pre-sowing seed treatment, the yield of winter wheat grain on the options without irrigation was 5.53-5.87 tha–1, depending on the variety under study (figure 1). the use of irrigation provided an increase in grain yield by 1.66-2.19 tha– 1 or 23.1-27.2%. panfilova a et al. (2023). not sci biol 15(2):11352 5 figure 1figure 1figure 1figure 1. influence of varietal characteristics and irrigation on the yield of winter wheat grain, tha–1 (average for 2020-2022 yrs) it should be noted that the ‘duma odes’ka’ variety responded better to irrigation in all the years of research, while on average for 2020-2022 yrs the grain yield of this variety was 8.06 tha–1, while in conditions without irrigation, plants of the ‘ozerna’ variety formed a slightly higher grain yield – 5.87 tha–1. irrigation is one of the means of sustainable development of regions with a lack of moisture supply for the growing season of plants and increasing crop productivity (ghane et al., 2009). research by martínez at al. (2020) irrigation sheet reductions of 1 to 0.8, 0.6 and 0.4 m decreased grain yield by 14.4, 37.6 and 76.8%, respectively. in limiting humidity (0.4 m) the number of spikes and biological yield decreased 63.7 and 73.3%. the data by zhou et al. (2018) the simulated grain yield based on different growing season rainfall categories was more affected by irrigation when the growing season precipitation was below 100 mm. two irrigations (75 mm each at the jointing stage and a thesis stage) resulted in the highest grain yield. the choice of variety plays an important role in increasing the productivity of agricultural crops, including winter wheat. we found that on average over the years of research, options for pre – sowing seed treatment and moistening conditions, slightly higher grain yields were formed by plants of the ‘duma odes’ka’ variety – 6.95 tha–1, which exceeded the indicators for the ‘ozerna’ variety by 0.17 tha–1 or 2.4%, the ‘ovidii’ variety by 0.44 tha–1 or 6.3%, and for the ‘anatoliya’ variety by 0.45 tha–1 or 6.5% (table 2). panfilova a et al. (2023). not sci biol 15(2):11352 6 table 2.table 2.table 2.table 2. winter wheat grain yield depending on varietal characteristics, pre-sowing seed treatment and moistening conditions, tha–1 (average for 2020-2022 yrs) variety (factor а) seed treatment (factor в) water treatment (control) azotofit-r phytocyde mycophrend-r organic-balance monophosphorus without irrigation (factor с) ‘ovidii’ 5.10 5.71 5.29 5.71 5.84 ‘duma odes’ka’ 5.46 6.08 5.76 5.95 5.90 ‘ozerna’ 5.37 6.07 5.81 5.94 6.16 ‘anatoliya’ 5.29 6.04 5.98 5.90 5.80 irrigation (factor с) ‘ovidii’ 6.73 8.15 7.31 7.85 7.44 ‘duma odes’ka’ 7.60 8.38 8.28 8.11 7.94 ‘ozerna’ 7.04 8.20 7.60 7.85 7.72 ‘anatoliya’ 6.77 7.48 7.21 7.21 7.29 lsd0.5: factor a: 3 – 11; factor b: 4 – 13; factor c: 67-87 research by djaman at al. (2018) it was found the grain yield was dependent on winter wheat variety, decreased with years, and varied from 1.84 to 7.09 tha–1. ‘tam107’ obtained the highest grain yield. based on the research results of wu and zhatova (2022) identified wheat variety that have high productivity parameters. these are variety such as ‘okhtyrchanka yuvileina’, ‘svitanok myronivskiy’, ‘melody odes’ka’, ‘kubok’, ‘zorepad’, ‘ovidii’, ‘shchedra niva’, ‘octave odes’ka’ and ‘slaven’. our studies determined that the use of biologics for pre-sowing seed treatment increased the yield of winter wheat grain compared to the control. thus, the use of the biological product azotofit-r provided the highest grain yield, which on average over the years of research, by variety and irrigation options amounted up to 7.01 tha–1, which was more than the control by 0.84 tha–1 or 12.0%. the yield of winter wheat grain was slightly lower when using phytocide, mycophrend-r and organic-balance monophosphorus biologics for pre – sowing seed treatment such as 6.66; 6.82 and 6.76 tha–1, which was respectively higher than the control by 0.49; 0.65 and 0.59 tha–1 or 7.4; 9.5 and 8.7%. on average, over the years of research, it was found that the highest yield was formed in all the variety we studied by combining the option of pre-sowing seed treatment with azotofit-r and irrigation. thus, the winter wheat variety ‘duma odes’ka’ on this variant of the experiment grain yield was 8.38 tha–1, yield of the variety ‘ozerna’ as 8.20 tha–1, the yield of ‘ovidii’ as 8.15, and yield of the variety ‘anatoliya’ as 7.48 tha–1, which was respectively more by 2.92; 2.83; 3.05 and 2.19 tha–1 or by 34.8; 34.5; 37.4 and 29.3% compared to the control version of the experiment (without seed treatment with biologics and without irrigation). the use of biologics in the cultivation of winter wheat also had a positive impact on its productivity in the research of other scientists. thus, almashova and skok (2022) found that the greatest increase in the yield of winter wheat grain was observed during three-time cultivation of crops with grain active–c in the tillering phase, flag leaf, grain filling (0.32 tha–1, 1.18%). at the same time, the greatest effect was observed when processing crops in the tillering phase, where the yield increase was 16% before the control. processing of crops in the flag leaf phase contributed to an increase in yield by 4%, and in the grain filling phase – 4.5%. research by markovska and hrechyshkina (2020) it was found the highest level of productivity was formed by plants of winter wheat of the maria variety against the background of applying mineral fertilizers at a dose of n30p30 for pre-sowing cultivation using foliar top dressing with organo-mineral fertilizer rost (2.0 l/ha) at the beginning of the spring vegetation recovery and in the flag leaf phase. so, on average over the years of research, the grain yield in this variant was 4.96 tha–1, the number of productive stems was 411 pcs/m2, number of grains in an ear was 27.6 pcs, the weight of 1000 grains were 51.5 g. panfilova a et al. (2023). not sci biol 15(2):11352 7 research by panfilova et al. (2020) it was determined that the higher grain yield and slightly betterquality indicators differed grain of the studied winter wheat variety with the joint use of pre-sowing application of n30p30 and foliar fertilizing of winter wheat crops twice during the vegetation season by escort-bio. the maximum grain yield in the experiment was formed by winter wheat plants of the variety zamozhnist in the background + escort – bio nutrition variant in the range from 3.76 to 6.28 tha–1 depending on the weather conditions of the year. conclusionsconclusionsconclusionsconclusions studies established that in all years of research, there is clearly a positive effect of using azotofit-r biologics for pre–sowing treatment of winter wheat seeds and irrigation for growing the ‘duma odes’ka’ variety, where the average grain yield over the years of research was 8.38 tha–1. studies established the efficiency of irrigation for the cultivation of all the studied variety of winter wheat. thus, on average, over the years of research and by the factor of pre – sowing seed treatment, the grain yield on irrigation applications was 7.19– 8.06 tha–1, which was higher than the indicators of options without irrigation by 1.66–2.19 tha–1 or 23.127.2%. the studied variety of winter wheat reacted differently to irrigation conditions. thus, under the conditions of natural moisture, the highest grain yield was formed by plants of the ‘ozerna’ variety (5.87 tha– 1), and on the irrigation option – ‘duma odes’ka’ (8.06 tha–1). the optimal combination of elements of cultivation technology developed to increase the yield of winter wheat makes it possible to obtain a high grain yield in the conditions of the southern steppe of ukraine, thus confirming the important scientific and practical significance of the work under study. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization, a.p. and m.k.; methodology, a.p., m.k. and n.m; validation, a.p. and m.k.; formal analysis, a.p. and m.k.; investigation, a.p., m.k. and n.m; resources, a.p. and m.k.; writing-original draft preparation, a.p.;writing-review and editing, a.p.; supervision, a.p., m.k. and n.m. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. panfilova a et al. 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(2023). not sci biol 15(2):11352 10 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11423 nsb el mekkaoui 2023.03.16.docx received: 29 dec 2022. received in revised form: 02 feb 2023. accepted: 10 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania el mekkaoui a et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11423 doi:10.15835/nsb15111423 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae phytochemical studies and phytochemical studies and phytochemical studies and phytochemical studies and in vitroin vitroin vitroin vitro evaluation of the antioxidant evaluation of the antioxidant evaluation of the antioxidant evaluation of the antioxidant activity of some medicinal and aromatic plants from moroccoactivity of some medicinal and aromatic plants from moroccoactivity of some medicinal and aromatic plants from moroccoactivity of some medicinal and aromatic plants from morocco anas el mekkaoui1,2*, mohamed khamar2, chaimae slimani3, abderrahman nounah2, essediya cherkaoui2, fatima benradi2, chaimae rais1 1national agency for medicinal and aromatic plants, laboratory of botany, p.o. box 159 taounate, 34025, 10, morocco; raischaimae18@gmail.com 2mohammed v university of rabat, higher school of technology, laboratory of civil and environmental engineering (lgce), p.o. box 227, salé, morocco; anasitsugo@gmail.com (*corresponding author); mohamed.khamar@um5.ac.ma; abderrahman.nounah@um5.ac.ma; essediya.cherkaoui@um5.ac.ma; fatima.benradi@um5.ac.ma 3sidi mohamed ben abdellah university, faculty of sciences and technologies, department of biology, laboratory of functional ecology and environmental engineering, p.o. box 2202, route d’imouzzer, fez, morocco; chaimae.slimani94@gmail.com abstractabstractabstractabstract the present work was carried out to evaluate the phenolic compounds and the antioxidant activities of some solvent extract (methanol, hydroethanol and aqueous) of several moroccan medicinal plants known for their high antioxidant properties. the extracts were obtained by sonication, then, the total phenolics and flavonoids compounds were determined using folin-ciocalteu and aluminium chloride. afterwards, the total antioxidant capacity and dpph scavenging methods were performed. results of phytochemical analysis showed that the total phenolics content were the highest in the hydroethanolic extract of arbutus unedo with 160.76 mg gae g-1dm, and the flavonoids content were the highest for the hydroethanolic extracts of inula viscosa with 489.77 mg qe g-1 dm. also, it can be noted that arbutus unedo, argania spinosa, and myrtus communis exhibited the most potent antioxidant activity respectively with 0.026; 0.043; 0.036 mg ml-1. keywords:keywords:keywords:keywords: antioxidant activity; medicinal plants; morocco; oxidative stress; phenolic compounds; radical scavenging introductionintroductionintroductionintroduction oxidative stress is a phenomenon that reflects an imbalance between the production of reactive oxygen species (ros) called oxidants, and their elimination by protective mechanisms called antioxidant systems, which can detoxify the reactive intermediates, or repair the resulting damage, causing toxic effects through the production of peroxides and free radicals (pizzino et al., 2017). in addition, some reactive oxidant species act as cellular messengers in redox signaling that can cause disruptions in normal cell signaling mechanisms (milkovic et al., 2019). https://www.notulaebiologicae.ro/index.php/nsb/index el mekkaoui a et al. (2023). not sci biol 15(1):11423 2 oxidative stress is thought to be involved in the development of atherosclerosis, neurodegenerative diseases such as alzheimer's and parkinson's, cancer, diabetes mellitus, and inflammatory diseases, as well as psychological diseases or aging processes (forman and zhang, 2021). the participation of oxidative stress, which is connected to the formation of reactive oxygen and nitrogen species by all aerobic organisms, including free radicals, is a common factor in the pathogenesis of the majority of chronic diseases (reza et al., 2010). these reactive molecular species have a major impact on intra and extra cellular signaling, and can start harmful metabolic processes. a sophisticated antioxidant defense has been established to counteract this damage, and dietary antioxidants play an important role in this defense (chib et al., 2020). antioxidants prevent oxidative stress in biological systems. because of the unpaired electron in their structure, antioxidants are able to neutralize free radicals or radical ions. they have the effect to remove the radical ions produced as a result of oxidation from the system without damaging the biological system (çalişkan and cengiz çalişkan, 2021). medicinal and aromatic plants (map) are inexhaustible natural sources of bioactive compounds, including antioxidants (lourenço et al., 2019). in light of its geographical location, morocco is said to have a large variety of map, with over 4,200 species and subspecies, of which 22% are endemic (rankou et al., 2013). the aim of our work is to exploit the potential of moroccan medicinal plants in order to highlight antioxidant activity which is closely related to the content of phenolic compounds (aryal et al., 2019) and demonstrate their possible use as alternative therapies against oxidative diseases. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant material the plant material used for the present study consists of seven medicinal and aromatic plants that were selected according to their known medicinal effects, their uses in traditional medicine, and the endemic character for some plants. the data related to these plants are represented in table 1. the leaves of these maps were sorted and dried for 72 h at 35 °c. thereafter, they were crushed and sieved to 300 μm to obtain a fine homogeneous powder, that will be used for extracts. table 1table 1table 1table 1. medicinal and aromatic plants studied species family gps data marrubium vulgare l. lamiaceae douar sahel boutaher (34°30.4718'n, 4°47.8572'o) inula viscosa l. asteraceae douar sahel boutaher (34°30.1870'n, 4°46.9117'o) retama monosperma l. fabaceae settat (33°0.4556'n, 7°34.9844'o) myrtus communis l. myrtaceae anpma (34°29.8993'n, 4°48.1756'o) arbutus unedo l. ericaceae tamesnit ratba (34°44.6810'n, 4°53.3705'o) argania spinosa l. sapotaceae agadir (30°26.1546'n, 9°27.3926'o) cannabis sativa cannabinaceae douar rkaiba (34°43.9322'n, 4°52.0309'o) preparation of the extract three extracts were prepared for each plant: a methanol, hydroethanol (20/80) and aqueous extract. to do this, 40 mg of dry matter was added to 10 ml of solvent, the extraction was made by ultrasound at 35 khz, the whole was then centrifuged for 30 min at 3000 rpm. the supernatant was recovered and stored at 4 °c. el mekkaoui a et al. (2023). not sci biol 15(1):11423 3 total phenolic content (tpc) the folin-ciocalteu technique was used to determine the total phenolic content of these maps (cheng and li, 2004). indeed, 200 μl of the extract was mixed with 1.5 ml of 10% folin-ciocalteu reagent. after 5 minutes, 1.5 ml of 5% sodium carbonate was added. the absorbance was measured at 725 nm after 2 hours of incubation. the concentration of total polyphenols was calculated based on a previous calibration curve performed with standard gallic acid. results are expressed as mg gallic acid equivalents per gram dry matter (mg gae g-1 dm). total flavonoids contents (tfc) the flavonoid contents were assessed in accordance with the method used barros et al., 2011. therefore, 0.3 ml of 5% nano2 were added to 1 ml of extract. after 5 minutes, 0.3 ml of 10% alcl3 were added. then, 2 ml of naoh 1 m were added, and the mixture's volume was subsequently raised to 10 ml using distilled water. the absorbance was measured at 510 nm. total flavonoids were calculated from a standard curve made with quercetin. results are expressed as milligrams of quercetin equivalents per gram of dry matter (mg qe g-1 dm) evaluation of the antioxidant activity dpph assay the purpose of this method was to determine which of the prepared extracts had the greatest antioxidant activity against dpph (2,2'-diphenyl-1-picrylhydrazyl). it is important to note that dpph is a stable free radical, and in the presence of antioxidants, the characteristic purple colour of dpph changes to yellow, and the absorbance is measured at 517 nm.... a) b) figure 1. figure 1. figure 1. figure 1. reaction of an antioxidant with dpph (molyneux, 2004); (a) diphenylpicrylhydrazyl + antioxidant-oh (purple color); (b) diphenylpicrylhydrazyl + antioxidant-o (yellow color) the antioxidant activity was tested according to the method described by brand-williams et al. (1995). this procedure consists in preparing dilutions of the order of 1/2, 1/4, 1/8 and 1/16 from a stock solution of 4 mg ml-1 of the studied extract. then, 1 ml of each dilution was added to 1 ml of dpph (0.004%). ascorbic acid was used as a reference molecule. the absorbance was measured at 517 nm by spectrophotometer, and the antioxidant activity was calculated according to the following formula: antioxidant activity %� � abs dpph � ��� �� �ℎ� ��� !"� abs dpph # 100 • abs dpph: absorbance of the solution of dpph • abs of the extract: value of the absorbance after the addition of the extract the regression curve of this activity allowed to determine the concentration that corresponds to 50% inhibition (the half maximal inhibitory concentration ic50505050: concentration of the tested sample or ascorbic acid necessary to reduce 50% of the dpph radical). a low ic50505050 value indicates a high capacity of the extract to act as a dpph scavenger (cheng and li, 2004). el mekkaoui a et al. (2023). not sci biol 15(1):11423 4 total antioxidant capacity (tac) the total antioxidant capacity was measured using the protocol described by prieto (prieto et al., 1999). in practice, 200 μl of extract were mixed with 3 ml of reagent solution (6m sulfuric acid, 280 mm sodium phosphate and 40 mm aluminum molybdate). the incubation was done at 95 °c for 90 min. after cooling, the absorbance was measured at 695 nm. the total antioxidant capacity was expressed as milligram ascorbic acid equivalent / gram dry matter (mg aae g-1 dm). statistical analysis the data obtained were subjected to analysis of variance (anova). statistical analysis was based on two-way anova followed by tukey's honestly significant difference test. the data were processed with the software "sys-tat 12". a test of comparison of the means was done each time there was a significant effect of factor studied by the anova results results results results total phenolic content figure 2 displays the results of the total phenolic contents. they ranged from 7.90 to 160.76 mg gae g1 dm. the highest concentration of phenols was observed in the hydroethanol extract for a. unedo. on the other hand, the aqueous extract of m. vulgare had the lowest content. the analysis of variance for total phenol content showed a highly significant difference between both the species and the solvents used in the present experimentation (df = 12, f-ratio = 10.843, p <0.001). figure 2figure 2figure 2figure 2. total phenolic content of the extracts 0 20 40 60 80 100 120 140 160 180 a. unedo i. viscoa r. monosperma a. spinosa c. sativa m. communis m. vulgare methanol extract hydroethanol extract aqueous extract mg gae g-1 dm el mekkaoui a et al. (2023). not sci biol 15(1):11423 5 total flavonoids contents the results of flavonoid contents were showed in figure 3. therefore, the concentration of flavonoids in the extract ranged from 36.01 to 489.77 mg qe g-1 dm. analysis of the results allowed us to highlight that the highest content was the ethanol extract of i. viscosa. however, the aqueous extract of r. monosperma, had the lowest concentration. statistically a highly significant difference was observed between the different species studied and the three extraction solvents used (dl = 12, f-ratio = 23.251, p <0.001). figure 3.figure 3.figure 3.figure 3. total flavonoid content of the extracts evaluation of antioxidant activity dpph method from the results, we found that all the extracts were able to reduce dpph free radicals. table 2 summarizes the ic50505050 values of the examined extracts and ascorbic acid. a. unedo recorded the lowest ic50505050 values for the three extraction solvents (methanol, hydroethanol, and aqueous extracts) with respectively 0.033; 0.026; and 0.034 mg ml-1, reflecting its potent antioxidant activity approaching that of ascorbic acid (ic50505050 = 0.01 mg ml-1). on the other hand, r. monosperma had the lowest anti-radical activity (ic50505050 = 0.957; 0.873 and 0.860 mg ml-1). the analysis of variance for ic50505050 showed a highly significant difference between the extracts and solvents used (dl = 12, f-ration = 101.246, p <0.001). 0 100 200 300 400 500 600 a. unedo i. viscosa r. monosperma a. spinosa c. sativa m. communis m. vulgare methanol extract hydroethanol extract aqueous extract el mekkaoui a et al. (2023). not sci biol 15(1):11423 6 table 2.table 2.table 2.table 2. ic50505050 values (mg ml-1) of tested extracts and ascorbic acid maps extract ic50 (mg ml-1) methanol hydroethanol aqueous a. unedo 0.033 ± 0.0004 0.026 ± 0.0006 0.034 ± 0.0007 i. viscosa 0.065 ± 0.0001 0.057 ± 0.0004 0.094 ± 0.0007 r. monosperma 0.957 ± 0.0101 0.873 ± 0.0045 0.86 ± 0.0495 a. spinosa 0.048 ± 0.0001 0.043 ± 0.0012 0.046 ± 0.0011 c. sativa 0.666 ± 0.0175 0.467 ± 0.0040 1.032 ± 0.0175 m. communis 0.037 ± 0.0002 0.036 ± 0.0002 0.038 ± 0.0001 m. vulgare 0.346 ± 0.0017 0.329 ± 0.0009 0.635 ± 0.0081 ascorbic acid 0.01 ± 0.0001 total antioxidant capacity the results of the total antioxidant capacity are showed in figure 4. the values obtained are expressed in terms of mg equivalent of ascorbic acid per gram of dry matter. according to these results, the evaluation of the total antioxidant capacity of the extracts showed a variability relative to the solvents. we found that the hydroethanol extracts present the highest antioxidant capacity, with 267.37 and 269.32 mg aae g-1 dm respectively for a. unedo and a. spinosa. in contrast, the lowest concentration belongs to r. monosperma for the aqueous extract, with a value of 29.37 mg aae g-1 dm. the statistical analysis of the total antioxidant capacity shows a highly significant difference between the studied plants and the different solvents used (dl = 12, f-ratio = 15.293, p <0.001). figure 4figure 4figure 4figure 4. total antioxidant capacity of the extracts 0 50 100 150 200 250 300 a. unedo i. viscosa r. monosperma a. spinosa c. sativa m. communis m. vulgare methanol extract hydroethanol extract aqueous extract mg aae g-1 dm el mekkaoui a et al. (2023). not sci biol 15(1):11423 7 discussiondiscussiondiscussiondiscussion organic solvent extraction is the most commonly used method for the preparation of plant extracts (zhang et al., 2018), due to the modification of the solvent influencing the yield/composition of the isolated molecules, it also offers a selective method for the extraction of bioactive compounds (kapadia et al., 2022). in terms of antioxidant activity, our results indicated that hydroethanol extracts have the strongest antioxidant power, a finding consistent with the study conducted by özbek et al. (2020). for the phenolic contents of arbutus unedo, our results ranged from 116.67 to 160.76 mg gae g-1 dm, which are higher compared to those reported by ait lhaj et al. (2022) with a highest result of 107.67 mg gae g-1 dm and habachi et al. (2022) with a highest result of 86 mg gae g-1 dm, who used different extraction methods and solvents for the leaves. the best ic50505050 value in our study for the antioxidant activity was 0.026 mg ml-1 obtained in the hydroethanol extract, which is higher than the value reported by bebek markovinović et al. (2022) of 0.076 mg ml-1, who used the same solvent. in the case of i. viscosa, the phenolic contents were found to range from 36.82 to 78.41 mg gae g-1 dm, consistent with the results of aydar et al. (2022) who obtained a value of 54.39 mg gae g-1 dm using a combination of ultrasonic and microwave extraction. our best ic50505050 value was 0.057 mg ml-1 obtained in the hydroethanol extract, which is lower than the result of yıldırım et al. (2022) who obtained 0.014 mg ml-1, while using chloroform as solvent. for r. monosperma, the phenolic contents were found to range from 24.7 to 32.44 mg gae g-1 dm, with an ic50505050 value of 0.86 mg ml-1. these results are lower compared to those reported by selaimia et al. (2020) of 155.61 mg gae g-1 dm and 0.118 mg ml-1, they used a diethyl ether solvent and a maceration method. for a. spinosa, we obtained for phenolic contents values between 99.02-106.21 mg gae g-1 dm, which is higher than what was reported by afrokh et al. (2023) with 47.75 mg gae g-1 dm. on the other side, our ic50505050 value of 0.043 mg ml-1 obtained in the hydroethanol extract is consistent with the ic50505050 obtained in the same study of 0.048 mg ml-1. m. vulgare, resulted in values between 7.9-13.43 mg gae g-1 dm for the phenolic contents, which is lower than what reported afrokh et al. (2023) with a value of 29.25 mg gae g-1 dm, whereas our ic50505050 of 0.329 mg ml-1 is higher than their result of 2.42 mg ml-1. for c. sativa, the phenolic contents were found to range from 14.07 to 27.88 mg gae g-1 dm, which is in agreement with the findings of aazza (2021) who reported a value of 19.07 mg gae g-1 dm. the best ic50505050 value obtained was 0.467 mg ml-1, which is higher compared to the results reported by benkirane et al. (2023) at 1.83 mg ml-1. finally, for the phenolic contents of m. communis the results were between 89.47-100.3 mg gae g-1 dm, which is higher compared to the results reported by hazrati et al. (2022) at 66.52 mg gae g-1 dm using a 24-hour hydro-ethanol maceration extraction method. the best ic50505050 value in our study was 0.036 mg ml-1 (hydroethanol extract), which is consistent with the findings of yangui et al. (2021) of 0.038 mg ml-1 where they used a hydromethanol solvent. the difference in phenol contents and antioxidant activity can be explained by the polarity of the solvent used in the extraction, knowing that the high solubility of phenols in polar solvents facilitates a better yield in extracts obtained from them (alara et al., 2021). this difference may also be due to several factors such as climate, soil, harvest period, storage condition, environmental factors (temperature, ph), or the extraction method (ben ahmed et al., 2017; sun et al., 2019; ngoune liliane and shelton charles, 2020; zeroual et al., 2021; zhang et al., 2022). furthermore, it has been established that antioxidant activity is positively correlated with the structure of phenols (osman et al., 2020). generally, phenols with a high number of hydroxyl groups present the highest antioxidant activity (heim et al., 2002) and that is due to their power to give more atoms to stabilize the free el mekkaoui a et al. (2023). not sci biol 15(1):11423 8 radicals (torres de pinedo et al., 2007). thus, the antioxidant effect is not only dose-dependent but also structure-dependent (rodríguez-bernaldo de quirós et al., 2010). therefore, a mixture of these plants could be considered and could accentuate even more their antioxidant effects, allowing them to be considered as a possible alternative to remedy oxidative diseases. conclusionsconclusionsconclusionsconclusions during this work, we were able to highlight the medical potential of these plants. the quantitative analysis of the methanol, ethanol and aqueous extracts showed the presence of flavonoid and polyphenols. furthermore, we were able to show that these plants have a good antioxidant activity especially a. unedo, a. spinosa and m. communis. additionally, these plants compounds have significant therapeutic effects with few side effects, their natural antioxidants may offer an alternative to conventional treatments for oxidative stress and could be considerate strong candidates for the prevention of free radical diseases like cancer, aging process, cardiovascular disease, and diabetes. it would be important to extend the range of this study parameters as well as the isolation, characterization, and identification of active compounds to valorise more these plants. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions data curation: aem, mk; methodology: aem, cs, cr; supervision: mk, cr, an, ec, fb; writing original draft: aem; writing review and editing: aem, cs. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences aazza s (2021). application of multivariate optimization for phenolic compounds and antioxidants extraction from moroccan cannabis sativa waste. journal of chemistry 2021:1-11. https://doi.org/10.1155/2021/9738656 afrokh m, tahrouch s, el mehrach k, fahmi f, ait bihi m, weber-ravn h, … tabyaoui m (2023). ethnobotanical, phytochemical and antioxidant study of fifty aromatic and medicinal plants. chemical data collections 43:100984. https://doi.org/10.1016/j.cdc.2022.100984 el mekkaoui a et al. 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(2023). not sci biol 15(1):11423 11 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11411 nsb saadaoui 2023.06.07.docx received: 16 dec 2022. received in revised form: 01 may 2023. accepted: 07 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania saadaoui e et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11411 doi:10.15835/nsb15211411 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae seed diversity and germination behavior in seed diversity and germination behavior in seed diversity and germination behavior in seed diversity and germination behavior in nerium nerium nerium nerium oleander oleander oleander oleander l.l.l.l. ezzeddine saadaoui1*, josé j.m. gómez2, kaouther ben yahia3, emilio cervantes2 1university of carthage, inrgref, lgvrf, regional station of gabès, tunisia; saad_ezz@yahoo.fr (*corresponding author) 2irnasa-csic. cordel de merinas 40, 37002, salamanca, spain; jjaviermg@gmail.com; ecervant@usal.es 3university of carthage, inrgref, lef, tunisia; adam.kaouther@yahoo.fr abstractabstractabstractabstract nerium oleander var. ‘villa romaine’, commonly called oleander, is an ornamental shrub that exists in tunisia in the wild and cultivated form. this plant is frequently propagated and produced in nurseries and used in urban plantings and on roadsides. the effects of nature of the plants (spontaneous or cultivated), climatic conditions (localities), and altitude (from 6 to 447 m) on seed shape, size, and germination of tunisian populations, were investigated. seeds were harvested from 23 locations belonging to six bioclimatic stages and distributed from north to south of tunisia. the seed parameters studied were area, roundness, j index, and germination rate. a significant variation was obtained between populations, categories (cultivated and spontaneous), bioclimatic zones and altitudes. seeds of cultivated plants were the smaller and showed the higher germination rates, compared with spontaneous populations, which had large seeds and a low germination rate. climatic conditions and geographical location had an effect on seed morphology; seeds in lower humid were small for both cultivated and spontaneous types, they also showed a low j index value and a high germination rate. on the other hand, in upper saharan, seeds were large and roundness and germination rate were the lowest. also, seeds from plants in higher altitudes were the smaller and presented the lower values of the j index compared with those collected from low-altitude sites. keywords:keywords:keywords:keywords: diversity; germination; nerium oleander l.; seed; shape introductionintroductionintroductionintroduction nerium oleander l., an evergreen shrub or small tree, is a unique species of the genus nerium from the apocynaceae family. even though being toxic in all its parts, this species is widely cultivated, resulting in one of the most poisonous among cultivated plants. it belongs to the tropical element of the mediterranean scrub flora, that is, a set of taxa of tropical origin that probably evolved before the onset of the mediterranean climate but now coexist with more recent, purely mediterranean elements (quezel, 1985). in tunisia, n. oleander grows both spontaneously and cultivated. its fruit (5-15 cm in length and 6-10 mm in diameter) consists of two follicles. the young unripe small fruit is green but it turns red when fully grown but still unripe. upon maturation, the fruit dries, turns brown, and its follicles twist to liberate the many small, plumed, winddispersed seeds (lev-yadunet al., 2009). the seed of n. oleander has an oblong shape and excluding https://www.notulaebiologicae.ro/index.php/nsb/index saadaoui e et al. (2023). not sci biol 15(2):11411 2 ornamentation, the 100 seed weight is 0.3 g. the length, width and thickness of the seed are 4.7, 1.5 and 0.7 mm respectively (demonty et al., 2014; figure 1). molecular analyses of this species by rapd and aflp show its high genetic variability (zibbu and batra, 2015). nerium oleander var. ‘villa romaine’ investigated in this work is characterized by its simple flowers, light pink and slightly fragrant. its presence in the arid regions of tunisia reflects its drought tolerance. the plant is propagated by seeds or cuttings. a germination rate above 82% and a rooting percentage of 100% were obtained for sowing and cuttings respectively (simion and anton, 2009). figure 1.figure 1.figure 1.figure 1. nerium oleander. plants growing in bou hedma (site number 19). flower, leaves, fruit, seeds. the hairs were removed from the seed for the morphological study seed morphology may give information useful in the phenotypic characterization and phylogenetic relationships between varieties and cultivars. an accurate description of seed shape may be done by comparison with geometric figures that resemble seed images. this method has been developed in the model plant (arabidopsis thaliana l., cervantes et al. (2010) as well as in the model legumes, lotus japonicus l. and medicago truncatula gaertn (cervantes et al., 2012). also, several studies have analyzed the diversity of silene species and populations with seed shape based on the comparison with geometric models, by comparing the seeds to the ellipse and cardioids shapes (martín-gómezet al., 2020, 2022a, 2022b; juan et al., 2022; rodríguezlorenzo et al., 2022) studied the diversity. in capparis spinosa l., the comparison of seed images with the cardioid curve was helpful to describe differences between two subspecies (saadaoui et al., 2013). in the euphorbiaceae jatropha curcas l. and ricinus communis l., seed shape was quantified by comparison with an ellipse; the form/production relationship has been verified (saadaoui et al., 2015, 2017; martín-gómez et al., 2016). we have established a protocol to analyze seed shape in n. oleander. the model applied for the quantitative geometric analysis has been an ellipse. quantification with a model allows the comparison of seed shape in populations grown in different climatic regions as well as between seeds proceeding from plants grown spontaneously or cultivated in 23 locations in tunisia. the main objectives of this work are to analyze the seed variability between populations, sites, and states (spontaneously or cultivated) and the effect of these characteristics on seed germination. we present in the study the variations in size, shape, and germination rate of seeds in plants of nerium oleander. saadaoui e et al. (2023). not sci biol 15(2):11411 3 materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant material mature seeds were obtained in the spring of 2019 from developed plants growing in 23 locations in tunisia (table 1). seeds were collected from five plants per location. ten fruits were harvested from each individual, the seeds of each population were pooled and 30 seeds per location were used in the analysis. a total of 708 seeds from 23 populations of nerium oleander var. ‘villa romaine’ grown in six different climatic regions throughout tunisia was analyzed (table 1). from the total, 570 seeds proceed from cultivated plants, growing on roadsides and 138 belong to spontaneous populations. cultivated plants were obtained by cuttings, from spontaneous plants, and after several generations. in nature, n. oleander is mainly propagated by seeds. table 1. table 1. table 1. table 1. geographic localization of studied populations population code population latitude (n) longitude (e) altitude (m) type bioclimate* 1 rimel 37°03’ 41.31’’ 9° 30’ 34.51’’ 149 cultivated lower humid 2 teskraya 37° 15’ 14.55’’ 9° 54’ 36.17’’ 6 spontaneous 3 bellif 37° 04’ 05.53’’ 9° 01’ 33.80’’ 77 4 ain snoussi 1 36° 49’ 45.95’’ 8° 55’ 50.29’’ 386 4’ ain snoussi 2 36° 49’ 12.72’’ 8° 55’ 27.49’’ 447 cultivated 5 sidi bou said 36° 51’ 10.47’’ 10° 19’ 08.57’’ 10 upper semiarid 6 grombalia 36° 35’ 26.66’’ 10° 28’ 24.49’’ 70 7 sidi khelifa 36° 14’ 49.02’’ 10° 25’ 29.13’’ 30 8 sousse 35° 46’ 52.35’’ 10° 36’ 52.35’’ 41 lower semiarid 9 msaken 35° 41’ 49.74’’ 10° 33’ 54.10’’ 46 10 monastir 35° 45’ 49.16’’ 10° 48’ 22.80’’ 33 11 kairouan 35° 40’ 30.0246’’ 10° 06’ 44.48’’ 54 upper arid 12 hbira 35° 10’ 55.48’’ 10° 17’ 05.80’’ 140 13 sfax 34° 49’ 07.24’’ 10° 42’ 17.21’’ 54 14 bou hedma 34° 29’ 20.41’’ 9° 26’ 41.90’’ 346 spontaneous 15 bir ali ben khelifa 34° 44’ 17.55’’ 10° 05’ 23.76’’ 171 cultivated lower arid 16 gafsa 34°26’ 06.27’’ 8° 46’ 50.95’’ 317 17 skhira 34°17'36.57" 10°03'56.27" 32 18 nahel 33° 52’ 28.07’’ 10° 03’ 51.07’’ 27 19 hamma 33° 52’ 50.56’’ 9° 46’ 24.98’’ 60 20 ghandri 33° 43’ 36.18’’ 10° 09’ 07.88’’ 43 spontaneous 21 zerkine 33° 41’ 00.70’’ 10° 15’ 29.94’’ 25 cultivated 22 medenine 33° 21’ 40.85’’ 10° 28’ 14.03’’ 112 23 kebili 33° 41’ 53.23’’ 8° 57’ 56.06’’ 33 upper saharan * bioclimates according to the classification of emberger (1955) image capture photographs of longitudinal views of seeds were taken with a digital camera nikon ds-fi1 adapted to a nikon ‘smz-1500’ stereo-microscope. composed images containing the model (ellipse; see later) and each seed were elaborated with the software image corel photo-paint x7. quantification of areas was done with image j (java image processing program). in this process, a microscopy photograph of graph paper was used to convert pixels into mm. saadaoui e et al. (2023). not sci biol 15(2):11411 4 quantitative morphology two magnitudes were used for the quantitative morphological analysis: roundness and j index. roundness, obtained with image j, is defined by ferreira and wayne (2010) as: � � 4 x area π major axis�� j index reflects the percent similarity of the seed image with a geometric figure used as a model. based on seed measurements and image comparisons, the model used for shape quantification is an ellipse with a relation between the major and minor axis of 3.7. the model was obtained by superimposing the profiles of all the seeds and selecting color mask (black) of higher intensity with corel photo-paint x7 (figure 2). the ratio between major and minor axis of 3.7 is also obtained as the mean value in all the seeds. quantification of the adjustment as done in each seed by superimposing the seed image with the model (ellipse), and the ratio of the areas in two regions: the common region in the ellipse and the seed image (c), and the total area occupied by the ellipse and the seed image (figure 2). the index of adjustment (j) is defined by: 100 (d) area (c) area (c) area j × + = where c represents the common region and d the regions not shared (total area is the sum of shared and non-shared). note that j ranges between 0 and 100, and decreases when the size of the non-shared region grows. it equals 100 when cardioid and seed image areas coincide, i.e., area (d) is zero. j index was calculated for a total of 708 seeds (30 seeds per population; except in the bellif population 3 of only 18 seeds). figure 2.figure 2.figure 2.figure 2. left: image of a seed (top) and model ellipse (bottom). right: the common region in the ellipse and the seed image (top), and the total area occupied by the ellipse and the seed image (bottom). j index is the ratio between both magnitudes germination tests thirty seeds from each location were deposited in water agar 1% in petri dishes. a total of 708 seeds were analyzed after 7 days of germination at 18 °c. twenty-five seeds per petri dish were tested, with four repetitions saadaoui e et al. (2023). not sci biol 15(2):11411 5 statistical analysis analysis of variance (anova) was applied to the comparison of magnitudes between populations and groups of populations (climates) as well as seed types (collected from spontaneous and cultivated plants) between the diverse populations. the magnitudes analyzed were seed area, roundness, j index, and germination rate. post-hoc analysis was carried out using the tukey test for samples of similar sizes such as, for example, comparisons between seed types in a population, the scheffé test for groups of populations (altitude, climatic regions, or comparison between spontaneous and cultivated populations; in general, samples are of different sizes). seed germination is a dynamic process. the analysis of seed germination in time was done with a glm (general linear model, christensen, 6; mccullagh and nelder, 1989). this method allows to work with qualitative variables (plant types or varieties; here the comparison between spontaneous and cultivated populations) and combines regression analysis throughout successive moments with anova involving values in all time points. glm was done with two fixed factors: plant source, with two plant types (spontaneous and cultivated), with 138 levels for spontaneous plant type, 570 levels for cultivated, and one random factor, seed. statistical treatment and graphics were done with spss® v. 23. for the analysis, the significance level p=0.05 was established; in addition, six digits of precision were used throughout the calculations. results results results results effect of population for all the morphological parameters analyzed (area, roundness and j index), the variation is significant between populations (p <0.05). ain snoussi 2, cultivated plants, showed the lowest area (4.44 mm2) and teskraya, spontaneous site, had the highest (7.73 mm2). for roundness, the highest value was at ain snoussi 2 (0.358) and the lowest was at kebili (0.257), belonging to lower humid and upper saharan climates, respectively. at the end, the highest j index (87.4) is at bou hedma and gafsa (two nearby sites) and the lowest is at 76 at ain snoussi 2 (table 2). table 2. table 2. table 2. table 2. comparison between populations for morphological parameters population n area (mm2) roundness j index rimel 30 7.22g.h 0.273a.b.c 86.1b.c teskraya 30 7.73h 0.297a.b.c 85.2b.c bellif 18 4.74a.b 0.294a.b.c 85.4b.c ain snoussi 1 30 6.15c.d.e.f 0.267a.b 84.1b.c ain snoussi 2 30 4.44a 0.358d 76.0a sidi bou said 30 7.00f.g.h 0.280a.b.c 84.3b.c grombalia 30 6.35c.d.e.f.g 0.293a.b.c 85.8b.c sidi khelifa 30 5.69b.c.d.e 0.293a.b.c 84.7b.c sousse 30 6.48d.e.f.g 0.283a.b.c 86.0b.c msaken 30 6.95f.g.h 0.267a.b 86.3b.c monastir 30 5.59b.c.d 0.283a.b.c 85.6b.c kairouan 30 5.37a.b.c 0.300a.b.c 83.1b.c hbira 30 5.35a.b.c 0.293a.b.c 85.1b.c sfax 30 6.31c.d.e.f.g 0.290a.b.c 83.4b.c bou hedma 30 6.55d.e.f.g 0.283a.b.c 87.4c bir ali ben khelifa 30 6.75f.g.h 0.312c 83.5b.c gafsa 30 4.76a.b 0.273a.b.c 87.4c skhira 30 6.03c.d.e.f 0.307b.c 82.6b nahel 30 6.40d.e.f.g 0.280a.b.c 87.3c hamma 30 6.63e.f.g 0.287a.b.c 84.6b.c ghandri 30 6.52d.e.f.g 0.293a.b.c 85.8b.c saadaoui e et al. (2023). not sci biol 15(2):11411 6 zerkine 30 6.34c.d.e.f.g 0.283a.b.c 84.3b.c medenine 30 6.29c.d.e.f.g 0.263a.b 83.9b.c kebili 30 6.66e.f.g 0.257a 85.3b.c n: number of seeds significant differences (at 0.05 significant level) are indicated by different letters (a,b,c,d,e,f,g and h) effect of the category (spontaneous /cultivated) a summary of results concerning the comparison between spontaneous and cultivated plants is presented in table 3; a significant difference (p <0.05) was observed between these two categories regarding for the area, j index, and germination rate. seeds of the cultivated plants are smaller, and have a lower j index and higher germination rate. on the other hand, the roundness and germination rate are without significant differences between spontaneous and cultivated sites. only location number 4 (ain snoussi, in the lower humid climate) contains spontaneous and cultivated plants, and also in this population seeds from cultivated plants are smaller, with higher roundness and lower j index values than the seeds obtained from spontaneous plants (figure 3). tabtabtabtable 3. le 3. le 3. le 3. comparison between spontaneous (s) and cultivated (c) plants for area, roundness, j index and germination % type n mean standard deviation standard error significance area (mm2) s 38 6.48 1.44428 0.12295 0.005 c 70 6.14 1.23909 0.05190 roundness s 38 0.286 0.04233 0.00360 0.638 c 70 0.288 0.05182 0.00217 j index s 38 85.6 4.4436 0.3783 0.012 c 70 84.5 5.3372 0.2236 germination (%) s 38 49 50 4.3 0.001 c 70 65 48 2.0 n: number of seeds saadaoui e et al. (2023). not sci biol 15(2):11411 7 figure 3.figure 3.figure 3.figure 3. representative seeds of the two populations grown in ain snoussi (cultivated (a) and spontaneous (b)) and kebili’s population(c) (scale is 5 mm). effect of bioclimates bioclimates have a significant effect (p <0.05) on the parameters of seed diversity. for the cultivated populations, a low area was recorded in lower humid and upper arid. however, the highest value was observed in the upper saharan (table 4 and figure 3). for roundness, the highest value was recorded in lower humid, and the lower value was in upper saharan. j index also varied. in fact, the lowest averages were in the lower humid, and high averages were observed in lower semi-arid, lower arid and upper saharan. for germination, the lowest germination rate is in the upper saharan (table 4). for the spontaneous populations, area, roundness, and germination rate were not significantly different. only the j index was significantly different. indeed, it was low in the lower humid and high in the upper and lower arid (table 5). table 4. table 4. table 4. table 4. comparison between climatic regions for cultivated plants bioclimate n area (mm2) roundness j index germination (%) lower humid 60 5.83a 0.316c 81.0a 75b upper semi-arid 90 6.35a. b 0.289b. c 84.9b 72b lower semi-arid 90 6.34a. b 0.278a. b 85.9b 67b upper arid 90 5.68a 0.294b. c 83.9a. b 72b lower arid 210 6.17a. b 0.286b 84.8b 59a. b upper saharan 30 6.66b 0.257a 85.3b 33a saadaoui e et al. (2023). not sci biol 15(2):11411 8 table 5. table 5. table 5. table 5. comparison between climatic regions for spontaneous plants bioclimate n area roundness j index germination (%) lower humid 88 6.43a 0.285a 84.8a 58 a upper arid 30 6.54a 0.283a 87.4b 43 a lower arid 30 6.52a 0.293a 85.8a. b 33 a effect of altitude table 6 shows a significant difference for the values of the area, as well as those of the j index according to the altitude; indeed, the area is 6.87 mm2 at low altitude and 5.47 mm2 at high altitude, in the same way the average values of j index are high (85.29) at altitude below 30 m and low (83.71) for altitudes above 300 m. on the other hand, these differences according to the altitudes are not significant for the roundness and germination rate. table 6. table 6. table 6. table 6. comparison between seeds grown in different altitudes parameters altitude n mean standard deviation standard error significance area (mm2) below 30 m 120 6.87 1.12 0.102 0.000 above 300 m 120 5.47 1.33 0.122 roundness below 30 m 120 0.28 0.050 0.004 0.147 above 300 m 120 0.29 0.059 0.005 j index below 30 m 120 85.29 4.49 0.410 0.037 above 300 m 120 83.71 6.94 0.634 germination (%) below 30 m 120 58 49.5 4.5 0.197 above 300 m 120 50 50.2 4.6 discussiondiscussiondiscussiondiscussion among the two magnitudes used to quantify seed shape in nerium oleander, roundness and j index, taking as the model an ellipse of a relationship between the major and minor axis of 3.7. the values of the j index based on this model give an accurate approach to seed shape useful and complementary to roundness values. nevertheless, the values obtained for j index are still lower than those obtained with other geometric models applied to a range of seed species. cardioid or cardioid-derived figures were applied to the model plant arabidopsis thaliana (l.) heynh (cervantes et al., 2010), as well as to the model legumes lotus japonicus and medicago truncatula gaertn (cervantes et al., 2012) and capparis spinosa l. (saadaoui et al., 2013). ellipsebased models were applied to the euphorbiaceae ricinus communis l. (martín gómez et al.2016; saadaoui et al., 2017) and jatropha curcas l. (saadaoui et al., 2015). in the majority of these cases j index values were around 90 reaching values over 95. to make shape comparisons between genotypes or treatments, we find that values for j index of 90 or upper provide better results. in general, those high values are obtained for seeds that present simple shapes, similar to geometric figures, and without appendixes or specialized structures for dissemination. seeds of n. oleander, in addition to the hairs that cover their surface, don’t adjust completely to the ellipse because they tend to have elongated, sharpened and curled shapes, resulting in complex structures, diverging from simple geometric models. these parameters may be related to the complexity of seed dispersion (herrera, 1991) for cultivated plants, extreme climates (lower humid and upper saharan) are characterized by extreme values for all three morphological parameters analyzed (j index, roundness, and area; table 4); this result points to the effect of climate on the development and morphology of the seed. there is no difference in seed saadaoui e et al. (2023). not sci biol 15(2):11411 9 area between the spontaneous populations, although aridity favors small seeds (peco et al., 2003; saadaoui et al., 2013). but seeds of the saharan site have an elongated shape which may assist them in hiding in small holes or apertures in the soil to adapt to the arid desert conditions (figure 3). j index showed a considerable difference between studied bioclimates. for cultivated plants, the lowest is in the upper humid and the highest is in the lower semi-arid, followed by upper saharan. also, for the spontaneous plants, the lowest j index is in upper humid and the highest values are in upper arid, followed by lower arid. a correlation was observed between the spontaneous and the cultivated for the bioclimate effect on the j. index. domestication reduces seed shape and j index, but the characters are variable with climatic regions. germination rate differs between cultivated sites; the highest values were recorded for upper humid, upper semi-arid, lower semi-arid, and upper arid and lower for lower arid and upper saharan. the latter showed the lowest germination rate (33%). this shows the direct effect of climatic conditions on cultivated plants and the quality of their seeds. indeed, abiotic stress, particularly elevated temperature and water stress will reduce seed yield and quality (hampton et al., 2016). seed size and shape is influenced by altitude. plants grown at higher altitudes produce seeds smaller and with lower values of the j index. bonnier (1890) in his classical experiments cultivating diverse plant species at varying altitudes showed that higher altitudes result in reduced plant size, which may also result in smaller seed size. similarly, in cactus (gymnocalycium monvillei (lem.) britton and rose), bauk et al. (2015) studied the effect of altitude on the seed parameters and concluded the presence of a relationship between altitude and seed height and shape. also, pluess et al. (2005) confirmed the effect of altitude on the seed size for several species studied in the swiss alps. rawat and bakshi (2011) studied pinus wallichiana and recorded a negative correlation between altitude, 1000 seed weight, and germination percentage. this correlation is positive with the germination period. but, for abies pindrow (silver fir), bhat et al. (2018) obtained a negative correlation between altitude and percentage germination. our results showed the effect of population, climate, and altitude on seed diversity, also, the plant culture acts on these variables. other factors may be influencing the seed size and shape of nerium oleander seeds. conclusionsconclusionsconclusionsconclusions this study was carried out at 23 nerium oleander var. 'villa romaine' sites in different bioclimates of tunisia. the analyses of variations within these localities by using morphological (area, roundness and j index) and physiological (germination) parameters showed high variability. these variables reflected differences in size, shape and physiological behavior. the observed variations seemed to be directly related to populations, their nature (spontaneous or cultivated), and/or bioclimatic conditions and altitudes. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization of research (ec, es, jjmg); designing of the experiments (ec, jjmg); contribution of experimental materials (es, ky, jjmg); execution of field/lab experiments and data collection (es, ky, jjmg); analysis of data and interpretation (ec, es, jjmg, ky); preparation of the manuscript (ec, es, jjmg, ky). all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. saadaoui e et al. 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(2023). not sci biol 15(2):11411 12 microsoft word 11338 nsb bolaji 2023.06.19.docx received: 01 sep 2022. received in revised form: 12 may 2023. accepted: 14 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania bolaji ao et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11338 doi:10.15835/nsb15211338 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae morphological characterization and cytological studies of the greenmorphological characterization and cytological studies of the greenmorphological characterization and cytological studies of the greenmorphological characterization and cytological studies of the green---stemmed and the redstemmed and the redstemmed and the redstemmed and the red----stemmed stemmed stemmed stemmed basella albabasella albabasella albabasella alba abolade o. bolaji1*, atanda s. oladejo2, oluwatobi i. adeniran1 1obafemi awolowo university, faculty of science, department of botany, ile-ife, nigeria; abolaji@oauife.edu.ng (*corresponding author); oluwatobiadeniran484@gmail.com 2obafemi awolowo university, faculty of agriculture, department of crop production and protection, ile-ife, nigeria; soladejo@oauife.edu.ng abstractabstractabstractabstract basella alba is an underutilized vegetable with ethnobotanical importance used for culinary as well as medicinal purposes in many parts of the world. morphological characterization and chromosome studies of the mitotic and meiotic cells of the green stemmed and the red-stemmed b. alba was carried out with a view to filling the knowledge gaps that exist in their morphological characterization and also to provide insightful information on their chromosome numbers and meiotic behaviour. the b. alba accessions studied were characterized with respect to their habit, leaves, inflorescence, fruits and seeds. mitotic and meiotic studies were carried out on the basella accessions using standard techniques. the morphological studies revealed significant differences between the green-stemmed and red-stemmed basella alba with respect to the green/red colour of their stems, colour of the flower bud apex, mean plant height at flower bud initiation, mean leaf length, mean leaf width, mean petiole length, mean flower per spike, mean fruit length and mean fruit diameter. the cytological study revealed a chromosome number of 2n=4x=44 for both the green-stemmed and the redstemmed b. alba studied. it also revealed the occurrence of chromosomal aberrations such as stickiness and precocious migration of chromosomes to the poles during meiosis, which could lead to irregular chromosome segregation that could result in chromosome instability and aberrant meiotic products within the species. keywords:keywords:keywords:keywords: bivalents chromosomes; descending dysploidy; malabar spinach; meiotic segregation; mitotic studies; underutilized vegetables introductionintroductionintroductionintroduction basella alba l. is the most common species in the family basellaceae (ozela et al., 2007) and it comprises the green-stemmed (basella alba var. alba) and purple-stemmed (basella alba var. rubra) basella alba forms. it is native to tropical southern asia and probably originated from india or indonesia (saroj et al., 2012). b. alba is particularly abundant in malaysia, philippines, thailand, nepal, india, tropical africa, the caribbean and tropical south america (palada and crossman, 1999). https://www.notulaebiologicae.ro/index.php/nsb/index bolaji ao et al. (2023). not sci biol 15(2):11338 2 basella alba is an underutilized vegetable often cultivated in home gardens (bolaji et al., 2022a) and used for culinary and medicinal purposes. it is a very popular vegetable in many communities of south-western nigeria and is also one of the chief sources of the major ingredients in the northern nigeria and north-eastern nigerian foods (izonfuo et al., 2006). it is useful as medicinal plant in the treatment of various ailments including anaemia (rahmatullah et al., 2010), pelvic inflammation disease, threatened abortion (focho et al., 2009), hypertension (olowokudejo et al., 2008), earache and sore throat (chatchawal et al., 2010; paul et al., 2011), gonorrhoea (singh et al., 2010), diabetes (shantha et al., 2016) and ulcer (dixit and goyal, 2011). chromosomes are key building blocks of eukaryotic genomes (tiang et al., 2012) and the cytogenetic characterisation of species require in-depth studies of the chromosome numbers and chromosomal behaviours of the species and their morphogenic varieties (silva et al., 2017). meiosis, a process by which sexually reproducing organisms reduce their genome from diploid to haploid (john, 1990) is a highly coherent and genetically controlled process (pessim et al., 2015) which involves the process of commitment and initiation, homologous chromosome pairing, synapsis, inter-homologous reciprocal recombination, disjunctive segregation and haploid gamete formation (murphy and bass, 2012). there were knowledge gaps concerning the morphological characteristics of the green-stemmed and the red-stemmed basella alba. previous reports had focused on their stem colour, leaf shapes, floral arrangement and fruit shape (palada and chang, 2003; adeyemi, 2007; kumar et al., 2013). previous cytological studies on the genus basella indicated varying reports on the chromosome numbers of the green-stemmed and the red-stemmed b. alba., with the reports varying from chromosome numbers 2n = 44 to 2n = 45 and 2n = 48 (hanson et al., 2005; silva et al., 2017). there is no known report of chromosome numbers of b. alba morphotypes from nigeria. there is also paucity of information about the meiotic behaviour of b. alba. the specific objectives of these study were therefore to characterize the green-stemmed and red-stemmed b. alba and establish their chromosome numbers as well as investigate their meiotic behaviours. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant source and identification the seeds of the basella alba accessions studied were collected from various locations in nigeria (table 1) and authenticated at the ife herbarium, department of botany, obafemi awolowo university, nigeria. the seeds were sown in polythene bags filled with sterilized top soil laid out in a completely randomized design and raised to maturity in the screen house of the department of botany, obafemi awolowo university ile-ife, nigeria. normal agronomic practices such as watering, weeding and staking were performed. morphological characterization of plants studied the qualitative and quantitative attributes of the green-stemmed and the red-stemmed basella alba accessions studied were characterized with respect to their habit, leaves, inflorescence, fruits and seeds. the quantitative data were collected in five replicates for thirty-eight (38) accessions for both the green-stemmed and the red-stemmed. the quantitative morphological attributes of the green-stemmed and red-stemmed b. alba were compared by subjecting the data obtained to the generalized linear model (glm) analysis of variance (anova) and the differences between the means that were significantly different at p<0.05 were evaluated by duncan multiple range test (dmrt) using system analysis software (sas) version 9.0. the means were presented as mean and standard error (mean ± se). bolaji ao et al. (2023). not sci biol 15(2):11338 3 table 1table 1table 1table 1. sources of the green-stemmed and red-stemmed basella alba studied accession basella alba form source location comment baiwo (10)* green-stemmed iwo 7.629444 °n 4.191111 °e cultivated in home gardens baife (8) green-stemmed ile-ife 7.523056 °n 4.515833 °e cultivated in school gardens baondo (5) green-stemmed ondo 7.236111 °n 5.239722 °e cultivated in home gardens baekiti (5) green-stemmed ekiti 7.616389 °n 5.218333 °e cultivated in home gardens baoyo (5) green-stemmed oyo 7.419167 °n 3.964722 °e cultivated in church gardens balag (5) green-stemmed ijede 6.942778 °n 4.191111 °e cultivated beside home briwo (5) red-stemmed iwo 7.629444 °n 4.191111 °e cultivated in home gardens brife (5) red-stemmed ile-ife 7.523056 °n 4.515833 °e cultivated in school gardens brondo (10) red-stemmed ondo 7.236111 °n 5.239722 °e cultivated in home gardens brekiti (7) red-stemmed ekiti 7.616389 °n 5.218333 °e cultivated in home gardens broyo (5) red-stemmed ogbomoso 8.146111 °n 4.259167 °e cultivated in school gardens brlag (6) red-stemmed ijede 6.942728 °n 3.098056 °e cultivated in home gardens *note: numbers in brackets represent number of plants within each of the accessions studied pollen viability and seed set study the pollen viability study was carried out on 100 pollen grains following the method of bolaji et al. (2022b). pollen grains from freshly dehisced anthers were harvested on to microscope slides and were stained with cotton-blue-in-lacto-phenol for 30 minutes. the well-formed and deeply stained pollens were considered as viable; while those with collapsed outline, partially stained or not stained were considered to be non-viable. the percentage of viable pollens were documented. the seed set study was carried out on 10 randomly selected plant. the percentage seed set was determined by dividing the number of seeds obtained by the number of seeds expected (i.e., number of flowers obtained) multiplied by 100, following the method of idowu and oziegbe (2017). mitotic chromosome studies young whole plants of the green-stemmed and the red-stemmed b. alba were placed in plastic bottles containing water and allowed to root. the root tips were excised and fixed in 1: 3 acetic-ethanol after four days between 9:00 a.m. and 11:00 a.m. the fixed root tips were left at room temperature for 48 hours before being used for preparation of mitotic cells. the root tips were hydrolysed in 18% hcl, squashed and stained with modified orcein for 15 minutes following the methods of bolaji et al. (2019). slides prepared were observed under the light microscope and chromosome counts were made for each of the basella forms. good mitotic spreads were photomicrographed using amscope mt microscope camera version 3.0.0.1 attached to light microscope. meiotic chromosome studies young flower buds were harvested into vias containing 1: 3 acetic-ethanol between 9:00 a.m and 11:00 am. the anthers were excised, squashed and stained in flp orcein (2g of orcein in 100 cm3 of solution containing equal parts of formic acid, lactic acid, propionic acid and distilled water). pollen mother cells were examined to investigate meiotic events and photomicrographs of good meiotic spreads were taken using amscope mt microscope camera version 3.0.0.1 attached to a light microscope. bolaji ao et al. (2023). not sci biol 15(2):11338 4 results results results results description of the green-stemmed and red-stemmed basella alba studied the green-stemmed (figure 1a) and red-stemmed (figure 1b) basella alba studied were perennial, herbaceous twinning, with glabrous stem (table 2). the mean plant height was 87.65 ± 5.32 cm for the greenstemmed, while it was 119.49 ± 6.99 cm for the red-stemmed. the red-stemmed had significantly greater plant height (table 3) than the green-stemmed at p = 0.0005. the mean stem diameter for both forms of b. alba were not significantly different. the green-stemmed had a mean stem diameter of 3.38 ± 0.09 cm, while the red-stemmed had 3.64 ± 0.15 cm. the mean number of primary branches was 5.26 ± 0.45 for the green stemmed and 4.34 ± 0.29 for the red-stemmed with no significant difference between them. the mean internode distance was 4.58 ± 0.23 cm for the green-stemmed and 5.05 ± 0.31 cm for the red-stemmed., with no significant difference between them. figure 1.figure 1.figure 1.figure 1. the green-stemmed and red-stemmed basella alba studied and their inflorescence, fruits and seeds; a: green-stemmed b. alba studied; b: red-stemmed b. alba studied; c: inflorescence of the greenstemmed b. alba studied; d: inflorescence of the red-stemmed b. alba studied; e: mature fruits of the green-stemmed basella alba studied;; f: mature fruits of the red-stemmed b. alba studied; g: seeds of the green-stemmed b. alba studied; h: seeds of the red-stemmed b. alba studied the leaves were green, simple, glabrous, succulent, cordate, margin entire, apex acute and round, venation pinnate, leaf arrangement opposite at seedling stage, alternate at mature stage, leaf attachment petiolate; leaves exstipulate for both forms of b. alba (table 2). the mean leaf length was 6.62 ± 0.37 cm for the green-stemmed and 7.75 ± 0.31 cm for the red stemmed, with the red-stemmed having significantly longer leaves than the green-stemmed at p = 0.0218 (table 3). the mean leaf width was 5.23 ± 0.19 cm for the greenstemmed and 6.67 ± 0.26 for the red-stemmed, with the red-stemmed having significantly wider leaves at p = 0.0001. the mean petiole length was 1.71 ± 0.06 cm for the green-stemmed and 1.95 ± 0.08 cm for the redstemmed with the red-stemmed having significantly longer petiole than the green-stemmed at p = 0.0172. bolaji ao et al. (2023). not sci biol 15(2):11338 5 table 2.table 2.table 2.table 2. comparison of morphological attributes of the green-stemmed and the red-stemmed basella alba studied characters green-stemmed basella alba red-stemmed basella alba habit perennial, herbaceous, twinning, stem green, glabrous, mean plant height at flower bud initiation 87.65 ± 5.32 cm, mean stem diameter 3.38 ± 0.09 cm, mean number of primary branches 5.26 ± 0.45, mean internode distance 4.58 ± 0.23 cm perennial, herbaceous, twinning, stem red, glabrous, mean plant height at flower bud initiation 119.49 ± 6.99 cm, mean stem diameter 3.64 ± 0.15 cm, mean number of primary branches 4.34 ± 0.29, mean internode distance 5.05 ± 0.31 cm leaves green, glabrous, succulent, cordate, margin entire, venation pinnate, leaf arrangement alternate, leaf attachment petiolate, mean length 6.62 ± 0.37 cm, mean width 5.23 ± 0.19 cm, petiole length 1.71 ± 0.06 cm red, glabrous, cordate, margin entire, venation pinnate, leaf arrangement alternate, leaf attachment petiolate, mean length 7.75 ± 0.31 cm, mean width 6.67 ± 0.26 cm, petiole length 1.95 ± 0.08 cm inflorescence racemose, flowers bisexual, pedicel sessile and glabrous, symmetry radial; one perianth with 5 segments joined at the base, perianth cream with pink apex, bracts attached to base of perianth, anther 5, pollen light yellow, powdery; filament white with adnate attachment; stigma simple, glabrous; style of unequal length, white, simple, glabrous; ovary superior, placentation basal, mean flower per spike 22.16 ± 0.93 racemose, flowers bisexual, pedicel sessile and glabrous, symmetry radial; one perianth with 5 segments joined at the base, perianth cream with deep purple apex, bracts attached to base of perianth, anther 5, pollen deep yellow, powdery; filament white with adnate attachment; stigma simple, glabrous; style of unequal length, white, simple, glabrous; ovary superior, placentation basal, mean flower per spike 16.45 ± 0.82 fruits drupe, glabrous, spherical, apedicelate on green spike; four lobed, one-seeded, unripe colour green, ripen colour deep purple, mean length 1.07 ± 0.03 cm, mean diameter 2.12 ± 0.02 cm, mean number of fruits per spike 12.21 ± 0.69 drupe, glabrous, spherical, apedicelate on green spike; four lobed, one-seeded, unripe colour green, ripen colour deep purple, mean length 1.15 ± 0.03 cm, mean diameter 2.25 ± 0.03 cm, mean number of fruits per spike 10.24 ± 0.74 seeds brown, spherical, mean length 0.66 ± 0.10 cm, mean diameter 1.25 ± 0.01 cm, mean number of seed per spike 12.21 ± 0.69 brown, spherical, mean length 0.64 ± 0.02 cm, mean diameter 1.26 ± 0.01 cm, mean number of seed per spike 10.24 ± 0.74 the inflorescence was racemose with each of the flowers consisting of one perianth with 5 segments joined at the base for both forms of b. alba (table 2). the perianth was cream with pink apex (figure 1c) in the green stemmed, while it was cream with deep purple apex (figure 1d) for the red-stemmed. bracts were attached to the base of the perianth in both forms. the flowers were bisexual, pedicel sessile and glabrous, symmetry radial; anther 5, pollen yellow, powdery; filament white with adnate attachment; stigma simple, glabrous; style of unequal length, white, simple, glabrous; ovary superior, placentation basal for both forms of b. alba studied. the mean flower per spike was 22.16 ± 0.93 for the green-stemmed while it was 16.45 ± 0.82 for the red stemmed, with the green stemmed having significantly higher number of flowers per spike than the red-stemmed at p = 0.0001 (table 3). bolaji ao et al. (2023). not sci biol 15(2):11338 6 table 3.table 3.table 3.table 3. comparison of morphometric characteristics between the green-stemmed and the red stemmed basella alba studied characters green stemmed basella alba red stemmed basella alba mean plant height at flower bud initiation (cm) 87.65 ± 5.32 b 119.49 ± 6.99 a mean stem diameter (cm) 3.38 ± 0.09 a 3.64 ± 0.15 a no. of primary branches 5.26 ± 0.45 a 4.34 ± 0.29 a mean internode distance (cm) 4.58 ± 0.23 a 5.05 ± 0.31 a mean leaf length (cm) 6.62 ± 0.37 a 7.75 ± 0.31 a mean leaf width (cm) 5.23 ± 0.19 b 6.67 ± 0.26 a mean petiole length (cm) 1.71 ± 0.06 b 1.95 ± 0.08 a mean flower per spike 22.16 ± 0.93 a 16.45 ± 0.82 b mean fruit length (cm) 1.07 ± 0.03 b 1.15 ± 0.03 a mean fruit diameter (cm) 2.12 ± 0.02 b 2.25 ± 0.03 a mean number of fruits per spike 12.21 ± 0.69 a 10.24 ± 0.74 a mean seed length (cm) 0.66 ± 0.10 a 0.64 ± 0.02 a mean seed diameter (cm) 1.25 ± 0.01 a 1.26 ± 0.01 a mean number of seed per spike 12.21 ± 0.69 a 10.24 ± 0.74 a *notes: different letters between cultivars denote significant differences (duncan test, p < 0.05) the fruits were berry, glabrous, spherical, apedicelate on green spike; four lobed, one-seeded, unripe colour green, ripen colour deep purple (figure 1e and figure 1f) for both forms of b. alba studied. the mean fruit length was 1.07 ± 0.03 cm for the green stemmed while it was 1.15 ± 0.cm for the red stemmed, with the red-stemmed having significantly longer fruits than the green-stemmed at p = 0.0398 (table 3). the mean fruit diameter was 2.12 ± 0.02 cm for the green-stemmed while it was 2.25 ± 0.03 cm for the red-stemmed with the red-stemmed having significantly wider fruits than the green-stemmed at p = 0.0002. the mean number of fruits per spike was 12.21 ± 0.69 for the green-stemmed, while it was 10.24 ± 0.74 for the red-stemmed with no significant difference between them. the seeds were brown (figure 1g and figure 1h), spherical in shape for both forms of b. alba studied. the mean seed length was 0.66 ± 0.10 cm for the green-stemmed, while it was 0.64 ± 0.02 cm for the redstemmed with no significant difference between them (table 3). the mean seed diameter was 1.25 ± 0.01 cm for the green-stemmed, while it was 1.26 ± 0.01 cm for the red-stemmed with no significant difference between them. the mean number of seed per spike was 12.21 ± 0.69 for the green-stemmed while it was 10.24 ± 0.74 for the red-stemmed with no significant difference between them. chromosome studies the study revealed a diploid chromosome number of 2n = 44 and haploid chromosome number of n = 22 for the green-stemmed (figure 2a, figure 2b, figure 2c) and the red-stemmed basella alba (figure 2d) studied. the meiotic chromosome study also revealed the occurrence of many quadrivalent chromosomes amidst bivalent chromosomes at diakinesis (figure 2c) and metaphase i (figure 2d). it also revealed the occurrence of chromosome stickiness (figure 2e) and precocious migration of chromosomes to the poles (figure 2e). the frequency of the chromosome stickiness was 2.5% in the green-stemmed b. alba; while it was 3.5% in the red-stemmed (table 4). the frequency of the precocious migration of chromosomes to the poles was 5.7% in the green-stemmed; while it was 6.3% in the red-stemmed. bolaji ao et al. (2023). not sci biol 15(2):11338 7 figure 2.figure 2.figure 2.figure 2. mitotic and meiotic chromosomes of the green-stemmed and red-stemmed basella alba studied; a: mitotic metaphase of green-stemmed b. alba showing 2n = 44 chromosomes; b: diakinesis of greenstemmed b. alba showing n = 22 bivalent chromosomes; c: diakinesis of green-stemmed b. alba showing n = 22(7iv+8ii); d: metaphase i of red-stemmed b. alba showing n = 22(9iv+4ii) chromosomes; e: diakinesis of green-stemmed b. alba showing chromosome stickiness and precocious migration to the pole (arrow) table 4.table 4.table 4.table 4. number of pollen mother cells analysed and percentage of abnormal cells affected by chromosome stickiness, laggards and precocious migration to the poles in the basella alba accessions studied accessions green-stemmed b. alba red-stemmed b. alba no. of pollen mother cells analysed 955 1255 cells with chromosome stickiness (%) 2.5 3.5 cells with precocious migration of chromosomes to the poles (%) 5.7 6.3 pollen viability (%) 93.70 94.5 seed set (%) 65.8 58.43 discussiondiscussiondiscussiondiscussion the green-stemmed and red-stemmed basella alba studied were similar with respect to many of their vegetative and reproductive attributes (table 3) and this is in support of their taxonomic classification as members belonging to same species as reported by roy et al. (2010), bolaji et al. (2022a), and bolaji et al. (2022b). however, there were significant differences between them with respect to the green/red colour of their stems, colour of the flower bud apex (i.e., colour of the perianth apex), mean plant height at flower bud initiation, mean leaf length, mean leaf width, mean petiole length, mean flower per spike, mean fruit length and mean fruit diameter. these significant differences further support their delimitation as varieties belonging to same species. this is corroborated by the reports of henry et al. (1987), roy et al. (2010) and bolaji et al. (2022a). chromosome counting from the floral and vegetative tissues of plants remains the most reliable method to establish their chromosome numbers (mayrose et al., 2020). in this study, the diploid chromosome count of bolaji ao et al. (2023). not sci biol 15(2):11338 8 2n = 44 and haploid chromosome count of n = 22 indicates a basic chromosome number of x = 11 thereby indicating a chromosome number of 2n = 4x = 44 for the green-stemmed and the red-stemmed basella alba studied. this is in line with the reports of silva et al. (2017) for basella alba species obtained from brazil. the chromosome numbers obtained for the green-stemmed and red-stemmed basella alba in this study contradicts those of hanson et al. (2005) who reported chromosome numbers 2n = 48, 41 and 44 for those obtained from england. it also contradicts the reports of sperling and bittrich (1993) who reported the basic chromosome number of x = 12 for b. alba. according to silva et al. (2017) the chromosome number 2n = 44 observed in b. alba may have been derived from descending disploidy from the number 2n = 4x = 48. the occurrence of many quadrivalent chromosomes amidst bivalent chromosomes at diakinesis and metaphase i further indicates that the green-stemmed and red-stemmed basella alba studied were tetraploids. the precocious migration of some of the chromosomes to the pole and stickiness of the chromosomes during meiosis could lead to irregular chromosome segregation and chromosome instability within the species. mendes-bonato et al. (2001) noted that though the cause and biochemical basis for chromosome stickiness is not known, it is characterised by intense clustering and in severe cases makes it impossible for the chromosomes to separate leading to formation of single or varying numbers of pycnotic nuclei that culminate into full chromatin degeneration. potapova and gorbsky (2017) also noted that the full consequences of chromosome segregation errors are vast in scope and could produce aneuploid or polyploid cells as well as progeny with altered chromosome content. it is however noteworthy that in this study the frequencies of the chromosome stickiness and precocious migration of chromosomes to the pole during meiosis was very low (table 4), hence could have been the reason why the pollen viability was still high and the percentage seed set was as well moderately high. conclusionsconclusionsconclusionsconclusions this study characterized the accessions of the green-stemmed and red-stemmed basella alba and established their chromosome as 2n = 4x = 44. the study also revealed significant differences between the green-stemmed and red-stemmed b. alba with respect to the green/red colour of their stems, colour of the flower bud apex, mean plant height at flower bud initiation, mean leaf length, mean leaf width, mean petiole length, mean flower per spike, mean fruit length and mean fruit diameter. the occurrence of many quadrivalent chromosomes amidst bivalent chromosomes suggest that they are tetraploids. the stickiness of the chromosomes and precocious migration to the poles during meiosis in the b. alba accessions studied could result in irregular segregation, which could possibly lead to chromosomal instability within the species. however, the frequencies of this aberrant chromosomal events were quite low in the b. alba accessions studied, hence, did not result in low pollen viability and low seed set. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization: aob; data curation: aob and oia; formal analysis: aob, aso and oia; investigation: aob, aso and oia; methodology: aob, aso and oia; project administration: aob, aso and oia; resources: aob, aso and oia; software: aob; supervision: aob; validation: aob; visualization: aob, aso and oia; writing original draft: aob and oia; writing review and editing: aob and aso. all authors read and approved the final manuscript. bolaji ao et al. 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(2023). not sci biol 15(2):11338 11 license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11351 nsb layachi 2022.03.16.docx received: 16 sep 2022. received in revised form: 28 jan 2023. accepted: 07 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania layachi r et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11351 doi:10.15835/nsb15111351 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae assessment of analgesic properties of alcohol and aqueousassessment of analgesic properties of alcohol and aqueousassessment of analgesic properties of alcohol and aqueousassessment of analgesic properties of alcohol and aqueous eeeextractsxtractsxtractsxtracts of of of of opuntia opuntia opuntia opuntia ficusficusficusficus----indicaindicaindicaindica flowersflowersflowersflowers rajaa layachi1a*, aziz mouhaddach1b, lamiae amallah1, mohammed benaziz2, fatima bouazza1, souad benaich3, rachida hassikou1, souad skalli1 1mohammed v university in rabat, faculty of sciences, plant and microbial biotechnologies, biodiversity, and environment center, morocco; r.layachi@um5r.ac.ma (*corresponding author); mouhaddach@gmail.com; l.amallah@hotmail.com; fatima.leader2020@gmail.com; r.hassikou@um5r.ac.ma; s.skalli@um5r.ac.ma 2sultan moulay slimane university, higher school of technology, pb170, 54000, khenifra, morocco; m.benaziz@usms.ma 3mohammed v university in rabat, faculty of sciences, physiology and physiopathology team, morocco; s.benaich@um5r.ac.ma a,bthese authors contributed equally to the work abstractabstractabstractabstract the cactus (opuntia ficus-indica (l.) mill.) belongs to the cactaceae family and it’s used in traditional folk medicine in treating a number of diseases and conditions. due to the remarkable biological activity and to the bioactive (phytochemicals) compounds of o. ficus-indica, it becomes the aim of many research studies. the current study aimed to evaluate the analgesic activity of various solvent fractions (aqueous and ethanol) prepared from the o. ficus-indica flowers. the centrally analgesic potential was evaluated using tail flick latency in tail immersion and hot plate methods in mice. morphine was used as a positive control at a dose of 3 mg kg1, s.c.. intra-peritoneal administration of the aqueous extract of o. ficus-indica flowers at the highest dose did not produce any toxicity symptoms, thus the median lethal dose (ld50) was estimated to be greater than 2,500 mg kg-1. the results of the pain behavior evaluation according to the gender approach of mice showed that the pain tolerance threshold is high in males compared to females. we found that various plant extracts at doses of 300, 500, and 1,000 mg kg-1 i.p., displayed significant and dose-dependent protective effects (p < 0.01, p < 0.001, and p < 0.0001) as measured by increased latency time compared to vehicle control. the maximum antinociceptive effect was with the ethanol extract (71%) at 60 minutes at a dose of 1,000 mg kg-1, which was equivalent to the effect of morphine (70%). the results suggested that o. ficus-indica might possess significant analgesic effects, supporting the use of this plant in traditional medicine. keywords:keywords:keywords:keywords: centrally analgesic; flowers; opuntia ficus-indica; morphine; traditional medicine introductionintroductionintroductionintroduction pain is one of the more complex pathological phenomena, this process involving the immune system, neurobiological processes and humoral systems (smith et al., 1943). in the normal state, prostaglandins and https://www.notulaebiologicae.ro/index.php/nsb/index layachi r et al. (2023). not sci biol 15(1):11351 2 other molecules participate in the genesis and remediation of this anomaly (sébastien, 2010). the current treatment for pain involves anti-inflammatory medications. due to their undesirable side effects, which limit their uses, there is more interest in natural compounds extracted from medicinal plants. these plants have analgesic and antipyretic activity and could constitute an alternative therapy because of their accessibility and low toxicity (queneau and ostermann, 2004; bussmann, 2011). the bibliographic survey reports that opuntia ficus-indica (l.) mill. (the plant name has been checked with http://www.theplantlist.org) is an important source of therapeutic agents, due to their pharmacological properties and their richness in bioactive molecules. o. ficus-indica has long been used in traditional medicine in various countries (park et al., 2001; mouhaddach et al., 2017). the data of the ethnobotanical survey carried out by mouhaddach et al. (2017) showed that the various parts of the o. ficus-indica plant can be used for the treatment of various health conditions including gastric disorders, kidney pain, back pain, and leg pain. cladodes (57%) were the most used part of the plant, followed by flowers (21%), seeds (11%) and roots (5%). several parts of the plant have been used to relieve pain, especially cladodes and flowers. the o. ficus-indica flowers exert a wide range of pharmacological activities, including antioxidant, antimicrobial (ammar et al., 2012), anti-ulcerogenic and healing activities (ammar et al., 2015; mouhaddach et al., 2017), as well as anti-inflammatory (ahmed et al., 2005; zou et al., 2005). o. ficus-indica has traditionally been used to reduce inflammation and previous studies have demonstrated its anti-inflammatory potential (shirazinia et al., 2019). more recently, the anti-inflammatory and the analgesic effects of cladodes methanol extract have also been investigated by siddiqui et al. (2016) and mouhaddach et al. (2017). within the available literature, few studies have focused on flowers compared to other parts of the plant (cladodes, fruits, and seeds). this study is the first report on analgesic effect of aqueous and alcoholic extracts of o. ficus-indica flowers via tail flick and hot plate tests. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant material o. ficus-indica flowers, spineless variety, were collected from the rhamna region (morocco) during the month of june 2015. then they were dried for one week (45 °c) and ground into a coarse powder with the help of a suitable grinder. the powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. preparation of aqueous and organic extracts the flower powder (50 g) mixed with distilled water (500 ml) was decocted at 100 °c for 15 minutes. then the mixture was filtered, lyophilized (using a phywe chrisa lyophilizer) and stored at 4 °c. at the time of its administration the extracts were dissolved in 0.9% saline (nacl). the organic flower extract was prepared via the soxhlet method using ethanol as the extraction solvent. animals adult male and female swiss mice (20-28 g, 2 months of age) obtained from the animal house, physiology and physiopathology laboratory, faculty of sciences, university mohammed v, rabat, morocco, were used throughout these experiments. they were housed in cages at room temperature and 12:12 h light/dark cycle. the animals were provided with pelleted diet and tap water ad libitum. all experiments were carried out according to strict adherence to ethical guidelines (zimmerman, 1983). layachi r et al. (2023). not sci biol 15(1):11351 3 analgesic activity method hot plate method mice of both genders were selected and divided into five groups designated as group-i, group-ii, groupiii, group-iv and group-v, consisting of eight mice (4♀ and 4♂) in each group for control, positive control and test sample group respectively. each group received a particular treatment, control group (0.9% nacl solution, i.p.), positive control (morphine 3 mg kg-1, subcutaneous, s.c.) and the test sample (ethanol and aqueous extracts of 300 mg kg-1, i.p., 500 mg kg-1, i.p., and 1,000 mg kg-1 i.p.). the animals were positioned on eddy’s hot plate kept at a temperature of 53±0.2 °c. a cut off period of 15 seconds was observed to avoid damage to the paw (franzotti et al., 2000). the latency (reaction) time was recorded when the mice licked their paws or jumped at 0, 30, 60, 90 and 120 minutes after intraperitoneal administration (i.p.) of the samples (eddy and leimback, 1953; toma et al., 2003). tail flick method central analgesic activity was assessed according to the method described by di stasi et al. (1988) mice in groups of eight (4♀ and 4♂) were held in position in a suitable restrainer with the tail extended. a 2-3 cm area of the tail was marked and immersed in a water bath thermo-statistically maintained at 53±0.2 °c. the withdrawal time of the tail from the hot water (in seconds) was noted at 0, 30, 60, 90, and 120 minutes as the reaction time or tail flick latency. the maximum cut-off time for immersion was 15 seconds to avoid tail injury. control mice were treated with vehicle (0.9% nacl solution, i.p.), while the reference group was given morphine subcutaneously (s.c.) (3 mg kg-1, s.c.). flowers extracts at doses of 300, 500, and 1,000 mg kg-1 were administered by intra-peritoneal injection. statistical analysis all values are expressed as mean ± s.e.m. (standard error of mean). data were analyzed by one-way anova (analysis of variance) followed by tukey’s multiple comparison tests. a level of p < 0.05 was considered statistically significant. results results results results acute toxicity of the aqueous extract the mice showed no change in general physical appearance and somatomotor skills during the observation period. no manifestations of tremors, convulsions, salivation, diarrhea, coma, or behaviors were observed. however, manifestations of sleep were observed in treated mice during the first 30 minutes after gavage. no other treatment-related changes or mortality were observed in mice over the 24-hour period following oral administration of a single dose of o. ficus-indica flower decoction at a dose of 2,000 mg kg-1. the median lethal dose (ld50) was therefore estimated to be greater than 2,500 mg kg-1. analgesic effect tail flick test tables 1 and 2 represent the results obtained via the tail immersion test to test the analgesic effect of aqueous and organic extracts of o. ficus-indica flowers. the results showed that the extracts at doses of 300, 500 and 1,000 mg kg-1, i.p. induce significant analgesic activity, demonstrated by an increase in latency time in seconds (table 1) compared to the control. the tail withdrawal latency time of control mice was 2.21±0.43 s. at 30 minutes after administration of the extracts, we found that the latency times of the mice of the batches receiving the two aqueous and ethanol layachi r et al. (2023). not sci biol 15(1):11351 4 extracts at doses of 300, 500 and 1,000 mg kg-1, i.p. as well as that of the mice of the batch receiving morphine at 3 mg kg-1, s.c., were significantly higher (p < 0.001) compared to the control group. the latency time of the batch that received the extract dosed at 1,000 mg kg-1, i.p., was significantly higher (p < 0.05) than that of the batch that received the extract at 500 mg kg-1, i.p. similarly, the batch which received the extract at a dose of 500 mg kg-1, i.p., turns out to have a significantly higher latency time (p < 0.05) than that of the batch which received the extract at 300 mg kg-1, i.p.. as for the percentage of pain inhibition, it was noted that the percentage of pain inhibition after 60 minutes with morphine at a dose of 3 mg kg-1 was 70%. this value was reached at a dose of 1,000 mg kg-1 of the aqueous extract. also, the pain inhibition percentage of the ethanol extract after 60 minutes was slightly higher at 71%. table 1.table 1.table 1.table 1. analgesic effect of aqueous and organic extracts of o. ficus-indica flower, latency in tail flick test in mice treatments doses mg kg-1 reaction time ± s.e.m. 0 min 30 min 60 min 90 min 120 min vehicle 0.2 2.21 ± 0.43 2.56 ± 0.54 2.06±0.14 2.56±0.61 2.74±0.33 morphine 3 2.78 ± 0.78 8.6 ± 0.95*** 9.38±1.55*** 9.03±1.23*** 6.29±0.89** ethanol extract 300 3.21 ± 0.54 4.55 ± 0.11*a 6.93±0.69**c 4.53±0.39*c 3.77±0.34c 500 3.06 ± 0.43 7.04 ± 0.88**b 8.37±0.73***b 6.98±0.35**c 4.74±0.37b 1,000 2.32 ± 0.47 7.20 ± 0.87***c 8.12±0.97***a 7.10±0.85***c 4.75±0.54b aqueous extract 300 2.04 ± 0.22 3.85 ± 0.12*c 5.39±0.15***c 4.90±0.21**c 3.93±0.21b 500 2.73 ± 0.59 5.63 ± 0.23**b 8.22±0.54***b 6.51±0.30**c 5.51±0.17b 1,000 2.71 ± 0.61 5.57 ± 0.15**b 8.65±0.27***b 6.57±0.18**c 5.77±0.16*b results are presented as mean ± sem (n=8), analyzed by one-way anova followed by tukey’s multiple comparison test (*): p < 0.01; (**): p < 0.001; (***): p < 0.0001 for difference in change from vehicle control group, (a): p < 0.05; (b): p < 0.001; (c): p < 0.001 for difference in change from standard reference group. table 2.table 2.table 2.table 2. pain inhibition percentage for different treatment types in the tail flick test treatments doses mg kg-1 inhibition percentage (%) 30 min 60 min 90 min 120 min morphine 3 68 70 69 56 ethanol extract 300 29 53 29 14 500 56 63 56 35 1,000 68 71 67 51 aqueous extract 300 47 62 58 48 500 51 68 58 50 1,000 68 70 69 56 hot plate test results from hot plate testing of latency time and inhibition percentage are presented in tables 3 and 4. morphine at dose of 3 mg kg-1, the aqueous and ethanol extract at doses of 300, 500 and 1,000 mg kg-1, showed a significant increase in the latency time. the latency time results at different doses showed a significant increase, hence a dose-dependent effect. the highest pain inhibition percentage (69%) for the aqueous extract was obtained by the highest dose 1,000 mg kg-1, while the percentage for ethanol extract was 65%. layachi r et al. (2023). not sci biol 15(1):11351 5 table 3.table 3.table 3.table 3. analgesic effect of aqueous and ethanol extracts of o. ficus-indica flowers on latency via the hot plate test treatments doses mg kg-1 reaction time ± s.e.m. 0 min 30 min 60 min 90 min 120 min vehicle 0,2 2.48±0.27 2.42±0.63 2.32±0.23 2.53±0.25 2.23±0.72 morphine 3 2.48±0.86 8.34±0.26*** 9.62±1.59*** 8.33±1.22*** 6.24±0.78** aqueous extract 300 2.34±0.25 3.44±0.19c 5.03±0.22**c 4.50±0.13*c 3.60±0.22c 500 2.22±0.28 3.72±0.22c 6.32±0.27**b 4.97±0.23**c 3.41±0.15c 1,000 2.43±0.19 4.88±0.15**c 7.92±0.18***b 5.46±0.20**c 3.84±0.18c ethanol extract 300 2.78±0.43 3.77±0.38c 6.50±1.16**c 4.64±0.38*c 3.63±0.28b 500 2.65±0.88 6.88±1.53**b 6.86±0.83**c 5.97±0.93**b 4.05±0.18b 1,000 2.66±0.85 5.81±0.71*c 7.54±0.43***b 6.18±0.62**c 4.25±0.35b results are presented as mean ± sem (n=8), analyzed by one-way anova followed by tukey’s multiple comparison test (*): p < 0.01; (**): p < 0.001; (***): p < 0.0001 for difference in change from vehicle control group, (a): p < 0.05; (b): p < 0.001; (c): p < 0.001 for difference in change from standard reference group. table 4.table 4.table 4.table 4. pain inhibition percentage for different treatment types in the tail flick test treatments doses (mg kg-1) inhibition percentage (%) 30 min 60 min 90 min 120 min morphine 3 70 74 70 60 aqueous extract 300 32 53 48 35 500 40 65 55 35 1,000 50 69 55 37 ethanol extract 300 26 57 40 23 500 61 61 56 34 1,000 54 65 57 37 pain behavior assessment according to the gender of mice pain inhibition results of aqueous extract of o. ficus-indica flowers obtained via experimental hot plate model in male and female mice are shown in figures 1, 2 and 3. the pain behavior results in male and female mice according to the hot plate test showed a significant difference (p < 0.05; p < 0.01). then, this difference was observed for all tested doses. finally, these results show that the pain tolerance threshold is high in males compared to females (the graphs have been represented with reference to the tail flick test model results). layachi r et al. (2023). not sci biol 15(1):11351 6 figure 1.figure 1.figure 1.figure 1. the percentage of pain inhibition according to mice gender after administration of the aqueous extract at 300 mg kg-1 figure 2.figure 2.figure 2.figure 2. the percentage of pain inhibition according to mice gender after administration of the aqueous extract at 500 mg kg-1 figure 3.figure 3.figure 3.figure 3. the percentage of pain inhibition according to mice gender after administration of the aqueous extract at 1,000 mg kg-1 0 10 20 30 40 50 60 70 30 min 60 min 90 min 120 min p a in i n h ib it io n p e rc e n ta g e (% ) reaction time (min) males females 0 10 20 30 40 50 60 70 80 30 min 60 min 90 min 120 min p a in i n h ib it io n p e rc e n ta g e (% ) reaction time (min) males females 0 10 20 30 40 50 60 70 80 30 min 60 min 90 min 120 min p a in i n h ib it io n p e rc e n ta g e (% ) reaction time (min) males females layachi r et al. (2023). not sci biol 15(1):11351 7 discussiondiscussiondiscussiondiscussion in the present study, we first examined the toxicity of the aqueous extract and also the anti-nociceptive potential of o. ficus-indica flower extracts (aqueous and ethanol) as a central analgesic using tail flick and hot plate test followed by the reaction to the painful behavior according to the gender of the mice. firstly, oral administration of the highest dose of 2,000 mg kg-1 of o. ficus-indica flower extracts was found to produce no symptoms of acute toxicity. although a slight sedation in the treated mice, no mouse mortality occurred during the 24 hours observation period. the extracts were found to be non-toxic at the highest dose (2,000 mg kg-1), thus, the ld50 was therefore estimated to be greater than 2,500 mg kg-1 according to the ocde decision tree (ocde, 2001). the highest dose dilutions were designated to assess analgesic activity, namely 300, 500 and 1,000 mg kg-1. the impact of gender on the response to an experimental nociceptive stimulus has been the topic of few studies. thus, the results obtained within the present work have shown that the pain tolerance threshold is higher in males compared to females. similarly, mogil et al. (2000) according that female rats are more sensitive to thermal stimuli compared to males. this peculiarity probably results from the dynamic interaction of psychological and physiological factors. concerning the analgesic impact study, the anti-nociceptive tests used in the current work were chosen to test the thermal cutaneous stimuli (tail flick and hot plate tests). sayyah et al. (2004) demonstrated that thermal stimuli are selectively inhibited by central analgesics however not peripheral analgesics in rats. we have used morphine in this study to test our hypothesis. the obtained results showed a significant difference compared to control for three doses tested, so the two extracts of o. ficus-indica flowers would have a central analgesic impact. therefore, these results justify their significant protective effect and dose-dependent response to the thermo-painful stimulus. the analgesic effect proven by the tested extracts was approximately comparable to that of morphine. indeed, at 3 mg kg-1 b.w., the pain inhibition of morphine was 70%. in the presence of flower extracts of o. ficus-indica at a dose of 1,000 mg kg-1, the inhibition percentage recorded is 70% for the aqueous extract and 71% for the ethanol extract, according to the tail flick test. in fact, the aqueous and ethanol extracts of o. ficusindica flowers caused a reduction of painful behavior in mice. these results showed that o. ficus-indica flowers used for pain treatment (mouhaddach et al., 2017) was justified. the obtained results by chahdoura et al. (2017) showed that the aqueous extract of opuntia microdasys flowers at a dose of 100 mg kg-1 produced a greater peripheral analgesic effect than that obtained with the positive control, lysine-aspirin (72% and 65.5% protection, respectively). these results are consistent with a few scientific studies reports that the plant has been used for their analgesic and anti-inflammatory actions. according to park et al. (1998), fruit extract, freeze-dried cladodes and stem extracts had analgesic and anti-inflammatory actions. considering the results prospected from ethnobotanical survey of o. ficus-indica, as well as those reported in the bibliographical review, we assumed that the richness of opuntia in phenolic compounds, particularly in flavonoids, could produce a significant analgesic effect. indeed, such a conclusion is supported by the phytochemical screening of o. ficus-indica which revealed the presence of bioactive molecules, mainly quercetin, nicotiflorin, narcissus, rutin, kaempherol, quercetin 3o-methylether and 2,3-dihydrokaempferol (lee et al., 2003; guevara-figueroa et al., 2010; ammar et al., 2017; abbas et al., 2022). kuti (2004) reported that quercetin was the dominant flavonol in all analyzed species belonging to four different cactus species, namely o. ficus-indica, o. lindheimeri, o. strepthacantha, and o. stricta, and that this molecule has a remarkable analgesic effect. more recently, ammar et al. (2017) reported that quinic acid was the predominant phenolic acid in the alcohol extract of opuntia flowers, while quercetin-3-o-rutinoside was the major flavonoid identified. it was followed by quercetin-3-o-galactoside, quercetin-3-o-rhamonoside and isorhamnetin-3-o-glucoside. also, the same study reports that this molecular combination produces a protective and anti-inflammatory effect layachi r et al. (2023). not sci biol 15(1):11351 8 according to the model of rat paw edema induced by carrageenan. in addition, fruit phytosterols have been identified as the main active anti-inflammatory component (park et al., 2001; izuegbuna et al., 2019). therefore, the freeze-dried aqueous extract (100-400 mg kg-1, i.p.) of opuntia dillenii fruits in hot plate assays in mice and rats possessed notable and dose-dependent analgesic activity (loro et al., 1999). furthermore, the obtained results in phenolic analysis are in agreement with previous studies reporting the presence of phenolic acids and flavonoids in the methanolic extract of o. ficus-indica flowers (yeddes et al., 2014), as well as in the methanol extract of o. microdasys flowers (chahdoura et al., 2014). quinic acid was the major phenolic acid identified in this study, which accounted for 12.4% of the total identified phenolic compounds. this compound is also present in moroccan o. ficus-indica with 3.65% of the total identified phenolic compounds (benayad et al., 2014; zeghbib et al., 2022). o. ficus-indica flowers extract was dominated by flavonol glycoside and the flavonol 3-o-glycosides (quercetin, kaempferol, and isorhamnetin) (de leo et al., 2010; kolniak-ostek et al., 2020). these bioactive molecules also showed that certain compounds possessed analgesic activity. also, flavonoids (kaempherol and isorhamnetin glycosides) are known to inhibit the prostaglandin synthetase enzyme, specifically endoperoxidase (ramaswamy et al., 1985; benattia et al., 219). bentley (1983) and negus et al. (2006) proved that the pain onset mechanism can be attributed to the release of substances in the intraperitoneal fluid such as prostaglandins (pge2, pgf2 α), serotonin, histamine, bradykinin which will stimulate nociceptive receptors located in the peritoneum. in addition, the chemical composition of opuntia flowers was characterized by the presence of flavonoids, saponins, tannins, and carbohydrates, contents (ennouri et al., 2014). thus, flavonoids are 5-lypo-oxygenase inhibitors, which allow inhibition of the inflammatory mediator’s effect (ferreira, 2002). as a result, the presence of flavonoids in the o. ficus-indica flowers extract might contribute to the analgesic effect. finally, we confirm that o. ficus-indica flowers possess a central analgesic activity, justifying their use in traditional medicine (mouhaddach et al., 2017). this effect can be explained by the presence of phenolic compounds and flavonoids inhibiting the pain process. other more advanced experimental models have made it possible to determine both the analgesic and anti-inflammatory activities of the aqueous extract obtained from opuntia microdasys flowers. these interesting results are used in native anti-cancer regimens and suggest that opuntia microdasys flowers could be explored for their pharmacological and antimutagenic profiles with potential utility of this plant within an herbal medicine or as a nutritional supplement. furthermore, a more thorough analysis would allow us to discern the precise doses required for both anti-inflammatory and analgesic activities and, on the other hand, to quantify this activity by precisely establishing the action mechanism of mediating molecules in this process. subsequently, it would be wise to seek a stable galenic formulation that can serve as a basis for this activity to enable design of a medicine to treat pain. conclusionsconclusionsconclusionsconclusions in the present study we demonstrated the analgesic property of the o. ficus-indica flowers extracts, using conventional pharmacological models. these findings support its usage within the traditional pharmacopoeia. these properties are most likely related to flavonoids and phenolic compounds highlighted in the bibliography. intra-peritoneal administration of o. ficus-indica flowers aqueous extract did not show any toxicity symptoms at the highest dose, thus the ld50 was estimated to be greater than 2,500 mg kg-1. the pain behavior results according to the gender approach of mice showed that the pain tolerance threshold is high in males compared to females. further experiments using purified extracts are needed to accurately identify the active compounds responsible for this analgesic effect and to understand their action mode. layachi r et al. (2023). not sci biol 15(1):11351 9 authors’ coauthors’ coauthors’ coauthors’ contributionsntributionsntributionsntributions ma: writing-original draft, sampling and analysis; lr: writing-original draft, methodology; al: investigation, resources; bm: statistical analysis, investigation; bf: validation, investigation; bs: formal analysis, investigation; hr: supervision, investigation, resources; ss: methodology, validation. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences abbas ey, ezzat mi, el hefnawy hm, abdel-sattar e (2022). an overview and update on the chemical composition and potential health benefits of opuntia ficus-indica (l.) miller. journal of food biochemistry 46:e14310. https://doi.org/10.1111/jfbc.14310 ahmed ms, el tanbouly nd, islam wt, sleem aa, el senousy as (2005). anti-inflammatory flavonoids from opuntia dillenii (ker-gawl) haw: flowers growing in egypt. phytotherapy research 19(9):807-809. https://doi.org/10.1002/ptr.1708.pmid:16220577 ammar i, ben amira a, khemakem i, attia h, ennouri m (2017). effect of opuntia ficus-indica flowers maceration on quality and on heat stability of olive oil. journal of food science and technology 54:1502-1510. https://doi.org/10.1007/s13197-017-2581-0 ammar i, ennouri m, attia h (2015). phenolic content and antioxidant activity of cactus (opuntia ficus-indica l.) flowers are modified according to the extraction method. industrial crops and products 64:97-104. https://doi.org/10.1016/j.indcrop.2014.11.030 ammar i, ennouri m, khemakhem b, yangui t, attia h (2012). variation in chemical composition and biological activities of two species of opuntia flowers at four stages of flowering. industrial crops and products 37:34-40. https://doi.org/10.1016/j.indcrop.2011.11.027 benattia kf, arrar z, dergal f, khabbal y (2019). pharmaco-analytical study and phytochemical profile of hydroethanolic extract of algerian prickly pear (opuntia ficus-indica.l). current pharmaceutical biotechnology 20(9). https://dx.doi.org/10.2174/1389201020666190620113129 benayad z, martinez-villaluenga c, frias j, gomez-cordoves c, essafi ne (2014). phenolic composition, antioxidant and anti-inflammatory activities of extracts from moroccan opuntia ficus-indica flowers obtained by different extraction methods. industrial crops and products 62:412-420. https://doi.org/10.1016/j.indcrop.2014.08.046 layachi r et al. 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licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11380 nsb bulu 2023.03.16.docx received: 15 nov 2022. received in revised form: 01 jan 2023. accepted: 01 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania bulu yi et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11380 doi:10.15835/nsb15111380 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae soil seed bank studies of soil seed bank studies of soil seed bank studies of soil seed bank studies of tithoniatithoniatithoniatithonia rotundifoliarotundifoliarotundifoliarotundifolia invaded fallow land and invaded fallow land and invaded fallow land and invaded fallow land and competitive association with competitive association with competitive association with competitive association with chromolaena odoratachromolaena odoratachromolaena odoratachromolaena odorata yetunde i. bulu*, rilwan o. ahmed, opefoluwa d. fadayomi, otitoloju kekere adekunle ajasin university, department of plant science and biotechnology, akungba-akoko, nigeria; yetunde.bulu@aaua.edu.ng (corresponding author); optimalrilwan@gmail.com; opeoluwafadayomi@yahoo.com; otito.kekere@aaua.edu.ng abstractabstractabstractabstract in nigeria, chromolaena odorata (l.) r. m. king and h. rob and tithonia rotundifolia (mill.) s. f. blake, are exotic invasive weeds, but a gradual decrease in the population of c. odorata with the rapid invasion of t. rotundifolia is now observed. therefore, this study investigated the adaptive features of each weed in competition, with a further determination of seedling recruitment from the soil seed bank of plots invaded by t. rotundifolia. field sampling was carried out in 10 plots: t. rotundifolia invaded plots and low or uninvaded plots in co-existing with other plants using 10 m x 10 m quadrats. the readily germinable seed species composition and the sorensen index of similarity between the seed bank and their above ground vegetation were determined. competition series experiment was also conducted for the two plants. mariscus alternifolius (227) and oldenlandia corymbosa (358) were the most abundant species in the seed bank while t. rotundifolia recruited 25 individuals. low similarity index existed between the seed bank and the standing vegetation of the invaded and uninvaded plots. t. rotundifolia had improved growth in heteroculture with c. odorata over when in monoculture. however, a reduction in growth of c. odorata occurred when in competitive association with t. rotundifolia compared to when in monoculture. t. rotundifolia had competitive advantage over c. odorata. keywords:keywords:keywords:keywords: invasive plant species; seedling recruitment; soil seed bank introductionintroductionintroductionintroduction in recent times, a lot of attention has been given to the harmful effect of invasive alien plant species on the functioning of ecosystems (zhang et al., 2019). among the mechanisms enhancing successful naturalization and invasion of a new environment by alien plant species, escape from their natural enemies (competitors, herbivores and pathogens) and ability to allocate resources to traits that promote greater competitive advantage, such as size and fecundity, are viewed as characters conveying advantage on them (joshi and vrieling, 2005). aside the formation of a mono-specific stand, the ability to establish a reserve of seeds in the soil seed bank in their new environment can contribute significantly to their successful naturalization and invasion potential (gioria et al., 2021). giving the function of the soil seed bank as source of propagules, genetic diversity https://www.notulaebiologicae.ro/index.php/nsb/index bulu yi et al. (2023). not sci biol 15(1):11380 2 and a mechanism of safeguarding against reproductive failure and unfavorable environmental conditions (larson and funk, 2016; gioria et al., 2021), early seed sets and their storage in the seed bank may give an alien invasive plant a hedge over the native plants. in response to invasion from an alien species, the seed bank may likewise serve a protective role to the native plants by preventing their extinction by functioning as source of seed storage and recruitments for species absent from the aboveground vegetation during the process of revegetation of the invaded community. so far, the effect of invasive plant species on plant communities and biodiversity has only been viewed by investigating changes in the above ground species richness with little information on the impact or significance of the seed bank. therefore, investigating the changes occurring in the seed bank of exotic invasive species and native species in invaded communities is essential to predict the recruitment potential of these species from the seed bank and in understanding the roles of the seed bank in invasion success. chromolaena odorata (l.) r. m. king and h. rob. and tithonia rotundifolia (mill.) s. f. blake are invasive alien weeds from mexico that are widely distributed in the south-western part of nigeria. c. odorata is a perennial herb that grows up to 2.5 m tall in open areas. it is hairy and glandular with leaves that are opposite, triangular to elliptical with serrated edges. the white to pale pink tubular flowers are in panicles of 10 to 35 flowers that form at the ends of branches. t. rotundifolia on the other hand is an erect annual herb with yellow or orange red blooms that contains between 8 and l0 ray florets and between 65 and 90 disk florets in its capitulum (adebowale and olorode, 2005; olorode et al., 2011). c. odorata was reportedly introduced into the country from sri lanka in 1937 and has currently reached an alarming population (uyi et al., 2013; uyi and igbinosa, 2013). on the other hand, t. rotundifolia is gaining increasing attention because of the formation of dense mono-specific stand and an ability to outcompete native vegetation (adebowale and olorode, 2005). the successful establishment and invasion of t. rotundifolia and c. odorata in new environment have been attributed to their fast growth rates, early maturity and large seed sets relative to native plant species. currently, t. rotundifolia is prominently observed in the country with a consequent gradual decrease in the population of c. odorata especially in the south western part of the country where it once dominated (adebowale and olorode, 2005). in this study, we hypothesized that t. rotundifolia outcompete c. odorata and form a large and persistent seed bank which will enhance its establishment and invasiveness in any new environment. this study therefore investigated the relative reciprocal performance of the two invasive species viz t. rotundifolia and c. odorata on each other. the study also examined the characteristics of the seed bank of t. rotundifolia invaded and uninvaded plots. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods vegetation analysis and soil seed bank determination soil seed bank studies were carried out at the screenhouse of the department of plant science and biotechnology (psb), adekunle ajasin university (aaua), nigeria. a recognisance study was first embarked upon in the northern part of ondo state, south western nigeria, to identify areas of vegetation cover with high population of t. rotundifolia in co-existence with c. odorata and other plant species. consequently, a field survey was embarked upon in fallow land vegetation along akure-akungba expressway in ondo state, nigeria (07°16ʼ34.0ʼn; 005°17ʼ48.9e to 07°27ʼ11.6ʼn; 005°43ʼ47.0e). sampling points were systematically placed at every 5 km distance along the expressway from akungba-akoko (07°16ʼ34.0ʼn; 005°17ʼ48.9e) to akure (07°27ʼ11.6ʼn; 005°43ʼ47.0e) between july and december, 2020. at each point, two sampling plots were selected in fallow land vegetation in the area; one dominated by t. rotundifolia (invaded plot) and another with low population of t. rotundifolia in co-existence with c. odorata and other plant species (uninvaded plot). a total of ten plots were identified with six designated as invaded plots (plots 1, 2, 3 4, 5 and 10) and four uninvaded plots (plots 6, 7, 8 and 9). in each sampling plot, a three 10 m x 10 m quadrat was laid out randomly bulu yi et al. (2023). not sci biol 15(1):11380 3 for determination of floristic composition with identification of plants to species level. species whose identities were in doubt were collected and taken to the department of psb herbarium for proper identification. three replicate soil samples were also collected each at depths of 0-15 cm and 15-30 cm, taken to the laboratory of the department of psb and air-dried. the soils were filled into perforated plastic pots and the direct germination method of thompson and grime (1979) was used to assess the readily germinable seed species composition. the relative abundance of plant species or families on the standing vegetation and seed bank was estimated as the percentage each species or families contributed to the total number of individuals present. the sorensen index of similarity between the above ground vegetation and their seed bank was estimated according to chao et al. (2005). plant competition experiment uniform seedlings (relatively equal size) of c. odorata and t. rotundifolia were collected from an open field around the sampling area with known history of clearing four weeks prior to the commencement of the experiment. the seedlings were transplanted into perforated polythene pots (30 cm by 25 cm) filled with top soil collected from the experimental field of the department of psb which was a nutrient-rich sandy-loam soil according to kekere et al. (2020). each pot replicated five times, contained a total of 6 seedlings in monoculture or heteroculture in a competition series experiment viz: t1 = 6 c. odorata: 0 t. rotundifolia; t2 = 5 c. odorata: 1 t. rotundifolia; t3 = 4 c. odorata: 2 t. rotundifolia; t4 = 3 c. odorata: 3 t. rotundifolia; t5 = 2 c. odorata: 4 t. rotundifolia; t6 = 1 c. odorata: 5 t. rotundifolia; and t7 = 0 c. odorata: 6 t. rotundifolia. the pots were arranged in a completely randomized experimental design in the screen house of the department of psb. the experiment lasted for 12 weeks. plant stem girth was measured with vernier calliper at the 2 cm point from the base of the stem while a meter rule was used to measure the height from the soil surface to the apical bud. plant leaves were counted and the leaf area determined with the leaf area meter (li-cor 300 model). at 12 weeks after planting, the plants were carefully uprooted and the primary roots counted with their lengths measured. the shoot lateral branches were also counted. fresh and dry mass of plant parts were measured and the moisture content calculated as ����� ��� � ��� ��� � ����� ��� � �100 while the relative growth rate was accounted for according to hoffmann and poorter (2002) with the formula: �� ����� �� �� � where w1 and w2 are the means of the natural logarithm of transformed plant weight. all data obtained were subjected to one-way anova. statistical means were separated with new duncan multiple range test at 95% level of significance using the statistical package for social sciences (spss software, version 24.0). resultsresultsresultsresults species composition of the standing vegetation forty-four plant species belonging to 15 families were identified in the ten plots of the standing vegetation. the plant habits as shown in table 1, comprised 26 herbs (59.10%), 10 shrubs (22.70%), 7 grasses (15.90%) and 1 climber (2.30%). on the invaded plots, plants belonged to six families (figure 1) contributed plant species to the above ground vegetation with the poaceae having the highest number of individuals (33.33%). each of the fabaceae, amaranthaceae and euphorbiaceae families had a relative abundance (ra) of 16.67%. other families had a ra of 8.33% and below. similarly, fourteen families were enumerated on the uninvaded plots. however, the fabaceae family had the highest ra (21.86%) followed by the poaceae and asteraceae with 12.50% each. meanwhile, the relative abundance of the cucurbitaceae, portulacaceae, euphorbiaceae and others ranged between 3.13% and 9.38%. bulu yi et al. (2023). not sci biol 15(1):11380 4 table 1table 1table 1table 1. plant species composition on the standing vegetation of the study plots s/n species family habit 1 2 3 4 5 6 7 8 9 10 1 aeschynomene indica fabaceae shrub + + 2 amaranthus viridis amaranthaceae herb + 3 andropogon tectorum poaceae grass + + 4 argeratum conyzoides asteraceae herb + 5 blumea aurita asteraceae herb + + 6 brachiaria jubata poaceae grass + 7 calopogonium spp fabaceae herb + + + + + 8 celosia argentea amaranthaceae herb + 9 centrosema pubescens fabaceae herb + + + 10 chamaecrista rotundifolia fabaceae shrub + 11 chromolaena odorata asteraceae shrub + 12 digitaria gayana poaceae grass + 13 cissus quadragullaris vitaceae climber + + 14 corchorus spp malvaceae herb + 15 desimodium spp fabaceae shrub + 16 eleutheranthera ruderalis asteraceae herb + 17 euphorbia alta euphorbiaceae herb + 18 euphorbia heterophylla euphorbiaceae herb + + 19 euphorbia hirta euphorbiaceae herb + 20 fleurya aestuans urticaceae herb + 21 gomphrena celosioides amaranthaceae herb + + 22 indigofera indica fabaceae shrub + 23 luffa cylindrical cucurbitaceae herb + 24 mariscus alterniphorus cyperaceae herb + + 25 melanthera scanden asteraceae herb + 26 mimosa invisa fabaceae shrub + + + 27 mitracapous villosus rubiaceaea herb + 28 oldenlandia corymbosa rubiaceae herb + + 29 panicum maximum poaceae grass + + + + + 30 paspalum scrobiculatum poaceae grass + 31 peperomia pellucida piperaceae herb + + 32 phyallathus amarus euphorbiaceae herb + + 33 physalis angulata solanaceae herb + 34 physalis micrantha solanaceae herb + 35 pupalia lappacea amaranthaceae herb + 36 rhynchelytrum repens poaceae grass + 37 richadia brasilensis rubiaceae herb + 38 senna hirsute fabaceae shrub + 39 sida acuta malvaceae shrub + + 40 spermacoce latifolia rubiaceae herb + + 41 sporobolus pyramidalis poaceae grass + 42 talinum trangularae portulacaceae herb + 43 tithonia rotundifolia asteraceae shrub + + + + + + 44 urena lobata malvaceae shrub + + key + = present; = absent plant species composition of soil seed bank seedling emergence from the soil seed bank at 0 15 cm and 15 – 30 cm depths are shown in figures 2 and 4. at 0-15 cm depth, there were 20 herbs, 10 grasses and 4 shrubs constituting 58.4% herbs, 29.4% grasses bulu yi et al. (2023). not sci biol 15(1):11380 5 and 11.8% shrubs respectively. the plants belonged to 14 families each on invaded and uninvaded plots with poaceae having the highest ra of 15% and 30% on invaded and uninvaded plots respectively. other families ranged between 10% and 5% for invaded plots, and 10% and 3.33 for uninvaded plots. the plant species composition from the soil seed bank at 15-30 cm depth is shown in figure 3. a total of 29 species composing of 18 herbs, 8 grasses and 3 shrubs were found, constituting 62.07%, 27.59% and 10.54% respectively. each of the invaded and uninvaded plots had plant species found in 13 families. poaceae had the highest ra of 28% on uninvaded plots and 35% on invaded plots. on the uninvaded plots, other families had 4-12% ra while 4-10% was recorded on the invaded plots. figure 1.figure 1.figure 1.figure 1. families of plant species in the standing vegetation of the studied plots figure 2.figure 2.figure 2.figure 2. families of plant species recruited from the seed bank at 0-15 cm depth seedling recruitment from the soil seed bank figure 4 shows the relative abundance of seedling recruited from the soil seed bank from the invaded and uninvaded plots at 0-15 cm and 15-30 cm depths. on the uninvaded plots, soils collected at 0-15 cm and 15-30 cm depths had the highest number of seedling recruits from seed bank at plot 7 (43.63 and 34.12 respectively) and lowest at plot 8 (3.73 and 2.56 respectively). on the invaded plots, seedling recruitment from the seed bank peaked at plot 3 (10.58 and 16. 97) and plot 5 (8.29 and 16.97) for soils collected at 0-15 and 1530 cm depths respectively. 0 5 10 15 20 25 30 35 r e la ti v e a b u n d a n c e family invaded uninvaded 0 5 10 15 20 25 30 r e la ti v e a b u n d a n c e family invaded uninvaded bulu yi et al. (2023). not sci biol 15(1):11380 6 figure 3.figure 3.figure 3.figure 3. families of plant species recruited from the seed bank at 15-30 cm depth at both the invaded and uninvaded plots, mariscus alternifolius and oldenlandia corymbosa were the most abundant species in the seed bank. the number of seedlings recruited from the soil seed bank from the invaded and uninvaded plots include o. corymbosa (73 and 513 seedlings), m. alternifolius (128 and 82), eleusine indica (24 and 11), panicum maximum (38 and 2), mitracarpus villosus (33 and 31), celosia leptostachya (80 and 3), and t. rotundifolia (13 and 12) respectively. figure 4figure 4figure 4figure 4. relative abundance of species recruited from the soil seed bank similarity in species composition (standing vegetation and seed banks) table 2 shows the sorensen similarity index between the soil seed bank at different depths (0-15 cm and 15-30 cm) on the invaded and uninvaded plots. high similarity index was recorded between 0-15 cm and 15-30 cm depths on the invaded plots at plot three (0.78), five (0.63) and ten (0.57) but there was no similarity in plot 1 as similarity index was 0.00. in addition, high similarity index ranging from 0.46 to 0.61 was recorded between 0-15 cm and 15-30 cm depths for plant species composition on the uninvaded plots. the similarity index in species composition between soil seed bank at both depths and the standing vegetation gave low values for both invaded and uninvaded plots. 0 5 10 15 20 25 30 35 40 r el at iv e ab u n d an ce r el at iv e ab u n d an ce r el at iv e ab u n d an ce r el at iv e ab u n d an ce family uninvaded invaded 0 5 10 15 20 25 30 35 40 45 50 1 2 3 4 5 6 7 8 9 10 invaded uninvaded invaded r el at iv e ab u n d ac e r el at iv e ab u n d ac e r el at iv e ab u n d ac e r el at iv e ab u n d ac e plots 0-15 cm 15-30 cm bulu yi et al. (2023). not sci biol 15(1):11380 7 table 2table 2table 2table 2.... similarity index in species composition at different soil depths and between standing vegetation plot no plot type similarity index 0-15 cm and 15-30 cm soil depth standing vegetation and 0-15 cm soil depth standing vegetation and 15-30 cm soil depth 1 invaded 0.00 0.29 0.00 2 invaded 0.22 0.20 0.44 3 invaded 0.78 0.17 0.00 4 invaded 0.36 0.40 0.22 5 invaded 0.63 0.00 0.00 6 uninvaded 0.61 0.20 0.07 7 uninvaded 0.54 0.37 0.37 8 uninvaded 0.57 0.40 0.31 9 uninvaded 0.46 0.14 0.00 10 invaded 0.57 0.14 0.00 growth parameters of c. odorata and t. rotundifolia under different levels of interspecific competition the height of c. odorata was significantly reduced by higher density of t. rotundifolia (table 3). for example, the lowest value of 33.00 cm and 33.25 cm were recorded at ratios 1:5 and 2:4 c. odorata and t. rotundifolia respectively. c. odorata however had the highest height (40.08cm) when the mixture of the two plants was of equal number. other growth parameters including stem girth (1.80 cm), number of leaves (16.58) and leaf area (33.04 cm) of c. odorata were highest when grown alone in pots than in competition with t. rotundifolia. the height of t. rotundifolia increased with increase ratio of c. odorata such that the highest height of 106.75 cm was recorded at t. rotundifolia and c. odorata density of ratio 1:5. except for the number of leaves where the highest value of 26.42 was recorded at t. rotundifolia and c. odorata 3:3 ratio, all other parameters including the stem girth (3.80 cm) and the leaf area (220.28 cm2) were highest at 1:5 ratio. in the absence of t. rotundifolia, c. odorata had significantly higher number of roots than when t. rotundifolia was present (table 4). a significant reduction in the number of roots was recorded in c. odorata with increased density of t. rotundifolia in the pots. a similar trend was recorded in the length of the roots of c. odorata where a significant high value of 15. 08 cm was obtained in the absence of t. rotundifolia. significantly higher value of 14.75 was recorded for the number of roots of t. rotundifolia when it occurred in equal density with c. odorata than when it occurred alone. a significant increase in the length of roots of t. rotundifolia (29.25 cm) was recorded when c. odorata and t. rotundifolia occurred at a ratio of 5:1 but this decreased when t. rotundifolia density increased with a corresponding reduction in the density of c. odorata. bulu yi et al. (2023). not sci biol 15(1):11380 8 table 3table 3table 3table 3. growth parameters of t. rotundifolia and c. odorata under different levels of interspecific competition growth param. plant species plant mixture (ratio) 6cr:0tr 5cr:1tr 4cr:2tr 3cr:3tr 2cr:4tr 1cr:5tr 0cr:6tr height (cm) cr 36.44±2.96d 35.05±2.74c 34.43±3.03b 40.08±2.88e 33.25±1.98a 33.00±2.38a 0.00 tr 0.00 106.75±15.32f 53.81±7.13d 68.83±9.22e 40.27±2.10a 49.36±8.62c 45.68±3.83b stem girth cr 1.80±0.05d 1.63±0.05c 1.58±0.04b 1.64±0.15c 1.59±0.08b 1.48±0.09a 0.00 (cm) tr 0.00 3.80±0.20f 3.01±0.15e 2.87±0.15d 2.41±0.09b 2.30±0.09a 2.58±0.14c no of cr 16.58±0.85f 13.20±1.47d 11.06±0.77b 14.25±1.29e 12.00±0.20c 9.00±1.91a 0.00 leaves tr 0.00 21.75±3.12e 21.13±0.55d 26.42±12.21f 17.81±0.34c 16.60±4.87b 10.83±0.24a leaf area cd 33.04±2.81e 23.79±1.88c 23.22±1.08c 27.52±17.56d 17.58±1.70b 16.39±1.71a 0.00 (cm2) tr 0.00 220.28±43.74f 92.70±18.42b 138.81±67.29e 98.63±6.12d 93.52±30.43c 58.43±5.98a where cr= chromolaena odorata, tr= tithonia rotundifolia. values are mean ± standard error for 5 replicates. means with similar alphabets in superscript on the same row are not significantly different from each other at p≥0.05 (duncan multiple range test). the root to shoot ratio (r:s) of c. odorata decreased with increasing density of t. rotundifolia (table 4). r:s of c. odorata was highest (0.20) in the absence of t. rotundifolia (6:0). this value was however not significantly different from the r:s of c. odorata when the ratio of the density of the two plants were 5:1 and 4:2 but differed significantly when the densities of c. odorata to t. rotundifolia were 3:3 (0.13), 2:4 (0.14) and 1:5 (0.15). t. rotundifolia equally had high r:s (0.49) in the absence of c. odorata which differed significantly from all other combination levels of the two plants. at high density of c. odorata, the r:s of t. rotundifolia was significantly low; 5cr:1tr (0.21) and 4cr:2tr (0.19) compared to r:s obtained at 2cr:4tr (0.42). table 4.table 4.table 4.table 4. root growth of c. odorata and t. rotundifolia under different levels of interspecific competition plant mixture (ratio) number of roots root length (cm) root: shoot ratio cr tr cr tr cr tr 6cr:0tr 9.33±1.02d 0.00 15.08±1.02d 0.00 0.20±0.11b 0.00 5cr:1tr 6.00±1.15c 11.90±0.55b 11.31±1.20c 29.25±2.80c 0.18±0.09b 0.21±0.09a 4cr:2tr 4.56±0.16a 11.00±1.08a 10.04±1.47b 23.13±2.73b 0.18±0.09b 0.19±0.08a 3cr:3tr 5.25±0.08b 14.75±0.55c 9.64±1.50b 22.15±1.69b 0.13±0.07a 0.25±0.09a 2cr:4tr 5.13±0.97b 11.56±1.16b 7.53±1.32a 18.93±2.09ab 0.14±0.08a 0.42±0.15bc 1cr:5tr 5.25±1.03b 11.90±0.55b 9.10±1.04b 15.49±0.63a 0.15±0.09a 0.38±0.08b 6tr:0cr 0.00 10.83±0.44a 0.00 16.23±1.51a 0.00 0.49±0.14c key: cr= chromolaena odorata, tr= tithonia rotundifolia. values are mean ± standard error for 5 replicates. means with similar alphabets in superscript on the same column are not significantly different from each other at p≥0.05 (duncan multiple range test). the total fresh mass (6.39 g) and dry mass (1.75 g) of c. odorata were significantly higher in the absence of t. rotundifolia (ratio of 6:0) than in its presence as shown in table 5. the fresh and dry biomass of c. odorata was significantly reduced when grown with t. rotundifolia compared to its monoculture. in contrast, t. rotundifolia in mixed culture with c. odorata (5cr:1tr) had high fresh (117.16 g) and dry (28.20 g) biomass which were significantly higher than the fresh (18.94 g) and dry (4.81 g) biomass of t. rotundifolia grown in monoculture (0cr:6tr). bulu yi et al. (2023). not sci biol 15(1):11380 9 table 5.table 5.table 5.table 5. fresh and dry mass, relative growth rate and moisture content of c. odorata and t. rotundifolia grown under interspecific competition plant mixture (ratio) total fresh mass (g) total dry mass (g) relative growth rate (mg/g) moisture content (%) cr tr cr tr cr tr cr tr 6cr:0tr 6.39±1.12b 0.00 1.75±0.45b 0.00 0.024±0.002b 0.00 72.57±10.98a 0.00 5cr:1tr 3.46±0.13a 117.16±20.24b 0.83±0.09a 28.20±9.00a 0.017±0.001a 0.032±0.004b 76.15±11.01a 75.93±11.00a 4cr:2tr 3.50±0.11a 108.72±22.28b 0.92±0.08a 23.92±8.12a 0.017±0.001a 0.029±0.002b 73.02±10.88a 75.71±10.89a 3cr:3tr 3.78±0.14a 105.28±22.81b 0.97±0.10a 25.41±8.44a 0.018±0.001a 0.031±0.004b 74.94±10.89a 75.89±11.00a 2cr:4tr 4.00±0.19a 25.64±9.00a 0.89±0.06a 5.51±0.33b 0.019±0.001a 0.014±0.001a 77.75±10.99a 78.51±11.47a 1cr:5tr 3.92±0.15a 21.62±9.18a 1.01±0.41a 5.13±0.35b 0.018±0.001a 0.012±0.001a 74.50±9.35a 76.27±11.23a 6tr:0cr 0.00 18.94±8.09a 0.00 4.81±0.22b 0.00 0.010±0.001a 0.00 74.60±10.54a key: cr= chromolaena odorata, tr= tithonia rotundifolia. values are mean ± standard error for 5 replicates. means with similar alphabets in superscript on the same column are not significantly different from each other at p≥0.05 (duncan multiple range test) discussiondiscussiondiscussiondiscussion in the study, herbaceous plants, grasses and few shrubby species were found in the above ground vegetation of the invaded and uninvaded plots. the species richness of the above ground vegetation of the uninvaded plots were higher than those of the invaded plots across the plots covered in the study. the low richness of the weedy flora on the invaded plots can be explained by the fact that the vegetation cover in these plots consisted of monoculture stand of t. rotundifolia which provided vegetation cover with minimal open spaces for the survival of other plant species. in this situation, other weedy species could not have thrived. many families contributed to the weedy species to the flora of the uninvaded plots. the prominent families in this study include the fabaceae, poaceae, asteraceae, rubiaceae, amaranthaceae, malvaceae, and solanaceae in a diminishing order of importance. on the other hand, the invaded plots had a total of six families contributing to the weed flora with the poaceae, fabaceae and euphorbiaceae listed as the major ones. the recruitment data from the seed bank indicated a considerably large number of families making contribution to the seed bank of the invaded plots. while 14 families contributed plant species at the 0-15cm depths, 13 families were recorded at the 15-30 cm depth. the dominant plant family on the standing vegetation i.e., the poaceae also made the highest contribution of seedlings to this soil seed bank. the poaceae also dominated the seed bank of the uninvaded plots. similar observation was reported by de andrade and miranda (2014) that poaceae (64%) are often the most abundant species in the soil seed bank following fire disturbance in savanna ecosystem. the absence of tree species in the standing vegetation of the plots could also account for their absence in the soil seed bank. the high density of herbaceous seeds in the seed bank may be related to species traits where an earlier study had reported that herbaceous seeds remain viable for a longer period of time in the soil than other seed types (ghersa and martinez-ghersa, 2000; wang et al., 2009). the result from this study shows that t. rotundifolia was either not found richly or totally absent in the seed bank of most of the invaded plots, though it was found in numerous populations in the standing vegetation. its absence from the seed bank may be as a result of rapid seedling emergence from the soil which will likely promote its establishment and spread (invasion) in a new environment. this result has shown that formation of a large or persistent seed bank may not be essential for successful invasion in a new environment as pointed out by gioria and pyšek (2016). contrarywise, many dominant plant species in the seed bank were absent in the above ground vegetation. notable examples from the invaded plots are oldenlandia corymbosa and mariscus alternifolius. although species composition in the above-ground and seed bank recruitments are often related such that the seed bank allows plant species that are lost from the vegetation to be re-established (bakker et al., 1996), a low similarity was observed between the species composition of the seed bank and aboveground vegetation on the invaded and uninvaded plots in this study. bulu yi et al. (2023). not sci biol 15(1):11380 10 this study has also shown that the negative effect of competition was more pronounced on c. odorata compared to t. rotundifolia as evident in their growth parameters. a significant reduction in the above and below ground growth parameters of c. odorata was observed while the growth of t. rotundifolia was facilitated when in interspecific competition with c. odorata as against when in monoculture. the possession of higher growth by t. rotundifolia than c. odorata increased its chance of high productivity which may confer advantage on its invasive potential. the ability of t. rotundifolia to produce larger leaf surface area when in competition with c. odorata must have given it better photosynthetic ability for more rapid growth and better canopy layer to suppress growth of c. odorata. the improved root system of t. rotundifolia might have been responsible for better competition for water and nutrient absorption than c. odorata. increased density of the two plants when in mixed culture caused significant reduction on the root/shoot ratio of one another compared to when grown separately in monoculture. the study reported increased in the biomass of t. rotundifolia when in mixed culture contrary to the reduced biomass of c. odorata. at monoculture stand of c. odorata, the growth was better than when in competition with t. rotundifolia. in the presence of high density of c. odorata, the growth t. rotundifolia was significantly improved over when in monoculture stands and/or reduce density of c. odorata. this must have accounted for the gradual decrease in the population of c. odorata with rapid invasion of t. rotundifolia currently being observed in fallow land areas previously occupied by the former. conclusionsconclusionsconclusionsconclusions the presence of t. rotundifolia affected the richness and relative abundance of other plant species in the environment. the general paucity of weedy species contributing to the above flora on the invaded plots can be explained by the direct effect of t. rotundifolia mono clonal stand disrupting the ability of other weedy species to germinate therefore their dominance in the seed bank. it is likely that many plants will be driven to extinction if the invasion by t. rotundifolia continually persist since native species present in the soil seed bank are unable to germinate and maybe exposed to predation or decomposition in the soil. t. rotundifolia has some adaptations for competitive advantage over c. odorata and other plant species in the study area. this was through rapid seedling emergence after seed set; more rapid root growth for better water and nutrient uptake; and formation of canopy cover to suppress the growth of c. odorata. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization: yib and ok; funding acquisition: all authors; data curation: roa and odf; formal analysis: all authors; writing review and editing: yib and ok. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. bulu yi et al. (2023). not sci biol 15(1):11380 11 acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences adebowale a, olorode o (2005). an overview of the invasive potential of tithonia species (asteraceae) in nigeria. science focus 10(3):65-69. bakker jp, poschlod p, strykstra rj, bekker rm, thompson k (1996). seed banks and dispersal: important topics in restoration ecology. acta botanica neerlandica 45:461-490. chao a, chazdon rl, colwell rk, shen tj (2005). a new statistical approach for assessing similarity of species composition with incidence and abundance data. ecology letters 8(2):148-159. https://doi.org/10.1111/j.14610248.2004.00707.x de andrade laz, miranda hs (2014). the dynamics of the soil seed bank after a fire event in a woody savanna in central brazil. plant ecology 215:1199-209. https://doi.org/10.1007/s11258-014-0378-z ghersa cm, martinez-ghersa ma (2000). ecological correlates of weed seed size and persistence in the soil under different tilling systems: implications for weed management. field crops research 67:141-148. https://doi.org/10.1016/s0378-4290(00)00089-7 gioria m, pyšek p (2016) the legacy of plant invasions: changes in the soil seed bank of invaded plant communities. bioscience 66:40-53. https://doi.org/10.1093/biosci/biv165 gioria m, carta a, baskin c, dawson w, essl f, kreft h, … pyšek p (2021). persistent soil seed banks promote naturalisation and invasiveness in flowering plants. ecology letters 24(8):1655-1667. https://doi.org/10.1111/ele.13783 hoffmann w, poorter h (2002). avoiding bias in calculations of relative growth rate. annals of botany 90(1):37-42. https://doi.org/10.1093/aob/mcf140 joshi j, vrieling k (2005). the enemy release and eica hypothesis revisited: incorporating the fundamental difference between specialist and generalist herbivores. ecology letters 8(7):704-714. https://doi.org/10.1111/j.14610248.2005.00769.x kekere o, babatunde af, akinbuwa o (2020). assessment of growth, yield and fruit nutritional composition of tomato and sweet pepper irrigated with abattoir wastewater. science letters 8(2):69-77. larson je, funk jl (2016) regeneration: an overlooked aspect of trait-based plant community assembly models. journal of ecology 104:1284-1298. https://doi.org/10.1111/1365-2745.12613 thompson k, grime jp (1979). seasonal variation in the seed banks of herbaceous species in ten contrasting habitats. journal of ecology 67:898-921. https://doi.org/10.2307/2259220 uyi oo, igbinosa ib (2013) the status of chromolaena odorata and its biocontrol in west africa. in: zachariades c, strathie lw, day md, muniappan r (eds). proceedings of the eighth international workshop on biological control and management of chromolaena odorata and other eupatorieae, nairobi. arc-ppri, pretoria, 1-2 november 2010, pp 86-98 wang j, ren h, yang l, li d, guo q (2009). soil seed banks in four 22-year-old plantations in south china: implications for restoration. forest ecology and management 258:2000-2006. https://doi.org/10.1016/j.foreco.2009.07.049 zhang l, li y, huang j, liu j, liu x (20190. evaluation of the short-term and long-term performance of biological invasion management in the china-myanmar border region. journal of environmental management 240:1-8. https://doi.org/10.1016/j.jenvman.2019.03.061 bulu yi et al. (2023). not sci biol 15(1):11380 12 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11409 nsb das 2023.03.16.docx received: 12 dec 2022. received in revised form: 22 jan 2023. accepted: 07 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania das k and singirikonda s (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11409 doi:10.15835/nsb15111409 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae elemental impact on antibacterial study of hydroalcoholic leaves elemental impact on antibacterial study of hydroalcoholic leaves elemental impact on antibacterial study of hydroalcoholic leaves elemental impact on antibacterial study of hydroalcoholic leaves extract of extract of extract of extract of belosynapsis vivipara belosynapsis vivipara belosynapsis vivipara belosynapsis vivipara kuntal das1*, sravani singirikonda2 1mallige college of pharmacy, department of pharmacognosy, #71, silvepura, chikkabanavara post, bangalore-560090, india; drkkdsd@gmail.com (*corresponding author); 2krupanidhi college of pharmacy, department of pharmacognosy, #12/1, chikkabelandur, carmelaram post, varthur, hobli, bangalore-560035, india; sravanisingirikonda@gmail.com abstractabstractabstractabstract belosynapsis vivipara (dalzell) c.e.c. fisch. (f: commelinaceae) is one of the rare plant species located throughout western ghats regions including karnataka. though the plant was described earlier but traditional uses and scientific evidences are still lacking. the aim of the present study was to identify the elemental content and to determine antibacterial potentiality of belosynapsis vivipara (bv) ethanol leaves extract. shade dried powdered material of bv leaves was estimated for elemental content using atomic absorption spectrophotometer (aas) followed by extraction by ethanol solvent (80%) in soxhlet apparatus for 13 hrs at 45 °c. extract was further used for anti-bacterial screening. in vitro antibacterial studies on the leaf extracts (25, 50 and 100 µg/ml) were carried out on medically important micro-organisms of escherichia coli, klebsiella pneumonia, pseudomonas aeruginosa (gram negative) and staphylococcus aureus, bacillus subtilis and streptococcus pyogenes (gram positive) against standard ampicillin (25 µg/ml). the powdered drug showed the presence of zinc, and copper in high amount less content of iron, whereas very less content of nickel and cobalt and absence of lead, mercury, arsenic and cadmium. the preliminary phytochemical screening revealed the presence of chemical constituents like alkaloids, flavonoids, phyto sterols, saponins and phenolics. furthermore, ethanol leaves extract of bv showed broad spectrum antibacterial efficacy against both gram positive and gram-negative microorganism along with the dose dependency effects. antibacterial activity was correlated with the elements and showed positive correlation. finally, belosynapsis vivipara (dalzell) c.e.c. fisch. leaves were established as an effective source against strong bacterial infection. keywords:keywords:keywords:keywords: antibacterial studies; elemental analysis; belosynapsis vivipara; mic; mbc; microorganisms introductionintroductionintroductionintroduction a significant contributor to chronic illnesses and mortality are bacterial infections. because of their efficacy and effectiveness, antibiotics have been the primary agent to treat for bacterial illnesses. the increasing use of antibiotics has, however, been directly linked to the emergence of bacterial strains that are multidrug resistant, according to a number of studies. in reality, the overuse of antibiotics has recently resulted in the development of super-bacteria that are resistant to almost all antibiotics. the prime classes of antibiotics are https://www.notulaebiologicae.ro/index.php/nsb/index das k and singirikonda s (2023). not sci biol 15(1):11409 2 mainly work under the mechanism of the production of cell walls, translational machinery, and dna replication machinery against the bacterial activities. unfortunately, each of these mechanisms is susceptible to development of bacterial resistance. the mechanisms of resistance include the expression of enzymes that alter or degrade antibiotics, such as beta-lactamases and aminoglycosides (poole, 2002), the modification of cellular components, such as the cell wall in the case of vancomycin resistance and ribosomes in the case of tetracycline resistance (jayaraman, 2009),and the expression of efflux pumps, which offer concurrent resistance against a number of antibiotics (knetsch and koole, 2011).the main cause of endangering public health are those related to antibiotic resistance and as a result microbial infections millions of deaths occur in each year worldwide. various strategies have been proposed in recent years to combat antibiotic resistance. combining failed antibiotics with other molecules has been one of the suggested methods to accomplish this, which appears to restore the desired antibacterial activity (brown, 2015). these compounds could be non-antibiotic medications with possible antibacterial capabilities that could open up new therapeutic possibilities (vandevelde et al., 2016). henceforth, in numerous studies natural products have been used to combat bacterial resistance in this instance, and phytochemicals have demonstrated strong effects (fazly bazzaz et al., 2018; shakeriet al., 2018). plant bioactive compounds can act alone or in combination with antibiotics to produce the synergistic antibacterial activity against a wide range of bacteria (fazly bazzaz et al., 2010; betts and wareham; 2014). thereafter, elements in the plant also play great role in formation of secondary metabolites and other physiological functions. many repots revealed the significant role of elements in physiological function and proper growth of the plant’s vis a vis improvement of the bioactive components in the plant body which further enhances the therapeutic or medicinal properties (kheyrkhah et al., 2018; lo piccolo et al., 2021). based on the concept, the present study was carried out with the herbal plant. there are many herbals reported with antimicrobial efficacy with their important bioactive compounds. due to the resistance towards antibiotics as well as unwanted health issues, the application of synthetic drugs are reduced and people inclined towards natural way of treatment using herbal plants though the responses are delayed but cures from root level and effective against broad spectrum microbes based on various mechanisms viz. potent free radical scavenger, by acts on destruction of cell membranes and cell walls, or blocking of nucleic acid and protein synthesis, or by enhancement of intracellular osmotic pressure (scheepmakeret al., 2019; parham et al., 2020; liang et al., 2022). oflate, belosynapsis vivipara (dalzell) c.e.c. fisch. (f: commelinaceae) is one of the rare plant species in india which distributed mainly in western ghats region especially maharashtra (sangali), karnataka (chikmagalur, coorg, hassan, mysore, shimoga), kerala (wayanad) and tamil nadu (anamalai hills). the leaves are look like spider legs and hence it is also known as spiderwort (kavade et al., 2012). the bioactive compounds and the therapeutic efficacies are not explored so far but currently the plant got immense attention due to its rarity. very recently, an anti-inflammatory and potent antioxidant activities were carried out for the said plant and revealed the presence of plant constituents like anthraquinones derivatives, phenolic nucleus, proteins, saponosides and flavonoids, saponin, alkaloids, steroids, tannins, proteins, coumarins (sasaneet al., 2021). there are many research activities still need to be carried out. based on the phytoconstituents present and antioxidant potentiality of the plant, first time the research extensively was carried out to establish element content in the plant, their impact on phytochemicals vis a vis potent antimicrobial efficacy. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant collection the belosynapsis vivipara (bv) plants were collected from wayanad, kerala, india (latitude: n 11° 42.5668'. longitude: e 76° 5.7322') (figure 1) and were identified and authenticated by dr. p.e. rajshekharan, principal scientist, indian institute of horticultural research, bengaluru, india. the plant was kept as das k and singirikonda s (2023). not sci biol 15(1):11409 3 herbarium in department of pharmacognosy, krupanidhi college of pharmacy, bengaluru (herbarium no: pcog/bv-436/leaves/kcp/2022-2023) for future reference. figure 1.figure 1.figure 1.figure 1. collection of bv leaves from wayanad, kerala, india preparation of plant material for extraction and for elemental content: the leaves were cleaned with running tap water followed by distilled water and spread on newspaper for drying. the leaves were separated from the root part and further dried under shade for 20 days and then powdered by mixer grinder to form coarse powder (sieve no. 44) (figure 2). figure 2.figure 2.figure 2.figure 2. bv leaves after cleaning with water das k and singirikonda s (2023). not sci biol 15(1):11409 4 about 500 g of powdered drug was separated for the extraction and 100 g of dried powder was kept aside for the elemental analysis. soxhlet extraction was performed using 80% ethanol solvent for extraction of the bv leaves for 9 hrs. further, the extracted solvent was filtered using muslin cloth and dried the solvent using rotary flash evaporator at 45 °c to procure viscous semi solid extract. the yield was calculated and kept for further investigation. in other hand, remaining 100 g of dried powder was used for determination of elements especially few heavy metals and some essential elements present in the leaves using atomic absorption spectrophotometer (aas). the powdered leaves were digested with three strong acid mixtures. triacid mixture was prepared by mixed with concentrated nitric acid, concentrated sulphuric acid, and 60% perchloric acid (100: 10: 40). fe, mn, mg, cu, zn, pb, cd, ni, as and cr were determined. 5 g of powdered plant samples were mixed with 15 ml of ternary acid mixture (previously prepared with three concentrated acids) and digested at 180 °c to 200 °c until dense white fumes evolved and formed residue. the residue was further diluted with glass distilled water and made up to definite volume in a volumetric flask. then the solution was ready for the analysis of fe, cu, mn, mg and zn and toxic heavy metals like cd, cr, pb, as and ni. phytochemical screening the crude extract was screened for presence of group of bioactive constituents present in bv extract through various chemical tests. all the tests were performed as per the standard method described in the previous literature (devi et al., 2014; das et al., 2018). antimicrobial activity microorganism used: escherichia coli atcc 8739, klebsiella pneumonia atcc43816, pseudomonas aeruginosa atcc 25619 (gram negative) and staphylococcus aureus atcc 29726, bacillus subtilis atcc 6633 and streptococcus pyogenes atcc 13813 (gram positive) were used as study organisms for the present investigation. all the organisms were procured from department of microbiology, bangalore university, bengaluru, india. broad spectrum antibiotic ampicillin (25 µg/ml) was used as standard and compared the result against the selected antibiotic in this study (das et al., 2011). all the organisms were maintained by sub cultured on nutrient agar medium in department of pharmaceutical microbiology, krupanidhi college of pharmacy, bengaluru, india. ampicillin stock was prepared as 25 µg/ml (w/v) concentration in sterile distilled water and used in the present study. minimum inhibitory concentration (mic) determination: the bacterium was injected into muller hinton culture medium for a 24-hour culture, and the result was a colony (hi media lab, india). extract concentrations of 100, 50, and 25 µg/ml were obtained for each of the two distinct plants that were gathered from various zones by adding 1.0 ml of the extract solution at concentrations of 200 g/ml to 1 ml of nutrient broth (das, 2014). 9 ml of nutrient broth containing standardized test organisms of bacterial cells were mixed with 0.1 ml of each concentration, and 0.5 mcfarland turbidity standard (1.0 x 108 cfu/ml) was inoculated into each test tube using the serial dilution procedure (das, 2014). after 24 hours of incubation at 37 degrees c, growth was found in the tubes, and depending on the mic value, a further dose was determined for the said activity. antibacterial activity of the bv extract was assessed by the agar well diffusion method (hussein et al., 2020) where each isolated microbe was subculture on the recommended specific media for each microorganism at 35-37 °c for 25 h. 100 mg of the bv extract were sterilized by filtration through a membrane filter. 6 mm discs were impregnated with sterile cock borer and 30 µl of bv extract was placed in the wells of agar plates inoculated with microbial culture (after dilution) including standard and then incubated all the plates 37 °c for 16 hours. the zone of inhibition (mm) was measured by using sliding calipers from the back of the inverted petri dishes (kaur et al., 2013). triplicate readings were taken to minimize the error. das k and singirikonda s (2023). not sci biol 15(1):11409 5 correlation study antimicrobial study was further correlated with the metal ion contents and determined the effect of metals on the activity. statistical analysis m-stat software was used for the elemental content in the bv leaves extract by taken mean values of three replicated set of data. further, antibacterial activities were expressed as the mean ± standard error of mean (sem) where values of ***p< 0.01 and * p < 0.05 were considered statistically significant. the plots for mic and antimicrobial activity determination were tabulated using microsoft excel and graph pad prism 5, respectively. results results results results bv leaves were extracted using ethanol solvent and yielded about 53.72 g of the extract. the per cent yield was calculated as 10.74 per cent. in case of elemental content, it was observed that leaves content mg, mn, fe, cu and zn with satisfactory amount. table-2, indicated that content of zn, fe and cu were higher whereas, as, cr and pb were not detected as specified condition (table-1). in the present study atomic absorption spectrophotometer (aas) was used with selected various wavelength for the detection of elements in the bv plant (tables 1 and 2). table 1.table 1.table 1.table 1. conditions of aas for bv leaves analysis elements wavelength (nm) intensity (ma) slit used (nm) mn 279.5 5 0.2 mg 285.2 4 0.5 fe 248.3 5 0.2 zn 213.9 5 1.0 cu 324.8 4 0.5 ni 232.0 4 0.2 cd 228.8 4 0.5 as 193.7 31 2.0 cr 357.9 7 0.2 pb 217.3 10 1.0 table 2.table 2.table 2.table 2. concentration of elements (mg/kg) in ethanolic leaves extract of bv plant plant extract content of hcontent of hcontent of hcontent of heavyeavyeavyeavy metals (mg/ kg)metals (mg/ kg)metals (mg/ kg)metals (mg/ kg) fe ni cu zn cd as cr pb mg mn bv leaves ethanol extract 2.08 ± 0.21 0.02 ± 0.14 3.73 ± 0.16 4.04 ± 0.11 0.01 ± 0.23 nd nd nd 3.21± 0.21 2.03± 0.40 mean ± sem (n = 3); nd = not detected simultaneously, various chemical tests were performed for the bv ethanol plant extract and revealed the presence of alkaloids, flavonoids, phyto sterols, saponins and phenolics (table 3). further, confirmed with the tlc for the extract based on the lcms study, showed the presence of particular alkaloids namely, betanine, betanidin, flavonoid like quercetin, and plant sterol like beta sitosterol. das k and singirikonda s (2023). not sci biol 15(1):11409 6 table 3.table 3.table 3.table 3. phytochemical screening of bv leaves extract chemical testschemical testschemical testschemical tests ethanol bv leaves extractethanol bv leaves extractethanol bv leaves extractethanol bv leaves extract alkaloids ++ glycoside + flavonoids ++ phenolics ++ tannins + steroidal compounds + phytosterols ++ saponins ++ proteins + resins - terpenoids + gum - (++) = prominently present; (+) = present; (--) = absent antimicrobial activity further investigated with the extract of bv plant and showed broad spectrum antimicrobial efficacy when compared with the standard antibiotics. initially, mic flowed by mbc were determined. the extract's mic value was established as the lowest concentration that, after 48 hours at 37 °c, totally inhibited bacterial growth. a quantity of liquid (5 µl) was removed from each plate well that showed no growth in order to determine the mbc, which was subsequently incubated at 37 °c for 24 hours. mbc was determined to be the lowest concentration at which there was no discernible bacterial growth during subculturing. mic determination: the result showed that the mic values varied from 10.6 to 24.2 μg/ml against all three positive organisms and 12.4 to 26.4 μg/ml against all three negative organisms for the ethanol bv leaves extract (figure 3). the mic value 12.4 μg/ml was obtained with extract against escherichia coli, and 10.6 μg/ml against staphylococcus aureus. mbc determination: minimum bactericidal effects were exhibited with various degrees for all the organisms. the lowest mbc (2.41 μg/ml and 2.33 μg/ml respectively) were obtained in this study with staphylococcus aureus, and escherichia coli respectively. figure 3.figure 3.figure 3.figure 3. mic and mbc of ethanol bv leaves extract ec = escherichia coli, kp = klebsiella pneumonia, pa = pseudomonas aeruginosa; sa = staphylococcus aureus, bs = bacillus subtilis, sp =streptococcus pyogenes mic 0 10 20 30 sa bs sp ec kp pa gram positive gram negative mic mbc µ g /m l das k and singirikonda s (2023). not sci biol 15(1):11409 7 bv ethanol leaves extract was studied for antibacterial activity and resulted significant dose dependent inhibition (p<0.01) against all the microorganisms and as compared standard ampicillin the results were lesser. ethanol leaves extract showed maximum inhibition against gram positive organisms than gram negative organisms. maximum inhibition showed for gram positive organisms straphyloccus aureus (18.5±0.04**) followed by bacillus subtilis (14.3±0.01**) and lowest activity showed for gram negative organisms escherichia coli (11.3±0.12*) followed by pseudomonas aeruginosa (9.7±0.31*) at the concentration of 100 µl/ml (figure 4; table 4). table 4.table 4.table 4.table 4. antimicrobial activity of ethanol bv leaves extract against various organisms plant plant plant plant extractextractextractextract conc conc conc conc (µg/ml)(µg/ml)(µg/ml)(µg/ml) zone of inhibition (mm)zone of inhibition (mm)zone of inhibition (mm)zone of inhibition (mm) sa bs sp ec kp pa bv extractbv extractbv extractbv extract 25252525 14.21±0.11** 12.7± 0.31** 9.21± 0.24** 9.36± 0.33** 7.32± 1.32** 8.62±0.36** 50505050 16.23±0.22** 13.4±0.10** 10.33±0.51** 10.3±0.52** 8.52± 1.10** 9.33±0.05** 100100100100 18.5±0.04** 14.3±0.01** 12.08±0.10** 11.3±0.20** 8.73± 2.11** 9.7±0.33** standardstandardstandardstandard 25252525 22.3±0.11 statistically significant: **p<0.01; *p<0.05 (one way anova); (n =3) ec = escherichia coli, kp = klebsiella pneumonia, pa = pseudomonas aeruginosa; sa = staphylococcus aureus, bs = bacillus subtilis, sp = streptococcus pyogenes st d s a b s s p e c p a k p 0 5 10 15 20 25 various microorganisms z o n e o f in h ib it io n ( m m ) figure 4.figure 4.figure 4.figure 4. antimicrobial activity of the bv ethanol leaves at conc. 100 µg/ml ec = escherichia coli, kp = klebsiella pneumonia, pa = pseudomonas aeruginosa; sa = staphylococcus aureus, bs = bacillus subtilis, sp = streptococcus pyogenes data represented three times n =3; (mean ± sem). correlation study of the metal ions with the plant extract and the antimicrobial activity was carried out and the positive correlation was observed and revealed the impact of elements on antimicrobial activity (table 5). thereafter, it was recorded that non-essential heavy metals were not detected which indicated the bv plant was safe and content no toxic metals. das k and singirikonda s (2023). not sci biol 15(1):11409 8 table 5.table 5.table 5.table 5. correlation study of elements with the antimicrobial activity fe mn mg zn cu sa bs sp ec kp pa fe 1 mn 0.981* 1 mg 0.980* 0.990** 1 zn 0.998** 0.988* 0.990** 1 cu 0.988* 0.962 0.983* 0.990** 1 sa 0.920 0.886 0.939 0.930 0.970 1 bs 0.998** 0.988* 0.990** 1.00*** 0.990** 0.930 1 sp 0.810 0.789 0.868 0.830 0.891 0.971 0.830 1 ec 0.981* 1.00*** 0.990** 0.988* 0.962 0.886 0.988* 0.789 1 kp 0.951 0.906 0.873 0.932 0.902 0.784 0.932 0.614 0.906 1 pa 0.996** 0.966* 0.976 0.994** 0.996** 0.947 0.994** 0.847 0.966* 0.938 1 statistically significant: ***p<0.001; **p<0.01; *p<0.05 (one way anova) ec = escherichia coli, kp = klebsiella pneumonia, pa = pseudomonas aeruginosa; sa = staphylococcus aureus, bs = bacillus subtilis, sp = streptococcus discussiondiscussiondiscussiondiscussion in the present study ethanol was used as the solvent is nontoxic with polarity index value of 5.2 (abarcavargas et al., 2016). thereafter, ethanol has high dielectric constant of 24.55 which capable to extract maximum bioactive components of the plant such as polyphenols, tannins, flavonols, terpenoids, and alkaloids (azmir et al., 2013). therefore, in the present study ethanol was used as solvent. detection and analysis of elements through atomic absorption spectrophotometer is most essential because the elements are responsible for the production, accumulation and enhancement of the bioactive component in plants (aziz et al., 2016). very few reports explained about role of elements on plant chemicals enhancement (singh et al., 2022) and productivity and thereafter, it was also required to know about the nonessential heavy metal content in the leaves because the plants were collected from the forest zone and there was chanced for contamination by the climatic conditions and variation of constituents. aas is a flexible, non-destructive analytical technology that is frequently used to identify minor and trace components in intricate biological materials (el-mesery et al., 2019). the results were compared with the standard limits of the nonessential heavy metals as per who guidelines, and was found to be within safe limits. various chemical constituents like zn, fe and cu were present prominently may be due to the presence of metals like mn, zn, cu and fe content in plant. previous literature reported that zn, cu and other metals influence the enhanced bioactive compounds in plant part (das and tribedi, 2015) and the same result reported in the present experiment. it was reported that the value of the lowest mbc obtained was not more four times higher than that of mic’s on the corresponding pathogens, it seems possible that the sample tested was possessed the antimicrobial activity (kowalska-krochmal and dudek-wicher, 2021). this value is not more than four times greater than that of the mic’s of the corresponding microorganisms. this data was supported by the previous literatures (meyer and lall, 2007; kuete et al., 2008). conclusionsconclusionsconclusionsconclusions microbiological resistance is becoming a bigger issue, and it is unclear how antimicrobial medications will be used in the future. as a result, the study also revealed that plant-based extracts might suppress bacteria. the result of the present investigation revealed the ethanol extract of bv leaves is significantly active against das k and singirikonda s (2023). not sci biol 15(1):11409 9 three gram positive and three-gram negative bacterial strains and suggested that bv leaves can be used for treatment of infection caused by broad spectrum microorganisms. further, content of elements were determined and resulted essential elements such as fe, mg, mn, cu and zn were present whereas toxic nonessential elements vis. cr, cd, ni, co, pb and as were not detected or may be present in very low limits which indicated the leaves of bv is safe for further applications. thereafter, when metal ions were correlated with the said activity, it showed positive correlation and established potential role in accumulation and availability of the bioactive constituents which causes potent antimicrobial activity. some of the observations have helped in identifying the active principle responsible for such said activity and first time reported as a developing plant source as drug for the therapeutic use in human beings. finally, in conclusion, the result clearly elaborated that belosynapsis vivipara has great potential to invade human pathogenic bacteria. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions kd has designed the work and written the whole manuscript. ss was performing the antimicrobial test along with design of graphs. both authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences abarca-vargas r, malacara cfp, petricevich vl (2016). characterization of chemical compounds with antioxidant and cytotoxic activities in bougainvillea x buttianaholttum and standl, (var. rose) extracts. antioxidants 5:45. https://doi.org/10.3390/antiox5040045 aziz s, saha k, sultana n, nur hp, ahsan ma, ahmed s, hossain mk (2016). comparative studies of elemental composition in leaves and flowers of catharanthus roseus growing in bangladesh. asian pacific journal of tropical biomedicine 6(1):50-54. https://doi.org/10.1016/j.apjtb.2015.10.003 azmir j, zaidul ism, rahman mm, sharif km, mohamed a, sahena f, omar akm (2013). techniques for extraction of bioactive compounds from plant materials: a review. journal of food engineering 117(4):426-436. https://doi.org/10.1016/j.jfoodeng.2013.01.014 betts jw, wareham dw. 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(2019). sense and nonsense of the secondary metabolites data requirements in the eu for beneficial microbial control agents. biology control 136:104005. https://doi.org/10.1016/j.biocontrol.2019.104005 shakeri a, sharifi mj, fazly bazzaz bs, emami a, soheili v, sahebkar a, asili j (2018). bioautography detection of antimicrobial compounds from the essential oil of salvia pachystachys. current bioactive compounds 14(1):8085. https://doi.org/10.2174/1573407212666161014132503 singh sk, wu x, shao c, zhang h (2022). microbial enhancement of plant nutrient acquisition. stress biology 2(3):114. https://doi.org/10.1007/s44154-021-00027-w tsuji bt, yang jc, forrest a, kelchlin pa, smith pf (2008). in vitro pharmacodynamics of novel rifamycin abi-0043 against staphylococcus aureus. journal of antimicrobial chemotherapy 62(1):156-160. https://doi.org/10.1093/jac/dkn133 vandevelde nm, tulkens pm, van bambeke f (2016). modulating antibiotic activity towards respiratory bacterial pathogens by co-medications: a multi-target approach. drug discovery today 21(7):1114-1129. https://doi.org/10.1016/j.drudis.2016.04.001 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11466 nsb nemiche 2023.06.19.docx received: 05 feb 2023. received in revised form: 10 mar 2023. accepted: 08 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania nemiche s et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11466 doi:10.15835/nsb15211466 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae effect of effect of effect of effect of ficus carica ficus carica ficus carica ficus carica extract on hematological parameters and extract on hematological parameters and extract on hematological parameters and extract on hematological parameters and antioxidant defense system in erythrocytes of antioxidant defense system in erythrocytes of antioxidant defense system in erythrocytes of antioxidant defense system in erythrocytes of aaaalbino rats exposed to nickel chloridelbino rats exposed to nickel chloridelbino rats exposed to nickel chloridelbino rats exposed to nickel chloride souhila nemiche1*, nadia ait hamadouche1, saïd nemmiche2 1university of oran 1 ahmed ben bella, faculty of nature and life sciences, department of biology, oran 31000, algeria; sbiochimie@outlook.fr (*corresponding author); naithamadouche@live.fr 2university of mostaganem, faculty of nature and life sciences, department of biology, mostaganem 27000, algeria; snemiche@hotmail.com abstractabstractabstractabstract heavy metals including nickel have adverse effects on hematological system and red blood cells antioxidant defense. the purpose of the study is to examine the possible corrective effect of fig (ficus carica l.) extract (fce) on hematological parameters and antioxidant enzyme activities in red blood cells of rats exposed to sub-lethal concentration of nickel. male wistar rats were exposed to nickel chloride (10 mg/kg) and then treated or no with fce (350 mg/kg) for 4-weeks. the intoxication induces alteration of haematological parameters and enhances blood haemolysis, generates oxidative stress induced decrease in the antioxidant enzyme activities and depletion in reduced glutathione levels in intoxicated group compared to the control group. the oral administration of fce increase significantly hemoglobin levels ((((hb), red blood cells count (rbc), hematocrit (ht) and decreases white blood cells counts (wbc), and platelets in f. carica treated groups, and also the extract increase the gsh-px and sod antioxidant activity, gsh reserves in red blood cells, and reduces lipids peroxidation. the present study concludes that fig fruits may exhibit potent antioxidant potential for erythrocytes and have a positive effect on hematological system. keywords:keywords:keywords:keywords: erythrocytes; fig; haematology; nickel chloride; oxidative stress introductionintroductionintroductionintroduction humans are exposed continuously or accidentally to natural and/or synthetic chemical pollutants that may interfere with their overall health status. nickel (ni) is a known heavy metal potentially toxic that can affect multiple organs in living systems (song et al., 2017). humans are exposed to ni via occupational and environmental exposure such as refining, mining, stainless steel industries, and battery manufacturing (schmidt et al., 2016). other environmental sources at low concentrations of nickel include tobacco (harasim and filipek, 2015) cooking utensils made of stainless steel and dental or orthopedic implants. nickel absorption, distribution and removal are influenced by factors such as the route of exposure, the physical form of the https://www.notulaebiologicae.ro/index.php/nsb/index nemiche s et al. (2023). not sci biol 15(2):11466 2 material (solid or powder) and the aerodynamic size of the nickel particles (fay et al., 2005). according to buxton et al. (2019) the distribution and removal of nickel depend on the route of administration and its binding to proteins. inhaled nickel is distributed mainly to the respiratory tract (lungs, nasal sinuses) and then to the kidneys (dunnick et al., 1989). orally absorbed nickel is distributed to the kidneys and then to the liver, brain, and heart (finke et al., 2015). the immediate toxic effects of ni exposure are the development of allergies manifested by contact dermatitis, respiratory tract irritation and neurological disorders (song et al., 2017). several studies have already indicated that a long-term exposure to nickel causes various toxic effects on different organs such as the lungs, liver, kidneys and cardiovascular system (buxton et al., 2019). the toxic effects of nickel may be prevented by some exogenous supplementation of antioxidant compounds. in recent years, scientific investigations have been a growing interest in the potential use of medicinal plant extracts to cure and prevent disease and protection against the effects of various xenobiotics (hao et al., 2015; kopeć et al., 2016). ficus carica l., also known as figs, is a fruit rich in natural bioactive compounds (nemiche et al., 2022; rodríguez-solana et al., 2018) with powerful antioxidant. this potential is related to the high presence of secondary metabolites. figs are considered as an important source of calcium and fiber, rich in sugar, iron, minerals, vitamins and amino acids. in addition, it is one of the highest sources of bioactive compounds such as phenolic acids, flavonoids, flavonols and flavanones, hydroxycinnamic acids, anthocyanins, and proanthocyanidins (pereira et al., 2017). f. carica l. has been incorporated into western pharmacopoeias and therapeutic guidelines for medicinal plants (barolo et al., 2014) for a long time. it has been reported that various parts (fruit, roots and leaves) and extracts of the fig tree can be used in the medical treatment of several diseases (menichini et al., 2012). fruits are also considered as a good laxative, expectorant and diuretic, and can be used against bleeding (solomon et al., 2006) or as a dietary supplement for diabetics (veberic et al., 2008). our study was aimed to evaluate the potential toxic effects of nickel chloride on blood components, mainly red blood cells (rbcs) and the possible corrective or protective effect of figs extract against nickel injuries. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant material fig (ficus carica l.) fruits ‘white’ variety was collected in a coastal region of western oran (algeria). they were identified taxonomically and authenticated by prof. hadjadj-aoul at the herbarium of botany directorate in ahmed ben-bella oran 1 university (voucher specimen n° lb 0695). the fruits were dried and ground into powder. the oliveira et al. (2009) method was used to prepare fig fruit extract (fce) as follows: 50 g of powder fig were boiled for 15 minutes, filtered, and then lyophilized (christ-alpha 2-4 lsc d-37520). animals a total of 48 male albino wistar rats weighing (165 ± 5 g) were used for this study. the animals were housed under standard conditions photoperiod (12-h light/dark cycle), temperature (23 ± 1 °c) and humidity (50 ± 15%), and maintained with free access to standard diet and water ad libitum. experimental design the rats were randomly divided into two lots: control lot (24 rats) considered as negative control received an intraperitonial (i.p) injection of 0.9 % saline solution, and intoxicated lot (24 rats) received 10 mg nicl2 kg-1 body weight (bw) by i.p injection three times a week. after 1 month of the experiment, 8 rats of each lot which represents respectively: nemiche s et al. (2023). not sci biol 15(2):11466 3 “group c1group c1group c1group c1” from control lot and “group ni1group ni1group ni1group ni1” from intoxicated lot were sacrificed. the rats remaining were divided into 4 groups of 8 rats each as follow: two groups from control lot: • group “c2”:group “c2”:group “c2”:group “c2”: served as a negative control group. • group “c+fc”:group “c+fc”:group “c+fc”:group “c+fc”: served as positive control and received 350 mg kg-1 fig “ficus carica” extract (fce) by gavage for 4 weeks. -two groups from intoxicated lot: • group “ni2”: group “ni2”: group “ni2”: group “ni2”: were given free access to water and food for 4 weeks after stopping the intoxication; • group “ni+fc”: group “ni+fc”: group “ni+fc”: group “ni+fc”: were treated with 350 mg fce kg-1 bw for 4 weeks after stopping the intoxication too. blood collection and hematological analysis the blood samples were stored in anticoagulant bottles containing edta (ethylene diaminetetra-acetic acid-na2) (sigma, st. louis, mo, usa) and used subsequently for hematological analysis. red blood cells count (rbc x 106 mm-3), white blood cells count (wbc x 10 mm-3), hemoglobin concentration (hb, g.dl-1), haematocrit (ht %), mean total platelet count (tpl %), mean corpuscular hemoglobin (mch), mean corpuscular volume (mcv) and mean corpuscular hemoglobin concentration (mchc) were measured by using fully automated hematology analyzer (biobase bk6200). erythrocytes separation the erythrocyte suspension was prepared as mentioned by beutler et al. (2011) for the evaluation of gsh, mda level, sod, gpx, and cat activities. lipid peroxidation determination red blood cells' (rbc) lipid peroxidation was analyzed in accordance with brown and duthie's method (1997). briefly, 100 µl of rbc were diluted in 900 µl of pbs buffer, added to 100 µl of h2o2 (1.15%) and then incubated for 60 min at 37 °c. a solution of 1 ml of 20% trichloroacetic acid (tca) was added. after centrifuging at 2,000 x g for 10 min, the supernatant sample was mixed with 100 µl of 2% butylated hydroxy toluene (bht). rbcs antioxidant capacity and assessment of haemolysis antioxidant activity for catalase was estimated at 240 nm by observing the decomposition of h2o2 and a decrease in absorbance as described by aebi (1984). the superoxide-dismutase (sod) activity was determined following the procedure described by marklund and marklund (1974). gsh-px (ec 1.11.1.9) activity in rbcs was assessed as described by rotruck et al. (1973). the concentration of gsh was measured according to the method of ellman (1959) and haemolysis was evaluated following the method of khan et al. (2015). statistical analysis the data were expressed as the mean ± sem in each group and analyzed by one-way analysis of variance (anova) followed by tukey's post-hoc test. all statistical analysis was carried out with spss statistical software (ver.23.0, spss inc., chicago, il, usa). the level of significance was set at p < 0.05. nemiche s et al. (2023). not sci biol 15(2):11466 4 results results results results hematological parameters the effect of nickel and fce on hematological parameters in different experimental groups is represented in table 1. nickel causes a significant (p<0.001) decrease of rbc, hb, and ht by -28%, -26%, 26% respectively in the ni1 group compared to control c1, in parallel an increase in the rate of wbc and platelet by +71%, +48% respectively was also observed in this group. the administration of the fig extract after stopping nickel intoxication to the ni+fc rat group shows a significant increase by +33%, +34%, +9% and 12% in rbc, hb, ht and mcv level respectively and decrease by -40% in platelet counts compared to the ni2 group. no significant variation was observed at the other parameters level of wbc, mch, and mchc. table 1.table 1.table 1.table 1. effect of nicl2 and fce on hematological parameters of different experimental groups parameters experimental groups c1 c2 c+fc ni1 ni2 ni+fc rbc (x 106 mm-3) 7.58 ± 0.14 8.38 ± 0.26 8.72 ± 0.05 5.50 ± 0.22*** 6.42 ± 0.07 8.58 ± 0.06### hb (g/dl) 12.72 ± 0.24 14.10±0.15 13.80±0.80 9.35±0.25*** 10.82±0.17 14.55±0.05### ht (%) 37.44 ± 0.74 41.26±0.56 43.15±0.68 27.53±0.12*** 41.85±0.39 45.72±0.43## mcv (fl) 49.83 ± 0.16 49.16±0.40 47.50±0.50 48.66±0.33 47.50±0.50 53.50±0.50### mch (pg) 32.08 ± 0.90 34.13±0.17 34.65±0.35 33.50±0.30 33.74±0.10 33.51±0.27 mchc (g/dl) 16.85 ± 0.33 16.94±0.37 16.55±0.05 16.40±0.28 16.42±0.16 16.95±0.23 wbc(x103mm-3) 3.04 ± 0.41 2.98±0.19 2.75±0.47 5.20±0.37** 4.12±0.09 3.02±0.07 platelet (103 cell/μl 684 ± 31 694±17.03 643.5±11.50 1012.5±16.50*** 791±47 598.33±10.91## each value is mean ± sem, n = 8 (tukey test, p < 0.05). the * depicts comparison with group “c1”; € depicts comparison with group “c2” (€p < 0.05, €€p < 0.01, €€€ p < 0.001); #depicts comparison with group “ni2” (#p < 0.05, ##p < 0.01, ### p < 0.001) rbcs antioxidant enzymes activities the level of erythrocyte antioxidant activity is represented in table 2. in the ni1 intoxicated group, the catalase activity shows a twofold-increase compared to control c1 group, while the activity of the gsh-px and sod recognize a significant decrease of 44% and 48% respectively in this group compared to control c1. after 1 month of stopping the intoxication and fce administration, a significant increase of +56% in sod activity is observed with a twofold-increase in gsh-px activity in rats of the ni+fc group treated with fce in comparison to rats of the ni2 group. also, in the control treated group we observe two-fold-increases in sod activity and significant enhancements of +58% in gsh-px activity in rate of c+fc group compared to control c1 group . tabletabletabletable 2222. effect of nicl2 and fce on antioxidant enzymes activities in rbcs of different experimental groups parameters experimental groups c1 c2 c+fc ni1 ni2 ni+fc cat (pmol/min/ml) 419.03 ± 39.4 602.43 ± 65.6 516.7 ± 13.3 856 ± 42.8*** 499.63± 10.2 417.14 ± 12.42 sod (u/ml) 1679 ±116 1869 ± 17.5 4178± 129€€€ 928 ± 29** 1156 ± 35.6 1807 ± 33.5# gsh-px (nmol/min/ml) 25.09±0.02 32.73±1.74 51.93 ±0.68€€ 10.38 ± 0.26* 14.92 ± 2.56 36.83 ± 5.74## each value is mean ± sem, n = 8 (tukey test, p < 0.05). the * depicts comparison with group “c1”; € depicts comparison with group “c2” (€p < 0.05, €€p < 0.01, €€€ p < 0.001); #depicts comparison with group “ni2” (#p < 0.05, ##p < 0.01, ### p < 0.001). nemiche s et al. (2023). not sci biol 15(2):11466 5 rbcs malondialdehyde levels the erythrocyte mda levels of different experimental groups are represented in figure 1. nickel induced a lipid peroxidation justified by the three-fold increase of the erythrocytes mda level in the rats of the ni1 group in contrast to the c1 group. however, we notice a significant decrease by 74% in the ni+fc group after fce administration in comparison to ni2 group. figure 1.figure 1.figure 1.figure 1. effect of nicl2 and fce extract on mda level of different experimental groups each value is mean ± sem, n = 8 (tukey test, p < 0.05). the *depicts comparison with group “c1”; #depicts comparison with group “ni2” (#p < 0.05, ##p < 0.01, ###p < 0.001). rbcs reduced glutathione (gsh) levels the reduced glutathione concentration in the animals of the different experimental groups is shown in figure 2. nickel induced a significant decrease by 49% in the erythrocyte glutathione levels in the ni1 group rats compared to the control c1. no significant variation was observed after fce administration in the erythrocyte glutathione reserves in the rats of the ni+fc group against the ni2 group figure 2.figure 2.figure 2.figure 2. the effect of nicl2 and fce on gsh levels of different experimental groups each value is mean ± sem, n = 8 (tukey test, p < 0.05). the * depicts comparison with group “c1 haemolyse the percentage haemolysis induced by nickel and fig extract in the different experimental groups is represented in the figure 3. in wistar rats, nickel chloride injection caused a significant increase (+78%) in 0 1 2 3 4 5 6 7 8 9 c1 c2 c+fc ni1 ni2 ni+fc m d a ( n m o l/ m l ) m d a ( n m o l/ m l ) m d a ( n m o l/ m l ) m d a ( n m o l/ m l ) experimental groupsexperimental groupsexperimental groupsexperimental groups *** ### 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 c1 c2 c+fc ni1 ni2 ni+fc g s h ( m m o l/ m l) experimental groups ** nemiche s et al. (2023). not sci biol 15(2):11466 6 haemolysis levels in ni1 group compared to control c1. in contrast, the treatment with figs extracts after stopping nickel intoxication induced a significant decrease (-24%) in haemolysis in rats of ni+fc group compared to the ni2 group. figure 3.figure 3.figure 3.figure 3. effect of nicl2 and fce on haemolysis (%) of different experimental groups each value is mean ± sem, n=8 (tukey test, p < 0.05). the * depicts comparison with group “c1”; #depicts comparison with group “ni2” (#p < 0.05, ##p < 0.01, ###p< 0.001). discussiondiscussiondiscussiondiscussion blood plays a decisive role in the regulation of vital processes. hence the need to keep its composition relatively constant and must also has the capacity to modify it in the most extreme stress situations due to the aggressions of various xenobiotics (schulz et al., 2012). heavy metals are toxic or carcinogenic elements in their nature and are considered as a danger to human health and the environment. the nickel has adversely affected peripheral white blood cells and red blood cells (méndez-gomez et al., 2008). evaluation of the hematological and oxidative profile proved to be a sensitive index for the study of the blood toxicity process of nickel. the objective of the current study is to investigate the sub-chronic toxicity of nickel chloride on hematological parameters of adult wistar rats and the possible corrective effects of fce as an antioxidant agent to reduce levels of the rbc toxicity. the present data showed that after 30 days of ni exposure, rats exposed exhibited a significant reduction in their rbc count, hb concentrations, ht values and increases in the rate of wbc and platelet count in ni1 group compared to control c1 group. adjroud (2013) reported that sub-cutaneous injection of 25 mg nicl2 kg-1 induced a significant decrease in the erythrocyte counts, haematocrit values, and hemoglobin concentration. significant variations in different blood parameters were also observed by ololade and oginni (2010) in the experimental fish after exposure to 4-12 mg nickel sulfate kg-1. nickel accumulation in the blood may be the cause of the decline in hematological parameters (demîr et al., 2005) and seemed to be associated with decreased iron absorption in wistar rats (cempel and janicka, 2002). the increase in white blood cells (wbc) count after nickel intoxication may be due to the inflammatory response as a defense mechanism. after one month of nickel stopping exposure, a slight significant improvement in hematological parameters in ni2 group compared to ni1 group has been noticed, but the fce administration showed an important significant increase in rbc counts, hb concentrations, ht values with a decrease in the rate of wbc and platelet count. these results are in agreement with results of said et al. (2017) who investigate the possible effect of f. carica fruit extracts against lead toxicity on hematological parameters of the fish nile tilapia (oreochromis niloticus). in fish exposed to 38.7 mg pb l-1 and received a diet supplemented with 600 mg figs kg-1, we notice an improvement in erythrocyte count, hb content, and ht values after 60 days of treatment. 0 10 20 30 40 50 60 70 80 90 c1 c2 c+fc ni1 ni2 ni+fc h e m o ly si s p e rc e n ta g e ( % ) experimental groups # ** nemiche s et al. (2023). not sci biol 15(2):11466 7 this research considers fig as chelating agent for lead ions. our findings are in agreement with fouad et al. (2019) who reported that prior to irradiation, oral administration of f. carica extract significantly increased wbc, plt, lymphocyte, and neutrophil counts. fathy et al. (2018) found similar results with rats treated with figs and dates extract for four weeks after exposure to adricin® (doxorubicin hydrochloride) and / or irradiation. the heamatotoxic effect of nickel is primarily due to the over-stimulation of metallothioneins and reactive oxygen species (ros) production that leads to erythrocyte oxidative damage. oxidative stress alters both the activity and content of all antioxidant defense system. our data on oxidative status indicated that exposure to nickel tends to inhibit sod and gsh-px activity and increases the catalase activity which prevent against h2o2 accumulation in the erythrocytes of ni1 intoxicated group. the data show also a decrease in rbcs-gsh level considered as the non-enzymatic first line of defense against oxidative stress induced by heavy metals and an augmentation in mda level in ni1 group compared to the c1 group. these results are in accordance with the study of gupta et al. (2006) who showed that nickel sulfate induced an increase in the malondialdehyde, glutathione levels and the activities of sod, gsh-px and cat in erythrocytes compared to untreated control rats. according to chen et al. (2003) and de luca et al. (2007), nicl2 induces a decrease in the activity of erythrocyte glutathione peroxidase and consequently an increase in oxidative stress. in accordance with study of kalahasthi et al. (2006) the level of mda in the ni-intoxicated group is the consequence of increased in rbcs lipid peroxidation. administration of f. carica extract restored the gsh content to normal levels with a decrease in mda and improved sod, catalase and gsh-px activities. many authors have already confirmed that f. carica have the capacity to improve the activity of antioxidant enzymes and the level of glutathione in serum and different organs. solomon et al. (2010) attributes the antioxidant effect of fresh figs to the presence of a powerful antioxidant called cyanidin-3-rhamnoglucoside (c3r), identified to inhibit lipid peroxidation and reduce oxidative stress. the treatment with the fc leaf extract alone or combined with ascorbic acid noticeably prevented the effect of lead induced-oxidative damage in rats (diab et al., 2018). the fig extract can induce an increase of the antioxidant enzyme level and chelating heavy metal ions, and consequently decreases oxidative stress. our results are in agreement with the work of elsayed et al. (2019) who found that administration of dried figs induced an increase in the level of reduced glutathione in animals exposed to diclofenac sodium. haemolysis is defined as destruction of erythrocytes with the release of their intracellular contents. red cells can haemolyse in the presence of chemical species or toxic element (shah et al., 2011). an excessive haemolysis can cause a reduction of red blood cells count and systematically a hemolytic anaemia. our results demonstrated that nickel has an important hemolytic power as shown by the increase in the hemolytic percentage in ni1 exposed group compared to control c1 group. little amelioration was observed after stopping intoxication in ni2 group compared to ni1 group, but an important significant decrease in hemolytic percentage was recorded after stooping intoxication and starting fce administration in ni+ fc group compared to ni2 group. our data show that fig fruits can protect from haemolysis induced by nickel chloride. according to asadi-samani et al. (2016), f. carica species is a medicinal plant that has been shown to be a hematopoietic agent due to its high antioxidant content, which contributes to the inhibition of the lysis of red blood cell membranes and the prevention of the harmful effects of free radicals. conclusionsconclusionsconclusionsconclusions on the basis of obtaining results, nickel ions induce haematotoxicity and changes in blood parameters. nickel toxicity in erythrocytes may occur through the oxidative stress pathway. also, the fce can exhibit potent antioxidant potential in red blood cells justified by an increase in glutathione reserves and activities of antioxidant enzymes. the effect produced by f. carica extract decreases significantly the haematotoxicity effect nemiche s et al. (2023). not sci biol 15(2):11466 8 of nickel. its richness and abundance in bioactive compounds make this fruit very important and can be considered as a way of reducing nickel toxicity in the blood. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) the animal experiments were conducted by minimizing the degree of suffering in accordance with the ethical principles and institutional guidelines of the national institutes of health guide for the care and use of laboratory animals (8th edition, 2011). the animal experiments were approved by the local ethical committee for the care and use of laboratory animals in the oran 1 university. ackackackacknowledgementsnowledgementsnowledgementsnowledgements we would like to thank “imen andalousi” and all the members of the biological analysis laboratory of “nouar fadila” maternity clinic for their help in the hematological analysis. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences adjroud o (2013) the toxic effects of nickel chloride on liver, erythropoiesis, and development in wistar albino preimplanted rats can be reversed with selenium pretreatment. environmental toxicology 28(5):290-298. https://doi.org/10.1002/tox.20719 aebi h (1984). catalase in vitro. in: methods in enzymology 105:121-126. https://doi.org/10.1016/s00766879(84)05016-3 asadi-samani m., kooti w, aslani e, shirzad h (2016). a systematic review of iran’s medicinal plants with anticancer effects. journal of evidence-based complementary & alternative medicine 21:143-153. https://doi.org/10.1177/215658721560087 barolo mi, mostacero nr, lópez sn (2014). ficus carica l. 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(2023). not sci biol 15(2):11466 11 license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11499 nsb lee s-y and heo j-y 2023.06.19.docx received: 02 mar 2023. received in revised form: 30 mar 2023. accepted: 05 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania lee s-y and heo j-y (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11499 doi:10.15835/nsb15211499 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae combined treatment with gibberellic acid and thidiazuroncombined treatment with gibberellic acid and thidiazuroncombined treatment with gibberellic acid and thidiazuroncombined treatment with gibberellic acid and thidiazuron improveimproveimproveimprovessss fruit quality of ‘red dream’ grapefruit quality of ‘red dream’ grapefruit quality of ‘red dream’ grapefruit quality of ‘red dream’ grape cultivarcultivarcultivarcultivar su-yeon lee, jae-yun heo* gangneung-wonju national university, department of plant science, gangneung 25457, republic of korea; gwnu.sylee@gmail.com; jyheo@gwnu.ac.kr (*corresponding author) abstractabstractabstractabstract this study was conducted to investigate the effect of treatment conditions of plant growth regulators on the marketability of ‘red dream’, a triploid grape. in order to achieve the purpose of this study, gibberellic acid 3 (ga3) 100 ppm single use treatment group was set as a control group. in addition, the fruit characteristics expressed when cytokinins such as thidiazuron (tdz) or forchlorfenuron (cppu) 2.5 ppm or 5.0 ppm mixed with ga3 100 ppm were compared with the control group. cluster weight was commonly higher in the mixed treatment of tdz or cppu than in the ga3 single use treatment, and it was found that the cytokinin mixed treatment could effectively induce the enlargement of ‘red dream’ berries. although the harvesting period was slightly delayed compared to ga3 single use treatment in the case of mixed treatment of ga3 and tdz or cppu, there was no inhibition of coloration, soluble solid sugar content and functionality, which are important indicators of marketability. in particular, in the ga3 100 ppm + tdz 5 ppm treatment group, where the enlargement of fruits was the largest, the occurrence rate of non-commercial berry was also found to be the lowest. therefore, it is expected to contribute to strengthening the marketability of ‘red dream’ if such plant growth regulator treatment condition is applied during cultivation. keywords:keywords:keywords:keywords: antioxidant activity; fruit characteristics; plant growth regulator; triploid grape introductionintroductionintroductionintroduction the cultivation area of grapes surveyed in korea was 14,655 ha in 2022, which ranks 4th among all fruit crops in terms of cultivation area (korean statistical agency, 2022). hence, grapes are regarded as a very important crop in korea (kim et al., 2021a), but the present grape cultivation area in korea is only 80% of what it was in the past 10 years. the competitiveness of the korean grape industry has also been weakened. recently, the possibility of strengthening the competitiveness of the grape industry is expected with the introduction of ‘shine muscat’ in korea. 'shine muscat' is a hybrid grape cultivar with a unique muscat scent and high quality. it has the advantage of being able to produce large seedless berries when treated with a plant growth regulator (pgr) twice before and after flowering (park et al., 2020). this has satisfied the needs of korean consumers who value high quality and convenience, and as a result, the cultivation area of ‘shine muscat’ is also rapidly increasing. https://www.notulaebiologicae.ro/index.php/nsb/index lee s-y and heo j-y (2023). not sci biol 15(2):11499 2 the problem is that many farms are selling ‘shine muscat’ fruits that have not reached the optimum harvest period in order to secure price competitiveness as the competition among farms intensifies due to the increase in the cultivation area of ’shine muscat’. apart from this fact, the changing pattern of grape cultivation in korea, such as the concentration of ‘shine muscat,’ can lead to a result that does not meet the characteristics of the consumer market that emphasizes diversity. if these problems intensify, the competitiveness of the korean grape industry is highly likely to weaken again. therefore, the introduction of various grape cultivars that can meet the needs of consumers is required in order to establish a sustainable grape industry system in korea. recently, many efforts have been made to breed new grape cultivars in korea, and various grape cultivars are being introduced to farmers (park et al., 2016; heo and park, 2017; roh et al., 2018; kim et al., 2020; park et al., 2022). among them, ‘red dream’, which is a triploid grape registered in 2016, has the advantage of being able to produce large seedless fruits with just one-time treatment of pgr at the full bloom stage (park et al., 2022). in addition, ‘red dream’ has high fruit quality and red skin that differentiates it from other cultivars, so it is expected to attract consumers' attention. currently, the production of seedless fruits of ‘red dream’ is carried out through gibberellic acid 3 (ga3) at a concentration of 100 ppm. however, there is a disadvantage that the occurrence rate of non-commercial berries is too high due to berry softness and cracking, which limits the expansion of the cultivation area of ‘red dream’. in some crop species where a similar problem occurred to 'red dream', it was solved through a mixed treatment of ga3 and cytokinin-based hormones (joshi et al., 2018; elmenofy et al., 2021). ‘red dream’ requires the treatment of pgr to produce fruits with commercial value in any case, and it can be most effectively used in the cultivation field if an appropriate pgr condition that can suppress the occurrence of non-commercial berries can be devised. the effect of pgr on fruit characteristics in fruit crops differs not only depending on the cultivar and type of mixed hormone but also on the treatment concentration (bons and kaur, 2020). therefore, sufficient prior research is needed to examine whether treatment of pgr can solve the problems occurring in 'red dream' and to find out the optimum treatment condition if it is valuable. in this study, we examined the fruit characteristics expressed in ‘red dream’ at harvest time after mixed treatment with different concentrations of thidiazuron (tdz) and forchlorfenuron (cppu) in ga3. from this, we aimed to evaluate the effectiveness of the mixed pgr treatment and obtain information on a practically useful pgr treatment condition for ‘red dream’. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods this study was conducted using 8 ten-year-old ‘red dream’ trees planted 3 meters apart in rows and vines at the vineyard of gangwondo agricultural research and extension services located in chuncheon, korea (37°94’73.80n, 127°75’44.30e) in 2022. for this study, ga3 (yooil company, korea), tdz (daeyu company, korea), and cppu (upi, korea) were used. the control group was treated with a single use of ga3 at 100 ppm. tdz or cppu mixed with ga3 at 2.5 ppm or 5.0 ppm was also applied to evaluate the effect of mixed pgr treatment. each pgr condition was applied to 2 inflorescences per tree (n=16) when they reached the full bloom stage. when the optimum harvest period was reached for each treatment cluster, they were harvested in the test field and immediately transported to gangneung-wonju national university located in gangneung, gangwon-do of korea. the effect of pgr treatment conditions on fruit characteristics in ‘red dream’ was evaluated by measuring and comparing the number of berries, weight of cluster and berry, berry firmness, occurrence rate of non-commercial berries, skin color, soluble solid sugar content, titratable acidity, and functionality, which are regarded as important factors in determining the marketability of grapes (heo and park, 2016). cluster weight was measured using an electronic scale (fy-3000, a&d, japan), and berry weight was calculated by dividing the cluster weight by the number of berries per cluster. berry firmness was measured with a fruit hardness tester lee s-y and heo j-y (2023). not sci biol 15(2):11499 3 (fht-05, landtek, china) after thinly removing the skin from the top of the berry and expressed in newtons (n). the occurrence rate of non-commercial berries was investigated by calculating the ratio with the total number of berries after counting the number of cracked and softened berries for each cluster. for measurement of skin color, hunter values were measured with a colorimeter (cr-400, konica minolta, japan), and then l (lightness) and hue angles, which are important in red grapes, were presented. for measurement of total soluble solid content (tss), we extracted juice from randomly selected 10 berries per each cluster. afterwards, it was measured with a digital refractometer (pal-1, atago, japan) and expressed in brix. titratable acidity (ta) was measured by diluting the fruit juice used for sugar content measurement by 39 times in distilled water and then measured with a digital titratable acidity meter (acid-2, atago, japan) and expressed as %. the effect of pgr treatment on functionality was evaluated by measuring and comparing the total phenol content, total flavonoid content, and ferric reducing antioxidant power (frap). for this experiment, 40 g of grape skin were randomly sampled with three replications for each treatment group and dried in a hot air dryer at 60 °c for 10 days. the dried samples were ground using a mortar and pestle to a fine powder. from the fine powder, 0.1 g was homogenized in 7 ml of 100% methanol for each sample. afterward, the homogenized solution was kept in a water bath (c-wba1, changshin science, korea) at 60 °c for 30 minutes. the extracted powder solution was centrifuged at 4 °c and 4500 rpm for 20 minutes (mega 17r, hanil, korea), and the supernatant was collected. the extraction was done twice, and the supernatants were combined. to measure the total phenolic content, the folin-ciocalteu method was used according to geleta et al. (2023). extracted samples of 25 µl (1 mg/ml) and 125 µl folin-ciocalteu reagent were mixed and then left to stand for three minutes. afterward, 37.5 µl sodium bicarbonate (20%), and 62.5 µl distilled water were added and kept in the dark at room temperature for one hour. the absorbance of the sample was read at 765 nm using a microreader spectrophotometer (mobi, microdigital, korea). gallic acid (0-100 µg) was used as the standard, and the result was expressed as mg gallic acid equivalent per gram dry weight (mg gae/g dw). for the measurement of total flavonoids, the aluminum chloride colorimetric method was applied with the method of chang et al. (2002) with slight modification. twenty-five microliters (1 mg/ml) of the extracted samples were mixed with 75 µl methanol, 5 µl aluminum chloride (10%), and 5 µl sodium acetate (1 m). they were then left to stand for 30 minutes in the dark at room temperature, and the absorbance of the sample was read at 415 nm. quercetin (0-250 µg) was used as the standard to construct the calibration curve. the result was expressed as mg quercetin per gram dry weight (mg qe/g dw). the ferric reducing antioxidant power (frap) assay was determined as described by benzie and strain (1996). frap reagent solution containing acetate buffer (300 mm, ph 3.6), 10 mm tptz (diluted in 40 mm hcl), and 20 mm fecl3 was prepared. three milliliters of frap reagent solution were mixed with 50 µl of the extracted sample. the mixture of samples with frap reagent was incubated at 37 °c for 4 minutes, and the absorbance of the sample was read at 593 nm at the microreader spectrophotometer. a calibration curve was constructed using different concentrations of trolox (0–100 µg), and the result was expressed as µm trolox/g dw. statistical comparison of fruit characteristics according to pgr treatment condition was performed using the analysis of variance and duncan's multiple range tests in the spss program (version 28, ibm, usa). results results results results and discussionand discussionand discussionand discussion table 1 shows the results of cluster weight, berry number, berry weight, berry firmness, and incidence of non-commercial berries of ‘red dream’ according to the pgr treatment conditions. the average berry weight of ‘red dream’ ranged from 9.1 to 10.5 g, and the number of berries was observed between 44.1 and 49.2. the weight and number of berries were generally high in the ga3 and cppu or tdz mixed treatment group, but no statistical significance was found. when the ga3 and cppu or tdz were applied together, excessive berry set was observed in another grape cultivar (kim et al., 2021b). the ga3 and cppu both promote fruit development and can enhance berry set, which is the process by which flowers are converted into berries. the lee s-y and heo j-y (2023). not sci biol 15(2):11499 4 tdz, on the other hand, can promote cell division and differentiation in berries, leading to the formation of more berries. when these types of pgrs are used in a mixed application, they can increase the rate of berry set and development, resulting in more fruit than usual. however, this can also lead to overcrowding and reduced fruit quality as the vine may not be able to support the growth of so many berries. this can also increase the risk of fungal diseases as the dense foliage and fruit can create a humid microclimate that is conducive to fungal growth. however, it was confirmed that such a problem did not occur even though the ga3 and cppu or tdz combined treatment with a relatively high concentration was applied in ‘red dream’. table 1table 1table 1table 1. effects of plant growth regulator treatment on fruit characteristics of ‘red dream’ treatmenttreatmenttreatmenttreatment conditionconditionconditioncondition cluster weight cluster weight cluster weight cluster weight (g)(g)(g)(g) berry numberberry numberberry numberberry number berry weight berry weight berry weight berry weight (g)(g)(g)(g) berry berry berry berry firmness (n)firmness (n)firmness (n)firmness (n) incidence of incidence of incidence of incidence of nonnonnonnon---commercial commercial commercial commercial berries (%)berries (%)berries (%)berries (%) gagagaga3333 100 ppm100 ppm100 ppm100 ppm 418.15±27.64b 44.12±3.49a 9.68±0.56a 6.28±0.14b 19.73±2.72a gagagaga3333 100 ppm + 100 ppm + 100 ppm + 100 ppm + cppu 2.5 ppmcppu 2.5 ppmcppu 2.5 ppmcppu 2.5 ppm 447.77±25.90ab 49.25±1.22a 9.12±0.54a 6.38±0.17ab 19.89±3.88a gagagaga3333 100 ppm + 100 ppm + 100 ppm + 100 ppm + cppu 5.0 ppmcppu 5.0 ppmcppu 5.0 ppmcppu 5.0 ppm 458.07±36.73ab 45.62±3.17a 10.02±0.30a 6.34±0.15ab 15.97±3.48ab gagagaga3333 100 ppm + 100 ppm + 100 ppm + 100 ppm + tdz 2.5 ppmtdz 2.5 ppmtdz 2.5 ppmtdz 2.5 ppm 456.33±21.24ab 46.25±2.19a 9.95±0.46a 6.49±0.13ab 14.28±3.09ab gagagaga3333 100 ppm + 100 ppm + 100 ppm + 100 ppm + tdz 5.0 ppmtdz 5.0 ppmtdz 5.0 ppmtdz 5.0 ppm 494.71±29.21a 48.00±4.34a 10.54±0.45a 6.55±0.11a 12.32±2.53b different letters indicate the significant differences between the mean at p < 0.05. the same letters denote no significant difference at p < 0.05. ± indicates standard error. mean cluster weight ranged from 418.1 to 494.7 g. it was found that cluster weight significantly increased in the ga3 and cppu or tdz mixed treatment group compared to the ga3 single use treatment group, which was presumed to be a result of the slight increase in the number and weight of berries. similar to cluster weight, berry firmness was higher in the ga3 and cppu or tdz combination treatment groups than in the ga3 single use treatment, regardless of the type and concentration. in particular, the highest cluster weight and berry firmness were observed in the ga3 100 ppm + tdz 5.0 ppm treatment group. it has been reported that the cppu induces enhanced cell durability and redirects the plant's energy and resources toward the development of berries, while the tdz promotes cell division and elongation by inducing cell proliferation (nisler, 2018; rojas et al., 2021). accordingly, larger berry firmness or size is frequently induced in grapes, as in the case of ‘red dream’ when the ga3 and cppu or tdz are treated together (el-abbasy et al., 2015). however, the ga3 and cppu combined treatment was more effective than ga3 and tdz mixed treatment for increasing berry weight and firmness in ‘shine muscat’, unlike our results (wang et al., 2019). these results indicate that the activity of mixed treatment with the ga3 and cppu or tdz can differ depending on the grape cultivar. it is assumed that the effect on berry weight and firmness was more effective in the highconcentration the ga3 and tdz mixed treatment group because the activity of tdz in berry cells of ‘red dream’ could be higher than that of cppu. the incidence rate of non-commercial berries was generally reduced in the ga3 and cppu or tdz mixed treatments compared to the ga3 single use treatment group (table 1). the lowest occurrence rate of non-commercial berries was observed in the ga3 100 ppm + tdz 5.0 ppm treatment group, which had the highest berry firmness by reducing berry softening and cracking. retamales et al. (1994) and stern et al. (2013) also found that berry firmness was increased due to intracellular structural changes that increased the thickness of the cell wall, which led to a decrease in the incidence of non-commercial berries when ga3 and cytokininbased pgrs were treated together. these results suggest that the combined treatment with ga3 and tdz in ‘red dream’ inhibited the occurrence of softening and cracking berries by inducing an increase in berry lee s-y and heo j-y (2023). not sci biol 15(2):11499 5 firmness. the use of pgrs like ga3, cppu, and tdz can promote cell division and expansion, leading to larger and firmer berries (wang et al., 2020; rojas et al., 2021). the combination of these pgrs can have a synergistic effect on increasing berry size and firmness, thereby reducing the incidence of non-commercial berries. additionally, firmer berries may also have a longer shelf life and better transportation properties, which can improve the overall quality of the grape crop. therefore, it seems necessary to obtain information on what kind of changes pgr treatment induces in the cell structure of ‘red dream’ berries in the future. the effect of pgr treatment on the coloration of ‘red dream’ is shown in table 2. in this experiment, it was confirmed that the l and hue angle values, which are important in red grape cultivars, did not show statistical significance in all pgr treatment groups. however, it was observed that the harvest period was delayed by about 3 to 7 days as the coloring was delayed in the ga3 and cppu or tdz mixed treatment group in this experiment. table 2table 2table 2table 2. effects of plant growth regulator treatment on coloration and harvest time of ‘red dream’ treatmenttreatmenttreatmenttreatment conditionconditionconditioncondition llll hue anglehue anglehue anglehue angle harvest timeharvest timeharvest timeharvest time gagagaga3333 100 ppm100 ppm100 ppm100 ppm 34.24±0.35a 41.29±3.55a september 19 gagagaga3333 100 ppm + cppu 2.5 ppm100 ppm + cppu 2.5 ppm100 ppm + cppu 2.5 ppm100 ppm + cppu 2.5 ppm 34.71±0.34a 42.68±3.54a september 22 gagagaga3333 100 ppm + cppu 5.0 ppm100 ppm + cppu 5.0 ppm100 ppm + cppu 5.0 ppm100 ppm + cppu 5.0 ppm 34.47±0.44a 41.76±3.45a september 22 gagagaga3333 100 ppm + tdz 2.5 ppm100 ppm + tdz 2.5 ppm100 ppm + tdz 2.5 ppm100 ppm + tdz 2.5 ppm 35.37±0.43a 42.85±1.08a september 22 gagagaga3333 100 ppm + tdz 5.0 ppm100 ppm + tdz 5.0 ppm100 ppm + tdz 5.0 ppm100 ppm + tdz 5.0 ppm 34.88±0.48a 42.34±2.69a september 26 the same letters denote no significant difference at p < 0.05. ± indicates standard error. it is presumed that more time is required for natural coloring with the mixed application of the ga3 and cppu or tdz in ‘red dream’. although they can be effective when used together in a mixed application, they can delay the harvest time of grapes. when used together, these compounds can result in larger fruit size than usual but still immature in terms of sugar content and flavor development. this is because the growth rate of the fruit is increased, but the maturation and ripening process is not necessarily accelerated. additionally, the cppu and tdz can both delay the ripening process of grapes by suppressing the production of ethylene, which is a hormone that plays a crucial role in fruit ripening (carvajal-millan et al., 2001; jung et al., 2015). in 'flame seedless', coloration was greatly inhibited when cytokinin-type hormones were treated, which not only delayed the harvest period but also interfered with the production of marketable berries (peppi and fidelibus, 2008). in 'red dream', the ga3 and cppu or tdz mixed treatment only caused a delay in the harvest time, but there was no difference in the final coloration. hence, it was estimated that the ga3 and cppu or tdz mixed treatment had no negative effect on the coloration of ‘red dream’. table 3table 3table 3table 3. effects of plant growth regulator treatment on total soluble solid content and titratable acidity of ‘red dream’ treatmenttreatmenttreatmenttreatment conditionconditionconditioncondition total soluble solid total soluble solid total soluble solid total soluble solid content (tss)content (tss)content (tss)content (tss) titratible acidity titratible acidity titratible acidity titratible acidity (ta)(ta)(ta)(ta) tss/ta tss/ta tss/ta tss/ta ratioratioratioratio gagagaga3333 100 ppm100 ppm100 ppm100 ppm 17.55±0.23ab 0.44±0.01b 40.16±0.60a gagagaga3333 100 ppm + cppu 2.5 100 ppm + cppu 2.5 100 ppm + cppu 2.5 100 ppm + cppu 2.5 ppmppmppmppm 17.07±0.19b 0.43±0.00b 40.18±0.38a gagagaga3333 100 ppm + cppu 5.0 100 ppm + cppu 5.0 100 ppm + cppu 5.0 100 ppm + cppu 5.0 ppmppmppmppm 18.65±0.35a 0.48±0.01a 39.16±0.95a gagagaga3333 100 ppm + tdz 2.5 100 ppm + tdz 2.5 100 ppm + tdz 2.5 100 ppm + tdz 2.5 ppmppmppmppm 16.77±0.17b 0.42±0.01b 39.84±0.86a gagagaga3333 100 ppm + tdz 5.0 100 ppm + tdz 5.0 100 ppm + tdz 5.0 100 ppm + tdz 5.0 ppmppmppmppm 17.87±0.28ab 0.44±0.00b 40.62±0.54a different letters indicate the significant differences between the mean at p < 0.05. ± indicates standard error. lee s-y and heo j-y (2023). not sci biol 15(2):11499 6 table 3 shows the effect of the ga3 and cppu or tdz mixed treatment on tss and ta. ta and tss ranged from 0.42 to 0.48 obrix and 16.8 to 18.7 %, respectively. ta was found to be significantly higher only in the ga3 100 ppm + cppu 5.0 ppm treatment group compared to other treatments. there was a significant difference in tss depending on the treatment group. the ga3 100 ppm + cppu 2.5 ppm and the ga3 100 ppm + tdz 2.5 ppm showed significantly lower values in tss, but other treatment groups had higher values than the ga3 single use treatment group. similarly, it has been reported that tss is affected by pgr treatment combination and condition (shin et al., 2019; wang et al., 2020; tyagi et al., 2021). this is presumed to be because sugar uptake and transport in grape berries based on treatment conditions could be regulated differently. however, it should be noted that the tss/ta ratio has a decisive effect on consumer preference for grapes (park et al., 2015). grapes with a high tss/ta ratio are considered sweeter and more flavorful, while grapes with a low ratio may be tart or bland. the tss/ta ratio can also affect the storage and shelf life of grapes (shahkoomahally et al., 2021). grapes with a high tss/ta ratio tend to have a longer shelf life, as the high sugar content can help preserve the fruit and prevent spoilage. on the other hand, grapes with a low tss/ta ratio may spoil more quickly, as the low sugar content can provide a favorable environment for microorganisms. interestingly, the tss/ta ratio ranged from 39.2 to 40.6, and there was no statistical significance in all treatment groups. hence, it was evaluated that the pgr treatment conditions used for this study would not have a significant difference in the berry quality perceived by consumers. table 4table 4table 4table 4. effects of plant growth regulator treatment on functionality of ‘red dream’ treatmenttreatmenttreatmenttreatment conditionconditionconditioncondition total phenol contenttotal phenol contenttotal phenol contenttotal phenol content ((((mg gae/g dry weight)mg gae/g dry weight)mg gae/g dry weight)mg gae/g dry weight) total flavonoid contenttotal flavonoid contenttotal flavonoid contenttotal flavonoid content (mg (mg (mg (mg qe/g dry weigh)qe/g dry weigh)qe/g dry weigh)qe/g dry weigh) ferric reducing ferric reducing ferric reducing ferric reducing antioxidant powerantioxidant powerantioxidant powerantioxidant power (µm trolox /g dry (µm trolox /g dry (µm trolox /g dry (µm trolox /g dry weigh)weigh)weigh)weigh) gagagaga3333 100 ppm100 ppm100 ppm100 ppm 5.64±0.17b 3.88±0.07bc 154.55±4.31b gagagaga3333 100 ppm + cppu 2.5 100 ppm + cppu 2.5 100 ppm + cppu 2.5 100 ppm + cppu 2.5 ppmppmppmppm 6.19±0.09a 4.09±0.02a 153.72±5.24b gagagaga3333 100 ppm + cppu 5.0 100 ppm + cppu 5.0 100 ppm + cppu 5.0 100 ppm + cppu 5.0 ppmppmppmppm 5.98±0.24ab 3.98±0.06ab 161.61±7.93ab gagagaga3333 100 ppm + tdz 2.5 100 ppm + tdz 2.5 100 ppm + tdz 2.5 100 ppm + tdz 2.5 ppmppmppmppm 6.11±0.06ab 3.81±0.05bc 177.35±8.34a gagagaga3333 100 ppm + tdz 5.0 100 ppm + tdz 5.0 100 ppm + tdz 5.0 100 ppm + tdz 5.0 ppmppmppmppm 5.71±0.12ab 3.72±0.09c 158.83±3.19ab different letters indicate the significant differences between the mean at p < 0.05. ± indicates standard error. the effect of pgr treatment on the functionality of ‘red dream’ is shown in table 4. consumers are increasingly interested in purchasing foods that offer health benefits beyond basic nutrition, and functional foods such as grapes can provide added value to the market. in addition, functional compounds can improve the sensory characteristics of the fruit, such as flavor and color, which can also enhance marketability. therefore, understanding and improving the functionality of grapes through practices such as pgr treatments can help to increase their marketability and ultimately benefit both producers and consumers. the total phenol content (tpc) was found to be significantly higher in the ga3 and cppu or tdz mixed treatment group than in the ga3 single use treatment group. the total flavonoid content was 4.09 mg qe/g in the ga3 100 ppm + cppu 2.5 ppm treatment group and 3.98 mg qe/g in the ga3 100 ppm + cppu 5.0 ppm treatment group, which was significantly higher than that of the ga3 single use treatment group with a value of 3.88 mg qe/g. however, it was found that there was no statistical difference in the ga3 and tdz mixed treatment compared to the ga3 single treatment. frap ranged from 153.7 to 177.35 µm trolox/g in the ga3 and cppu or tdz mixed treatment group and was higher overall compared to the ga3 single use application treatment group. as a result, it was confirmed that the content related to functionality was generally higher in the ga3 and cppu or tdz mixed treatments than in the ga3 single use treatment. it is presumed that the content of lee s-y and heo j-y (2023). not sci biol 15(2):11499 7 the functional substance would have increased as the activity of the cell increased. as we found, it has been reported that the mixed treatment of ga3 and tdz in 'pione' significantly increased tpc compared to the single treatment of ga3 (lee et al., 2003). antioxidants are compounds that help to protect cells from damage caused by free radicals, which are unstable molecules that can damage dna and other molecules in the body (martemucci et al., 2022). grapes are known as a rich source of antioxidants, including polyphenols such as resveratrol and flavonoids (wijekoon et al., 2022; zhou et al., 2022). cytokinin has been shown to increase the synthesis of polyphenols in grapes, which can contribute to higher antioxidant activity (tyagi et al., 2022). cytokinin can also help to regulate the expression of genes that are involved in the synthesis of antioxidants, leading to higher levels of these compounds in the fruit. gibberellin, on the other hand, has been shown to increase the expression of genes that are involved in the synthesis of flavonoids, which can also contribute to higher antioxidant activity in grapes (jadhav et al., 2020). when cytokinin and gibberellin are applied together, they can work synergistically to enhance the production of both polyphenols and flavonoids, leading to higher antioxidant activity compared to gibberellin alone. this is because the two hormones can target different pathways involved in antioxidant synthesis and work together to increase the overall output of these compounds. apart from this fact, kaplan et al. (2019) reported that when pgrs are treated at inappropriate concentrations, useful functional substances such as flavonoids were not accumulated properly. although only three functionality related parameters were measured in this experiment, similar or higher values were observed in all parameters when the results of the ga3 and cppu or tdz combined treatment groups were compared with the ga3 single use treatment group. this result strongly indicates that the functionality was also not impaired by the ga3 and cppu or tdz mixed treatment in ‘red dream’. overall, the combined treatment of ga3 and cppu or tdz in 'red dream' is effectively applied in suppressing the incidence of non-commercial berries and increasing cluster enlargement without significantly impairing fruit quality compared to the ga3 single use treatment. in particular, the ga3 100 ppm + tdz 5.0 ppm treatment group was confirmed to have the highest cluster weight and berry firmness. in addition, the incidence rate of non-commercial berry was also reduced by 37.6% compared to the ga3 single use treatment. hence, it was estimated to be the most effective treatment condition to supplement the cultivation problems of ‘red dream’ among pgr treatment combinations used in this experiment. conclusionsconclusionsconclusionsconclusions in this study, we investigated the effect of various pgr combinations on the fruit quality of newly bred ‘red dream’ grape. the results showed that the ga3 and cppu or tdz mixed treatment resulted in significantly higher cluster weight and lower incidence rate of non-commercial berries compared to the ga3 single use treatment group. the functionality was also higher overall in the mixed treatment group. these findings suggest that the mixed treatment of ga3 and cppu or tdz can enhance the marketability of 'red dream' grape, possibly by increasing the activity of cells and the synthesis of antioxidants such as polyphenols and flavonoids. however, it is important to note that inappropriate concentrations of pgrs can negatively affect the tss. our result suggests that the mixed treatment of ga3 and cppu or tdz can be an effective strategy for enhancing the marketability of ‘red dream’ grape. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions syl: conceptualization; 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contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11554 nsb henouda 2023.06.20.docx received: 28 apr 2023. received in revised form: 05 jun 2023. accepted: 13 jun 2023. published online: 20 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania henouda s et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11554 doi:10.15835/nsb15211554 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae study of the effect of study of the effect of study of the effect of study of the effect of moringa oleiferamoringa oleiferamoringa oleiferamoringa oleifera leaves powder in southwestern leaves powder in southwestern leaves powder in southwestern leaves powder in southwestern algerian diabetic patients: a pilot clinical trialalgerian diabetic patients: a pilot clinical trialalgerian diabetic patients: a pilot clinical trialalgerian diabetic patients: a pilot clinical trial sarra henouda1*, saida karouche2, amina attou1, ahmed boulal1 1university of ahmed draia, faculty of science and technology, laboratory of saharan natural resources, adrar 01000, algeria; sarra.henouda@univ-adrar.edu.dz (*corresponding author); ami.attou@univ-adrar.edu.dz; ahm.boulal@univ-adrar.edu.dz 2larbi ben m’hidi university, department of natural and life sciences, laboratory of natural substances, biomolecules and biotechnological applications, oum el bouaghi, algeria; saidabmc86@yahoo.fr abstractabstractabstractabstract moringa oleifera lam. leaves are commonly used for diabetes worldwide. to date, there has been no research study done to investigate its effect on lipid and carbohydrate profile in algerian diabetic patients. this pilot clinical study aimed to evaluate its long term-effect on lipid and carbohydrate profile in algerian diabetic patients in preparation for a larger trial. 44 diabetic patients from adrar city were administrated with 3600 mg of mo leaves powder twice a day at breakfast and at 7 p.m. for a period of 90 consecutive days, along with their regular hypoglycemic medications, in order to evaluate their serum lipid (tc, c-hdl, c-ldl and tg) and carbohydrate profile (blood sugar and hba1c), weight, bmi and blood pressure, across five time-points (on days 0, 3, 7, 30, and 90). the results showed that oral administration of moringa oleifera powder had a statistically significant effect on blood sugar (hba1c), ldl-c, hdl-c levels in diabetic patients (p<0.05). moringa oleifera leaf powder seemed reduced ldl-c, and hba1c and elevated hdl-c, in diabetic patients. no side effect was reported by any participant. however, it did not have a statistically significant effect on weight, bmi and blood pressure. the data from the present clinical trial provide persuasive, although preliminary evidence supporting the therapeutic potential of moringa oleifera leaf powder for managing chronic hyperglycemia and dyslipidemia in algerian patients with diabetes. a more extensive trial is necessary to determine the moringa oleifera leaf powder optimal dose and evaluate if its effect results into long-term advantages. in addition, further investigations are required to clear the underlying mechanisms involved with these effects. keywords:keywords:keywords:keywords: algeria; diabetic; leaves powder; moringa oleifera abbreviations:abbreviations:abbreviations:abbreviations: 95% ci: 95% confidence interval; bg: blood glucose; bmi: body mass index; hba1c: hemoglobin a1c; hdl-c: high-density lipoproteins; idf: international diabetes federation; ldl-c: low density lipoproteins; mo: moringa oleifera; sbp/dbp: systolic/diastolic blood pressure; sd: standard deviation; tc: total cholesterol; tg: triglycerides; w: weight. https://www.notulaebiologicae.ro/index.php/nsb/index henouda s et al. (2023). not sci biol 15(2):11554 2 introductionintroductionintroductionintroduction diabetes is a chronic metabolic disorder that affects lipid, carbohydrate, and protein functions, resulting in persistent hyperglycemia, arising from abnormal insulin secretion, insulin action, or both, causing slow damage (weyer et al., 1999), which leads over time to severe damage to the heart, blood vessels, eyes, kidneys and nerves (who, 2023). the incidence of diabetes has increased exponentially over the years and is projected to rise above 783 million by 2045 in adults between the age of 20-79 (vs. 463 and 537 million in 2019 and 2021, respectively), likewise, the mortality death was found to increase and approximately 6.7 million adults are estimated to have died as a result of diabetes or its complications in 2021 (saeedi et al., 2019; idf 2021). according to the idf (2021), the prevalence of diabetes in algeria has been increased to 7.4% in 2021 in adults aged 20-79 years. in addition, algeria is one of the top ten countries in the world for both the number of children with type 1 diabetes and the number of new cases of type 1 diabetes. algeria ranks among the top ten countries globally in terms of both the number of children with type 1 diabetes and the number of new cases of type 1 diabetes. therefore, enhancing the management of diabetes is still a major public health concern at the national level. the who appraised that more than 80% of people worldwide are estimated to use traditional medicine (who, 2023). in the last years, the high cost of prescription medications, particularly for impoverished populations, especially in developing countries, and in particular, their detrimental side effects have led to an increase in herbal medicine use (nicolle et al., 2011; yuan et al., 2016). in rural areas, financial barriers to accessing medications and to achieving optimal health are even greater; most of the population in the adrar region cannot easily access modern medicines and prefer to use herbal medicine as the first line treatment. therefore, there will always be the need to search for cost-effective and efficient hypoglycemic agents with fewer side effects. mo (family: moringaceae) (olson, 2002) is one of the medicinal plant widely used in recent years. various parts of this plant such as the leaves, roots, seed, fruit have been extensively studied for their many therapeutic potential such as antitumor, antiurolithiatic, antifertility, hypotensive, analgesic, antipyretic, antiepileptic, hypocholesterolaemic, antifungal, antibacterial, antifungal, aphrodisiac, cholagogue, antioxidant, hepatoprotective, immunomodulattors, cardiotonic and as cardiac and circulatory stimulants (karadi et al., 2006; martín et al., 2013; al-asmari et al., 2015; saini et al., 2016; mabrok and mohamed, 2019). furthermore, mo is used worldwide (in bangladesh (kadir et al., 2012), south africa (semenya et al., 2012), senegal (dièye et al., 2008; yousefian, 2012), nigeria (popoola and obembe, 2013), ugandan rural (kasolo et al., 2010) as a popular remedy for diabetes. the leaves are the most explored part of the tree due to the presence of various nutrients such as essential amino acids, minerals, vitamins and carbohydrates (vergara-jimenez et al., 2017). they are also characterized by a low calorific value, which makes them suitable for the diet of overweight people (gopalakrishnan et al., 2016). bioactive compounds that are present in its leaves are alkaloids, flavonoids, phenolic compounds, tannins, saponins, lignin, amides, glycosides, carotenoids (kashyap et al., 2022), which play an important role in stabilizing blood pressure and bg levels in diabetic patients (padayachee and baijnath, 2020). mo leaves non-toxicity in vivo has been confirmed in many studies (mbikay, 2012). no acute or subacute toxicity was reported following treatment with mo leaves, even at doses of 2000 mg/kg (adedapo et al., 2009), according to barichella et al. (2019), a daily dose of 14 g was safe and well accepted. hence, the plant is relatively safe both for nutritional and medicinal uses (adedapo et al., 2009). no report contradicting this evidence was found in the scientific literature. in addition, it should be noted that the plant is already used as food. medicinal plants local knowledge remains poorly documented in scientific literature. mo one of these medicinal plants which is used for antidiabetic purpose in the area. the present study is a scientific approach to reestablish the traditional uses of mo in algerian south eastern and to evaluate its hypoglycemic, henouda s et al. (2023). not sci biol 15(2):11554 3 hypolipidemic, hypotensive and weight-loss potentialities. we aimed to conduct a pilot approach of the mo leaves powder effect on the lipid (tc, c-hdl, c-ldl and tg) and carbohydrate profile (bg and hba1c) among algerian south eastern diabetic patients. another objective of the present study was to test whether it was possible to recruit and retain patients to such a study for further studies. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods collection and preparation of plant materials moringa is the most popular plant found in southern algeria (boulal et al., 2019; boulal et al., 2020). the moringa oleifera l. leaves used in our study, which were identified by botanists from our university, were collected from mid-march to early april 2021-2022 from the adrar region, located in the central part of the sahara in southwestern algeria (figure 1). they were washed with tap water, shade dried, milled and ground into powder with the help of a mixer. the measured powder (3600 mg/day for each patient) was stored in hermetically sealed glass jars and kept in a dry place. figure 1.figure 1.figure 1.figure 1. presentation of the mo leaves collection site (boulal et al., 2021) patients and ethical issues we recruited 44 diabetic volunteers from adrar city, 1200 km south of algeria. diabetic patients were excluded if they had any major cardiac, respiratory, renal, liver or mental disease; or if they had any allergy to mo leaves powder. eligible patients had to be ≥18 years old. all patient detailed data regarding personal and medical history of diabetes were gathered on individual interview. clinical characteristics were collected from medical records. all selected patients were informed about study objectives and allowed an opportunity to ask questions, they were ensured of the confidentiality of information, and then they gave their signed informed consent for inclusion before participating in the study. the study was conducted in accordance with the declaration of helsinki, and the protocol was approved by the ethics committee of the hospital where the participants were recruited. the study was carried out from february 2021 to june 2022. study on long-term effect of mo on serum biochemical parameter levels, weight, bmi, and blood pressure diabetic patients were asked to take 3600 mg of mo leaves powder twice a day, in the morning with breakfast and in the evening at 7 p.m. for 90 days. participant’s hypoglycemic medications were not altered and they were instructed to maintain their usual nutritional habits throughout the study. on days; 0, 3, 7, 30, and 90 the blood samples were collected in order to assess the lipid (tc, c-hdl, c-ldl and tg) and henouda s et al. (2023). not sci biol 15(2):11554 4 carbohydrate profile (bg and hba1c). the body weight, bmi and hta of each patient were also measured. patients were asked about any symptoms. assay of serum biochemical parameters levels fasting bg, c-hdl, c-ldl and tg were determined using automated chemistry analyzer////mindray® bs-240. assay of hba1c levels hba1c was determined using automated hplc method/medconn® (mq-2000 pt). statistical analysis data were analysed using spss 26 (ibm). continuous variables are presented as means and standard deviations, whereas categorical variables are presented as numbers and frequencies. normality and homogeneity checks were carried out on the residuals using shapiro-weilk test and levene/bartlett test. repeated measures anova or freidman test were used to determine the effect of mo powder on serum biochemical parameters levels, weight, bmi and hta across time-points (d0, d3, d7, d30, and d90) according to application condition, then post-hoc comparisons were used to determine the source of significant differences where appropriate. dependent sample t-test was used to compare the effect of mo powder on hba1c rate. p < 0.05 was considered statistically significant with 95% confidence intervals (cis). results results results results participants a total of 44 diabetic patients were enrolled in this study. sociodemographic characteristics of diabetic participants are summarized in table 1. the current study diabetic group included more women (sex ratio; 0.375). the mean age of the women was higher than that of the men (52.41 ± 14.895 vs. 47.50 ± 14.463 years). furthermore, the percentage of subjects currently smoking or having previously smoked was low compared to those who never smoked (6.8 % vs. 93.2%, respectively). smoking habits did not appear to be a significant contributor to variations in serum biochemical parameters levels of lipid (tc, c-hdl, c-ldl and tg) and carbohydrate profile (bg and hba1c), and to variations in weight, bmi and bp, on d0, d3, d7, d30, and d90 , in this cohort (p>0.05). the percentage of patients who never attended school was relatively high compared to those who were schooled (18.2% vs. 81.8%, respectively). however, it was feasible to recruit and retain patients to this study, even if it was not easy, despite the high number of participants with a low educational level (≤ 9 years of school: 75%), but they participated significantly in the group. no major side effects were reported by any subject except for some gastric issues seen during the first few days of treatment. effect of mo powder on bg bg of diabetic participants was on average about 0.29 g/l lower after 90 days of mo powder administration, the results are summarized in table 2. however, the results for the repeated measures anova indicate that no significant time effect of mo leaves powder on bg levels for diabetic patients (bgd0: 1.89 ± 0,652 (0.90-3.56 g/l) vs. bgd90: 1.60 ± 0.573 (0.71-2.99 g/l) p>0.05). effect of mo powder on hba1c rate (%) table 2 shows the differences among baseline measurement of hba1c diabetic patients, which was taken at the start of the study (d0) and a final reading that was taken on the ninetieth day (d90) (hba1cd0 : 10.03 ± 2.187 (6.20 15.10) vs. hba1cd90 : 9.20 ± 1.702 (6.5-13.10), p=0.000). mo powder leaves has a henouda s et al. (2023). not sci biol 15(2):11554 5 statistically significant effect on hba1c values of diabetic patients. the present results indicate that long-term administration of mo powder leaves decreases hyperglycemia in diabetic patients. effect of mo powder on tc in the present study group, tc values progressed downwardly over time, but the change was not statistically significant. thus, mo powder leaves administration has no statistically significant effect on patients’ serum tc levels (tcd0: 1.71 ± 0.454 (0.92-3.09) vs. tcd90: 1.67 ± 0.398 (1.03-3.01), p>0.05) (table 2). table 1.table 1.table 1.table 1. participant’s characteristics mean age in years (sd) 51.07 ± 14.776 (29-84) gender men n, (%) women n, (%) sex ratio 12 (27.3) 32 (72.7) 0.375 family status, n (%) married 36 (81.8) single 1 (2.3) divorced 2 (4.5) widow 5 (11.4) profession, n (%) active 19 (43.18) inactive 25 (56.81) regular exercise, n (%) yes 12 (27.3) no 32 (72.7) education level, n (%) never attended school 8 (18.2) mosque 7 (15.9) primary/middle school 18 (40.9) secondary 7 (15.9) university 4 (9.1) smoking status, n (%) never smoking 93.2% ever smoking 2.3% current smoking 4.5% diabetes duration (months) 78.55 ± 58.668 (2-300) side effect, n (%) yes 5 (11.36) gastric problem (nausea) no 39 (88.63) henouda s et al. (2023). not sci biol 15(2):11554 6 table 2table 2table 2table 2.... effect of mo powder leaves on day 90 serum biochemical parameters levels in diabetic subjects r: reference values; nd: non-diabetic; cd: controlled diabetes; uc: uncontrolled diabetes. ldl-c (a, d3 vs d30 p = 0.001**; b, d7 vs d30 p = 0.027*; d0 vs d30 p =0.002**. hdl-c (a, d3 vs d30 p=0.002**; b, d7 vs d30 p=,027*; c, d0 vs d30; p = 0.01*; d, d3 vs d90 ; p=0.000*** ;e, d7 vs d90 ; p=0,006** ; f, d0 vs d90 ; p = 0.03*) *p< 0.05; ** p<0.01; *** p<0.0001 effect of mo powder on ldl-c during the twelve weeks mo powder leaves treatment, ldl-c levels of diabetic patients were measured on d0, d3, d7, d30, and d90, and the results are summarized in table 2. normality checks were carried out on sample data, which were normally distributed. repeated-measures anova with a greenhouse-geisser correction showed that mean ldl-c concentrations differed significantly across five time points [f(3.179, 111.256) = 8.683, p=0.000] (ldl-cd0: 0.97 ± 0.364 (0.30-1.89) vs. ldl-cd90 : 0.81 ± 0.423 (0.01-1.64)). post hoc analysis using the bonferroni correction revealed that ldl-c values were statistically significantly decreased by 0.414 g/l between d3 and d30 ((95% ci, p = 0.001), then reduced by 0.239 g/l between d7 and d30 (95% ci, p = 0.027), finally decreased by an additional 0.341 g/l on d30 (95% ci, p =0.002). no other differences are significant. effect of mo powder on hdl-c diabetic participants used mo powder leaves for 90 consecutive days. their hdl-c was measured before the special diet, after 3, 7, 30 and 90 days. normality checks were carried out on data, which were approximately normally distributed. a repeated measures anova with a greenhouse-geisser correction was performed to compare the effect of mo powder leaves on hdl-c values of diabetic participants. the results (table 2) show that there was a statistically significant effect of mo powder leaves on hdl-c concentrations (f(ddl=1.65, 61.061)=10,402, p = 0.000) (hdl-cd0: 0.43 ± 0.139 (0.17-0.99) vs. hdl-cd90 : 0.55 ± 0.234 (0.30-1.37)). post hoc test using the bonferroni correction showed an increased hdl-c levels between the initial assessment (d0) and follow-up assessment 30 days later by an average of 0.253 g/l (0.43 ± 0.139 vs 0.68±0.422, p =0.01 respectively) and then increased by 0.123 g/l between d0 and d90 (p = 0.03). therefore, we can conclude that the results of repeated measures anova indicate a significant temporal effect of treatment with mo powder leaves on increasing hdl-c levels of diabetic patients. biochemical parameters (g/l) days blood sugar (0.6-1.10) r tc (< 2.00) r ldl-c (< 1.50) r hdl-c (> 0.55) r tg (0.35-1.35) r hba1c (%) nd < 6 cd < 7 ud > 8 d0 1,89 ± 0,652 (0.90 -3.56) 1.71 ± 0.454 (0.92-3.09) 0.97 ± 0.364 (0.30-1.89) 0.43 ± 0.139 (0.17-0.99) 1.16 ± 1.121 (0.32-6.75) 10.03 ± 2.187 (6.20-15.10) d3 1.82 ± 0.581 (0.733.27) 1.64 ± 0.393 (0.91-2.66) 1.04 ± 0.383 **(0.24-2.30) 0.37 ± 0.134 (0.13-0.90) 1.06 ± 0.926 (0.26-5.30) / d7 1.78 ± 0.628 (0.70-3.15) 1.68 ± 0.439 (0.62-2.80) 0.87 ± 0.429 * (0.05-2.35) 0.41 ± 0.199 (0.06-0.93) 1.15 ± 0.882 (0.26-5.18) / d30 1.80 ± 0.640 (0.71-3.09) 1.63 ± 0.383 (0.82-2.45) 0.63±0.399 a (0.01-1.34) 0.68±0.422 abc (0.17-2.17) 1.11±0.789 (0.14-4.80) / d90 1.60±0.573 (0.71-2.99) 1.67 ± 0.398 (1.03-3.01) 0.81 ± 0.423 (0.01-1.64) 0.55 ± 0.234 def (0.30-1.37) 1.33 ± 0.676 (0.58-4.00) 9.20 ± 1.702 ***(6.5-13.10) henouda s et al. (2023). not sci biol 15(2):11554 7 effect of mo powder on tg the mo powder leaves treatment has no statistically significant effect on serum tg levels in diabetic patients (tgd0: 1.16 ± 1.121 (0.32-6.75 g/l) vs. tgd90: 1.33 ± 0.676 (0.58-4.00 g/l), p>0.05 (=0.052) (table 2). it should be noted that diabetic patients with moderate to severe hypertriglyceridemia at d0 had significantly decreased serum tg levels with mo powder administration by the end of the study (tgd0: 4.06 vs. tgd90: 1.86 and tgd0: 6.75 vs. tgd90 4 g/l, respectively). effect of mo powder on weight change in body weights in diabetic patients is shown in table 3. mo powder leaves has no statistically significant effect on patients’ weights (wd0: 74.69 ± 12.044 (45-105 kg) vs. wd90: 73.69 ± 11.839 (50-101kg), p>0.05). however, administration of mo powder leaves for 90 days improved the weight gain in diabetic patients who were underweight. effect of mo powder on bmi the study cohort showed a non-statistically significant bmi reduction at the end of the study, following oral administration of mo leaves powder (bmid0: 28.47 ± 4.701 (16.53-39.45) vs. bmid90: 28.07 ± 4.487 (18.37-38.21) p>0.05) (table 3). effect of mo powder on blood pressure patients in this cohort presented non-significant sbp/dbp reductions, following oral administration of mo leaf powder, at the end of the study (bpd0: 134.87 ± 22.927/78.46 ± 11.130 (100-190/60-100 mm hg) vs. bpd90: 126.41 ± 15.302/97.18 ± 117.315 (90-160/50-800 mm hg), p>0.05, table 3). table table table table 3333.... effect of mo on day 90 weights, bmi and hta in diabetic patients discussiondiscussiondiscussiondiscussion we successfully enrolled and maintained participation of diabetic patients in a trial that explored the effects of mo leaves powder on serum biochemical parameters levels and also on weight, bmi and blood pressure, for 90 days across five time points (d0, d3, d7, d30, and d90). the choice of the posology (sissoko et al., 2020) and the duration of its administration was based on scientific evidence, the advice of the diabetologists following the diabetic participants as well as the traditional healers in the region. no adverse effects were reported. based on various searches, this is the first scientific report evaluating the potential of mo leaves powder as antidiabetic, hypolipidemic, weight loss and antihypertensive agent originated from algeria. wight (kg) bmi (kg/m2) hta (mm hg) d0 74.69 ± 12.044 (45-105) 28.47 ± 4.701 (16.53-39.45) 134.87 ± 22.927/78.46 ± 11.130 (100-190/60-100) d3 74.72 ± 12.087 (45-105) 28.48±4.737 (16.53-39.86) 131.54 ± 20.843/76.67 ± 11.773 (100-190/50-100) d7 73.79±12.254 (45-105) 28.12±4.736 (16.53-39.45) 131.79 ± 22.580/76.67 ± 10.345 (100-190/50-100) d30 73.51 ± 11.573 (49-101) 28.01 ± 4.473 (18-39.04) 128.97 ± 21.126/74.36 ± 10.207 (100-190/60-100) d90 73.69 ± 11.839 (50-101) 28.07 ± 4.487 (18.37-38.21) 126.41 ± 15.302/97.18 ±11 7.315 (90-160/50-800) henouda s et al. (2023). not sci biol 15(2):11554 8 hypoglycemic effect at 90-day follow-up, treatment with mo leaves powder resulted in a lower glycemic response in diabetic patients, with no statistically significant differences. there was a trend, though not significant, toward a difference in reducing the rise in bg. these data, stemming mainly from the results of small sample study, suggest information on the hypoglycemic potential of mo leaf powder, require larger sample size to confirm this effect. a larger study of diabetic patients is needed to confirm this effect, and to investigate whether a larger dose would have a greater effect. however, daily administration of mo leaf powder for a period of 90 days had a statistically significant effect on hba1c values (p = 0.000), indicating that mo medication can induce with time better carbohydrate balance. it would also be interesting to evaluate its effect on postprandial bg levels in diabetic patients. several studies in diabetic rats have shown that mo leaf extracts have antidiabetic effect (kar et al., 2003; momoh et al., 2013), which might be mediated through the stimulation of insulin release leading to enhanced glucose uptake and glycogen synthesis (olayaki et al., 2015). among the mo leaves investigated compounds, alpha glucosidase inhibitor is a very crucial one in regulating carbohydrate and lipid metabolism (raptis and dimitriadis, 2001; adisakwattana and chanathong, 2011). moreover, qualitative phytochemical analysis of mo leaves has shown that they are also composed of proteins, dietary fibers, micro-minerals, flavonoids and triterpenoids, which are very effective in reducing blood sugar (leone et al., 2015). a daily intake of mo leaves powder may help to prevent hyperglycemia and hyperlipidemia. hypolipidemic effect the results of this study showed that the administration of mo powder leaves (3600 mg, twice a day) to diabetic patients for a period of 90 days seemed decrease the serum ldl-cholesterol values (p=0.004), which exhibited significant hypolipidemic effect on the 3rd, 7th, 30th days after treatment, suggesting that the mo powder leaves has role in management of hyperlipidemia on chronic administration that does not reach three months. but not those of total cholesterol and tg, this could be due to the reduced number of patients compared to studies which have confirmed its important role in the reduction of these two lipid parameters. however, treatment of diabetic patients with mo powder leaves in the present study showed a significant increase in hdl levels. hypercholesterolemia is one of the major risk factors for coronary and cardiovascular diseases in diabetic patients (ravi et al., 2005). in spite of the availability of several anti-hyperlipaemic agents, there is increase in their incidence and risk of congestive heart failure. therefore, there is still considerable interest in the evaluation of new synthetic and herbal anti-hyperlipidemic agents. preclinical studies suggest that mo powder leaves may have a lipid-lowering activity (ghasi et al., 2000; mehta et al., 2003), which could reduce this risk. increased hdl values are one of the required criteria of a good cholesterol-lowering agent that associated with a lower risk of heart disease. the hypolipidemic effect of mo leaves is due to the presence of the sterols, particularly β-sitosterol that appears to be a bioactive leaf phytoconstituent responsible for its hypolipidemic potential (rajanandh and kavitha, 2010). β-sitosterol lowers the cholesterol level by lowering plasma ldl concentration and by inhibiting the cholesterol reabsorption from endogenous sources in association with simultaneous increase in its excretion into faeces in the form of neutral steroids (ghasi et al., 2000). for the more, the mo leaves contain vitamin c, flavonoids and triterpenoids that are very effective to regulate lipogenesis in liver and control bg and cholesterol levels (leone et al., 2015). the change in the lipid profile with mo powder leaves treatment may contribute to the decreased incidence of atherosclerosis, cardiovascular and coronary heart disease. further investigations are needed to confirm these results and to specify the appropriate dose and duration of treatment to have its ideal cholesterol-lowering effect. in addition, exploration of circulating biochemical inflammatory markers should be performed, as the balance of pro and anti-inflammatory cytokines could assess the risk of complications, monitor disease progression, and guide treatment decisions of both diabetes and atherosclerosis (barbu et al., 2022). henouda s et al. (2023). not sci biol 15(2):11554 9 mo leaves are particularly interesting as a potential dietary intervention for diabetic patients with no risk of toxicity. giving mo leaf powder in capsules might be more convenient. pending the results of further research, it is best to encourage diabetic patients to incorporate mo into their daily diet if they are not already doing so. limitations this was a small pilot study due to logistical difficulties and the mentality of patients in the region because they are not used to participating in this type of study. this is the first pilot study in the adrar region. conclusionsconclusionsconclusionsconclusions the results of this study concluded that mo leaves powder, administered twice a day, seemed to reduce serum biochemical parameters levels in diabetic patients, particularly it had a significant effect in lowering longterm high blood sugar levels (hba1c rate). it seems that the mo leaf powder has long-term effect in diabetic patients. further investigations and clinical studies should be done with more diabetic patients to confirm the results obtained, and studying other parameters influencing the lipid and glycemic profile as well as the study of the reduction of long-term complications of diabetes. intervention to prevent diabetes should target both abnormalities defects in insulin secretion and insulin action. additionally, the bioactive constituent should also be identified. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization: sh, sk and ab; data curation: sh and aa; formal analysis: sh and aa; funding acquisition: sh; investigation: sh and aa; methodology: sh, sk, aa and ab; project administration: sh and ab; resources: sh and ab; supervision; sh, sk, aa and ab; validation; visualization: sh, sk and ab; writing original draft: sh, sk and aa; writing review and editing: sh, aa and ab. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) this study received approval no. 61/2021 from the ethics committee of tililane hospital. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. henouda s et al. 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(2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11360 doi:10.15835/nsb15111360 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae impact of magnetization of irrigation water on growth, yield and impact of magnetization of irrigation water on growth, yield and impact of magnetization of irrigation water on growth, yield and impact of magnetization of irrigation water on growth, yield and nutritional qualities of nutritional qualities of nutritional qualities of nutritional qualities of tomato under deficit irrigationtomato under deficit irrigationtomato under deficit irrigationtomato under deficit irrigation modupe r. baiyeri1, kamorudeen o. yusuf2*, rasheed o. obalowu1, ganiyu saad3, ibrahim k. banjoko4 1kwara state polytechnic, ilorin, department of agricultural and bio-environmental engineering, institute of technology, ilorin, nigeria; baiyerimodupe@gmail.com; abrashaola@gmail.com 2university of ilorin, department of agricultural and biosystems engineering, ilorin, nigeria; yusuf.ok@unilorin.edu.ng (*corresponding author) 3kwara state polytechnic, ilorin, department of electrical and electronics engineering, institute of technology, ilorin, nigeria; saadlekan@gmail.com 4kwara state polytechnic, ilorin, department of agricultural technology, ilorin, nigeria; frome2ukay@gmail.com abstractabstractabstractabstract tomato is a vegetable crop commonly grown in nigeria and consumed by most people. it contains vitamins and vital nutrients that are essential for good health. this study was conducted to determine the effect of magnetization of irrigation water on the growth, yield and nutritional qualities of tomatoes under deficit irrigation. tomato seeds (‘roma vf’ and ‘uc 82b’ varieties) were planted in 96 buckets (11 liters capacity), 48 buckets for magnetized water (mw) and 48 buckets for non-magnetized water (nmw), grown in a greenhouse and harvested 82 days after planting. the irrigation water was treated with 30 pieces of 10×25×50 mm neodymium magnets (1.2 tesla). the tomato plants were subjected to deficit irrigation to determine the effect on the performance of tomatoes at 100% (1 liter), 80% (0.8 liters), 60% (0.60 liters) and 50% (0.50 liter) water requirement. each treatment was replicated 6 times for both mw and nmw. the mw increased the growth of ‘roma vf’ and ‘uc 82b’ varieties by 5.44-38.10% and the stem girth by 21.13-49.01%. mw increased the yield of ‘roma vf’ variety by 110.00%, 36.00%, 6.26% and 24.00% for 100%, 80%, 60% and 50% of water application, respectively but increased the yield of ‘uc 82b’ variety by 56.52% for 100% water application. mw also improved vitamin a and c content by 7.89-27.94% and 0.45-19.06%, respectively. the concentrations of lead were slightly higher in the tomato irrigated with mw than in the nmw but values of other heavy metals were not consistent and very close for both mw and nmw. keywords:keywords:keywords:keywords: available water; deficit irrigation; drip irrigation; magnetized water; tomato; vitamin c; paired t-test introductionintroductionintroductionintroduction tomato (solanum lycopersicum l.) is a vegetable crop commonly grown in nigeria and consumed by most people in the country. it contains good nutritional values such as vitamins a, c, k, e and other vital nutrients essential for good health, prevent some diseases and proper functioning of the body systems. tomato https://www.notulaebiologicae.ro/index.php/nsb/index baiyeri mr et al. (2023). not sci biol 15(1):11360 2 is a protective food because it contains a high amount of antioxidants like lycopene, an antioxidant pigment responsible for tomato’s red colour and protects the body against diseases (singh et al., 2022). in nigeria, there is inadequate availability of fertilizer to improve soil fertility for growing crops, including tomatoes, inadequate availability of water during the dry season for irrigation and poor water quality typically affect the yield of tomatoes. erba et al. (2013) reported that tomato fruit's variety and ripening stage were the main factors affecting the nutritional values of tomatoes. ugonna et al. (2015) reported that nigerian tomato farmers are having problems with improved technology for growing tomatoes, inadequate and expensive inputs (such as hybrid seeds, fertilizer, insecticides, and herbicides), high postharvest losses, inadequate processing and marketing infrastructure, low yield and productivity of tomatoes. water availability to meet the evapotranspiration requirement typically affects growth of tomato plants, photosynthesis rate, fruit production and quality of tomato (sánchez-rodríguez et al., 2011; chand et al., 2020). ali et al. (2021) reported that tomatoes contain nutritional values such as minerals, vitamins, proteins, essential amino acids, monounsaturated fatty acids, carotenoids containing lycopene and other food essential compounds. ali et al. (2021) also pointed out that tomatoes have carotenoid containing lycopene and tomato-based food products to protect body against cancer, cardiovascular diseases, cognitive function and osteoporosis. therefore, there is a need for continuous research to improve the yield and nutritional qualities of tomatoes against the factors militating production, especially in some developing countries. irrigation water with magnetic field to become magnetized water could accelerate plant growth, enhance crop yield, save irrigation water and reduce the effect of deficit irrigation on crop growth and yield (kareem, 2018; abd-ellateef and mutwali, 2020; abou el-yazied, 2011). surendran et al. (2016) reported that magnetization of irrigation water improved cow pean’s growth rate and yield using magnetic flux densities of 1800 2000 g when the water was allowed to flow through the magnetic field for 10 minutes and a flow rate of 2 liters/s. kareem (2018) reported that irrigating tomato plant with magnetized water increased the water use efficiency (water productivity) by 78% and the yield of tomatoes by 52.68%. sadeghipour and aghaei (2013) also pointed out that magnetized water increased water use efficiency of cowpea by 22%. fakhri et al. (2018) reported that magnetized water increased tomato seeds' germination rate, accelerated plant growth, and increased tomato's fresh and dry weight. rawabdeh (2014) pointed out that magnetic treatment of irrigation water increased the uptake of essential elements (n, p and k) compared to plants irrigated with nonmagnetized water. magnetic treatment of irrigation water is a promising technology for improving crop productivity and it is a non-chemical method which could not cause soil degradation or pollute the environment. water is a paramagnetic substance with positive and negative charges and the orientation of the molecules of water changes under the influence of a magnetic field. babu (2010) reported that magnetized water has properties of reduced surface tension, smaller water molecules with tiny water clusters and increased solubility of the magnetized water to dissolve more macro and micro-elements which could make more nutrients available for plant growth. in addition, the magnetization of irrigation water enhanced tomato plants' absorption rate of water from the soil for evapotranspiration compared to non-magnetized water (yusuf and ogunlela, 2017). the effect magnetization of water by the magnetic field on the water structure depends on the magnetic flux density, time of exposure of magnetic induction, the flow rate of water through the magnetic field and temperature of the water with the range of magnetic flux density varied from 0.1 to 0.8 t (cai et al., 2009). tomato is a vegetable fruit that is commonly consumed globally by many people and consumption of vegetable is one of the sources of intake of heavy metals, which can cause cancer and other diseases to man (sultana et al., 2021). high concentrations of heavy metals such as cadmium, chromium, copper, lead, manganese, nickel and zinc in tomatoes could make them toxic for human consumption, which could affect the nutritional quality of the tomato (hellen and othman, 2014; sultana et al., 2021). tomato plants and other vegetable plants could easily absorb heavy metals from the soil and retain them in edible tomato fruits (hellen and othman, 2014; sultana et al., 2021). magnetization of irrigation water that enhanced better baiyeri mr et al. (2023). not sci biol 15(1):11360 3 absorption of plant nutrients from the soil could also increase the uptake of heavy metals by the tomato plant and affect the quality of the tomato. therefore, there is also a need to check the effect of magnetization of irrigation water on the concentration of heavy metals in tomato fruits and compare it with the fao/who permissible limits of heavy metals in vegetables to avoid the consumption of tomatoes that cause cancer and other diseases to man. drip irrigation is a method of irrigation in which water is applied directly to the point where the plant is planted or near the plant through an emitter or dripper fitted to a plastic pipe drop by drop (ranjan and sow, 2020). deficit irrigation is a system of irrigation in which the water requirement by the plant is not supplied. still, a certain percentage of water requirement, like 50%, 60%, 70%, 80% or 90% is applied to use the little available water for crop production. deficit irrigation (inadequate supply of water to plant) is normally practiced where water is limited to reduce the cost of irrigation water and is better practiced during the plant's vegetative growth. efereres and sorian (2006) defined deficit irrigation as the application of water below the evapotranspiration requirements of a plant. with the system of deficit irrigation, water demand for irrigation could be reduced and the water is saved which could be diverted for other uses. however, with deficit irrigation, the plant would under-go water shortage which could affect the growth rate and yield, especially during the flowing or fruiting stage. the objectives of this study were to determine the impact of magnetization of irrigation water on the growth, yield and nutritional qualities of ‘roma vf’ variety and ‘uc 82b’ variety tomatoes. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods location of the study the study was conducted at the institute of technology, kwara state polytechnic, ilorin, nigeria. ilorin lies on latitude 8°30n and longitude 4°35e, about 340 m above the mean sea level (akpenpuun and busari, 2016). ilorin is in the southern guinea savannah of the ecological zone of nigeria, with a mean annual rainfall of about 1,300 mm. the wet season starts at the end of march and ends in october while the dry season starts in november and ends in march (ogunlela, 2001). the mean minimum and maximum temperatures of ilorin are 18 °c and 38 °c, with a mean relative humidity of 77.50% and a daily mean sunshine hour of 7.1 h. description of greenhouse and buckets used for growing the tomato the greenhouse is a simple garden shed 10 m in length, 8 m wide, 3 m in height at the centre and 2 m high on both sides constructed using wood. the top of the greenhouse was covered with transparent nylon (2 mm in thickness) and 1 m of the sides of the greenhouse were also covered with the nylon from the top to prevent rain water from reaching the tomato during the experiment. in addition, all the sides of the greenhouse from bottom to 1 m high were covered with a screen (wire-mesh) to prevent the entrance of birds, rodents, goats and insects into the greenhouse. in addition, a door was provided for entrance and exit. the area covered with wire-mesh provides ventilation for the plant and there is an opening at the top of the greenhouse to exit of vapour and ventilation. the garden shed (greenhouse) is shown in figure 1. ninety-six (96) buckets were used to grow the two varieties of tomato (‘roma vf’ variety and ‘uc 82b’ variety) with 24 buckets for each variety for the magnetized and non-magnetized water. the bucket has a capacity of 11 liters, 263 mm in diameter and 235 mm in height. the bottom of each bucket contains five (5) holes drilled with 5 mm drill to allow excess water drainage from the bucket. the 96 buckets were filled with sandy loam to a depth of 210 mm and 25 mm to top was left in each bucket. the soil used in this study was obtained from the same place that is rich in organic matter and thoroughly mixed to have homogenous soil fertility. a total of 13 kg of the soil was put in each bucket giving a total of 1,248 kg of soil for experiment. the buckets were arranged with 8 rows and 12 buckets on a row with 6 buckets for ‘roma vf’ variety and 6 buckets baiyeri mr et al. (2023). not sci biol 15(1):11360 4 for ‘uc 82b’ variety as shown in figure 2. the physico-chemical properties of the soil are presented in table 1. figure 1figure 1figure 1figure 1. pictorial of the greenhouse figure 2figure 2figure 2figure 2. arrangement of the buckets with the soil and germinated tomato plants in the greenhouse table 1table 1table 1table 1. physico-chemical properties of the soil soil parameters value sand content (%) 78.00 silt content (%) 17.78 clay content (%) 4.22 bulk density (g/cm3) 1.262 ph 6.77 organic matter (%) 5.78 organic carbon (%) 2.95 nitrogen (%) 0.82 phosphrus (%) 10.58 calcium (mg/kg) 5.94 magnesium (mg/kg) 1.24 sodium (mg/kg) 6.39 potassium (mg/kg) 8.32 electrical conductivity (µs/cm) 511.27 baiyeri mr et al. (2023). not sci biol 15(1):11360 5 magnetization of the irrigation water magnetization of irrigation water means producing magnetized water with magnetic field for the tomato plants. the magnetized water was produced during the irrigation when the water flowed through the magnetic treatment unit (20 cm by 50 cm rectangular metallic pipe and 100 cm long) that was fixed into the pipe to supply water to the tomato plants in the greenhouse. neodymium magnet (n52 model) which is powerful with magnetic flux density ranging from 1.2 1.5 tesla (12,000 – 15,000 gauss), was used to produce the magnetized. thirty (30) pieces of neodymium magnets (10 × 25 × 50 mm) were used to produce the magnetized water. fifteen (15) pieces of the magnet were arranged on one side of the rectangular metallic pipe and the remaining 15 pieces of the magnet were arranged on the other side of the rectangular pipe as shown in figure 3. figure 3figure 3figure 3figure 3. pictorial view of the arrangement of the magnets on the rectangular metallic pipe when the water flows through the pipe, the magnetic field will flow across the water at a right angle to obey fleming’s right angle rule which states that the flow of water must be perpendicular to the flow of the magnetic field. if the flow of water is not perpendicular to the direction of the magnetic field but parallel to it or flowing in the same direction, the force of the magnetic field would not act on the water and there would be no effect of the magnetic field on the water. the irrigation water would not be magnetically treated. the magnetic flux density inside the rectangular magnetic treatment unit through which the water flows was measured to be 2,350 g using a gaussmeter (with model gm-2 by alpha lab inc). the water was treated through the magnetic field for 60 s during irrigation. the chemical properties of water before and after magnetization that were treated for 60 s, 120 s and 180 s are presented in table 2. table 2. table 2. table 2. table 2. chemical properties of the water before and after magnetization water parameters t1 t2 t3 t0 ph 8.000 7.450 7.950 8.100 n (%) 0.053 0.070 0.044 0.079 na + (mg/l) 1.695 1.680 1.715 1.720 k + (mg/l) 0.415 0.390 0.385 0.350 ca 2+ (mg/l) 1.070 0.665 0.645 0.480 mg 2+ (mg/l) 0.460 0.335 0.330 0.315 fe2+ (mg/l) 0.320 0.325 0.275 0.475 pb2+ (mg/l) 0.010 0.025 0.030 0.015 mn2+ (mg/l) 0.055 0.055 0.040 0.015 zn2+ (mg/l) 0.020 0.025 0.020 0.010 baiyeri mr et al. (2023). not sci biol 15(1):11360 6 cd2+ (mg/l) nd nd nd nd cr2+ (mg/l) nd nd nd nd t1 = magnetized water treated for 60 s, t2 = magnetized water treated for 120 s, t3 = magnetized water treated for 180 s, t0 = control (non-magnetized water), nd = not detected planting of tomato seed, irrigation of the tomato plant and determination of some plant parameters two varieties of tomato seeds (‘roma vf’ and ‘uc 82b’) were planted at about 20 mm depth in the buckets filled with sandy loam on the 24th of july, 2020. germination of the tomato started with 80% of water application on the 29th july, 2020 (5 days after planting). the quantities of water applied to the tomato plants in the buckets as the four treatments at 2 days irrigation interval were 100% of the water requirement (1 liter), 80% of the water requirement (0.80 liters), 60% of the water requirement (0.60 liters) and 50% of water requirement (0.50 liters). the plant growths were determined weekly for 5 weeks 14 days after planting for the two varieties of the tomato plant irrigated with magnetized water and non-magnetized water. first, the growth was determined by measuring the height of the plant from the soil base in the bucket to the terminal bud using a tape rule. next, the diameter of the stem (stem thickness) of the tomato plants was measured carefully 30 mm above the soil base using a vernier caliper. the diameters of the stem of the tomato plants were measured three (3) times, 19 days, 49 days and 71 days after planting. the pictorial view of the tomato plant 6 weeks after planting is shown in figure 4. figure 4. figure 4. figure 4. figure 4. pictorial view of the tomato plant 6 weeks after planting determination of yield of the tomatoes the tomato fruit was harvested 82 days after planting on the 13th of october, 2020. all the tomato fruits were harvested including the ripped and unripped two varieties of tomato plants because of the lockdown and order by the government that people should stay at home to prevent the spread of covid-19 in 2020. the yield of the tomato was determined based on the weight of the tomato fruits harvested from each treatment. the tomato fruits from each treatment were weighed on a weighing balance and recorded. determination of nutritional qualities and concentration of heavy metals in the tomatoes the protein, carbohydrate, crude fibre, moisture content, iron, ash, vitamin a and vitamin c contents of the tomato fruits were determined using the standard methods given by aoac (2000). the concentrations of some selected heavy metals were determined from the tomato fruits irrigated with magnetized water or nonmagnetized water to know if the magnetized water increased the uptake of heavy metals from the soil that could be above the permissible limit. the chromium, copper, iron, lead, manganese, nickel and zinc were determined using the standard methods given by apha (2005) and aoac (2000). baiyeri mr et al. (2023). not sci biol 15(1):11360 7 statistical analysis by paired t-test for the growth, stem diameter and yield of the tomato paired t-test was used to check if the effect of magnetized water were significant on the growth, stem diameter (stem girth) and yield of the tomato. first, the mean difference between the results of magnetized water and the control results was determined. then, the mean, the standard deviation, the standard error and the t-test values were determined using equations (1), (2), (3) and (4), respectively as given by (montgomery, 1998; yusuf et al., 2022). the data of the yield of tomato for the computation of the paired t-test was presented in table 3. n d d  = (1) ( ) 1 2 2 − − =  n dnd δ (2) n er δ δ = (3) er cal d t δ = (4) where d is the mean of the difference from the data x1 and x2, σd is the summation of d, n is the number of observations, δ is the standard deviation, δer is the standard error and tcal is the calculated value of the t-test (α = 0.05/2 = 0.025). table 3table 3table 3table 3. mean height of tomato plant (‘roma vf’ variety) for computation of the paired t-test tomato plant height mw (t1) tomato plant height nmw(t0) d = t1 t0 d2 58 42 16 256 230 192 38 1,444 303 265 38 1,444 825 722 103 10,609 983 894 89 7,921 n = 5 ∑d = 284∑d = 284∑d = 284∑d = 284 ∑d∑d∑d∑d2222 = 21,674= 21,674= 21,674= 21,674 t0 and t1:were defined in figure 2 8.56 5 284 ==d (1) ( ) 225.37 15 8.565674,21 2 = − − =δ (2) 648.16 5 225.37 == er δ (3) 412.3 648.16 8.56 == cal t (4) similarly, t2 versus t0 and others were calculated using the same method with the equations (1) to (4). baiyeri mr et al. (2023). not sci biol 15(1):11360 8 results results results results growth and stem diameter of the tomato plant the growths of the tomato plants after germination, measured 14 days after planting for 5 weeks are presented in tables 4 and 5. the growth rate in term of the heights of the plant 42 days after planting for ‘roma vf’ variety at 100%, 80%, 60% and 50% water application was 983 mm, 938 mm, 1,029 mm and 906 mm, respectively for tomato plant irrigated with magnetized water while the corresponding heights of tomato plant irrigated with non-magnetized water were 894 mm, 957 mm, 864 mm and 877 mm and shown in table 4. from table 5, the growths of the tomato plant for ‘uc 82b’ variety at 42 days after planting with magnetized water for 100%, 80%, 60% and 50% water application were 983 mm, 954 mm, 977 mm and 906 mm, respectively while the corresponding heights for tomato plants irrigated with non-magnetized water were 866 mm, 903 mm, 828 mm and 775 mm. magnetized water increased the growth rate of the tomato plants more than the tomato irrigated with non-magnetized water for ‘roma vf’ and ‘uc 82b’ varieties. the results of statistical analysis by paired t-test to know the effect of the magnetized water or magnetization of irrigation water on the growth of tomato plants for the two varieties are presented in table 6. the stem thickness (diameter of the stem) of the tomato plant for ‘roma vf’ variety is presented in table 7 and the results of statistical analysis by paired t-test are presented in table 8. the stem diameter of the tomato plant irrigated with magnetized water 71 days after planting for 100%, 80%, 60% and 50% water application were 79.7 mm, 75.4 mm, 60.2 mm and 40.2 mm, respectively but the corresponding heights of the tomato plant irrigated with non-magnetized water were 57.2 mm, 50.6 mm, 49.7 mm and 31.6 mm as shown in table 7. table 4table 4table 4table 4. . . . mean height of tomato plant (‘roma vf’ variety) irrigated with magnetized and non-magnetized water day after planting (days) height of tomato plant irrigated with mw (mm) height of tomato plant irrigated with nmw (mm) 100% 80% 60% 50% 100% 80% 60% 50% 14 58 59 133 112 42 72 60 55 21 230 226 239 231 192 206 224 214 28 303 315 363 323 265 292 311 276 35 825 796 813 722 722 770 734 703 42 983 938 1,029 906 894 957 864 877 mw = magnetized water, nmw = non-magnetized water 100% = irrigation applied at 100% of available water, 80% = irrigation applied at 80% of available water 60% = irrigation applied at 60% of available water, 50% = irrigation applied at 50% of available water table 5. table 5. table 5. table 5. mean height of tomato plant (‘uc 82b’ variety) irrigated with magnetized water and nonmagnetized water day after planting (days) height of tomato plant irrigated with mw (mm) height of tomato plant irrigated with nmw (mm) 100% 80% 60% 50% 100% 80% 60% 50% 14 93 46 50 47 48 59 49 16 21 249 233 239 229 206 230 217 238 28 289 298 291 345 289 314 299 319 35 812 782 814 722 690 764 721 668 42 983 954 977 906 866 903 828 775 mw, nmw, 100%, 80%, 60% and 50% were defined in table 4. baiyeri mr et al. (2023). not sci biol 15(1):11360 9 table 6. table 6. table 6. table 6. value of t-test for the growth of the tomato plant treatment degree of freedom calculated value of t table value of t at α ≤ 0.05 effect of the treatment on the growth of tomato plant ‘roma vt’ variety t1 versus t0 4 3.412 2.776 significant t2 versus t0 4 0.801 2.776 not significant t3 versus t0 4 2.236 2.776 not significant t4 versus t0 4 4.298 2.776 significant ‘uc 82b’ variety t1 versus t0 4 2.781 2.776 significant t2 versus t0 4 0.703 2.776 not significant t3 versus t0 4 1.704 2.776 not significant t4 vs t0 4 1.993 2.776 not significant table 7table 7table 7table 7. . . . mean stem diameter of tomato plant (‘roma vf’ variety) irrigated with magnetized water and non-magnetized water day after planting (days) stem diameter of tomato plant irrigated with mw (mm) stem diameter of tomato plant irrigated with nmw (mm) 100% 80% 60% 50% 100% 80% 60% 50% 19 39.0 33.2 30.1 29.0 37.4 30.2 30.2 20.0 49 63.3 58.7 56.2 40.2 50.6 49.7 42.3 40.7 71 79.7 75.4 60.2 40.2 57.2 50.6 49.7 31.6 mw, nmw, 100%, 80%, 60% and 50% were defined in table 4 table 8table 8table 8table 8. . . . values of t-test for the stem diameter of the tomato plant (‘roma vt’ variety) treatment with degree of freedom calculated value of t table value of t at α ≤ 0.05 effect of the treatment on the growth of tomato plant t1 versus t0 2 2.034 4.303 not significant t2 versus t0 2 1.888 4.303 not significant t3 versus t0 2 1.942 4.303 not significant t4 versus t0 2 1.838 4.303 not significant yield of the tomato fruits the yields of the tomato for ‘roma vf’ and ‘uc 82b’ varieties harvested once, 82 days after planting were presented in table 9, and the results of statistical analysis of the t-test of the effect of magnetized water on the yield were presented in table 10. the mean yield of tomatoes with ‘roma vf’ variety irrigated with magnetized for 100%, 80%, 60% and 50% water application were 315 g, 170 g, 170 g and 155 g, respectively while the corresponding values for the ‘roma vf’ variety irrigated with non-magnetized water were 160 g, 125 g, 169 g and 125 g. the yields of the tomato irrigated with magnetized water were all higher than those irrigated with non-magnetized water at all levels of water applied. for the ‘uc 82b’ variety, only the yield of tomato plant irrigated with magnetized water with 100% water application was higher at 150 g than the tomato irrigated with non-magnetized water with 100% water application with 115 g. the yields of tomato irrigated with non-magnetized water for 80%, 60% and 50% produced higher yields than those irrigated with magnetized water, as shown in table 9. the effect of magnetization of irrigation water for growing tomatoes was not statistically significant on the yield of the two varieties of tomatoes as shown in table 10. baiyeri mr et al. (2023). not sci biol 15(1):11360 10 table 9table 9table 9table 9. mean yield of tomato (‘roma vf’ and ‘uc 82b’ varieties) irrigated with magnetized water and non-magnetized water (corrected version of the results with red colour with too low yield) awp (%) tomato yield irrigated with mw (g) tomato yield irrigated with nmw (g) ‘roma vf’ variety ‘uc 82b’ variety ‘roma vf’ variety ‘uc 82b’ variety 100 315 180 150 115 80 170 155 125 210 60 170 210 160 255 50 155 130 125 185 awp = percentage of available water applied during the irrigation mw = magnetized water, nmw = non-magnetized water table 10. table 10. table 10. table 10. values of t-test for the yield of the tomatoes treatment degree of freedom calculated value of t table value of t at α ≤ 0.05 effect of the treatment on the growth of tomato plant ‘roma vt’ variety 3 1.790 3.182 not significant ‘uc 82b’ variety 3 0.949 3.182 not significant nutritional values and concentrations of heavy metals in the tomato fruits the nutritional qualities of the tomato were assessed based on the percentage contents of vitamin a, vitamin c, carbohydrate, protein, crude fat, fibre and the concentrations of the heavy metals in the tomato fruits harvested from the tomato plants irrigated with magnetized and non-magnetized water. the percentage of vitamin a, vitamin c, carbohydrate, protein, crude fat, fiber and moisture content are presented in tables 11 and 12 for the ‘roma vf’ variety and ‘uc 82b’ variety, respectively. the tomato fruit irrigated with 50% of the water requirement for ‘roma vf’ variety and the tomato fruits irrigated with 80% and 50% of water requirements for ‘uc 82b’ variety were mistakenly disposed of before the time of the nutritional analysis was done for the tomato and results were not included in the tables 11, 12, 13 and 14. based on the results presented in tables 11 and 12 for the nutritional values, the values of vitamin c with the tomato plant irrigated with magnetized water for 100%, 80% and 60% were 116.40, 116.20 and 112.10 mg/kg, respectively. in comparison, the corresponding values for the control (non-magnetized water) with 100%, 80%, 60% and 50% water application were 110.10, 97.62, 111.60 and 88.76 mg/kg, respectively. vitamin c and vitamin a contents were higher for the tomato plants irrigated with magnetized water than for tomato plants irrigated with non-magnetized water (control), as shown in table 11. tomato is a vegetable fruit and an essential source of vitamin c and magnetization of irrigation water increased the vitamins a and c contents of the tomato for the varieties as shown in tables 11 and 12. the carbohydrate and protein contents of tomatoes irrigated with magnetized water and non-magnetized water had almost the same values for the two varieties of tomatoes. the concentrations of the heavy metals in the tomato fruits are presented in tables 13 and 14 for the ‘roma vf’ variety and ‘uc 82b’ variety, respectively. magnetization of irrigation usually increases the uptake of some elements from the soil. still, it did not increase the uptake of the heavy metals more than the tomato irrigated with non-magnetized water. therefore, the values of the heavy metals (cr2+, cu2+, fe2+, mn2+, ni2+ and zn2+) assessed from the tomato fruits below the permissible limits of fao/who except for the concentration of lead (pb2+) that were above the permissible limits of fao/who for the two varieties as shown in tables 13 and 14. baiyeri mr et al. (2023). not sci biol 15(1):11360 11 table 11table 11table 11table 11. nutritional values of the tomato fruit (‘roma vf’ variety) to 2 decimal places instead 3 d.p parameters in the tomato fruit tomato fruit irrigated with mw tomato fruit irrigated with nmw 100% 80% 60% 50% 100% 80% 60% 50% moisture content (%) 88.70 90.42 88.56 89.26 88.63 90.02 90.00 crude fat (%) 0.37 0.49 0.42 0.46 0.31 0.39 0.43 fibre (%) 0.72 0.72 0.42 0.78 0.55 0.49 0.54 ash (%) 3.70 4.03 3.66 2.67 3.50 2.75 2.40 protein (%) 3.79 2.86 4.01 3.70 2.88 2.64 4.11 carbohydrate (%) 97.28 98.51 97.44 96.88 95.87 96.29 97.47 vitamin a (mg/kg) 0.87 0.82 0.75 0.68 0.76 0.64 0.60 vitamin c (mg/kg) 116.40 116.20 112.10 110.10 97.62 111.60 88.76 mw = magnetized water, nmw = non-magnetized water table 12table 12table 12table 12. . . . nutritional values of the tomato fruit (‘uc 82b’ variety) parameters in the tomato fruit tomato fruit irrigated with mw tomato fruit irrigated with nmw 100% 80% 60% 50% 100% 80% 60% 50% moisture content (%) 90.83 89.07 89.36 89.25 88.68 90.38 crude fat (%) 0.36 0.42 0.43 0.45 0.60 0.52 fibre (%) 0.66 0.42 0.54 0.59 0.64 0.66 ash (%) 2.84 2.61 3.51 3.24 4.16 3.48 protein (%) 2.91 3.18 3.79 2.09 2.97 2.57 carbohydrate (%) 97.61 95.87 97.59 95.62 97.05 97.61 vitamin a (mg/kg) 0.84 0.69 0.67 0.75 0.65 0.60 vitamin c (mg/kg) 116.20 112.30 110.20 98.00 111.00 88.76 table 13table 13table 13table 13. concentration of heavy metals in the tomato fruit (‘roma vf’ variety) conc. heavy metals in the tomato fruit fao/who (mg/kg) tomato fruit irrigated with mw tomato fruit irrigated with nmw 100% 80% 60% 50% 100% 80% 60% 50% chromium (mg/kg) 2.30 0.403 0.541 0.324 0.349 0.487 0.358 0.430 copper (mg/kg) 73.00 0.592 0.843 0.548 0.840 0.745 0.742 0.664 iron (mg/kg) 425.50 2.187 0.758 0.980 1.243 1.452 0.897 1.501 lead (mg/kg) 0.30 2.430 2.005 1.246 1.002 1.240 1.008 0.986 manganese (mg/kg) 1.552 1.860 2.055 2.544 2.345 1.756 1.652 nickel (mg/kg) 63.00 0.326 0.528 0.436 0.547 0.428 0.522 0.380 zinc (mg/kg) 100.00 0.261 0.243 0.248 0.450 0.345 0.320 0.302 table 14.table 14.table 14.table 14. concentration of heavy metals in the tomato fruit (‘uc 82b’ variety) conc. heavy metals in the tomato fruit fao/who 2001 (mg/kg) tomato fruit irrigated with mw tomato fruit irrigated with nmw 100% 80% 60% 50% 100% 80% 60% 50% chromium (mg/kg) 2.30 0.346 0.467 0.298 0.314 0.422 0.347 copper (mg/kg) 2.00 0.486 0.497 0.651 0.750 0.740 0.678 iron (mg/kg) 1.00 1.576 0.997 0.984 1.027 1.752 2.050 lead (mg/kg) 0.50 2.054 1.552 0.755 0.984 1.026 1.320 manganese (mg/kg) 5.00 2.455 2.097 2.025 1.548 1.755 0.902 nickel (mg/kg) 63.00 0.428 0.278 0.406 0.505 0.249 0.415 zinc (mg/kg) 1.50 0.264 0.262 0.400 0.259 0.310 0.252 baiyeri mr et al. (2023). not sci biol 15(1):11360 12 discussiondiscussiondiscussiondiscussion water is essential for tomato production, and the quantity of water applied below the water requirement of the tomato plant during irrigation affects the growth rate, stem diameter and yield of the tomato as shown in tables 4, 5, 7 and 9. magnetization of irrigation water increased the growth rate of the tomato plants for both the ‘roma vf’ variety and ‘uc 82b’ variety as shown in tables 4 and 5. the growth rate results were in agreement with the study of fakhri et al. (2018) that magnetized water increased the germination rate of tomato seeds and enhanced better growth of the tomato plant. in addition, the results of high growth rate and bigger stem diameter of tomato plant irrigated with magnetized water were in agreed with the results obtained by yusuf and ogunlela (2015) that magnetization of irrigation increased the growth rate and stem diameter of tomato plant. kareem (2018) also stated that magnetized water increased the growth rate, saved irrigation water and reduced the effect of water shortage (deficit irrigation) on plant growth. the effect of magnetization of irrigation water was statistically significant on the growth of the tomato plant for the ‘roma vf’ and ‘uc 82b’ varieties when 100% of water requirement was supplied, also significant at 50% water requirement for ‘roma vf’ variety but not significant for 80% 60% and 50% as shown in table 6. magnetizing irrigation water is a promising technology to improve crop yield, especially in some developing countries where fertilizers to improve soil fertility are rarely available. table 9; increased the yield of the tomato for the ‘roma vf’ variety at 82 days when the tomato fruits were all harvested. the result obtained for ‘uc 82b’ variety revealed that magnetization of irrigation water only increased the yield at 100% water requirement. however, 80%, 60% and 50% water requirements produced higher of tomato fruits when harvested 82 days after planting. the results of the yield obtained in this study were in agreement with the results of kareem (2018) that magnetized water increased the water use efficiency (water productivity) by 78%. in addition, it increased the yield of tomatoes by 52.68%. surendran et al. (2016) also reported that magnetized water enhanced the growth rate and increased the yield of cowpea. however, the effect of magnetized water was not significant on the yield for the varieties as presented in table 10. in tables 11 and 12 for the nutritional values of the tomato fruits, it was observed that magnetized water increased vitamin a and vitamin c. this was in agreement with the results yusuf and ogunlela (2016) that magnetization irrigation water increased the vitamin a and c contents of tomatoes. furthermore, rawabdeh (2014) reported that magnetized water increased the uptake of some essential elements (n, p and k) compared to plants irrigated with non-magnetized water. therefore, this could also improve the tomato's protein, vitamins a and c contents. the nutritional quality of the tomato could be affected by the concentration of heavy metals in the fruits. tables 13 and 14 show that the magnetization of irrigation water did not increase the concentrations of chromium, copper, iron, manganese, nickel and zinc compared to the tomato irrigated with nonmagnetized water. the concentrations of heavy metals in the tomato that could cause cancer and other diseases in men were below the permissible limits of fao/who (2001). the concentrations of lead (pb2+) in the two varieties of tomato irrigated with magnetized water and non-magnetized water were above the permissible limits of fao/who. the result was in agreement with yusuf and ogunlela (2016) that magnetized water increased the concentration of lead in the tomato. conclusionsconclusionsconclusionsconclusions magnetization of irrigation water increased the growth rate of the tomato plant. magnetized water increased the stem diameter and the growth rate and development of the tomato plant at various levels of water application (100%, 80%, 60% and 50% water requirement applied) compared to tomato plants irrigated with non-magnetized water. magnetized water for irrigating tomato plant increased the yield of ‘roma vf’ variety tomatoes by 110.00%. furthermore, it increased the yield of the ‘uc 82b’ variety by 56.62% when the water baiyeri mr et al. (2023). not sci biol 15(1):11360 13 requirement was fully supplied during the irrigation. in addition, tomato plants irrigated with magnetized water had higher yields under deficit irrigation at 80%, 60% and 50% water requirements applied. magnetized water also increased the vitamin a and vitamin c contents of tomatoes. it did not increase concentrations of heavy metals (chromium, copper, iron, manganese, nickel and zinc) in the tomato fruits. still, it slightly increased the concentration of lead (pb2+) in the tomato fruits. from this study, it is recommended that further studies should be carried out to determine the uptake of lead (pb2+) and its concentration in tomatoes irrigated with magnetized water. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements the authors appreciate the effort of the rector of the kwara state polytechnic ilorin for his efforts and support to get the grant of one million, six hundred and fifty thousand naira (n1,650,000 which is equivalent to 3,795 euro or usd 4290 in 2020 at the average official exchange rate of n434.78 per euro or n 384.62 per us dollar in 2020 when the grant was given to the research team) from the tertiary education trust fund (tetfund abuja, nigeria. the authors also appreciate the director of the institute of technology, kwara state polyechnic, ilorin and the authors also grateful to the head of departments of agricultural and bioenvironmental engineering, electrical and electronics engineering and department of agricultural technology for allowing member (s) of their staff to participate in the research. baiyeri mr et al. (2023). not sci biol 15(1):11360 14 conflconflconflconflict of interestsict of interestsict of interestsict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences abd ellateef mm, mutwali em (2020). effect of magnetized water on germination and some growth characters of broad bean (vicia faba l.), asian journal of agriculture and food sciences 8(4):98-102. https://doi.org/10.24203/ajafs.v8i4.6145 abou el-yazied aa, shalaby oa, el-gizawy am, khalf sm, el-satar a (2011). effect of magnetic field on seed germination and transplant growth of tomato. journal of american science 7(12):306-312. akpenpuun td, busari ra (2016). impact of climate on the yield of major tuber crops in kwara state, nigeria. global journal of agricultural science 16:61-65. http://dx.doi.org/10.4314/gjass.v16i1.8 ali my, sina aai, khandker ss, neesa l, tanvir em, kabir a, khalil mi, gan sh (2021). nutritional composition and bioactive compounds in tomatoes and their impact on human health and disease: a review. foods 10:45. https://dx.doi.org/10.3390/foods10010045 aoac (2000). official methods of analysis of the association of official analytical chemists. 15th edition, arlington, virginia, usa, 2000. apha (2005). standard methods for the examination of water and waste water, 21st edition. american public health association, washington. babu c (2010). use of magnetic water and polymer in agriculture. tropical research 8(806):d001. cai r, yang h, he j, zhu w (2009) the effects of magnetic fields on water molecular hydrogen bonds. journal of molecular structure 938:15-19. https://doi.org/10.1016/j.molstruc.2009.08.037 chang kt, weng ci. (2006). the effect of an external magnetic field on the structure of liquid water using molecular dynamics simulation. journal of applied physics 100: 043917. https://doi.org/10.1063/1.2335971 efereres e, sorian ma (2006). deficit irrigation for reducing agricultural water use. journal of experimental botany 113. https://doi.org/10.1093/jxb/erl165 erba d, casiraghi mc, ribas a, cáceres r, marfà o, castellari m (2013). nutritional value of tomatoes (solanum lycopersicum l.) grown in greenhouse by different agronomic techniques journal of food composition and analysis 31:245-251. https://doi.org/10.1016/j.jfca.2013.05.014 fao/who (2001). food standards programme: joint codex committee on contaminants in foods. fifth session, the hague, netherlands, fao/who, 21-25 march 2011, 1-90. hellen le, othman oc (2014). levels of selected heavy metals in soil, tomatoes and selected vegetables from lushoto district-tanzania, international journal of environmental monitoring and analysis 2(6):313-319. https://doi.org/10.11648/j.ijema.20140206.13 kareem nsa (2018) evaluation of magnetizing irrigation water impacts on the enhancement of yield and water productivity for some crops. journal of agricultural science and technology a 8:270-283. https://doi.org/10.17265/2161-6256/2018.05.002 montgomery dc, runger gc, hubele nf (1998). engineering statistics. john wiley and sons, inc, new york, pp 135248. ogunlela ao (2001). stochastic analysis of rainfall event in ilorin, nigeria. journal of agricultural research and development 1(1):39-49. https://doi.org/10.4314/jard.v1i1.42189 ranjan s, sumit s (2020). drip irrigation system for sustainable agriculture. newsletter 2(12):57-69. rawabdeh h, shiyab s, shibli r (2014). the effect of irrigation by magnetically chlorophyll and macroelements uptake of pepper (capsicum annuum l.). jordan journal of agricultural sciences 10(2):205-214. sánchez-rodríguez e, rubio-wilhelmi mm, blasco b, constán-aguilar c, romero l, ruiz jm (2011). variation in the use efficiency of n under moderate water deficit in tomato plants (solanum lycopersicum) differing in their tolerance to drought. acta physiologiae plantarum 33:1861-1865. https://doi.org/10.1007/s11738-011-0729-5 baiyeri mr et al. (2023). not sci biol 15(1):11360 15 sultana r, chamon as, mondol mn, tasnim, i (2021). metal concentration in commonly sold fruit vegetables in dhaka city market and probable health risk. dhaka university journal of biological sciences 30(1):35-47. https://doi.org/10.3329/dujbs.v30i1.51807 ugonna cu, jolaoso ma, onwualu ap (2015). tomato value chain in nigeria: issues, challenges and strategies. journal of scientific research and reports 7(7):501-515. yusuf ko, ogunlela ao (2016). effect of magnetically treated water on the quality of tomato, kathmandu university journal of science, engineering and technology 12(2):29-33. https://doi.org/10.3126/kuset.v12i2.21519 yusuf ko, ogunlela ao (2017). effect of magnetic treatment of water on evapotranspiration of tomato. arid zone journal of engineering, technology and environment 13(1):86-96. the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11345 nsb dib 2023.03.16.docx received: 08 sep 2022. received in revised form: 21 nov 2022. accepted: 06 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania dib m and benbott a (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11345 doi:10.15835/nsb15111345 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae ftir analysis and study of some physicochemical parameters and ftir analysis and study of some physicochemical parameters and ftir analysis and study of some physicochemical parameters and ftir analysis and study of some physicochemical parameters and antioxidant activity of antioxidant activity of antioxidant activity of antioxidant activity of opuntia ficus indicaopuntia ficus indicaopuntia ficus indicaopuntia ficus indica seed oil seed oil seed oil seed oil from tebessa region, algeriafrom tebessa region, algeriafrom tebessa region, algeriafrom tebessa region, algeria mouna dib, amel benbott* larbi ben m’hidi university, department of natural and life sciences, laboratory of natural substances, biomolecules and biotechnological applications, oum el bouaghi, algeria; malika1959@yahoo.fr (*corresponding author); dibmouna1991@yahoo.com abstractabstractabstractabstract opuntia ficus-indica is well-known in algeria for its nutritional and therapeutic properties. our study aims to determine the physicochemical properties, the functional groups and the antioxidant activity of cactus seed oil and thus contribute to the valorization of seeds. the extraction of prickly pear seed oil with cold-press provided a yield of 4.26± 0.93%. the evaluation of the its physicochemical parameters by the norms of afnor yielded the following results: density: 0.908± 0.00030 kg/dm; ri: 1.4714±0,00010; av: 1.4± 0.09 mg koh/g; iv: 114.08± 0.67 g i 2 /100 g; sv: 188.94± 0.58 mg koh / g; es: 187.54±0.49 mg koh/g; pv: 1.56 g o2 /g; chlorophyll number: 1.076 and carotenoids number: 0.315. ftir spectrum was used to determine the functional groups and type of associated vibrations in the extract oil, which was scanned in the range (4000-400) cm -1. the ftir results showed that the oil extract contains fatty acid methyl esters, revealing functional groups with ranges of properties, hc = o, -(ch2) n -, c-o, c = c and c = o in the spectrum. the analysis of antioxidant activity using dpph and frap methods showed that the ofi seed oil had a significant activity in capturing free radicals (ic50= 0.050 mg/ml), (ec50 = 0.123 mg/ml) respectively. we conclude from this current study that this oil can be used in folk medicine. keywords:keywords:keywords:keywords: antioxidant activity; ftir; opuntia ficus indica; physicochemical characteristics; seed oil introductionintroductionintroductionintroduction opuntia ficus indica (ofi) belongs to the family cactaceae (el-mustafa et al., 2014), native to the arid and semi-arid regions of mexico, but has moved from central and south america to the mediterranean basin, africa, the middle east, asia and india (shetty, 2012). in algeria, ofi is grown on several hectares in souk ahras and tebessa (neffar et al., 2011). for use in human food as fresh fruit for livestock feed or for fencing (chougui et al., 2013). opuntia species have been used in traditional medicine to treat chronic diseases, particularly diabetes, obesity, cardiovascular disease (santos-díaz et al., 2017), rheumatism, asthma, hypercholesterolemia, and hypertension (osuna-martınez et al., 2014). the plant has other biological activities including antioxidant, anticancer, anti-inflammatory, anti-allergic, hypocholesterolemic, and anti-aging activities (benayad et al., https://www.notulaebiologicae.ro/index.php/nsb/index dib m and benbott a (2023). not sci biol 15(1):11345 2 2014; ammar et al., 2015; barba et al., 2017; fiad et al., 2020; izuegbuna et al., 2019), these biological activities are due to the high level of active compounds such as polyphenols, vitamins (belhadj slimene et al., 2020), polyunsaturated fatty acids and amino acids (al-mustafa et al., 2014). research by andreu-coll et al. (2020) and khaled et al. (2020) shows that the seeds contain the highest concentration of bioactive compounds such as catechins, epicatechin, and ferulic acid. the oil extracted from the seeds is rich in tocopherols and unsaturated fatty acids with a predominance of linoleic acid (kadda et al., 2021). this study focused on the oil yield, physicochemical characteristics and antioxidant activity of cold-press ofi seed oil from tebessa cultivars. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant collection and identification during the prickly pear fruits harvest season, which represent the stage of full maturity of the plant, the fruits with a contrasting yellow-orange color, were picked at the end of ripening in august 2022 from the ouenza region (southeast of tebessa, algeria). the plant was identified by prof. y. halis researcher in scientific and technical research center on arid regions. to obtain ofi seeds, the fruits were washed with running water to remove impurities, then peeled to recover the pulp, after which we separated the seeds from the pulp using water and sieves while gradually reducing the pores by rinsing abundantly to get rid of the mucilage. finally, flat and very hard seeds were obtained, which were dried in a well-ventilated place away from light. oil extraction the extraction method chosen for our work is cold-pressure extraction, in which the seeds are passed through a screw press (ibg monforts oekotec), at a temperature of 30 °c, and this process does not require chemicals. this type of extraction is mainly used to produce virgin edible oils. the resulting oil is filtered to reduce phospholipids, which is desirable from a carbonation point of view. the oil extraction yield (y) was calculated according to akbari et al. (2019), through the following equation: y ( %) = mass of oil extracted(g) mass of seed powder used to make the extraction (g) x100 the oil is collected in a dark glass bottle, and stored in the refrigerator at 4 ℃ awaiting analysis. determination of the physicochemical characteristics of seed oils twelve (12) physicochemical parameters were studied, including ph, refraction index, rotational capacity, mixing with ethanol, soluble impurities content, water content (h2o%), acid values, saponification index, iodine index, peroxide value, ester index chlorophyll and carotenoids. ph the ph was measured directly in the sample using a ph meter form (adwa ad1000) density according to the methods (afnor, 1978), after cleaning the pycnometer with ethanol and acetone their empty weight corresponds: m0 two grams of distilled water were put in the pycnometer for 30 min, then this mass was weighed at 20 °c, this weight represents: m1 dib m and benbott a (2023). not sci biol 15(1):11345 3 two grams of oil were for 30 min in a water bath at 20 °c. the pycnometer filled with oil represents: m2 the density is calculated by the following formula: d= (m2_m0)/ (m1_m0) refractive index (ri) the refractive index of a material is one of the most important optical parameters. each oil has a specific refractive index measured at a certain temperature, less than 2 ml of sample is necessary and the result is delivered in a couple of seconds (hu et al., 2021). rotational capacity (rc) rotational capacity is the property that some chemicals exhibit in the plane polarization deviation of polarized light (hagen et al., 2016). in order to evaluate the rotation angles of ofi seed oil, we used a polar marker: karl kolb, fitted with a 1 cm cell filled with an ethanolic solution of oil at a rate of 0.2 g in 100 ml of solvent. the observed rotation angle is read directly on the instrument allowing to determine the value of the rotational force of our oil. insoluble impurity content this process involves the quantitative determination of dust and other insoluble foreign matter in nhexane. where a test sample containing an excess of n-hexane is treated and then filtered the resulting solution, washing the filter and litter with the same solvent, drying at 103 °c and then weighing the content (iso 663:2017) water content (h2o%) according to the method of gortzi et al. (2008), it was measured by drying at 105 °c for 24 hours in an oven model memmert and weighting the difference between the dry and wet samples. the results were expressed in percent (%). acidity value (av) measurement it is determined using the standardized ethanol method nf t 60-204 (afnor, 1993). approximately 10 g of the oil sample is weighed into the conical flask. the test portion is dissolved in approximately 100 ml of previously neutralized ethanol, carefully brought to near boiling point before use. titration is carried out, stirring vigorously with the 0.1 n potassium hydroxide solution until the phenolphthalein turns pink. saponification value (sv) measurement the saponification value is determined by taking 1.0 g of oil sample in a conical flask to which is added 15 ml 1 n koh and 10 ml of distilled water and heated under a reserved condenser for 30-40 min to ensure that the sample was fully dissolved. after this sample was cooled, phenolphthalein was added and titrated with 0.5 m of hcl until a pink endpoint was reached. a blank was determined with the same time conditions (zahir et al., 2014) iodine value (iv) measurement the iodine number is defined as the number of grams of iodine fixed per 100 g of fat. it was determined according to wijs’ method (wolff, 1968). according to the experimental protocol used, we add to the fatty substance in solution in chloroform an excess of iodine chloride, called wijs' reagent. after a few minutes of dib m and benbott a (2023). not sci biol 15(1):11345 4 reaction, potassium iodide and water are added distilled. the released iodine is titrated with a titrated solution of sodium thiosulfate (0.1n) in the presence of starch paste. peroxide value (pv) measurement the peroxide index of a fatty substance is the number of micrograms of active oxygen of the peroxide contained in one gram of oil or the number of mill equivalents of active oxygen per kilogram of fatty substance and oxidizing potassium iodide with release of iodine under the conditions of the nf t 60-220 method (afnor, 1993). determination of ester index (ei) the ester index is the number of milligrams of potassium hydroxide necessary to saponify the esters contained in one gram of fatty substance. the esterification index is given by the following relationship: esterification index = saponification index acid index (aoac, 1984) determination of chlorophyll and carotenoids the analysis of the pigments (chlorophylls and carotenoids) was determined according to mínguezmosquera et al. (1991) method. the absorbance of a flask filled up with7.50 g of oil mixed with 25 ml of pure cyclohexane was measured relative to that of the solvent at 670 nm for chlorophylls and at 470 nm for carotenoids. the pigment content was determined by the following formulas: chlorophyll content (mg/kg) = (a 670 x 106) / (613 x 100 x d) carotenoid content (mg/kg) = (a 470 x 106) / (2000 x 100 x d) d: represents the thickness of the cell (1 cm) fourier transform infrared (ftir) spectroscopy fourier transform infrared (ftir) spectroscopy was used to identify the functional groups of ofi seed oil. infrared spectra of the extract were recorded using a jasco ft ir-4700 fourier instrument. the analysis was performed as described by cebi et al. (2020) using an attenuated total reflection (atr) cell, in a spectral range of 4000-400 cm-1 with a resolution of 4 cm-1. biological activities dpph radical scavenging assay the varied extracts were tested for the antioxidant potential by using the technique outlined by benouchenne et al. (2020) with some modification. briefly, a 0.4 mm of dpph solution was prepared in methanol, 160 μl of this solution was added to 40 μl of sample diluted in methanol at different concentrations. after 30 min of incubation in the dark, the absorbance was measured at517nm. the results were given as 50% inhibition concentration (ic50) and compared with ascorbic acid. the low absorbance value indicates a higher free radical scavenging activity. the following equation was used to calculate the capacity to trap the dpph radical. inhibition ( %) = absorbance of control − absorbance of the sample absorbance of control x100 ferric reducing antioxidant power frap this method is based on the ability of extracts to reduce ferric iron (fe) to ferrous iron (fe 2+), established by ozgen et al. (2006). it consists of mixing 1 ml of each solution of extracts or the standard antioxidant (ascorbic acid) at different concentrations with 1 ml of phosphate buffer (0.2 m, ph 6. 6) and 1 ml of a 1% solution of potassium ferricyanide [k3fe (cn)6]). the mixture obtained is incubated at 50 °c for dib m and benbott a (2023). not sci biol 15(1):11345 5 20 min, and then 1 ml of 10% trichloroacetic acid (ccl3cooh) is added to stop the reaction. the total mixture was centrifuged at 3000 rpm for 10 minutes. after which, 1 ml supernatant was withdrawn from the test tubes and was mixed with 1 ml of distilled water and 0.5 ml of 0.1% iron chloride (fecl3) solution. the absorbance of the reaction mixture is read at 700 nm against a blank, which contains all reagents except fecl3. the reducing power of iron in the samples tested compared to the standards used is calculated according to the following formula: reducing power = absorbance of fecl3 − absorbance of fecl3 in the presence of the extract or standard absorbance of fecl3 x100 data processing the analysis of the results was carried out by microsoft® office excel 2010, and microcap origin 6.0 professional for the graphs. results results results results yield estimation and determination of some physicochemical properties of the oil we remember that the oil was extracted from the dry seeds of ofi by cold pressing; we got a yellow oil that smells good. we recovered a large amount of oil, and the yield obtained is estimated at 4.26 ± 0.93% (table 1). table 1.table 1.table 1.table 1. the oil extraction yield of ofi seeds type of measures analytical methods yield (%) color physical state of room temperature oil extraction cold-presse 4.26 ± 0,93 yellow liquid the results for the physicochemical properties of the oil from ofi seeds are presented in table 2. table 2table 2table 2table 2. physicochemical characteristics of the oil extraction physicochemical parameters measured seed oil of ofi ph 5.69 refraction index (nd, 20 °c) 1.4714± 0,00010 insoluble impurities content (g/100g) 1.22 ± 0,44 water content (h2o%), 0.325± 0,094 density (kg/dm3) 0.908± 0,00030 acid values (mg koh/g of oil) 1.4± 0,09 saponification index (mg koh/g of oil) 188.94± 0,58 iodine index (g i 2 /100 g of oil) 114.08± 0.67 peroxide value (meq o2/kg of oil) 1.56± 0.26 ester index (mg koh/g of oil) 187.54±0.49 chlorophyll (mg/kg) 1.076 carotenoids (mg/kg) 0.315 results are expressed as mean ± standard deviation ftir analysis ftir analysis spectra of ofi oil extract are shown in figure 1. in this study, oil analysis was performed using a ftir spectrophotometer with wave number 400-4000 cm-1 and scanned in absorption form. the result of the ftir analysis can confirmed that the sample contains functional groups, as the c-h stretch is shown in dib m and benbott a (2023). not sci biol 15(1):11345 6 table 3 related to the c-h aliphatic fatty acid stretch. the strong density of about 1742.50 cm-1 shows the co stretching of esters typical, and thus are common in fatty acid methyl esters spectral. the peak at 1462.81 cm-1 correspond to the asymmetric stretching of -ch3 present in the oil spectrum, in addition to other groups shown in the table 3 and figure 1. table 3table 3table 3table 3. frequencies of functional groups expected from ofi oil analysis wavelength number (cm-1) types of vibration assignment 3007.80 asymmetrical stretching =c-h (alkenes) 2921.75, 2953.92 asymmetrical stretching c-h 2852.57 symmetrical stretching c-h methylene (alkane) 1742.50 stretching c = o ester carbonyl in triglycerides and fame 1462.81 asymmetric stretching −ch3 1377.14 asymmetric stretching o−ch2 1158.36 stretching o-ch3 1098.25 stretching c-o absorption 721.25 bending of alkenes and overlapping of rocking vibration of methylene =c-h olefinic (alkene) group and –(ch2 ) n methylene groups (cis disubstituted alkenes and aromatic) figure 1. figure 1. figure 1. figure 1. ftir spectrum of ofi oil analysis dpph free radical-scavenging activity the profiles of anti-radical activity shown in figure. 2 yielded that the oil extracts of ofi had antioxidant activity, causing the reduction of dpph • radical to its non-radical form dpph-h; this results in a change from purple to yellow which has a visible absorption band at 517 nm. the oil extract from the seeds, at a concentration of 100 μg/ml., showed the highest effective percentage in removing dpph with an inhibition percentage of 84%, which is lower than that of the standard used (ascorbic acid) with an inhibition rate of 92%. dib m and benbott a (2023). not sci biol 15(1):11345 7 figure 2. figure 2. figure 2. figure 2. dpph free radical scavenging activities of 0fi oil extracts and ascorbic acid the ic50 inhibitory concentration is inversely proportional to the antioxidant capacity of a compound; it expresses the quantity of antioxidant required to reduce the concentration of the free radical by 50%. the smaller the ic50 value, the greater the antioxidant activity of a compound. the ic50 values for the oily and control extract are shown in figure 3. an oil extract shows very important antioxidant activity (ic50= 0.050 mg/ml) but remains weak compared to that of dpph-restoring ascorbic acid with an ic50 of 0.019 mg/ml. according to these results, ascorbic acid remains the most effective antioxidant compared to the oil extract of the studied plant. figure 3.figure 3.figure 3.figure 3. ic50 values of ascorbic acid and oil extracts iron reducing power test: frap (ferric reducing antioxidant power) by this test, the ability of the oil extract of ofi to transform ferric iron into ferrous iron was evaluated. this mechanism is known as an indicator of electron-donating activity, characteristic of anti-oxidant capacity. potassium ferricyanide is reduced to ferro-cyanide by polyphenols. in fact, the combination of ferric ions with ferro-cyanide leads to forming a complex of a green colour, which absorbs uv at 700 nm. furthermore, there is a proportionality relationship between the measured od and the antioxidant activity ((hinneberg et al., 2006).). the reducing power of iron by the oil extract is illustrated in figure 4. 0 20 40 60 80 100 12,5 25 50 100 in h ib it io n p er ce n ta ge s concentration µg/ml ascorbic acid oil extract 0 10 20 30 40 50 60 ascorbic acid oil extract 19,21 50,29 ic 5 0( ic 5 0( ic 5 0( ic 5 0( μμ μμ g /m l) g/ m l) g/ m l) g/ m l) extracts (extracts (extracts (extracts ( μμμμg/ml)g/ml)g/ml)g/ml) dib m and benbott a (2023). not sci biol 15(1):11345 8 figure 4. figure 4. figure 4. figure 4. iron-reducing power of ascorbic acid and oil extracts through the experimental results shown in figure 4, it was found that there is a competitive relationship between increasing the concentration and the reversibility of different extracts, due to the presence of reversible compounds (antioxidants) such as vitamin c. they were the basis of reference for this study, showing good reversibility of many free radicals. the highest absorption value was 1.3 at a concentration of 1 mg/ml, while the highest absorption value was estimated for the oily extract 1.19 at the same concentration. compared with standard, the ec50 of oil (ec50: 0.123 mg/ml) are very closer to ascorbic acid (ec50: 0.063mg/ml) (figure 5). figure 5figure 5figure 5figure 5. ec50 values of ascorbic acid and oil extracts discussiondiscussiondiscussiondiscussion yield assessment seed oil extraction the extracted oil is yellow in colour. this indicates that they contain pigments that allow them to obtain this color. our results showed that the seed oil content from ofi in the tebessa region was estimated at 4.26 ± 0.93%, a value close to that reported by nounah et al. (2021), where the productivity of ofi in the sidi ifni region of morocco was estimated to be (4.1%), but it is considered low compared to the productivity obtained by de wit et al. (2017), whose oil contents in cultivars ranged between 5.65% and 8.09% in south africa. 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 0,2 0,4 0,6 0,8 1 1,2 a bs or ba n ce a t 70 0n m concentration (mg/ml) oil extract ascorbic acid 0,123 0,063 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 oil extract ascorbic acid e c 50 ( m g/ m l) extracts dib m and benbott a (2023). not sci biol 15(1):11345 9 physicochemical characteristics of ofi seeds oil iodine value (iv) ofi seed oil from tebessa shows a high iv (114.08) due to its high content of unsaturated fatty acids. the value in this study is higher than those reported by khémiri and bitri (2019) which was estimated 108.52 (cactus pear seed oils). de wit et al. (2021) reported slightly higher iodine values ranging between 120.58 (tormentosa) and 127.43 (zastron), and it is lower than that of ofi and o. megacantha in morocco (132.81132.73) (ettalibi et al., 2021). de wit et al. (2017) showed that the iodine index could show the stability of the oil, because a high degree of unsaturation indicates a high susceptibility to oxidation. we conclude that lower iodine value indicates greater resistance to oxidation due to increased saturation. peroxide value (pv) pv is a very useful criterion for assessing the first stages of oxidative deterioration of an oil. the value of the peroxide index found in this study is of the order of 1.56 ± 0.26 meq o2/kg, this value is a good indicator of the relative oxidation stability of the extracted oil. our results show that the high iodine value and oxidation stability of the seed oil support the good qualities of edible oil. saponification index (si) the saponification index of a fatty substance is higher if the carbon chain of the fatty acid is short, the si of the obtained oil was estimated as (188.94 ± 0.58 mg koh/g) which is close to that of cottonseed oil (189198), and sunflower (188-194), which are commonly used in food (aïssi, 2009). the saponification value determined is similar to that reported by zine et al. (2013) (186.63), and is higher than that obtained by r'bia et al. (2017) (175.20). density the density of the obtained oil was estimated as 0.908 which is close to that determined by özcan and al juhaimi (2011) (0.907), and it is higher than that reported by r'bia et al. (2017) (0.805) in its results. acidity acidity is related to the amount of free fatty acids, which informs about the quality of conservation of an edible oil. the values of the acid for the samples of ofi oil is 1.4 mg koh / g. this value is comparable with that reported by r’bia et al. (2017); khémiri and bitri (2019). pigments contents the chlorophyll content (1.076 mg/kg) of the oil studied is strictly greater than that of carotenoids (0.315 mg/kg). the chlorophyll content is less than 2 ppm. this low content is desired to avoid the pro-oxidant action of chlorophyll pigments and to ensure good preservation of the oils (boulfane and coll, 2015). the chlorophyll and carotenoid contents of this oil is close to those reported by marhri et al. (2022) (1.52 mg/kg0.61 mg/kg), and it is lower than those found by other studies (el mannoubi et al., 2009) (2.403 mg/kg -8.01 mg/kg) (khémiri and bitri, 2019) (10. 52mg/kg 4.75mg/kg). ester index (ei) the ei value of ofi oil, estimated at (163.50 ± 0.19) mg koh/g oil, is close to the saponification index value (186.59 ± 0.63) mg koh/g oil. dib m and benbott a (2023). not sci biol 15(1):11345 10 ftir fourier transforms infrared (ftir) spectroscopy is an ideal method for analysis of complex mixtures such as edible fats and oils and other secondary metabolites extracted from specific part of plants (muchtaridi et al., 2019). the major functional groups identified here are the frequency of 3007.80 cm-1 that indicates the presence of the unsaturated olefin double bond group with a medium intensity (zhang et al., 2012). this was closely followed by the saturated aliphatic groups of 2921.75 cm-1 with its asymmetrical stretching and strong intensity as well as 2852.57 cm-1 with its associated symmetrical stretching and strong intensity (coates, 2000). the ir regions of 2953.92 ;2852.57, 1742.50 cm-1 are attributed to the fatty acid chain length and the absorption of free fatty acids (ffas) occurring within the range of 1720–1700 cm-1, which is used to estimate the hydrolysis of triglycerides to ffas (bertran et al., 1999). there is another major absorbance near 1742.50 cm-1 due to the ester carbonyl functional group of the triglycerides. the ch asymmetrical bending vibration of 1462.81 cm-1 can be ascribed to –ch3 methyl of the alkane group. the frequency of 1418.02 cm-1 corresponded with the in plane bending of the vinyl c – h or cis = c – h bond of the olefinic group (coates, 2000; zhang, 2012) and it is associated with a strong intensity. the peak at 1377.41 cm-1 can be attributed to the glycerol group o−ch2 (mono-, diand triglycerides), which is present in the refined oil spectrum and should be absent in the fame spectrum (dube et al., 2004) the frequency stretch of 1296.27 cm-1 to 1031.21 cm-1 was assigned to the c – o ester carbonyl group with its characteristic strong intensity and stretching vibration. the bands of 1236.53 and 1158.36 cm-1 have been proved to be related to the proportion in the sample of saturated acyl groups (guillen et al., 1997). the frequency of band 1098.25 cm-1 does not generally suffer large variations except in oil samples rich in oleic acyl groups. as the oxidation process advances, the frequency of this band diminishes and reaches a minimum value (liang et al., 2013). the frequency range of 997.58– 602.42 cm-1 was ascribed to the = c – h trans or cis – disubstituted alkene group. thus, it can be said that the functional groups present here are ch2 and ch3 of the saturated aliphatic compounds, the c = o of the dimerized carboxylic acid group, the c – o ester carbonyl group and the trans or cis – disubstituted alkene group (oyerinde and bello, 2016). antioxidant activity according to the literature, the results of our antioxidant activity are much lower (ic50= 0.050 mg/ml) than those reported by other authors, where berraaouan et al. (2015) demonstrated the antioxidant activity of cold-pressed ofi oil with the dpph assay (ic50= 0.96 mg/ml). karabagias et al. (2020) showed that pure prickly pear seed oil has a high anti-oxidation activity with a percentage of (84 ± 0.010%), which is similar to that obtained. in addition, the present results are consistent with those of ramírez et al. (2017), who reported significant antioxidant activity with respect to seed oil obtained from two mexican prickly pear. ofi seed oil obtained, showed high reducing power with lower ec50 than the oil extracted from different spice seeds of o. microdasys (1.11 mg/ml) and o. macrorhiza (0.60 mg/ml); produced by chahdoura et al. (2015). in another study, chougui et al. (2015) showed that prickly pear peels were more resistant to oxidation than the reference with vitamin e. conclusionsconclusionsconclusionsconclusions we conclude from the physical and chemical study that cold-pressed ofi seed oil can be converted into edible oil with relative oxidation stability (peroxide value = 1.56 ± 0.26 meq o2/kg), ), and low acidity (1.4 mg koh/g), which indicates a low decomposition point for high content of unsaturated fatty acids (high iodine value 114.08). the low content of chlorophyll (1.076 mg/kg) ensures that the oils are well preserved. dib m and benbott a (2023). not sci biol 15(1):11345 11 ftir spectroscopy specifically indicates the presence of the olefinic group with a frequency (3007.80 cm-1 and 1418.02 cm-1), and the triglyceride group is shown in the carbonyl region (1742.50 cm-1). ofi seed oil exhibits good antioxidant power and is useful in many diets, in pharmacology and in the cosmetic industry. more studies are needed to identify the main compound responsible for the antioxidant activity of prickly pear seeds. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions the ab took the lead in methodology, evidence organization, resource sourcing and oversight, dm participated in the design and also provided critical feedback on the manuscript and assisted in its revision and editing. both authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements we thank prof. y. halis researcher in scientific and technical research center identified the plant for arid areas (strc, biskra). for their help in identification of plant. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. 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distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations. dib m and benbott a (2023). not sci biol 15(1):11345 15  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11546 nsb bacila 2023.06.26.docx received: 05 apr 2023. received in revised form: 16 jun 2023. accepted: 20 jun 2023. published online: 26 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania băcilă i et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11546 doi:10.15835/nsb15211546 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae epidemiology of epidemiology of epidemiology of epidemiology of enterococcus faeciumenterococcus faeciumenterococcus faeciumenterococcus faecium isolates sampled from different isolates sampled from different isolates sampled from different isolates sampled from different sources in romania using mlst technique and eburst algorithmsources in romania using mlst technique and eburst algorithmsources in romania using mlst technique and eburst algorithmsources in romania using mlst technique and eburst algorithm ioan băcilă1a, endre jakab2,3,5b, dana şuteu1*, octavian popescu4,5,6 1national institute of research and development for biological sciences, institute of biological research, department of experimental biology; 48 republicii st., 400015 cluj-napoca, romania; ioan.bacila@icbcluj.ro; dana.suteu@icbcluj.ro; dana.suteu@icbcluj.ro (*corresponding author) 2babeș-bolyai university, faculty of biology and geology, hungarian department for biology and ecology, 5-7 clinicilor st., 400006 cluj-napoca, romania 3babeș-bolyai university, centre for systems biology, biodiversity and bioresources, 5-7 clinicilor st., 400006 cluj-napoca, romania; endre.jakab@ubbcluj.ro; 4institute of biology bucharest of the romanian academy, 296 splaiul independenței st., bucharest, 060031, romania 5babeș-bolyai university, molecular biology centre, interdisciplinary research institute on bio-nano-sciences, 42 treboniu laurian st., cluj-napoca, 400394, romania 6babeș-bolyai university, emil g. racoviță institute, 5-7 clinicilor st., cluj-napoca, 400006, romania; opopescu.ubbcluj@gmail.com a,bthese authors contributed equally to the work abstractabstractabstractabstract enterococcus faecium is emerging as an important cause of multidrug resistance and hospital acquired infections, special attention being paid to the vancomycin resistant species. therefore, the characterization of pathogenic strains/isolates plays an important role in the epidemiology of infectious diseases. the enterococcal rate was determined from wastewaters in cluj-napoca area. as presence of e. faecium was detected, a number of isolates from wastewater, birds and humans were epidemiologically analyzed according to the mlst website. comparisons were performed against a collection of available isolates, with multiple origins, contained in the mlst database. out of the enterococcus isolates collected from wastewater, 11 were identified as e. faecalis (40.74%); 8 as e. casseliflavus (29.62%); 5 as e. faecium (18.50%); 2 as e. gallinarum (7.40%) and one isolate as e. durans. based on the mlst data and using the eburst algorithm, the isolates of e. faecium sampled from romania were split in three groups: one group comprised isolates from human hosts and wastewater (cj316, 106/6, cj197, cj22, 129/6, cj117, cj24, 284/7, and 43/7), while the second (g9, g10-2, g7, g3-2, and g9-1) and the third group (g8, g6, and 40/7) originated from bird hosts. the rest of the isolates were not joined in a particular group, assuming the lack of a phylogenetic bond between these isolates. the obtained data suggested the existence of at least two phylogenetic lines of e. faecium in romania: a line that had mainly human host prevalence, while in the other line the animal hosts dominated. keywords:keywords:keywords:keywords: eburst; enterococcus faecium; epidemiology; mlst https://www.notulaebiologicae.ro/index.php/nsb/index băcilă i et al. (2023). not sci biol 15(2):11546 2 introductionintroductionintroductionintroduction bacteria of the genus enterococcus (formerly the ‘fecal’ or lancefield group d streptococci) are ubiquitous microorganisms. they occur in large numbers in different types of soil, surface waters, vegetables, plant material and foods, especially those of animal origin (giraffa, 2014), but have a predominant habitat in the gastrointestinal tract of humans and animals. e. faecium and e. faecalis are the predominant gram-positive cocci in human stools, while e. faecium is the prevalent species in production animals like poultry, cattle, and pig, and e. mundtii and e. casseliflavus are found in plant sources (klein, 2003). from an ecological point of view, the distribution of enterococcus species varies throughout europe: in spain and the uk e. faecalis and e. faecium are the most commonly isolated species from both clinical and environmental sources, in sweden e. faecium has a lower incidence and e. hirae a higher isolation rate, whereas in denmark e. hirae is the dominant species, isolated mainly from slaughtered animals (kühn et al., 2003). enterococcus species play an important role in food industry (dairy production, storage of meat and vegetables foulquié moreno et al., 2006) or as probiotics to treat diarrhea and improve immunity (franz et al., 2011). nevertheless, some species of enterococcus, such as e. faecalis and e. faecium, were also reported to be associated with many infections, including urinary tract infection, bacteremia, endocarditis, neonatal infection and infection of the central nervous system (o’driscoll and crank, 2015). a much bigger problem is represented by the fact that, with the extensive use of antibiotics, enterococcus species have developed resistance to many antibiotics (zhong et al., 2017). currently, e. faecium is emerging as an important cause of multidrug resistance and hospital acquired infections, special attention being paid to vre (vancomycin resistant enterococcus) (lebreton et al., 2014; adegoke et al., 2022; correa-martínez et al., 2022; toc et al., 2022). even if e. faecalis is responsible for about 80% of all enterococcal infections in humans, while e. faecium causes only about 20% of the infections, e. faecium represents the majority of the vre (sievert et al., 2013). ecology and epidemiology studies of enterococcus have reported that e. faecalis and e. faecium are being regularly isolated from cheese, fish, sausages, minced beef, and pork (klein, 2003; foulquié moreno et al., 2006). the characterization of pathogenic strains plays an important role in the epidemiology of infectious diseases, generating the necessary information for the identification, tracking and intervention against epidemics (tavanti et al., 2005). mlst (multilocus sequence typing) is one of the techniques used in global epidemiology, aiming to identify strains with a high pathogenicity and, thus, providing an improved picture of the activity of bacteria within environment and human populations. studies on microbial populations using the mlst technique are generally intended to estimate the genetic diversity (usually counted as the relative contribution of recombination and mutations per allele or per locus), as well as evaluate the relative impact of genetic dispersion and natural selection in the evolutionary history of these pathogens (stefani and agodi, 2000; pérez-losada et al., 2005). mlst is based on the sequence of housekeeping genes which exhibit in each strain a distinct numerical allelic profile, abbreviated to a unique identifier: the sequence type (st). the relatedness between two strains can be then inferred by differences between the allelic profiles (francisco et al., 2009). the possible patterns of evolutionary descent obtained within mlst can be further analyzed by eburst. eburst represents an advanced algorithm that involves partitioning the large number of mlst data sets into groups of related, non-overlapping sts, clonal complexes, and then discerns the best-fit pathway within each clonal complex to the founder genotype (spratt et al., 2004). thus, following a set of very simple rules, eburst can be used to find out how the diversification of bacterial clones has occurred and to provide evidence for the emergence of clinically relevant clones (feil et al., 2004). the aims of this study were i) – to determine the enterococcal ratio in wastewater from the cluj-napoca area; ii) to determine using mlst the genetic relatedness of randomly selected e. faecium isolates from wastewaters, birds and humans, and confront the results against an international mlst database. băcilă i et al. (2023). not sci biol 15(2):11546 3 materials and methodsmaterials and methodsmaterials and methodsmaterials and methods bacterial isolates sampled from wastewater in cluj-napoca area. cultures and identification between june 2006 and august 2008, 27 water samples were collected from the someşeni wastewater treatment plant in cluj-napoca, romania. residual water samples were isolated in pre-sterilized containers and analyzed within 6 hours of isolation, according to international standards. the water samples divided into 6/100 ml portions were subjected to filtration on a 0.45 μm pore diameter membrane. to ease the filtration process and to reduce the number of colonies, the samples were diluted with h2o uv/up, the initial dilution being 1:4, followed by a subsequent dilution of 1:9. after the filtering process, the samples were placed directly on the culture medium in petri dishes. two types of selective culture media were used. the filters were initially placed on petri dishes containing m enterococcus agar (mea) (fluka, buchs, switzerland), because the sodium azide, captan and nalidixic acid contained in this formula, are known to inhibit the growth of many species of bacteria and fungi, conferring selectivity to the medium. after casting the plates, 300 μl of 1% ttc solution (2,3,5-triphenyl tetrazolium chloride) were added to the surface of the medium. within the bacterial cell, ttc was reduced to insoluble formazan, which conferred a dark pink color to the colonies. cultivation of filters on mea was performed in the oven, for 48 hours at a temperature of 41 °c. as a safety measure, with respect to the identity of the cultivated microorganisms, cultivation was carried out on a second type of medium, namely esculin iron agar (fluka, buchs, switzerland). this second medium was used to confirm the identity of the colonies based on the ability of enterococci to hydrolyze esculin. the procedure consisted in transferring the m enterococcus agar filter membranes onto esculin iron agar and incubating them in the oven at 41 °c for 20 minutes. the hydrolysis of esculin resulted in black or dark brown colonies, confirming the presence of enterococci. for species identification of enterococcal isolates (table 3) the following methods were used: classical biochemical identification by observing hemolysis pattern on blood agar and two standardized methods: api® 20 strep from biomérieux (a kit includes strips that contain up to 20 miniature biochemical tests for manual identification of microorganisms) and vitek (a fully automated system which performs bacterial identification). dna extraction of the bacterial isolates sampled from wastewater in cluj-napoca area the bacterial isolates were cultivated on lb (luria-bertani) liquid media overnight at a temperature of 37 °c. in order to disrupt the cell walls, a digestion with lysozyme (sigma, st. louis, usa) and proteinase k (promega, madison, usa) was performed, following the protocol described in băcilă et al. (2007). dna purification were performed using wizard genomic dna purification kit (promega, madison, usa), according to the manufacturer’s recommendations. dna quality was estimated on a 1% agarose gel stained with ethidium bromide, and dna concentration was quantified using a nanodrop 1000 spectrophotometer (thermo fisher scientific, wilmington, usa). identification of enterococcus faecium isolates by pcr the predominant species in cultures are: e. faecalis and e. faecium. to distinguish between the two species of enterococci, one fragment of the ddl gene was amplified within pcr. the amplification reaction was carried out on isolates from wastewater (one sample from bucharest was added to the 27 samples previously collected from cluj-napoca area), as well as on isolates from other sources: human and birds (table 1). as positive controls, the strains e. faecium attc 35667 and e. faecalis attc 51299 were used. the pcr program was the following: initial denaturation for 2 min at 94 °c, followed by 30 cycles of 1 min at 94 °c, 1 min at 54 °c, and 1 min at 72 °c; and a final extension for 10 min at 72 °c. the specific primers were: ddlf1: 5’-gcaagccttcttagaga-3’ and ddlf2: 5’-catcgtgtaagctaacttc-3’ (dutka-malen et al., 1995). băcilă i et al. (2023). not sci biol 15(2):11546 4 mlst technique and dna sequencing applied on enterococcus faecium isolates sampled from different sources and different parts of romania a number of 32 isolates of e. faecium with different origins were analyzed with mlst and eburst. the samples were taken from the wastewater (five isolates including one from bucharest and four out of the 27 samples previously collected from cluj-napoca area), from birds (12 isolates collected from their faeces) and from humans (15 isolates collected from hospitalized persons: samples of secretions of infected wounds, blood or peritoneal fluid) (table 1). sequence types of e. faecium isolates were determined employing a mlst scheme, performing amplification of housekeeping genes by pcr and subsequent sanger sequencing of the pcr products, as previously described by homan et al. (2002). table 1table 1table 1table 1. list of enterococcus faecium isolates used in mlst analysis e. faeciume. faeciume. faeciume. faecium isolate isolate isolate isolate code code code code originoriginoriginorigin sampledsampledsampledsampled ssssampling ampling ampling ampling locationlocationlocationlocation year of isolationyear of isolationyear of isolationyear of isolation g5 birds faeces cluj 2007 g6 birds faeces cluj 2007 g7 birds faeces cluj 2007 g8 birds faeces cluj 2007 g9 birds faeces cluj 2007 g10 birds faeces cluj 2007 g12 birds faeces cluj 2007 g2-2 birds faeces timişoara 2007 g3-2 birds faeces timişoara 2007 g5-2 birds faeces timişoara 2007 g9-1 birds faeces timişoara 2007 g10-2 birds faeces timişoara 2007 105/6 human blood bucharest 2006 106/6 human blood bucharest 2006 113/6 human blood bucharest 2006 129/6 human blood bucharest 2006 156/6 human blood bucharest 2006 40/7 human blood bucharest 2007 43/7 human blood bucharest 2007 283/7 human blood cluj 2007 284/7 human blood cluj 2007 cj117 human wound secretion cluj 2008 cj197 human peritoneal fluid cluj 2008 cj316 human peritoneal fluid cluj 2008 18/7 human blood oradea 2006 19/7 human wound secretion oradea 2006 41/6 human blood timişoara 2006 cj14 wastewater cluj 2008 cj20 wastewater cluj 2008 cj22 wastewater cluj 2008 cj24 wastewater cluj 2008 b176 wastewater bucharest 2003 băcilă i et al. (2023). not sci biol 15(2):11546 5 the typing scheme used regions of seven structural genes (gdh glucose-6-phosphate dehydrogenase; purk 5-(carboxyamino) imidazole ribonucleotide synthase; pst phosphate atp-binding cassette transporter; atpa atp synthase, alpha subunit; gyd glyceraldehyde-3-phosphate dehydrogenase; adk adenylate kinase; ddl d-alanine ligase). these genes are well-conserved structures in the bacterial genome, so the likelihood of mutations over time is rather low, allowing them to be successful tools in the accurate genetic characterization of certain pathogenic bacterial species, as precise identification of infectious agent strains is essential for both epidemiological studies and public health decisions. the sequences of each amplified gene fragment were compared to all previously identified (alleles) sequences for that locus, thus determining the number of alleles for all seven loci. combining the number of alleles from all the loci defined the allelic profile of the strain. each different allelic profile was considered a st-sequence type. such a st represented a suitable and clear label for each isolate. characterization of bacterial isolates was achieved by comparing the sequences obtained with the sequences existing in the databases. the pcr method is particularly precise, safe and time-efficient, allowing a large number of samples to be analyzed in a relatively short time. the difficulty encountered was the setting of optimal temperatures for the amplification of genetic material. optimal boost temperatures varied in a fairly wide range, starting at 47 °c for the purk gene and reaching 59 °c for the ddl gene (table 2). the primers required to amplify fragments belonging to the seven structural genes are also presented in table 2. table 2table 2table 2table 2. primers used for gene amplification in mlst technique and their annealing temperature namenamenamename sequence (5’sequence (5’sequence (5’sequence (5’----3’)3’)3’)3’) annealing temperature (annealing temperature (annealing temperature (annealing temperature (°c) gdh1 5’-ggc gca cta aaa gat atg gt-3’ 52 gdh2 5’-cca aga ttg ggc aac ttc gtc cca-3’ purk1 5’-gca gat tgg cac att gaa agt-3’ 47 purk2 5’-tac ata aat ccc gcc tgt ttc/t-3’ psts1 5’-ttg agc caa gtc gaa gct gga g-3’ 50 psts2 5’-cgt gat cac gtt cta ctt cc-3’ atpa1 5’-cgg ttc ata cgg aat ggc aca-3’ 48 atpa2 5’-aag ttc acg ata agc cac gg-3’ gyd1 5’-caa act gct tag ctc caa tgg c-3’ 58 gyd2 5’-cat ttc gtt gtc ata cca agc-3’ adk1 5’-tat gaa cct cat ttt aat ggg-3’ 58 adk2 5’-gtt gac tgc caa acg att tt-3’ ddl1 5’-gag aca ttg aat atg cct tat g-3’ 59 ddl2 5’-aaa aag aaa tcg cac cg-3’ the amplification reaction was performed in a 50 μl volume including: 1x pcr buffer; 1.5 mm mgcl2; 0.2 mm of each dntp; 1 μm forward primer; 1 μm reverse primer; gotaq polymerase 1.25 u; and 6 μl genomic dna. the following amplification program was used: initial denaturation for 3 min at 94 °c, followed by 35 cycles of 30 sec at 94 °c, 30 sec at 47-59 °c, and 30 sec at 72 °c; and a final extension for 5 min at 72 °c. prior to sequencing, a purification step with promega wizard sv gel and pcr clean-up system (promega corporation, madison, usa) was carried out according to the manufacturer’s manual. sequencing of both strands was performed using dtcs quick start master mix; 3.2 μl primer (1 pmol/µl); 0.5-10 μl of template dna; and 0-9.5 μl h2o uv/up. the following thermal cycle parameters have been used: 96 °c 2 min; 40 cycles of 96 °c 20 sec, 50 °c 20 sec, 60 °c 4 min. excess primers and labelled ddntps were removed by purification with dna clean & concentrator™ (zymo research, orange, usa) according to the standard recommended by the manufacturer. the samples (total volume 20 μl) were prepared prior to sequencing by băcilă i et al. (2023). not sci biol 15(2):11546 6 adding 20 μl of hidi formamide and then were loaded onto ceq 8000 genetic analyzer (beckman-coulter, fullerton, usa). data analysis after performing the sequencing reactions, the sequences were analyzed using bioedit (hall, 1999) and eburst (http://www.eburst.mlst.net). the purpose of eburst algorithm was to identify, based on sequence data, groups of isolates with related genotypes in population, and, then, to establish the founding genotype of each group. in the next step, the algorithm determined also the descendants of the founding genotype, which would be able to represent foundational genotypes for other subgroups of bacterial isolates. the graphical form of the analyses performed by the eburst algorithm was a radial diagram centered on an ancestral founding genotype. results results results results species identification of enterococcal isolates sampled from wastewater in cluj-napoca area the identification of enterococcal species isolated from wastewater was quite difficult to achieve and required both a constant repetition of the performed analyses and a combined use of several analysis methods. 27 isolates were identified in the collected samples of wastewater and the results obtained by a classical method have been confirmed by at least one standardized method (table 3). table 3table 3table 3table 3. identification of enterococcal isolated sampled from wastewater isolateisolateisolateisolate classical identification classical identification classical identification classical identification (biochemical series)(biochemical series)(biochemical series)(biochemical series) apiapiapiapi®®®® 20 strep 20 strep 20 strep 20 strep (biomérieux)(biomérieux)(biomérieux)(biomérieux) vitekvitekvitekvitek cj01 e. casseliflavus e. casseliflavus cj02 e. faecalis e. faecalis 99.9% cj03 e. faecalis e. faecalis 99.9% cj04 e. faecalis e. faecalis 99.9% cj05 e. faecium e. faecium 94.3% cj06 e. faecalis e. faecalis 99.9% cj07 e. faecalis e. faecalis 99.9% cj08 e. faecalis e. faecalis 99.9% cj09 e. faecalis e. faecalis 99.9% cj10 e. faecalis e. faecalis 99.9% cj11 e. faecalis e. faecalis 99.9% cj12 e. faecalis e. faecalis 99.9% cj13 e. casseliflavus e. casseliflavus 99.9% cj14 e. faecium e. faecium 97.5% e. faecium 94.26% cj15 e. casseliflavus e. casseliflavus 99.9% e. casseliflavus 99.9% cj16 e. faecalis e. faecalis 99.8% e. faecalis 99% cj17 e. casseliflavus e. casseliflavus 99.9% cj18 e. casseliflavus e. casseliflavus 99.9% cj19 e. casseliflavus e. casseliflavus 91.56% cj20 e. faecium e. faecium 94.8% e. faecium 99% cj21 e. casseliflavus e. casseliflavus 97.41% cj22 e. faecium e. faecium 97.8% e. faecium 98.5% cj23 e. durans e. durans 93.8% e. durans 99% băcilă i et al. (2023). not sci biol 15(2):11546 7 cj24 e. faecium e. faecium 97.9% e. faecium 99% cj25 e. gallinarum e. gallinarum 99% cj26 e. gallinarum e. gallinarum 85.66% cj27 e. casseliflavus e. casseliflavus 98.06% the percent indicate the degree of confidence in accuracy of the results the dominance of e. faecalis and e. faecium in the wastewater system was documented by previous studies that have examined species distributions of enterococcus in wastewater (blanch et al., 2003; moore et al., 2008). moreover, ferguson et al. (2013) in a study performed on marine water and wastewater, found that e. faecalis was the dominant species in a ratio of over 90%, while e. faecium was present in a ratio of about 5%, the last 5% being represented by other enterococcal species. nevertheless, in the present case, the species hierarchy differed from expectance (figure 1). e. faecalis was placed on the first position with 11 identified isolates (40.74% from the total of analyzed isolates), on the second place was the species e. casseliflavus represented by eight isolates (29.62%), e. faecium was represented by only five isolates (18.50%), whereas the rest of the species were identified as e. gallinarum two isolates (7.40%) and e. durans one isolate (3.70%). figure 1.figure 1.figure 1.figure 1. enterococcal ratio of species (%) isolated from wastewaters identification of the enterococcus faecium species by pcr this genetic identification method for species of enterococcal isolates was based on the specific amplification of a d-alanine-d-alanine ligase-encoding fragment (ddl). following migration of the pcr products on 1% agarose gel, in case of e. faecium isolates amplification of a 550 bp fragment was obtained, whereas no amplification was visible for e. faecalis isolates (figure 2). băcilă i et al. (2023). not sci biol 15(2):11546 8 figure 2figure 2figure 2figure 2. agarose gel pattern showing amplification of a ddl gene fragment which signaled the presence of e. faecium 1: e. faecium cj14; 2: e. faecium cj20; 3: e. faecium cj22; 4: e. faecium cj24; 5: e. faecium attc 35667 (positive control); m: molecular marker 1 kbp (axygen biosciences, union city, usa); 6: e. faecium g5; 7: e. faecium 106/6; 8: e. faecium 283/7; 9: e. faecalis attc 51299 (positive control); 10: e. faecalis cj02; 11: e. faecalis cj12; 12: negative control (no dna) the isolates codes are the same as in tables 1 and 3 the amplification reaction was performed post species identification by classical, biochemical, and automated phenotypic methods (table 3), as a confirmation of the previous results. inference of the phylogenetic relationships between enterococcus faecium isolates sampled from romania using the mlst approach and eburst algorithm allelic variation and genetic diversity of enterococcus faecium isolates a number of 32 isolates of e. faecium from different sources were included in the mlst assay (table 1). for these isolates a variable number of unique locus alleles was obtained, ranging from four unique alleles for the adk gene to 12 unique alleles for the atpa gene (table 4). table 4table 4table 4table 4. allelic variation in seven structural genes from enterococcus faecium isolates locuslocuslocuslocus length of sequence (bp)length of sequence (bp)length of sequence (bp)length of sequence (bp) number of unique allelesnumber of unique allelesnumber of unique allelesnumber of unique alleles gdh 530 9 purk 492 6 pst 583 6 atpa 556 12 gyd 395 7 adk 437 4 ddl 465 6 following the analysis of 32 isolates of e. faecium, the existence of 31 sts was established. isolates 41/6 and 105/6 exhibited the same allelic profile. both isolates originated from humans, being sampled from the same medium blood. surprisingly, isolates 105/6 was isolated in bucharest, while isolates 41/6 was isolated in timișoara. a number of five isolates showed 100% similarity for six of the analyzed loci, with differences appearing only in atpa locus. these isolates were: cj22, 106/6, 197, cj316, and 113/6. the source of these isolates differed: isolate cj22 originated from wastewater and was isolated in cluj-napoca, isolates 106/6 and 113/6 were of human origin and have been collected from blood in bucharest, the other two isolates, cj197 and cj316 were sampled from peritoneal fluids collected from patients hospitalized in cluj-napoca. băcilă i et al. (2023). not sci biol 15(2):11546 9 another number of five isolates of e. faecium presented 100% similarity for five out of seven analyzed loci. of these, isolates 43/7 and 129/6 were sampled in bucharest from blood. the other three isolates derived from cluj-napoca, cj24 isolate being collected from wastewater, while cj117 and 284/7 isolates are of human origin, collected from plague and blood. there was also a batch of eight isolates which exhibited a 100% similarity for four out of seven loci, despite the fact that sources of origin are quite heterogeneous: isolates 18/7 and 19/7 are of human origin, being collected in oradea from blood, respectively plaque discharge; isolates cj20 and 283/7 were taken from cluj-napoca, but from different sources (wastewater and human blood); isolate b176 was sampled from wastewater in bucharest; the remaining three isolates are of animal origin, being sampled from poultry faeces collected in timișoara (g3-2 and g9 isolates) and cluj (g10). the rest of the isolates showed 100% similarity for three or less of the analyzed loci. assortment of enterococcus faecium isolates sampled from romania based on the mlst data (sequence data, allele profiles) and subsequent analysis with eburst algorithm, the division of e. faecium isolates sampled from romania revealed three groups. the first group included cj316, 106/6, cj197, cj22, 129/6, cj117, cj24, 284/7, and 43/7 isolates (figure 3a); the second group included g9, g10-2, g7, g3-2, and g9-1 isolates (figure 3b); and the third group comprised g8, g6, and 40/7 isolates (figure 3c). the rest of the isolates (g5-2, g5, g2-2, 283/7, b176, 156/6, 41/6, cj20, 19/7, 18/7, cj14, g10, and g12) were not joined by eburst in a particular group, assuming the lack of a phylogenetic bond between these isolates. the lack of framing of these isolates was due to the small number of common loci. figure 3figure 3figure 3figure 3. grouping of enterococcus faecium isolates using eburst algorithm. a) isolates cj316, 106/6, cj197, cj22, 129/6, cj117, cj24, 284/7, and 43/7; b) – isolates g9, g10-2, g7, g3-2, and g9-1; c) isolates g8, g6, and 40/7 inference of phylogenetic relationships between enterococcus faecium isolates sampled from romania and from other parts of the world using the mlst database and the eburst algorithm the next step after determining the phylogenetic relationships between the e. faecium isolates sampled from romania, was to assign these isolates to the general group of e. faecium isolates sampled from different parts of the world. the isolates collected from romania were introduced into the mlst database, and after applying the eburst algorithm, the sequent ratio revealed the division of the isolates into 20 distinct groups (data not shown). subsequently, a radial dendrogram of the group 1 mlst was generated (figure 4). băcilă i et al. (2023). not sci biol 15(2):11546 10 the following romanian isolates were included in group 1 mlst: cj316, 106/6, cj197, cj22, 129/6, cj117, cj24, 284/7, and 43/7. isolates cj24, 106/6, cj22, cj197, and cj316 represented slvs (single locus variants), whereas the other four isolates were dlv (double locus variants). the other romanian isolates were distributed into two separate mlst groups, respectively group 5 mlst and group 11 mlst (data not shown). this grouping confirmed the results obtained by applying the eburst algorithm exclusively for isolates from romania, when these isolates formed groups 2 and 3 (figure 3 b, c). discussiondiscussiondiscussiondiscussion enterococcus species distribution in wastewater the dominance of e. faecium and e. faecalis is well documented in previous studies which examined the distribution of enterococcus species in wastewater systems (sinton and donnison, 1994; blanch et al., 2003; moore et al., 2008). similarly, the prevalence of e. casseliflavus is also consistent with clinical human fecal samples (ruoff et al., 1990; stern et al., 1994). nevertheless, the enterococcal rate is very variable, depending on various factors such as: degree of urbanization, number of hospitals, and so on. while performing a study on a hospital wastewater treatment plant, karimi et al., (2016) found that out of 315 enterococci isolates, 162 (51.42%) were identized as e. faecium, 87 (27.61%) as e. hirae, 35 (11.11%) as e. faecalis, 11 (3.5%) as e. gallinarum, 7 (2.22%) as e. casselieavus, and 4 (1.26%) as e. avium. băcilă i et al. (2023). not sci biol 15(2):11546 11 figure 4figure 4figure 4figure 4.... dendrogram of group 1 of enterococcus faecium isolates from mlst database. green color is used to highlight the isolates sampled from romania included in this group. assortment of enterococcus faecium isolates sampled from romania a firstfirstfirstfirst groupgroupgroupgroup of enterococcal isolates was formed after eburst analysis around cj316 isolate (figure 3a). this isolate was sampled from peritoneal fluid of human source collected in cluj-napoca. interestingly, among the slvs belonging to this isolate, the isolate cj197 (sampled from the peritoneal fluid collected from patients hospitalized in cluj-napoca) was noted, indicating a significant relationship between these two isolates. another interesting connection was based on the observation that other two slvs of the cj316 isolate are collected from the wastewaters from cluj-napoca, cj22 and cj24. this observation led to the assumption of a connection between the isolates collected in the hospital environment and those taken from the environment (here represented by wastewaters). băcilă i et al. (2023). not sci biol 15(2):11546 12 other close connection, in terms of origin, within this first group was also observed on the dlv level, involving two isolates sampled in cluj, namely the isolate cj117, sampled from human wound secretion, and isolate 284/7, collected from human blood. the other three slvs of the group were represented by the isolates 43/7, 106/6, and 129/6. these isolates were also connected by the place of origin, all three isolates being taken from samples collected from patients from bucharest, two from the blood (106/6 and 129/6) and 43/7 from sputum. a second groupsecond groupsecond groupsecond group of e. faecium consisted of five isolates, all of animal origin, sampled from poultry faeces taken from cluj and timișoara. according to the dendrogram (figure 3b), the central isolate of this group was g9, sampled from cluj. the group contained three slvs, two of them being isolated in timișoara and one in cluj. the only dlv was represented by the isolate g9-1, collected in timișoara. the central isolate of the third groupthird groupthird groupthird group (figure 3c) is the g8 isolate, of animal origin. the group had two slvs represented by the isolates g6 (sampled in cluj from bird faeces) and 40/7 (collected from human blood in bucharest). based on data obtained by querying the mlst database and the use of the eburst algorithm, some general conclusions regarding the local epidemiology could be drawn for the romanian isolates of e. faecium. thus, for 17 isolates, straightforward connections in terms of their origin were established. however, for 14 isolates (45.65%) the phylogenetic pattern could not been so easily explained. some possible answers could include (but are not limited to) the high genetic variability of bacterial isolates (martínez-carranza et al., 2018), the high degree of genetic recombination (shikov et al., 2022), the horizontal genetic transfer between bacterial isolates (heuer and smalla, 2007), and genetic dispersion (oh et al., 2010). inference of phylogenetic relationships between enterococcus faecium isolates sampled from romania and from other parts of the world subsequent to the eburst analysis of 1387 isolates of e. faecium sampled from different parts of the globe, 20 groups of isolates were obtained. the main group was constituted of 1135 isolates. the founder genotype of this group was an isolate with a st=17 (figure 4) and an allelic profile of 1 1 1 1 1 1 1. according to the database, the first isolate with st=17 was collected from a patient in the united kingdom in 1992. this lineage (st=17) was characterized by ampicillin resistance, pathogenicity island, and was associated with hospital outbreaks (willems et al., 2005). the distribution of these isolates (st=17) was higher in europe, probably due to the existence of more extensive studies: 47 isolates in germany, 23 isolates in the uk, 20 isolates in netherlands, 15 isolates in italy, 4 isolates in france, 3 isolates in greece, 2 isolates in denmark and poland, whereas only one isolate was isolated from hungary, norway, and serbia. by comparison, few isolates with st 17 were identified on other continents: 11 isolates on the australian continent, 4 isolates in the north america (usa), while in south america (brazil) only one isolate (willems et al., 2005). the source of e. faecium st=17 isolates was in an overwhelming proportion the hospitalized patients. still, there were also some exceptions, in australia (two cases) and in the uk and france (one case each), where the source of the isolates was represented by people in outpatient treatment. furthermore, in two cases found in 2001 in the usa, the isolates were taken from the environment. concerning the romanian isolates, the following isolates were included in group 1 mlst: cj316, 106/6, c197, c222, 129/6, c117, cj24, 284/7, and 43/7. the isolates cj24, cj22 (both from cluj wastewater), 106/6 (from human blood in bucharest), cj197, and cj316 (both from human peritoneal fluid collected from patients hospitalized in cluj) represent slvs emerging directly from the founding genotype st=17. the other four isolates represented dlvs, but were not joined in the same subgroup. the isolates cj117 (human wound secretion, cluj) and 129/6 (human blood, bucharest) were part of the same dlv. isolates 284/7 (human blood, cluj) and 43/7 (human sputum, bucharest) belonged to two different dlvs. băcilă i et al. (2023). not sci biol 15(2):11546 13 the presence of five lines of e. faecium sampled from romania as slv, pertaining directly to the st=17 complex, is particularly alarming because st=17 is a predictor of subsequent bacteremia in hospitalized patients carrying vref and has been frequently isolated from nosocomial settings (kim et al., 2021). conconconconclusionsclusionsclusionsclusions out of 27 enterococcus isolates sampled from wastewater, 11 isolates were identified as e. faecalis (40.74%); 8 isolates as e. casseliflavus (29.62%); 5 isolates as e. faecium (18.50%); 3 isolates as e. gallinarum (7.40%) and one isolate as e. durans. by applying the eburst algorithm for e. faecium isolates sampled from romania, three groups were delineated mainly on origin source. thus, group 1 joined isolates from human sources and environment (wastewater), whereas groups 2 and 3 comprised isolates originated mainly from birds. this pattern suggested the existence of at least two phylogenetic lines of e. faecium isolates in romania: a line that had mainly the human host prevalence, and a second line with mainly animal host prevalence. the first phylogenetic line highlighted the existence of a clear connection between the isolates of human origin and those of wastewater origin. this connection was most likely determined by the uncontrolled spills in flowing water networks, which underlined a direct link between the hospital environment and the surrounding environment (wastewater in the present case). the results of our analyses pointed out that the degree of isolate differentiation revealed by mlst and eburst was high enough to be used in epidemiological investigations. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions the contributions of authors to the manuscript are as follows: conceptualization: op, ib; field work: ib, ej; data curation: ib, dș, ej; formal analysis: ib; funding acquisition: op; investigation: ib, ej; methodology: ib; project administration: op; writing original draft: ib; writing review and editing: ib, dș, ej, op. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements the authors are thankful to the staff of someșeni wastewater treatment laboratory from cluj-napoca and the cantacuzino institute from bucharest for help in collecting and identifying enterococcal samples. this work was partly supported by ceex 28/2005 from the national authority for scientific research, romania, as well as by the core project bioresgreen, subproject bioclimpact no. 7n/03.01.2023, code 23020401. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. băcilă i et al. 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(2023). not sci biol 15(2):11546 16 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11459 nsb beppe 2023.03.16.docx received: 26 jan 2023. received in revised form: 09 mar 2023. accepted: 10 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania beppe gj et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11459 doi:10.15835/nsb15111459 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae antidepressant and anxiolyticantidepressant and anxiolyticantidepressant and anxiolyticantidepressant and anxiolytic----like activities of the like activities of the like activities of the like activities of the dichloromethane/methanol extract of dichloromethane/methanol extract of dichloromethane/methanol extract of dichloromethane/methanol extract of crateva adansoniicrateva adansoniicrateva adansoniicrateva adansonii in mice in mice in mice in mice exposed to chronic mild stressexposed to chronic mild stressexposed to chronic mild stressexposed to chronic mild stress galba j. beppe1*, nanou g. allah-doum1, bertrand p. barga1, alice i. folefack1, alain b. dongmo2 1university of maroua, faculty of science, department of biological science, p.o. box 814 maroua, cameroon; beppe840@gmail.com (*corresponding author); allahdoumgael14@gmail.com; bertrandbarga90@gmail.com; folefackens2018@gmail.com 2university of douala, faculty of science, department of animal biology, p.o. box 24157 douala, cameroon; alainberd@yahoo.fr abstractabstractabstractabstract crateva adansonii (ca) is traditionally used in the treatment of epilepsy and memory loss. this work aims to evaluate the antidepressant and anxiolytic activities of the dichloromethane/methanol extract of ca trunk bark in a chronic unpredictable stress-induced depression (ucms) model in mice. after exposure of mice to ucms for 42 days, anhedonia was assessed using the sucrose preference test, antidepressant effects by the forced swim and caudal suspension tests, anxiolytic effects by the light/dark compartment (ldb) and open arena (of) tests. oxidative stress parameters malondialdehyde (mda), superoxide dismutase (sod), catalase (cat), and reduced glutathione (gsh) were assessed. the results showed that multiple administrations of c. adansonii extract (150 and 300 mg/kg, resulted in a significant increase from 38.5% to 64.9% (p<0.001) in sucrose intake and a decrease from 47 seconds to 14 seconds in the immobility time in the forced swim test compared to the ucms group. the extract significantly (p<0.001) reversed the time spent in the dark box at 150 mg/kg, and the number of groomings at 150 and 300 mg/kg compared to the ucms group in the ldb and of test. there was also a significant (p<0.001) improvement in sod, gsh, and a reversal of mda. the extract of ca improved symptoms of depression and anxiety in mice treated with different dose. the effects observed would be due to the presence in the extract of polyphenols such as flavonoids. these effects would justify the use of this extract in traditional medicine. keywords:keywords:keywords:keywords: antidepressant; antioxidant; anxiolytic; chronic stress; crateva adansonii introductionintroductionintroductionintroduction depression is considered a psychiatric disorder characterized by sadness, loss of interest, and enjoyment, feelings of guilt, disturbed sleep, appetite, feeling tired, and lack of concentration (moreno, 2017). it is considered a major public health problem; nearly one in five people have experienced, are experiencing, or will experience depression in their lifetime (pelluet, 2019). despite advances in the detection of this disease and the https://www.notulaebiologicae.ro/index.php/nsb/index beppe gj et al. (2023). not sci biol 15(1):11459 2 discovery of new therapeutic strategies for its treatment, depression leads to numerous complications (roopa et al., 2019). this pathology affects approximately 9% of the world's population and evolves in 20 to 30% of cases to relapse and then recurrence (senova et al., 2019). depression is linked to high mortality, contributes to suicide, disruption of relationships, memory impairment, and loss of work time, and often leads to abuse of certain medications (halverson, 2010). anxiety, which is often considered one of the symptoms of depression, is an emotion provoked by an observed or experienced threat, which most often leads to avoidance or evasion (waraich et al., 2004). permanent exposure to stress is the real cause of neuropsychiatric disturbances (ricardo et al., 2010). these disturbances begin at the molecular level through stimulation of the hypothalamicpituitary-adrenal complex or oxidative stress. indeed, the involvement of stress and glucocorticoids in behavioral aspects is very important as they act on several vital components such as sleep (born et al., 1989), mood disorders (papadopoulou et al., 2015), food intake (ulrich et al., 2015) or social behaviors (cavigelli and caruso, 2015). the stress axis regulates stress, and its non-regulation results in depression and its associated disorders (prévôt, 2015). several therapies are now used in the treatment of nervous system pathologies in general and depression in particular. antidepressants currently used as treatment act through one or more of the following mechanisms: either through inhibition of serotonin or norepinephrine and dopamine reuptake, antagonism of serotonergic or noradrenergic presynaptic inhibitory receptors, or inhibition of monoamine oxidase (mazelin, 2019). however, these classes of drugs have many serious side effects such as hallucinations, memory impairment, anxiety, and even depression (sehonou and dodo, 2018). basic and clinical research is very interested in exploring new therapeutic targets, and new molecules acting on the central nervous system. crateva adansonii dc is a tree widespread in the sahelian and sudanian zones. this plant is used in traditional medicine to treat epilepsy. its bactericidal, anti-inflammatory actions and effects against yellow fever, hemorrhoid, indigestion, and gastritis have been scientifically proven (zingue et al., 2016b). this study was undertaken to investigate the neuroprotective effects of dichloromethane/methanol (dcm/meoh) extract of c. adansonii dc. bark on chronic mild stress induced in mice. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods chemical substances clomipramine (anafranil®) was obtained from alfa-sigma (france), and was dissolved in 2% ethanol. it was administered orally at a dose of 20 mg/kg, and a volume of 10 ml/kg. plant material and extraction protocol the bark of the trunk of crateva adansonii (capparaceae) was collected in april 2020 in the locality of moutourwa in the far north of cameroon. it was recognized by professor todou gilbert, a botanist at the university of maroua, and authenticated at the national herbarium of cameroon by comparison with a sample that was there under the reference number hnc 36359. the bark was dried and ground. the powdered bark of c. adansonii (2000 g) obtained was recollected in 5 l of dcm/meoh mixture (v/v : 1/1) for 72 h at room temperature and the obtained macerate was filtered with whatman paper n°4. the filtrate obtained was concentrated in a rotavapor (buchi r-300) at 60 °c. a dry extract (33.2 g) was obtained, representing an extraction yield of 1.66%. quantitative phytochemical analysis of dichloromethane/methanol extract of c. adansonii trunk bark determination of total polyphenols total polyphenols were performed according to the method of folin-ciocalteu (mahmoudi et al., 2013). the extract was dissolved in methanol at a concentration of 10 g/l. the assay consisted of taking a volume of 0.5 ml of the solution or standard solution with 1 ml of 1/10th folinciocalteu. after 5 minutes, beppe gj et al. (2023). not sci biol 15(1):11459 3 1 ml of 7.5% sodium carbonate was added to the solution, the tubes were then placed in the dark for 30 minutes at a temperature of 37 °c, and the absorbance was measured at a wavelength of 750 nm. the concentrations of polyphenols in the sample were determined from a calibration range established with gallic acid (0-125 μg/ml). determination of flavonoids in an acidic medium, and the presence of aluminum chloride (alcl3), flavonoids give a red coloration with an absorption maximum of 430 nm. the assay consisted in mixing 1 ml of extract dissolved in methanol at a concentration of 10 g/l or the standard solution with 1 ml of alcl3, after shaking, 2 drops of acetic acid were added and then after a second shaking, the absorbance was measured at a wavelength 430 nm. the concentrations of flavonoids in the sample were determined from a calibration range established with quercetin (0-100μg/ml) (mimica-duckic, 1999). determination of tannins in acidic media, tannins react with vanillin to form a complex that exhibits an absorption maximum at 500 nm. the assay consisted in mixing a volume of 0.2 ml of methanolic extract at a concentration of 10 g/l or of the standard solution with 2 ml of reagent (1 g of vanillin/100 ml concentrated hcl), the mixture was shaken and incubated at 30 °c for 5 min then the od was read at 500 nm. flavonoid concentrations in the sample were determined from a calibration range established with catechin (0-50 μg/ml) (bainbridge et al., 1996). evaluation of the in vitro antioxidant activity of the dichloromethane/methanol extract of the bark of the trunk of c. adansonii ferric reducing antioxidant power (frap) assay the ferric reducing power was determined according to the method recommended by benzie and strain (1996). the assay consisted of mixing a volume of 0.1 ml of extract dissolved in methanol (10g/l) or standard solution with 1 ml of fe(iii)-tptz solution (acetate buffer/tptz/fecl3=10:1:1), the mixture was stirred and then 5 min after, the od was read at 593 nm. the reducing powers of the sample were determined from a calibration range established with vitamin c (0-125 µg/ml). an increase in the absorbance corresponded to an increase in the reducing power of the extracts tested (hubert, 2006). evaluation of the antiradical activity with dpph 2,2-diphenyl-2-picrylhydrazyl (dpph) is a stable purplish free radical that absorbs at 517 nm. this method is based on measuring the ability of antioxidants to scavenge the dpph radical (sun et al., 2005). the assay consisted of mixing a volume of 0.2 ml of extract dissolved in methanol (10 g/l) or standard solution with 2 ml of dpph solution, the mixture was stirred, and then 5 min after, the od was read at 517 nm. the antioxidant powers of the sample were determined from the calibration range established with trolox (0-125 µg/ml). animal material and experimental protocol thirty male mice aged 8-12 weeks and weighing between 20-30 g were randomly distributed into 5 groups of 6 animals each. they were housed in plastic cages and acclimated for two weeks before the start of the experiment. the animals had free access to food and water, except on days when food and water deprivation were used as stressors. animals were subjected to ucms daily for 42 days and 30 minutes after treatment administration. preliminary tests were carried out in order to choose the different doses. a normal control group received 2% ethanol (10 ml/kg, p.o), a negative control group received 2% ethanol (10 ml/kg, p.o), a positive control group received clomipramine (20 mg/kg, p.o) and two test groups that received c. adansonii beppe gj et al. (2023). not sci biol 15(1):11459 4 extract at different doses (ca 150 and 300 mg/kg; p.o). only animals in the normal control group were not subjected to ucms. behavioural tests were started after 30 days of treatment. ucms were induced according to the method described by kuegong et al. (2020) with a slight modification (table 1). table 1.table 1.table 1.table 1. chronology of exposure to stressors and their duration weekweekweekweek mondaymondaymondaymonday tuesdaytuesdaytuesdaytuesday wednesdaywednesdaywednesdaywednesday thursdaythursdaythursdaythursday fridayfridayfridayfriday saturdaysaturdaysaturdaysaturday sundaysundaysundaysunday week 1 weighing social stress (4h) litter removal (2h) night (3h) restraint (2h) day (6h) changes to litter social stress (4h) restraint (2h) wet bedding (6h) tilt (3h) social stress (4h) food deprivation (24h) sound stimulation (2h) week 2 weighing restraint (3h) tilt (2h) mouse feces (2h) wet bedding (12h) forced swimming (10 min) litter changes bedding social stress (4h) restraint (1h30) sound stimulation (2h) cold bath (5min) water deprivation (24) day/night alternation every 30 minutes (6h) day/night alternation every 30 minutes (6h) week 3 weighing insulation (24h) insulation (24h) social stress (4h) bath (2h) wet litter (12h) litter changes restraint (6h) food deprivation (24h) sound stimulation (3h) week 4 weighing wet litter (4h) cold bath (5min) social stress (4h) forced swim at 45°c (5min) change of bedding day/night alternation (4h) wet litter (24h) changes in bedding bath (2h) day (12h) day/night alternation every 30 minutes (4h) week 5 weighing wet litter (4h) forced swimming (10 min) tilt (3h) social stress (3h) restraint (2h) litter removal (4) mouse feces (2h) litter change cold bath (10 min) day/night alternation every 30 minutes (6h) food deprivation (24h) week 6 weighing restraint (4h) forced swimming 45°c (5 min) sound stimulation (2h) insulation (24h) restraint (1h) wet bedding (6h) tilt (3h) night (3h) sucrose preference test forced swimming test tests of the light/dark compartment box and open field test sacrifice beppe gj et al. (2023). not sci biol 15(1):11459 5 behavioural tests depression tests sucrose preference test anhedonia allows the effectiveness of the ucms protocol to be monitored. for each isolated mouse, two water bottles were provided, one containing tap water and the other a 1% sucrose solution. the positions of the water bottles were changed 12 h later to avoid the lateralization effect (liu et al., 2016). the sucrose solution was prepared in advance, and the solution temperature was equal to room temperature (chang et al., 2012). the water provided to the animals was at room temperature. the sucrose preference was determined as follows: sucrose preference (%) = (amount of 1% sucrose solution) / (volume of sucrose solution + volume of water) × 100 forced swimming test the forced swimming test is used to measure depressive-like behavior in animals (porsolt, 1979). each animal is placed in a device consisting of a transparent glass cylinder (30 cm high × 20 cm diameter and 20 cm deep) containing water maintained at 25 ± 2 °c (kitada et al., 2017). the aim of the test is to leave the animal for 6 minutes and attempt to climb the wall or remain immobile. a resigned or depressed animal showing behavioral despair will spend more time immobile than a control animal (beppe et al., 2015). the animal is then removed from the cylinder, dried with a towel, and placed under a heat lamp until recovery. anxiety tests lighted/dark box test the lighted/shadowed box test was carried out according to rebai's (2017) method with small modifications. the lighted/obscure box (45 × 27 × 27 cm) was made of polywood and consisted of two pieces connected by an opening (7.5 × 7.5 cm) located at floor level in the center of the partition. the floor was divided into 9 × 9 cm squares and covered with plexiglas. the small room (18 × 27 cm) was painted black and the large room (27 × 27 cm) white. lighting was provided by a 60-watt table lamp located 40 cm above the center of the white chamber (foyet et al., 2012). during the test, mice were individually placed in the center of the light room with their backs opposite to the light room, and the behaviors observed were latency, number of transitions, time spent in the lightroom, and time spent in the darkroom. open field test the open space arena was constructed of white polywood and square in shape measuring 72 × 72 cm with a height of 36 cm. three red lines were drawn with a marker and were visible through the clear plexiglas floor delineating the central zone, intermediate zone, and peripheral zone (foyet et al., 2012). mice were placed one at a time in the open field box for 6 min, and the behaviors recorded were time spent in the central square, number of lines crossed, the number of dressings, number of groomings, and time spent at the edge of the arena. after each trial, the mouse was removed and returned to its cage, and the entire field was cleaned with a 70% ethanol solution before the next animal was tested. biochemical analysis of oxidative stress parameters brain sample preparation on the last day of the experiment, the mice were anaesthetized and decapitated. the whole brain was removed for biochemical studies. part of the brain was ground and homogenized with 0.1m phosphate buffer (ph 7.4), the homogenate was centrifuged (3000 rpm for 15 min at 4 °c), and the supernatant was collected and stored at -20 °c for biochemical analyses. beppe gj et al. (2023). not sci biol 15(1):11459 6 determination of malondialdehyde (mda) the determination of the amount of mda in the hippocampus was performed following the technique described by wilbur et al. (1959). in each test tube, 0.5 ml of an iron chloride solution (2 mm) was added to 0.5 ml of the homogenate. the reaction medium was incubated for 1 h at room temperature and then centrifuged at 1000 rpm for 10 min. subsequently, 100 μl of supernatant was mixed with 500 μl of 1% phosphoric acid and 500 μl of thiobarbituric acid in a 1% tca solution. the mixture was homogenized by vortexing, passed through a boiling water bath for 20 min, cooled in an ice bath, and centrifuged at 3000 rpm for 10 min. the supernatant was collected again, and the absorbance was read at 532 nm against the blank. the amounts of mda were estimated and expressed as mm/mg of an organ. superoxide dismutase (sod) assay the assay of sod was performed according to the principle described by mishra in 1972, which is based on the ability of sod to inhibit or retard the auto-oxidation of adrenaline to adrenochrome in basic media. for this, 140 μl of the homogenates were added to 1660 μl of carbonate buffer (ph=10.2) followed by 200 μl of freshly prepared adrenochrome (0.3 mm). auto-oxidation was then measured by od reading at 480 nm at t=30 s and t=90 s. sod activity was expressed in units/mg of an organ. determination of reduced glutathione (gsh) the gsh assay is performed according to the method described by ellman in 1959, which is based on the reaction of 2,2-dinitro-5,5-dithiodibenzoic acid (dtnb) with the sh group of glutathione to form a yellow complex, the yellow complex absorbing at 412 nm. in each assay, 200 µl of homogenate and 3 ml of ellman's reagent are added to the tube. after homogenisation, the mixture is incubated at room temperature for 60 min. prepare blank tubes under the same experimental conditions, replacing the homogenate with phosphate-buffered saline (0.2 m, ph 7.4, pka 7.2). read the absorbance of each tube at 412 nm against the blank and the amount of mda expressed in mm/mg of organ. histology of the tissues histological analysis of the brain (hippocampus) was assessed using 5 µm sections of paraffin-embedded tissue. coronal slices were obtained from the brain (left hemisphere) in the hippocampal region using the mouse brain atlas with the following coordinates (anterior/posterior d 2.0 mm, medial/lateral d 1.5 mm and dorsal/ventral ap d 2.0 mm) (smith and bruton, 1977). after hematoxylin-eosin staining, micrographs of brain sections were evaluated using a digital camera (scientico, haryana, india) attached to a light microscope. statistical analysis the results obtained were expressed as mean ± sem. data were analyzed by one-way anova (force swimming test, light and dark box, and open arena test), and two-factor anova (tps) followed by dunnett's and bonferroni's post-tests, respectively. all analyses were performed using graph pad prism version 8.0.1 for windows. results were considered significant for p<0.05. results results results results total polyphenols content (tpc), total flavonoids content (tfc), and total tannins of the dcm/meoh extract of c. adansonii bark the phytochemical screening performed on the dcm/meoh extract of c. adansonii showed that flavonoids are the most abundant polyphenols, with an amount of 211.47 ± 11.66 mg quercetin equivalent beppe gj et al. (2023). not sci biol 15(1):11459 7 (eq) /g dry extract compared to tannins with an amount of 152.96 ± 5.12 mg catechin eq /g dry extract (table 2). table 2.table 2.table 2.table 2. total phenol, total flavonids and total tanin quantity of the dcm/meoh extract of c. adansoni compounds concentration in the dcm/meoh of c. adansonii total phenol total flavonoids total tanin 384.02 � 6.27 (mg ega/100 g dw) 211. 47 � 5.38 (mgeq/100 g dw) 152. 96 � 5.12 (mg ec/100 g dw) results are expressed as mean ± mse.mgega/100 g dw: milligram equivalent gallic acid per 100 g dry weight; mg eq/100 g dw: milligram equivalent quercetin per 100 g dry weight; mg ec/100 g d w: milligram equivalent catechin per 100 g dry weight. in vitro antioxidant activity of the dcm/meoh extract of c. adansonii bark the antioxidant potential of the dcm/meoh extract from the bark of the trunk of c. adansonii was measured by two tests (dpph and frap) and is correlated with their contents in total phenols and flavonoids. figure 1 below shows the reducing power of iron and dpph of the dcm/meoh extract of the bark of the trunk of c. adansonii. the results revealed an inhibition of 60.36% for the dpph test and 42.630% of the frap test, they are lower compared to butylhydroxytoluene (bht) which showed 75.453% inhibition. figurefigurefigurefigure 1111. in vitro antioxidant activity at dpph and frap of the dcm/meoh extract of c. adansonii effects of dcm/meoh extract of c. adansonii bark on sucrose preference and immobility time in the forced swimming test after 30 days of treatment, ucms induced a significant decrease (p<0.001) in sucrose preference (figure 2a) and significantly increased (p<0.001) immobility time (figure 2b) of animals in the negative control group compared to animals in the normal control group. the administration of the extract at the doses of 150 and 300 mg/kg caused a significant increase (p<0.001) in sucrose preference and a significant decrease (p<0.001) in the immobility time of the animals compared to the negative control group. the immobility time thus decreased from 46,8�0,689 s in the ucms control group to 13,9�0,688 s in the test group treated at a dose of 150 mg/kg. similarly, there was a significant increase (p<0.001) in sucrose preference and a significant decrease (p<0.001) in immobility time in animals in the positive control group compared to animals in the negative control group. 75,453 60,336 42,63 0 10 20 30 40 50 60 70 80 90 bht ca-dpph ca-frap bht ca-dpph ca-frap beppe gj et al. (2023). not sci biol 15(1):11459 8 figure 2.figure 2.figure 2.figure 2. effect of dcm/meoh extract of c. adansonii on sucrose preference (a) and immobility time in the fst (b) of mice before and after 30 days of treatment each bar represents the mean ± mse, ucms: chronic mild unpredictable stress; clomip + ucms: positive control receiving clomipramine (20 mg/kg); ca + ucms: animals receiving the dcm/meoh mixture extract (v/v: 1/1) of c. adansonii at doses of 150 and 300 mg/kg. ***p < 0.001 compared to ucms group. ###p < 0.001 compared to the normal group. effects of dcm/meoh extract of c. adansonii bark on time spent in the ldb and number of groomings in the oft figure 3 below represents the time spent in the dark compartment in the ldbt (figure 3a) and the number of grooming in the oft (figure 3b) of the animals after 30 days of treatments. ucms induced a significant (p<0.001) increase in the time spent in the dark compartment, increasing this time from 122�1.56 s in the normal control group to 164�1.55 s in the ucms control group, and from the grooming count of 3.00�0.0632 in the normal control group to 6.00�0.0816 in the ucms control group. in the ucms extracttreated groups, there was a significant (p<0.001) decrease in the time spent in the dark compartment (105� 1.87 s) at the dose of 150 mg/kg and the number of groomings (3.00�0.0816 s; 3.80�0.122 s) at the doses of 150 and 300 mg/kg respectively compared to the ucms control group. clomipramine induced a significant decrease (p<0.001) in the time spent in the dark compartment of the positive control group compared to the negative control group. beppe gj et al. (2023). not sci biol 15(1):11459 9 figure 3.figure 3.figure 3.figure 3. effect of dcm/meoh extract of c. adansonii on time in the ldbt (a) and number of groomings in oat (b) after 30 days of treatment each bar represents the mean ± mse, ucms: chronic mild unpredictable stress; clomip + ucms: positive control receiving clomipramine (20 mg/kg); ca + ucms: animals receiving the dcm/meoh mixture extract (v/v: 1/1) of c. adansonii at 150 and 300 mg/kg. ***p < 0.001 compared to ucms group. ###p < 0.001 compared to the normal group. effect of dcm/meoh extract of c. adansonii bark on malondialdehyde (mda) concentration, superoxide dismutase (sod) activity, and reduced glutathione (gsh) concentration figure 4 below shows the concentration of mda (figure 4a), sod activity (figure 4b) and gsh concentration (figure 4c) in the hippocampal homogenate of mice after 30 days of treatments. ucms induced a significant increase (p<0.001) in mda concentration and a significant decrease (p<0.001) in sod activity in negative control animals compared to normal control animals. the extract caused a significant decrease (150 mg/kg, p<0.001; 300 mg/kg, p<0.01) in mda concentration and a significant increase (p<0.001) in sod activity and gsh concentration at doses of 150 and 300 mg/kg in treated animals compared the control group. similarly. there was also a significant (p<0.01) decrease in mda concentration and a significant (p<0.001) increase in sod activity in positive control animals compared to negative control animals. beppe gj et al. (2023). not sci biol 15(1):11459 10 figure 4.figure 4.figure 4.figure 4. effects of dcm/meoh extract of c. adansonii on mda concentration (a), sod activity (b) and gsh concentration (c) in the hippocampus of mice after 30 days of treatment each bar represents the mean ± mse, ucms: chronic mild unpredictable stress; clomip + ucms: positive control receiving clomipramine (20 mg/kg); ca + ucms: animals receiving the dcm/meoh mixture extract (v/v: 1/1) of c. adansonii at doses of 150 and 300 mg/kg. ***p < 0.001 compared to ucms group. ###p < 0.001 compared to the normal group. effects of dcm/meoh extract of c. adansnii bark on hippocampal microarchitecture the hippocampal microarchitecture depicted in figure 5 shows that the negative control exhibits several histopathological changes in the hippocampus, marked by a decrease in the density of neuronal cells in ammon's corns (ca1, ca2, and ca3), leukocyte infiltration (ca1), and vacuolization of gyrus dente cells compared to the normal control. the extract at doses of 150 and 300 mg/kg reversed all hippocampal structures, close to those of the normal control. similarly, clomipramine caused a restructuring of all hippocampal structures. beppe gj et al. (2023). not sci biol 15(1):11459 11 figure 5.figure 5.figure 5.figure 5. microphotographs of the gyrus dente (x100) and ammon horns 1, 2 and 3 (x200) of the hippocampus of male mice; hematoxylin-eosin staining. a = normal control; b = negative control; c = positive control; d, e = batches receiving dcm/meoh extract of c. adansonii at doses of 150 and 300 mg/kg, respectively; gd = gyrus dente; ca1, 2, 3 = ammon horns 1, 2, and 3; pn = neuronal loss; va = neuronal vacuolation; il = leukocyte infiltration. discussiondiscussiondiscussiondiscussion the current study aimed to demonstrate to evaluate the effects of a dcm/meoh extract of c. adansonii bark on depression and anxiety in mice exposed to chronic unpredictable mild stress (ucms). sucrose preference and forced swimming tests were used to assess antidepressants, while the caudal suspension and dark/light compartment tests were used to assess anxiolytic effects. anhedonia is one of the primary symptoms of depression in humans and the sucrose preference test is an indicator of anhedonic behavior (rygula et al., 2005). we observed that exposure of mice to ucms for 42 days induced a change in behavior by a significant decrease in sucrose preference. pretreatment with dcm/meoh extract of ca at doses of 150 and 300 mg/kg significantly (p< 0.001) increased sucrose preference in animals. the extract may act by stimulating the reward and motivation centers, resulting in increased dopamine levels in the animals' brains (murray et al., 2008). the dcm/meoh extract of c. adansonii could therefore have an antidepressant effect. numerous studies have shown that there is a comorbidity between depression and anxiety, giving them common symptoms (koprdová et al., 2016). the light and dark box test is widely used to assess the effect of drugs on the general behavior and excitability level (foyet et al., 2014). beppe gj et al. (2023). not sci biol 15(1):11459 12 in this test, anxiety is generated by the conflict between the desire to explore and the fear of the lighted, unfamiliar space (crawley, 1985). in this study, the ucms induced a state of anxiety in mice that was reflected in a significant increase in time spent in the dark compartment. chang et al. (2012) showed that ucms contributed to the development of anxiety in animals in the elevated cross-maze test. pretreatment with the dcm/meoh extract of c. adansonii at the 150 mg/kg dose reportedly inhibited amygdala hyperactivity while protecting the prefrontal cortex, resulting in a significant decrease in time spent in the dark box of animals exposed to ucms. the extract might act through a non-selective antagonism mechanism at 5-ht1 and 5ht2 receptors which are involved in anxiety-like behaviors (bourin and hascoёt, 2010; rynn et al., 2003). in this work, ucms also induced a significant increase in grooming. this is thought to be due to a decrease in serotonin concentration in the limbic system and dysfunction of the gabaergic system (švob et al., 2016). gaba increases chlorine ion transport in the intracellular medium, inducing hyperpolarization of the latter, making the cell refractory to certain stimuli thus decreasing hyperactivity in the central nervous system (lopes et al., 2012). pretreatment with dcm/meoh extract of c. adansonii at doses 150 and 300 mg/kg protected the serotonergic and gabaergic systems of the central nervous system, resulting in a significant decrease in the number of grooming events. these results confirm that the dcm/meoh extract of c. adansonii could have an anxiolytic activity. glucocorticoids accelerate cellular metabolism, which consequently increases free radical formation via the mitochondrial electron transport chain (mcintosh et al., 1998). excessive generation of free radicals such as reactive oxygen and nitrogen species can potentially damage fatty acids, proteins, and dna through oxidative stress (hazel et al., 2021). chronic stress can also lead to the production of substances that can activate glutamate nmda receptors thus leading to excitotoxicity and consequently oxidative stress (banasr et al., 2010). pretreatment with dcm/meoh extract of c. adansonii protected the mouse brains from the subcortical low intensity (ucms)-generated free radicals, resulting in a significant decrease (at 150 mg/kg and 300 mg/kg) in lipid peroxidation, and a significant increase in sod activity and gsh levels at 150 and 300 mg/kg. the analysis of the antioxidant activity in vitro showed that the extract has more anti-radical activity than reducing activity, with a percentage of inhibition to dpph of 60.36%. the improvement of enzymatic and non-enzymatic defense against free radicals would be due to the presence in the creteva adansonii extract of some secondary metabolites such as flavonoids. flavonoids act mainly as primary antioxidants, stabilizing peroxide radicals but can also deactivate reactive oxygen species and inhibit lipoxygenase or chelate metals (sarni-manchado and cheynier, 2006). hypersecretion of corticosteroids following chronic stress leads to impaired brain function and inhibition of hippocampal stem cell proliferation, resulting in reduced production of new neurons (gold, 2015). ucms resulted in alterations in the various structures of the hippocampus. pretreatment with the dcm/meoh extract of c. adansonii protected the hippocampus from the neurotoxic effects of ucms: this, therefore, supports the neuroprotective effect of the extract. flavonoids in the extract may protect the brain in several ways, including protecting vulnerable neurons, enhancing existing neuronal function, and stimulating neuronal regeneration (vauzour et al., 2010). conclusionsconclusionsconclusionsconclusions this study revealed that the dcm/meoh extract of c. adansonii has antidepressant effects and anxiolytic activities. moreover, the extract decreased lipid peroxidation and increased the activity of sod and the level of gsh. these antidepressant, anxiolytic, and antioxidant effects would be due to the presence of phenolic compounds (flavonoids) in the extract. these results would justify at least partially the use of this extract in traditional medicine. studies are underway to determine the possible mechanisms of action of this extract. beppe gj et al. (2023). not sci biol 15(1):11459 13 authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions nganganganga----d: id: id: id: investigated the traditional healers, to choose the plant, bpbbpbbpbbpb: provided an extract and proposed the methodology, gjb: gjb: gjb: gjb: validated the methodology and wrote the manuscript. aifaifaifaif: analyzed the data and revised the english version of the manuscript, abdabdabdabd: corrected the protocol and brought expertise to the whole manuscript. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) animals were handled according to the guidelines of the cameroon bioethics committee (reg. no. fwa-irb00001954). the protocol was approved by the ethics committee of the faculty of sciences of the university of maroua (ref. no. 14/0261/uma/d/fs/vd-rc). each animal was tested in only one behavioral test and tests were made to minimize animal suffering. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences bainbridge z, tomlins k, wellings k, westby a (1996). methodes of assessing quality characteristics of non-gain starch staples. natural ressources institute, university of greenwich, pp 75. banasr m, chowdhury gm, terwilliger r, newton ss, duman rs, behar kl, sanacora g (2010). glial pathology in an animal model of depression: reversal of stressinduced cellular, metabolic and behavioral deficits by the glutamatemodulating drug riluzole. molecular psychiatry 15(5):501-511. https://doi.org/10.1038/mp.2008.106. beppe gj, alain bd, foyet hs, dimo t, marius m, hritcu l (2015). the aqueous extract of albizia adianthifolia leaves attenuates 6-hydroxydopamin-induced anxiety, depression and oxidative stress in rat amygdala. complementary and alternative medicine 15:374-387. https://doi.org/10.1186/s12906-015-0912-0 benzie if, strain jj (1996). the ferric reducing ability of plasma (frap) as a measure of "antioxidant power": the frap assay. analytica biochemistry 239(1):70-76. https://doi.org/10.1006/abio.1996.0292 born j, spath-schwalbe e, schwakenhofer h, kern w, fehm hl (1989) influences of corticotropin-releasing hormone, adrenocorticotropin, and cortisol on sleep in normal man. journal of clinical endocrinology and metabolism. 68:904-911. https://doi.org/10.1210/jcem-68-5-904 bourin m, hascoёt m (2010). implication of 5-ht2 receptor subtypes in the mechanism of action of the gabaergic compound etifoxine in the fourplate test in swiss mice. behavioural brain research 208:352-368. https://doi.org/10.1016/j.bbr.2009.11.046 cavigelli sa, caruso mj (2015). sex, social status and physiological stress in primates: the importance of social and glucocorticoid dynamics. philosophical transactions: biological sciences 370(1669):20140103. https://doi.org/10.1098/rstb.2014.0103 beppe gj et al. 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(2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11462 doi:10.15835/nsb15111462 rererereviewviewviewview articlearticlearticlearticle.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae pathophysiological aspects of renal stone formation and stone typespathophysiological aspects of renal stone formation and stone typespathophysiological aspects of renal stone formation and stone typespathophysiological aspects of renal stone formation and stone types rabie kachkoul1*, ghita benjelloun touimi2, ghita el mouhri1, radouane el habbani1, anissa lahrichi1 1sidi mohammed ben abdellah university, faculty of medicine and pharmacy, laboratory of biochemistry, bp 1893, km 22, road of sidi harazem, fez, morocco: rabie.kachkoul@usmba.ac.ma (*corresponding author); elm.ghita@gmail.com; radouane500@gmail.com; anissafmpf@hotmail.fr 2sidi mohammed ben abdellah university (usmba), faculty of medicine and pharmacy of fez, laboratory of human pathology biomedicine and environment, fez, morocco; ghita.benjellountouimi@usmba.ac.ma abstractabstractabstractabstract urinary stone formation is one of the oldest and most widespread diseases known to man. the disease has a multifactorial etiology that includes anatomic, environmental, genetic, infectious, metabolic, nutritional, and most importantly socio-economic factors. it is caused by a biochemical imbalance in urine between stoneforming inhibitors and promotors in a process known as lithogenesis. the mechanisms underlying the formation and development of urinary stones are not fully understood, but it can be said that they generally begin by increased urinary supersaturation of lithiasis promoters in the urine, followed by nucleation and aggregation. subsequently, the crystals combine with other crystals in solution to form agglomerates that accumulate in the kidney. free radical-mediated oxalate-induced renal membrane damage promotes crystal retention at the surface of the renal papilla, as well as crystal nucleation at lower supersaturation levels. in addition, stone type identification is of great interest in guiding the physician to an effective diagnosis, which allows to determine the causes in order to treat the disease and prevent recurrence. in this context, this present study reviews current knowledge on the pathophysiological aspects of kidney stone formation as well as the type of stones. keywords:keywords:keywords:keywords: kidney stone disease; pathophysiological aspects; stone formation; stones type introductionintroductionintroductionintroduction kidney stone disease is a crystalline concretion of mineral and organic components formed in the kidneys or in the urinary tract. about 80% of kidney stones contain calcium oxalate (caox) as the main mineral phase, mainly mixed with calcium phosphate (cap) and sometimes uric acid (khan, 2014). urolithiasis represents a growing urological disorder of human health, affecting approximately about 2 to 20% of the population, with variations observed across different countries (hesse et al., 2003). according to estimates, within the first 5 years following the discovery of the first stone, the likelihood of recurrence ranges between 30 to 50%, while in the subsequent 10 years, the rate increases to approximately 50 to 60% (curhan, 2007; safarinejad, 2007; daudon, 2008; arumuham and bycroft, 2016). this pathology has been recognized https://www.notulaebiologicae.ro/index.php/nsb/index kachkoul r et al. (2023). not sci biol 15(1):11462 2 since the earliest times and is an integral part of the human history (el lekhlifi et al., 2014). the archaeological and paleontological documents clearly show that this pathology is one of the oldest human diseases. the first documented urinary and bladder stones were identified by shattock in 1905 (modlin, 1980), found in egyptian mummies and dated to about 4400 bc. -jc for the first stone and from 4800 bc. -jc for the second (modlin, 1980). the urolithiasis management involves drug treatments and/or surgical intervention to extract the stones by the techniques such as extracorporeal shock wave lithotripsy (eswl), ureteroscopy (urs), percutaneous nephrolithotomy (pcnl) and open surgery. this latter becoming relatively uncommon, although there is some use laparoscopic/robotic surgery for stone removal (geraghty et al., 2017, 2023). moreover, endoscopic stone removal and eswl have revolutionized the treatment of urolithiasis, this latter technique show a success rate for kidney stones with minimum complications rate, but depend on the stone’s characteristics, patient and other parameters (chakit et al., 2023). the use of urs has increase over the past decade due to massive innovations in minimally invasive surgery and recommended for intra-renal stones of 1-2 cm (geraghty et al., 2017). on the other hand, these methods are expensive and can cause acute kidney injury and decline in kidney function, moreover, do not prevent the likelihood of new stones formation (agawane et al., 2019; sikarwar et al., 2017). however, various therapies, including thiazide diuretics and alkaline citrate, are used in an attempt to prevent the recurrence of stones induced by hypercalciuria and hyperoxaluria, but the scientific evidence for their effectiveness is less convincing (bashir and gilani, 2011; sikarwar et al., 2017). currently, there is no satisfactory drug to cure and/or prevent the recurrence of kidney stones, which remains a serious health problem for humans (alelign and petros, 2018). therefore, a better understanding of the mechanisms involved in the formation of stones is a necessity for prevention and the development of treatment methods. in this context, this work provides a synthesis on the pathogenesis and pathophysiological aspects of urolithiasis, as well as the type of stones. the pathogenesis of kidney stone disease is a very complicated phenomenon that implies physiological, physicochemical, biological, biochemical and genetic aspects that act individually or in synergy. it is important to note that there are not many studies that investigate and examine all these variables together. pathogenesis and physiopathology of urolithiasispathogenesis and physiopathology of urolithiasispathogenesis and physiopathology of urolithiasispathogenesis and physiopathology of urolithiasis stone formation brings together all biological and physicochemical processes that occur from supersaturated urine and lead to the formation of a stone in the urinary tract. this process is called “lithogenesis” and involves several phases that occur sequentially or simultaneously (figure 1) ( daudon et al., 2008; aggarwal et al., 2013). these phases can be classified into two stages. the first one concerns certain initial phases of lithogenesis, which may be called crystallogenesis. it’s corresponds to the formation of crystals from substances initially dissolved in the urine and is not in itself a pathological process (daudon et al., 2008). while the second stage includes the retention and development of stones in the urinary tract, known as calculogenesis. however, understanding the lithogenesis mechanisms involved in the formation of each stone is an essential step in defining effective rules for treatment and recurrence prevention (daudon et al., 2000, 2008). kachkoul r et al. (2023). not sci biol 15(1):11462 3 figure 1.figure 1.figure 1.figure 1. schematic representation of various cellular and extracellular events during urinary stone formation. (opn: osteopontin, ha: hyaluronic acid, sa: sialic acid, mcp-1: monocytic chemotactic protein-1.) (aggarwal et al., 2013) several possible hypothetical models have been proposed to explain the mechanism of urinary stone initiation and formation. no single model can rationalize the evidence observed in all patients with this disease, as many factors can interfere (khan et al., 2016). nevertheless, the chemical processes of crystal nucleation and growth are essential for the initiation and development of all stone’s types (robertson et al., 1971). however, stone formation is caused by an abnormal combination of factors affecting the thermodynamic driving force (supersaturation) and kinetic processes (flow control), involved in the crystallization of various minerals (khan et al., 2016). urine supersaturation in solutions like urine, the process of crystallisation is primarily driven by supersaturation. as a simple definition, upon addition of a salt to a solvent, it will dissolve until it reaches a certain concentration, at which point no further dissolution is possible. this concentration is known as the saturation point. an additional dose of salt will lead to crystallization as long as the temperature, pressure and ph remain unchanged (aggarwal et al., 2013). indeed, in the living environment, the latter parameter (ph) is the most important factor influencing the solubility of the substance, as the pressure and temperature can be considered constant (daudon et al., 2008). in the majority of individuals, urine is almost always metastable to calcium oxalate, whether they develop stones or not. additionally, at certain times, it may also be metastable to other components, such as uric acid, urates and calcium phosphate. although the urine of patients with calcium is often more supersaturated with calcium oxalate and calcium phosphate than in normal individuals. nevertheless, even in this case (supersaturation), nucleation of calcium oxalate, or phosphates does not usually kachkoul r et al. (2023). not sci biol 15(1):11462 4 occur, and the crystals produced do not grow and aggregate to a sufficient size to be retained in the kidney on the basis of size alone (kok and khan, 1994). this result is probably due to the presence of crystal formation inhibitors, particularly during the nucleation, growth or aggregation stage (ratkalkar and kleinman, 2011; verkoelen, 2006). supersaturation (ss) is expressed as the ratio between the solute’s concentration (c) and its solubility product (miller et al., 2007; ratkalkar and kleinman, 2011). the point at which saturation of a solution is reached and crystallisation begins, is commonly referred to as the thermodynamic solubility product (ksp) (figure 2). subsequently, as the concentration of the solute continues to increases, it eventually reaches a level where it exceeds the maximum solubility and can no longer remain dissolved. at this point, crystals begin to form in the urine, which is called the formation product (kf) (basavaraj et al., 2007). on the other hand, if crystallisation inhibitors were unable to act, the end result would be nephrolithiasis (carvahlo and nakagawa, 1999; miller et al., 2007; ratkalkar and kleinman, 2011; aggarwal et al., 2013). at ss values less than 1, the crystals of a substance dissolve, whereas, at ss values greater than 1, crystals can form and develop. although increasing urine volume is an obvious way to reduce ss. in addition, and for unclear reasons, coe et al. (2005) found that sodium intake and urinary calcium excretion increased with increasing urine volume, partially offsetting the drop in ss. however, the most important determinants for calcium oxalate (caox) ss are urine calcium and oxalate concentrations, whereas for calcium phosphate (cap), urine calcium concentration and ph are crucial. the latter parameter also represents a primary determinant of uric acid (ua) ss (coe et al., 2005). the ss range between the solubility product and the point of crystal formation (kf), is called the metastable zone (figure 2), where the concentration of the salt is greater than its solubility and precipitation is inevitable. the amount of salt required to produce a solid phase is called the upper limit of metastability (ulm) (coe et al., 2005; miller et al., 2007). figure 2.figure 2.figure 2.figure 2. saturation states of a solution (miller et al., 2007) when ss exceeds kf, the urine enters an unstable zone. due to the high level of supersaturation, the kinetics of crystallization enable the formation and growth of crystals, will allow the formation and growth of crystals in the time necessary for the urine transit through the kidney. knowing that the transit time of urine through the nephrons during usual diuresis is between 1 and 3 minutes (daudon et al., 2000). when the urine is below the kps for a particular solute, this latter cannot crystallise. kachkoul r et al. (2023). not sci biol 15(1):11462 5 crystalline germination-nucleation nucleation is the initial process of crystallisation, in which occurs at a critical level of ss, the reunion of a small number of molecules (a few tens or hundreds) to form a crystal nucleus, which can then grow by subsequent deposition from the solution. this is called primary nucleation when the crystals of the precipitation phase are not involved, or secondary nucleation when nuclei form on pre-existing crystal surfaces. primary nucleation can be divided into homogeneous and heterogeneous nucleation (kavanagh, 2011). homogeneous nucleation occurs spontaneously when the supersaturation is sufficient. this is difficult to achieve in practice as all foreign particles and surface defects must be excluded. nucleation that occurs on an existing surface, such as cell membranes, cell debris, other crystals, red blood cells, and urine streams is called heterogeneous. the latter occurs at a lower ss level than homogeneous nucleation ( miller et al., 2007; kavanagh, 2011; aggarwal et al., 2013; alelign and petros, 2018). damage to renal tubule cells can favour the crystallisation of caox crystals by providing substances for their heterogeneous nucleation (khan, 2006; aggarwal et al., 2013). in vitro cellular degradation subsequent to renal tubular cells injury produces numerous membrane vesicles, that have been observed to serve as effective nucleators of calcium crystals, including cap and caox. indeed, incubation of the proximal tubular border membrane in a metastable solution of calcium oxalate shows the association of the latter with cellular degradation products (khan et al., 1990; fasano and khan, 2001). however, the stone matrix contains both membrane vesicles and lipids, cell membrane phospholipids are proposed to promote crystal nucleation (khan et al., 2002). it is interesting to note that the damaged but intact membranes cells also showed the ability of caox crystals to nucleate. direct nucleation on the cell surface may also promote crystal retention in the tubule (lieske and deganello, 1999). on the other hand, oxalate is widely distributed in plant-based foods as potassium, sodium and ammonium oxalate (water soluble form) and insoluble calcium oxalate. oxalate has been shown to be toxic to renal epithelial cells of cortical origin. it has been observed that exposure of renal epithelial cells to oxalate results in disruption of normal renal epithelial activities (aggarwal et al., 2010), alterations in gene expression (khan et al., 2014), impaired mitochondrial function (cao et al., 2004), formation of reactive oxygen species (ros) (thamilselvan et al., 2003; yu et al., 2011) and consequently a decrease in cell viability (aggarwal et al., 2010). oxalate-induced membrane damage is mediated by peroxidation of lipids and proteins via ros generation with altered biochemical reactions, including depletion of the antioxidant defense system and the calcium pump failure. calcium and oxalate accumulate and then precipitate in the presence of membrane fragments to form stones (khan, 2005; ahmed et al., 2018). according to khan’s research in 2004, the interaction between kidney cells and crystals leads to the generation of reactive oxygen species (ros), which in turn cause diverse cellular responses (khan, 2004). however, crystals deposition is a relatively common phenomenon in the kidneys and often associated with inflammation. calcium phosphate (cap) and calcium oxalate (caox) crystals can cause tubulointerstitial damage and inflammation. urate crystals associated with chronic gout cause an intense inflammatory reaction. cystine crystals in homozygous cystinuria patients also trigger an inflammatory response (khan, 2004). furthermore, exposure of renal epithelial cell to crystals leads to increased synthesis of osteopontin (opn), bikunin, heparin sulfate, monocyte chemoattractant protein 1 (mcp-1) and prostaglandin e2. these compounds are known for their participation in inflammatory processes and in extracellular matrix production. this exposure also results in a significant increase in the release of lactate dehydrogenase (ldh) (cell injury indicator) and cellular malondialdehyde (mda) content (lipid peroxidation indicator) (thamilselvan et al., 2003). the deposition of calcium oxalate crystals in the kidneys of rats also activates the renin-angiotensin system (khan, 2004). in parallel, in vitro pretreatment of renal epithelial cells with diphenyleneiodium (dpi) (nadph oxidase inhibitor), leads not only to a reduction in ros, mcp-1 and opn production, but also to a reduction in cell damage induced by oxalate and calcium oxalate crystals (umekawa et al., 2005). vitamin e effectively restores cellular antioxidants and prevents lipid peroxidation kachkoul r et al. (2023). not sci biol 15(1):11462 6 (thamilselvan et al., 2003). in addition, citrate and vitamin e reduce free radicals production induced by shock wave lithotripsy treatment (delvecchio et al., 2005). the study by umekawa et al. (2004) reported that the kidneys of hyperoxaluric rats treated with candesartan, an angiotensin ii type i receptor inhibitor, had less crystalline calcium oxalate deposits, reduced opn expression, and lower mda levels compared to untreated rats (umekawa et al., 2004). concerning the mechanism of action (figure 3), calcium oxalate crystals adhere to epithelial cells and nadph oxidase generates superoxide (o2•-), which will activate cyclophilin d. the opening of the mitochondrial permeability transition pore (mptp) associated with mitochondria collapse, generates oxidative stress, activation of the apoptotic pathway and a high opn expression. in contrast, cyclosporin a and nmethyl-4-isoleucine cyclosporine (nim811), a novel selective inhibitor of cyclophilin d activation, blocks mptp opening by inactivating cyclophilin d, opn expression and renal calcification (niimi et al., 2012; 2014, yasui et al., 2017). figure 3.figure 3.figure 3.figure 3. proposed pathway for the explanation of renal epithelial cell exposure to crystals: (1) com crystals attach to renal tubular cells; (2) nadph oxidase generates o2•-; (3) cyclophilin d activation and mptp opening accompanied by mitochondrial collapse; (4) blocking of mptp opening through inactivation of cyclophilin d by cyclosporin a (5) o2•release by mitochondria; (5-a) decrease in sod; (5-b) increase in 8-ohdg, (5-c) increase in 4-hne and mda; (6) cytochrome c release by mitochondria, caspase 3 activation; (7) apoptosis activation and cell damage (8) modification of phosphididylserine distribution in renal tubular cells membranes; (9) increased crystal binding and crystallization (niimi et al., 2012) cytochrome c, an important apoptosis-inducing protein, that is also released during mitochondrial collapse, thereby activating caspase-9 and caspase-3. these events trigger the onset of apoptosis, cell injury and alter the phosphatylserine distribution in renal tubular cells membranes (kohri et al., 2012; niimi et al., 2012). however, during this process, ros are also released from the intramembrane compartment into the cytosol, further damaging the renal tubular cells (yasui et al., 2017). kachkoul r et al. (2023). not sci biol 15(1):11462 7 crystal growth once a crystal nucleus reaches a critical size and the relative supersaturation remains greater than 1, the overall free energy is reduced by adding new crystalline components to the nucleus (aggarwal et al., 2013). however, crystal growth is determined by molecule size and shape, material physical properties, ss levels, ph, and any defects that may form in the crystal structure (basavaraj et al., 2007). furthermore, the process of stone growth is slow and takes time, often longer than the transit time of urine through the nephron, making the risk of intra-renal crystalline retention by crystals size very low (daudon et al., 2008). crystals aggregation-agglomeration aggregation is a crucial step in stone formation, where the crystals in solution stick together to form larger particles. in this process, large particles (several tens or, more rarely, several hundred microns) are formed in a very short time, less than the passage time of urine through the kidney (daudon et al., 2008; tsujihata, 2008; alelign and petros, 2018). however, the phenomenon of electrostatic attraction as a function of crystal surface charge has been implicated to explain this action (miller et al., 2007; daudon et al., 2008). crystal agglomeration is favoured by viscous bonds, involving urinary macromolecules (lipids, polysaccharides, and other materials derived from cells), which have several binding sites and act as a kind of glue. the latter secondarily promotes the attachment of new crystals to the former by relating them to each other, contributing to stone architecture and thus leading to retention of particles in the urinary tract (basavaraj et al., 2007; miller et al., 2007; daudon et al., 2008; ratkalkar and kleinman, 2011; aggarwal et al., 2013). yet, the interactions between macromolecules and crystals are complex and depend on several factors such as ph, ionic strength, inhibitors and promoter’s concentration. all of these factors can alter the conformation of these macromolecules, their affinity for crystals and their efficiency in exerting their inhibitory action (daudon et al., 2008). crystal retention stone’s development needs crystals formation in the tubular fluid, followed by crystals retention and accumulation in the kidneys (verkoelen and verhulst, 2007). three pathways of stone formation and growth have been proposed by evan (2010) (figure 4b). the first hypothesis, called the free particle model, indicates that crystal nuclei are formed by homogeneous nucleation in the nephron lumen, under conditions of increasing supersaturation of salts in the ultrafiltrate. as the nuclei grow, they become lodged in the distal nephron’s lumen, leading to tubular segment obstruction. the minor calyx of the renal collecting system could be the site of the free particles formation (evan, 2010). kachkoul r et al. (2023). not sci biol 15(1):11462 8 figure 4.figure 4.figure 4.figure 4. illustration of crystal retention pathways: a) according to (khan et al., 2016) [1: free particle mechanism (randall’s plug); 2: fixed particle mechanism (randall’s plate)]. b) according to (evan, 2010) [1: free particle mechanism; 2: fixed particle mechanism; 3: randall’s plaque] the second hypothesis, called the fixed particle model, where crystal nuclei form in the nephron lumen and then adhere to the apical surface of the tubular epithelium. once the step of crystal attachment to the cells has occurred, the nuclei would be fixed in position and exposed to the potentially supersaturated ultrafiltrate, which would facilitate the growth of these crystals (evan, 2010). the third pathway suggests that crystals in urine may become fixed to a site of interstitial calcium phosphate crystalline deposition (randall’s plaque), following the loss of normal urothelial coverage of a renal papilla (figure 4b). an anchored nest of urinary crystals could form an overgrowth on the interstitial plaque, thus allowing the fixed stone to develop (evan, 2010). however, khan et al. (2016) suggest two pathways (figure 4a) and combine the first two pathways mentioned above by (evan, 2010) into the free particle mechanism (randall’s plug), while the second is of fixed particle (randall’s plaque) (khan et al., 2016). stone growth stone growth velocity initiated by crystal retention is very variable and depends on the urine supersaturation level, and hence on the nature of the metabolic, genetic abnormalities and dietary faults. growth is not the only spontaneous mode of evolution that a stone retained in the urinary tract can undergo. in fact, other phenomena can also occur that lead to changes in the crystalline phases. they result from the instability of some hydrated forms which tend to evolve progressively over time towards thermodynamically more stable and less hydrated forms (daudon et al., 2008). stone formation promoters and inhibitors. inhibitors are substances that decrease the initiation of supersaturation, nucleation, crystal growth, aggregation velocity or any other process necessary for stone formation (basavaraj et al., 2007). inhibitors can act either directly by binding to the crystal surface and preventing crystal development, or indirectly by acting on the urinary environment (alelign and petros, 2018). there are at least four types of inhibitors in urine (table 1). small organic and inorganic anions such as citrate and pyrophosphates, multivalent metal cations such as magnesium and macromolecules such as osteopontin and tamm-horsfall protein (basavaraj et al., 2007; ratkalkar and kleinman, 2011; alelign and petros, 2018). some inhibitors may play two opposing roles. kachkoul r et al. (2023). not sci biol 15(1):11462 9 they inhibit one stage of crystal formation while also promoting another stage. for example, tamm-horsfall protein and glycosaminoglycans promote crystal nucleation, but inhibit crystal aggregation and growth (miller et al., 2007). table 1.table 1.table 1.table 1. inhibitors and promoters of crystallisation (basavaraj et al., 2007; miller et al., 2007; daudon et al., 2008; ratkalkar and kleinman, 2011; aggarwal et al., 2013; alelign and petros, 2018) low molecular weight low molecular weight low molecular weight low molecular weight inhibitorsinhibitorsinhibitorsinhibitors macromolecular inhibitorsmacromolecular inhibitorsmacromolecular inhibitorsmacromolecular inhibitors promoterspromoterspromoterspromoters cationscationscationscations zn2+ fe3+ mg2+ anionsanionsanionsanions citrate isocitrate phosphocitrate pyrophosphate aspartate glutamate hippurate proteinproteinproteinprotein tamm-horsfall protein nephrocalcin uropontin/osteopontin bikunin urinary prothrombin fragment 1 fibronectin calprotectin lithostathine inter-a-inhibitor glycosaminoglycans chondroitin sulphate heparin sulphate keratan sulfate dermatan sulfate hyaluronic acid calcium sodium oxalate urate ammonium cystine tamm-horsfall protein low urine ph low urine flow xanthine myeloperoxidase albumin annexin ii hyaluronic acid however, the promoters participate in the formation of insoluble species in urine and reduce the formation product of the supersaturated solution. this product may also be reduced due to the absence of endogenous inhibitors or by their opposite effects as a result of structural defects or other interfering substances (basavaraj et al., 2007; ratkalkar and kleinman, 2011). they are around ten in number (table 1) and very often associate in pairs or in threes to form a crystallizable substance, which itself can be present in several crystalline species (daudon et al., 2008). stone’s type stone’s type stone’s type stone’s type the identification of stone type has a major interest in guiding the physician towards an accurate diagnosis, which allows to determine the causes in order to treat and prevent recurrence of the disease. for these reasons, a morpho-constitutional analysis should be performed at least once during the lithiasis history and repeated in the event of recurrence, as the causes may change (courbebaisse et al., 2017). this analysis allows us to determine the chemical composition, the crystalline form as well as the stone structural characteristics. these data reveal the specific disease causes. for example, calcium oxalate stones, which are dominated by dihydrate form called weddellite, are essentially linked to hypercalciuria contexts, while those composed mainly of the monohydrate form known as whewellite are associated with hyperoxaluria, and alterations in stone structure reflect the severity of this condition (daudon et al., 2008). according to cotton et al. (2014), more than 70 of the chemical species have been identified in the composition of urinary stones and may be mineral or organic, including at least 25 of drug origin (cotton et al., 2014). moreover, there are six basic morpho-constitutional categories of urinary stones that can be further split into a variety of morphological subtypes, each corresponding to a particular etiological or pathophysiological condition (daudon et al., 2016a). kachkoul r et al. (2023). not sci biol 15(1):11462 10 oxalo-calcic stones globally, oxalo-calcic stones are the most frequent in the world and morocco country. according to studies carried out in the region of fez (morocco), the percentage of these stones is 60.98% (el habbani et al., 2016) and 56.25% in children (el lekhlifi et al., 2016). 66.6% in the rabat-salé (morocco) (bouatia et al., 2015) and 58.5% in the middle atlas (oussama et al., 2000). regarding the stone’s structure, there two principal types, namely calcium oxalate monohydrate (com) (figure 5a), that can be further categorized into five different morphological aspects (subtype types ia, ib , ic, id and ie) and characterised by a compact structure, hard and resistant to extracorporeal shock wave lithotripsy (doré, 2005). while the second type which is calcium oxalate dihydrate or weddellite (cod) (figure 5b), exhibits 3 subtypes (iia, iib, iic) ( doré, 2005; lechevallier et al., 2008; aguilar-ruiz et al., 2012; castiglione et al., 2015; daudon et al., 2016a). however, calcium oxalate crystals are mainly found in three different forms which are com, cod and the trihydrate form called caoxite (cot). the latter form is very rare (daudon, 2013, 2015, 2016b). yet com is the most thermodynamically stable form, has a higher affinity for renal tubular cells, and therefore responsible for stones formation in kidney (verkoelen et al., 1995; wesson et al., 1998). phosphate stones these stones type can be divided into two major groups which are calcium phosphates and ammonium magnesium phosphates. the most well-known are brushite (figure 5g), carbapatite (figure 5e) and struvite (figure 5f). the latter two are associated with a chronic urinary tract infection by urease-positive bacteria (proteus, klebsiella pneumoniae, pseudomonas aeruginosa staphylococcu saureus, aspergillus fumigatus, enterobacters). the latter produce the urease which is necessary to cleave urea into ammonia and co2, making the urine more alkaline at a ph generally higher than 7 and promotes their precipitation in insoluble ammonium-based products. this stone type is also called coralliform stones, because it can grow rapidly and fill pyelocalicial cavities (puigvert, 2002; rieu, 2005; bruyere et al., 2008). figure 5.figure 5.figure 5.figure 5. different types of urinary stones: a) calcium oxalate monohydrate; b) calcium oxalate dihydrate; c) anhydrous uric acid; d) ammonium urate; e) carbapatite; f) struvite; g) brushite; h and i) cystine; j) protein; k) n-acetylsulfadiazine; l) metabolites triamterene (cloutier et al., 2015; daudon et al., 2016a; estrade et al., 2017). kachkoul r et al. (2023). not sci biol 15(1):11462 11 uric stone uric stones have a high frequency in morocco with percentages of 19.8 and 18.42% in the regions of rabat and fez respectively (bouatia et al., 2015; el habbani et al., 2016). diets rich in purines, especially those containing animal protein, such as meat and fish, lead to hyperuricosuria, low urine volume, and low urine ph below 5.05, which promotes the uric acid stone formation (figure 5c; d) (alelign and petros, 2018). in contrast, the alkalisation of urine by bicarbonate intake converts uric acid, which is very poorly soluble in ionized form, into very soluble urates. moreover, a low-fructose diet (such as sodas, fruit juices, honey, maple syrup, etc.) and in purines (offal, game, poultry, cold meats, anchovies, crustaceans, etc.) must be provided to reduce the risk formation of this stones type. cystine stones cystine stones are uncommon and result from an abnormal transport of dibasic amino acids in the proximal tube. this hereditary disorder with autosomal recessive or incompletely recessive transmission depending on the genetic form, results in urine leakage of these amino acids, of which cystine is the least soluble. this leads to intratubular cystine crystallisation in the excretory tract (traxer et al., 2008). cystine stone (figure 5h; i) is hard and more resistant to eswl, producing large blocks that can obstruct the excretory tract (doré, 2005). its medico-surgical management is essential to limit the risk of lithiasis recurrence and to preserve the patient’s renal function (traxer et al., 2008). drug-induced stones drug stones are rare and can be classified into two categories. the first is the result of a drug’s urinary crystallisation, or a poorly soluble metabolite whose urinary excretion is important. the second includes all those related to the drugs metabolic effects. this type of stone appears in patients treated with a high dose and long term, and by the intervention of other factors such as urine ph, diuresis (servais et al., 2006). protein stones stones composed principally of protein are infrequent. they can be found mainly in three clinical settings. the first is chronic pyelonephritis linked to a urinary tract infection, the second concerns proteinuria observed in glomerular kidney disease, hematuria where bleeding caused by crystals and the third corresponds to the situation of end-stage renal disease (daudon et al., 2016a). conclusionsconclusionsconclusionsconclusions this review work has highlighted the process of urinary stone formation, which includes all the biological and physicochemical pathways produced from supersaturated urine and lead to the development of a stone in the urinary tract. the studies on this process must be deepened because many aspects of the kidney stones formation remain unclear and sometimes contradictory. this will lead to the development of a new prevention strategy, as well as the development of drugs against this disease. authorsauthorsauthorsauthors’’’’ contributionscontributionscontributionscontributions rk: conceptualization, wrote the paper, collect and interpret data; gbt, gem, reh, mm and al: contribution to writing the manuscript, correction of the final manuscript. all authors read and approved the final manuscript. kachkoul r et al. (2023). not sci biol 15(1):11462 12 ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences agawane sb, gupta vs, kulkarni mj, bhattacharya ak, koratkar ss, rao vk (2019). patho-physiological evaluation of duranta erecta for the treatment of urolithiasis. journal of ayurveda and integrative medicine 10(1):4-11. https://doi.org/10.1016/j.jaim.2017.08.001 aggarwal a, tandon s, singla sk, tandon c (2010). diminution of oxalate induced renal tubular epithelial cell injury and inhibition of calcium oxalate crystallization in vitro by aqueous extract of tribulus terrestris. international brazilian journal of urology 36(4):480-488. https://doi.org/10.1590/s1677-55382010000400011 aggarwal kp, narula s, kakkar m, tandon c (2013). nephrolithiasis: molecular mechanism of renal stone formation and the critical role played by modulators. biomed research international 2013:1-21. https://doi.org/10.1155/2013/292953 aguilar-ruiz j, arrabal-polo ma, sierra m, arrabal-martin m (2012). application of mineralogical techniques in the study of human lithiasis. ultrastructural pathology 36(6):367-376. https://doi.org/10.3109/01913123.2012.729879 ahmed s, hasan mm, khan h, mahmood za, patel s (2018). the mechanistic insight of polyphenols in calcium oxalate urolithiasis mitigation. biomedicine & pharmacotherapy 106:1292-1299. https://doi.org/10.1016/j.biopha.2018.07.080 alelign t, petros b (2018). kidney stone disease: an update on current concepts. advances in urology 2018:1-12. https://doi.org/10.1155/2018/3068365 arumuham v, bycroft j (2016). the management of urolithiasis. surgery (oxford) 34(7):352-360. https://doi.org/10.1016/j.mpsur.2016.04.007 basavaraj dr, biyani cs, browning aj, cartledge jj (2007). the role of urinary kidney stone inhibitors and promoters in the pathogenesis of calcium containing renal stones. eau-ebu update series 5(3):126-136. https://doi.org/10.1016/j.eeus.2007.03.002 bashir s, and gilani ah (2011). antiurolithic effect of berberine is mediated through multiple pathways. european journal of pharmacology 651(1-3):168-175. https://doi.org/10.1016/j.ejphar.2010.10.076 bouatia m, benramdane l, oulad bouyahya idrissi m, draoui m (2015). an epidemiological study on the composition of urinary stones in morocco in relation to age and sex. african journal of urology 21(3):194-197. https://doi.org/10.1016/j.afju.2015.02.006 bruyere f, traxer o, saussine c, lechevallier e (2008). infection et lithiase urinaire. progres en urologie 18(12):10151020. https://doi.org/10.1016/j.purol.2008.09.015 cao lc, honeyman tw, cooney r, kennington l, scheid cr, jonassen ja (2004). mitochondrial dysfunction is a primary event in renal cell oxalate toxicity. kidney international 66(5):1890-1900. https://doi.org/10.1111/j.1523-1755.2004.00963.x kachkoul r et al. 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(2023). not sci biol 15(1):11462 16 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11402 nsb zeouk 2023.03.16.docx received: 05 dec 2022. received in revised form: 02 feb 2023. accepted: 07 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania zeouk i (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11402 doi:10.15835/nsb15111402 rererereviewviewviewview articlearticlearticlearticle.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae from skin infection to invasive diseases: a descriptive review of from skin infection to invasive diseases: a descriptive review of from skin infection to invasive diseases: a descriptive review of from skin infection to invasive diseases: a descriptive review of staphylococcus aureusstaphylococcus aureusstaphylococcus aureusstaphylococcus aureus, focusing on panton, focusing on panton, focusing on panton, focusing on panton----valentine leucocidin and valentine leucocidin and valentine leucocidin and valentine leucocidin and methicillinmethicillinmethicillinmethicillin----resistant strainsresistant strainsresistant strainsresistant strains ikrame zeouk national agency of medicinal and aromatic plants, laboratory of pharmaceutical industry, taounate 34025, morocco; ikramezeouk20@gmail.com abstractabstractabstractabstract despite advances in scientific research, staphylococccus aureus remains a pyogenic and toxigenic bacterium involved in different infections, it endowed with the capacity to infect several biotopes and cause a wide range of infections ranging from skin diseases to other serious pathologies such as pneumonia, meningitis, sepsis, osteomyelitis and infectious endocarditis. moreover, the emergence of resistant strains constitutes a serious public health problem. thus, the development of new active compounds from natural sources such as medicinal plants is urgently needed. to this end, the aim of our review was to describe the state of art of infections caused by s. aureus, its pathogenesis, treatment and to provide a synthesis about studies reporting a bio guided isolation of most promising compounds selected for their anti-staphylococcal activity. keywords:keywords:keywords:keywords: bio guided fractionation; natural products; resistance; staphylococcus aureus introductionintroductionintroductionintroduction the emergence of antibiotic resistance constitutes a serious public health problem. predictive models have estimated that by 2050, antimicrobial resistance will be the main cause of global death with more than 10 million deaths per year, including around 5 million in asia, 4 million in africa, 400.000 in europe and 300.000 in north america (o’neill, 2014). the additional cost attributed to antibiotic resistance will increase to 1.5 billion euros (eur-rc, 2011) in europe, and in the united states it would be around 55 billion $ (cdc, 2013). the world health organization (who) has sounded the alarm on the consequences of antibiotic resistance, thus it has placed a global plan action that defines several objectives mainly the development of alternative treatment (oms, 2016). however, the problem of resistance persists and in 2020 the same organization has declared reemergence of resistant microorganisms and has insisted on the development of new therapeutic molecules (oms, 2022). staphylococcus aureus is among the pathogens resistant to treatment, it is an opportunistic and cosmopolitan bacterium that colonizes the skin and mucous membranes of humans. within this species there are toxigenic variants such as those expressing the panton-valentine leucocidin toxin (pvl) which aggravates the pathology and make s. aureus more toxicogenic (nakaminami et al., 2020; https://www.notulaebiologicae.ro/index.php/nsb/index zeouk i (2023). not sci biol 15(1):11402 2 petraitiene et al., 2020). s. aureus causes a major public health problem despite advances in scientific research, it endowed with the capacity to infect several biotopes and cause a wide range of infections ranging from skin diseases to other serious pathologies such as pneumonia (grousd et al., 2019), meningitis (aguilar et al., 2010), sepsis (bawazir and mustafa, 2020), osteomyelitis (weiss et al., 2020), and infectious endocarditis (selton-suty et al., 2012). transmission of s. aureus is both community-based and nosocomial (chow et al., 2020). in addition, strains of s. aureus have developed multidrug resistance to different families of antibiotics. today, more than 90% of s. aureus strains produce a penicillinase that limits the action of beta-lactams. moreover, methicillin, the first semi-synthetic penicillin not susceptible to penicillinases, quickly disappointed the medical world with the emergence of strains resistant to methicillin (mrsa). in 2019, who has developed a new indicator of antimicrobial resistance to track the frequency of sepsis caused by mrsa and the median rate reported by 25 countries was 12.11% (oms, 2022). in fact, the treatment of mrsa is based on glycopeptides (vancomycin). however, less susceptible strains to glycopeptides have been isolated. these isolates are called visa (vancomycin-intermediate s. aureus), or more generally gisa (glycopeptide-intermediate s. aureus). among the newest antibiotic on the market, linezolid (zyvox), it was introduced to the north american market in 2000. as early as 2001, cases of mrsa resistant to linezolid have been reported (tsiodras et al., 2001; rouard et al., 2018). faced with the emergence of resistance to current antibiotics, strategies are now in place to renew therapeutic biomolecules. pharmaceutical companies have turned away from natural products to synthetic chemistry. thus, tens of billions of dollars have been invested in research and development (r&d), however, these new methods of discovering bioactive molecules seem to have reached certain limits (gershell and atkins, 2003; butler, 2004), which may explain the interest in researching new compounds from natural sources such as medicinal plants that are endowed with a secretion of different secondary metabolites (bérubé-gagnon, 2006). the aim of the present review was to describe the state of art of infections caused by s. aureus, its pathogenesis, treatment and to provide a synthesis about studies reporting a bioguided isolation of most promising compounds selected for their antistaphylococcal activity. staphylococcus aureusstaphylococcus aureusstaphylococcus aureusstaphylococcus aureus the essential reservoir for s. aureus is humans where it lives mainly in the commensal state in moist skin areas (perineum, armpits), mucous membranes and nasal cavities (freeman-cook and cook, 2006). however, these bacteria can become formidable pathogens, after an alteration of the normal skin architecture, an invasive infection could be induced (archer, 1998). the mode of transmission of staphylococcus spp. is broader. in other words, there is not a definite cycle of transmission. air, dust, bedding, blankets, medical equipment, food, and hands are all disseminators of s. aureus emphasizing intraand human-to-human transmission (robinson et al., 2019). it is now accepted that s. aureus is one of the most dangerous bacteria transmitted by both community and nosocomial ways, leading to the spread of many serious infections difficult to treat. clinical manifestationsclinical manifestationsclinical manifestationsclinical manifestations infections caused by s. aureus can range from skin infections, which are easy to treat, to other invasive pathologies, which are much more difficult to treat or even fatal. s. aureus can infect the skin, respiratory, digestive, urinary and reproductive tracts and can cause serious infections of the heart and bones (figure 1). zeouk i (2023). not sci biol 15(1):11402 3 figure figure figure figure 1111. skin colonization by staphylococcus aureus and the resulting infections (by zeouk, 2022) skin infections skin infections skin infections skin infections according to moran et al. (2006), s. aureus is responsible of 76% of skin and soft tissue infections (moran et al., 2006b). the main skin infections caused by s. aureus include among others impetigo, folliculitis, boil, and skin abscess (figure 2). figure 2. figure 2. figure 2. figure 2. common skin infections caused by s. aureus: a, impetigo ; b, folliculitis (selk and wood, 2019); c, boil; d, skin abscess zeouk i (2023). not sci biol 15(1):11402 4 the clinical profile of these infections is different; impetigo is manifested as pustules, vesicles, or delicate bubbles at the superficial level, most often around the mouth or nose, on the chin or behind the ears. it can also affect the trunk, buttocks, or hands. these lesions quickly become inflamed and form honey-colored scabs (figure 2, a). it is a contagious infection that affects mainly children, but also adults with immune deficiency (lawrence and nopper, 2012). folliculitis begins as small papules, with a pustule centered by a hair associated with perifollicular erythema (figure 2, b). all parts of the body can be affected, such as the thighs, perineum, arms, back, and eyelid (selk and wood, 2019). the deep and necrotizing form of folliculitis with involvement of the pilosebaceous follicle in its entirety, is called a boil (figure 2, c) which is manifested as a painful inflammatory papule or nodule centered around a pustule (del giudice, 2020). the boil looks similar to a skin abscess (figure 2, d) which is also presented as a nodule or an erythematous mass, sometimes with a central pustule and spontaneous drainage of pus, but which does not have a privileged location (kobayashi et al., 2015). thus, del giudice (2020) has insisted on considering skin infections caused by s. aureus as a complex group of diseases with a very varied clinical spectrum. moreover, skin abscesses are the most common and dangerous manifestation of s. aureus, because when they are pyogenic, they can develop in deeper tissues like the underlying muscles, then bacteria can spread to form abscesses throughout the internal organ system (kobayashi et al., 2015). in a recent retrospective study, rochet et al. (2020) have shown that the rate of patients visits for skin abscesses in the emergency department has increased significantly and that s. aureus was the pathogen involved in 73% of these infections (rochet et al., 2020). moreover, ismail et al. (2020) have described a large epidemic of skin abscesses among gold mine employers in south africa. s. aureus pvl was responsible for this epidemic, due to cross-contamination resulting from poor hygiene practices. invasive infections caused by invasive infections caused by invasive infections caused by invasive infections caused by s. aureuss. aureuss. aureuss. aureus s. aureus is responsible for pneumonia which can have a serious prognosis, especially those corresponding to a nosocomial infection (nosocomial pneumonia) (grousd et al., 2019). in a recent study in healthcare workers, colonization of saliva by s. aureus was the main cause of pneumonia and the oral environment acts as a potential reservoir for lower respiratory tract infection and development of pneumonia (chiang et al., 2020). another pathology caused by s. aureus is meningitis, a retrospective study has shown that when caused by s. aureus, meningitis has devastating clinical consequences and high mortality rates (36%) due to limited treatment options (aguilar et al., 2010). other more serious diseases are osteomyelitis and endocarditis. it was reported that during osteomyelitis, s. aureus presented 60.5% of positive cases in culture (weiss et al., 2020). in addition, another study has confirmed the high prevalence of staphylococcal osteomyelitis with 85.1% of cases (silago et al., 2020). according to urish and cassat (2020), treatment of osteomyelitis has remained largely unchanged over the past decades and focuses on personalized antibiotic therapy with correction of medical co-morbidities (urish and cassat, 2020). thus, the majority of cases of osteomyelitis have been associated with large, deep purulent collections requiring surgical debridement (kok et al., 2018), which increasingly complicates the patients care. furthermore, s. aureus is the main causative agent of infectious endocarditis associated with high death rates and embolic events (selton-suty et al., 2012). this infection has been noted to be fatal in 20% to 65% of cases, and surviving patients suffer permanent sequelae from local invasion of cardiac structures (murray, 2005). in addition to organic damage, the ubiquitous nature of s. aureus increases the risk of its passage into the blood from different primary foci by causing sepsis with distinct origins, namely cutaneous, urinary, dental and others. indeed, mrsa sepsis was first reported as the underlying etiology of acute esophageal necrosis (bawazir and mustafa, 2020). in all described pathologies in the present work, one infection could be the complication of the other which is a pathological behavior frequently observed in s. aureus. in this sense, albayati et al. (2020) have reported that s. aureus skin and soft tissue infections cause an unusual complication zeouk i (2023). not sci biol 15(1):11402 5 of persistent bacteremia which itself leads to complicated endocarditis in addition to an affinity of the involved strain for vertebral osteomyelitis (al-bayati et al., 2020). several studies have documented the risk of endocarditis as a complication of bacteremia and have confirmed that it is a serious complication (bouchiat et al., 2015; andersen et al., 2020). furthermore, grillo et al. (2020) have shown that the bacteremia itself can develop from urinary tract infections due to probing and catheterization, which leads to significant mortality considering that in many cases, the strains of mrsa were the most identified. therefore, it is necessary to ensure the complete eradication of s. aureus infections and especially those of the skin. indeed, the ability of all these infections to be life-threatening or not depends on several factors, in particular the virulent nature of the strain in question, the host's immune response and the intensity of resistance to the clinical protocol. several studies have confirmed that the rate of infections worldwide gradually increases with increasing drug resistance and that clinical anti-infective treatment of resistant strains has become more difficult, especially with an immune response modulated by the bacterium (guo et al., 2020; scudiero et al., 2020). antiantiantianti----biotherapy and emergence of resistance biotherapy and emergence of resistance biotherapy and emergence of resistance biotherapy and emergence of resistance beta-lactams, aminoglycosides, and macrolides are among the main antibiotic families used in the treatment of s. aureus infections (taylor, 2013). however, s. aureus has a remarkable ability to acquire resistance to different antibiotics with first the appearance of resistance to penicillin which has been so prevalent that the antibiotic is no longer effective, more than 90% of the strains are resistant (lowy, 2003). this resistance has increased because, under massive and selective use of the antibiotic, certain strains of s. aureus express penicillinases able to hydrolyze the active beta-lactam site of the drug (khoshnood et al., 2019). to this end, scientists have developed a new semi-synthetic penicillin able to resist against hydrolysis of staphylococcal beta-lactamases, methicillin (pottinger, 2013). however, s. aureus has quickly developed several resistant strains (mrsa) characterized by a modified penicillin binding protein (pbp2a/c) and which loses its affinity for most antibiotics belonging to the beta-lactam family (gajdács, 2019). this broad-spectrum resistance is mainly due to the meca and mecc genes, located on staphylococcal chromosomal cassette mec (sccmec) (paterson et al., 2014). although mrsa is the major pathogen identified in nosocomial pathologies, it is the most isolated germ also during community infections. this spread lead moran et al. (2006) to suggest that among all s. aureus infections, a main proportion is caused by mrsa (moran et al., 2006b). this suggestion has been widely confirmed in most of the skin, internal and visceral infections previously described. for example, in a study of military personnel with skin and soft tissue infections, 70% of the strains were resistant to methicillin (landrum et al., 2012). therefore, alternative anti-mrsa treatment recommendations were developed namely vancomycin which has become the mainstay of antibiotic therapy for many forms of mrsa infections (diaz et al., 2017). its activity is due to its binding to cell wall precursors, not to pbp2 (pottinger, 2013). however, a phenomenon of gradual increase in mics (minimum inhibitory concentrations) has been reported in many s. aureus isolates leading to the emergence of visa (vancomycin intermediate-resistant s. aureus), vrsa (vancomycin resistant s. aureus) and hetero-vrsa (ahmad et al., 2018). because of the rapid emergence of resistance in s. aureus strains and the spread of invasive mrsa infections, other antibiotics have been used. in a recent review, guo et al. (2020) have documented the antimrsa antibiotic therapy and have reported that daptomycin is effective in the treatment of skin infections and bacteremia caused by mrsa. unfortunately, the use of this antibiotic is limited in pneumonia because its mechanism of action is based on the destruction of the electrical potential of the plasma membrane without inhibiting the lipoteichoic acid, thus in the respiratory system, its activity is blocked by the alveolar surfactant zeouk i (2023). not sci biol 15(1):11402 6 (taylor and palmer, 2016). moreover, roch et al. (2017) have described a clinical case with mrsa infection in which the strain isolated was resistant to daptomycin (roch et al., 2017). indeed, mrsa have not developed resistance only to daptomycin but also to tetracycline and ciprofloxacin (lai et al., 2017). recently, yamashita et al. (2019) have conducted a comparative study between daptomycin, vancomycin and azithromycin using a mouse model of mrsa pneumonia. they have demonstrated that treatment with azithromycin after 24 h of infection was effective, showing significantly longer survival and a low bacterial load in the lungs. therefore, they suggested that this antibiotic may be a potential prophylactic agent for mrsa pneumonia (yamashita et al., 2019). despite intensified efforts to find an effective treatment, mrsa is still a major cause of mortality and morbidity around the world. in addition, the concomitant emergence of resistance is to be expected. according to vestergaard et al. (2019), multidrug resistance of mrsa has considerably complicated the difficulties of scientific research (vestergaard et al., 2019b). virulence factorsvirulence factorsvirulence factorsvirulence factors the pathogenicity of s. aureus depends on many virulence factors in addition to the immune defenses of the host. these factors mainly include exoproteins such as secreted toxins (exotoxins) which disrupt host cells and interfere with immune responses, and surface proteins which play various roles in pathogenesis such as adhesion, but which is not a direct cause of toxicity towards host tissues (vincenot et al., 2008). expression of virulence factors by to expression of virulence factors by to expression of virulence factors by to expression of virulence factors by to s. aureuss. aureuss. aureuss. aureus the pathophysiology of s. aureus infections begins with the colonization of surfaces producing different kinds of adhesion molecules, adhesins. among the most important molecules is protein a which is a key factor in infections establishment (palmqvist et al., 2002). depending on the state of the host cell, protein a can act either by disguising s. aureus from the host's immune system by allowing it to resist against phagocytosis, or by completely deactivating the humoral immune response, or by inducing inflammatory cytokines and chemokines (falugi et al., 2013; gonzalez et al., 2019). in addition to protein a, some strains can form a microcapsule or develop a viscous polysaccharide substance called slime (baselga et al., 1993). after attachment to the tissues of the host cell, s. aureus secretes several enzymes involved in different mechanisms such as the degradation of the host tissues, which promotes the extension of the infectious focus. the main extracellular enzymes include, among others, proteases, catalases, deoxyribonucleases, lipases, phosphatases, hyaluronidases and coagulases ... for example, lehman et al. (2019) have shown that during the proliferation of skin abscesses, staphylococcal proteases played an important role in the digestion of peptides and amino acids necessary for the nutrient metabolism of s. aureus. in addition, the overexpression of these proteases was involved in the elevated pathogenesis of fak (fatty acid kinase) during skin infection (ridder et al., 2020), while treffon et al. (2020) have concluded that the two superoxide dismutases (soda and sodm)typical of s. aureus compensates the survival of this bacterium during the destruction of leukocytes, which confirms that the interaction between these two enzymes is at the origin of the virulence and the persistence of s. aureus in the respiratory tracts and during cystic fibrosis, in addition to triggering inflammatory reactions and the fight against oxidative stress (treffon et al., 2020). other enzymes allow s. aureus to fight oxidative reactions, such as catalase which converts hydrogen peroxide into water and oxygen (mandell, 1975). at the same time, s. aureus produces around forty exotoxins that make up about 10% of the total secretome, many of which have same functions due to remarkable structural similarity. according to their functions, several studies have shown that exotoxins fall into three main groups namely cytotoxins which act on the membranes of host cells leading to cell lysis and inflammation, zeouk i (2023). not sci biol 15(1):11402 7 toxic enzymes which damage host cells, and superantigens involved in the massive production of cytokines inducing proliferation of t and b cells (tam and torres, 2018). major toxins include hemolysins, leukocidins, exfoliatins, enterotoxins, and toxic shock toxin. the toxin known as panton-valentine leukocidin (pvl) is known worldwide for its potential role in virulence and for its involvement in invasive infections. thus, although only 5% of s. aureus strains produce pvl, it was widely studied. indeed, pvl only causes cytotoxic changes in human monocytes and rabbits, because the cytotoxic activity towards these cells is highly specific and targets receptors coupled to human g proteins and those of rabbits (spaan et al., 2015), therefore, these models are a good approach to better understand the complexity and pathology mediated by pvl. in this context, several studies have developed animal models on rabbits and have shown that individuals infected with wild-type pvl+ strains have developed more severe infections and higher mortality rates compared to individuals infected with pvlstrains (diep et al., 2010; lipinska et al., 2011). moreover, to assess the production of pvl during human infections, nakaminami et al. (2020) have conducted a recent study in which they have demonstrated that pvl-producing strains are widely distributed in skin infections and that the severity of these infections in patients infected with pvl+ is greater than that in patients infected with pvl(nakaminami et al., 2020). this severe progression of infections caused by pvlproducing strains has been confirmed in the literature (petraitiene et al., 2020). currently, duployez et al. (2020) have reported a fatal case of a young adult with covid-19, where the complication of viral infection was related to necrotizing pneumonia caused by s. aureus producing pvl (duployez et al., 2020). thus, the pathogenesis of s. aureus involves a multitude of virulence factors that do not occur at the same time, but their production is finely coordinated, which explains the increased pathogenicity of s. aureus and the variation in its clinical profile. according to jenul and horswill (2019), the regulation of virulent factors by s. aureus is subject to a complex network that integrates the host and signals derived from the environment (jenul and horswill, 2019). one of the most studied regulatory systems is the agr gene which is a "quorum sensing system" and which allows s. aureus to discover the density of its own population and to translate this information into a specific gene expression model in order to control the expression of its genes (butrico and cassat, 2020). interaction hostinteraction hostinteraction hostinteraction host--- s. aureuss. aureuss. aureuss. aureus in s. aureus infection, the pathogen is recognized by cells in the infected tissue. many cells can fulfill this role, macrophages, monocytes and neutrophils are major players in the control of staphylococcal infections (accarias, 2014). indeed, the composition of the wall of s. aureus itself is also involved in the recognition and initiation of the host's immune defense. among these components, lipoproteins, lipoteichoic acid (lta) and peptidoglycan (pgn) are the predominant (leemans et al., 2003). several studies have documented the interaction between s. aureus wall components and the host's immune response. the synergy between lta and pgn leads to the production of a cascade of cytokines and chemokines allowing the recruitment of inflammatory cells in the host. protein a with lta can also stimulate the release of these inflammatory mediators(gómez et al., 2004; wu et al., 2020). nevertheless, s. aureus can limit phagocytosis and to attenuate the pro-inflammatory responses of the host, which favors its persistence in the microenvironment, especially by modulating macrophages, or even inducing necroptosis in these cells by the previously described virulence factors (patou et al., 2008). therefore, s. aureus will be able to survive and disseminate in phagocytes (horn et al., 2017). these interactions when regulated and the secreted cytokines are adjusted to balance between pro and anti-inflammatory ones, host cells successfully phagocytose and destroy the bacteria. however, s. aureus like any other infectious agent can also trigger an exaggerated immune response, during infection, if for example zeouk i (2023). not sci biol 15(1):11402 8 two cytokines are secreted in a high amount, they can participate in a fatal outcome (vom berg et al., 2013). indeed, it has been shown that pvl seems to have a major impact in the amplification of the immune responses of the host, huang et al. (2020) have shown in a pneumonia model that s. aureus pvlwas responsible for the severity of the disease by increasing the expression of pro-inflammatory cytokines (huang et al., 2020). in addition to the increased production of cytokines, pvl was able to modulate the host's immune response by decreasing the expression of tfnα (yoong and pier, 2012). in addition to immune responses, the metabolic pathway plays an important role in the interactions between s. aureus and the host. lopez et al. (2017) have found that using the fak enzyme complex, s. aureus is able to detect specific cis-unsaturated fatty acids which are very abundant in host tissue and which s. aureus is unable to produce (lopez et al., 2017). in the same context, potter et al. (2020) have conducted a comprehensive analysis of the metabolic needs of s. aureus during osteomyelitis, and they showed that the biosynthesis of aspartate represents a key metabolic node for the survival of staphylococci during infection, this biosynthesis has been greatly favored by the host's nutrient medium (potter et al., 2020). furthermore, delekta et al. (2018) have shown that during infection, human lipoprotein particles provide a viable source of exogenous fatty acids for s. aureus (delekta et al., 2018). another study conducted by lehman et al. (2019) has shown that during abscesses, collagen abundant at the site of infection in the host can serve as a nutrient reservoir for s. aureus overgrowth (lehman et al., 2019). antiantiantianti----s. aureuss. aureuss. aureuss. aureus plant extracts and isolated active compoundsplant extracts and isolated active compoundsplant extracts and isolated active compoundsplant extracts and isolated active compounds plant extracts are an important source for the identification of active compounds against s. aureus (table 1). the chemical diversity of these compounds is quite remarkable. chabán et al. (2019) have conducted a bioguided fractionation of the extract prepared from the aerial part of lepechinia meyenii, a species of the lamiaceae family, selected from an ethnobotanical study. the ethanolic extract has been shown to be the most effective with mics ranging from 62.5 to 500 μg/ml against strains of s. aureus resistant and sensitive to methicillin. chemical fractionation resulted in the identification of carnosol, rosmanol and carnosic acid as active compounds with mics ranging from 7.8 to 62.5 μg/ml against 15 strains of mrsa and 11 strains of mssa (chabán et al., 2019). zheng et al. (2019) were interested in the study of different parts of the garcinia esculenta species belonging to the clusiaceae family and they have identified a new xanthone, (±) garciesculenxanthone c with bacteriostatic effect against mrsa, mssa and visa (zheng et al., 2019). table 1. table 1. table 1. table 1. some effective crude extract fractionated against s. aureus plantsplantsplantsplants famillyfamillyfamillyfamilly extractextractextractextract phytochemical phytochemical phytochemical phytochemical families of isolated families of isolated families of isolated families of isolated compoundscompoundscompoundscompounds referencesreferencesreferencesreferences lepechinia meyenii (walp.) (arial part) lamiaceae ethanolic diterpenes phenols and polyphenols (chabán et al., 2019) garcinia esculenta (leave and twigs) clusiaceae ethanolic xanthones, biphenyles (zheng et al., 2019) syzygium antisepticum (leave) myrtaceae acetone/methanol/ water sesquiterpenes (yuan and yuk, 2018) pterocarpus erinaceus (peel and root) fabaceae methanol/ dichloromethane triterpenoides and steroides (tittikpina et al., 2018) acacia polyacantha (leave, peel and root) fabaceae methanolic sterols, triterpenes, saponines, and flavonoïds (ashu et al., 2020) zeouk i (2023). not sci biol 15(1):11402 9 sesquiterpenes have also shown significant activity against s. aureus. yuan and yuk (2018) have characterized β-caryophyllene as the main compound in the active extract of syzygium antisepticum. this sesquiterpene has induced damage to s. aureus membrane (yuan and yuk, 2018). in the same year, tittikpina et al. (2018) have characterized active compounds in the extract of pterocarpus erinaceus of the fabaceae family. these compounds were identified as friedeline, 2,3 dihydroxypropyloctacosanoate, and β-sitosteryl-β-dglucopyranoside, and they have showed interesting activity against mrsa with a mic of 4 µg/ml (tittikpina et al., 2018). several studies have confirmed the richness of fabaceae in species with antistaphylococcal activity (ashu et al., 2020; guidi et al., 2020). recently, the bioguided fractionation of boswellia dalzielii has confirmed the effectiveness of this process in the purification of compounds active against s. aureus. the crude extract has exhibited moderate activity (mic = 250 µg/ml), the fractions have exhibited good activity with mics ranging from 7.8 to 125 µg/ml, while the purified compounds from these fractions have exhibited promising activity with a mic value of 3.125 µg/ml (tegasne et al., 2020). further studies have considered bioguided fractionation for the discovery of compounds able to inhibit biofilm formation by s. aureus. in this context, manilal et al. (2020) have reported the antistaphylococcal activity of moringa stenopetala selected among three other species. its ethanolic extract has shown a bacteriostatic effect against mrsa by inhibiting its growth in the preformed matrix of the biofilm. chemical analysis of this extract revealed 12 active compounds belonging to different chemical classes (manilal et al., 2020b). moreover, biofilm formation by s. aureus has been shown to be highly influenced by frangula alnus extract rich in flavonoids and anthraquinones such as catechin and emodin (đukanović et al., 2020). recently, our team has conducted a bioguided fractionation of the ethanolic extract of rhamnus alaternus, the purification procedure allowed us to identify the same families anthraquinone and flavonoids, emodin being the most active compound without cytotoxicity towards murine macrophages (zeouk et al., 2021). conclusionsconclusionsconclusionsconclusions s. aureus represents a causative agent of different serios diseases that constitutes an increasing health problem. current antibiotics are not satisfactory despite the advances in science. the multiplicity of diseases caused by s. aureus is mainly related to the strains as well as the immune response of the infected host. thus, the development of new antibiotics with a noticeable effect and reduced toxicity remains an urgent need especially from natural antimicrobial agents, which could constitute a promising therapeutic approach. poincianella pluviosa (peel) fabaceae ethanolic polyphenols (guidi et al., 2020) boswellia dalzielii (peel) burceraceae methanolic terpenoides and triterpenes (tegasne et al., 2020) moringa stenopetala (leave) moringinaceae ethanolic several families (manilal et al., 2020a) frangula alnus (peel) rhamnaceae ethyl acetate phenols, flavonoids and anthraquinones (đukanović et al., 2020) rhamnus alaternus (leave) rhamnaceae ethanolic anthraquinones flavonoids (zeouk et al., 2021) zeouk i (2023). not sci biol 15(1):11402 10 authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions the author read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences accarias s (2014). impact du phénotype des macrophages résidents sur la nature de la réponse inflammatoire précoce lors d’une infection par staphylococcus aureus. aguilar j, urday-cornejo v, donabedian s, perri m, tibbetts r, zervos m (2010). staphylococcus aureus meningitis case series and literature review. medicine (baltimore) 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licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11437 nsb boukelloul 2023.03.16.docx received: 08 jan 2022. received in revised form: 08 feb 2023. accepted: 10 mar 2023. published online: 16 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania boukelloul i et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11437 doi:10.15835/nsb15111437 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae antagonism and plant growth promoting traits of actinomycetes antagonism and plant growth promoting traits of actinomycetes antagonism and plant growth promoting traits of actinomycetes antagonism and plant growth promoting traits of actinomycetes isolated from the rhizosphere of halophyte isolated from the rhizosphere of halophyte isolated from the rhizosphere of halophyte isolated from the rhizosphere of halophyte atriplex halimusatriplex halimusatriplex halimusatriplex halimus l.l.l.l. inas boukelloul1,2, lamia aouar2*, mohammed chekara bouziani1,3, amar zellagui1,2, mouna derdour4, youcef necib5 1university of oum el bouaghi, department of natural sciences and life, po box 358, oum el bouaghi 04000, algeria; inasbouka@gmail.com; zellaguiuniv@yahoo.com 2university of oum el bouaghi, laboratory of biomolecules and plant breeding, department of natural sciences and life, po box 358, oum el bouaghi 04000, algeria; aouar.lamia@univ-oeb.dz (*corresponding author) 3university mentouri constantine 1, laboratory of genetic biochemistry and plant biotechnology, constantine, algeria; bouziani25@yahoo.fr 4national center for biotechnology research, uv 03 po box e73 constantine 25000, algeria; moonlmd@hotmail.fr 5university mentouri constantine 1, laboratory of microbiological engineering and applications, constantine, algeria; youcefnecib@yahoo.fr abstractabstractabstractabstract biocontrol is considered as an effective alternative to the application of agrochemicals, which are harmful to the environment, human, and animal health. in this study, twenty-six strains of actinomycetes were isolated from rhizospheric arid soil of the halophyte atriplex halimus l. ῾guettaf’ in biskra province, algeria. the six isolates that have inhibited at least three phytopathogenic fungi among the five tested (fusarium oxysporum, alternaria alternata, fusarium solani, aspergillus flavus and botrytis cinerea) were selected, and have been tested in vitro against phytopathogenic bacteria (pectobacterium carotovorum and streptomyces scabies). they were also evaluated for their ability to hydrolyze phosphate, elaborate siderophores, produce indole-3acetic acid (iaa), and to antagonize s. scabies in vivo (on radish seedlings). based on the physicochemical analyses, soil samples were categorized as alkaline and extremely-saline. the antagonism results revealed varying antifungal potential among the selected isolates (act11, act16, act17, act18, act23 and act24), about 50% were able to inhibit the growth of f. solani and a. flavus, followed by 33.33% of those having antagonized f. oxysporum, while a. alternata was found to be the most sensitive. only act18 has antagonized s. scabies in vitro with an inhibition diameter zone of 19 ± 0.41 mm. however, in vivo trials showed that four isolates have counteracted s. scabies. among them, act18 and act24 have significantly and positively affected the root surface (p = 0.0062) and prevented common scab. iaa was detected in all selected isolates with act24 being the highest producer (77.45 μg ml−1). additionally, degradation ability revealed that four isolates were able to hydrolyze phosphate while three exhibited the capacity of elaborating siderophores. the six isolates were assigned to streptomyces genius according to their morphological, physiological and chemotaxonomical traits. based on this study, streptomyces sp. act18 and streptomyces sp. act24 that tolerate 7.5% nacl concentration, https://www.notulaebiologicae.ro/index.php/nsb/index boukelloul i et al. (2023). not sci biol 15(1):11437 2 prevent common scab and exhibit some plant growth attributes, may be considered as promising biocontrol agents to be applied in arid and saline soils. kekekekeywords:ywords:ywords:ywords: actinomycetes; antagonism; atriplex halimus; pgpr; rhizospheric soil introductionintroductionintroductionintroduction field crops production is affected by plant diseases, fungi are the main cause of damage and quality degradation of harvested products. in this regard, a range of fungal species cause diseases that affect all parts of the plants, including f. oxysporum and f. solani, which are considered as the causal of plant wilting diseases (sudiana et al., 2020). on the other hand, b. cinerea is one of the most widespread phytopathogenic fungi, it causes a gray mold disease that affects many crops (de angelis et al., 2022). diseases caused by alternaria spp. are very common such as leaf blight, which attacks a variety of crops leading to a reduction in their marketability (wang et al., 2020). aspergillus species are the cause of post-harvest diseases on the crop, altering the features and nutritional value of the products (rios-muñiz and evangelista-martínez, 2022). in addition to fungal damage, bacteria are responsible for 24% of the 14% overall yield damage engendered by phytopathogens, including s. scabies, p. carotovorum, agrobacterium tumefaciens, erwinia amylovora and xanthomonas spp. (aouar et al., 2020; le et al., 2022). common radish scab disease is caused by several streptomyces spp., mainly induced by s. scabies, it does not affect yield, but it badly impacts marketability (kang et al., 2022). usually, the control of the phytopathogenic fungi is undertaken by agrochemicals products which have harmful consequences on the environment, human and animal health. quite recently, considerable attention has been paid to the implementation of antagonistic microorganisms to suppress plant diseases engendered by phytopathogenic bacteria and fungi, with the purpose of avoiding the use of chemical control methods and overuse of fungicides (suárez-moreno et al., 2019). actinobacteria and other bacterial genera such as pseudomonas and bacillus have been greatly investigated for their biocontrol potential (vurukonda et al., 2021). in the last few years, actinobacteria has been gaining an important interest for their essential role in controlling plant pathogens against both bacteria and fungi (lee et al., 2021). streptomyces is considered as the remarkable genius of actinobacteria. they have the potential to be employed as a biopesticide and to promote plant growth (nonthakaew et al., 2022). they constitute a diversified group of gram-positive, spore-forming bacteria with comparatively sizeable genomes and a high g/c percentage of more than 70%. streptomyces represent about 80% of all antibiotic producers in comparison to other genera. they are also well recognized for their capacity to produce a variety of active substances with agricultural uses, including phytohormones (aouar et al., 2020). iaa is the main plant hormone assigned to the auxins class; it plays an essential role in plant growth by enhancing cell proliferation, root formation and elongation (vurukonda et al., 2021). as plant growth promoting bacteria (pgp), streptomyces species display several pgp traits; they can directly improve plant growth by solubilization of minerals like phosphorus and siderophores secretion (alibrandi et al., 2021). atriplex (chenopodiaceae) is a halophyte genus with significant phenotypical diversity. it comprises numerous halophytes and is largely spread in arid and saline ecosystems around the world. it has been found that the rhizospheric soil of halophytes is a potential source of plant growth-promoting bacteria (chaudhary et al., 2022). atriplex halimus l. has been the subject of numerous recent studies around the world, by exploring its related endophytic, rhizospheric, and non-rhizopspheric microbial populations (tahtamouni et al., 2016; bona et al., 2021). from the algerian arid rhizosphere of atriplex halimus, dif et al. (2022) isolated gramnegative bacteria able to promote tomato growth through phosphate solubilization, phytohormones and siderophores production. boukelloul i et al. (2023). not sci biol 15(1):11437 3 arid and saline soils may be promising ecosystems to isolate new actinomycetes strains with antagonistic potential against soil-born phytopathogenic fungi and bacteria. biocontrol agents from this ecosystem could be interesting as they are already adapted to the salinity and aridity conditions. the diversity of actinomycetes colonizing the arid-salty ecosystem of atriplex halimus l. has not been extensively explored. to the author’s knowledge, in algeria few or no studies have been focused on the investigation of actinomycetes possibly pgpr and antagonistic, isolated from the rhizosphere of this halophyte, widely distributed in the algerian arid regions and sahara. in this paper, we explore the isolation of actinomycetes from the rhizosphere of atriplex halimus l. collected from the arid zone in biskra province. collected soils from the three rhizospheric sites of the halophyte were analyzed for some physico-chemical properties. selected isolates were identified according to their morphological, physiological, and chemotaxonomical traits, and characterized for their ability to inhibit phytopathogenic agents (fungi and bacteria) in vitro and s. scabies in vivo (in growth poutches). also, they were explored for their pgpr features: iaa production, siderophores release, and phosphate solubilization. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods study site, soil samples collection and their physico-chemical traits actinomycetes strains were isolated from the rhizospheric soil of three different sites of the halophyte atriplex halimus l. ̔ guettaf᾿, collected from the region of biskra, located in the south-eastern of algeria. this zone is distinguished by an annual index of aridity of de martonne i = 0.34 that corresponds to a hyperarid climate (i < 5) (lebourgeois and piedallu 2005). the locations of the three explored sites are: site-1 /chaiba (34°78'20''n, 5°04'97''e), site-2 /chegga (34°45'19''n, 5°89'18''e) and site-3 /el feidh, (34°46'15''n, 6°57'06''e). from each site, the root systems of atriplex halimus l. (roots and soil surrounding the halophyte) were taken from the soil and gently scraped to separate rhizospheric soil, remove debris (roots and stones) and collect about 100-150 g. the soil samples were then filled into sterile container and transported to the laboratory for analysis (aouar et al., 2020). phs were measured directly, on 1: 2.5 soil to water mixtures. conductivity was measured in the limpid filtrate obtained by adding 1:5 distilled water to each soil sample (khenaka et al., 2019). in order to calculate the humidity rate, ten grams of the soil were oven-dried for two days at 105 °c to reach a steady weight. each dried sample was burnt for 16 h at 450 °c, the difference between the dry weight and the ash weight yields the organic matter content (lee and hwang, 2002). for each test, the experiments were repeated thrice. isolation of actinomycetes one selective media gba (agar 20 g; starch 20 g; meat extract 5 g; peptone 10 g; glycerol 20 g; caco3 3 g) was used for actinomycetes isolation. the latter was mixed with 75 µg ml-1 amphotericin b to prevent filamentous fungi growth and 10 µg ml-1 of polymixin to inhibit gram-negative bacteria. the sampled soil was dried in the laboratory at room temperature for 24 hours. for each sample, 1 g of soil was introduced into a tube containing 9 ml of sterile physiological water. then, the tubes were vortexed for 4 to 5 minutes. these suspensions were considered as the stock solutions. decimal dilutions were ranging from 10-1 to 10-6. afterward, an aliquot of 0.1 ml of 10-3 to 10-6 dilutions was poured on the surface of gba plates and incubated for 21 days at 28 °c. experiments were performed in triplicate. the actinomycetes colonies were identified according to their macroscopic and microscopic aspects. regular observations were conducted, during the incubation period, to determine the appearance of the isolated colonies. the examined macroscopic characters included: growth rate, spore mass color, as well as their size, shape, texture, and border (regular, irregular, jagged). actinomycetes were recognised by their typical filamentous and powdery appearance. these observations were assisted by using optical microscope at 10x boukelloul i et al. (2023). not sci biol 15(1):11437 4 magnification (williams and cross, 1971). colonies exhibiting the macroscopic characteristics of actinomycetes were subjected to gram staining. then, strains were maintained on isp2 slants at 4 °c, and stored at 20 °c in 20% glycerol for long-term use (aouar et al., 2012). in vitro antagonism trials against pathogens the phytopathogenic fungi: b. cinerea, used in this study was obtained from the laboratory of mycology at the faculty of sciences, university of bejaia. a. alternata and f. oxysporum were acquired from the laboratory of mycology at the national center for biotechnology research in constantine (algeria). additionally, more phytopathogenic fungi (f. solani and a. flavus) and bacteria (s. scabies and p. carotovorum) were procured from the laboratory of biomolecules and plant improvement, university of oum el bouaghi, algeria (table 1). table 1.table 1.table 1.table 1. origins and plants diseases of pathogenic fungi and bacteria strains pathologies host plants phytopathogenic fungi: f. oxysporum, f. solani a. flavus a. alternata b. cinerea fusariosis wilting, yellowing brown spot, black rot grey rot solanum lycopersicum l. phytopathogenic bacteria: p. carotovorum s. scabies fire blight common scab pyrus communis solanum tuberosum l., raphanus sativus l. in vitro antagonism assays involved a confrontation of actinomycetes isolates and plant pathogens: a. alternata, f. oxysporum, f. solani, a. flavus and b. cinerea on patato dextrose agar (pda). they were performed as reported by aouar et al. (2020). thus, actinomycete discs were taken and cultured on pda plates for 4 days at 30 °c. then, agar plugs were cut from fungi colonies (aged of 5 days), placed on the pda plate at 3 cm distance from the actinomycetes disc and incubated at 25 °c for 5 days in the dark. another fungus culture without actinomycete disc was realized to be served as control. after the incubation period, colonies diameters of essays and controls were scored. each test was repeated in five replicates. the antibacterial efficiency against p. carotovorum and s. scabies was assessed by the disc diffusion procedure (aouar et al., 2020). the antagonistic strains were first grown on isp2 at 30 °c for 7days. discs from each antagonist were deposited on muller-hilton agar plates previously inoculated with the phytopathogenic bacteria. the prepared petri dishes were stored at 4 °c for 4 h, before incubation at 30 °c for 24 h. antibacterial activity was evaluated by measuring the diameter of the inhibition zone encircling the actinomycete discs in millimeters (mm). each trial was performed in triplicates. morphological, physiological and chemotaxonomy characterization identification of actinomycetes was conducted using standard procedures of morphological, physiological, and chemotaxonomic characteristics, as described by shirling and gottlieb (1966) and bergey's manual of systematic bacteriology (goodfellow et al., 2012). morphological characterization was based on macroscopic description of cultures streaked on isp2 medium for 21 days at 28 ± 2 °c. it includes colony morphology, produced pigments, color of spore mass, and reverse of the colony. microscopic morphology was examined by observation of mycelium growth using cover-slip method. thus, spore chains morphology was determined by direct microscopic examination (100x) (a minimum of 10 microscope fields should be examined). their morphology may be verticillate or simple: rectiflexibles (rf), retinaculum apertum (ra) (open loops) and spirals (s). boukelloul i et al. (2023). not sci biol 15(1):11437 5 the selected isolates were evaluated on isp2 medium as a basal medium for three physiological features, the capacity to grow at different temperatures (15 °c, 25 °c, 30 °c, 40 °c and 50 °c), as well as the tolerance to nacl concentrations (2.5, 5, 7.5 and 10%) and phs (5, 7, 9, 11 and 13) by incubation at 28 ± 2 °c for 5 days. the rating scale for all these tests was estimated according to a range from 0 to 3, in the following way: 0 = no growth; 1 = slight growth; 2 = moderate growth and 3 = good growth (sreevidya et al., 2016). diaminopimelic acid (dap) isomers (l-dap or meso-dap) were revealed according to the staneck and roberts (1974) protocol through analysis of the total cell acid hydrolysate by thin layer chromatography. to carry out this test, 500 μl of 6n hcl was added to cryotubes containing glass beads and a seed loop of each isolate taken from isp2 actinomycetes cultures. the cryotubes were vortexed for 6 minutes and incubated in a thermoblock at 100 °c for 4 hours. then, they were centrifuged at 4,000 rpm for 5 minutes and the supernatant was dried in a thermoblock at 100 °c. once dry, it was dissolved in 500 μl of sterile distilled water. this step was repeated twice. then a volume of 3 μl of each sample and 1 μl of the control (1.9 mg of dap standard in 10 ml of water) were applied to the tlc cellulose plate, which were placed in a tlc separation chamber containing 50 ml of the solvent system: methanol-water-6 n hcl-pyridine (33.3: 11: 1.6: 4.1 v/v). after migration, the plates were allowed to dry and sprayed with ninhydrin (in 0.2% acetone w/v) and dried at 100 °c for 5-10 minutes before reading. the diaminopimelic acid stains were olive green in color turning to yellow and l-dap migrates further than meso-dap. analysis of the major predominant sugars in the cell wall of actinomycetes is of taxonomic importance was performed using thin layer chromatography. to conduct this analysis, 0.1 ml of 0.25 n hcl was added to each cryotube. subsequently, from the pure cultures of each isolate on isp2 medium, a seeding loop was added to each cryotube and autoclaved for 15 minutes at 121 °c. after cooling to room temperature, 1 μl of the standard preparation of the sugars (arabinose and galactose) and 3 μl of each cell wall extract were deposited on the cellulose tlc plate, which was introduced in a tlc chamber containing solvent system: n-butanolh2o-pyridine-toluene (10: 6: 6: 1 v/v) and allowed to migration until the front was 1 cm from the end of the plate. subsequently, it was dried and sprayed in the gas extractor chamber, with the aniline and phthalate reagent (aniline 0.093 g, phtalic acid 0.166 g and water-saturated butanol 100 ml). finally, it was incubated at 105 °c for 4 minutes. aldo-pentoses and aldo-hexoses give a bright red and brown color, respectively (hasegawa et al., 1983; goodfellow et al., 2012). indole-3-acetic acid (iaa) detection and quantification iaa production was assessed according to the procedure reported by khenaka et al. (2019) and benadjila et al. (2022) with slight modifications. for each isolate, a volume of 10 µl of spore suspension (108 ufc ml-1) was inoculated on yeast malt extract medium (ym) supplemented with two concentrations of ltryptophan (l-trp) (1.25 and 5 mm) and agitated on rotary shaker (125 rpm) for 4 days at 30 °c in darkness. the cultures were then centrifuged at 11,000 rpm for 15 min. a volume of 100 µl of each supernatant was mixed with 100 µl of salkowski's reagent (1 ml of 0.5 m fecl3 in 50 ml of 35% hclo4) and kept for 30 min in the dark. the deviation of the color to the pink showed the production of iaa. its quantification was performed at 530 nm according to an od standard curve. each test was performed in five replicates. phosphate solubilization phosphate solubilization essay was conducted using pikovskaya medium (pvk) complemented with ca3 (po4)2 as described by khenaka et al. (2019). after 8 days of incubation, the development of a clean circle around the colonies indicates phosphate solubilization. halo diameters are calculated by deducting the colony diameter from the whole diameter. the phosphate dissolution capacity was determined positive as the halo diameter ≥ 5 mm. the experiment was repeated five times. boukelloul i et al. (2023). not sci biol 15(1):11437 6 siderophores secretion the selected isolates were tested for their siderophores production potential by the universal cas assay. prior to undertaking the experiment, the glassware was washed with 3 mol l-1 hydrochloric acid to eliminate iron. this test was carried out using a modified procedure described by arora and verma (2017). cas agar was obtained by adding 100 ml of cas reagent to 900 ml of sterilized lb agar medium. the inoculated plates were incubated at 30 °c for 5-7 days. in addition, an un-inoculated plate was considered as control. the appearance of an orange halo around the colony was regarded as positive. five repetitions were performed. in vivo antagonistic trails towards s. scabies on radish seedlings experiment was carried out in growth pouches (125 x 75 mm, mega international) with radish seedlings (raphanus sativus) according aouar et al. (2021). for inoculums preparation, 10 µl of spore stock (108 ufc ml-1) of s. scabies and antagonists were grown in 50 ml of tryptic soy broth (tsb) under agitation for 48 h at 30 °c. after centrifugation, the obtained pellets were suspended in 5 volumes of fresh tsb. on the other hand, sterilized radish seeds were germinated on water plate agar in darkness at 25 °c for 48 h. in each pouch previously sterilised and filled with 2 ml saline, six seedlings were placed. negative control consisted only of seedlings and saline solution; positive control was prepared by mixing 1 ml of the pathogen inoculum (s. scabies) and 1 ml of saline solution. confrontation test consists of mixing 1 ml of each inoculum (antagonist and pathogen) at the same time, followed by the incubation in a growth chamber for 6 days at 21 °c and 68% humidity. after that, pouches have been emptied of their liquid and scanned, before being analysed with whinrhizo software (v. 2002c) for measurement of the seedling’s roots surface. trials were carried out in five replicates. data analysis each experiment was carried out in replicates and data were expressed as mean ± standard deviation. the data of the physico-chemical analysis of the soil samples and the iaa production were analysed using student’s test. while a one-way analysis of variance (anova) was performed on the antagonism measurements followed by tukey's hsd post-hoc test for the antagonism against fungi and least significant difference (lsd) for the in vivo antagonism against s. scabies using spss software (version 23). results were considered statistically significant at the p < 0.05. results results results results physico-chemical characterization of the soil samples and actinomycetes isolation the phs values are summarized in table 2. rhizospheric samples presented phs that range between 8.03 and 8.28. the collected soil from el feidh region presented the highest values of conductivity (5.54 ms cm-1) compared with those of chegga and chaiba (3.19 and 4.65 ms cm-1, respectively). from the three samples, 26 actinomycetes were isolated. the isolation process was achieved following two to three consecutive inoculations until the pure actinomycete colonies were obtained. overall, 12 isolates were isolated from chegga's soil (46.15%), 9 isolates from chaiba's soil (34.62%), while only 5 isolates were obtained from el feidh's soil (19.23%). boukelloul i et al. (2023). not sci biol 15(1):11437 7 table 2.table 2.table 2.table 2. physicochemical properties of the sampled soils and number of isolates sites soil characteristics isolates ph moisture content (%) total organic matter ec ms cm-1 site1/chaiba 8.03 ± 0.03 b 12.60 ± 0.03 b 3.20 ± 0.01 b 4.65 ± 0.04 b 9 (34.62%) site2/chegga 8.28 ± 0.02 a 11.80 ± 0.02 c 5.54 ± 0.02 a 3.19 ±0.02 c 12 (46.15%) site3/el feidh 8.25 ± 0.04 a 19.23 ± 0.07 a 2.24 ±0.005 c 5.54 ± 0.03 a 5 (19.23%) ec: conductivity of aqueous extracts 1/5th. different letters between sites denote significant differences (student test, p < 0.05). the twenty-six isolates were evaluated for their potential to inhibit in vitro plant pathogenic fungi. f. oxysporum, f. solani, a. alternata, a. flavus and b. cinerea. confrontation has allowed the screening of six promoting isolates (act11, act16, act17, act18, act23, and act24) this isolates showed a potential inhibitory effect against the growth of at least three fungi. among the previously mentioned isolates, 50% were able to inhibit the growth of f. solani, and a. flavus, while 33.33% antagonized f. oxysporum. accordingly, these six isolates were selected for further experiments (figure 1; figure 2). it is worth mentioning that the origins of these isolates are as follows: isolates act11, act24 and act17 are from chaiba’s sample, isolates act16 and act23 are from chegga’s soil, while act18 is isolated from el feidh’s sample. boukelloul i et al. (2023). not sci biol 15(1):11437 8 figure 1.figure 1.figure 1.figure 1. in vitro bioassay activity of actinomycetes isolates in mm against fungi; (a): a. flavus; (b): f. solani; (c): b. cinerea; (d): a. alternata; (e): f. oxysporum data are reported as the mean ± sd of five parallel measurements. values with different superscripts (a, b, c, d, or e) in the same column differ significantly (anova test, p < 0.05). figure 2.figure 2.figure 2.figure 2. antifungal and antibacterial activity of actinomycete isolates against phytopathogenic fungi and bacteria; (a): a. alternata; (b): f. oxysporum; (c): f. solani; (d): p. carotovorum boukelloul i et al. (2023). not sci biol 15(1):11437 9 the in vitro assays performed against phytopathogenic bacteria revealed only two antagonistic isolates. results are shown in table 3. interestingly, isolate act18 has inhibited both tested bacteria. nevertheless, s. scabies was more resistant than p. carotovorum, which showed an area of 21 mm (figure 2). however, act24 has antagonized only p. carotovorum (20 mm). table 3.table 3.table 3.table 3. inhibition zone diameters (mm) of the phytopathogenic bacteria by the actinomycetes pathogens diameter of inhibition zone (mm) act11 act16 act17 act18 act23 act24 s. scabies n.i n.i n.i 19.00 ± 0.41 n.i n.i p. carotovorum n.i n.i n.i 21.66 ± 0.47 n.i 20.00± 0.82 n.i.: no inhibition. genera determination according to morphology, physiology and chemotaxonomy all the isolates are fast growing, which suggest the lack of mycolic acids in their cell walls. the cell wall hydrolysate of all the isolates, revealed l-dap isomer as the main component of the peptidoglycan. furthermore, no characteristic sugar (galactose or arabinose) has been noticed in the whole-cell hydrolysate. actinomycetes colonies are distinguished by their typical filamentous aspect. the morphological description of the six isolates is summarized in table 4. except for act24, the colonies were dry and showed sporulated aerial mycelium, with varied pigmentation ranging from beige, purple, gray or pink. similarly, the reverse of the colonies showed brown, beige, and yellow pigmentation. for act18 and act23 colonies, the aspects were rough in comparison with the smooth surface of the others. among the six isolates, only act11 has produced diffusible pigments. moreover, all isolates were gram-positive. moreover, microscopic observation of the cover-slip cultures revealed long chains with three types: spiral (s), retinaculum apertum (ra) or rectiflexibile (rf). physiological traits are shown in table 5. the entire isolates grew between ph 5 and 11 with an optimum growth at ph 7. in contrast, no growth was noted at ph 13. regarding the ability to develop at different temperatures, the five isolates were able to grow under a range of temperatures between 25 °c and 40 °c, none of the isolates show growth in high temperature (50 °c). moreover, three isolates resisted well at 7.5% of nacl concentration. tabltabltabltable 4.e 4.e 4.e 4. morphological and physiological characteristics of the selected isolates features/isolates isolates act11 act16 act17 act18 act23 act24 morphological characteristics: colony surface smooth smooth smooth rough rough smooth spore mass color beige purple gray beige pink colony reverse yellow beige brown beige beige brown diffusible pigments yellow chain morphology ra ra rf s/ra rf ra physiological characteristics nacl 2.5% 5% 7.5% 10% 3 3 3 0 3 3 2 0 3 3 2 0 3 3 3 0 3 3 1 0 3 3 3 0 boukelloul i et al. (2023). not sci biol 15(1):11437 10 ra: retinaculum apertum; 0 = no growth; 1 = slight growth; 2 = moderate growth and 3 = good growth. indole-3-acetic acid production in this investigation, the overall measurements of iaa are summarized in table 5. they ranged from 4.18 to 77.45 µg ml-1 and all isolates need l-trp as precursor. isolate act24 was the best producer when the medium was supplemented with 5 mm of l-trp. furthermore, statistical analysis revealed that iaa production was positively correlated (p ˂ 0.05) with l-trp concentrations for isolates act17, act18, act23 and act24. conversely to isolates act11 and act16, which did not show any correlation with l-trp concentration (p = 0.179, p = 0.986, respectively). table 5.table 5.table 5.table 5. results of pgpr traits isolate iaa production (µg ml-1) phosphate solubilization (mm) siderophores production 1.25 mm 5 mm act11 19.82 ± 0.73 a 18.76 ± 0.57 a act16 14.37 ± 0.40 a 14.39 ± 1.70 a 13.67 ± 0.94 + act17 16.27 ± 0.27 b 29.55 ± 0.98 a 11.67 ± 0.47 act18 4.18 ± 0.59 b 25.03 ± 3.07 a 12.33 ± 0.47 + act23 7.79 ± 0.38 b 57.85 ± 1.63 a act24 10.39 ± 0.36 b 77.45 ± 1.42 a 16 ± 0.82 + data are reported as the mean ± sd of five parallel measurements. for each isolate, values with different superscripts (a, b) differ significantly (student test, p < 0.05) solubilization of phosphate and siderophores production among the six tested isolates, clear zones were scored for four isolates on pvk plates (table 5). diameters were ranging from 11.67 ± 0.47 to 16 ± 0.82 mm. the isolate act24 was the most active. relating to the cas assay, three isolates (act16, act18 and act24) have showed ability to produce siderophores (table 5). in vivo bioassay against s. scabies on radish seedling root surface were estimated by winrhizo software, values were then analysed by anova followed by lsd test, which provide results shown in table 6. root surface of negative and positive controls were 8.13 and 3.69 cm2, respectively. from the six isolates, only act18 and act24 has inhibited significantly (p = 0.0062) the pathogen in vivo, this antagonistic activity was revealed by the increase of the root surface of the seedlings compared to the positive control in which seedlings were inoculates only by the s. scabies (figure 3). moreover, ac18 and act24 allowed the disappearance of common scab symptom, which was characterised by root temperatures 15 °c 25 °c 30 °c 40 °c 50 °c 1 3 3 1 0 2 3 3 2 0 2 3 3 2 0 1 3 3 2 0 3 3 3 1 0 1 3 3 2 0 phs 5 7 9 11 13 1 3 2 1 0 1 3 2 2 0 1 3 3 2 0 1 3 2 2 0 1 3 2 2 0 1 3 2 2 0 boukelloul i et al. (2023). not sci biol 15(1):11437 11 necrosis. isolates act16, act17 were also able to inhibit s. scabies in vivo (p < 0.05) but with a reduced effect, values were 6.21 and 5.82 cm2, respectively. table 6.table 6.table 6.table 6. biocontrol of actinomycetes against s. scabies on radish seedlings data are reported as the mean ± sd of five parallel measurements. values with different superscripts (a, b, c) in the same column differ significantly (lsd test, p < 0.05). figure 3.figure 3.figure 3.figure 3. in vivo confrontation against s. scabies on radish seedling; (a): negative control; (b): positive control; (c): seedlings inoculation with both s. scabies and act18 discussiondiscussiondiscussiondiscussion it has been discovered that microorganisms were more numerous in the soil immediately adjacent to the roots (the rhizosphere) than in the loose soil distant from the roots (ding et al., 2019). the major influences that the rhizospheric microorganisms have, nowadays, become an important tool to preserve plant health by an eco-friendly approach. it has been established that actinobacteria are the major microorganism in the rhizosphere and are the most ideal for controlling plant pathogens (putrie et al., 2020). for these reasons, rhizospheric actinomycetes have been chosen to be explored for antagonists screening. as indicated by sreevidya et al. (2016) salinity and ph ranges in the rhizosphere are crucial criteria for the competitivity and persistence of microorganisms. furthermore, resistance to high salinity and ph should be a criterion for the screening of microorganisms for biological control. in this regard, arid rhizospheric soil of the halophyte atriplex halimus l. has been chosen for actinomycetes isolation. this plant is well adapted to arid soil; it was collected from biskra region, which is characterized by a hyperarid climate. regarding to physicchemical results and referring to the ph and ec soil interpretation scale (richards, 1954), soils samples are categorized as alkaline (ph≥ 7) and very salty (ec varies from 2.4 to 6). these results are confirmed by the hyperarid bioclimatic stage of the biskra region based on the de martonne aridity index. similarly, the phs essays root surface (cm2) negative control 8.13 ± 1.15 a positive control (s. scabies) 3.69 ± 0.44 c co-inoculation of s. scabies with: act18 8.05 ± 0.66 a act16 6.21 ± 1.26 ab act17 5.82 ± 1.44 b act11 3.91 ± 0.55 c act23 3,76 ± 0.13 c act24 7.88 ± 1.03 a boukelloul i et al. (2023). not sci biol 15(1):11437 12 values are approximately close to those obtained by khenaka et al. (2019), who studied samples from rhizospheric soil of the same region, and revealed that ph varied from 7.9 to 8. furthermore, according to the scale of lee and hwang (2002), the percentages of organic matter of the three soils were very low (less than 4%). the chegga soil gave the higher number of isolates, it is worth noting that this soil sample is characterized by a relatively low conductivity, compared to the others samples, furthermore, it has a low moisture rate. these findings are in accordance with those of lee and hwang (2002), which reported that the rate of organic matter is a very important factor; it directly affects the distribution of actinomycetes. thus, this group of bacteria mostly colonizes soils rich in organic matter, but conversely, they prefer soils with low moisture content. the twenty-six isolates displayed sensitivity towards at least three pathogens. a. alternata was recorded as the most sensitive towards all antagonistic isolates, followed by b. cinerea. similarly, wang et al. (2020) have demonstrated that a. alternata was also very sensitive towards streptomyces lydicus m01. with regard to f. oxysporum and b. cinerea, rios-muñiz and evangelista-martínez (2022) have also reported the in vitro sensitivity of these pathogens when facing rhizospheric streptomyces sp. cacis-2 15ca isolate. from our findings, it appears that the two isolates streptomyces sp. act18 and streptomyces sp. act24 are of interest, since they showed both antibacterial and antifungal activities, so they have a broad spectrum, especially act18 that has inhibited gram-negative and gram-positive bacteria in addition to fungi. similarly, the study conducted by le et al. (2022) has revealed that streptomyces sp. an090126, isolated from agricultural soil in korea, showed a broad-spectrum against various phytopathogenic bacteria and fungi. our results are promising, because according to lee and hwang (2002), in most cases antagonists with in vitro antifungal activity are also active in vivo. following the recommendations of goodfellow et al. (2012), the presence of the l-dap isomer and the lack of characteristic sugars suggest the presence of the parietal i chemotype, which is typical of streptomyces and related genera. all isolates presented the characteristic odor of wet soil, typical of actinomycetes, especially of the genus streptomyces, as specified by other studies. considering spore mass color, the isolates could be classified into four actinomycetes series: beige, gray, purple and pink. in general, microscopic morphology showed un-fragmented vegetative mycelium, abundant aerial mycelium, and well-developed bearing long chains of spores either ra, rf or s type. according to the descriptions provided by bergey’s manual of systematic bacteriology (goodfellow et al., 2012), this being a morphological characteristic of the genus streptomyces. thereby, the combination of macroscopic and microscopic morphology, and the parietal chemotype type i allow us to assign these isolates to the genus streptomyces. in actinomycetes group, chemotaxonomical, morphological, and physiological characters are of interest, they may allow the determination of the genus. similar studies, relying on such features to identify streptomyces genus, have been already reported by several researchers (qadir and atalan, 2019; aouar et al., 2020). in the present investigation, all isolates were assigned to streptomyces genus, which is compatible with earlier studies that reported the prevalence of the streptomycetes among soil-borne actinomycetes. in addition, rhizospheric streptomycetes display an antagonistic potential and produce antifungal substances due to the high input of organic matter from plant root exudates (lee and hwang, 2002). all the isolates were able to grow in a ph ranged from 5 to 11, in addition to being able to tolerate a temperature up to 40 °c and to have a tolerance to nacl till 7.5%. these results were expected since these isolates come from an arid soil. interestingly, act18 and act24 showed broad-spectrum activity and exhibit a good growth at 7.5% nacl. these strains are originated from soils with the highest conductivity values. it has been demonstrated in several studies that actinomycetes comprise species that are resistant to high nacl levels (dif et al., 2022). considering this capacity, sreevidya et al. (2016) have demonstrated the capacity of rhizospheric streptomyces isolates to grow at 8% nacl and exclusively the isolate sai-13 to tolerate a concentration of 10% nacl. such characteristics can be considered as advantages for a potential biocontrol agent. boukelloul i et al. (2023). not sci biol 15(1):11437 13 iaa belongs to the group of phytohormones, and it is commonly regarded as the main native auxin. it is a signaling molecule involved in cell division and root extension (vurukonda et al., 2021). moreover, several studies have reported the capacity of rhizospheric streptomyces to synthesize iaa and other indolic derivates in liquid media (khenaka et al., 2019; oleńska et al., 2020; ali et al., 2021). in our investigation, all isolates demonstrate the ability to produce iaa in medium amended with l-trp, which is certainly synthesized via ltrp-dependent pathway. the measured iaa concentrations are greater than those obtained by ashwini et al. (2018) and djebaili et al. (2020) that ranged between 6.88-20.22 µg ml-1 and 7.44-21.4 µg ml-1, respectively. all tested isolates were positive for iaa production; similar high percentage (100%) has also been obtained in other studies (sreevidya et al., 2016; aouar et al., 2020). however, djebaili et al. (2020) have scored a lower rate of 64%. hence, it seems that iaa production is an attribute widely encountered among rhizospheric actinomycetes. phosphorus is an extremely important element for plant growth. all over the world, soils are enriched with inorganic p in the form of chemical fertilizers to assist crops production. nerveless, its overuse deteriorates soil quality. thus, the employment of phosphate-solubilizing microorganisms enhances the soil fertility by converting insoluble p to soluble p (qin et al., 2015). it has been reported that actinomycetes phosphatesolubilizing ability was less explored compared to the other pgpr traits (sudiana et al., 2020). phosphate solubilization assess revealed that among the six tested isolates, four (67%) were able to dissolve p and exhibit clear zones surrounding the colonies. the obtained results are compatible with those of djebaili et al. (2020) which recorded 79% of positive strains. current research has focused on siderophores production by actinomycetes. the detection of siderophores production by three isolates was expected for such stains originating from arid soils. it has been shown that streptomyces sp. from several crop rhizospheric soils have this capacity (khenaka et al., 2019; aouar et al., 2020; warrad et al., 2020). the ability of biocontrol agents to secrete siderophores in appropriate quantities may limit the fe3+ accessibility to the pathogen and may induce plant resistance (rana et al., 2019). our result revealed the siderophores elaboration by three isolates. these findings are of interest, because they have established a positive correlation between plant development and siderophores secretion, and as a result this factor must be included in the selection process of pgp agents (sreevidya et al., 2016; ebrahimi-zarandi et al., 2021; dif et al., 2022). among actinomycetes genera, streptomyces is recognized as saprophytic and the most important. species associated to this genus are recognized for their capacity to produce a large number of different secondary metabolites. nevertheless, some species are phytopathogenic and engender plant disease; s. scabies is the type of strains of this group. it provokes common scab, which is a root necrosis of plants such as potato and radish due to the inhibition of cellulose synthesis (beaudoin et al., 2021). in this investigation, we tested the ability of the six isolates to oppose in vivo against s. scabies and to prevent root necrosis. results showed that the comparison of the root growth of the negative and the positive controls, revealed a significant decrease (p < 0.05) which indicated a root necrosis induced by s. scabies. such findings were reported by previous studies that have carried out on radish seedlings by legault et al. (2011) and aouar et al. (2020) and aouar et al. (2021). however, the co-inoculation of antagonistic isolates act18, act24, act16, and act17 with the pathogen contributes to the re-appearance of the roots and the disappearance of the symptoms of common scab (more or less significantly). interestingly, the results revealed that act18 that had antagonized s. scabies in vitro, affected significantly and positively the root surface. the findings were not expected for act16, act17 and act24 since they did not show antagonistic activity in vitro, it is possible that they acted by other mechanisms rather than antibiosis, especially that act16 and act24 were capable to produce siderophores and solubilize phosphate or it could involve other mechanisms. laboratory experiments conducted by suárez-moreno et al. (2019) revealed that streptomyces strains carry several important features for biocontrol, which have also been related to plant-growth promotion. moreover, according to parasuraman et al. (2022) in vivo antagonism may boukelloul i et al. (2023). not sci biol 15(1):11437 14 also involve the induction of the plant defence, through direct and indirect mechanisms. these mechanisms include solubilization of plant nutrients, such as iron and phosphorus, nitrogen fixation and production of various plant hormones such as auxins, cytokines, and ethylene or reducing the harmful effects of plant pathogens by producing antibiotics and siderophores. conclusionsconclusionsconclusionsconclusions the investigation that assesses the potential of six streptomyces sp. strains isolated from the rhizosphere soil of atriplex halimus l. ῾guettaf’ for the in vitro antagonism against some phytopathogenic fungi and bacteria and the exhibition of some pgpr attributes, leads to the screening of two interesting strains: streptomyces sp. act18 and streptomces sp. act24, showing efficient growth under physiological conditions of high temperature, ph and nacl. moreover, they revealed a broad spectrum of the in vitro antagonistic activity when facing to plant pathogens. in addition to their potential to counteract s. scabies in vivo, by preventing common scab and enhancing roots development of the radish seedlings. these abilities probably involved one or several mechanisms, especially since streptmyces are known to produce substrates of agrochemical interest. interestingly, these two strains displayed some pgpr traits: iaa, siderophores production and phosphate solubilization. based on the attributes mentioned above, the use of such strains in arid and saline soils may represent a sustainable solution to protect crops and enhance agriculture rather than agrochemical products. however, they need more advanced evaluation under field conditions to be proposed as biocontrol agents. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization: la; funding acquisition: yn, az; resources: mcb, md; software: la, ib, az, mcb and md; supervision: la; validation: ib, la, az; writing: ib and la; review and editing la, ib. all authors read and approved the final manuscript. ethicalethicalethicalethical approvalapprovalapprovalapproval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this work was supported by directorate-general for scientific research and technological development, algeria (dgrsdt) through the ibtikar funding project. authors would like to thank mr. salem beghou the responsible of the pedagogical laboratories of the university of oum el bouaghi. thanks are due also to dr chawki bensouici from national center for biotechnology research (crbt) for his assistance. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. boukelloul i et al. 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(2016). diversity of endophytes across the soil-plant continuum for atriplex spp. in arid environments. journal of arid land 8:241-253. http://dx.doi.org/10.1007/s40333-015-0061-9 vurukonda ss, giovanadri d, stefani e (2021). growth promotion and biocontrol activity of endophytic streptomyces spp. prime archives in molecular sciences, 2nd edition 1:1-55. http://hdl.handle.net/11380/1248582 wang m, xue j, ma j, feng x, ying h, xu h. 2020. streptomyces lydicus m01 regulates soil microbial community and alleviates foliar disease caused by alternaria alternata on cucumbers. frontiers in microbiology 11:942. http://dx.doi.org/10.3389/fmicb.2020.00942 warrad m, hassan ym, mohamed msm , hagagy n, al-maghrabi oa, selim s, … abd elgawad h (2020). a bioactive fraction from streptomyces sp. enhances maize tolerance against drought stress. journal of microbiology and biotechnology 30(8):1156-1168. http://dx.doi.org/10.4014/jmb.2003.03034 williams st, cross t (1971). actinomycetes. in: booth c (ed). method in microbiology. academic press, london pp 295-334. the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. (microsoft word 11508 nsb rasc\363n-solano 2023.06.19.docx) received: 06 mar 2023. received in revised form: 02 apr 2023. accepted: 14 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania rascón-solano j et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11508 doi:10.15835/nsb15211508 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae efficient use of timber resources in mexico: historical efficient use of timber resources in mexico: historical efficient use of timber resources in mexico: historical efficient use of timber resources in mexico: historical development and current challengesdevelopment and current challengesdevelopment and current challengesdevelopment and current challenges joel rascón-solano1, oscar a. aguirre-calderón2, wibke himmelsbach2*, juan a. nájera-luna3, eduardo alanís-rodríguez2, javier jiménez-pérez2, eduardo treviño-garza2 1autonomous university of nuevo león, faculty of forestry sciences, doctoral program in sciences with orientation in natural resources management, national highway km 145, zip 67700. linares, nuevo león, mexico; #85, km. 145, linares, nuevo león, zip 67700, nuevo león, méxico; forestal_rascon@hotmail.com 2autonomous university of nuevo león, faculty of forestry sciences, national highway nacional #85, km. 145, linares, nuevo león, zip 67700, nuevo león, méxico; oscar.aguirrecl@uanl.edu.mx; wibke.himmelsbach@uanl.edu.mx (*corresponding author); eduardo.alanisrd@uanl.edu.mx; javier.jimenezpr@uanl.edu.mx; eduardo.trevinogr@uanl.edu.mx 3division of postgraduate studies and research, technological institute of el salto, tecnológico street 101 col. la forestal zip 34942, durango, méxico; jalnajera@itelsalto.edu.mx abstractabstractabstractabstract the use and consumption of wood has always captured the attention of forest researchers, mainly from the point of view of forest management and the sustainable processing of raw materials. for this reason, the wood industry has not only been concerned with the maintenance of forests, but also with efficient processing. the objective was to identify, analyse and discuss the main elements that influence the efficient use of natural timber resources at a global, regional and local level, providing a historical and current perspective of the industrial forestry sector in mexico. historically, the wood industries worldwide have been concerned with applying intensive silvicultural treatments in native and artificial forest stands, in search of satisfying the demand of a growing market. in this context, industries evolved as processes needed to be more efficient. therefore, efforts were made to reduce and take advantage of forest residues, at the same time various historical events generated additional needs. at present, the integration of silviculture and forest transformation allow to increase and improve the quality of the products generated. through the application of intensive silvicultural treatments, products of a single species are obtained. however, it is possible to direct production to a specific market as the forest mass develops. for its part, selective treatments in uneven-aged forests maintain a constant production of diverse products for different specialized markets. in mexico, it is imperative to analyse and generate innovative or competitive products derived from wood to improve the efficiency of forest harvesting. keywords:keywords:keywords:keywords: forest residues; intensive silviculture; selective silviculture; transformation and processing; wood industry; wood products https://www.notulaebiologicae.ro/index.php/nsb/index rascón-solano j et al. (2023). not sci biol 15(2):11508 2 introductionintroductionintroductionintroduction historically, forests have provided human society with essential goods and services for livelihood and income generation. long before modern wood-cutting techniques and tools were developed, humans gathered food, medicine, and other resources that met their basic needs in forests (sheppard et al., 2020), ecosystems where natural resources with important economic, cultural and ecological values are still extracted (cardenas et al., 2018). for this reason, the forestry sector is usually economically important in countries endowed with forests (lundmark et al., 2021; rascón-solano et al., 2022a; hernández et al., 2023). currently, some activities are challenged by low-value wood products (townsend et al., 2019), high costs of extraction of wood or limitations in operational productivity (holzfeind et al., 2021), and changes in the used and installed capacity of forest industries (fuentes et al., 2006). on the other hand, the need to establish strict policies and regulations regarding the use of forest resources in a sustainable manner (ayalamendivil and sandoval, 2018) and the structure, competitiveness, and diversification of the market (coelho junior et al., 2019) are factors that add complexity to industrial activities. in this sense, hurmekoski and hetemäki (2013) emphasized that the success of the forestry sector in meeting these challenges depends on improving its performance and competitiveness, as well as on the development of new products, improved technologies, and the ability to adapt to changing market conditions. it is important to mention that the natural potential and proper management of forests allows the increase in the harvest of raw material, to ensure the growing demand of the timber industry (tymendorf and trzciński, 2020). in this regard, silvicultural treatment is one of the most important approaches in forest management (martínez-meléndez et al., 2021). for years, various silvicultural treatments have been implemented around the world to maximize productive yields (halbritter, 2015), with the goal of supplying, through sustainable forest management, the tangible needs of forest products required by society (rojas, 1995). accompanied by the above, supply chain planning in the forest products industry covers a wide range of operations and decisions strategic and operational (bredström et al., 2010). since forest resources are of great global and national importance, strong forest governance and a balance of interests between timber harvesting and the forest industry are necessary (trishkin et al., 2014). to what has been described above, it is added that the business environment of the forest industries has undergone drastic changes (lähtinen and toppinen, 2008). due to these changes, maintaining competitiveness and acceptable business success, especially in traditional sawmilling, is more challenging than ever before (lähtinen et al., 2008). changes in production and consumption patterns (hetemäki and hurmekoski, 2016), industrial processes, final product demands and management practices (lauri et al., 2021) are factors that challenge the sustainability of the current forestry industry (borz et al., 2021). based on the above, the objective was to identify, analyze and discuss the main elements that influence the efficient use of natural timber resources at a global, regional and local level, providing a historical and current perspective of the industrial forestry sector in mexico. it assumes that the availability of resources, forest management methods, industrial capacity and the available market are factors that determine the sustainable use of timber resources in mexico. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods despite the socioeconomic importance of the timber industry in mexico, the information available on this activity is mostly fragmented and incomplete. for this reason, for this review, information from 90 investigations related to forest management oriented to timber harvesting and the timber industry was considered. preference was given to studies that presented precise information on the activities carried out in the development of the timber activity and it was discussed how processes and products can be adapted to the rascón-solano j et al. (2023). not sci biol 15(2):11508 3 conditions of the forestry sector in mexico at an international level. a total of 23 works published between the years 1914 and 2021 were selected, these investigations mention how the historical management of forests for timber purposes has been given. we consider a total of 14 papers published between 1930 and 2022 that describe the historical evolution of the timber industries. in relation to the use of solid waste resulting from the transformation of wood, 6 publications covering the year 1926 to 1997 were selected. we analyzed 16 articles to describe the challenges and opportunities for mexico in relation to intensive forestry and the products to be generated in the industry, works published between 2008 and 2022 were considered. finally, to describe the products generated from mixed forest and the challenges presented for mexico, 31 investigations published between 1999 and 2022 were reviewed. results results results results and discussionand discussionand discussionand discussion historical development of silviculture for industrial purposes the management of forest resources during the last two centuries has been mainly oriented towards the generation of sustainable production schemes for goods with market value, focused on wood (aguirre, 1997). for this reason, the wood industry has not only been concerned with sustaining forests, but also with the efficient transformation (ahec, 1995). however, dávalos (1996) indicates that the waste of timber forest resources has had and will have unfortunate consequences for forest sustainability. timber industries worldwide have been based mainly on the application of intensive silvicultural treatments in native and planted forests (kruger and everard, 1997; zwolinski, 1998) by integrating tree management and improvement practices (daniels, 1984), seeking to satisfy the demands of a growing market (clapp, 1995). initially, it was suggested that natural forests have the capacity to produce the raw material for all the wood industries (korstian, 1914; guthrie, 1925; cox, 1976). however, the intensive management of forest plantations has played an increasingly important role for timber production (sedjo, 1984) and bioenergy products (anderson et al., 1983). in mexico, there have been historically quite stringent government restrictions to control logging on private land (meyer, 1939). however, the first experiences of timber harvesting began at the beginning of the 20th century in the state of chihuahua (estrada and rodríguez, 2021), where clearcutting was practiced to install the railroad tracks that passed through the forest massifs (musalem, 1982). while forest management was already widespread in europe during the 1980s, mexican silviculture was not yet highly developed (moreno, 1988). in contrast, mexican timber harvesting has focused mainly on low-intensity selective felling (islas et al., 1988), followed by intensive management with the treatment of regeneration cuttings (rodríguez, 1997). furthermore, harvesting has been based on the implementation of the mexican forest management method (mfmm) for virgin forests (ramírez-maldonado and romahn de la vega, 1999) and the mexican method of management of irregular forests (mmmif) for uneven-aged stands (gadow and puumalainen, 2000), both cases with selective felling. additionally, for even aged forests, intensive felling has been applied. in this regard, the silvicultural development method (sdm) (valencia, 1992) and to a lesser extent the silvicultural conservation and development system (sicodesy) were applied (ramírez-maldonado and romahn de la vega, 1999). primarily, forestry management has been implemented for commercial purposes from a principle of environmental and ecosystemic sustainability (dávalos, 1996; aguirre-calderón, 2015). historical background of the timber industry the use and consumption of wood has always captured the attention of forest researchers, mainly from forest management and the transformation of raw materials. the first literary reference to a working sawmill comes from a fourth-century roman poet, ausonius, who describes a watermill cutting wood (ritti et al., 2007). later, in the 11th century, hydraulic and animal-drawn sawmills were in widespread use in the islamic world, from spain and north africa to central asia (lucas, 2005). rascón-solano j et al. (2023). not sci biol 15(2):11508 4 the first sawmills were powered by wind power, around the 13th century. in germany the first sawmills powered by hydraulic power appeared as early as 1322 (peterson, 1973). works such as those carried out by boissiere (1991) and szasz (1991) indicate, that in central and eastern europe, the beginnings of timber were based on the production of charcoal for the forges and the carving of logs for medieval construction until the 16th century. during the industrial revolution at the end of the 18th century and the middle of the 19th century, wood undoubtedly acquired greater relevance, as it was closely related to some of the most significant processes of economic change (de molina and alier, 2001). part of this change was the construction of tracks and the manufacture of wagons, the production of crates for agricultural products, and urbanization to provide support and cladding for the construction of buildings (zapata, 2002). in north america, during european colonization in the mid-17th century, the first sawmills installed in virginia state simply adapted the circular saw to mechanical power, generally driven by water currents to speed up the process. later on, forestry companies integrated steam-powered double-disc sawmills that were implemented in the 18th century. thereafter, in the 19th century, the band saw industries were powered by electric motors (peterson, 1973). the first sawmills in mexico were put into operation in the pine forests of the state of michoacán at the end of the 19th century (musalem, 1982). as for the north of the country, in 1880, the first sawmill was built in coahuila to supply the mining industry. shortly after, the first timber industry was installed in the state of durango in 1893 (aylor, 1930). for its part, the state of chihuahua began the transformation of pine wood in sawmills on railcars intended to increase the length of the train tracks. subsequently, there was the presence of american lumber companies in 1905, to which the state government granted a concession for the installation of sawmills, a paper mill and a furniture factory (estrada and rodríguez, 2021). additionally, zavala (1996), tamarit et al. (2021) and rascón-solano et al. (2022a) indicates that the degree of technological advancement and adaptation of the wood industry in mexico has been slow or even lagged behind in some regions of the country (escárpita, 2002). origin of solid waste processing regarding the management and transformation of waste from timber processing, courtnage (1926) reports that portable sawmills were 10 % less efficient than static ones. besides, they produced significant amounts of waste that were not used or transformed into other products, due to high transportation costs. later in germany, at the end of the 1940’s, the particle board industry emerged, due to the shortage of sawn timber and the need to rebuild cities during the postwar period (borgin, 1958). sawing cuttings are processed to obtain a wide variety of wood products, including pallets, boxes for agricultural products, and pulp for papermaking (aylor, 1930). gisborne (1929) mentions that the pulp industry was expected to replace the sawmill industry because it was more efficient and generated less volume of waste, since the bark was the only waste, this transition has not occurred in mexico. as for the bark, which constitutes up to 20% of round wood, it was regularly used as fuel for sawmills and boilers for drying wood (citation). moreover, bark was used to produce soil fertilizer and soil nutrients (ahec, 1995). subsequently, at the end of the 20th century, there is practically no waste in the timber industry anymore, thanks to the increasing research to optimize wood processing (kauman, 1997). furthermore, the development and introduction of machines using lasers and scanners, have contributed significantly to the improvement of quality control and the efficient use of wood supplies (ahec, 1995). current challenges in the intensive silviculture and the timber industry of mexico regarding the management of forest resources, different silvicultural interventions provide a wide range of forest products. figure 1 shows a hypothetical example of the various industrial products that can be obtained from the application of intensive silvicultural treatments in even-aged pine stands, such as the treatment of seed trees using the silvicultural development method. once regeneration has developed, the first stand intervention is carried out to adequately distribute the trees, increase forest productivity and reduce competition for resources by managing stand density through thinning. at this stage, the forest generates rascón-solano j et al. (2023). not sci biol 15(2):11508 5 biomass that must be used to generate bioenergy through pellets. thus, short (3 to 6 years) and medium (5 to 7 years) rotation plantations have been included in bioenergy generation programs (santangelo et al., 2015; ouyang et al., 2022), with the main objective of producing fuel to generate heat and electricity (aylott et al., 2008). however, this alternative has not yet been widely explored in mexico and other latin american countries (borrego-núñez et al., 2022). development stage of the forestdevelopment stage of the forestdevelopment stage of the forestdevelopment stage of the forest figure 1.figure 1.figure 1.figure 1. potential primary wood products as a result of different silvicultural treatments in even-aged coniferous forest stands of mexico. the mentioned years correspond to the time period between the silvicultural interventions. the dbh (diameter at breast height) mentioned is reached at the point in time of intervention. 1) tree regeneration (non-existent raw material); 2) biomass and material enabled for broomstick; 3) biomass and material enabled for broomstick, impregnated stakes for the agricultural sector and impregnated beam; 4) biomass, impregnated stakes for the agricultural sector, thin diameter sawing logs , secondary products, fibers for boards, particles for boards and bark for bioenergy; 5) biomass, thin diameter sawing logs, secondary products, fibers for boards, particles for boards and bark for bioenergy; 6) biomass, thin diameter sawing logs, thick diameter sawing logs, pole for electrification, fibers for boards, particles for boards and bark for bioenergy; and 7) biomass, roundwood for boards, pole for electrification, thick diameter sawing logs, thin diameter sawing logs, secondary products, fibers for boards, particles for boards and bark for bioenergy when presenting the commercial diameters, a first thinning is carried out (stage 3), which consists of reducing the density in conglomerate groups. this stage has a greater number of products that can be extracted from the forest. for instance, it is possible to generate rustic beams for construction and stakes for agricultural use, while the preservation of the products allows their prolonged use. as described by román et al. (2014), individuals with dimensions similar to those shown, have the potential to produce poles, firewood and rural constructions. furthermore, juvenile wood is associated with shrinkage and a high probability of warping and cracking in the final products (ruano and hermoso, 2021; ruano et al., 2022). therefore, they are not regularly integrated into traditional industrial processes. hence, logging residues cannot be easily transformed rascón-solano j et al. (2023). not sci biol 15(2):11508 6 into products such as sawn wood. however, they fulfill ecological functions such as maintaining adequate levels of biomass in the process of being reintegrated into the soil, when left or returned to forest stands (villelasuárez et al., 2018). secondary wood products for processing are obtained at the earliest during the second or third thinning (stages 4 and 5), when the forest stand has reached a certain maturity. during these operations, raw materials are obtained for the thin-diameter sawmill industry, medium-density fiberboard and particleboard industry, as well as for the supply of cellulose pulp to the paper industry. in addition, impregnated beams can be the product of silvicultural interventions during these stages. in addition, small-sized logs have the potential to produce pallets in secondary sawmills (taipe and rivas, 2021; haro et al., 2015). according to eisenbies et al. (2021) and latterini et al. (2022), the demand for lignocellulosic biomass has increased for different paper industrial purposes. conventional wood products are obtained from even-aged stands during the fourth thinning. in northern mexico (stage 6), this treatment is applied when the forests have an average age of 50 years approximately. with the harvested individuals that present the best phenotype, priority should be given to the generation of poles for electrification and telecommunications. in accordance with corvalán (2020), this product requires a specific silvicultural scheme to satisfy the market. the main industries that must be benefited with supply are medium density fiber boards and the sawmill industries of thin and thick diameters. referring to this, mature forests as potential producers of raw material for the manufacture of fiberboard and sawmilling (grigsby et al., 2015); borz et al., 2021). however, lauri et al. (2021) mention that this mainly depends on the demand of the market. during the seventh stage of forest management to supply the forestry industry (stage 7), the aim is to promote the emergence of natural regeneration or to assist the forest through planting and subsequently, remove the upper layer made up of mature trees and cultivate this juvenile tree stand. firstly, with regeneration felling, a large volume of biomass from large individuals is planned to be removed; secondly, regeneration release logging plans to remove all adult individuals. high-quality raw material is obtained from these treatments, since intensive silviculture promotes self-pruning and the development of straight stems in the stock (ruano and hermoso, 2021). priority should be given to the plywood industry due to the high quality of the raw material, followed by the sawmill industry for thick and thin diameters. kallakas et al. (2020) and fekiač et al. (2021) mention that the main industry to be supplied at the end of the shift from intensively managed forests is plywood, due to the high quality of the logs and a low percentage of knots. finally, the secondary products of the sawmill and the production of particle boards based on sawdust and chips will benefit from the primary transformation. in this way, mirski and dziurka (2011) indicate that chipboards are an essential element in improving the integral use of wood. harvesting residues, such as the tops of the trees and crowns, have the potential to be transformed into pellets for bioenergy. villela-suárez et al. (2018) mention similar functions for harvesting residues. the bark obtained after the transformation has an energetic potential. ahec (1995) proposes to use bark as fuel for the boilers that dry the wood or to produce fertilizers (ayipio et al., 2021). additionally, the bark is also an important source of nutrients for the forest, in addition, it is captured carbon that has the ability to integrate into the soil. current challenges in the uneven-aged silviculture and the timber industry of mexico unlike even-age management methods, selection treatments in uneven-aged stands have the potential or deficiency, depending on the manager or industrial interest, to produce a wide variety of raw materials, as diverse as the tree stand is complex. the hypothetical example shown in figure 2 contains a common structure of uneven-aged forests in northern mexico, where the genera pinus and quercus predominate, followed by the genera juniperus, arbutus and occasionally the genus alnus (rascón-solano et al., 2022b). these types of forests offer a solution to climate change by storing carbon, providing ecosystem services and sustainable products (lo monaco et al., 2022; başkent and kašpar, 2022; saklaurs et al., 2022). on the other hand, they have the rascón-solano j et al. (2023). not sci biol 15(2):11508 7 potential to produce soft and hard wood, with which it is possible to supply different markets at the same time. likewise, the presence of trees in different stages of development supplies different industries interested in the raw materials contained therein. figure 2.figure 2.figure 2.figure 2. potential primary wood products as a result of selective silviculture in uneven-aged mixed pineoak forest stands in mexico. 1) regeneration (non-existent raw material); 2) biomass; 3) biomass and broomstick enabled fabrication; 4) biomass broomstick enabled fabrication, impregnated agricultural poles and impregnated beams; 5) biomass, impregnated beams, thin diameter sawing, secondary products, fibers for boards, particles for boards and bark for bioenergy or gardening; 6) biomass, thin diameter sawing, secondary products, fibers for boards, particles for boards and bark for bioenergy or gardening; 7) biomass, thin diameter sawing, thick diameter sawing, secondary products, fibers for boards, particles for boards and bark for bioenergy or gardening; 8) biomass, round logs for boards, thick and thin diameter sawing, secondary products, fibers for boards, particles for boards and bark for bioenergy or gardening; 9) biomass, fibers for boards, charcoal and domestic fuel; 10) biomass and domestic fuel; 11) biomass, thick and thin diameter sawing, secondary products, charcoal and domestic fuel; 12) biomass, domestic fuel and impregnated agricultural poles; 13) biomass, impregnated agricultural poles, thick diameter sawing and thin diameter sawing; and 14) biomass, thick diameter sawing and thin diameter sawing. as previously mentioned, regeneration in establishment does not have an industrial potential. nevertheless, it will become the main source of biomass in this type of stands. additionally, it is important to mention that the genus pinus has been widely used in various countries, and the products obtained from its management are similar or even the same as those indicated in this study. on the other hand, figure 2 shows species that have not been used commercially, such as the genus arbutus, because their stems tend to have an irregular shape that is difficult to handle in the industry. however, individuals of this species have the potential to be destined mainly for the energy industries, for the production of composite pellets with other genera from the same ecosystem. tovar-rocha et al. (2014) mention that despite the fact that arbutus is a genus of ecological importance, it has been little studied and its wood is mainly used for the manufacture of handicrafts. for this reason, there is a lack of management of this genus. sub-indices 9 and 12 in the figure 2 illustrate the dead softwood and hardwood trees in these forest stands, due to competition, climate change and the attacks by pests and diseases (pimienta et al., 2007; trigueros et al., 2018). the plans to use this raw material are rascón-solano j et al. (2023). not sci biol 15(2):11508 8 intended to supply the sawmill industries with logs. in this regard, löwe et al. (2022) found that the use of wood from trees infested with bark beetles without the presence of mold and fungi is not problematic for construction purposes. hence, the transformation and commercialization of sawn wood from dead trees is feasible (mackes and eckhoff, 2015; loeffler and anderson, 2018). with the branches and residues of harvesting, domestic fuels are mainly produced, when these residues are not used, they are integrated as biomass in the soil. several authors mention that firewood is the main fuel for the communities, depending on the product to supply and cover their daily needs (quiroz-carranza and orellana, 2010; mozo and silva, 2022). currently, there is the possibility of redirecting the raw material to bioenergy and board industries that have promising growth on a global scale. with regard to the industrial management of hardwoods such as the quercus genus, historically the raw material has been destined for the sawmill industry. in the early stages of development of these species it is not possible to supply the sawmills. however, rogers et al. (2022) indicate that silvicultural approaches to hardwoods have varied widely, to meet both, diversifying ecological silvicultural objectives and traditional production. hence, these juvenile individuals have the potential to be transformed into energy and charcoal. suchomel et al. (2011) mention that the growing demand for juvenile hardwood for bioenergy is an important economic alternative. subsequently, when the individuals reach commercial size, it is pertinent to supply the sawmilling industry. in this context, recent studies have pointed out that the marketing of hardwoods in the form of sawn timber is an important economic opportunity (nicholls and bumgardner, 2018; hassler et al., 2019). the potential for the use of this wood type has not been increased in mexico, due to the complexity of processing and the technological limitations of the industries (zavala, 2003). currently harvested genera of low commercial interest such as juniperus, have been consumed by local markets as a priority for the generation of agricultural poles. similarly, vaughan and mackes (2017) mention that this alternative is a further future opportunity to use juniper wood in road railing systems, because it has excellent resistance to natural decay (ffolliot et al., 1999). the technological wood properties of this conifer species are still largely unknown. however, juniper has the potential to produce high quality sawtimber when individuals reach maturity or supply the bioenergy sector, so its wood properties should be further studied. forest industries have enrolled in certification of forest management and the chain of custody due to a perceived increase in demand for certified products, and the need to increase the productive efficiency of forest land (bond et al., 2014; sjølie et al., 2015; hyytiä, 2022). thus, global practices for optimizing the use of forest resources, based on appropriate distribution of products and performance planning of all products to be produced, are directly related to international forest certification guidelines. at the individual tree level (considering mature trees from 25 to 30 meters high), it is possible to obtain product for various industrial uses. mainly, the production of plywood is obtained from the base of the tree (4.88 meters length), because it is where the largest diameter of the log and the highest quality free of defects occur (fekiač et al., 2021). highand medium-quality lumber is derived from the second and third section of the log (9.75 meters length, as larger diameter logs tend to produce a higher volume of quality lumber (borz et al., 2021; orozco et al., 2017). the fourth chop section has adequate quality to produce medium density fiberboard (4.88 meters length), the dimensional characteristics and quality of the log result in better economic benefits (grigsby et al., 2015). pulp for papermaking is obtained from the penultimate section of the log (4.88 meters length), mainly because the inferior dimensions and low quality of the wood do not advance to the final product (eisenbies et al., 2021; latterini et al., 2022). biomass production for energy generation originates at the tip of the tree, because according to (villela-suárez et al., 2018), it is a product that is generally not used and has energy potential. for mexico and other countries in latin america, it is essential to increase certified forest areas (hernández et al., 2023). in addition, research should focus on the development of new wood-derived products, analyzing them and putting them into practice. thus, there is great potential to serve attractive rascón-solano j et al. (2023). not sci biol 15(2):11508 9 international markets, including textiles, solid and liquid biofuels, chemical products, bioplastics, and packaging (hurmekoski et al., 2018). conclusionsconclusionsconclusionsconclusions over time, the timber industry was able to improve its processing procedures and increase productivity, due to technological progress. for instance, relevant historical events motivated the professionals of the time to look for alternatives, to diversify production, as well as to make efficient use of available resources, especially solid waste from forest management and wood processing. the availability of resources is crucial for the diversification of wood production and the residues from forest harvesting and wood processing have a high potential to produce alternative products. the management methods largely determine the industry that can be supplied. the use of even-age forest stands is an alternative to focus on high production for one or two specific markets. on the other hand, complex tree structures have the potential to supply a wide variety of products to several markets at the same time. besides, these types of forests have a high potential for carbon storage, the provision of ecosystem services and the generation of sustainable products. in turn, the installed industrial capacity delimits the production of timber products, and industrial diversification is again one of the factors determining the efficient use of wood resources in each forest region. the production and market sectors must drive the future of the timber industry in mexico, based on success stories and international programmes. domestic and foreign investment needs to be promoted for the development of new products and manufacturing capabilities that can offer highly engineered wood and pulp products. forestry research institutions should develop advanced processes and emerging products that can be attractive to the market and that allow the transformation of wood harvesting residues. this revision work allows to exemplify in a simple way the different products that the wood industries of mexico can produce. it is based on coniferous forests that are management in a large part of the country, considers the most representative species in this type of ecosystem and provides recommendations regarding the sustainable use of the various tree structures found, guiding the diverse use in relation to the stage of forest development. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization, j.r.s. and o.a.a.c.; methodology, j.r.s., o.a.a.c. and w.h.; validation, j.a.n.l.; formal analysis, e.a.r., j.j.p. and e.t.g..; investigation, j.r.s. o.a.a.c. and w.h.; resources, j.r.s.; writingoriginal draft preparation, j.r.s., w.h.; writing-review and editing, o.a.a.c. and j.a.n.l.; supervision, o.a.a.c. all authors read and approved the final manuscript. ethical apethical apethical apethical approvalprovalprovalproval (for researches involving animals or humans) not applicable. rascón-solano j et al. 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(2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11442 doi:10.15835/nsb15211442 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae wickerhamomyces anomaluswickerhamomyces anomaluswickerhamomyces anomaluswickerhamomyces anomalus wo2 inoculation protects wo2 inoculation protects wo2 inoculation protects wo2 inoculation protects suaeda suaeda suaeda suaeda fruticosafruticosafruticosafruticosa from salt stress by the stimulation of the production of from salt stress by the stimulation of the production of from salt stress by the stimulation of the production of from salt stress by the stimulation of the production of carotenoids and sugars and the augmentation of sodium translocationcarotenoids and sugars and the augmentation of sodium translocationcarotenoids and sugars and the augmentation of sodium translocationcarotenoids and sugars and the augmentation of sodium translocation chahrazed aibeche1*, warda sidhoum2, 3, omar khelil4,5, fadila cherifi1, kheira errouane1, nawel selami1, slimane choubane4, 5, abderrahmane senina1, chahinez mardhi1, abderrezak djabeur1 1laboratory of production and valorization of plants and microorganisms (lp2vm), department of biotechnology, faculty of natural and life sciences, university of science and technology of oran mohamed boudiaf usto-mb, b.p. 1505, el-mn’aour, oran 31000, algeria; chahrazed.aibeche@univ-usto.dz (*corresponding author); n_selami@yahoo.fr; errouane_80@yahoo.fr; f.cherifi@yahoo.com; abdou-seni2010@live.fr; chahinezmardhi22@gmail.com; sidjabeur@yahoo.fr 2laboratory of microorganisms biology and biotechnology, department of biotechnology, university of oran 1 ahmed ben bella, algeria 3abdelhamid ibn badis university, department of biology, faculty of natural science and life, mostaganem, algeria; warda.sidhoum@univ-mosta.dz 4higher school of biological sciences of oran (essbo), p.o. box 1042 saim mohamed, emir abdelkader estate (ex-inessmo) 31000 oran, algeria 5laboratory of aquaculture and bioremediation (aquabior), university of oran 1 ahmed ben bella, p.o. box 1524, el m'naouer 31000 oran, algeria; khelil-omar@hotmail.fr; slimane.choubane@gmail.com abstractabstractabstractabstract the objectives of this study were to test the strain wickerhamomyces anomalus wo2 as a plant growthpromoting yeast (pgpy), to evaluate the effect of its inoculation on the growth and physiological performance of suaeda fruticosa subjected to salt stress, and to understand by which mechanism the yeast can protect the plant from high salinity. the results showed that the strain is halophilic, and grows at salt concentrations of up to 15%. salinity caused decreases in chlorophyll a, b, and t in both inoculated and non-inoculated s. fruticosa. a significant increase in carotenoids was observed in w. anomalus wo2 inoculated plants. inoculation enhanced the production of proteins, polyphenols and flavonoids at 1% of salinity, and sugars at all concentrations of nacl. although na+ and k+ were higher in the leaves of non-inoculated plants compared to inoculated ones, the correlation of sodium translocation factor (tf) with salinity was very strong positive only in the inoculated plants. the production of carotenoids had a very strong positive relationship with salinity in inoculated plants, and a very strong negative correlation in non-inoculated plants. soluble sugars were very strongly positively correlated with salinity in both inoculated and non-inoculated plants. however, a strong positive correlation of sugars with carotenoids was observed only in inoculated plants. this strain proved to be a promising candidate as a pgpy under salt stress. this work amends the pgpy bank with new strains having interesting abilities to resist high concentrations of nacl and which can be used in the future as a biofertilizer. https://www.notulaebiologicae.ro/index.php/nsb/index aibeche c et al. (2023). not sci biol 15(2):11442 2 keywords:keywords:keywords:keywords: carotenoids; growth; inoculation; pgpy; salt stress; sodium translocation; suaeda fruticose; sugars; wickerhamomyces anomalus introductionintroductionintroductionintroduction due to unsuitable agricultural practices and global warming, more and more lands around the world are suffering from degradation problems caused by the accumulation of salt. in fact, salinization is one of the most serious threats to agriculture worldwide. this problem is further aggravated in arid and semi-arid regions (minhas et al., 2020). according to fao (2021), 424 million hectares of arable lands are affected by salinity. high salt concentrations disrupt plants survival by inhibiting various physiological and metabolic processes, which affects crop yields and threatens food security (egamberdieva et al., 2019). in this worrying context, some solutions focus on limiting salt devastating effects. thus, plant growth promoting microbes (pgpms) are considered as a promising alternative to overcome this problem. plant growth promoting microbes (pgpms) are microorganisms present in the rhizosphere. previous studies have already focused on the isolation of halotolerant pgpms from the rhizosphere of halophyte plants (fu et al., 2016; etesami et al., 2018). these plants are not only a niche for the growth of pbpms, but they also have the ability to sequester salts from salt contaminated soils (etesami et al., 2018). suaeda fruticosa (syn. s. vera forssk. ex j.f. gmel.) is a leaf succulent halophyte that grows in arid and semiarid saline habitats or salt marshes (hameed et al., 2012). this plant species is widely distributed in halomorphic ecosystems in algeria (bahi et al., 2020). it has a strong ability to accumulate and sequester nacl (hameed et al. 2012), and shows the capacity to accumulate large amounts of heavy metals in its tissues without symptoms of toxicity. this ability makes it an excellent candidate for revegetation and remediation of metal-contaminated areas and saltaffected soils (sidhoum and fortas, 2019). plant growth promoting microbes have a positive effect on plant physiology and can protect plants from biotic and abiotic stress (chennappa et al., 2019). among the best known and most widely used pgpms, rhizobacteria and mycorrhizal fungi, are arousing enormous interest due to their ability to improve plant growth and health under saline conditions (nadeem et al., 2014). compared to rhizobacteria and mycorrhizal fungi, the use of yeast as growth promoters still in an early stage. yeasts can be very useful, due to their production of molecules of interest and their ability to tolerate polluted and saline conditions (shruthi et al., 2022). as plant growth promoters, some yeasts have demonstrated a great ability in terms of indole acetic acid production. species belonging to hanseniaspora, pichia, candida, sporidiobocus, meyerozyma, symmetrospora, rhodotorula were isolated from various sources (citrus peel, citrus leaves, citrus pulp and soil) (shruthi et al., 2022). other plant growth promoting yeasts (pgpys) have been isolated from plant leaves (fu et al., 2016), grapes (fernandez-san millan et al., 2020) and water tanks (gomes et al., 2015). this indicates that pgpys can be isolated from unconventional sources. saline habitats such as seas and salt lakes are an ideal place to isolate halotolerant microorganisms that could be used as pgps. recently, krishnan et al. (2020) and srinivasan et al. (2022) reported the isolation of the yeast w. anomalus from seaweed in indian coast and its applications as pgp. in addition to a broad-spectrum antimicrobial activity against a variety of species, w. anomalus exhibits a multitude of applications with biotechnological importance (padilla et al., 2018). this strain was applied in the production of fuel chemicals, in food and beverage production and in biocontrol for cereal grain preservation (padilla et al., 2018). although all the mentioned performances, little has been reported on w. anomalus environmental applications, except for its tolerance to some toxic metals such as chromium and cadmium (breierová et al., 2002; martorell et al., 2012), elimination of lead from water (aibeche et al., 2022), and aromatic hydrocarbons degradation (hesham et al., 2006), thus highlighting its potential role in environmental bioremediation. aibeche c et al. (2023). not sci biol 15(2):11442 3 considering the lack of studies that relate the application of w. anomalus as a pgp, the objective of this study was to evaluate the effect of w. anomalus wo2 inoculation on the growth and physiological performance of the halophyte plant suaeda fruticosa under saline stress and to understand by which mechanism the yeast can protect the plant from high salinity. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods strain and culture conditions the yeast strain w. anomalus wo2 was used in the present study. it was isolated from water samples of dayet oum ghellaz lake (province of oran north west of algeria) and registered in genbank database with the accession number mt331854. this strain is characterized by its ability to eliminate several heavy metals (copper, zinc, lead, cadmium, chromium) at high concentrations (aibeche et al., 2022). the strain activation was carried out on yepd broth for 72 h at 28 °c under continuous shaking at 150 rpm. effect of nacl on yeast growth the salinity tolerance of the studied yeast was evaluated by cultivating the strain in 200 ml flasks with 50 ml of yeast extract peptone dextrose medium (yepd) supplemented with 0%, 3%, 5%, 10%, 15% and 20% of nacl and incubated at 28 °c under continuous shaking at 150 rpm. growth was monitored by optical density at 620 nm each 24 h for 96 hours. the experiment was performed in triplicate. plant material sampling and experimental design the seeds of s. fruticosa were collected from the lakeside of telamine lake (35°42’50”n 0°23’30’’w) located in the district of gdyel (province of oran, algeria). the proximity of telamine lake to the industrial zone ii of hassi amer (7 km) makes it vulnerable to pollution hazards (sidhoum and fortas, 2019). the collected seeds were surface sterilized with a 12° sodium hypochlorite solution for 10 min and subsequently rinsed with distilled water. the seeds were sown in 250 ml capacity plastic pots containing a sterile cultivation substrate prepared by homogenously mixing peat and sand (w/w). the experiment was carried out in the experimental greenhouse of the university of sciences and technology of oran, under natural light conditions. after 6 weeks of growth, uniformly developed seedlings were selected for inoculation. twenty germinated seedlings were inoculated by 20 ml of w. anomalus wo2 suspension grown in yepd medium, prepared at a final concentration of 107 ufc ml-1, after 24 h of growth at 28 °c under continuous shaking (150 rpm). twenty non-inoculated controls received the same volume of medium in the absence of yeast. after one week of cultivation, each group of plants (inoculated and non-inoculated) was divided into four sub-groups and were irrigated every three days for one month with 75 ml of water with separate solutions of nacl at concentrations of 0%, 1%, 3%, and 5% (w/v). morphological, physiological and biochemical growth parameters at the end of the experiment, shoot lengths, fresh and dry shoot weights were measured. the shoots water content was calculated as described by turner (1981) using the following formula: wc (%) = (f.w-d.w/f.w)100 where f.w = fresh weight and d.w = dry weight. for chlorophyll and carotenoid determination, fresh biomass (leaves) were homogenized in 80% icecold acetone in the dark. the mixture was then centrifuged at 10000 rpm for 10 min at 4 °c and the supernatant was used for the immediate determination of pigments. absorbances of the solutions were determined spectrophotometrically at 663, 645 and 480 nm. concentrations of total chl (t-chl), chl a, chl aibeche c et al. (2023). not sci biol 15(2):11442 4 b, and total carotenoids were calculated using the formula described by arnon (1949). chlorophyll and carotenoid concentrations were expressed as milligrams per gram of fresh weight. for the evaluation of total protein content of fresh leaves, an amount of 1 g of different samples of suaeda fruticosa leaves were ground in a mortar in 5 ml of 0.06 m phosphate buffer (ph 7). the mixture was then centrifuged at 10000 rpm for 10 min at 4 °c. homogenates were used for protein quantification according to the method described by bradford (1976), using bovine serum albumin (bsa) as a standard protein. the absorbance of the mixtures was measured spectrophotometrically at 595 nm. the concentration of total polyphenol of plant leaves was determined according to the method of folinciocalteu (singleton et al., 1999). 0.5 g of fresh leaves was mixed with 50 ml of 70% methanol; the mixture was then incubated at room temperature for 24 h under continuous stirring. after that, the solution was centrifuged at 3800 rpm for 20 min, the supernatant was collected and evaporated, then 5 ml of 70% methanol were added to the residues. 100 μl of each test solution was mixed with 500 μl of folin-ciocalteu (0.1 m). after 5 minutes, 400 µl of aqueous sodium carbonate na2co3 (7.5 %) was added to the mixture and incubated for 2 hours and the measurement was done at 760 nm. total polyphenols values were expressed as milligram gallic acid equivalent per dry weight material (mg gae g-1 dw). flavonoids were extracted by stirring 0.5 g of fresh leaves stirred in 5 ml of 80% methanol for 24 hours. the mixtures were filtered through a screen cloth, then centrifuged at 5000 rpm for 5 min. the total flavonoid content of each extract was measured at 510 nm using the aluminium chloride colorimetric method (chang et al., 2002). the results are expressed in mg of catechin equivalent per 100 g of sample. total soluble sugar contents were determined using the anthrone reagent method (yemm and willis, 1954). extraction of sugars was done by submersion of 100 mg of fresh leaves in 5.25 ml of 80% (v/v) ethanol at 25 °c for 20 h. total soluble sugars were analysed by reacting 2 ml of ethanolic extract, diluted 10 times previously in 80 % ethanol, with 4 ml freshly prepared anthrone (2 g anthrone + 1000 ml of h2so4) in water bath at 92 °c for 8 minutes and the cooled samples were read at 585 nm. for na+ and k+ contents estimation, 100 mg of dried sample was digested by nitric acid (lambert, 1975). the k+ and na+ contents were determined by flame photometer (jenway pfp7). the translocation factor (tf), bioconcentration factor (bcf) and bioaccumulation factor (baf) values were used to evaluate the potential of plants for phytoextraction and phytostabilization of sodium in soil according to baker (1981). the factors were calculated by the following formula: bcf = na� na� � baf = na ��� na� � tf = na ��� na� where, naleaf, naroot, and nasoil are sodium concentrations in leaves, in roots, and in soil respectively. data analysis statistical analysis was performed using the spss software program (spss 23.0). each treatment was replicated 3 times unless otherwise specified, and the data were presented as the mean value ± standard deviation (± sd). the relationship between the studied parameters was investigated by calculating the correlation coefficient r. data were analysed by two-way analysis of variance (anova) and glm (generalized linear model). to determine the statistical significance of differences (p< 0.05) between means, the tukey test was performed. aibeche c et al. (2023). not sci biol 15(2):11442 5 results results results results effect of nacl on yeast growth the strain w. anomalus wo2 exposed to increasing nacl concentrations showed its best growth after 24 h of incubation at 0, 3 and 5% of nacl (figure 1). yeast growth continued to progress to reach its maximum at 72 h. at 10% of nacl, growth was slightly inferior compared to lower concentrations and it reached its maximum after 96 h. however, the growth of the yeast was very low at 15% of nacl in the first 48 h, although it started to grow again to reach its maximum after 96 h of incubation. finally, no growth was recorded for the yeast cultivated at 20% of nacl. figure 1.figure 1.figure 1.figure 1. growth of wickerhamomyces anomalus wo2 in the presence of 0 %, 3 %, 5 %, 10 %, 15 % and 20 % nacl concentrations at 28 °c effect of w. anomalus wo2 inoculation on plant growth parameters under nacl stress after 4 weeks of growth under different nacl concentrations, the plants of s. fruticosa exhibited differences in growth parameters, but no mortality was detected at any concentration. fresh and dry shoots weight (figure 2 a, b) were at their maximum at 1% of nacl, with a slight superiority in the inoculated plants. with increasing salinity, fresh and dry shoot weights progressively declined. furthermore, shoot length (figure 2 c) was around 20 cm for most concentrations, with a slight superiority for the inoculated plants. the maximum height was reached at 1% of nacl in the inoculated plants (22.20 cm ± 1). as for shoots water content (figure 2 d), an increase in values was noted as concentrations of salt increased until 3% of nacl, with no differences registered between inoculated and non-inoculated plants. shoots water content showed an important decrease at 5% of nacl for non-inoculated plants (81.72% ± 4.5), while the yeast-inoculated plants maintained a high-water content (87.09% ± 5.8). 0 0,5 1 1,5 2 2,5 0 h 24 h 48 h 72 h 96 h o d 6 2 0 time (h) 0% 3% 5% 10% 15% 20% aibeche c et al. (2023). not sci biol 15(2):11442 6 figure 2.figure 2.figure 2.figure 2. effect of wickerhamomyces anomalus wo2 inoculation on growth parameters of suaeda fruticosa under salt stress; (a)(a)(a)(a) dry weight; (b)(b)(b)(b) fresh weight; (c)(c)(c)(c) shoot length; (d)(d)(d)(d) water content (wc) effect of w. anomalus wo2 inoculation on photosynthetic pigments under nacl stress the effect of salinity stress on photosynthetic pigments was studied (figure 3). under non-saline conditions, plants appeared green and healthy compared to the salt-stressed plants. salt stress significantly decreased the concentrations of chlorophyll a, b and t in both inoculated and non-inoculated s. fruticosa. in the range from 0% to 5% of nacl, the values varied from 17.63 mg g-1 ± 1 to 7.19 mg g-1 ± 0.65, from 7.82 mg g-1 ± 1.03 to 3.62 mg g-1 ± 0.58 and from 25.47 mg g-1 ± 1.87 to mg g-1 mg g-1 ± 0.56 for chlorophylls a, b and t respectively in non-inoculated plants. as for inoculated plants, the values of chlorophylls a, b and t went from 17.57 mg g-1 ± 1.16 to 8.18 mg g-1 ± 0.25, from 8.18 mg g-1 ± 1.06 to 2.75 mg g-1 ± 1.03 and from 25.74 mg g-1 ± 1.57 to 10.93 mg g-1 ± 0.77. also, a decrease in carotenoid contents was observed in non-inoculated plants (figure 3 d), with concentrations ranging from 2.95 mg g-1 ± 0.83 to 1.26 mg g-1 ± 0.24. however, a significant increase was noticed in inoculated plants where the concentrations of carotenoid ranged from 2.61 mg g-1 ± 0.6 to 7.89 mg g-1 ± 1.15 which represents an augmentation of 627% (figure 3 d). according to the glm results, inoculation had no significant effect on the chlorophylls (total, a and b) contents, while it significantly increased the content of carotenoids at 1, 3 and 5% of nacl (table 1). a strong negative correlation was observed between salinity and chl a, b and t in both non-inoculated and inoculated plants (table 3). however, for carotenoids, a very strong negative correlation was observed in non-inoculated plants (r= -0.82), whereas a very strong positive correlation was obtained in inoculated plants (r= 0.94) (tables 3 and 4). 0 5 10 15 20 0 1 3 5 f re sh w e ig h t (g ) nacl concentration (%) (a)ni i 0 0,5 1 1,5 2 2,5 0 1 3 5 d ry w e ig h t (g ) nacl concentration (%) (b)ni i 0 5 10 15 20 25 30 0 1 3 5 s h h o t le n g h t (c m ) nacl concentration (%) (c)ni i 70 75 80 85 90 95 100 0 1 3 5 w c ( % ) nacl concentration (%) (d)ni i aibeche c et al. (2023). not sci biol 15(2):11442 7 table 1. table 1. table 1. table 1. significance of sources of variation for inoculation (i) and salt stress [nacl] effects on morphological and physiological growth parameters using generalized linear model (glm) fresh weight (g)fresh weight (g)fresh weight (g)fresh weight (g) dry weight (g)dry weight (g)dry weight (g)dry weight (g) shoot length (cm)shoot length (cm)shoot length (cm)shoot length (cm) wc (%)wc (%)wc (%)wc (%) iiii 3.26 ns 0.48 ns 1.46 ns 3.62 ns [nacl][nacl][nacl][nacl] 28.10 *** 9.79*** 1.56 ns 10.60*** i*[nacl]i*[nacl]i*[nacl]i*[nacl] 1.04 ns 0.519 ns 0.025 ns 2.40 ns chl a (mg gchl a (mg gchl a (mg gchl a (mg g---1111)))) chl b (mg gchl b (mg gchl b (mg gchl b (mg g---1111)))) chl t (mg gchl t (mg gchl t (mg gchl t (mg g---1111)))) carotenoid (mg gcarotenoid (mg gcarotenoid (mg gcarotenoid (mg g---1111)))) iiii 0.86 ns 0.045 ns 0.17 ns 33.87*** [nacl][nacl][nacl][nacl] 104.19*** 17.83*** 85.48*** 13.38*** i*[nacl]i*[nacl]i*[nacl]i*[nacl] 0.519 ns 0.42 ns 0.01 ns 39.80*** protein (mg gprotein (mg gprotein (mg gprotein (mg g---1111) ) ) ) polyphenol (mg gpolyphenol (mg gpolyphenol (mg gpolyphenol (mg g---1111)))) flavonoid (mg gflavonoid (mg gflavonoid (mg gflavonoid (mg g---1111) ) ) ) sugar (mg gsugar (mg gsugar (mg gsugar (mg g---1111) ) ) ) iiii 7.07* 311.57*** 7651.64*** 559.94*** [nacl][nacl][nacl][nacl] 2.39 ns 138.06*** 6388.37*** 13.82*** i*[nacl]i*[nacl]i*[nacl]i*[nacl] 1.78 ns 181.57*** 6109.88*** 11.62*** wc: water content. chl a: chlorophyll a. chl b: chlorophyll b. chl t: chlorophyll t. significance levels: ns indicates no significant. * p< 0.05. ** p< 0.01. *** p<0.001. figure 3.figure 3.figure 3.figure 3. wickerhamomyces anomalus wo2 inoculation effect on chlorophyll a (a)(a)(a)(a), chlorophyll b (b)(b)(b)(b), total chlorophyll (c)(c)(c)(c) and carotenoid content (d)(d)(d)(d) in leaves of suaeda fruticosa under salts stress effect of w. anomalus wo2 inoculation on biochemical parameters under nacl stress the response of s. fruticosa seedlings to nacl stress in the presence of of the halophilic yeast strain w. anomalus wo2 was investigated by determining the biochemical status of the plants, including protein, polyphenol, flavonoid and total sugars. the results are presented in table 1 and figure 4. both salinity and inoculation affected significantly polyphenols, flavonoids and sugar foliar contents according to the glm results (table 1). hence, the assayed microbial inoculations affected significantly the foliar sugar concentrations of plants grown under non-stressing conditions. 0 5 10 15 20 25 30 0 1 3 5 c h lo ro p h y ll a ( m g / g ) nacl concentration (%) (a) ni i 0 5 10 15 20 25 30 0 1 3 5 c h lo ro p h y ll b ( m g / g ) nacl concentration (%) (b)ni i 0 5 10 15 20 25 30 0 1 3 5 c h lo ro p h y ll t ( m g / g ) nacl concentration (%) (c)ni i 0 2 4 6 8 10 0 1 3 5 c a ro ti n o id ( m g / g ) nacl concentration (%) (d)ni i aibeche c et al. (2023). not sci biol 15(2):11442 8 the inoculation induced significant differences in protein content, and had a highly significant effect on polyphenols, flavonoids and sugar contents. this treatment increased highly the biochemical parameters at 1% nacl concentration. thus, the increasing of salt concentration (3% and 5%) in inoculated plants induced the production of sugar and the diminution of other parameters (figure 4). the correlation analysis revealed a weak between polyphenols and flavonoids with salinity, regardless of of the presence of yeast (tables 3 and 4). as for protein content, a very strong positive relationship with salinity was found in non-inoculated plants (r= 0.99). the amount of soluble sugars strongly positively correlated with salinity in both inoculated and non-inoculated plants (r= 0.94 and 0.90 respectively) (tables 3 and 4). figure 4.figure 4.figure 4.figure 4. effect of wickerhamomyces anomalus wo2 inoculation on growth parameters of suaeda fruticosa under salt stress; (a)(a)(a)(a) protein content; (b)(b)(b)(b) polyphenol content; (c)(c)(c)(c) flavonoid content; (d)(d)(d)(d) carbohydrate content effect w. anomalus wo2 inoculation on bioconcentration (bcf), bioaccumulation (baf) and translocation (tf) of nacl to investigate the mechanism underlying salt resistance of s. fruticosa seedlings, the amounts of na+ and k+ accumulated in tissues and rhizospheric soil were measured. the k+ and na+ concentrations in the rhizospheric soil were relatively similar with all external nacl treatments. nevertheless, in inoculated plants, an increase in na+ was observed with the increase of nacl. na+ and k+ tissue concentrations were higher in leaves compared to roots (figure 5). in non-inoculated plants, na+ increased remarkably with nacl with higher nacl concentrations in both leaves and roots. however, in inoculated plants, na+ concentrations remained unchanged in leaves and slightly decreased in roots at higher nacl levels. regarding k+ concentration in roots, a decrease was noticed in non-inoculated plants, while its concentration fluctuated in inoculated ones. in the leaves, k+ presented changing concentrations for both inoculated and non-inoculated plants with the increasing of nacl concentrations. although, potassium accumulations in all tissues of s. fruticosa seedlings were reduced by external nacl but increased in all leaves and roots of inoculated plants at higher nacl concentrations (table 2, figure 5). 20 30 40 50 60 70 80 0 1 3 5 p ro te in c o n te n t (m g / g ) nacl concentration (%) (a) ni i 0 5 10 15 20 25 30 0 1 3 5 p o ly p h e n o l c o n te n t (m g g a e / g ) nacl concentration (%) (b) ni i 0 2 4 6 8 0 1 3 5 f la v o n o id c o n te n t (m g e c / g ) nacl concentration (%) (c) ni i 0 5 10 15 20 0 1 3 5 t o ta l so lu b le s u g a rs ( m g / g ) nacl concentration (%) (d) ni i aibeche c et al. (2023). not sci biol 15(2):11442 9 similarly, a significant increase in the na+/k+ ratio was noticed in the rhizospheric soil of inoculated seedlings as the nacl concentrations increased, while the ratio was relatively unchanged in non-inoculated plants. concerning roots tissue, the na+/k+ ratio increased with increasing salt concentrations in inoculated seedlings, unlike the non-inoculated ones where the ratio increased until 3% nacl and then declined afterward. in the leaves, the na+/k+ ratio increased in both inoculated and non-inoculated plants, reaching its maximum at 3% nacl, and then decreased at 5%. figure 5.figure 5.figure 5.figure 5. effects of wickerhamomyces anomalus wo2 inoculation on na+, k+ and na/k ratio in soil, root and leave of suaeda fruticosa under different concentrations of nacl solution (0%, 1%, 3% and 5%) table 2. table 2. table 2. table 2. significance of sources of variation for organ (org), inoculation (i) and salt stress [nacl] effects on k, na and na/k, bcf, baf, tf ratio using generalized linear model (glm) kkkk mg g-1 na na na na mg g-1 na/kna/kna/kna/k bcfbcfbcfbcf bafbafbafbaf tftftftf orgorgorgorg 23076.57*** 2252.64*** 1197.30*** / / / iiii 1.45 ns 10.24** 25.43*** 15,39*** 2,16 ns 13,76*** [nacl][nacl][nacl][nacl] 589.87*** 105.54*** 185.03*** 9,04*** 7,76** 3,76* org * iorg * iorg * iorg * i 159.20*** 15.52*** 1.73 ns / / / org * org * org * org * [nacl][nacl][nacl][nacl] 595.84*** 35.46*** 54.11*** / / / i * [nacl]i * [nacl]i * [nacl]i * [nacl] 155.05*** 8.107*** 11.15*** 27,08*** 15,63*** 6,58** org*i*[nacl]org*i*[nacl]org*i*[nacl]org*i*[nacl] 131.21*** 10.50*** 52.68*** / / / significance levels: ns indicates no significant. * p< 0.05. ** p< 0.01. *** p<0.001. bcf: bioconcentration factor; baf: bioaccumulation factor; tf: the translocation factor. the effect of w. anomalus wo2 inoculation on bioconcentration (bcf), bioaccumulation (baf) and translocation (tf) factors of sodium in s. fruticosa plants under salt stress is represented in figure 6. the highest bcf value was obtained at 1% nacl (9.7% ± 1) in inoculated plants. a continuous increase of this factor was noticed for the non-inoculated plants across all salt concentrations, associated to a decrease in the 0 10 20 30 40 50 ni i ni i ni i soil root leave n a ( µ g / g ) na+ 0% 1% 3% 5% 0 0,5 1 1,5 ni i ni i ni i soil root leave k ( µ g / g ) k+ 0% 1% 3% 5% 0 20 40 60 80 100 120 140 160 180 200 ni i ni i ni i soil roots leaves n a / k r a ti o na/k 5% 3% 1% 0% aibeche c et al. (2023). not sci biol 15(2):11442 10 inoculated plants. for baf, the highest value was noted at 1% nacl (14.77% ± 1) in yeast inoculated plants. for the translocation factor (tf), it was superior in the non-inoculated plants at 0, 1, 3% nacl (1.88% ± 0.12, 1.93% ± 0.13, 2.05% ± 0.14 respectively). however, at 5% nacl the tf of yeast inoculated plants became higher (1.93% ± 0.14). a continuous increase proportional to salt concentration was noticed in inoculated plants (table 2, figure 6). figure 6.figure 6.figure 6.figure 6. wickerhamomyces anomalus wo2 inoculation effect on bioconcentration (bcf)(bcf)(bcf)(bcf), bioaccumulation (baf)(baf)(baf)(baf) and translocation (tf)(tf)(tf)(tf) factors of sodium in suaeda fruticosa plants under salt stress the relationship between salinity and bcf, baf and tf was studied. the results are presented in tables 3 and 4. bcf and baf had very strong positive correlation with salinity in non-inoculated plants (r= 0.91 and 0.89 respectively). whereas the correlation was strong negative in inoculated plants (r= -0.90 and 0.76 respectively). as for translocation factor (tf), the correlation was very strong positive only in inoculated plants (r= 0.97). relationship between pigments and biochemical parameters the relationship between pigments and biochemical parameters is represented in tables 3 and 4. in non-inoculated plants, there was a very strong positive correlation between chlorophylls a, b, and t with carotenoids (r= 0.94, 0.92 and 0.94 respectively), while the correlation was weak negative in inoculated plants. the correlation between pigments (chl a, b and t) and protein content was strong negative in inoculated plants and weak negative in non-inoculated plants. also, protein content correlated negatively with carotenoid content in non-inoculated plants (r= -0.72). there was a weak correlation between pigments and polyphenols in both inoculated and non-inoculated plants, contrary to flavonoids in non-inoculated plants, where a strong negative correlation with pigments was observed. 0 3 6 9 12 15 18 0 1 3 5 b io c o n c e n tr a ti o n f a c to r nacl concentration (%) bcfni i 0 3 6 9 12 15 18 0 1 3 5 b io a c c u m u la ti o n f a c to r nacl concentration (%) bafni i 0 0,5 1 1,5 2 2,5 3 0 1 3 5 t r a n s lo c a ti o n f a c to r nacl concentration (%) tfni aibeche c et al. (2023). not sci biol 15(2):11442 11 a strong negative correlation between total soluble sugars with chl a, b and t (r= -0.80; -0.92 and -0.85 respectively) and a strong positive correlation with carotenoid were noticed in inoculated plants (r= 0.76). while, the relationship was moderate negative in non-inoculated plants (r= -0.52). relationship between bioconcentration (bcf), bioaccumulation (baf) and translocation (tf) of nacl with the studied parameters the relationship between bcf, baf with chl a, chl b and chl t was very strong negative only in noninoculated plants (table 3). in inoculated plants (table 4), the correlation was moderate positive with bcf and weak positive with baf. the correlation was very strong negative between carotenoids and bcf and baf regardless of yeast inoculation. while no correlation was noticed between pigments and tf in non-inoculated plants, a very strong negative correlation was registered after yeast inoculation with chl a, b and t (r= -0.88, 0.96 and -0.91 respectively). in the opposite, tf was very strongly positively associated with carotenoid content (r= 0.84). in non-inoculated plants (table 3), the relationship was very strong positive between protein content and bcf and baf (r=0.88 and 0.82), while it was moderate negative with tf (r= -0.59). the correlation was absent with bcf, weak positive with baf and moderate positive with tf in inoculated plants (r= 0.53) (table 4). polyphenols correlated only with baf where a strong positive correlation was obtained for both plant groups (tables 3 and 4). as for flavonoids, in non-inoculated plants, the relationship was weak positive with bcf and moderate positive with baf and tf (table 3). whereas in inoculated plants (table 4), it was strong positive with bcf, very strong positive with baf and weak negative with tf. finally, for soluble sugar content, the correlation was very strong to strong positive with bcf and baf respectively and very strong negative with tf in non-inoculated plants (table 3). while the opposite was noticed in inoculated plants where the correlation was very strong to strong negative with bcf and baf respectively and very strong positive with tf (table 4). table 3. table 3. table 3. table 3. correlation coefficient between different parameters in non-inoculated plants saltsaltsaltsalt chlchlchlchl chl bchl bchl bchl b chl tchl tchl tchl t carotcarotcarotcarot protprotprotprot polypolypolypoly flavflavflavflav sugsugsugsug bcfbcfbcfbcf bafbafbafbaf tftftftf saltsaltsaltsalt 1.00 chl achl achl achl a -0.80 1.00 chl bchl bchl bchl b -0.73 0.99 1.00 chl tchl tchl tchl t -0.78 1.00 1.00 1.00 carotcarotcarotcarot -0.82 0.94 0.92 0.94 1.00 prot.prot.prot.prot. 0.99 -0.70 -0.61 -0.68 -0.72 1.00 polyphpolyphpolyphpolyph -0.14 0.43 0.48 0.45 0.64 -0.02 1.00 flavflavflavflav 0.31 -0.78 -0.84 -0.80 -0.78 0.15 -0.81 1.00 sugarsugarsugarsugar 0.90 0.60 -0.51 -0.58 -0.52 0.94 0.30 -0.03 1.00 bcfbcfbcfbcf 0.91 -0.87 -0.82 -0.86 -0.75 0.88 0.34 0.37 0.90 1.00 bafbafbafbaf 0.89 -0.97 -0.93 -0.96 -0.87 0.82 0.97 0.59 0.78 0.97 1.00 tftftftf -0.49 0.12 0.03 0.09 -0.06 -0.59 -0.37 0.51 -0.82 -0.59 -0.37 1.00 chl a: chlorophyll a. chl b: chlorophyll b. chl t: chlorophyll t. carot: carotinoid content. prot: protein content. poly: polyphenols. flav: flavonoids; sug: sugar; bcf: bioconcentration factor; baf: bioaccumulation factor; tf: the translocation factor. p values were considered significantly (p ≤ 0.05, *; p ≤ 0.01, **). aibeche c et al. (2023). not sci biol 15(2):11442 12 table 4.table 4.table 4.table 4. title title title title title title title saltsaltsaltsalt chl achl achl achl a chl bchl bchl bchl b chl tchl tchl tchl t carotcarotcarotcarot protprotprotprot polypolypolypoly flavflavflavflav sugsugsugsug bcfbcfbcfbcf bafbafbafbaf tftftftf saltsaltsaltsalt 1.00 chl achl achl achl a -0.74 1.00 chl bchl bchl bchl b -0.86 0.97 1.00 chl tchl tchl tchl t -0.79 1.00 0.99 1.00 carotcarotcarotcarot 0.94 -0.53 -0.66 -0.58 1.00 prot.prot.prot.prot. 0.30 -0.85 -0.74 -0.82 0.01 1.00 polyphpolyphpolyphpolyph -0.48 -0.23 -0.02 -0.15 -0.68 0.68 1.00 flavflavflavflav -0.35 -0.32 -0.08 -0.23 -0.45 0.63 0.91 1.00 sugarsugarsugarsugar 0.94 -0.80 -0.92 -0.85 0.76 0.49 -0.30 -0.31 1.00 bcfbcfbcfbcf -0.90 0.43 0.64 0.51 -0.86 0.00 -0.10 0.71 -0.87 1.00 bafbafbafbaf -0.76 0.17 0.40 0.25 -0.80 0.27 0.96 0.87 -0.71 0.96 1.00 tftftftf 0.97 -0.88 -0.96 -0.91 0.84 0.53 -0.62 -0.16 0.96 -0.81 -0.62 1.00 chl a: chlorophyll a. chl b: chlorophyll b. chl t: chlorophyll t. carot: carotinoid content. prot: protein content. poly: polyphenols. flav: flavonoids; sug: sugar; bcf: bioconcentration factor; baf: bioaccumulation factor; tf: the translocation factor. p values were considered significantly (p ≤ 0.05, *; p ≤ 0.01, **). discussiondiscussiondiscussiondiscussion soil salinization is becoming a major agricultural problem worldwide, mainly in arid and semi-arid regions. the increase in the level of salinity leads to negative impacts on soil properties and plant physiology. the objective of this study was to evaluate the effect of w. anomalus wo2 inoculation on s. fruticosa growth under salt stress. the yeast w. anomalus wo2 is a halophilic strain in this study, the yeast strain w. anomalus wo2 showed a great ability to grow at high salt concentrations. being able to withstand salt concentrations of up to 15%, makes this strain a promising candidate to be applied in organic farming and the bioremediation of salt-affected soils. similar studies have discussed the halophilic nature of w. anomalus. bonatsou et al. (2015) reported that 16.32% was the minimum inhibitory concentration of salt for the strain w. anomalus y18. also, praphailong and fleet (1997) stated that w. anomalus did not grow at 20% of nacl, while it grew at 15% which is in concordance with the current study. therefore, based on this halophilic trait, this strain was selected for the study of the effect of its inoculation on s. fruticosa growth parameters under salt stress. nacl affects plant growth parameters regardless of yeast inoculation the obtained results about the effect of w. anomalus wo2 inoculation on plant growth parameters under nacl stress, revealed that, under 1% of nacl, the fresh and dry shoot weights and shoot lengths of s. fruticosa were at their maximum. no significant difference was noticed between inoculated and non-inoculated plants. this indicates that the optimal concentration of salinity for the growth of s. fruticosa is 1%. however, this halophyte plant is able to survive and develop even at salinities up to 5% of nacl (855.58 mm) for one month. the high tolerance of s. fruticosa to salinity up to 5.85% (1000 mm) was also observed in previous studies (khan et al., 2000; bankaji et al., 2016). the water content increased until 3% of salinity concentration, which is superior to the results obtained by khan et al. (2000), where they reported, maximum shoot tissue water content at 1.17% (200 mm) of nacl. this parameter showed an important decrease at 5%, which is in concordance with the result of khan et al. (2000) who observed a decreased in wc at higher salt concentrations. the present findings indicated that the yeast-inoculated plants had higher water content at 5% of nacl, suggesting it may be one of the mechanisms aibeche c et al. (2023). not sci biol 15(2):11442 13 of the plant’s defence system against high salt stress. similarly, chauhan et al. (2019) observed that the inculcation of rice with bacillus amyloliquefaciens has improved water content under salt stress. inoculation enhances carotenoids content under salt stress regarding the effect of salinity on pigments, salt irrigation led to a decrease in chlorophyll a, b and t in inoculated and non-inoculated plants. previous studies have reported a decrease in chlorophyll content in s. prostrata, s. persica and s. europaea with increasing soil salinity (aghaleh et al., 2009; akcin et al., 2016). the decrease in chlorophyll content in salt-stressed plants is associated with an increase of chlorophyllase activity responsible for chlorophyl degradation (santos, 2004). also, high salinity induces ethylene production in plants, which inhibit chlorophyll biosynthetic pathway (fahad et al., 2015). unlike non-inoculated plants, a significant increase in carotenoids was observed in plants inoculated with w. anomalus wo2. inoculation of plants with pgpms has been associated with an increase in carotenoids content (katsenios et al., 2021). in a similar study, the yeast yarrowia lipolytica fh1 increased carotenoids production in maize seedlings under salt stress (gul jan et al., 2019). salinity at 1% boosts protein, polyphenols and flavonoids in inoculated plants despite the negative effects of salinity on plant growth parameters, the obtained results demonstrated that the inoculation of s. fruticosa plants with the w. anomalus wo2 improved protein, polyphenols, and flavonoids thus enhancing plant tolerance against salt stress. although the difference between inoculated and non-inoculated plants was not as great, the increase in total soluble proteins content could be attributed to the growth hormones produced by yeast and the direct stimulation of proteins synthesis (gaballah and gomaa, 2004; stino et al., 2009; khalil and ismael, 2010; agamy et al., 2013), providing plants with essential nutrient elements required for protein formation (hayat, 2007). the present investigation demonstrated that higher amounts of phenolic compounds and flavonoids have been observed in inoculated plants under 1% of salt stress. thanks to the inoculation with the yeast, phenolic compounds and flavonoids production increased by 334% and 2136% respectively compared to noninoculated plants at the same concentration of salt (1%). recently, muhammad et al. (2019), reported that increased levels of these compounds was observed in wheat inoculated by trichoderma reesei. indeed, improved flavonoids and phenols production may help the plants in various aspects, including growth, reproduction, resistance to pathogens and the protection against abiotic stress (mona et al., 2017). in wheat and jointed goatgrass, polyphenols and flavonoids were observed to be involved against salt stress-induced oxidative stress (kiani et al., 2021). inoculation of the plants enhance sugar production at all concentrations of salt compared with non-inoculated plants, the soluble sugar concentrations in inoculated plants were significantly higher in leaves at all nacl concentrations. at higher concentrations of salt, total soluble sugars were increased by 3.6 times indicating a role of sugars in salt-stress tolerance. these results are in accordance with the study of feng et al. (2002) which demonstrated that higher accumulation of soluble sugars in mycorrhizal plant tissue, especially in roots, could make mycorrhizal plants more resistant to osmotic stress induced by exposure to salt. several studies have demonstrated that the accumulation of sugars and polyols was stimulated by salt stress in different plant species. a strong correlation has been established between the accumulation of sugars and the level of tolerance to salinity (hanana et al., 2011). aibeche c et al. (2023). not sci biol 15(2):11442 14 yeast inoculation protects the plant from salt stress at high concentrations in the present study, the results showed that the presence of w. anomalus wo2 at higher concentrations of salt induced na+ accumulation in the soil, which caused a decrease of na+ accumulation factors (baf, bcf). whereas its accumulation always took place in the leaves of non-inoculated plants. also, the sodium translocation factor (tf) increased as the salt concentration increased in inoculated plants. this sequestration of salt outside the plant is very important to avoid salt toxicity. it should be noted that the yeast species used in this study have certain properties that could have a direct positive effect on plant development and salt tolerance. kasim et al. (2016) demonstrated that w. anomalus produces exopolymers in the extracellular space in stressful growth environment. as a response to the presence of nickel and cadmium stress, w. anomalus produces exoglycoproteins and phosphomannans as components of exopolymers (breierová et al., 2002; breierová et al., 2008). these exopolymers serve as an effective protective barrier against the penetration of heavy metal ions into the cells (breierová et al., 2008). under stressful conditions such as ion toxicity and osmotic stress caused by water limitation, some microorganisms develop biofilm as a strategy to increase the chances of survival (enebe et al., 2018). the properties of this biofilm which functions as a barrier, depends on its composition and structure (breierová et al., 1996). the decrease in na+ content in the roots of the inoculated plants under high salt stress could be explained by the ability of pgpms to induce changes in the expression of the plant genes that control the na+ content or to actively participate in this ionic readjustment (lanza et al., 2019). in this situation, pgpms can accumulate na+ inside their cells, or actively remove it outside the roots of the plant (lanza et al., 2019). at higher nacl levels, the na+ accumulation factors (baf, bcf) decrease in inoculated plants was accompanied with an increase of k+ concentration. many studies on the mycorrhizal effect revealed that mycorrhizal plants can increase k+ uptake while decreasing na+ accumulation in the cytoplasm under saline conditions compared to non-mycorrhizal plants (giri et al., 2007; hammer et al., 2011). to reduce cytosolic na+ concentration, some halophytes have developed a mechanism of ion compartmentation by sequestering excessive cytosolic na+ into the central vacuole, which alleviates the na+ toxicity, thus maintains ion homeostasis in saline conditions (yamaguchi et al., 2013; flowers et al., 2015). the relationship between the studied parameters according to the results of the correlation between the different parameters, yeast plays a central role in the protection of the plant against salt stress via the stimulation of the production of carotenoids. the translocation factor also seems to play an important role. in the presence of yeast, the more the salinity increased, the more this parameter increased and the more the bcf and baf decreased. it seems also that there is a close relationship between flavonoids and bcf and baf. in the absence of the yeast, the plant exhibited a different behaviour. the more salt was present, the more the plant produced protein and the more bcf and baf increased. although sugar production correlated positively with salinity, regardless of whether the plant was inoculated or not. the obtained results proved that there was also a direct relationship between carotenoid content and sugar production and between sugar production and tf in inoculated plants. whereas in the absence of yeast, there was an inverse relationship between sugar production and tf. carotenoids are known to be involved in protecting plants from salt stress (li et al., 2020). gul jan et al. (2019) reported that the accumulation of carotenoids increased maize seedlings salt stress tolerance after plant inoculation with the yeast yarrowia lipolytica fh1. chlorophyll had a negative relationship with salinity in both inoculated and non-inoculated plants. under these stressful conditions, carotenoids protected s. fruticosa by playing a role of accessory light-harvesting pigments, by extending effectively the range of light absorption which is a known role of carotenoids (young, 1991). another role of carotenoids is the protection of plants from the accumulation of reactive oxygen species (ros) at the cellular level (young, 1991). when the plant is exposed to salt stress, ros are generated at concentrations that may adversely affect the survival of the plant aibeche c et al. (2023). not sci biol 15(2):11442 15 (latowski et al., 2011). to better adapt to these conditions, the plant produces carotenoids such as xanthophyll which is among the most important ros scavenging mechanisms (latowski et al., 2011). another adaptation strategy consists in synthesizing osmoprotectants, mainly sugars, and accumulating them in the cytoplasm and organelles. the key role of soluble sugars during stress includes carbon storage, osmo-protection, and hunting of the reactive oxygen species (gupta and huang, 2014, hanana et al., 2011). these osmolytes, usually hydrophilic in nature, are poorly charged but polar and highly soluble molecules, suggesting that they can adhere to the surface of proteins and membranes to protect them from dehydration (hanana et al., 2011). moreover, in non-inoculated plants, it has been demonstrated through the correlation study, that proteins have close relationship with sugar content. in this study translocation of sodium had a crucial role in yeast inoculated plants. it had a direct relationship with carotenoid and sugar productions. during salt stress, the energetic cost of na+ sequestration in the vacuoles increases. it was observed in this study how salt stress was accompanied by the decrease in chlorophyll levels. indeed, the more the chlorophyll level decreased, the more the plant produced sugars. this is due to salt stress which reduces photosynthesis causing the plant to accumulate high content of sugars which could represent transient carbon storage (sellami et al., 2019). sugars also play an important role as a signalling molecule. at the genomic level, high glucose concentrations modify significantly the positive or negative expression of genes (han et al., 2015). solfanelli et al. (2006) discussed the role that sugars play as a signalling molecule in flavonoid biosynthetic pathways. this may explain why sugar production was closely correlated with carotenoid production. indeed, sugar might act as signalling molecule in the biosynthesis of carotenoid in s. fruticosa in order to protect the plant from salinity. the expression of the genes involved in the carotenoid pathway has been observed previously in tomato and tobacco plants exposed to high concentrations of salts (ann et al., 2011; li et al., 2020). conclusionsconclusionsconclusionsconclusions w. anomalus wo2 is a salt-tolerant strain that has been successfully used as a plantgrowth-promoting yeast (pgpy). its wide range tolerance of salinity makes it a promising strain with potential applications in the field of soil remediation and sustainable agriculture. inoculation of the halophyte plant s. fruticosa with the w. anomalus wo2 increased the tolerance of the plant to salt stress through the high increase of carotenoid production. this substance that compensates for the loss of chlorophyll caused by salinity, was not produced in the non-inoculated plants. w. anomalus wo2 also increased the tolerance of s. fruticosa by the excessive production of sugars which play an osmo-protective role and act as signalling molecules that trigger salt stress tolerance mechanisms. increased sodium translocation is another mechanism by which inoculated plants resisted to high salt levels. these promising results indicate the possibility of using w. anomalus wo2 as pgpy to solve the problem of salinity affected soils, and to increase the tolerance to salt stress in high consumption plants in countries with arid and semi-arid climates. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions ca, ns and adj designed and supervised the study; ca, ws, fc, ke, as and cm performed the laboratory experiments and bioassays, ca, ws, ok, sch, sn and adj writing original draft, reviewed and edited the draft. all authors read and approved the final manuscript. aibeche c et al. (2023). not sci biol 15(2):11442 16 ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements the authors acknowledge the financial support of the algerian general directorate of scientific research and technological development (dgrsdt) and the algerian ministry of higher education and scientific research (mesrs). conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences agamy r, hashem m, alamri s (2013). effect of soil amendment with yeasts as bio-fertilizers on the growth and productivity of sugar beet. african journal of agricultural research 7(49):6613-6623. https://doi.org/10.5897/ajar12.1989 aghaleh m, niknam v, ebrahimzadeh h, razavi k (2009). salt stress effects on growth, pigments, proteins and lipid peroxidation in salicornia persica and s. europaea. biologia plantarum 53(2):243-248. https://doi.org/10.1007/s10535-009-0046-7 aibeche c, selami n, zitouni-haouar f e h, oeunzar k, addou a, kaid-harche m, djabeur a (2022). bioremediation potential and lead removal capacity of heavy metal-tolerant yeasts isolated from dayet oum ghellaz lake water (northwest of algeria). international microbiology 25(1):61-73. https://doi.org/10.1007/s10123-021-00191-z akcin a, yalcin e (2016). effect of salinity stress on chlorophyll, carotenoid content, and proline in salicornia prostrata pall and suaeda prostrata pall. subsp. prostrata (amaranthaceae). brazilian journal of botany 39(1):101-106. https://doi.org/10.1007/s40415-015-0218-y ann bm, devesh s, gothandam km (2011). effect of salt stress on expression of carotenoid pathway genes in tomato. journal of stress physiology and biochemistry 7(3):87-94. bahi k, miara md, hadjadj-aoul s (2020). approche diachronique de la flore des bassins fermés halomorphes de la région d’oran (no algérie) diachronic analysis of the flora of the halomorphic closed basins in the region of oran (nw algeria). bulletin de la société royale des sciences de liège. https://doi.org/10.25518/0037-9565.9763 baker a j (1981). accumulators and excluders‐strategies in the response of plants to heavy metals. journal of plant nutrition 3(1-4):643-654. https://doi.org/10.1080/01904168109362867 bankaji i, cacador i, sleimi n (2016). assessing of tolerance to metallic and saline stresses in the halophyte suaeda fruticosa: the indicator role of antioxidative enzymes. ecological indicators 64:297-308. https://doi.org/10.1016/j.ecolind.2016.01.020 bonatsou s, benítez a, rodríguez-gómez f, panagou e z, arroyo-lópez f. n (2015). selection of yeasts with multifunctional features for application as starters in natural black table olive processing. food microbiology 46:66-73. https://doi.org/10.1016/j.fm.2014.07.011 bradford mm (1976). a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analytical biochemistry 72(1-2):248-254. https://doi.org/10.1016/00032697(76)90527-3 breierová e, stratilová e, šajbidor j (1996). production of extracellular polymers by yeast-like genera dipodascus and dipodascopsis under nacl stress. folia microbiologica 41(3):257-263. https://doi.org/10.1007/bf02814627 aibeche c et al. 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(2023). not sci biol 15(2):11442 20 license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11502 nsb ben rabeh 2023.06.19.docx received: 00 xxx 2022. received in revised form: 00 xxx 2023. accepted: 08 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania ben rabeh s et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11502 doi:10.15835/nsb15211502 rererereviewviewviewview articlearticlearticlearticle.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae environmental stress tolerance, hydroenvironmental stress tolerance, hydroenvironmental stress tolerance, hydroenvironmental stress tolerance, hydro----distilldistilldistilldistilled essential oils ed essential oils ed essential oils ed essential oils characteristics and biological activities of characteristics and biological activities of characteristics and biological activities of characteristics and biological activities of eucalyptus torquataeucalyptus torquataeucalyptus torquataeucalyptus torquata luehm.luehm.luehm.luehm. sonia ben rabeh1,2*, kaouther ben yahia3, samir dhahri4, souda belaïd1,2, imen chemlali1,2, chokri ben romdhane4, mehrez romdhane2, ezzeddine saadaoui4 1university of gabes, faculty of science gabes, gabes, tunisia; soniabenrabeh.a@gmail.com (*corresponding author); belaidsouda95@gmail.com; chemlali.imen@gmail.com 2university of gabes, national engineering school of gabes, laboratory of energy, water, environment and processes, gabes, tunisia; mehrez.romdhane@univgb.tn 3university of carthage, i national institute for rural engineering, water and forestry (inrgref), lef, tunisia; kaoutherbenyahia01@gmail.com 4university of carthage, national institute for rural engineering, water and forestry (inrgref), lgvrf, tunisia; dhahri.samir@iresa.agrinet.tn; benromdhanechokri@gmail.com; saad_ezz@yahoo.fr abstractabstractabstractabstract eucalyptus has become one of the most widely planted genera in the world because of its tolerance to a wide range of soil types and climates, as well as for its many industrial, commercial and medicinal uses. eucalyptus torquata luehm. is a plantation species frequently planted in semi-arid and arid regions for its ecological, forestry, ornamental and melliferous interests. based on literature, drought tolerance of this species was mostly directed to adaptation mechanisms. physiological investigations reveal the importance of stomatal closure and increased solute contents suggesting that osmotic adjustment is one of the main responses to drought in e. torquata. on the other hand, it showed low sensitivity to salt stress. this paper also highlights the immense benefits of e. torquata which contains essential oils with variable chemical composition and rich essentially in 1,8-cineole, torquatone, α-pinene, trans-myrtanol, α-eudesmol, β-eudesmol, globulol, transpinocarveol and aromadendrene. these oils, as well as the methanol and aqueous extracts possess a wide variety of bioactivities of great importance which are particularly valuable as antibacterial and antifungal agents also have a strong toxicity against insects and mites in addition to antiproliferative and cytotoxic effects against different types of cancer cells. keywords:keywords:keywords:keywords: biological activities; chemical composition; coral gum; essential oil; eucalyptus introductionintroductionintroductionintroduction eucalypts (eucalyptus spp.) are endemic to australia; however, its few species are indigenous to neighboring countries. the genus eucalyptus comprises more than 800 species and hybrids, which includes ben rabeh s et al. (2023). not sci biol 15(2):11502 2 shrubs and flowering trees. it is the most valuable genus and it is found in almost all parts of the world due to human introduction (chemlali et al., 2022). it has been broadly cultivated in many countries to utilize as wood for a diverse range of products and grow across a large range of climatic environments and soil types. eucalypts are known for their adaptation to arid conditions and are considered drought, salt and heat tolerant compared to other trees (teulières et al., 2007). the mild climate is the most preferred for most species of eucalyptus, they are highly distributed where there are warm summers, temperate winters, moderate rainfall, dry atmosphere and plenty of sunlight (najum rasheed et al., 2005).the distribution of eucalyptus all over the world also the dominance in australia means ecological importance of this genus which provide food and habitat resources for a diverse range of fauna (vuong et al., 2015). eucalypts are one of the most bio-economic plant species with high potential to grow in salt-affected soil with arid climatic condition also capable of providing an economic return in the future, it used for ornamental purposes, afforestation, providing feedstock for the pulp and paper industries or to obtain timber and gum, also a nectar resources for honey and known by cosmetic and medicinal values (saadaoui et al., 2022). in some countries dried eucalyptus leaves are used as tobacco and smoked for asthma (sefidkon et al., 2008), aqueous extracts are used for aching joints, bacterial dysentery, ringworms, tuberculosis, etc. (sefidkon et al., 2010). also, hot water extracts of dried leaves are traditionally used as analgesic, anti-inflammatory, and antipyretic remedies for the symptoms of respiratory infections, such as cold, flue and sinus (silva et al., 2003). eucalyptus has been prized as a rich source of essential oils that’s more useful as it is easily extractable and has advantages as its superior quality and is regarded as safe and non-toxic by the united states food and drug authority (fda). eucalyptus oils are volatile organic compounds found in fruits, flowers, bark, seeds, wood and roots, while, these compounds are mainly extracted from foliage (boland et al., 1991) because in the leaves that oils were most plentiful and more than 300 species of this genus contain volatile oils in their leaves (pino et al., 2002). eucalyptus essential oils contain terpenoids, phenolic, flavonoids and alkaloids that possess many bioactivities that could be grouped into three classes viz: perfumery, industrial and medicinal (abiri et al., 2021). in fact, essential oils rich in 1,8-cineole are utilized as pharmaceuticals, whereas those rich in citronellal, citral and geranyl acetate are used in perfumery (dhakad et al., 2018). under natural conditions, essential oils from the leaves of eucalyptus known to provide allelopathic property to this plant (may and ash, 1990) and defense to eucalyptus leaves against attack by harmful insects, and thus acts as a natural pesticide (batish et al., 2008; üstüner et al., 2018; gallon et al., 2020; sadraoui-ajmi et al., 2022). in fact, many researchers reviewed the biological properties of eucalyptus essential oils including anti-microbial, fungicidal, antiviral, insecticidal/insect repellent, herbicidal, acaricidal and nematicidal also the anti-tumour and cytotoxic activities (zhang et al., 2010; vuong et al., 2015; barbosa et al., 2016; dhakad et al., 2018; salehi et al., 2019; abiri et al., 2021; chandorkar et al., 2021). the importance and the commercial uses of essential oils of eucalyptus have increased the research on their extraction, exploring their chemical compositions and bioactivities. in fact, eucalyptus torquata luehm. commonly known as coral gum or coolgardie gum is an attractive tree with a small to medium size growing to 6-8 m and a spread of some 5 m, presenting a single trunk, a greyish green foliage and the blade has a lanceolate shape. the length of leaves around 90-120 mm and wide of 15-20 mm, the profuse flowers are reddish-pink or coral colored and hang decoratively on reddish stems. flowering is very conspicuous and occurs in spring to summer (al-snafi, 2017). it is a fast-growing tree known for tolerance to drought, often hybridising in cultivation with another commonly grown arid zone species, e. woodwardii to give a hybrid e. torwood. it recommends planting e. torquata in protected areas as an ornamental tree in the public and private gardens due to its beautiful flowers and its medium size (el-juhany and al alshaikh, 2015). the researchers were also exploring the potency of e. torquata by valuing their extracts and essential oils which show different biological activities such as antibacterial, antifungal (ashour, 2008), cytotoxic (ashour, 2008; bardaweel et al., 2014; lahmadi et al., 2021) and pesticidal activity (ebadollahi et al., 2017; el finti et al., 2022; ebadollahi et al., 2022). also, e. torquata considered an important sources of nectar ben rabeh s et al. (2023). not sci biol 15(2):11502 3 and pollen for honeybees as showing an abundant flowering of long period and good quality of pollen and nectar for the nutrition of bee; for these reasons, it is frequently planted in arid regions (eisikowitch et al., 2012; saadaoui et al.,2022 ). the purpose of this study is to provide the readers with information concerning the tolerance and behavior of e. torquata under drought and salt stress, also, exploring the potency and diversity of extracts and essential oils of this species in terms of chemical composition and biological activities. evaluation of the tolerance evaluation of the tolerance evaluation of the tolerance evaluation of the tolerance of of of of e. torquatae. torquatae. torquatae. torquata to drought and salt stressto drought and salt stressto drought and salt stressto drought and salt stress drought and the salinization of soil are a widespread environmental problems and an important factors determining plant productivity and distribution (teulières, 2010).for landscape applications like reclamation of dry and arid saline lands, eucalyptus is a good choice, it’s a versatile woody species that develops an extensive deep root system and presents the challenge of finding a good compromise between adaptation to specific environmental conditions and productivity (teulières et al., 2007). responses to drought drought is the second productivity-limiting stress after cold to find subsequently biotic and abiotic stresses, it was suggested that the availability of water is the important determining factor for the distribution of eucalyptus (li and wang, 2003). in fact, among 117 eucalyptus species introduced in tunisia, e. torquata is considered a drought-resistant species (khouja et al., 2001; saadaoui et al., 2017). australian native plants nursery (2015) mentioned that e. torquata is tolerant of extended dry periods (el-juhany et al., 2008), also, in saudi arabia it was classified among the high tolerating species to drought (el-juhany and al al-shaikh, 2015). in the mediterranean arid regions, e. torquata showed a high tolerance level and flower abundance also in the southern provinces of north africa (chemlali et al., 2022). mechanisms employed for drought resilience of e. torquata were investigated by souden et al. (2020) which reported physiological and biochemical responses of this species subjecting to a dehydration period followed by rehydration. it reported that e. torquata was less resilient to drought than e. camaldulensis. nevertheless, common responses were shown during the dehydration phase including lowering cell water potential from -1mpa to -4.9mpa after 28 days and to 7.1mpa after 45 days of no irrigation which was restored with 88% after rewatering. in the face of water stress, lowering the water potential of the cells by the plant, help to maintain the water content of the cells and, consequently, the turgor (white et al., 2000). other physiological responses for e. torquata are observed including the early closure of stomata which starting from -3.5mpa to prevent water loss, the net photosynthesis was decreased to achieve less than 2 μmol.m⁻2.s⁻1. the chlorophyll fluorescence parameters fv/fm (maximum photochemical efficiency of psii) was decreased and after 30 days of re-watering, e. torquata restored the structural and functional integrity of its photosynthetic machinery. changes in xylem conductivity under water deficit also showed which conducted to minimal xylem embolism for e. torquata and the value of ψ xylem which induced 50% plc (ψ50) is-4.6mpa, obviously, the level of xylem cavitation decreased after rehydration (souden et al., 2020). it has been shown also that in case of drought and/or salinity, osmotic adjustment is the key to the adaptation of plants at the cellular level this by the accumulation of organic and inorganic solutes which helps to reduce the water potential without reducing the actual water content (sanders and arndt, 2012). for e. torquata, water stress induced accumulation of soluble sugars (glucose and fructose) and cyclitols (pinitol, myo-inositol) for its osmotic adjustment (souden et al., 2020). these adaptive traits are the key factor in the determination of e. torquate drought resistance. ben rabeh s et al. (2023). not sci biol 15(2):11502 4 responses to salinity another major stress for plants is the salinity of soils. in fact, the exposure to salt stress triggers many common reactions in eucalyptus species which have developed several strategies to cope with these challenges (assareh, 2016). however, three strategies for achieving greater salt tolerance: damage prevention, homeostasis establishment and growth regulation (zhu, 2001). how e. torquata deal with and respond to salinity stress has been reported by balti et al. (2021) and the study showed that e. torquata was the salt-sensitive even at lower salt concentrations (80 mm nacl) among other species such as e. gomphocephala and e. loxophleba. salt stress induces certain biochemical and physiological changes in e. torquata, also visible symptoms mainly by the development of necrotic spots in leaves after exposure to 170 mm of nacl for 30 days which indicates saltinduced damage at cellular level. slower growth was not observed for e. torquata indicating the inability of growth modulating under salt stress. changes in photosynthesis also observed, salt stress majorly affect optimal protein function in the photosynthetic electron transport chain (petc). the chlorophyll fluorescence-based psii-related parameters calculated showed lower values, in addition to that, a decline in chlorophyll and carotenoids contents in leaves has been observed. the k+/na+ ratio for e. torquata declined significantly than other species mainly for e. loxophleba which have the ability to selectively increase k+ amounts over na+ (balti et al., 2021). moreover, nacl salinity causes a significant effect on na+, k+ and cluptake and their distribution, in this, higher levels of external na+ interfere with k+ acquisition limiting plant k uptake. therefore, one of the important physiological mechanisms for salinity tolerance is the k+ selective absorbance (nasim et al., 2008). germination also is strongly influenced by osmotic pressure caused by salts in the soil solution (madsen and mulligan, 2006). mechergui et al. (2019) reported that seeds of e. torquata were not able to germinate at up to 9, 12 and 15 g.l⁻1 nacl that means that the salinity levels influenced significantly the percentage of germination. for the responses of e. torquata to salt stress in relation to growth, it reported that this species showed low survival percentages and volume growth to age 20 years under salt water irrigation (el-juhany and al al-shaikh, 2015). all these results suggest a good drought tolerance of this forest species; however, it shows a relative sensitivity to the presence of nacl in the growing medium and soils. yields of yields of yields of yields of e. torquatae. torquatae. torquatae. torquata on essential oils on essential oils on essential oils on essential oils the essential oils of e. torquata may be obtained from different plant parts; however, as observed in table1, the highest was found in the leaves whose production was much higher than that in the trunk bark. hydro distillated leaves of e. torquata ranged 1.15-3% of essential oil. similar essential oils yield (1.21-3.1%) has been reported for e. globulus, as the principal source of eucalyptus oil in the world (derwich et al., 2009; mossi et al., 2011; mulyaningsih et al., 2011; harkat-madouri et al., 2015). the geographical origin also highly affects this production; in this, good extraction yields were observed for plants from tunisia. in fact, several studies reviewed the parameters that can influence the total essential oil content of plant including part of plant (silva et al., 2011), geographic origin (gilles et al., 2010; almas et al., 2018), the seasonal variations (silva et al., 2011), the phenological stage (salem et al., 2018), method of extraction (ben hassine et al., 2010; herzi et al., 2013; chamali et al., 2021), rainfall and harvesting regime (gilles et al., 2010). ben rabeh s et al. (2023). not sci biol 15(2):11502 5 table 1. table 1. table 1. table 1. essential oil yields in e. torquata obtained by hydro distillation in tunisia, iran, morocco and cyprus originoriginoriginorigin part usedpart usedpart usedpart used harvest period/sample crushed harvest period/sample crushed harvest period/sample crushed harvest period/sample crushed or notor notor notor not essential oils essential oils essential oils essential oils yields (%)yields (%)yields (%)yields (%) referencesreferencesreferencesreferences tunisia trunk bark february 2019/dry sample ground into a fine powder 0.006 (lahmadi et al., 2021) leaves january 2005/dry leaves boorishly crushed 3.2 (elaissi et al., 2010) january 2007/dry leaves medially crushed 1.86 (ben hassineet al., 2010) iran dry leaf powder 1.15 (ebadollahi et al., 2017) morocco april 1991 1.17 (zrira et al., 1994) cyprus march 2021/ dry leaves crushed into tiny pieces 1.6 (yiğit hanoğlu et al.,2022) chemical profiling of chemical profiling of chemical profiling of chemical profiling of e. torquatae. torquatae. torquatae. torquata essential oilsessential oilsessential oilsessential oils essential oils obtained from eucalyptus are usually rich in monoterpenes and in some cases sesquiterpenes. nevertheless, the chemical profile and main components of oils from eucalyptus varied significantly between species. mostly, the main components were the oxygenated monoterpenes 1,8-cineole and the monoterpene hydrocarbons α-pinene with various percentages dependent on the specific species (goldbeck et al., 2014; ishnava et al., 2013). other compounds also are detected as major component in eucalyptus oils as example; limonene in e. crebra oils, citronellal in e. citriodora oils (ghaffar et al., 2015) and p-cymene in e. oleosa (chamali et al., 2019). in tunisia, most of eucalyptus species oils showed that the oxygenated monoterpenes constituted the major fraction as the 1,8-cineole was the major component (elaissi et al., 2010; elaissi et al., 2011a, 2011b, elaissi et al., 2011; elaissi et al., 2012, elaissi et al., 2012; sebei et al., 2015; limam et al., 2020; ameur et al., 2021). a wide number of terpenes have been identified in the leaves essential oil of e. torquata, using analyses by gc-fid, gc or gc-ms (table 2). in spite of current variations of the origin of the analyzed plants, there is consistence that 1,8-cineole and α-pinene are a characteristic compound of this species. 1,8-cineole was isolated in concentrations between 11 and 70% also the α-pinene obtained with concentration between 10 and 20%. other compounds detected such as trans-pinocarveol, α-terpineol and borneol from chemical class of oxygenated monoterepenes. the oils also contain considerable amount of the monoterpene hydrocarbons pcymene, also the aromadendrene and alloaromadendrenefrom chemical class of sesquiterepene hydrocarbons. α-eudesmol, β-eudesmol, γ-eudesmol and globulol are the main oxygenated sesquiterpenes. a high percentage of torquatone also was detected. this last compound forms a member of acylphloroglucinols which was a class of specialized metabolites with relatively high content in eucalyptus with diverse structures and bioactivities (singh et al., 2009; yao et al., 2021). torquatone was first isolated from e. torquata and e. caesia benth growing in australia with 25 and 50% of the essential oil fraction respectively (bowyer and jefferies, 1959). it derivative also from the essential oils of number of eucalyptus species and absent in others and present with relative high concentration in e. torquata (ghisalberti, 1996; bignell et al., 1997a, 1997b; elaissi et al., 2010; yiğit hanoğlu et al., 2022). the chemical formula of torquatone is c16h24o4; there is 4,6-trimethoxy-3,5-dimethyl-1-(3methylbutyroyl)-benzene (menut et al., 1999; figure 1). when the composition of twelve eucalyptus essential oils (e. torquata luehm, e. woodwardi maiden, e. stricklandii maiden, e. occidentalis endl, e. brockwayi c. a. gardn, e. salmonophloia f. muell, e. gillii maiden, e. oldfieldii f. muell, e. largiflorens f. muell, e. loxophleba benth, e. sargentii maiden, e. gracilis f. muell,) are compared, the high percentage of torquatone is recorded ben rabeh s et al. (2023). not sci biol 15(2):11502 6 for e. torquata with 42% also the low percentage of sesquiterpenes hydrocarbons and oxygenated monoterpene with a low quantity of 1,8-cineole (12%) and a relative high amounts of α-pinene (10.5%), α-eudesmol (2.9%), β-eudesmol (10.1%) and γ-eudesmol (1.3%). eucalyptus woodwardii oil had resembling chemical characteristics to e. torquata oil essentially in major compounds detected (elaissi et al., 2010; ben amor, 2021). torquatone 1,8-cineole α-pinene trans-myrtanol figure 1. figure 1. figure 1. figure 1. chemical structure of major elements of essential oil of e. torquata plants cultivated in different countries produce essential oils with variable composition as can be seen from table 2. torquatone is detected as a major component of e. torquata leaves essential oils from tunisia (42%), australia (42%) and cyprus (29%) while totally absent in oils from iran and morocco. the same for αeudesmol, β-eudesmol and γ-eudesmol that are not detected in iranian species. an intra-specific variation is also recorded and explained by geographical, environmental and climatic variations which affect the chemical composition of essential oils. also, it was proven that essential oils of different plant parts have different chemical composition (table 2). the trunk bark essential oil of e. torquata growing in tunisia has a completely distinct chemical profile compared to the leaf essential oils. the 1,8-cineole was totally absent and the major constituents being the oxygenated monoterpenes (84.7%), with trans-myrtanol (73.4%) and myrtenol (4.7%) as the main components. the apocarotene cis-β-ionone and the fatty acid nonanoic acid also identified in significant percentages of 3.9% and 2.4% respectively, the sesquiterpene hydrocarbons were represented with only 2% with γ-maaliene as the main component (1.3%) (lahmadi et al., 2021). therefore, there are notable quantitative and qualitative differences in e. torquata essential oils compositions; it is mentioned in literature that these differences are attributed to several exogenous factors: harvest time, seasonal factors, soil composition, geographical position and the method of drying of plants. endogenous factors are involved including genetic makeup and the ontogenetic development stage (marzoug et al., 2011; zandi-sohani and ramezani, 2015). since the chemical composition of the eucalyptus essential oils is directly associated with their biological activities, the following discussion will be focused on such activities of e. torquata. the specific and different composition in e. torquata can only act on these activities. ben rabeh s et al. (2023). not sci biol 15(2):11502 7 table 2. table 2. table 2. table 2. major components of e. torquata essential oils obtained by hydro distillation from tunisia, iran, australia, morocco and cyprus originoriginoriginorigin part usedpart usedpart usedpart used major components (%)major components (%)major components (%)major components (%) identification identification identification identification methodmethodmethodmethod referencesreferencesreferencesreferences tunisia trunk bark trans-myrtanol (73.4), myrtenol (4.7), (e)-β-ionone (3.9), nonanoic acid (2.4), α-terpineol (1.9), decanoic acid (0.9), γmaaliene (1.3), cis-myrtanol (1.2), β cyclocitral (0.8), geranylacetone (0.8), (e)-ocimenol (0.7) (gc-ei-ms) (lahmadi et al., 2021) leaves torquatone (42), 1,8-cineole (12), αpinene (10.5), β-eudesmol (10.1) transpinocarveol (5.1), α-eudesmol (2.9), pcymene (2), globulol (2), γ -eudesmol (1.3), aromadendrene (1.1), δ-cadinol (0.9) (gc (ri) and gc/ms) (elaissi etal., 2010) iran 1,8-cineole (69.6), α-pinene (9.5), terpinen-4-ol (0.8), α-terpineol (1.1), alloaromadendrene (7.8), aromadendrene (4.5), limonene (1.5), p-cymene (0.7) gc-fid and gcmass (nikbakht et al.,2015) 1,8-cineole (66.9), α-pinene (13.9), trans-pincarveol (6.3), p-cymene (4.2) (gc and gc/ms) (sefidkon et al., 2010) 1,8-cineole (28.57), α-pinene (15.74), globulol (13.11), alloaromadendrene (7.26) α-terpineol (2.64), , , , epiglobulol (2.50), p-cymene (2.46), transpinocarveol (2.09), viridiflorol (1.86), endo-borneol (1.72), neoalloocimene (1.53), terpineol-4 (1.51), ledene (1.39), α-gurjunene (1.25), , , , δ-selinene (1.04) (gc-ms) (ebadollahi et al., 2017) 1,8-cineole (24.2), α-pinene (20) globulol ((((8.4), aromadendrene (7.8), αterpineol (2.5), cubeban-11-ol (2.4), trans-sabinol (2), alloaromadendrene (1.8) (gc-ms) (ebadollahi et al., 2022) australia torquatone (42), 1,8-cineole (11.2), αpinene (10.2), α-eudesmol (10.2), βeudesmol (11.1), γ-eudesmol (4.8) (gc-fid and gcms) (baranska et al., 2005) morocco 1,8-cineole (46,9), α-pinene (16,7), bornéol (10.8), 4-terpinéol (3,2), globulol (1.6), p-cymene (1.3) (gc-fid) (zrira et al., 1994) cyprus torquatone (29.2), 1,8-cineole (18.8), αpinène (18.6), β-eudesmol (10.3), αeudesmol (6.8) (gc and gc-ms) (yiğit hanoğlu et al., 2022 ben rabeh s et al. (2023). not sci biol 15(2):11502 8 biological activitiesbiological activitiesbiological activitiesbiological activities antimicrobial activity of e. torquata essential oils eucalyptus essential oils endowed antimicrobial action against a large spectrum of bacteria and fungi which consist to its therapeutic properties as a promising alternative to drugs for several diseases and disorders (zhang et al., 2010; barbosa el al., 2016; dhakad et al., 2018). additionally, the possible interactions of eucalyptus essential oils with conventional antimicrobial agents was studied that could lead to new treatment strategies involving reduced antibiotic doses and for higher therapeutic efficacy (knezevic et al., 2016; scazzocchio et al., 2016; al-qaysi el al., 2020). the bioactivity of eucalyptus essential oils may be due to their monoterpene components; in fact, antimicrobial activity could be attributed to the presence of compounds such as 1,8-cineole, α-pinene, β-pinene and limonene (dhakad et al., 2018). however, the interactions of different constituents may be responsible for the total bioactivity of eucalyptus essential oils that can potentially lead to additive, synergistic, or antagonistic effects (mulyaningsih et al., 2010). eucalyptus torquata essential oils marked antimicrobial activities against a large spectrum of bacteria based on agar diffusion method and the microdilution method (table 3) the bioassays confirm that grampositive bacteria are more sensitive compared to gram-negative ones. indeed, leaves, stems and flowers essential oils of e. torquata exhibited a moderate to high antibacterial activity against staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis and bacillus subtilis with inhibition zones in the range of 10 and 22 mm of diameter and against the two bacteria klebsiella pneumoniae and proteus mirabilis with inhibition zones in the range of 8 and 10 mm of diameter while not active against salmonella typhi. also, e. torquata flowers essential oil demonstrated antibacterial action against pseudomonas aeruginosa with inhibition zone of 11mm (ashour, 2008; bardaweel et al., 2014) this last bacteria was resistant to essential oils obtained from several eucalyptus species and from other plants (elaissi et al., 2011; wilkinson and cavanagh, 2005). in other hand, pseudomonas aeruginosa with escherichia coli are resistant to leaves essential oils of e. torquata grown in egypt while susceptible to that grown in jordan with inhibition zones of 11 and 9 mm respectively (ashour, 2008; bardaweel et al., 2014). minimum inhibitory concentration (mic) of leaves essential oils of e. torquata from jordan was calculated by the microdilution method, norfloxacin 1 mg/ml was used as reference controls for antibacterial activity. the mic values for bacillus subtilis, staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas aeuriginosa are 198, 201, 197, 204 and 217 μg/ml respectively (bardaweel et al., 2014). e. torquata essential oils also cause growth inhibition of some fungal species, oils from flowers, stems and leaves of e. torquata from egypt and jordan exhibited a moderate to high antifungal activities against mycelial fungi aspergillus flavus and aspergillus nigeralso against the yeast candida albicans. flowers essential oil from egypt showed the maximum zone inhibition against aspergillus flavus and candida albicans with inhibition zones of 17 and 15 mm respectively, while the leaves essential oil is active with inhibition zones of 10 and 14 mm respectively. nevertheless, essential oils from jordan are active against aspergillus flavus and candida albicans with inhibition zones of 10 mm and with mic values of 198 and 192 μg/ml respectively (ashour, 2008; bardaweel et al., 2014). as a result, the difference in the chemical composition of e. torquata essential oils shown previously could be the cause of the difference in their biological and therapeutic activities. antimicrobial activities of e. torquata extracts hence, there is an urgent need to find alternative antimicrobial agents for the treatment of resistant pathogenic microorganisms. the use of plant-based antimicrobials has several advantages over synthetic chemicals since the lower incidence of numerous side effects, low toxicity for mammals and high degradability ben rabeh s et al. (2023). not sci biol 15(2):11502 9 (raja, 2014). eucalyptus species are known to be a rich source of bioactive compounds, including phenolic, flavonoid, terpenoids, tannins, phloroglucinol and cardiac glycosides, which had potential antimicrobial activities (luís etal., 2016; elansary et al., 2017; bhuyan et al., 2017; sabo and knezevic, 2019). indeed, phenolic compounds are those which contribute significantly to the antioxidant activities of plant extracts (siramon and ohtani, 2007; ghaffar et al., 2015). two studies conducted in morocco revealed that aqueous extracts of powdered waste from e. torquata ( leaves, stems, twigs and other parts) contained total polyphenols amounts of 73.48 and 76.68 mg gae/g dw and flavonoids content of approximately 58 mg re/g dw in which significant antioxidant capacity has been investigated for these extracts (bouhlali et al., 2020; bouhlali et al., 2021). additionally, methanol and aqueous extracts from leaves, stems and flowers of e. torquata showed antibacterial action against different medically bacteria such as staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis, bacillus subtilis and escherichia coli with different inhibition zones in the range of 7 and 25 mm, also, it marked an antifungal activity against the yeast candida albicans with inhibition zones in the range of 9 and 14 mm of diameter (ashour, 2008). in another way, the growing interest in the use of natural plant products in the biological control of plant disease through the use of biological methods has been a great challenge for agriculture for a long time. in fact, eucalyptus species possess fungicidal properties against large spectrum of phytopathogenic fungi (zhou et al., 2016; gakuubi et al., 2017; abdelkhalek et al., 2020). also, aqueous extracts from e. torquata waste (leaves, branches, twigs) showed an antifungal activity against two fungal pathogens fusarium oxysporum f. sp. albedinis and mauginiella scaettae in a dose dependent manner and more stronger than extracts from other plants such as acacia cyanophylla, cupressus atlantica, nerium oleander and schinus molle (bouhlali et al., 2020; bouhlali et al., 2021). these soil-borne fungal pathogens caused a serious threat to date palm (phoenix dactylifera l.) in morocco. “bayoud” disease and inflorescence rot are the principal enemy of palm trees caused by these pathogens and researchers suggest the use of this plant to control these diseases. it reported that at every concentration tested, e. torquata extract showed the strongest inhibition activity on fungal mycelia growth of pathogens. at a dose of 4% of extract, the spore germination of fusarium oxysporum inhibited with 79.21% after 7 days of incubation and strong sporulation reductions is shown with 44.97% of extract after 10 days of incubation (bouhlali et al., 2020). 100% inhibition of spore germination of mauginiella scaettae at a low concentration of 1% of e. torquata extracts after 24h of incubation and a great reduction in sporulation by 88.05% at a dose of 4%. the inhibitory effect of these extracts is related to their composition; moreover, content in polyphenols and flavonoids in aqueous extracts of e. torquata are found to be correlated with this antifungal activity as well as their antioxidant properties (bouhlali et al., 2021). ben rabeh s et al. (2023). not sci biol 15(2):11502 10 table 3. table 3. table 3. table 3. biological properties of e. torquata essential oils and extracts e. torquatae. torquatae. torquatae. torquata effectseffectseffectseffects plant part plant part plant part plant part (extract or oil)(extract or oil)(extract or oil)(extract or oil) tested organism tested organism tested organism tested organism and /or cell lineand /or cell lineand /or cell lineand /or cell line effects and/or effects and/or effects and/or effects and/or related mechanisms related mechanisms related mechanisms related mechanisms aaaa referencesreferencesreferencesreferences antimicrobial antimicrobial antimicrobial antimicrobial activityactivityactivityactivity leaves essential oil from jordan b. subtilis, s. aureus s. epidermidis p. aeuriginosa e. coli c. albicans, a. flavus growth inhibition izd mic 11 198 12 201 10 197 9 204 11 217 10 192,198 (bardaweel et al., 2014) leaves, stems and flowers essential oils from egypt b. subtilis s. aureus s. epidermidis e. faecalis k. pnemounia p. mirabilis c. albicans a. flavus a. niger 11-15 10-16 19-22 12-19 8-10 8-9 10-15 14-17 12-15 (ashour, 2008) flowers essential oil from egypt p. aeuriginosa 11 leaves, stems and flowers methanolic extract from egypt s. aureus s. epidermidis e. faecalis b. subtilis, e. coli c. albicans 16-17 10-16 13-19 13-15 10-11 leaves, stems and flowers aqueous extract from egypt s. aureus s. epidermidis e. faecalis b. subtilis c. albicans 10-13 9-13 18-24 11-14 9-10 anticanceranticanceranticanceranticancer activityactivityactivityactivity stems and leaves essential oils from egypt mcf7 human breast adenocarcinoma cells notable cytotoxic effect with ic50 values of 1.34(stem oil) and 5.22 μg/ml (leaf oil) leaves essential oil from jordan nine mammalian cell line (mcf-7, hela, caco, t47d, bjab, raji, a498, pc3 and caki) cytotoxic effect with ic50 values ranging from 33 and 115 μg/ml after exposure time of 48 h probably involved cell death by apoptosis (bardaweel et al., 2014) trunk bark essential oil from tunisia two human cell lines mda-mb-231 and sw620 inhibitory effect on cell proliferation with ic50 values of (lahmadi et al., 202) ben rabeh s et al. (2023). not sci biol 15(2):11502 11 40.66 and 26.71 μg/ml after 48h insecticidal activityinsecticidal activityinsecticidal activityinsecticidal activity aqueous extract of leaves from morocco d. opuntiae 65% mortality in the population of adults females and 50% of nymphal stage after three application of extract (60%) (el finti et al., 2022) leaves essential oil from iran r. dominica fumigant toxicity after 72h with lc50 and lc90 values of 31.567 and 105.017 μl/l of air respectively ebadollahi et al., 2022) acaricidal activityacaricidal activityacaricidal activityacaricidal activity leaves essential oil from iran t. urticae strong fumigant toxicity against female adults (lc50 = 3.59 (μl/l air after 24h) (ebadollahi et al. (2017) aizdizdizdizd – inhibitory zone diameter (mm), micmicmicmicminimal inhibitory concentration (μg/ml), ic50ic50ic50ic5050% inhibitory concentration, lc50lc50lc50lc50-50% lethal concentration, lc90lc90lc90lc90 90% lethal concentration. anticancer activity the cytotoxic effect of extracts and components isolated from different species of eucalyptus has been studied by several researchers. anti-tumor properties of phenolics, terpenoids (monoterpenes, sesquiterpenes, diterpenoids and triterpenoids) derived from eucalyptus plants have been discussed by abiri et al. (2021) which explain the broad spectrum of toxicity, antitumor properties, and mechanisms against cancerous cell lines of eucalyptus-derived essential oil which can be a promising green anti-cancer drugs. also, bardaweel et al. (2014) demonstrated the cytotoxic effect of essential oils from e. torquata gorwn in jordan which reported that it was varied and highly cell line dependent; in fact, various cytotoxicity levels have been observed on the cancer cell lines treated with essential oil after exposure time of 48 h at 37 °c based on mtt assay. it reported that the ebv-negative burkitt’s lymphoma bjab cell and the human burkitt’s lymphoma raji cell line are the most sensitive cell lines with ic50 values of 33 and 39 μg/ml respectively. although, cytotoxic proprieties also observed against the human breast adenocarcinoma mcf7 cell line (ic50 values of 115 μg/ml), the human ductal breast epithelial tumor cell line t47d (ic50 values of 82 μg/ml), the human clear cell renal cell carcinoma caki cell (ic50 values of 94 μg/ml), the human kidney carcinoma cell line a498 line (ic50 values of 87 μg/ml), the human prostate cancer pc3 cell line (ic50 values of 108 μg/ml), the human colon adenocarcinoma caco-2 cell line (ic50 values of 108 μg/ml) and the human epithelial carcinoma hela cell line (ic50 values of 91 μg/ml). also, the lactate dehydrogenase (ldh) activity and the decrease in dna content of cell line treated indicated that the cytotoxic activity of e. torquata essential oils probably mediated through induction of cell death by apoptosis (bardaweel et al., 2014). the trunk bark essential oil of e. torquata grown in tunisia displayed significant antiproliferative effect against two human cancer cell lines: breast carcinoma cell lines mda-mb-231 and colorectal cancer cell lines sw620 which demonstrated inhibitory effect on the tested cell lines proliferation in a dose-dependent manner after 48 h of incubation using crystal violet staining (cvs) assay, the highest cytotoxic activity of essential oil is observed at 100μg/ml and it's shown that colon carcinoma cells are more sensitive against essential oils (with ic50 values of 26.71 μg/ml) than breast mda-mb-231 (with ic50 values of 40.66 μg/ml) (lahmadi et al., 2021). according to the protein-staining sulphorhodamine b (srb) assay for cell growth, essential oils of e. torquata from egypt (extracted from stem with ic50 value of 1.34 μg/ml and leaves with ic50 value of 5.22 ben rabeh s et al. (2023). not sci biol 15(2):11502 12 μg/ml) have a cytotoxic effect on the human breast adenocarcinoma cell line (mcf7) and failed to exert a considerable effect on human hepatocellular carcinoma cell line (hepg2) (ashour, 2008). these studies increase the attention in exploring this species and improving the therapeutic opportunities against cancer. e. torquata as pesticide attacks and infection by pests (especially weeds, pathogens and animal pests) are the largest competitor of agricultural crops that severely reduce crop productivity (oerke, 2006). however, the excessive use of synthetic pesticide residue in food, accumulated in the environment and increasing health hazards to humans in addition to the increasing risk of pesticide resistance (pimentel et al., 1992), is thus pertinent to explore the pesticidal activities of natural products. eucalyptus species are known to be a rich source of bioactive compounds that allow it to act directly as natural pesticide (radwan et al., 2000; shukla et al., 2002; batish et al. 2008; anita et al., 2012; barbosa et al., 2016; adak et al., 2020). absolutely, e. torquata has been shown insecticidal properties against the cochineal, dactylopius opuntiae, an insect that highly damaging the cactus plants in morocco, , , , it found that three applications of aqueous extract (60%) of e. torquata leaves are needed to reduce mealybug populations also caused the death of 65% of females and 50% of nymphal stages of dactylopius spp. after 72 h after spraying with e. torquata extract which could be an alternative for the control of wild cochineal (el finti et al., 2022). also, e. torquata essential oils showed a great insecticidal potential on the adults of rhyzopertha dominica, an insect pest of stored products, in which significant fumigant toxicity against insect which was augmented by increasing the concentration of e. torquata essential oils and the exposure time. the lc50 value decreased with increasing exposure time from 37.728 (µl/l of air) after 24 h to 31.567 (µl/l of air) after 72 h of exposure with essential oil. in fact, sublethal biochemical disruption has been shown in treated insects, including the reduction of energy content resulting from the significant decrease of the protein and glycogen contents. in other hand, an inhibition of digestive amylase and protease enzyme activities, also, a significant decreases in the relative growth rate of insects (ebadollahi et al., 2022) which confirm that e. torquata extract and essential oils can be used to control insect pests. also, it found that possess acaricidal properties as reported by ebadollahi et al. (2017) which demonstrated that e. torquata leaves essential oils have strong toxicity against the adult females of tetranychus urticae koch and the observed lc50 values in the fumigation test was 3.59 μl/l air after 24 h. conclusionsconclusionsconclusionsconclusions with many eucalyptus species adapted to arid conditions, e. torquata is considered drought tolerant. the corresponding tolerance mechanisms developed by this species were demonstrated in this review. to assume, drought resistance of e. torquata was manifested by stomatal closure to prevent water loss. osmotic adjustment was a coping strategy to water stress by increasing the accumulation of solutes, including soluble sugars (glucose and fructose) and cyclitols (pinitol and myo-inositol) in addition to that, the resilience to xylem embolism. however, salt stress may have a negative impact on e. torquata which appears sensitive to the presence of nacl that acts in particular on photosynthesis. this paper showed also the large variability in yields and chemical composition that exists among e. torquata hydro distillated essential oils from several origins. the majority of oils produced are rich in 1,8-cineole, α-pinene and torquatone. it can be concluded that e. torquata derived essential oils are a rich resource of active phytochemicals which can possess a wide range of biological activities; from the present review, it is clear that possess potent antimicrobial capacity and exhibit an advances anticancer and biocidal effects. ben rabeh s et al. 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(2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11365 doi:10.15835/nsb15211365 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae multivariate analysis of the salinitymultivariate analysis of the salinitymultivariate analysis of the salinitymultivariate analysis of the salinity----induced alterations in morphology, induced alterations in morphology, induced alterations in morphology, induced alterations in morphology, physiology, physiology, physiology, physiology, nodulation, and yield in twonodulation, and yield in twonodulation, and yield in twonodulation, and yield in two contrasting mungbean varietiescontrasting mungbean varietiescontrasting mungbean varietiescontrasting mungbean varieties ganesh d. mankar1, uttam r. wayase1, deepak b. shelke2, kiran b. raskar1, pankaj s. mundada3, mahendra l. ahire4, tukaram d. nikam5, rajkumar b. barmukh1* 1post graduate research centre, modern college of arts, science and commerce (autonomous), shivajinagar, pune 5, affiliated to savitribai phule pune university, pune 7, ms, india; gdmbotany@gmail.com; botanica5050@gmail.com; kiranraskar1488@gmail.com; barmukhbotany@moderncollegepune.edu.in (*corresponding author) 2department of botany, amruteshwar arts, commerce and science college, vinzar, velhe, pune 4112213, ms, india; dpk.shelke1@gmail.com 3yashavantrao chavan institute of science, department of biotechnology, satara 415001, ms, india; mundada.pankaj77@gmail.com 4yashavantrao chavan institute of science, department of botany, satara 415001, ms, india; mlahire@gmail.com 5savitribai phule pune university, department of botany, pune 411007, ms, india; tdnikam@unipune.ac.in abstractabstractabstractabstract changes were estimated in the morphology, physiology, photosynthesis, nodulation, and yield in two mungbean varieties ‘pkv akm 12-28’ and ‘vbn (gg)3’ under salt stress (0, 75, 100, and 125 mm nacl) for 15, 30, and 45 days. multivariate modelling was used to analyse results to explore complex data and to visualize time and concentration-dependent modulations. principal component analysis showed modulations in morpho-physiological attributes such as shoot length, root length, the number of secondary branches, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight and leaf area; photosynthetic attributes such as chlorophyll ‘a’, chlorophyll ‘b’, total chlorophyll, total carotene and total anthocyanine content; nodulation attributes such as nodules per plant, size of the nodule, and fresh weight per nodule, and yield attributes such as number of pods per plant, fresh weight per pod, and seed characteristics such as the number of seeds per pod and fresh weight per 1000 seeds are key traits affected by salt stress and can be used as indicators. discriminant analysis identifed modulations in morpho-physiological attributes such as root length, leaf area, root fresh weight, shoot fresh weight, shoot dry weight, shoot length and photosynthetic attributes such as chlorophyll-a content, and mean nodule weight as discriminating variables at different salt concentrations. besides, it identified modulations in morpho-physiological attributes such as root length, root fresh weight, photosynthetic attributes such as total anthocyanin content and total chlorophyll content, nodulation attribute such nodule size and nodule weight, and yield attributes such as pod number and number of seeds per pod are discriminating variables at various durations of salt stress. principal component analysis and discriminant analysis identifed ‘pkv-akm 12-28’ as salt-tolerant and ‘vbn (gg)3’ as salt-susceptible varieties. multiple correlation analysis identified significant correlations among morphological, physiological, photosynthetic, nodulation and yield parameters. https://www.notulaebiologicae.ro/index.php/nsb/index mankar gd et al. (2023). not sci biol 15(2):11365 2 keywords:keywords:keywords:keywords: mungbean; nodulation; physiological; salt stress; yield abbreviations:abbreviations:abbreviations:abbreviations: chla: chlorophyll ‘a’ content, chlb: chlorophyll ‘b’ content, dpt: day post salt treatment, fw-1000: fresh weight per 1000 seeds, fwp: fresh weight per pod, hca: hierarchical cluster analysis, , la: leaf area, mca: multiple correlation analysis, nn: number of nodules per plant, np: number of pods per plant, ns: size of the nodule, nsp: number of seeds per pod, nw: fresh weight per nodule, pca: principal component analysis, rdw: root dry weight, rfw: root fresh weight, rl: root length, rtwc%: root tissue water content, sb: secondary branches, sdw: shoot dry weight, sfw: shoot fresh weight, sl: shoot length, sti: salt tolerance index, stwc%: shoot tissue water content, tac: total anthocyanin content, tcc: total carotene content, tchl: total chlorophyll content introductionintroductionintroductionintroduction mungbean, vigna radiata (l.) r. wilczek (fabaceae) is one of the valuable dietary pulse crops playing a vital role in fulfilling the food requirements of the ever-increasing population in the world and especially in india (ram and singh, 1993). in india, 3.72 million hectares of agricultural land is under mungbean cultivation, giving 1.56 million tons of production (ali and gupta, 2012). mungbean is used as food, fodder, and green manure and also in cosmetics and pharmaceuticals industries (tang et al., 2014). it is an important source of proteins, vitamins, antioxidants, and minerals (randhir et al., 2004) and has a significant role in ensuring the nutrition security of developing countries such as india (dhingra et al., 1991). soil salinity is one of the significant abiotic environmental stress factors responsible for limiting agricultural productivity in many regions of the world (panta et al., 2014). salinity has already affected 20– 50% of agricultural land, and it is increasing by nearly 10% annually (xu et al., 2011; shrivastava and kumar, 2015). fao (2015) reported that 800 million hectares of land and 32 million hectares of agricultural land are under salinity worldwide. saline soils are dominated mainly by na+ and clions; both are toxic to plants and are considered the most important ions (hasegawa, 2013). salinity limits plant growth and development by disrupting the osmotic and ionic balance in the form of water stress, nutritional stress, oxidative stress, and ion toxicity (arif et al., 2020). salinity affects the shoot and root length, fresh and dry biomass, and leaf area in most crops (sarabi et al., 2016; raza et al., 2017; shelke et al., 2017). moreover, it adversely affects many essential cellular and metabolic processes like photosynthesis (hamani et al., 2020). furthermore, reduction in photosynthetic capacity under salinity depends on salinity type, duration of treatment, species, and plant age (sultana et al., 1999; steduto et al., 2000; hester et al., 2001; koyro, 2006). salinity also limits plants' growth and development, which ultimately affects yield parameters such as pod and seed characters in most of the crops (ahmed, 2009). also, salinity limits the plant productivity in legumes by hampering the nodulation process by affecting nodule number, nodule size, and fresh nodule mass (elahi et al., 2004). the effect of nacl stress in plants has been studied generally through morphological and physiological responses in plants and conventional visual perception of their variations or changes (elahi et al., 2004; ghosh et al., 2015; sehrawat et al., 2015; muchate et al., 2016; shelke et al., 2017; rahneshan et al., 2018). however, interpretations and conclusions based on conventional approaches are less conclusive because of the complicated nature of morphological and physiological responses and their interrelationships. moreover, traditional data analysis can extract only quantitative data characteristics and does not interpret conceptual descriptions of dependencies among data variables and the underlying reasons (michalski and kaufman, 1997). multivariate analysis tools such as principal component analysis (pca), discriminant analysis (da), pearson’s multiple correlation analysis (mca) enables the accurate analysis and interpretation of vast and complex datasets. these tools adequately analyse and interpret complex interrelationships among parameters in mankar gd et al. (2023). not sci biol 15(2):11365 3 environmental, biological, chemical, and ecotoxicological studies (mujunen et al., 1996; simeonov et al., 2003; singh et al., 2004; sinha et al., 2009a, 2009b; shelke et al., 2017). in the present study, the effects of salt stress levels and exposure time on morpho-physiological, photosynthetic, nodulation, and yield parameters in previously screened salt-tolerant ‘pku-akm 12-28’ and susceptible ‘vbn (gg)3’ mungbean varieties were evaluated. furthermore, the effect of these changes on yield under salinity was also evaluated. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods plant materials, growth, and salt treatment certified and disease-free seeds of mungbean [vigna radiata (l.) r. wilczec] varieties ‘pku-akm 1228’ and ‘vbn (gg)3’ were procured from pulses research unit, dr. panjabrao deshmukh krishi vidyapeeth, akola, and national pulses research centre, tamil nadu agricultural university, respectively. plants were grown in the botanical garden of the modern college of arts, science, and commerce, shivajinagar, pune-5. the potting mixture was prepared from the sandy clay loam soil collected from charholi village in pune district (ms). plants were grown in non-perforated 35 cm × 20 cm plastic pots. each pot contained a 15 kg soil and farmyard manure in a 3:1 ratio. fifteen seeds were sown in each pot. thinning of plants was done fifteen days after sowing to maintain six plants per pot. the salt stress was given to fifteen-day seedlings through hoagland nutrient medium (hoagland and arnon, 1950) containing 0.75, 100, and 125 mm nacl (equivalent to 0.3, 7, 8, and 9 dsm-2 ec, respectively). to maintain the desired ec of the potting mixture, 300 ml respective salt solution was added to each pot on every alternate day until the experiments were concluded. each treatment was replicated in three pots. the following data was collected/analyses were performed on two plants per pot on the 15th, 30th, and 45th day after the salt stress treatments began. morpho-physiological growth analysis the data was collected on the plant morphological parameters that included shoot length (sl), root length (rl), the number of secondary branches (sb), shoot fresh weight (sfw), root fresh weight (rfw), shoot dry weight (sdw), root dry weight (rdw), shoot tissue water content (stwc%), root tissue water content (rtwc%), and leaf area (la) (shelke et al., 2017). photosynthetic pigments analysis chlorophyll content was estimated by arnon’s (1949) method. the carotenoid content was estimated by maclachlan and zalik’s (1963) method. the anthocyanins were estimated by mancinelli’s (1984) method. nodulation parameters analysis after harvesting the pods, plants were uprooted. the number of nodules per plant (nn), size of the nodule (ns), and fresh weight per nodule (nw) were measured (elahi et al., 2004). yield parameters analysis after completion of the plant’s life cycle, plant pod characteristics such as the number of pods per plant (np), fresh weight per pod (fwp), and seed characteristics such as the number of seeds per pod (nsp) and fresh weight per 1000 seeds (fw-1000) were measured (ahmed, 2009). mankar gd et al. (2023). not sci biol 15(2):11365 4 statistical analyses all the experiments were performed with three replicates in a completely randomized block design (crd). the data were presented as a mean ± standard deviation (table 1a-1d). for multivariate modelling, the morpho-physiological dataset consisted of 12 variables, photosynthetic datasets consisted of 5 variables, the nodulation dataset consisted of 3 variables, and the yield dataset consisted of 4 variables. these data sets were subjected to multivariate modelling through principal component analysis (pca) in the past statistical package (hammer et al., 2001). the discriminant analysis (da) was performed in statistica v 10.0 software using the standard, forward stepwise and, backward stepwise modes (sinha et al., 2009a). the correlations between morpho-physiological and photosynthetic, nodulation, and yield parameters of the nacl stressed plants at different salt stress levels and durations were determined using pearson’s correlation method in spps v 20 software (chunthaburee et al., 2015; shelke et al., 2017). results results results results effect of salt stress on morpho-physiological parameters the variations in the morpho-physiological parameters in ‘pku-akm 12-28’ and ‘vbn (gg)3’ at different concentrations and exposure of nacl stress are presented in table 1a. the analysis of variance (anova) showed a differential response of mungbean varieties to nacl. at 15 dpt (day post salt treatment), increase in salt concentration significantly reduced plants sl, rl, la, sb, sfw, rfw, rdw, sdw, stwc and rtwc. noteworthy similar trends were observed for 30 and 45 dpt. at 15 dpt in ‘vbn(gg)3’ sl (18%, 22% and 25%), rl (14%, 18% and 30%), la (33%, 42% and 49%), sb (0%, 5% and 33%), sfw (2%, 23% and 31%), rfw (11%, 40% and 66%), sdw (42%, 56% and 81%), rdw (27%, 40% and 61%), stwc (11%, 12% and 21%), and rtwc (8%, 0% and 9%), were significantly reduced under 75, 100 and 125 mm nacl respectively compared to control. while, at 30 dpt sl (11%, 23%, and 27%), rl (11%, 14%, and 40%), la (17%, 20% and 32%), sb (0%, 13% and 13%), sfw (24%, 25% and 41%), rfw (20%, 33% and 44%), sdw (12%, 58% and 67%), rdw (27%, 43% and 61%), stwc (5%, 13% and 13%), and rtwc (3%, 5% and 8%) were decreased under 75, 100 and 125 mm nacl respectively. further 45 dpt, the salt’s effect was more pronounced resulting in reduced sl (22%, 28%, and 37%), rl (11%, 17%, and 39%), la (39%, 33% and 52%), sb (23%, 28% and 37%), sfw (12%, 27% and 32%), rfw (23%, 32% and 42%), sdw (39%, 62% and 73%) rdw (19%, 71% and 72%), stwc (21%, 32% and 40%), and rtwc (5%, 46% and 42%) under 75, 100 and 125 mm nacl respectively. effect of salt stress on photosynthetic parameters photosynthetic pigment content was affected in both the varieties with increasing salinity and exposure duration (table 1b). at 15 dpt in ‘vbn(gg)3’, chl ‘a’ (26%, 28% and 45%), chl ‘b’ (33%, 40% and 30%), tchl (28%, 32% and 40%), tcc (56%, 35% and 42%), tac (2%, 6% and 9%), was significantly reduced under 75, 100 and 125 mm nacl respectively compared to control. while, at 30 dpt even more reduction in chl ‘a’ (24%, 52% and 60%), chl ‘b’ (25%, 44% and 64%), tchl (24%, 49% and 59%), tcc (26%, 35% and 57%), tac (25%, 36% and 41%) was observed under 75, 100 and 125 mm nacl. at 45 dpt, the salt’s effect was more pronounced and profound reduction in chl ‘a’ (49%, 72% and 74%), chl ‘b’ (55%, 69% and 79%), tchl (51%, 71% and 75%), tcc (57%, 69% and 89%), tac (44%, 59% and 69%) was observed under 75, 100 and 125 mm nacl. effect of salt stress on nodulation parameters increasing salinity and exposure duration also affected nodulation parameters in both varieties (table 1c). at 15 dpt in ‘vbn(gg)3’ nn (52%, 76% and 100%), ns (20%, 37% and 100%), nw (46%, 61% and mankar gd et al. (2023). not sci biol 15(2):11365 5 100%) was reduced significantly under 75, 100 and 125 mm nacl respectively compared to control. while, at 30 dpt, further reduction in nn (28%, 48% and 66%), ns (36%, 58% and 100%), nw (49%, 59% and 100%) was observed. after 45 dpt, the effect was more pronounced and severe reduction in nn (43%, 64% and 87%), ns (20%, 51% and 65%), nw (30%, 47% and 70%) was observed under 75, 100 and 125 mm nacl stress. however, both genotypes differed significantly for nodulation parameters under 75, 100 and 125 mm nacl at 15, 30 and 45 dpt compared with their controls. the ‘pku-akm 12-28’ was less harmed for these parameters than ‘vbn(gg)3’ at all salt concentrations and exposure durations. thus, though both genotypes differed significantly in their responses on morpho-physiological, photosynthetic, and nodulation parameters at all salt concentrations and exposure durations, these parameters were less affected in ‘pku-akm 12-28’ than ‘vbn(gg)3’. mankar gd et al. (2023). not sci biol 15(2):11365 6 table 1a.table 1a.table 1a.table 1a. effect of nacl stress on morpho-physiological traits in vigna radiata varieties ‘pku akm 12-28’ and ‘vbn (gg)3’ variety nacl (mm) exposure duration (days) coding shoot length (cm) root length (cm) number of secondary branches leaf area (cm2) shoot fresh weight (gm) root fresh weight (gm) shoot dry weight (gm) root dry weight (gm) shoot tissue water content (%) root tissue water content (%) pku akm 12-28 0 15 pc0e1 34.60 ± 2.28 23.10 ± 2.61 4.00 ± 0.00 33.94 ± 4.66 6.79 ± 0.59 0.414 ±0.050 1.419 ± 0.126 0.158 ± 0.019 79.09 ± 0.93 61.11 ± 8.80 30 pc0e2 37.20 ± 1.56 28.12 ± 2.37 5.20 ± 1.10 40.34 ± 9.95 8.59 ± 0.88 1.104 ±0.229 2.111 ± 0.202 0.250 ± 0.032 75.08 ± 4.72 76.67 ± 5.45 45 pc0e3 43.93 ± 2.11 31.40 ± 3.86 7.20 ± 0.45 62.34 ± 4.06 9.61 ± 0.92 1.689 ± 0.262 3.650 ± 0.243 0.710 ± 0.027 61.83 ± 3.34 57.33 ± 5.15 75 15 pc1e1 34.76 ± 2.93 28.82 ± 2.00 4.00 ± 0.00 28.97 ± 6.20 6.42 ± 0.89 0.733 ± 0.045 1.287 ± 0.121 0.179 ± 0.021 79.90 ± 2.83 75.52 ± 2.84 30 pc1e2 36.92 ± 2.40 32.16 ± 1.26 4.80 ± 0.45 42.87 ± 6.95 9.26 ± 1.03 1.496 ± 0.153 2.101 ± 0.221 0.430 ± 0.013 77.10 ± 3.49 71.02 ± 3.00 45 pc1e3 41.22 ± 1.26 34.26 ± 2.06 6.40 ± 0.55 49.45 ± 3.06 9.48 ± 0.76 1.787 ± 0.254 3.151 ± 0.156 0.957 ± 0.029 66.59 ± 3.17 45.54 ± 8.41 100 15 pc2e1 33.28 ± 3.25 18.72 ± 2.72 4.40 ± 0.55 28.35 ± 6.48 5.57 ± 1.15 0.486 ± 0.994 1.147 ± 0.292 0.146 ± 0.018 78.14 ± 9.14 69.53 ± 5.96 30 pc2e2 36.54 ± 1.40 26.72 ± 1.16 4.40 ± 0.55 36.06 ± 1.92 7.74 ± 1.77 0.994 ± 0.197 1.183 ±0.197 0.309 ± 0.011 84.24 ± 3.62 68.01 ± 6.25 45 pc2e3 39.06 ± 1.39 27.26 ± 1.47 5.20 ± 0.45 42.24 ± 3.68 8.51 ± 0.87 1.527 ± 0.271 3.064 ± 0.137 0.545 ± 0.025 63.67 ± 4.49 63.14 ± 8.18 125 15 pc3e1 31.32 ± 0.31 19.90 ± 3.63 3.75 ± 0.50 23.71 ± 2.82 4.49 ± 0.78 0.324 ± 0.089 0.995 ± 0.189 0.137 ± 0.035 77.27 ± 6.08 54.42 ±19.73 30 pc3e2 35.66 ± 1.59 21.78 ± 1.20 4.20 ± 0.45 32.11 ± 1.96 6.40 ± 1.20 0.756 ± 0.120 1.295 ± 0.119 0.154 ± 0.012 79.17 ± 4.38 79.12 ± 4.73 45 pc3e3 35.54 ± 1.09 23.26 ± 2.98 5.40 ± 0.55 37.49 ± 1.76 7.69 ± 0.53 1.287 ± 0.173 1.607 ± 0.278 0.274 ± 0.026 78.90 ± 4.75 78.38 ± 3.57 vbn (gg)3 0 15 vc0e1 30.54 ± 1.96 21.20 ± 2.63 3.60 ± 0.55 31.18 ± 2.12 5.80 ± 1.08 0.443 ± 0.061 1.296 ± 0.146 0.146 ± 0.018 77.30 ± 2.76 66.91 ± 3.50 30 vc0e2 34.40 ± 2.90 26.16 ± 1.07 4.60 ± 0.55 37.00 ± 3.56 8.73 ± 0.85 0.969 ± 0.100 1.980 ± 0.178 0.234 ± 0.017 77.02 ± 4.18 75.62 ± 3.61 45 vc0e3 43.92 ± 1.65 28.90 ± 2.14 7.00 ± 0.00 54.99 ± 4.38 9.39 ± 1.72 1.560 ± 0.110 3.554 ± 0.237 0.695 ± 0.019 60.68 ±10.46 55.23 ± 4.07 75 15 vc1e1 24.88 ± 1.04 18.20 ± 1.80 3.60 ± 0.55 21.01 ± 5.61 5.64 ± 1.38 0.394 ± 0.056 0.746 ± 0.061 0.107 ± 0.011 86.14 ± 3.53 72.64 ± 3.05 30 vc1e2 30.54 ± 2.59 23.20 ± 4.82 4.60 ± 0.55 30.65 ± 3.40 6.62 ± 1.03 0.774 ± 0.101 1.747 ± 0.160 0.169 ± 0.014 72.97 ± 5.88 77.92 ± 2.24 45 vc1e3 34.26 ± 0.89 25.64 ± 2.27 5.40 ± 0.55 33.29 ± 2.00 8.25 ± 0.59 1.207 ± 0.191 2.176 ± 0.312 0.562 ± 0.035 73.56 ± 4.04 52.32 ± 9.60 100 15 vc2e1 23.99 ± 1.07 17.30 ± 2.10 3.40 ± 0.55 17.95 ± 1.78 4.46 ± 0.83 0.264 ± 0.035 0.572 ± 0.041 0.087 ± 0.008 86.68 ± 3.51 66.92 ± 3.00 30 vc2e2 26.62 ± 1.61 22.48 ± 2.57 4.00 ± 0.00 29.69 ± 3.20 6.52 ± 0.62 0.644 ± 0.075 0.834 ± 0.071 0.132 ± 0.012 87.07 ± 1.86 79.16 ± 3.41 45 vc2e3 31.72 ± 1.10 23.90 ± 1.70 5.00 ± 0.00 36.92 ± 2.22 6.85 ± 0.92 1.053 ± 0.148 1.339 ± 0.148 0.199 ± 0.018 80.09 ± 3.89 80.86 ± 2.95 125 15 vc3e1 22.94 ± 1.26 14.88 ± 1.54 2.40 ± 0.55 15.89 ± 2.62 3.98 ± 0.82 0.148 ± 0.026 0.240 ± 0.044 0.057 ± 0.013 93.79 ± 1.43 60.58 ± 12.78 30 vc3e2 25.18 ± 1.31 15.56 ± 0.99 4.00 ± 0.00 25.28 ± 2.66 5.18 ± 0.71 0.543 ± 0.120 0.647 ± 0.094 0.092 ± 0.012 87.39 ± 1.86 82.11 ± 5.96 45 vc3e3 27.56 ± 1.34 17.66 ± 2.49 4.40 ± 0.55 26.27 ± 3.19 6.38 ± 0.59 0.899 ± 0.177 0.948 ± 0.111 0.191 ± 0.021 85.12± 1.10 78.31 ± 3.76 mankar gd et al. (2023). not sci biol 15(2):11365 7 table 1b.table 1b.table 1b.table 1b. effects of nacl stress on photosynthetic pigments in vigna radiata varieties ‘pku akm 12-28’ and ‘vbn (gg)3’ the data represented mean ± standard deviation variety nacl (mm) exposure duration (days) coding chlorophyll ‘a’ content (µg/gm fw) chlorophyll ‘b’ content (µg/gm fw) total chlorophyll content (µg/gm fw) total caroteonoid content (µg/gm fw) total anthocyanine content (µg/gm fw) pku akm 12-28 0 15 pc0e1 25.62 ± 0.38 13.19 ± 0.32 39.43 ± 0.53 9.67 ± 0.28 0.479 ± 0.011 30 pc0e2 32.70 ± 0.61 17.02 ± 0.47 50.51 ± 1.10 13.12 ± 0.33 0.843 ± 0.056 45 pc0e3 36.98 ± 2.62 15.99 ± 2.43 53.85 ± 4.91 20.77 ± 0.34 1.383 ± 0.046 75 15 pc1e1 25.54 ± 0.69 13.20 ± 0.83 39.36 ± 1.54 9.15 ± 0.42 0.568 ± 0.010 30 pc1e2 28.73 ± 0.61 14.35 ± 1.00 43.76 ± 1.62 12.56 ± 0.44 0.635 ± 0.037 45 pc1e3 25.06 ± 0.64 11.51 ± 1.08 37.17 ± 1.74 14.52 ± 0.34 0.980 ± 0.065 100 15 pc2e1 26.53 ± 0.73 14.08 ± 0.27 41.25 ± 1.01 7.59 ± 0.37 0.424 ± 0.010 30 pc2e2 26.56 ± 0.69 14.24 ± 0.63 41.45 ± 1.15 11.95 ± 0.50 0.732 ± 0.041 45 pc2e3 19.33 ± 0.72 9.94 ± 0.63 29.74 ± 1.26 9.76 ± 0.41 0.676 ± 0.035 125 15 pc3e1 19.93 ± 0.30 9.11 ± 0.96 29.51 ± 1.20 7.27 ± 0.39 0.458 ± 0.028 30 pc3e2 24.86 ± 0.47 13.87 ± 0.50 39.32 ± 0.82 7.66 ± 0.43 0.660 ± 0.045 45 pc3e3 17.68 ± 0.46 8.19 ± 1.19 26.29 ± 1.64 6.72 ± 0.35 0.738 ± 0.027 vbn (gg)3 0 15 vc0e1 24.25 ± 0.35 12.97 ± 0.74 37.80 ± 1.03 9.43 ± 0.49 0.464 ± 0.036 30 vc0e2 29.41 ± 0.64 16.92 ± 0.63 47.04 ± 1.08 11.76 ± 0.41 0.781 ± 0.039 45 vc0e3 36.08 ± 1.58 15.64 ± 1.24 52.59 ± 2.86 18.77 ± 0.57 1.143 ± 0.035 75 15 vc1e1 18.03 ± 0.70 8.67 ± 0.90 27.13 ± 1.61 4.14 ± 0.25 0.474 ± 0.016 30 vc1e2 22.32 ± 0.57 12.72 ± 1.37 35.58 ± 1.95 8.69 ± 0.57 0.585 ± 0.026 45 vc1e3 18.27 ± 0.74 6.98 ± 0.88 25.68 ± 1.64 8.02 ± 0.50 0.641 ± 0.049 100 15 vc2e1 17.55 ± 0.32 7.71 ± 0.70 25.68 ± 1.03 6.08 ± 0.51 0.490 ± 0.036 30 vc2e2 14.00 ± 0.19 9.41 ± 0.72 23.75 ± 0.91 7.68 ± 0.25 0.495 ± 0.031 45 vc2e3 10.09 ± 0.72 4.78 ± 0.54 15.12 ± 1.13 5.71 ± 0.36 0.466 ± 0.026 125 15 vc3e1 13.28 ± 0.98 9.04 ± 2.42 22.64 ± 1.77 5.49 ± 0.22 0.421 ± 0.026 30 vc3e2 12.96 ± 0.40 6.00 ± 0.64 19.28 ± 0.40 5.04 ± 0.30 0.463 ± 0.019 45 vc3e3 9.46 ± 0.46 3.26 ± 0.41 12.94 ± 0.74 2.04 ± 1.45 0.356 ± 0.020 mankar gd et al. (2023). not sci biol 15(2):11365 8 table 1c.table 1c.table 1c.table 1c. effect of nacl stress on nodulation traits in vigna radiata varieties ‘pku akm 12-28’ and ‘vbn (gg)3’ the data represented mean ± standard deviation table 1d.table 1d.table 1d.table 1d. effect of nacl stress on pod and seed traits in vigna radiata varieties ‘pku akm 12-28’ and ‘vbn (gg)3’ at 45 days after salt treatment the data represented mean ± standard deviation variety nacl (mm) exposure duration (days) codings number of nodules/ plant size of nodule (mm) fresh weight / nodule (mg) pku akm 12-28 0 15 pc0e1 11.00 ± 1.22 2.65 ± 0.32 12.41 ± 1.84 30 pc0e2 14.20 ± 0.84 3.11± 0.54 15.79 ± 0.94 45 pc0e3 25.80 ± 1.30 5.28 ± 0.18 18.24 ± 0.72 75 15 pc1e1 11.00 ± 2.45 2.22 ± 0.25 10.94 ± 1.63 30 pc1e2 12.00 ± 2.24 2.94 ± 0.33 13.22 ± 0.99 45 pc1e3 18.60 ± 2.07 4.34 ± 0.25 16.29 ± 0.89 100 15 pc2e1 9.40 ± 1.82 1.53 ± 0.28 7.40 ± 0.71 30 pc2e2 11.60 ± 2.07 2.68 ± 0.05 10.91 ± 0.57 45 pc2e3 15.80 ± 0.84 3.53 ± 0.28 14.69 ± 1.14 125 15 pc3e1 5.40 ± 0.55 1.23 ± 0.29 6.15 ± 0.16 30 pc3e2 11.00 ± 1.58 1.85 ± 0.27 8.67 ± 0.46 45 pc3e3 12.20 ± 0.84 2.62 ± 0.34 11.71 ± 1.29 vbn (gg)3 0 15 vc0e1 9.20 ± 0.45 2.17 ± 0.18 13.30 ± 1.93 30 vc0e2 11.20 ± 1.30 2.79 ± 0.20 14.09 ± 0.81 45 vc0e3 22.80 ± 0.84 4.89 ± 0.35 17.82 ± 0.72 75 15 vc1e1 4.40 ± 0.55 1.73 ± 0.14 7.11 ± 0.77 30 vc1e2 8.00 ± 0.71 1.78 ± 0.26 7.16 ± 1.30 45 vc1e3 13.00 ± 0.71 3.91 ± 0.44 12.53 ± 0.78 100 15 vc2e1 2.20 ± 0.45 1.37 ± 0.10 5.24 ± 0.56 30 vc2e2 5.80 ± 1.30 1.16 ± 0.29 5.71 ± 0.97 45 vc2e3 8.20 ± 0.84 2.38 ± 0.60 9.44 ± 0.71 125 15 vc3e1 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 30 vc3e2 3.80 ± 0.45 0.00 ± 0.00 0.00 ± 0.00 45 vc3e3 3.00 ± 1.00 1.72 ± 0.21 5.25 ± 0.76 variety nacl (mm) coding number of pods/plant fresh weight /pod (gm) number of seeds/pod fresh weight of 1000 seeds (gm) pku akm 12-28 0 pc1 4.00±0.71 1.21±0.24 11.20±0.84 58.83±7.73 75 pc2 3.20±0.45 1.16±0.10 10.80±0.45 58.60±10.33 100 pc3 2.80±0.45 0.70±0.06 11.20±0.84 59.02±6.35 125 pc4 2.00±0.00 0.41±0.17 7.00±1.58 50.67±7.69 vbn (gg)3 0 vc1 3.80±0.45 0.97±0.07 5.62±2.00 52.12±8.21 75 vc2 2.40±0.55 0.44±0.13 6.02±0.55 37.80±6.39 100 vc3 1.40±0.55 0.28±0.02 4.29±1.00 31.23±4.88 125 vc4 1.20±0.45 0.04±0.01 0.00±0.00 0.00±0.00 mankar gd et al. (2023). not sci biol 15(2):11365 9 effect of salt stress on yield parameters in ‘pku-akm 12-28’ at 45 dpt of 75, 100, and 125 mm nacl stress (table 1d), np was reduced by 20, 30, and 50% respectively, whereas in ‘vbn (gg)3’, these reductions were by 36.84, 63.16, and 68.42% respectively. at 75 mm nacl, fwp was drastically decreased in vbn (gg)3 by 54.34%. as the salt concentration increased to 125 mm nacl, fwp was reduced by 65.72% and 95.46% in ‘pku-akm 12-28’ and ‘vbn (gg)3’, respectively. seed parameters such as nsp and fw-1000 were affected differently in these two mungbean varieties under salinity. at 75 mm nacl, spp and sw-1000 were decreased significantly in ‘vbn (gg)3’ (16 and 22.47% respectively). as the salt concentration increased to 125 mm nacl, spp, and sw-1000 were decreased by 37.50% and 13.86%, respectively in ‘pku-akm 12-28’. moreover, seed formation did not occur in ‘vbn (gg)3’ at 125 mm nacl. thus, ‘pku-akm 12-28’ was less affected by salt stress than ‘vbn(gg)3’. principal component analysis pca was performed on the normalized dataset to evaluate: a) the plant-nacl interactions, b) differences in responses on parameters under nacl stress levels, and c) dependence among variables and the factors that insuence them. pca of the entire morphophysiological data set (table 1a) produced ten pcs, with the first two pcs explaining about 90.69% of the total variance in the morphophysiological data set. the loadings and scores of the frst two pcs (pc1 vs. pc2) are presented in figure 1. the frst two pcs represent maximum variance and resect the main groupings in the data set. pc1 accounting for 80.87% of the total variance was positively correlated (loading > 90%) with sl, rl, sb, la, sfw, rfw, sdw, and rdw. pc2 accounting for 9.82% of the total variance was positively correlated (loading > 86%) with rtwc. the scores show a visible differentiation between the tissue responses at different salinity levels and exposure time in both varieties. scores of the pc1 in pku-akm 12-28 were higher than those of ‘vbn(gg)3’. the score in pc1 was highest in ‘pku-akm 12-28’ for 75 mm nacl exposure for 45 days. on the contrary, it was least in ‘vbn (gg)3’ exposed to all nacl concentrations for 45 days. it indicates ‘pku-akm 12-28’ has a higher tolerance level than ‘vbn (gg)3’ under salinity. thus, differences in changes in the morphological variables under all salt concentrations and exposure durations suggest intrinsic differences in two mungbean varieties. figure 1.figure 1.figure 1.figure 1. pca scores and loadings of the frst two pcs obtained from the morpho-physiological dataset of mungbean varieties mankar gd et al. (2023). not sci biol 15(2):11365 10 pca of the photosynthetic dataset (table 1b) gave five pcs, with the first two pcs explaining 98.03% of the total variance. figure 2 shows the photosynthetic data sets' loadings and scores of the frst two pcs (pc1 vs. pc2). the pc1 (86.76% variance) showed high positive loadings (>90%) on chl-a, chl-b, tchl, and tcc and positive loadings (>83%) on tac. on the other hand, the second pc accounted for 11.27% of the total variance. the corresponding scores in the plot, along with the loadings, suggest that at all nacl concentrations for a short exposure (e1, 15 days), chl-a, chl-b, tchl, and tcc were affected and reduced by a greater extent in ‘vbn (gg)3’ as compared to ‘pku-akm 12-28’. however, both varieties showed increased tac at 75 mm nacl and decreased at 125 mm nacl. moderate exposure (e2, 30 days) brought significant changes in chl-a, chl-b, tchl, and tcc. these parameters were more affected in ‘vbn (gg)3’ than ‘pku-akm 12-28’. likewise, the tchl was more affected in ‘vbn (gg)3’ at 125 mm nacl at moderate exposure (e2). exposure for a longer duration (e3, 45 days) and higher salt concentration was associated with a remarkable reduction in all photosynthetic parameters in ‘vbn (gg)3’ than ‘pku-akm 12-28’. hence, scores of ‘vbn (gg)3’ (vc1e3, vc2e3, vc3e3) lay more on the negative side as compared to scores of ‘pku-akm 12-28’ (pc1e3, pc2e3, pc3e3) for pc1. moreover, the scores of ‘vbn (gg)3’ were more negative compared to ‘pku-akm 12-28’ at all salt concentrations and exposure durations. it indicates that ‘pku-akm 12-28’ has a higher salinity tolerance than ‘vbn (gg)3’. figure 2.figure 2.figure 2.figure 2. pca scores and loadings of the frst two pcs obtained from the photosynthetic dataset of mungbean varieties pca of nodulation dataset (table 1c) yielded three pcs, with the first two pcs explaining 98.26% of the total variance. the loadings and scores of the frst two pcs (pc1 vs. pc2) of the nodulation data set are given in (figure 3). it may be noted that pc1 (95.11% variance) showed high positive loadings (>97%) on all nodulation parameters studied in the present investigation. on the other hand, the second pc accounted for 3.15% of the total variance. the corresponding scores in combination with the loadings suggested that at short exposure (e1), at 75 mm nacl, nn and nw was drastically decreased in ‘vbn (gg)3’ compared to ‘pkuakm 12-28’. moreover, at 125 mm, nodules were not formed in ‘vbn (gg)3’ at a short exposure period. hence, scores of ‘vbn (gg)3’ (vc1e1, vc2e1, vc3e1) lie more on the negative side compared to scores of ‘pku-akm 12-28’ (pc1e1, pc2e1, pc3e1) for pc1. however, the moderate exposure (e2) brought significant changes in nn, ns, and nw. the ns and nw were negligible in ‘vbn (gg)3’ compared to ‘pkuakm 12-28’. exposure to higher duration (e3) and higher salt concentration was associated with remarkable changes in nn, ns, and nw, which was more reduced in ‘vbn (gg)3’ than ‘pku-akm 12-28’. thus, ‘pkumankar gd et al. (2023). not sci biol 15(2):11365 11 akm 12-28’ showed a higher tolerance level than vbn (gg)3 under saline conditions, resulting in better nodulation. figure 3.figure 3.figure 3.figure 3. pca scores and loadings of the frst two pcs obtained for the nodulation dataset of mungbean varieties at 45 days exposure time (e3), pca was performed on the normalized yield dataset (table 1d) that gave four pcs, with the first two pcs explaining about 97.01% of the total variance in the yield data set. the loadings and scores of the frst two pcs (pc1 vs. pc2) of yield data set given in (figure 4) the pc1 (87.48%) showed high positive loadings (>91%) on all yield parameters studied in the present investigation. on the other hand, the second pc accounted for 9.53% of the total variance. the corresponding scores in combination with the loadings suggested that at more prolonged exposure (e3), salinity influenced all yield parameters as salt concentration increased. however, at 75 and 100 mm nacl, all yield parameters were significantly reduced in ‘vbn (gg)3’ than ‘pku-akm 12-28’. hence, the scores of (vc3 and vc2) and (pc2 and pc3) are present at negative and positive quadrant with respect to pc1. in contrast, a higher salt concentration (125 mm) was associated with a remarkable reduction in all yield parameters in ‘vbn (gg)3’. hence, the score (vc4) is present in the negative quadrant with respect to pc1 and pc2. thus, for yield output, ‘pku-akm 12-28’ has a higher salinity tolerance than ‘vbn (gg)3’. mankar gd et al. (2023). not sci biol 15(2):11365 12 figure 4.figure 4.figure 4.figure 4. pca scores and loadings of the frst two pcs obtained for the yield dataset of mungbean varieties discriminant analysis variations in morpho-physiological, photosynthetic, nodulation, and yield parameters at different nacl concentrations in mungbean varieties were further investigated through discriminant analysis (da). discriminant analysis at different nacl concentrations the category variables (y) were four concentrations of nacl to which mungbean varieties were exposed. standard, forward and backward stepwise modes of da were performed, and the discriminant functions (dfs) (table 2a) and classification matrices were obtained (table s1a). for morphophysiological parameters, the standard and forward stepwise da constructed dfs that included all ten parameters and six parameters, respectively, and rendered the corresponding cms assigning 91.66% and 83.83% cases correctly. da with forward stepwise mode includes variables obtained from more significant to insignificant changes. in contrast, backward stepwise mode removes the beginning with less significant changes until no considerable changes are observed. forward stepwise da showed rl, la, rfw, sfw, sdw, and sl. thus, the da results suggest that rl, la, rfw, sfw, sdw, and sl are the most signifcant parameters to differentiate among the four sets of the plant responses observed at the four nacl-induced stress levels. both ca and da identified morphological parameters that significantly changed under different salt stress levels. the box and whisker plots of selected parameters (forward step mode) showing different responses identified by da are presented in (figure s1a). all the chosen parameters showed observable variations in morpho-physiological characters at different nacl concentrations. the rl and rfw showed a similar variation pattern that increased at 75 mm nacl and again decreased at 100 and 125 mm nacl. la, sfw, sdw, and sl also showed a similar pattern of variation. for photosynthetic parameters, the standard and forward stepwise da modes constructed dfs (table 2b) that included all five and only one parameters, respectively, and rendered the corresponding cms (table s1b), assigning 62.50% and 50% cases correctly. these results suggest that chla is a signifcant parameter to discriminate the four sets of the plant responses corresponding to the four nacl concentrations. figure s1b shows box and whisker plots of the selected parameters showing responses at different nacl concentrations identified by da. chl ‘a’ showed significant variation under different salinity levels. for nodulation parameters, the standard and forward stepwise da modes constructed dfs (table 2c), including all three, and one parameter, respectively, and rendered the corresponding cms (table s1c), assigning 50% and 58.33% cases correctly. thus, the da results suggest that the nw variable is a signifcant parameter to discriminate between the four sets of the plant responses corresponding to different nacl concentrations. as identified by da, box and whisker plots of selected parameters showing different responses mankar gd et al. (2023). not sci biol 15(2):11365 13 to nacl concentration are given in (figure s1c). nw showed variations in nodulation under salinity at different nacl concentrations. for yield parameters, the standard and forward stepwise da modes constructed dfs (table 2d), including all four, and two parameters, respectively, and rendered the corresponding cms (table s1d), assigning 87.50% and 75% cases correctly. thus, the da results suggest that np and nsp variables are signifcant parameters to discriminate between the four sets of the plant responses corresponding to different nacl concentrations. as identified by da, box and whisker plots of selected parameters showing different nacl concentration responses are presented in (figure s1d). np and nsp showed variations in nodulation changes under salinity at different nacl concentrations. table 2atable 2atable 2atable 2a. classifcation functions for discriminant analysis (da) of nacl stress levels and morphophysiological parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficient a c0 c1 c2 c3 sl 24.09 24.31 25.47 26.84 rl 4.06 5.25 3.38 1.98 sb 95.04 95.70 92.42 90.80 la -11.30 -12.90 -12.29 -12.48 sfw -75.71 -82.88 -87.28 -93.62 rfw -363.18 -325.42 -296.43 -261.47 sdw 711.44 692.20 690.40 691.55 rdw -223.60 -168.99 -196.43 -211.50 stwc 54.47 53.54 53.99 54.67 rtwc 4.27 4.68 4.44 4.32 constant -2902.09 -2804.36 -2804.61 -2837.42 forward da mode rl 1.051 1.817 0.554 -0.429 la 2.537 1.136 1.429 1.125 rfw -198.965 -144.388 -128.863 -100.970 sfw 40.750 31.071 28.189 23.523 sdw 19.767 13.535 5.836 -0.906 sl -0.580 -0.062 0.811 1.788 constant -146.082 -98.446 -87.125 -72.126 backward da mode no variables in the model a discriminant function coefficient for different concentrations of nacl table 2b.table 2b.table 2b.table 2b. classifcation functions for discriminant analysis (da) of nacl stress levels and photosynthetic parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficient a c0 c1 c2 c3 chl-a -156.233 -25.4380 -223.567 44.4849 chl-b -153.414 -25.3860 -218.584 43.5800 chl-t 153.607 25.6656 218.931 -42.9237 tcc -1.034 -1.4601 -1.180 -2.1963 tac 14.550 18.6754 16.735 28.7089 constant -18.310 -11.7295 -9.381 -9.6374 forward da mode mankar gd et al. (2023). not sci biol 15(2):11365 14 chl-a 1.0141 0.7560 0.62507 0.53799 constant -17.0231 -10.0771 -7.32761 -5.78752 backward da mode no variables in the model adiscriminant function coefficient for different concentrations of nacl table 2c.table 2c.table 2c.table 2c. classifcation functions for discriminant analysis (da) of nacl stress levels and nodulation parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficients a c0 c1 c2 c3 nn -0.2851 -0.47787 -0.29071 -0.02407 ns -2.2465 0.52571 -0.50152 -0.90021 nw 2.1207 1.30428 1.17356 0.67328 constant -11.4343 -6.76885 -4.79501 -2.54175 forward da mode nw 1.1444 0.83974 0.66667 0.39683 constant -10.1268 -6.09233 -4.35242 -2.43724 backward da mode no variables in the model adiscriminant function coefficient for different concentrations of nacl table 2d.table 2d.table 2d.table 2d. classifcation functions for discriminant analysis (da) of nacl stress levels and yield parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficients a c0 c1 c2 c3 np 41.9003 26.3729 18.4692 19.5117 fwp -6.7078 -8.3712 -14.2649 -13.9229 nsp -4.0862 -2.0917 -0.9653 -1.2877 fws-1000 -0.1388 -0.0944 -0.0283 -0.0428 constant -58.4046 -23.8890 -12.9079 -12.6333 forward da mode np 38.9620 23.5019 14.9170 15.8907 nsp -4.7786 -2.7054 -1.5894 -1.9453 constant -57.2681 -22.9129 -10.8943 -10.6946 backward da mode no variables in the model adiscriminant function coefficient for different concentrations of nacl discriminant analysis at different salt exposure periods the effect of the duration of salt stress in the mungbean varieties was also analysed through discriminant analysis. the category variables (y) were the three exposure durations (e1, e2, and e3). for morphophysiological parameters, the standard and forward stepwise da modes constructed dfs (table 3a) that included all ten parameters of standard da mode, three parameters of forward da mode, and two parameters backward da mode and rendered the corresponding cms assigning 100%, 95.83%, and 87.50% cases correctly (table s2a). backward stepwise da showed that the two variables rl and rfw followed rfw, mankar gd et al. (2023). not sci biol 15(2):11365 15 rl and, rtwc in the forward stepwise da. the box whisker plots of selected parameters showing responses at three durations of salt stress are given in figure s2a. the rl and rfw showed observable variations in the plants exposed to nacl stress for different durations. these results suggest higher salt stress tolerance in ‘pkuakm 12-28’ than ‘vbn (gg)3’. for photosynthetic parameters, the standard, forward, and backward stepwise da modes constructed dfs, including all 5, 3, and 2 parameters, respectively (table 3b) and rendered the corresponding cms (table s2b), assigning 87.50%, 87.50%, and 79.16% cases correctly. thus, the da results revealed that tac and chl-t (figure s2b) variables are the most critical parameters to distinguish the plant’s responses at different salt stress durations for nodulation parameters, da modes constructed dfs, including all 3 and 2 parameters, respectively (table 3c), and rendered the corresponding cms (table s2c), assigning 70.83% and 70.83% cases correctly. thus, the da results revealed that ns and nw (figure s2c) variables are the most critical parameters to discriminate between the three sets of exposure duration. table 3atable 3atable 3atable 3a. classifcation function for discriminant analysis (da) of nacl stress exposure duration and morpho-physiological parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficient a e1 e2 e3 sl 27.82 27.24 26.61 rl 14.59 13.26 11.85 sb 76.85 78.42 85.09 la -16.73 -16.59 -16.69 sfw -106.03 -102.05 -103.13 rfw -412.56 -383.93 -336.05 sdw 679.07 668.86 663.07 rdw 60.77 66.44 39.23 stwc 53.60 53.08 52.76 rtwc 7.25 7.45 6.98 constant -2896.19 -2863.60 -2803.71 forward da mode rfw -52.9685 -24.8113 7.9188 rl 4.7909 3.7403 2.007 rtwc 1.2531 1.4998 1.3714 constant -80.3519 -92.8168 -76.9834 backard da mode rl 4.0180 2.8152 1.1612 rfw -60.4579 -33.7754 -0.2775 constant -29.6968 -20.2488 -16.3136 adiscriminant function coefficient for different exposure time to nacl mankar gd et al. (2023). not sci biol 15(2):11365 16 table 3b.table 3b.table 3b.table 3b. classifcation function for discriminant analysis (da) of nacl stress exposure duration and photosynthetic parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficient a e1 e2 e3 tcc -2.0453 -2.150 -1.7515 tac 15.5846 28.852 43.5428 chl-t 77.6689 98.227 89.2248 chl-b -76.3725 -95.990 -89.2785 chl-a -79.1170 -100.831 -91.1556 constant -8.8371 -11.723 -10.9816 forward da mode tac -3.98361 8.32545 26.84189 chl-b 1.07203 2.03613 -0.19708 chl-a -0.05132 -0.75577 -0.21431 constant -5.50456 -8.05605 -8.55061 backward da mode tac -5.89820 3.58689 26.82064 chl-t 0.33578 0.21606 -0.20508 constant -5.22112 -6.32339 -8.55060 adiscriminant function coefficient for different exposure time to nacl table 3c.table 3c.table 3c.table 3c. classifcation function for discriminant analysis (da) of nacl stress exposure duration and nodultion parameters in the mungbean varieties under salinity standard da mode linear discriminant functions for groups coefficients a e1 e2 e3 nn -0.22221 0.03228 -0.27848 ns 0.18621 -0.12711 7.62617 nw 0.54314 0.42797 -0.75725 constant -2.64157 -3.14643 -7.67020 forward da mode ns -0.68209 -0.00097 6.53798 nw 0.50589 0.43338 -0.80393 constant -2.52641 -3.14400 -7.48934 backward da mode no variables in the model adiscriminant function coefficient for different exposure time to nacl multiple correlations variations in the morphophysiological, photosynthetic and nodulation parameters in mungbean varieties exposed to different levels and durations of nacl stress were evaluated through pearson’s correlation (table s3a and figure 5). variation in sl was positively correlated with la (r=0.91**), sfw (r=0.85**) and sdw (r=0.89**) while variation in rl was positively correlated with sb (r=0.75**), sfw (r=0.89**). the sb showed a positive correlation (r=0.90**) with la, rfw, and sdw. la was positively correlated with sfw (r=0.90**) and sdw (r=0.93**). furthermore, sfw was positively correlated with rfw (r=0.93**). very high positive correlations were observed among variations in chl-a, chl-b, and tchl. variation in tcc was positively correlated with chl-a (r=0.88**), chl-b (r=0.76**) and tchl (r=0.85**). the variation in tac was positively correlated (r=0.90**) with tcc. the photosynthetic parameters significantly correlated with sl, mankar gd et al. (2023). not sci biol 15(2):11365 17 rl, sb, la, sfw, rfw, sdw, and rdw. more than (r=0.90**) strong positive correlation was observed in variation of nn, ns, and nw under salinity. nn, ns, and nw were positively correlated with all morphological and photosynthetic parameters except rtwc and stwc, which are negatively correlated. figure 5.figure 5.figure 5.figure 5. pearson’s correlation among variations in the morpho-physiological, photosynthetic and nodulation parameters in mungbean plants exposed to different concentrations nacl for different durations. (correlations significant at p<0.05 are boxed.) at 45 days (e3) exposure time (table s3b and figure 6), the variations in np and fwp were correlated significantly positively with each other by more than (r=0.90**) under salt stress. while variation in nsp and sw-1000 was significantly positively correlated with each other by more than (r=0.90**) under salt stress. variations in seed parameters np and fwp were significantly positively correlated with seed parameters by more than 72 and 80% under salt stress, respectively. moreover, more than (r=0.90**) positive correlation was observed in nn, ns, nw, np and fwp. variation in nsp showed positive correlation with variation in nn (r=0.75*), ns (r=0.68), nw (r=0.80*), np (r=0.71*) and fwp (r=0.80*) under salinity. moreover, variation in fw-1000 was positively correlated with nn (r=0.83**), ns (r=0.75*), nw (r=0.90**). changes in all pod and seed parameters showed a significant positive correlation with changes in morphological, photosynthetic, and nodule parameters except rtwc and stwc, which were negatively correlated under salinity. thus, a decrease in morpho-physiological, photosynthetic, and nodulation parameters directly affect yield parameters. mankar gd et al. (2023). not sci biol 15(2):11365 18 figure 6. figure 6. figure 6. figure 6. pearson’s correlation among variations in the morpho-physiological, photosynthetic and nodulation and yield parameters in mungbean plants exposed to different concentrations nacl at 45 days after salt treatment. correlation is significant at p<0.05 are boxed. discussiondiscussiondiscussiondiscussion soil salinity limits the plant’s growth and development through osmotic and ionic stress and reduces productivity (arif et al., 2020). mungbean [vigna radiata (l.) r. wilczek], one of india's economically important and significant dietary crop plants is susceptible to salt stress (ghosh et al., 2015). its production in the last decades was influenced mainly by its susceptibility to different biotic and abiotic stresses at various stages of growth, including the soil salinity (sehrawat et al., 2015). the salinity equivalent to about 50 mm nacl can reduce the yield by more than 60% (abd-alla et al., 1998). it is also suspected that increasing soil salinity will result in ∼50% loss of arable land by the mid-21st century (hasanuzzaman et al., 2012). soil salinity mainly alters various morphological, photosynthetic, nodulation, and yield parameters, the extent of which varies with severity and duration of stress, and ultimately limits crop productivity (elahi et al., 2004; munns 2005; ahmed 2009; shelke et al., 2017). differential responses to salinity at the varietal level were observed for morphology, photosynthesis, nodulation, and yield (rao et al., 2002; singla and garg, 2005; kumar and singh, 2012; chunthaburee et al., 2015; sarabi et al., 2016; shelke et al., 2017). therefore, the present study was aimed to investigate morpho-physiological, photosynthetic, nodulation, and yield modulation in two mungbean varieties, ‘pkv akm 12-28’ and ‘vbn (gg)3’, under various salt regimes and exposure durations. soil salinity has significantly affected the morphological parameters such as shoot and root length, total biomass, plant height, and leaf growth in many crops (dolatabadian et al., 2011; morales et al., 2012). in the present investigation, modulations in sl, rl, sb, sfw, rfw, sdw, rdw, stwc%, rtwc%, and la with the increasing nacl concentrations of 75, 100, and 125 mm were observed. principal component analysis (pca) of the morphological dataset suggests that the parameters with significant loadings in the first principal component (pc1) are related to plant stress and may be considered ‘stress factor.’ a close association of sl, rl, sb, la, sfw, rfw, sdw, and rdw showed a nearly 90% positive correlation with pc1, which indicates a reduction in these parameters under salinity. pca finds structure in a multivariate dataset, identifies the most relevant parameters, uncovers the variance of a large dataset of inter-correlated variables and transform them mankar gd et al. (2023). not sci biol 15(2):11365 19 into a smaller set of (uncorrelated) independent variables (principal components), and can discriminate samples of diverse biological groups (singh et al., 2004; sinha et al., 2009b; 2009a; chunthaburee et al., 2015; shelke et al., 2017). a simultaneous interpretation of the scores and loadings suggests significant changes in morphological parameters at all levels of salt stress. however, the plant’s defense system is suppressed at higher salt concentrations and more prolonged exposures. it was observed that ‘vbn(gg)3’ is more affected under salinity stress than ‘pku-akm 12-28’. the reduction sl, rl, sb, la, sfw, rfw affects the absorption and transport of water and nutrient allocation from root to shoot, ultimately affecting plants' growth and development (satti and lopez, 1994; sherif et al., 2007). our results are in line with those of (kamrul et al., 2018) and (rahman et al., 2016). the da is used to identify the variables which discriminate between two or more naturally occurring groups. it constructs a discriminant function (df) for each group by analysing raw data (singh et al., 2004; sinha et al., 2009a; 2009b). in the present investigation, the da results indicate rl, la, rfw, sfw, sdw, and sl to be the most significant parameters to discriminate the four sets of the plant responses corresponding to four concentrations of nacl. in contrast, the rl and rfw were the most critical parameters to distinguish among the three levels of stress exposure durations. salinity impairs the synthesis of plant pigments (taïbi et al., 2016). also, it reduces photosynthesis either due to a reduction in green pigments or inhibition of their synthesis (najar et al., 2019). in pca, a highly positive correlation of chl-a, chl-b, chl-t, tcc, and tac loadings to pc1 indicate that these photosynthetic parameters are closely related and affected under the increasing concentration of nacl. the distribution pattern of scores and loading plots suggests a remarkable reduction in the photosynthetic parameters during moderate and more prolonged salt stress exposures. during the moderate exposure duration (30 days), significant induction in the defense mechanism was observed, worsened by prolonged exposure of 45 days. these results suggest an impaired stress defense mechanism with increasing levels of salt stress. these results align with those in p. vulgaris (turan et al., 2007) and vigna subterranean (taffouo et al., 2010). an increase in the anthocyanin content was observed in the present investigation at a low salinity level. it could induce an active protective response under saline stress (chutipaijit et al., 2009). salinity also affected carotenoid contents in both varieties. these results corroborate with reports in p. vulgaris (gadallah, 1999) and maize and wheat genotypes (singh et al., 2008). the da results indicate chl-a to be the most significant attribute to discriminate the four sets of the plant responses corresponding to four concentrations of nacl stress. these results also reveal the tac and chl-t as the most critical parameter to discriminate the three levels of stress exposure durations. in the present study, the photosynthetic pigments viz., chlorophyll a, b, and total chlorophyll, carotenoid, and anthocyanins decreased with increasing salinity. a more significant reduction was observed in ‘vbn (gg)3’ as compared to ‘pku akm 12-28’. a reduction in nodulation parameters was observed with increasing salinity. leguminous plants can fix atmospheric nitrogen through symbiotic association with soil bacteria (rhizobium spp.) which form nodules on the roots of these plants. it is essential to understand the optimum conditions required for nitrogen fixation to provide full benefits to the plant (kijne et al., 1995). legume-rhizobium symbiosis may get affected under salinity by inhibiting the bacterial infection process, reducing the survival of rhizobia, disrupting nodule development and function, or decreasing plant growth (singleton and bohlool, 1984). in the pca analysis of the nodulation dataset, a close association of nn, ns, and nw was observed in pc1, which is indicative of a decrease in nodule number, nodule size, and nodule weight under nacl stress. scores indicated that salinity significantly affects plants at moderate and highest salinity and exposure time. however, it was affected more in ‘vbn (gg)3’ than ‘pku-akm 12-28’. these results corroborate earlier reports in other plant species. (elsheikh and wood, 1995) reported adverse effects of salinity on growth and nodulation in soybean and that nodulation was more sensitive than plant growth under saline conditions. a significant reduction was observed in the nodule number, nodule size, and nodule biomass under salinity in soybean (singleton and bohlool, 1984), sesbania sesban (mahmood et al., 2008). mirza and tariq (1993a,1993b) also reported adversely mankar gd et al. (2023). not sci biol 15(2):11365 20 affected nodulation of cicer arietinum and trifolium alexandrinum under salt stress. the da results in the present investigation indicate nw to be the most significant parameters to discriminate between the four sets of the plant responses corresponding to four concentrations of nacl stress. these results also reveal that ns and nw are the most critical parameters to discriminate the three levels of stress exposure durations. our results revealed a decrease in yield (pod and seed) parameters as salt concentration increased. salinity adversely affects the economic yield of the crop (sarin, 1975). reduced pod number may be one of the main parameters to measure the quantitative yield. pca revealed that studied yield parameters had a close association with each other and showed more than 90% positive correlation with pc1 under salinity, thus indicating that all these parameters are significantly reduced under salinity. furthermore, nodulation in ‘vbn (gg)3’ was affected more than ‘pkv-akm 12-28’. gill (1979) observed similar results in barley yield under salinity. reduction in yield under salinity has been reported in many crops such as rice, cotton, bean, barley, and wheat (keating and fisher, 1985). reduced dry matter and grain yield were reported in sorghum cultivars by maas et al. (1986). elahi et al. (2004) also reported a reduction in pod number and pod fresh mass under increasing salinity levels in mungbean. the da results indicate np and nsp to be the most significant parameters to discriminate between the four sets of the plant responses corresponding to four concentrations of nacl stress. furthermore, mca revealed a significant positive correlation between photosynthetic and growth parameters. taiz and zeiger (1998) have suggested that plant growth depends on photosynthesis. therefore, environmental stresses affecting photosynthesis, in turn, reduce growth. fisarakis et al. (2001) remarked that a decline in photosynthesis under salinity inhibits vegetative growth. significant positive correlations were observed among photosynthetic and yield parameters. less green leaves, leaf expansion, production, and senescence result in less photosynthetic activity, which may be why the yield is reduced under salinity (ahmed, 2009). there was observed a positive correlation between nodulation and yield parameters. it indicates that the effects of salinity on nodulation can ultimately reduce yield and production. our result corroborates with the observations of rao et al. (2002), who suggested that the grain production in legumes is reduced because of their low salt tolerance in combination with the high sensitivity of the symbiotic nitrogen fixation process under stress. applying a multivariate modelling technique to analyse the effects of salt stress on morphophysiological, photosynthetic nodulation, and yield parameters in two mungbean varieties thus demonstrated the grouping of variables and their interrelationship. this technique also identified significant differences in variables responsible for differential behaviour. it also identified significant parameters responsible for differential behaviour. such an interpretation is not possible by using conventional methods. conclusionsconclusionsconclusionsconclusions these analytical tools revealed differential patterns for morpho-physiological, photosynthetic, nodulation, and yield changes in the mungbean varieties ‘pku-akm 12-28’ and ‘vbn(gg)3’. these tools extracted the patterns of variations in significant morpho-physiological, photosynthetic, nodulation, and yield parameters and their inter-relationships under nacl stress. the multivariate modelling approach identified morpho-physiological (rl, la, rfw, sfw, sdw, sl, rl, rfw) and photosynthetic (chl-a, chl-t, and tac), nodulation (nn, ns, and nw), and yield (np, fwp, nsp and fw-1000) as critical parameters that can discriminate tolerant varieties. this analysis also identified positive correlations among yield and other morpho-physiological, photosynthetic, nodulation parameters. moreover, it also revealed the level of salt tolerance in selected mungbean varieties and confirmed ‘pku-akm 12-28’ as salt-tolerant and ‘vbn(gg)3’ as salt susceptible variety. this multivariate modelling approach can be used to understand the complex datasets on nodulation and yield, their interrelationships, and visualization of relationships among other variables. mankar gd et al. (2023). not sci biol 15(2):11365 21 authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions gdm: performed the experiments, collected and analysed the data and prepared draft manuscript, urw: analysed data and prepared draft manuscript, kbr: performed the experiments and collected data, dbs: analysed data and prepared draft manuscript, psm and mla: analysed data and prepared draft manuscript, tdn: designed the experiments and rbb: designed the experiments, analysed the data, and finalized the manuscript. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements we are grateful to dr. neeta m. patil, head, department of botany, and principal dr. r. s. zunjarrao, for providing the required research infrastructure. this work was supported by council of scientific and industrial research, new delhi, ms, india, by awarding the csir-srf fellowship to mr. ganesh mankar (grant number08/658(0001)/2017-emr-i). conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences abd-alla mh, vuong td, harper je (1998). genotypic differences in dinitrogen fixation response to nacl stress in intact and grafted soybean. crop science 38:72-77. https://doi.org/10.2135/cropsci1998.0011183x003800010013x. ahmed s (2009). effect of soil salinity on the yield and yield components of mungbean. pakistan journal of botany 41:263-268. ali m, gupta s (2012). carrying capacity of indian agriculture: pulse crops. current science 102:874-881. arif y, singh p, siddiqui h, bajguz a, hayat s (2020). salinity induced physiological and biochemical changes in plants: an omic approach towards salt stress tolerance. plant physiology and biochemistry 156:64-77. https://doi.org/10.1016/j.plaphy.2020.08.042 arnon di (1949). copper enzymes in isolated chloroplasts. polyphenoloxidase in beta vulgaris. plant physiology 24:115. https://doi.org/10.1104/pp.24.1.1 chunthaburee s, dongsansuk a, sanitchon j, pattanagul w, theerakulpisut p (2015). physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage. saudi journal of biological sciences 23:467-477. https://doi.org/10.1016/j.sjbs.2015.05.013 chutipaijit s, cha-um s, sompornpailin k (2009). differential accumulations of proline and flavonoids in indica rice varieties against salinity. pakistan journal of botany 41:2497-2506. dhingra kk, dhillon ms, grewal ds, sharma k (1991). performance of maize and mungbean intercropping in different planting patterns and row orientations. indian journal of agronomy 36:207-212. dolatabadian a, modarres ss, ghanati f (2011). effect of salinity on growth, xylem structure and anatomical characteristics of soybean. notulea scientia biologicae 3:41-45. https://doi.org/10.15835/nsb315627 mankar gd et al. 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(2023). not sci biol 15(2):11365 25 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. (microsoft word 11353 nsb ch\341vez-mendoza 2023.03.17.docx) received: 20 sep 2022. received in revised form: 08 mar 2023. accepted: 15 mar 2023. published online: 17 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania chávez-mendoza c and sánchez e (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11353 doi:10.15835/nsb15111353 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae antioxidant capacity and nutraceutical compounds content of six antioxidant capacity and nutraceutical compounds content of six antioxidant capacity and nutraceutical compounds content of six antioxidant capacity and nutraceutical compounds content of six common bean (common bean (common bean (common bean (phaseolus vulgaris phaseolus vulgaris phaseolus vulgaris phaseolus vulgaris l.l.l.l.) varieties ) varieties ) varieties ) varieties harvested in morelos, mexicoharvested in morelos, mexicoharvested in morelos, mexicoharvested in morelos, mexico celia chávez-mendoza, esteban sánchez* centro de investigación en alimentación y desarrollo a. c. av. cuarta sur #3820. fracc. vencedores del desierto, c.p. 33089, delicias, chihuahua, méxico; celia.chavez@ciad.mx; esteban@ciad.mx (*corresponding author) abstractabstractabstractabstract common bean is considered one of the most important legumes in the world. it is the main source of protein, calories, b vitamins, minerals, polyphenols and other elements, which collectively give it a high nutraceutical value. in mexico a great agrobiodiversity exists in the production of this grain, which implies the need to generate information regarding its nutritional quality as a tool to apply future genetic improvement programs. the purpose of this study was to characterize the antioxidant capacity (ac) and nutraceutical content of six bean varieties produced in morelos state, mexico. grain morphometric characteristics, color (l, a*, b* chroma and °hue), nutritional quality, ac (dpph), nutraceutical compounds content, micro and macro nutrients were determined. a significant effect (p < 0.05) of variety on almost all the variables evaluated except for phytic acid, p, k, ca, c, s and h was observed. lower lightness was obtained in varieties with darker colors such as ‘negro’/102 (24.96), ‘negro’/104 (26.85) and ‘sangre de toro’ (32.41) and higher lightness in lighter colored varieties such as ‘peruano’ bean (69.21), ‘pinto’ (65.94) and ‘flor de mayo’ (50.14). nutritional and nutraceutical quality of the latter genotype stood out, as it had the highest crude fiber content (5.71 %), total phenols (4.24 mg gae g-1), flavonoids (1.99 mg ce g-1), ac (96.76% inhibition), and a high protein content (23.29%). results also exhibited significant correlation (p < 0.05) between total phenols and flavonoids with ac. it is concluded that the nutritional characterization carried out on bean varieties from important producing areas in mexico provide a valuable database for genotype selection with high functional and nutritional character, either to be grown for direct consumption, future biofortification or breeding programs. keywords:keywords:keywords:keywords: antioxidant capacity; bioactive compounds; micronutrients; mexican bean; morelos introductionintroductionintroductionintroduction nutraceutical products are defined as chemical or biological substances that can be found as natural components of food or added to it, and that are particularly beneficial, both in disease prevention and in improvements in physiological functions of the organism. the consumer's and the general population's interest in obtaining optimal diets to maintain good health and prolong lifespan has led to an increase in natural food markets in which this type of product has priority (pérez, 2006). common bean is considered one of the most important legumes in the world. it is the main source of protein, calories, b vitamins, minerals, polyphenols https://www.notulaebiologicae.ro/index.php/nsb/index chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 2 and other elements, which collectively give it a high nutraceutical value (herrera-hernández et al., 2018). this legume is considered a functional food and has gained great interest for study due to its high content of bioactive compounds such as enzyme inhibitors, lectins, phytates, oligosaccharides and phenolic compounds that may have metabolic roles in humans and animals that consume them frequently. its antioxidant capacity, antimutagenic and antiproliferative effect have been associated with the presence of phenolic compounds, and this could explain the numerous scientific studies that have suggested that the consumption of this food is related to several beneficial health effects, such as reduction of coronary heart disease, protective effects against cancer, and decrease of diabetes and obesity risk (gálvez et al., 2007). the antioxidant activity of beans is due to the presence of phenolic acids and flavonoids, mainly tannins, both raw and cooked, gallic, vanillic, pcoumaric, ferulic, sinapic and chlorogenic acid, which are of great importance as precursors in the synthesis of phenolic compounds in plants (huber et al., 2016). legumes are also an excellent source of micronutrients. they are a source of se, thiamine, niacin, folate, riboflavin and pyridoxine. they also contain vitamin e and a as well as fe and zn, although the fe content can vary greatly depending on the variety, for example, white beans contain almost twice as much fe as black beans. however, most of the fe contained is tightly bound to phytates, which reduce absorption and may contribute to fe deficiencies in countries where beans and other legumes are a staple food (mudryj et al., 2014). nevertheless, beans have the potential to treat fe deficiency anemia and other diseases associated with micronutrient deficiencies that affect a large number of people around the world. in this regard, biofortification of this crop is a technique that has been launched with the rationale that high mineral content grains will increase the supply and availability of non-heme fe in various human populations. such nutritional improvement focuses on both increasing nutrient content and reducing the anti-nutritional factors contained in the plant, such as oxalates, phytates and tannins, which together affect the bioavailability of these nutrients to consumers (diaz et al., 2010). common beans are a staple food in many latin american and african countries. mexico is considered the center of origin of this legume, which has been consumed since pre-hispanic era (espinosa-alonso et al., 2006). this country has the widest variety of beans and is accepted as the center of origin of the common bean, since 47 out of 52 species classified in the phaseolus genus were identified in mexico (silva-cristobal et al., 2010). in mexico, there is a great diversity of this crop, in which color is one of the attributes that determine consumption preferences in different mexican regions, such as yellow in the northwest, beige with brown and cream spots in the northeast, black in the south and various specific colors in the central region (espinosaalonso et al., 2006). it is known that the coat color is attributed to the presence and quantity of polyphenols such as flavonol glycosides, condensed tannins and anthocyanins which function to protect the seed against predators and pathogens (beninger et al., 1999; takeoka et al., 1997). since there are several factors that influence the nutritional quality of the bean grain, and due to the wide agrobiodiversity that exists in mexico, the objective of this study was to characterize the antioxidant capacity and nutraceutical content of six bean varieties from morelos, mexico, in order to select varieties that have the potential to be bio-fortified with micronutrients, with a focus on improving bean nutritional quality, thus contributing to the nutrition of the population, especially the most unprotected whose food base is legumes. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods sample preparation six common bean varieties representative of morelos state, mexico, were used for the study were used, which are presented in table 1. seeds were harvested in 2018 and collected and analysed in the same year. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 3 table 1.table 1.table 1.table 1. bean varieties grown in morelos state, mexico used in the study variety origin laboratory work classification number color photography ‘sangre de toro’ tlayacapan morelos 98 red ‘peruano’ cuautla morelos 100 yellow ‘flor de mayo’ cuautla morelos 101 spotted ‘negro’ cuautla morelos 102 black ‘pinto’ cuautla morelos 103 pinto ‘negro’ cuautla morelos 104 black for the analysis, 100 grains of each variety were used. samples were ground to a fine powder which was stored in polyethylene bags and kept in a desiccator until analysis. the determinations were made in triplicate. morphometric characteristics determination of beans seed weight. one hundred seeds of each bean variety were placed in a petri dish and weighted using an analytical scale (and company limited, milpitas, ca, u.s.a.). the result was reported in g of 100 seeds. length, width and thickness. these were determined using a digital vernier (steren®, azcapotzalco, mexico city, mexico). one hundred seeds of each variety studied were used for the test. results were expressed in mm. color determination for color evaluation, 100 seeds of each variety were taken and placed in a glass petri dish until the container was full, and using a portable colorimeter konica minolta dp-400 (minolta co. ltd. osaka, japan) the cielab system color coordinates (l*, a* and b*) were obtained from the surface of these samples. where parameter l* represents brightness, which varies from 0 (black) to 100 (white); a* can have either positive (red) or negative (green) values; and b* represents yellow when the value is positive and blue when it is negative. color coordinates l*, a* and b* were used to obtain the ciel*c*hº color space, where c represents chroma or color saturation and hº is the hue angle or hue representing color according to the angle on the 360 chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 4 ° color wheel, with red-purple at 0 °, yellow at 90 °, blue-green at 180 ° and blue at 270°, counter clockwise (mcguire, 1992). chroma and hue were calculated using the following formulas (mcguire, 1992): �∗ = (�∗2 + �∗2)1/2 (1) °h= 360° + [(arctan (b*/a*)) /6.2832]*360 (2) when a*>0 and b*<0 or °h= [(arctan (b*/a*)) /6.2832]*360 (3) when a>0 y b>0 or °h= 180 + [(arctan (b*/a*)) /6.2832]*360 (4) when a<0 y b<0 nutritional quality analysis of bean ash content determination ash analysis was performed using the method proposed by mexican norm nmx-f-f066-s-1978 (1978). one g of sample was weighed in a crucible and kept at constant weight (100 ºc for 2 h) then placed in a desiccator and taken to a muffle (felisa®) where it was kept at 600 ºc to carbonize the sample until calcination was reached. results were expressed as percentage of ash. fat content determination fat content in the bean samples was determined using the golfish method proposed by the association of official analytical chemist (aoac) (2000). goldfish flasks were dried and kept to constant weight in an oven. the goldfish labconco® grease extractor was then assembled and the sample was placed inside filter paper and covered with absorbent cotton, to be introduced into the equipment. the solvent (petroleum ether) was added and kept under reflux for 2.5 h. after the extraction time was over, the solvent was recovered by distillation, retaining only the fat. finally, the flask with fat was weighed and expressed as a percentage. moisture content determination moisture was obtained by the open-capsule drying method proposed by the aoac (2000). for the analysis, 1 g of sample was taken from each bean variety, which was weighed in an aluminium capsule that was previously dried at 75 ºc until constant weight. each capsule with the sample was weighed and placed in an oven (felisa® st. livonia, michigan, u.s.a) for 12 h at 75 ºc. then, the capsule was removed from the oven and placed in a desiccator. afterwards, its weight was taken. moisture content was expressed as a percentage. fiber content determination crude fiber was determined using the method proposed by mexican norm nmx-f-90-s-1978 (1978). the analysis was performed on the previously defatted sample. first, the sample weight was taken and recorded, then each sample was transferred to a beaker for fiber determination and 200 ml of 1.25% sulfuric acid and 1 ml of isoamyl alcohol as defoamer were added to each beaker. mixture was kept at boiling point for 30 min. afterwards, it was rinsed to remove sulfuric acid and isoamyl alcohol residues and at the same time to neutralize the mixture. subsequently, 200 ml of 1.25% sulfuric acid was added, kept boiling for 30 min and then rinsed in glass fiber until neutralized. thereafter, fiberglass with the sample in the capsule was placed in an oven (felisa) and left to dry for 12 h until sample was completely dry. once the drying process was finished, the capsule with the fiberglass and sample was weighed, and then the fiber percentage of each bean variety was obtained by weight difference. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 5 carbohydrate content determination carbohydrates were obtained by difference with ash, fat and moisture content obtained and reported as a percentage. protein content determination to quantify protein concentration, the flash 2000 organic elemental analyzer (thermo scientific® corporation, cambridge, uk) was used, whose procedure is based on the dumas method proposed by reussicalvo et al. (2008). first, a tin capsule was taken and placed on a microbalance (mettler toledo®, columbus, ohio, usa), then 9 µg of vanadium pentoxide and 3 µg of the bean sample were weighed. finally, sample was placed in the flash 2000 autosampler for analysis. protein concentration was expressed as a percentage. energy determination energy contained in each sample was measured as the sum of calories contained in carbohydrates, fat and protein as described in the mexican official norm nom-051-scfi/ssa1-2010 (2010). energy expressed in kcal 100-1 g-1. mineral analysis micronutrient determination one g of finely ground sample was weighed on an analytical balance (and company limited, milpitas, ca, u.s.a.). afterwards, acid digestion of the dehydrated plant tissue was performed by adding 25 ml of triacid mixture (1000 ml nitric acid, 100 ml hydrochloric acid and 25 ml sulfuric acid), and placed in a digester (labconco® corporation, kansas city, mo, u.s.a.). after digestion, sample was filtered and volumetrically diluted with tridistilled water in a 50 ml flask. finally, samples were poured into polypropylene tubes for further analysis. fe, zn, mn, cu and ni concentration was determined by atomic absorption spectrophotometry (atomic absorption spectrophotometer ice 3000 thermo scientific® corporation, cambridge, uk) and concentration was expressed in ppm. macronutrient determination magnesium (mg), potassium (k), calcium (ca) were quantified by atomic absorption spectrophotometry (atomic absorption spectrophotometer ice 3000 thermo scientific) in the same way as micronutrients were obtained and expressed as a percentage. phosphorus (p) was determined by colorimetry using the ammonium metavanadate-molybdate method. 500 µl of the digested sample was taken for minerals, 1 ml of phosphorus reagent (ammonium metavanadate-molybdate) and 3.5 ml of tridistilled water was added; it was stirred in a vortex (vwr, thorofare, new jersey, u.s.a.) and allowed to stand for 1 h. after that time, the samples were read in a uv/vis spectrophotometer (jenway spectrophotometer, jenway limited®, essex, england) at a wavelength of 430 nm; a calibration curve was performed with a phosphorus standard (ion chromatography standard (ic) acculontm reference standard, new haven, connecticut, u.s.a.) and results were expressed as dry weight percentage (%). c, h, s and n organic compounds determination c, h, s and n were determined by the dumas method proposed by reussi-calvo et al. (2008). first, a tin capsule was taken and placed on a microbalance (mettler toledo®, columbus, ohio, usa). nine µg of vanadium pentoxide and 3 µg of the bean sample were weighed, placed in a capsule and then closed. finally, sample was placed in the flash 2000 organic elemental analyzer (thermo scientific® corporation, cambridge, uk) and the compound concentration was reported as a percentage. three replicates were performed for the analysis. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 6 antioxidant capacity determination (2, 2-diphenyl-1-picrylhydrazyl (dpph)) the analysis was carried out using the method proposed by hsu et al. (2003). extract was obtained by maceration of 1 g of finely ground seed in 5 ml of 80% methanol, which was centrifuged (allegra® refrigerated centrifuge, beckman coulter, inc.; fullerton, california, u.s.a.) at 6000 rpm for 10 min at 4 ºc. once extract was centrifuged, 0.5 ml of the supernatant was taken and mixed with 2.5 ml of freshly prepared 0.1 mm dpph solution. thereafter, sample was incubated (boekel scientific incubator) for one h in dark conditions and at room temperature. after this time, samples were read in a uv/vis spectrophotometer (genesys 10s, thermo scientific® corporation, cambridge, uk) at a wavelength of 517 nm. resulting antioxidant capacity was reported as percentage inhibition. nutraceutical compound determination total phenols extraction and quantification phenolic compounds were extracted using the colorimetric method proposed by singlenton and rosi (1965). for analysis, 0.5 g of ground bean seed was mixed with 2.5 ml of methanol, 2.5 ml of chloroform, and 1.25 ml of 2% nacl solution (j. t. baker, state of mexico, mexico) and macerated to obtain an extract. subsequently, mixture was homogenized, then centrifuged (allegra® refrigerated centrifuge, beckman coulter, inc.; fullerton, california, u.s.a.) at 5000 rpm for 10 min and three phases were obtained, a methanol phase, which contains the phenolic acids, interphase containing the proteins precipitated by nacl and the chloroform phase constituted by dissolved lipids. for the reaction, 750 µl of 2% na2co3 was placed in a test tube and mixed with 250 µl of folin-ciocalteau reagent (sigma-aldrich, st. louis, mo, usa), 1375 µl of deionized water and 250 µl of enzyme extract. finally, mixture was incubated at room temperature for 60 min. quantification was obtained using a standard curve of gallic acid (10-100 µg ml-1) at an absorbance of 725 nm. results are shown in mg of gallic acid equivalents per g of sample (mg gae g-1) (dry weight). flavonoid content determination flavonoid analysis was performed according to the method proposed by zhishen et al. (1999). extract was obtained by macerating 0.5 g of ground seed with 5 ml of 85% methanol. subsequently, it was centrifuged (allegra® refrigerated centrifuge, beckman coulter, inc.; fullerton, california, u.s.a.) at 4000 rpm for 10 min. then an aliquot of 250 µl was placed in a test tube, and 75 µl of nano2 (j. t. baker, state of mexico, mexico) was added, the mixture was homogenized in a vortex (vwr, thorofare, new jersey, u.s.a.) and allowed to stand for 5 min. afterwards, 150 µl of alcl3 (sigma-aldrich, st. louis mo, usa) and 500 µl of naoh (j.t. baker, state of mexico, mexico) were added and diluted to a final volume of 2.025 ml with tridistilled water. absorbance was then measured at 510 nm in a uv/vis spectrophotometer (genesys 10s, thermo scientific® corporation, cambridge, uk). results obtained were expressed as mg catechin equivalents (ce) per g of sample (mg ce g-1) based on dry weight. anthocyanin determination anthocyanin content was determined by ph differential according to the method proposed by wrolstad et al. (2005). 0.5 g of finely ground bean was mixed with 5 ml of methanol (j.t. baker, estado de mexico, mexico). mixture was centrifuged (allegra® refrigerated centrifuge, beckman coulter, inc.; fullerton, california, u.s.a) at 4000 rpm for 10 min. after the centrifugation time, 2 phases of the sample were obtained; 0.5 ml of the first phase were taken and placed in a test tube, then 2 ml of potassium chloride (kcl) (j.t. baker, estado de méxico, méxico) were added, homogenized in a vortex (vwr, thorofare, new jersey, u.s.a.) and its absorbance was obtained in a uv/vis spectrophotometer (genesys 10s, thermo scientific® corporation, cambridge, uk) at 460 nm. subsequently, 0.5 ml of the second phase was taken and deposited in a test tube, 2 ml of sodium acetate was added, homogenized in the vortex and the absorbance chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 7 reading was taken in the uv/vis spectrophotometer. results were expressed as mg cyanidin-3-glucoside (c3g) g-1 (dry weight)-1. phytic acid determination for phytic acid determination, the method proposed by mckie and mccleary (2016) was used, first total phosphorus was obtained by the ammonium metavanadate-molybdate method in an absorption range of 430 nm and a potassium phosphate standard curve. once the results were obtained, the following formula was applied: phytic acid = (total phosphate) /0.282 (5) where 0.282 is a conversion factor from total phosphorus to phytic acid. results were expressed in g of phytic acid per 100 g (g 100-1 g-1) based on dry weight of sample. statistical analysis data was subjected to an analysis of variance in a completely randomized one-factor design to evaluate the effect of bean variety on the different variables studied, as well as an analysis of comparison of means using the tukey test and an analysis of correlation between variables performed with sas statistical package (sas institute, inc; cary, nc, usa) means were accepted as significantly different at a 95% confidence interval (p ≤ 0.05). results were reported as mean± standard deviation. results results results results and discussionand discussionand discussionand discussion morphometric characteristics of bean seeds table 2 shows weight, length, width and thickness of bean seeds of the evaluated varieties. statistical analysis showed a significant difference (p ≤ 0.05) between varieties for the four variables mentioned above. seed width ranged from 5.5 to 9.1 mm, with the lowest value for ‘flor de mayo’ and the highest for ‘sangre de toro’, the latter being the only variety with a statistical difference compared to other varieties. bean thickness ranged from 4.9 to 6.8 mm, with ‘pinto’ being the lowest and ‘peruano’ the highest, although the latter variety was statistically equal to ‘flor de mayo’, ‘negro’/102 and ‘negro’/104. in addition, kernel length ranged from 10.8 to 17.3 mm, with ‘negro’/102 being the shortest variety and ‘sangre de toro’ the longest. finally, the weight of 100 seeds ranged from 23.06 to 53.42 g, with ‘negro’/102 being the shortest and ‘sangre de toro’ the heaviest. in summary, ‘sangre de toro’ had the highest values for width, length and weight among all analysed varieties, while ‘negro’/102 was the shortest and lightest. results obtained from dimensions of the bean varieties analysed in the present work are lower than those reported by herrera-hernández et al. (2018) in bean varieties grown in zacatecas, mexico. likewise, the weight of 100 seeds was higher than reported by mederos and reynaldo (2007) in ‘cuban bean’ varieties with black and red coat. according to the classification of aguirre and gómez-aldapa (2010), ‘sangre de toro’ and ‘peruano’ varieties are classified as large (>40 g) and ‘flor de mayo’, ‘negro’/102, ‘pinto’ and ‘negro’/104 as medium (26 to 40 g). other studies have shown great variability in the weight of 100 seeds, for example pliegomarín et al. (2013) found a large amplitude of this variable in seeds collected in central valleys of oaxaca, mexico, with intervals ranging from 11.2 to 74.8 g of 100 seeds, being out of the range of what was found in this study, with very small genotypes such as ‘negro delgado’ with a weight of 11.2 g of 100 seeds coming from zaachila and ‘frijolon’ with 74.8 g weight with the same origin. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 8 table 2.table 2.table 2.table 2. morphometric characteristics of several common bean varieties from morelos state, mexico variety/ classification number* width (mm) thickness (mm) length (mm) weight (g of 100 seeds) ‘sangre de toro’/98 9.1 ± 0.01a 6.4 ± 0.02ab 17.3 ± 0.09a 53.42 ± 3.59a ‘peruano’/100 8.2 ± 0.05b 6.8 ± 0.06a 13.6 ± 0.04b 44.41 ± 1.01b ‘flor de mayo’/101 5.5 ± 0.02b 5.5 ± 0.02bc 12.6 ± 0.05bc 30.97 ± 0.18c ‘negro’/102 7.3 ± 0.02 b 5.5 ± 0.02bc 10.8 ± 0.03d 23.06 ± 0.33d ‘pinto’/103 7.5 ± 0.07b 4.9 ± 0.03c 13.1 ± 0.09bc 29.07 ± 0.36c ‘negro’/104 7.4 ± 0.02b 5.6 ± 0.05bc 11.46 ± 0.04cd 30.29 ± 0.84c data correspond to mean ± sd. different letters per column indicate significant statistical difference between varieties. tukey test (p ≤ 0.05). *laboratory work classification. bean seed color characteristics bean color is an important aspect for consumer taste so its evaluation is necessary (aguirre-santos et al., 2011). table 3 presents the colour characteristics l*, a*, b*, chroma and hue of common bean seeds from morelos, mexico, the statistical analysis showed significant difference (p ≤ 0.05) between varieties in all five variables analysed. luminosity ranged from 24.96 to 69.21, with ‘negro’/102, ‘negro’/104 and ‘sangre de toro’ being the bean varieties with the lowest luminosity corresponding to darker bean colors. while ‘peruano bean’, ‘pinto’ and ‘flor de mayo’ had the highest l* value corresponding to lighter colors of all varieties analysed. these values correspond to those reported by chávez-mendoza et al. (2019) in bean varieties from different regions of mexico. they are also similar to those reported by aguirre and gómez-aldapa (2010) for ‘pinto saltillo’, ‘bayo victoria’ and ‘negro san luis’ bean varieties with 7.125, 57.1 and 21.85, respectively. regarding the a* value, it ranged from 0.71 to 26.043. where ‘peruano’ and ‘negro’/104 beans had the lowest values for this color characteristic, with no statistical difference between them, indicating a lower tendency to red and a higher tendency to green. in addition, ‘sangre de toro’ had the highest a* value of all varieties analysed, which coincides with its strong red color. on the other hand, b* value was within a range of -1.42 to 33.44. ‘negro’/102 and ‘negro’/104 had negative values indicating a greater tendency to blue color, while ‘peruano’ bean had the highest b* value with a more yellow tendency. other studies reported negative values of b* for dark-skinned beans such as ‘negro puebla’, ‘negro 151’, ‘negro 152’, ‘negro querétaro’, ‘negro san luis’, ‘negro sinaloa’, ‘negro veracruz’, ‘medellín’, ‘nayarit 80’, ‘jamapa’, ‘negro perla’, ‘merentral’, ‘altiplano’ and ‘negro puebla 152’, which places them in the third quadrant of the tri-stimulus hunter l scale, a* b* corresponding to blue-green coloration (salinas-moreno et al., 2005). regarding chromaticity, it showed a range from 1.77 to 33.45, with ‘negro’/102 and ‘negro’/104 having the lowest values or lowest color saturation, whereas the ‘peruano bean’ had the highest color clarity or chromaticity while ‘flor de mayo’ and ‘negro’/104 were in the intermediate range for this variable. this range was higher than that reported by aguirre and gómez-aldapa (2010) in ‘negro san luis’, ‘pinto saltillo’ and ‘bayo victoria’ varieties. hue angle was within the range of 11.75 ° and 126.64 °, with ‘sangre de toro’ variety having the lowest value, which is coincidentally in the red tone of the color wheel (mcguire, 1992), while ‘negro’/102 variety had the highest angle with a bluish-green tone, equal to the coloration reported by aguirre and gómez-aldapa (2010) for ‘negro san luis’ bean, which corresponds to the third quadrant of the tristimulus scale. likewise, ‘peruano’ and ‘pinto’ varieties showed a shade closer to yellow, since their hue angle was close to 90 °, as reported by mcguire (1992). chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 9 table 3.table 3.table 3.table 3. seed color characteristics of several common bean varieties from morelos state, mexico variety/ classification number* l a* b* chroma ° hue ‘sangre de toro’/98 32.41 ± 0.09d 26.04 ± 1.03a 5.42 ± 0.20d 26.60 ± 1.04b 11.75 ± 0.25f ‘peruano’/100 69.21 ± 1.1a 0.71 ± 0.68d 33.44 ± 1.07a 33.45 ± 1.07a 88.78 ± 1.75c ‘flor de mayo’/101 50.14 ± 0.71c 14.81 ± 0.51b 11.46 ± 0.59c 18.73 ± 0.58c 37.74 ± 1.62e ‘negro’/102 24.96 ± 0.40f 1.05 ± 0.11d -1.42 ± 0.09e 1.77 ± 0.05d 126.64 ± 4.49a ‘pinto’/103 65.94 ± 0.67b 6.21 ± 0.24c 16.46 ± 0.06b 17.59 ± 0.05c 69.32 ± 0.08d ‘negro’/104 26.85 ± 0.60e 0.91 ± 0.07d -1.80 ± 0.12e 2.03 ± 0.07d 116.99 ± 3.55b data correspond to mean ± sd. different letters per column indicate significant statistical difference between varieties. tukey (p ≤ 0.05). *laboratory work classification. nutritional analysis table 4 reports the nutritional composition of the bean varieties analysed in the present study. statistical analysis showed significant differences (p ≤ 0.05) between varieties in protein, ash, fat, moisture, carbohydrate, fiber and energy content. protein concentration ranged from 18.03 to 26.92%. ‘sangre de toro’ and ‘pinto’ varieties had the lowest content, ‘flor de mayo’, ‘negro’/102 and ‘negro’/104 had intermediate values and ‘peruano’ bean showed the highest concentration. the results obtained in most of the varieties analysed in this study are similar to those reported by armendáriz-fernández et al. (2019) in bean varieties harvested in oaxaca, mexico. as well as to those found by herrera-hernández et al. (2018) in varieties harvested in zacatecas, mexico. they also coincide with the results obtained by peña-betancourt and conde-martínez (2012) on wild bean varieties (‘durango atypical’ and ‘typical’, ‘oaxaca chico’ and ‘tlaxcala atypical’ and ‘typical’). also, commercial beans such as ‘flor de mayo’, ‘peruano’, ‘garbancillo’ and ‘flor de junio’. mederos (2006), on the other hand, indicates that protein in beans ranges between 16 and 30% and that varieties most consumed in latin america have an average concentration of 20%, which is in agreement with results obtained in the present study. according to the same author, bean protein has a high lysine and phenylalanine plus tyrosine content, so it fulfils all the minimum requirements recommended by the food and agriculture organization (fao) or the world health organization (who). ash content ranged from 4.0 to 4.96%. ‘flor de mayo’ and ‘negro’/104 varieties had the lowest concentration with no statistical difference between them, followed by ‘pinto’, ‘negro’/102, and ‘peruano’ with intermediate values, and finally ‘sangre de toro’ with the highest concentration among all six varieties analysed. the obtained range was higher than reported by herrera-hernández et al. (2018) in varieties harvested in zacatecas, mexico and slightly lower than results obtained by armendariz-fernández et al. (2019) in bean varieties harvested in the state of oaxaca, mexico. aguirre and gómez-aldapa (2010) reported slightly lower results in ‘negro san luis’, ‘pinto saltillo’ and ‘bayo victoria’ varieties, noting that ash content may vary depending on cultivar genetics and soil characteristics. fat concentration was within the range of 0.96 to 1.64%. ‘negro’/104 had the lowest value for this variable while ‘peruano’ had the highest content of all evaluated variables, with no statistical difference between ‘sangre de toro’ and ‘negro’/102. these results are similar to those reported by aguirre and gómez-aldapa (2010) on ‘negro san luis’, ‘pinto saltillo’ and ‘bayo victoria’ varieties corresponding to a range of 0.92 to 1.71%. in addition, they are higher than those reported by fernandez and sanchez (2017). lipid fraction of bean is the smallest, and is constituted by a mixture of acylglycerides whose predominant fatty acids are monounsaturated and polyunsaturated (ulloa et al., 2011). moisture content of common bean was within the range of 10.29 and 14.58%, being ‘negro’/104 the variety with the lowest moisture content and ‘negro’/102 with the highest value. results obtained in this last variety were much higher than those reported in other black beans such as ‘negro san luis’, which had a chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 10 moisture content of 11.95 % (aguirre and gómez-aldapa, 2010), but were similar to those found in some cuban varieties (mederos and reynaldo, 2007); however, the remaining varieties analysed in the present work were much lower than the values reported by the latter authors. other studies have shown results different from those found in this work such as the one conducted by armendariz-fernández et al. (2019) who reported that ‘sangre de toro’ and ‘peruano’ varieties had a lower moisture content, while the ‘flor de mayo’ variety had a higher amount (12.8%) than that observed in this study. meanwhile, herrera-hernández et al. (2018) reported much lower moisture values in bean varieties produced in zacatecas, mexico, ranging from 6.14 to 7.42%. peña-betancourt and conde-martínez (2012) found much lower results in wild and improved bean varieties, suggesting that the lower moisture content of wild varieties was due to increased temperature and lack of irrigation at the production site, in addition to prolonged storage. aguirre and gómez-aldapa (2010) reported that moisture content is related to seed age and postharvest handling, as well as to processing methods and conditions. carbohydrates constitute the main fraction in legume beans (mederos, 2006), 100 g of raw beans provide 52 to 76 g depending on variety (ulloa et al., 2011). carbohydrate concentration obtained in the present study was between 51.21 and 61.53%, where ‘pinto’ variety had the highest content, while ‘peruano’ had the lowest. no statistical difference was obtained between the concentration of ‘negro’/104 (58.28%) and ‘sangre de toro’ (58.01%), nor between ‘flor de mayo’ (53.58%) and ‘negro’/102 (53.67%), which had intermediate values for this variable. other studies have shown values different from these results; thus, armendariz-fernández et al., (2019), reported a higher carbohydrate content in ‘flor de mayo’ (55 %) and ‘peruano’ (56.6%) varieties, while in ‘sangre de toro’ beans the found value was lower (55.2%). for their part, herrera-hernández et al. (2018) reported a higher range (57.16% to 65.79%) of carbohydrate content in common bean varieties grown in zacatecas, mexico, including ‘flor de mayo’, ‘negro’, ‘pinto saltillo’ bean, among others. while fernandez and sanchez (2017) found much lower values than those obtained in the present study in different varieties of beans produced and consumed in mexico purchased in a local market in delicias, chihuahua, mexico, such as ‘bayo’, ‘pinto’, ‘negro’, ‘alubia’, ‘flor de mayo’ and ‘peruano’, where the latter was the exception as it showed a higher concentration than that obtained in this study. bean carbohydrates consist mainly of starch and other polysaccharides (dietary fiber) with small but significant amounts of oligosaccharides; starch represents more than 50% of seed weight and is the dominant carbohydrate in the human diet, hence the importance of this legume (mederos, 2006). bean is also a good source of fiber which ranges in value from 14-19 g 100-1 g-1 of the raw food, from which up to half may be of the soluble form. the main chemical components of fiber in beans are pectins, pentosans, hemicellulose, cellulose and lignin (ulloa et al., 2011). half a cup of beans provides between 5.2 and 7.8 g of total fiber (messina, 2014). fiber content in the present study ranged from 3.21 to 5.71%, ‘flor de mayo’ variety had the highest value while ‘negro’/104 bean had the lowest. these results were far higher than those reported by herrera-hernández et al. (2018) in genotypes grown in zacatecas, mexico and to those reported by armendariz-fernández et al. (2019) on varieties produced in oaxaca, mexico. finally, the energy content of the analysed beans was between 318.39 and 334.6 kcal, with ‘negro’/104 being the variety with the highest energy content and ‘negro’/102 the lowest. similar results were reported by armendariz-fernández et al. (2019) on several bean varieties produced in oaxaca, mexico, including ‘sangre de toro’, ‘bayo’, ‘peruano’ among others. likewise, results were lower than those reported by herrerahernández et al. (2018) in bean varieties produced in zacatecas, mexico. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 11 table 4.table 4.table 4.table 4. nutritional composition of several common bean varieties from morelos state, mexico variety/ classification number* protein (%) ash (%) fat (%) moisture (%) carbohydrates (%) crude fiber (%) energy (kcal 100-1g-1) ‘sangre de toro’/98 18.03 ± 0.13d 4.96 ± 0.03a 1.53 ± 0.04a 13.45 ± 0.04b 58.01 ± 0.15b 3.40 ± 0.02c 320.41 ± 0.23d ‘peruano’/10 0 26.92 ± 1.87a 4.22 ± 0.02c 1.64 ± 0.05a 11.93 ± 0.02c 51.21 ± 0.10d 3.81 ± 0.02b 328.36 ± 0.13c ‘flor de mayo’/101 23.29 ± 1.40b 4.0 ± 0.02d 1.11 ± 0.07cd 11.53 ± 0.02d 53.58 ± 0.12c 5.71 ± 0.02a 320.59 ± 0.39d ‘negro’/102 21.72 ± 1.15bc 4.26 ± 0.01c 1.47 ± 0.07ab 14.58 ± 0.03a 53.67 ± 0.12c 3.4 ± 0.02c 318.39 ± 0.55e ‘pinto’/103 19.66 ± 1.33cd 4.45 ± 0.05 b 1.26 ± 0.13bc 10.63 ± 0.02e 61.53 ± 0.15a 3.31 ± 0.04d 332.74 ± 0.80b ‘negro’/104 22.71 ± 0.66bc 4.05 ± 0.05d 0.96 ± 0.05d 10.29 ± 0.01f 58.28 ± 0.13b 3.21 ± 0.03e 334.6 ± 0.14a data correspond to mean ± sd. means with the same letter between rows indicate that there is no statistical difference between varieties. tukey test (p ≤ 0.05). *laboratory work classification. mineral content analysis micronutrient content table 5 shows the micronutrients present in the common bean varieties produced in morelos state, mexico. statistical analysis showed a significant difference (p < 0.05) between varieties in the content of all the microelements analyzed. iron (fe) concentration ranged from 89.225 to 136.416 ppm, with ‘flor de mayo’ and ‘sangre de toro’ varieties having the lowest fe content and no significant difference between them, while ‘pinto beans’ had the highest concentration. these results are higher than those reported by akond et al. (2011a) on 14 genotypes from the international center for tropical agriculture and the united states of america. as well as those obtained by armendáriz-fernández et al. (2019) on bean varieties produced and consumed in oaxaca, mexico. on the other hand, they are lower than those obtained in most of the bean varieties analyzed by chávezmendoza et al. (2019), who found that this micronutrient is present in greater proportion in the coat than in the bean cotyledon. fe deficiency has been mainly related to anemia. this micronutrient is also part of a large number of enzymes involved in energy production and in the proper functioning of the immune response in humans. beans contribute approximately 40% of fe to the diet of people who base their diet on this legume and corn (mederos, 2006), hence the importance of studying this micronutrient in the different genotypes produced in mexico, which serves as a basis for biofortification and genetic improvement studies. zinc (zn) content ranged from 24.82 to 35.78 ppm, ‘flor de mayo’ variety had the lowest value, while ‘peruano’ had the highest. these results were similar to those reported by armendáriz-fernández et al. (2019) in bean varieties produced and consumed in the state of oaxaca mexico. some studies report that zn content in beans is one of the highest among vegetables, almost equal to that found in dairy products, although lower than that found in meat. evaluations carried out on bean collections reveal ranges in the content of this micronutrient from 21 to 54 ppm with an average of 35 ppm (mederos, 2006), which coincides with the results found in this work. importantly, this microelement is associated with decreased oxidative stress in cells and improved immune cell function. in addition, its deficiencies have been shown to cause dna damage in peripheral blood cells in rats (mudryj et al., 2014). nickel (ni) concentration in analyzed varieties ranged from 2,113 to 6,783 ppm with ‘negro’/102 bean having the lowest value and ‘sangre de toro’ having the highest. these data are similar to those reported by chávez-mendoza et al. (2019) in different bean varieties, where a higher presence of this micronutrient was observed in the cotyledon than in the coat, with ‘bayo’ having the highest value at 8.62 ppm, while ‘negro’ and ‘flor de mayo’ beans had the lowest, coinciding with what was reported in the present study. manganese (mn) content ranged from 9.036 to 18.82 ppm, with ‘negro’/102 having the lowest concentration of this micronutrient, while ‘pinto’ had the highest value. these results were similar to those reported by chávez-mendoza et al. (2019) in several bean varieties, including ‘flor de mayo’, ‘pinto saltillo’, chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 12 ‘negro san luis’, ‘negro 8025’, ‘negro jamapa’, ‘higuera azufrado’ among others. however, they are lower than those obtained by herrera-hernández et al. (2018) in some varieties grown and consumed in zacatecas, mexico, including the ‘flor de mayo’ variety in which a higher content of this microelement was obtained (25.45 pm) than that obtained in the present work. lastly, cu concentrations of the varieties studied ranged from 3.48 to 6.69 ppm. ‘sangre de toro’ had the lowest content of this micronutrient while ‘pinto’ had the highest. these results are much lower than those recorded by herrera-hernández et al. (2018) in bean varieties grown in zacatecas state, mexico. summarizing, ‘pinto’ had the highest concentration of fe and mn, ‘peruano’ had the highest concentration of cu and zn, while ‘sangre de toro’ had the highest ni content and the lowest amount of fe and cu. the average concentration of these micronutrients in the analysed seed in descending order was as follows: fe > mn > zn> cu > ni. this behaviour is similar to that reported by chávez-mendoza et al. (2019) in several bean varieties, with the exception of manganese, which, unlike the present study, was in lower concentration than zinc. observed differences in mineral content among the varieties analysed in this study can be attributed to the genotype and the environment in which they are produced. previous studies have shown that the concentration of these compounds in beans has varied as a function of genetic material, crop management and storage conditions (espinoza-garcía et al., 2016). table 5.table 5.table 5.table 5. micronutrient concentration in common bean varieties produced in morelos state, mexico variety/ classification number* fe (ppm) zn (ppm) ni (ppm) mn (ppm) cu (ppm) ‘sangre de toro’/ 98 89.22 ± 1.65c 32.97 ± 3.70ab 6.78 ± 0.21a 13.27 ± 0.64bc 3.48 ± 0.42b ‘peruano’/ 100 101.40 ± 3.24bc 35.78 ± 5.10a 3.28 ± 0.46c 11.05 ± 0.41cd 6.69 ± 0.50a ‘flor de mayo’/ 101 91.06 ± 6.49c 24.82 ± 0.66b 2.45 ± 0.33cd 10.54 ± 0.65cd 3.95 ± 0.68b ‘negro’/ 102 99.76 ± 5.83bc 31.85 ± 1.133ab 2.11 ± 0.30d 9.036 ± 1.43d 6.09 ± 0.56a ‘pinto’/ 103 136.41 ± 10.01a 31.81 ± 3.48ab 5.23 ± 0.46b 18.82 ± 1.83a 4.21 ± 0.68b ‘negro’/ 104 113.43 ± 6.09b 34.38 ± 0.90a 2.49 ± 0.28cd 14.491 ± 0.73b 4.39 ± 0.38b data correspond to mean ± sd. different letters per column indicate significant statistical difference between varieties. tukey (p ≤ 0.05). *laboratory work classification. macronutrient determination ca, mg and k are the main cations in common bean. there is greater availability of ca than mg or k (suárez-martínez et al., 2016). table 6 shows the p, k, mg and ca contents obtained in the bean samples of the different varieties analysed. statistical analysis showed a significant difference (p < 0.05) between varieties in mg concentration but not in p, k and ca content. phosphorus, plays a relevant role in various metabolic processes vital to all living organisms such as being part of macromolecular structures, in energy generation and metabolic regulation, hence the importance of its availability in free form as inorganic p, which can be accessed by organisms' cells, as well as other nutrients that are chelated (rodriguez-blanco et al., 2018). p concentration in bean genotypes evaluated was within a range of 0.149 to 0.194%, although no statistical difference was observed between varieties for this variable, ‘negro’/104 bean had the lowest concentration of this macronutrient while ‘negro’/102 had the highest. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 13 results obtained in the present study were higher than those described by armendáriz-fernández et al. (2019) in common beans produced in oaxaca, mexico, where ‘peruano’ and ‘sangre de toro’ had the highest concentrations with 0.17% for both, and varieties such as ‘negro michigan’ and ‘bayo bola’ had concentrations as low as 0.01%. fernandez and sanchez (2017) meanwhile reported higher p values than those obtained here in ‘pinto’, ‘peruano’ and kidney bean. as for k concentration, it ranged from 1.464 to 1.815%. these results are higher than those recorded by armendáriz-fernández et al. (2019) in common bean produced in the state of oaxaca, mexico. they also exceed the concentrations of this macronutrient obtained by fernandez and sanchez (2017) in beans commonly consumed in mexico such as ‘bayo’, ‘pinto’, ‘peruano’, ‘negro’, kidney bean, ‘flor de mayo’ and green bean. ca, mg and cu are deficient in developed and developing countries which is attributable to the low availability of mineral elements in the soil and/or the low ability of plants to store them in their tissues, associated at the same time with the scarce availability of food of animal origin (araméndiz-tatis et al., 2016). for the present study, the mg content in the analysed beans ranged from 0.169 to 0.201%, with the ‘negro’/104 bean having the lowest concentration, while the ‘negro’/102 bean had the highest amount. results were much higher than those reported by herrera-hernández et al. (2018) in common bean varieties produced in zacatecas, mexico, in which ‘patola’ and ‘japanese’ beans had the highest concentrations of this macronutrient with a concentration of 0.16% in both genotypes. fernandez and sanchez (2017) also found lower concentrations than those obtained here, in bean varieties produced and consumed in mexico. ca belongs to the group of minerals that should always be part of our diet. it is the most abundant mineral element in our body, as it is an important part of the skeleton and teeth. it accounts for about 2% of body weight and is an essential cellular component for maintaining and/or performing the various specialized functions of virtually all cells in the body. these functions, non-skeletal, we can divide them into structural and properly regulatory (martinez, 2016). in this study, ca content ranged from 0.154 to 0.273%. these results are similar to those covered by herrera-hernández et al. (2018) on common bean varieties produced in zacatecas, mexico. other studies have disclosed lower concentrations than those obtained in the present investigation in ‘pinto’, ‘flor de mayo’ and ‘peruano’ beans (fernandez and sanchez, 2017). in summary, ‘negro’/102 had the highest mg concentration while ‘negro’/104 had the lowest. moreover, all the varieties analysed had the same p, k and ca contents according to the statistical analysis. table 6.table 6.table 6.table 6. macronutrient concentration (%) in common bean varieties produced in morelos state, mexico variety/ classification number* p k mg ca ‘sangre de toro’/ 98 0.180 ± 0.009a 1.815 ± 0.04a 0.192 ± 0.002ab 0.228 ± 0.073a ‘peruano’/ 100 0.182 ± 0.026a 1.655 ± 0.048a 0.188 ± 0.006bc 0.1545 ± 0.022a ‘flor de mayo’/ 101 0.170±0.015a 1.659 ± 0.033a 0.173 ± 0.003d 0.218 ± 0.0651a ‘negro’/ 102 0.194 ± 0.084a 1.605 ± 0.315a 0.201 ± 0.002a 0.273 ± 0.025a ‘pinto’/ 103 0.175 ± 0.059a 1.464 ± 0.080a 0.179 ± 0.001dc 0.245 ± 0.034a ‘negro’/ 104 0.149 ± 0.012a 1.687 ± 0.062a 0.169 ± 0.001d 0.239 ± 0.058a data correspond to mean ± sd. different letters per column indicate significant statistical difference between varieties. tukey test (p ≤ 0.05). *laboratory work classification. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 14 organic compound determination c, s, n, h table 7 presents the c, s, n and h contents obtained in the common bean samples of the different varieties analysed. statistical analysis showed significant difference (p < 0.05) between varieties in n concentration, but not in c, s, and h content (p > 0.05). carbon concentration in the analysed samples ranged from 40.441 to 42.349%, s from 0.040 to 0.082% and h from 6.153 to 6.391% with no significant statistical difference between varieties for the three elements. these results are lower than those shown by chávez-mendoza et al. (2019) in several bean varieties, in which no significant difference was observed between varieties in the cotyledon, as occurred in the present investigation; however, these authors did observe significant differences in the content of these elements in the seed coat. on the other hand, paredes et al. (2009) found c and s concentrations higher than those found in the present study in chilean, nuevo granada, durango and mesoamerican breeds. nitrogen content ranged from 2.926 to 4.0%, with ‘sangre de toro’ variety having the lowest concentration and ‘peruano’ the highest. these results are lower than those obtained by chávez-mendoza et al. (2019) in several bean varieties, both in coat and cotyledon, including ‘flor de mayo’, ‘pinto saltillo’, ‘negro jamapa’, ‘negro san luis’, ‘negro 8025’, among others. summarizing, it was observed that ‘sangre de toro’ variety presented the lowest values in n concentration, while ‘peruano’ bean and ‘flor de mayo’ had the highest amount of this compound. furthermore, all varieties analysed had statistically the same c, s and h concentration. table 7.table 7.table 7.table 7. c, s and n concentration (%) in common bean varieties produced in morelos state, mexico variety/ classification number* c s n h ‘sangre de toro’/98 40.441 ± 0.53a 0.087 ± 0.018a 2.926 ± 0.12c 6.153 ± 0.203a ‘peruano’/100 41.150 ± 0.11a 0.097 ± 0.010a 4.000 ± 0.21a 6.332 ± 0.064a ‘flor de mayo’/101 41.418 ± 0.69a 0.094 ± 0.023a 3.660 ± 0.28ab 6.315 ± 0.166a ‘negro’/102 41.625 ± 1.00a 0.086 ± 0.019a 3.542 ± 0.232abc 6.391 ± 0.176a ‘pinto’/103 42.349 ± 2.74a 0.040 ± 0.036a 3.068 ± 0.43bc 6.513 ± 0.49a ‘negro’/104 41.598 ± 0.52a 0.082 ± 0.015a 3.542 ± 0.13abc 6.347 ± 0.098a data correspond to mean ± sd. different letters per column indicate significant statistical difference between varieties. tukey test (p ≤ 0.05). *laboratory work classification. antioxidant capacity and nutraceutical compounds antioxidant capacity the antioxidant capacity obtained in the analysed bean varieties is presented in figure 1. statistical analysis showed significant difference (p ≤ 0.05) between varieties in this variable, which ranged from 73.59 to 96.76% inhibition. ‘peruano’ bean had the lowest antioxidant capacity, with no statistical difference with ‘pinto’ and ‘negro’/104, while the highest value was for the ‘flor de mayo’ variety, which was statistically equal to ‘sangre de toro’ and ‘negro’/102. study by chávez-mendoza et al. (2019) also revealed ‘flor de mayo’, ‘negro 8025’ and ‘negro san luis’ as the varieties with the highest antioxidant capacity, finding differences in this variable between cotyledon and coat, with the latter having the highest percentage of inhibition. these authors noted that these varieties had the highest phenolic content, which could explain their higher antioxidant activity, as was the case in the present study, where ‘flor de mayo’, ‘sangre de toro’ and ‘negro’/102 had the highest concentration of total phenols, flavonoids and anthocyanins. silva-cristobal et al. (2010) also disclosed that the antioxidant capacity of legumes depends on the total polyphenol content. aguilera et al. (2011) point out that the antioxidant activity is directly related to the polyphenol structure, such as the number of hydroxyl groups, degree of glycosylation etc. gonzález de mejía et al. (1999) found that phenolic compounds extracted from the coat of common bean inhibit the mutagenicity induced by chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 15 benzopyrene and 1-nitropyrene (premutagenic and mutagenic agents, respectively), hence the importance of studying these compounds present in common bean. on the other hand, armendariz-fernández et al. (2018) reported lower results in bean varieties grown in oaxaca, mexico also finding that ‘sangre de toro’ and ‘flor de mayo’ variety along with two other varieties, had the highest values of antioxidant ability with 82.1% and 79.1% respectively. some authors have stated that ‘flor de mayo’ is the preferred variety for consumption in central mexico (cardador-martínez et al., 2002), which confirms the importance of this variety for mexican consumers. figure 1figure 1figure 1figure 1. antioxidant capacity of common bean varieties grown in morelos state, mexico. means with the same letter are not significantly different, tukey test (p ≤ 0.05). *laboratory work classification. nutraceutical compounds table 8 shows the nutraceutical compounds evaluated in common bean varieties produced in morelos state, mexico. statistical analysis showed significant difference (p ≤ 0.05) between varieties in the content of total phenols, flavonoids and anthocyanins, but not in the phytic acid concentration (p > 0.05). total phenol content ranged from 1.76 to 4.24 (mg gae g-1). statistical analysis showed significant differences between varieties in the content of this compound. ‘negro’/104 was the variety with the lowest phenol concentration along with ‘negro’/102, whereas ‘flor de mayo’ had the highest content, followed by ‘sangre de toro’, while ‘pinto’ and ‘peruano’ beans had intermediate concentrations. other studies have found that these compounds are found in greater amounts in the bean coat than in the cotyledon; and have reported results similar to those obtained in the present work in some common mexican bean varieties, such as ‘flor de mayo’, ‘negro 8025’, ‘negro san luis’, ‘pinto saltillo’ among others (chávez-mendoza et al., 2019). likewise, the results obtained were superior to those found by espinosa-alonso et al. (2006) in wild and weedy mexican bean germplasm materials from chiapas, chihuahua, durango, guerrero and jalisco, as well as in ‘pinto’ and ‘negro jamapa’ beans. the total phenol level obtained in the bean of the present study exceeds that observed in wild berry species of the genus vaccinium, which are among the most important sources of these bioactive compounds in fruits with a content of 0.81 to 1.70 mg gae g-1 (taruscio et al. 2004), which denotes the importance of consumption of the common bean studied in this work. according to rodriguez et al. (2021) the differences in phenolic composition between varieties may be related to the color of the seed coat, however, other studies have suggested that this variability is due more to genotype than to color, as well as to environmental conditions under which it is produced. a b a a ab ab 0 20 40 60 80 100 120 ´sangre de toro´/98 ´peruano´/100 ´flor de mayo´/101 ´negro´/102 ´pinto´/103 ´negro´/104 a n ti ox id an t c ap ac it y of c om m on b ea n v ar ie ti es (% i n h ib it io n ) bean variety/clasification number chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 16 phytic acid is bound to minerals and does not allow mineral availability due to its chelating property, it has been reported to inhibit the absorption of fe, zn, ca, mg and mn (gupta et al., 2015). however, it is also considered an antioxidant and anti-carcinogen with potential human health benefits. this accounts for 60 to 85% of the total p in the seed (akond et al., 2011b). in the present study, phytic acid concentration ranged from 0.149 to 0.194 g 100-1 g-1, with no significant difference between varieties. similar results were disclosed by iniestra-gonzález et al. (2005) on a group of 16 mexican common bean varieties with different bean types, ‘negro’, ‘pinto’, ‘crema’ or ‘bayo’, ‘azufrado’, ‘flor de mayo’ and ‘blanco’. while they were much lower than those obtained by díaz-batalla et al. (2006) in wild and cultivated varieties of mexican common raw and cooked beans and by de paula et al. (2018) in different genotypes of colombian cowpea, in which the control ‘criollo córdova’ showed the maximum value with 12.27 mg g-1. on the other hand, the results obtained were superior to those shown by akond et al. (2011a) in several common bean genotypes from usa, brazil and the international center for tropical agriculture. although phytic acid is considered an anti-nutrient, it does not represent a problem and can positively affect human health if a diverse diet is consumed in which micronutrient intake and bioavailability are high (diaz-batalla et al., 2006). flavonols quercetin and kaemferol are the most important flavonoids in foods and their consumption has been linked to an inverse association between lung cancer and risk of cardiovascular disease (díaz-batalla et al., 2006). their presence affects the flavour and color of common bean (yang et al., 2018). in the present research, flavonoids were present in a concentration ranging from 0.275 to 1.991 mg ce g-1. ‘peruano’ bean had the lowest concentration, which showed no significant difference with ‘negro’/104, ‘negro’/102 and ‘pinto’ bean. whereas ‘flor de mayo’ had the highest content of these nutraceutical compounds, whose concentration was statistically equal to that obtained in the ‘sangre de toro’ variety. result obtained in ‘flor de mayo’ was slightly higher than that reported by herrera-hernández et al. (2018) in this same variety but grown in zacatecas, mexico, in general the analysed varieties in this study had a higher content of flavonoids than those evaluated by those authors such as ‘bayo’ bean, ‘flor de junio’, ‘reata’, ‘canario’, ‘pinto saltillo’, ‘negro’ among others. other studies have shown that there is a difference in the content of these nutraceutical compounds between the seed coat and the whole grain (aquino-bolaños et al., 2016). these authors disclosed results lower than those found in the present work in seed samples from 26 common bean populations collected in several rural communities in the states of oaxaca, guerrero, puebla, tlaxcala and estado de méxico, presenting a range in whole seed from 0.10 to 0.78 mg ce g-1. rodriguez et al. (2021) observed that color influences seed flavonoid content in genotypes of spanish origin, with those of white coat having the lowest amount and those of red color the highest, which coincides with the results obtained in this study with the ‘sangre de toro’ variety. other lines that showed high concentrations of these nutraceutical compounds were pink, brown and black, which does not coincide with the results found in the present work. anthocyanins constitute one of the most important groups of natural pigments and are responsible for many of the colors of fruits and vegetables, as well as flowers; in beans they are present in higher amounts in black or blue-violet seeds (guevara-lara et al., 2006). anthocyanins in the samples analysed ranged from 0.763 to 2.400 mg c3g g-1. no significant difference was found between ‘pinto’ and ‘negro’/104 beans, which had the lowest concentration of these compounds, while ‘sangre de toro’, ‘peruano’ and ‘flor de mayo’ showed the highest values with no statistical difference between them. results were higher than those shown by guevara-lara et al. (2006) in wild bean and weed samples from chiapas, chihuahua, durango, guerrero, jalisco, michoacán, morelos, nayarit, oaxaca, sinaloa and zacatecas. likewise, values obtained also exceeded the results presented by reynoso-camacho et al. (2007) in ‘pinto zapata’, ‘flor de mayo’, ‘anita’ and ‘white tlaxcala’ beans, but were lower than those obtained in the ‘flor de junio marcela’ variety. they were also higher than the values reported by herrera-hernández et al. (2018) in several common bean varieties grown in the state of zacatecas mexico. differences in the content of these bioactive compounds are due to factors such as genotype and place of origin. in contrast to the present study, rodriguez et al. (2021) found the highest concentration of monomeric anthocyanins in black coat bean samples with an average of 4.40 mg c3g g-1. this chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 17 same result was obtained by salinas-moreno et al. (2005) in 15 mexican black bean varieties. whereas aquinobolaños et al. (2016) stated that the highest concentration of this bioactive compound was present in the cream-pinkish varieties, with similarity to that obtained in ‘flor de mayo’ variety of the current study. meaning that the color may be independent of the anthocyanin content in the common bean. in summary, of all the varieties analysed, ‘negro’/104 beans had the lowest concentration of total phenols, flavonoids and anthocyanins, while ‘flor de mayo’ and ‘sangre de toro’ had the highest values. table 8table 8table 8table 8. nutraceutical compounds of common bean varieties produced in morelos state, mexico variety/ classification number* total phenols (mg eag g-1) phytic acid (g100-1 g-1) flavonoids (mg ce g-1) anthocyanins (mg c3g g-1) ‘sangre de toro’/98 2.89 ± 0.40ab 0.18 ± 0.009a 1.579 ± 0.10a 2.400 ± 0.11a ‘peruano’/100 2.24 ± 0.83b 0.182 ± 0.02a 0.275 ± 0.07b 2.147 ± 0.22a ‘flor de mayo’/101 4.24 ± 0.57a 0.170 ± 0.01a 1.991 ± 0.09a 2.134 ± 0.18a ‘negro’/102 1.97 ± 0.57b 0.194 ± 0.08a 0.518 ± 0.12b 1.599 ± 0.07b ‘pinto’/103 2.19 ± 0.28b 0.175 ± 0.05a 0.638 ± 0.27b 0.763 ± 0.15c ‘negro’/104 1.76 ± 0.26b 0.149 ± 0.01a 0.416 ± 0.16b 1.133 ± 0.20c data correspond to mean ± sd. different letters per column indicate significant statistical difference between varieties. tukey test (p ≤ 0.05). *laboratory work classification correlation analysis table 9 shows the pearson correlation coefficients obtained between color variables, nutraceutical compounds and antioxidant ability. table 9.table 9.table 9.table 9. pearson correlation coefficients between color variables, nutraceutical compounds and antioxidant capacity of different varieties of common bean grown in morelos state, mexico. l a* b* chroma °hue af ac tf flavonoids anthocyanins l 1 a* -0.13 1 b* 0.92** -0.13 1 chroma 0.69* 0.42 0.82** 1 °hue 0.70* 0.29 0.58* 0.60* 1 af 0.05 0.03 0.08 0.10 0.15 1 ac -0.38 0.44 -0.56* -0.36 -0.01 0.11 1 tf 0.14 0.64* 0.06 0.31 0.34 -0.21 0.50* 1 flavonoids -0.10 0.83** -0.16 0.23 0.28 0.04 0.61* 0.86** 1 anthocyanins -0.04 0.54* 0.22 0.57* 0.08 0.10 -0.08 0.429 0.49* 1 *significant linear correlation (p < 0.05); **highly significant linear correlation (p < 0.0001). °hue = hue angle. fa = phytic acid. ac = antioxidant capacity. tf = total phenols. brightness showed a highly significant positive correlation with b* value (r=0.92), as well as significant with chroma (r=0.69) and hue angle (r=0.70). likewise, a* value was highly positively correlated with flavonoids (r=0.83) and significantly with total phenols (r=0.64) as well as anthocyanins (r=0.54). as for b* value, it showed a highly significant positive linear correlation with chroma (r=0.82) and significant with hue angle (r=0.58), it also had a significant negative correlation (r=-0.56) with antioxidant capacity. on the other hand, chroma correlated positively and significantly with hue angle (r=0.60) and anthocyanins (r=0.60). while phytic acid showed no correlation with any of the color variables and nutraceutical compounds evaluated; total chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 18 phenols were highly positively correlated with flavonoids (r=0.86) and significantly correlated with antioxidant capacity (r=0.50) whereas flavonoids were significantly and positively correlated with anthocyanins (r=0.49). the results of the present investigation revealed an important contribution of nutraceutical compounds such as total phenols, flavonoids and anthocyanins in the a* value of common bean grown in the producing region of morelos state, mexico. some authors have suggested that the color of the coat of this crop is closely related to the content of several phenolic compounds, prominent among them being flavonoids (yang et al., 2018) likewise, in the current work a good correlation was observed between total phenols and flavonoids with antioxidant capacity, this result has been obtained by other authors (rodriguez et al., 2021; mastura et al., 2017). however, contrary to what was obtained by other authors (aquino-bolaños et al., 2016), in this study no linear correlation (p > 0.05) was observed between anthocyanins and antioxidant capacity. conclusionsconclusionsconclusionsconclusions the current research showed that genotype has a significant effect on antioxidant capacity, and nutraceutical compound content in bean seed. obtained colour values coincided with the coloration perceived in the different varieties evaluated. thus, lower lightness was observed in varieties with darker colours such as ‘negro’/102, ‘negro’/104 and ‘sangre de toro’ as well as higher lightness in lighter coloured varieties such as ‘peruano’ bean, ‘pinto’ and ‘flor de mayo’. furthermore, the highest a* value corresponded to the varieties with a greater tendency to red coloration, such as ‘sangre de toro’, and the highest b* value corresponded to those with a greater tendency to yellow coloration, such as ‘peruano’. on the other hand, the relationship between color and nutraceutical compound content was not very clear in the present study, since the only component that had a significant positive correlation with these was the a* value, which may indicate that the content of these compounds in the seed depends more on the genotype than on the seed coat color. in terms of nutritional and nutraceutical quality, of all the varieties studied, ‘flor de mayo’ beans had the highest crude fiber content, total phenols, flavonoids, anthocyanins and antioxidant ability, and, after ‘peruano’ beans, the highest protein content. the correlation found between flavonoids and total phenols with antioxidant capacity shows these nutraceutical compounds as important indicators of antioxidant capacity of the seeds evaluated. finally, the study showed that the bean varieties produced in morelos state, mexico, may be of interest from a functional and nutritional point of view. likewise, these varieties are favourable lines to be biofortified or used in genetic improvement programs in the future, for benefiting the population whose basic source of protein is this legume or those with scarce resources. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions both authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. chávez-mendoza c and sánchez e (2023). not sci biol 15(1):11353 19 acknowledgementsacknowledgementsacknowledgementsacknowledgements we thank to consejo nacional de ciencia y tecnología (conacyt)-méxico (national council of science and technology, mexico) for the support granted in the "convocatoria proyectos de desarrollo científico para atender problemas nacionales 2015" (call for scientific development projects to address national problems 2015), to project 1529 "biofortificación de cultivos agrícolas básicos, clave para combatir la desnutrición" (biofortification of basic agricultural crops, key to fight malnutrition). conflict ofconflict ofconflict ofconflict of interestsinterestsinterestsinterests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences aguilera y, estrella i, benitez v, esteban rm, martín-cabrejas ma (2011). bioactive phenolic compounds and functional properties of dehydrated bean flours. food research international 44:774-780. https://doi.org/10.1016/j.foodres.2011.01.004 aguirre sea, gómez-aldapa ca (2010). evaluación de las características fisicoquímicas en la especie de frijol phaseolus vulgaris de las variedades pinto saltillo, bayo victoria y negro san luis [evaluation of the physicochemical characteristics in the bean species phaseolus vulgaris of the varieties pinto saltillo, bayo victoria and negro san luis]. in: proceedings of the xii congreso nacional de ciencia y tecnología de alimentos, guanajuato, méxico pp 27-28. aguirre-santos ea, rodríguez-miranda j, rosales-serna r, castro-rosas 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the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chemistry 64:555-559. https://doi.org/10.1016/s0308-8146(98)00102-2 the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material 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through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania rawani a (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11325 doi:10.15835/nsb15111325 rererereviewviewviewview articlearticlearticlearticle.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae potential biological control agents for the control of vector potential biological control agents for the control of vector potential biological control agents for the control of vector potential biological control agents for the control of vector mosquitoes: a reviewmosquitoes: a reviewmosquitoes: a reviewmosquitoes: a review anjali rawani university of gour banga, malda, department of zoology, laboratory of parasitology, vector biology, and nanotechnology, west bengal, india; micanjali@gmail.com abstractabstractabstractabstract mosquitoes are a major cause of lethal vector-borne diseases like dengue, malaria, filariasis, chikungunya, and japanese encephalitis, among other diseases. in a developing country like india, mosquito-borne diseases are significant threats to familiar people as in certain places, there remains low sanitation. larval and pupal life stages of mosquitoes are mostly confined to tropical and temperate waterbodies and often form a significant proportion of biomass waterbodies. due to rebound vectorial capacity, resistance to chemical insecticides, and harmful environmental effects, the vector control program has shifted to using biological control agents. these methods are target-specific, eco-friendly, cost-effective, and can be easily deployed. so, the present review is focused on collating and updating the information on the use of aquatic predators, bacterial strains such as bacillus sp. and actinobacterial, algae, and fungi, which are widely used for control of adult mosquitoes in their variety of natural habitats. this review also covers the predation of larvivorous fish and botanical insecticides. keywords:keywords:keywords:keywords: aquatic predators; biological control; botanical pesticides; larvivorous fishes; microbial agents; vector mosquitoes introductionintroductionintroductionintroduction mosquitoes are medically important insects, not only as nuisance biters, but also act as a vector of some harmful pathogens causing dreadful diseases of humans like malaria, dengue, yellow fever, chikungunya, zika, and japanese encephalitis among other diseases (chandra et al., 2008a). in 124 tropical and subtropical countries, around 55% of the population is at risk of these diseases (beatty et al., 2007). malaria is transmitted by ten anopheline species in india of which six are of primary importance. the primary vectors in rural areas are anopheles culicifacies and in urban areas is anopheles stephensi. malaria affects 36% of the world’s population, i.e., 0.241 billion people in 107 countries (world malaria report, 2021). according to who (1981), in the south east asian region, 2.5 million (85.7%) people are at risk of malaria. india alone contributes about 70% of the total cases among the 2.5 million reported cases in south east asia (kondrachine, 1992). approximately 102 million cases of filariasis are primarily transmitted by culex quinquefasciatus. nearly 1,100 million people are living in areas endemic to lymphatic filariasis were in most cases either have patent microfilaraeimeia or chronic filarial disease (michael et al., 1996). aedes aegypti is responsible for dengue fever and yellow fever in https://www.notulaebiologicae.ro/index.php/nsb/index rawani a (2023). not sci biol 15(1):11325 2 india (who, 2017). dengue infection is endemic in over 100 countries worldwide and each year it causes nearly 100 million cases of dengue fever, 500000 cases of dengue haemorrhagic fever and 24,000 deaths (gibbons and vaughn, 2002; guha-sapir and schimmer, 2005). the world health organization embrace vector mosquito control as the only measures to prevent or control such diseases. although interest in biological control of mosquitoes agents was large at the beginning of the 20th century, it is stopped since discovery of the insecticidal properties of the ddt in 1939. but this insecticide has the deleterious effects for health and environment, so alternatives to chemical insecticide become necessary. currently, the most important measures to control these diseases are mosquito control and personal protection from mosquito bites. vector control strategies mainly include the use of chemical insecticides, plant-based insecticides, and biological control agents (poopathi and tyagi, 2006). in the past several decades, a number of chemical insecticides have been effectively used to control mosquitoes. constant use of man -made insecticides for mosquito control disrupts natural biological control systems and lead to re-emergence of mosquito populations. it also resulted in the development of resistance in target organisms, harmful effects on non-target organisms and human health problems and subsequently this initiated a search for alternative control measures (das et al., 2007). these problems have prompted researchers to look for alternative vector control agents with high efficacy and low or no adverse effects on environment and human health. the development of new strategies includes natural insecticides which is an important tool to counter the evolution of resistance among the target organisms without affecting the non-target organisms (cetin and yanikoglu, 2006). besides the natural insecticides, biocontrol agents are also given importance in order to combat the population of mosquito as these are target specific, nontoxic to environment, find easy application in the field, cost effective, lack infectivity and pathogenicity in mammals including man and has little and no evidence of resistance in target mosquito species. the use of biological control agents such as predatory fish, bacteria, algae, fungus and aquatic insects had shown promising results to control mosquito populations (murugesan et al., 2009). the present review, work, is the assemblage of a wide range of biological control agents along with their updated information. the objective of the present review is to compile the all-biological control agents which have been reported so far for the control of vector mosquitoes. aquatic insects and aquatic insects and aquatic insects and aquatic insects and copepodscopepodscopepodscopepods mosquito's life cycle includes stages: egg, larva, pupa, and adult. the first three stages are aquatic, i.e., confined to water bodies provided a high chance for predation to aquatic insects and thus helped in the mosquito control program. aquatic insects mainly belong to orders such as coleoptera, diptera, hemiptera, and odonata and are keen to prey upon mosquito larvae. many of these predators may be shows polyphagous habits, which means they are feeding on a broad range of prey species (generalist predator); some are oligophagous, having a restricted range of prey; or monophagous, having a minimal range of prey; these are also known as specialist predators. generally, mosquito larvae feeders belong to the polyphagous type (collins and washino, 1985). these predators also predate the prey in various ways depending on their mouthparts. some predators of order odonata have chewing mouthparts, so they eat their prey. however, predators like beetle larvae and hemiptera, having a sucking mouth part, suck the prey's body fluid (hemolymph). so, in general, mosquito larvae and pupae are predated by almost all aquatic insect belongs to aquatic coleoptera (especially dytiscidae), hemiptera (especially notonectids), and odonata, have been observed to consume mosquito larvae as a part of their natural food habits (peckarsky, 1980). the backswimmer, notonecta undulata (hemiptera, notonectidae), has been shown to prey on the second instar of mosquito larvae (ellis & borden, 1970). besides the wide range of aquatic predators from the phylum arthropoda (mainly belonging to orders such as coleoptera, diptera, hemiptera, and odonata), cyclopoid copepods are a diverse group of small crustaceans and have often been used as effective biological control agents against different species of mosquitoes, such as anopheles (roa et al., 2002), aedes (riviere and thirel, 1981) and culex (marten et al., 1984). being microcrustaceans cyclopoid copepods (cyclopoids: a subclass of copepods) are not harmful to rawani a (2023). not sci biol 15(1):11325 3 human health (who, 2009) and have been used to control mosquito larvae of public health importance in artificial containers and under laboratory/field laboratory conditions in some countries like australia, thailand, vietnam, sri lanka and to some extent in the americas (marten and reid, 2007a, 2007b; vu et al., 2005). however, predation might occur at any life stage; most research focused on mosquito larval and pupal stages as they are easy to locate. in a single habitat, all three stages are confined. so, field experiments with aquatic insects become easy to carry out. predating immature stages of mosquitoes seems to be a significant component of mosquito mortality. the major aquatic predator and their prey mosquito species are listed in tables 1 and 2. table 1. table 1. table 1. table 1. list of aquatic predaceous insects and their prey name of predatorname of predatorname of predatorname of predator order: order: order: order: family of predatorfamily of predatorfamily of predatorfamily of predator mosquito prey mosquito prey mosquito prey mosquito prey (larval stage)(larval stage)(larval stage)(larval stage) referencesreferencesreferencesreferences acilius sulcatus coleoptera: dytiscidae cx. quinquefasciatus chandra et al., 2008b agabus erichsoni coleoptera: dytiscidae ae. communis nilsson and soderstrom, 1988 colymbetes paykulli coleoptera: dytiscidae culex sp. lundkvist et al., 2003 dytiscus marginicolis coleoptera: dytiscidae culiseta incidens lee, 1967 lestes congener odonata: lestidae culiseta incidens lee, 1967 lacconectus punctipennis coleoptera: dytiscidae ae. albopictus sulaiman and jeffery, 1986 rhantus sikkimensis coleoptera: dytiscidae cx. quinquefasciatus aditya et al., 2006 toxorhynchites splendens diptera: culicidae cx. quinquefasciatus aditya et al., 2006 aeshna flavifrons odonata: aeshnidae cx. quinquefasciatus mandal et al., 2008 coenagrion kashmirum odonata: coenagrionidae cx. quinquefasciatus mandal et al., 2008 ischnura forcipata odonata: coenagrionidae cx. quinquefasciatus mandal et al., 2008 rhinocypha ignipennis odonata: chlorocyphidae cx. quinquefasciatus mandal et al., 2008 sympetrum durum odonata: libellulidae cx. quinquefasciatus mandal et al., 2008 brachytron pratense odonata: aeshnidae an. subpictus chatterjee et al., 2007 crocothemis servilia odonata: libellulidae ae. aegypti sebastian et al., 1990 enallagma civile odonata: coenagrionidae cx. tarsalis miura and takahashi, 1988 labellula sp. odonata: libellulidae ae. aegypti bay, 1974 orthemis ferruginea odonata: libellulidae ae. aegypti sebastian et al., 1980 orthemis ferruginea odonata: libellulidae an. pharoensis cordoba and lee, 1995 tramea torosa odonata: libellulidae an. pharoensis lee, 1967 trithemis annulata odonata: libellulidae an. gambiae el rayah, 1975 abedus indentatus hemiptera: belostomatidae cx. annulirostris washino, 1969 anisops sp. hemiptera: notonectidae cx. annulirostris shaalan et al., 2007 diplonychus sp. hemiptera: notonectidae cx. annulirostris shaalan et al., 2007 diplonychus indicus hemiptera: belostomatidae ae. aegypti venkatesan and sivaraman, 1984 buenoa scimitar hemiptera: notonectidae cx. quinquefasciatus rodríguez-castro et al., 2006 diplonychus rusticus heteroptera: notonectidae cx. quinquefasciatus saha et al., 2007 diplonychus annulatus heteroptera: notonectidae cx. quinquefasciatus saha et al., 2007 anisops bouvieri heteroptera: notonectidae cx. quinquefasciatus saha et al., 2007 rawani a (2023). not sci biol 15(1):11325 4 enithares indica hemiptera: notonectidae anophiline, culicine and aedine wattal et al., 1996 notonecta sellata hemiptera: notonectidae cx. pipiens fischer et al., 2012 notonecta hoffmani hemiptera: notonectidae cx. pipiens murdoch et al., 1984; chesson, 1989 anisops breddini hemiptera: notonectidae ae. aegypti weterings et al., 2014 notonecta undulate hemiptera: notonectidae ae. aegypti quiroz-martinez and rodriguez-castro, 2007 anopheles barberi diptera: culicidae cx. pipiens petersen et al., 1969 anopheles gambiae diptera: culicidae an. gambiae koenraadt and takken, 2003 bezzia expolita diptera: ceratopogonidae ae. aegypti hribar and mullen, 1991 chaoborus crystallinus diptera: chaoboridae ae. aegypti bay, 1974 ochthera chalybesceens diptera: ephydridae an. gambiae minakawa et al., 2007 toxorhynchites amboinensis diptera: culicidae ae. aegypti digma et al., 2019 toxorhynchites brevipalpis diptera: culicidae aedes sp. gerberg and visser, 1978 toxorhynchites brevipalpis conradti diptera: culicidae ae. africanus sempala, 1983 toxorhynchites kaimosi diptera: culicidae ae. africanus sempala, 1983 toxorhynchites rutilus rutilus diptera: culicidae ae. aegypti padgett and focks, 1981; focks et al., 1982 toxorhynchites guadeloupensis diptera: culicidae ae. aegypti honório et al., 2007 anisops sardea hemiptera: notonectidae cx. quinquefasciatus mondal et al., 2014 lacotrephes griseus heteroptera: nepidae cx. quinquefasciatus ghosh and chandra, 2011 ranatra elongata heteroptera: nepidae cx. quinquefasciatus saha et al., 2020 ranatra filiformis heteroptera: nepidae cx. quinquefasciatus saha et al., 2020 lacotrephes griseus heteroptera: nepidae cx. quinquefasciatus saha et al., 2020 table 2. table 2. table 2. table 2. list of aquatic predaceous copepods (subphylum: crustacea; subclass: copepoda) and their prey species name of predatorname of predatorname of predatorname of predator mosquito prey (larval stage)mosquito prey (larval stage)mosquito prey (larval stage)mosquito prey (larval stage) referencesreferencesreferencesreferences mesocyclops sp. ae. aegypti vu et al., 2012 mesocyclops sp. ae. aegypti vu et al., 2005 mesocyclops sp. ae. aegypti kay et al., 2002 mesocyclops thermocyclopoides ae. aegpyti soto et al., 1999 mesocyclops darwini ae. aegpyti marten et al., 1994a,b cyclopoid an. aquasalis roa et al., 2002 mesocyclops leuckartipilosa ae. aegypti ae. polynesiensis riviere and thirel, 1981 macrocyclops albidus mesocyclops longisetus aedes sp anopheles sp. marten et al., 1997 mesocyclops australiensis aedes sp. brown et al., 1991 mesocyclops leuckuarti aedes marten, 1984 rawani a (2023). not sci biol 15(1):11325 5 mesocyclops scarassus cyclops varicans cyclops languides anopheles sp. ranathunge et al., 2019 cyclops varicans cyclops languides cyclops vernalis mesocyclops leukart mesocyclops scarassus ae. aegypti ae. albopictus udayanga et al., 2019 macrocyclops albidus mesocyclops leukarti ae. koreicus baldacchino et al., 2017 cyclopoid sp. aedes sp. russell et al., 2021 cyclopoid sp. aedes japonicus linus et al., 2019 calanoid copepod aedes sp. cuthbert et al., 2018 fungifungifungifungi the search for effective mosquito pathogens that can be used in mosquito control operations has been going on for several years. both laboratory and field studies have been carried out on those fungi that have shown mosquito larvicide efficacy. many species of fungi are currently being considered for use in the microbial control of mosquito larvae. several fungus species of the genus coelomomyces, lagenidium, metarhizium, culicinomyces, entomophthora, etc., have a potential biocidal effect against mosquito larvae (scholte et al., 2004). however, some researchers have reported the mode of action of these fungal species, which depicts that they generally affect the cuticle and abdomen of the mosquito larvae (butt et al., 2013). at first, the fungal species spores' adhesion occurs at the mosquito larvae's cuticle. then the spore becomes generated, penetrating the cuticle where the growth and development occur in the hemocoel (brian, 2009). the saprophytic feeding starts, fungal re-emergence, and ultimately the larva dies. they are also the possible routes of invasion by the fungus to cause the mortality of larvae (seye et al., 2009). these are the main sites of infection when treated with fungal formulations (bukhari et al., 2011). the attachment and growth of the fungus into the perispiracular valves of the siphon tube causes the blockage of air intake through respiration, thus leading to the death of the larvae (butt et al., 2013). in table 3, there is information about the essential fungi which have been reported so far in the mosquito control program. table 3.table 3.table 3.table 3. list of fungal species reported against mosquito larvae fungal speciesfungal speciesfungal speciesfungal species mosquito hostmosquito hostmosquito hostmosquito host referencesreferencesreferencesreferences beauveria bassiana culex sp. aedes sp. anopheles sp. clark et al., 1968 beauveria tenella aedes aegypti aedes dorsalis pinnock et al., 1973 crypticola clavulifera aedes aegypti frances et al., 1989 coelomomyces indicus anopheles arabiensis anopheles culicifacies anopheles gambiae anopheles indificus service, 1977 service, 1977 muspratt, 1963 whisler et al., 1999 coelomomyces lairdi anopheles punctulatus maffi and nolan, 1977 rawani a (2023). not sci biol 15(1):11325 6 c. psorophorae var. psorophorae ochlerotus taeniorhynchus aedes cinereus aedes vexans federici and roberts, 1975; popelkova, 1982; mitchell, 1976; goettel, 1987a c. stegomyiae var. stegomyiae aedes aegypti aedes albopictus aedes scutellaris shoulkamy et al., 1997; lucarotti and shoulkamy, 2000; laird et al., 1992; ramos et al., 1996; padua et al., 1986; laird, 1967 coelomomyces punctatus anopheles crucians anopheles quadrimaculatus pillai and rakai, 1970 conidiobolus destruens culex pipiens mietkiewski and van der geest, 1985 coelomomyces polynesiensis aedes polynesiensis pillai and rakai, 1970 coelomomyces maclaeyae aedes polynesiensis pillai and rakai, 1970 coelomomyces numularius anopheles squamosus ribeiro and da cunha ramos, 2000 coelomomyces pentangulatus culex erraticus ribeiro and da cunha ramos, 2000 coelomomyces angolensis culex guiarti ribeiro, 1992 culicinomyces bisporales aedes kochi sigler et al., 1987 culicinomyces spp. (unidentified) anopheles amictushilli culex quinquefasciatus sweeney, 1978a culicinomyces clavisporus aedes aegypti aedes rubrithorax aedes atropalpus epactius anopheles farauti anopheles stephensi cooper and sweeney, 1982 frances, 1986 couch et al., 1974 sweeney, 1978b couch et al., 1974 culicinomyces clavisporus culex erraticus culex territans culex quinquefasciatus couch et al., 1974 entomophthora culicis an. stephensi culex pipiens culex spp kramer, 1982; roberts, 1974, roberts and strand, 1977 entomophthora conglomerata culex pipiens roberts, 1974 entomophthora coronata culex quinquefasciatus lowe et al., 1968; low and kennel, 1972 entomophthora musca aedes aegypti steinkraus and kramer, 1987 eryinia conica aedes aegypti culex restuans cuebas-incle, 1992 fusarium oxysporum aedes detritus culex pipiens husan and vago, 1972; breaud et al., 1980 fusarium culmorum culex pipiens badran and aly, 1995 fusarium dimerum culex pipiens badran and aly, 1995 leptolegnia sp. (unidentified) aedes albopictus anopheles gambiae mansonia titillans mansonia dyari fukuda et al., 1997 nnakumusana, 1986 lord and fukuda, 1990 lord and fukuda, 1990 rawani a (2023). not sci biol 15(1):11325 7 leptolegnia chapmanii aedes aegypti culex quinquefasciatus anopheles albimanus anopheles quadrimaculatus mcinnis and zattau, 1982; lord and fukuda, 1990; mcinnis and zattau, 1982 mcinnis and zattau, 1982 leptolegnia caudata anopheles culicifacies bisht et al., 1996 lagenidium giganteum aedes aegypti anopheles freeborni culex pipiens rueda et al., 1990; golkar et al., 1993 kerwin et al., 1994 golkar et al., 1993; kerwin et al., 1994 metarhizium anisopliae aedes aegypti aedes albopictus culex quinquefasciatus culex pipiens anopheles stephensi ramoska et al., 1981; daoust et al., 1982 ravallec et al., 1989 ramoska et al., 1981; lacey et al., 1988 daoust and roberts, 1982 pythium carolinianum aedes albopictus culex quinquefasciatus su et al., 2001 pythium sierrensis anopheles freeborni culex tarsalis ochlerotus triseriatus uranotaenia anhydor clark et al., 1966 pythium sp. culex tigripes culex quinquefasciatus nnakumusana, 1985 paecilomyces lilacinus aedes aegypti agarwalda et al., 1999 pythium sp. culex tigripes culex quinquefasciatus nnakumusana, 1985 smittium morbosum anopheles hilli aedes albifasciatus sweeney, 1981d garcia et al., 1994 tolypocladium cylinddrosporum aedes aegypti aedes vexans culiseta inornata culex tarsalis ochlerotus triseriatus goettel, 1988 goettel, 1987b goettel 1987b soares, 1982 nadeau and biosvert, 1994 trichophyton ajelloi anopheles stephensi culex quinquefasciatus mohanty and prakash, 2000 verticillium lecanii ochlerotus triseritus ballard and knapp, 1984 zoophthora radicans aedes aegypti dumas and papierok, 1989 algaealgaealgaealgae mosquito larvae in their aquatic environment feed on microorganisms, small aquatic animals such as rotifers, and other small particulate matter. however, some mosquito larvae also partially or substantially depend on algal mass as a part of their diet (merritt et al., 1992a). mosquito larvae filter water and algae from the column, and the study suggested that in the gut, after the feeding, algae are generally presented in proportion to their abundance along with the microflora and microfauna (ranasinghe and amarasinghe, 2020). coggeshall (1926) experimented with a pond with high algae production where anopheles sp. is the foremost breeder and found an abundance of algae in their gut and a high population of anopheles sp. in the pond. rawani a (2023). not sci biol 15(1):11325 8 though an abundance of algae in mosquito breeding sites favours their development, some of the algal sp. might be responsible for larval mortality. purdy (1924) revealed that some algal species could kill mosquito larvae. he found that in the california rice field, there was a dense layer of filamentous algae (blue-green algae) tolypothrix sp., the larvae of culex sp. and anopheles sp. were absent. however, larvae of these mosquito species are found in the nearby pond where these blue-green algae were absent. the blue-green algae of order chlorococcales are ill-digested by mosquito larvae. some of the algae of this order is entirely indigestible (marten, 2007). some toxins (such as photometabolite toxic produced by blue-green algae anabaena sp.; microcystins toxic produced by oscillatoria agardhii and anabaena circinalis) released by these algae might be responsible for larval mortality, the same toxin that causes algal bloom in the pond that kills fish and cattle (ingram and prescott, 1954). so, algae are not enough, but their toxins might be a point of interest in developing insecticides to control the mosquito population. in table 4, there is a list of algae that showed the mosquito larvicide activity. table 4table 4table 4table 4. list of algal species reported as mosquito larvicide algal algal algal algal speciesspeciesspeciesspecies mosquito speciesmosquito speciesmosquito speciesmosquito species referencesreferencesreferencesreferences anabaena unispora aedes aegypti griffin, 1956 anabaena circinalis aedes aegypti griffin, 1956 aulosira implexa culex tarsalis gerhardt, 1953 chlorella ellipsoidea culex quinquefasciatus, dhillon and mulla, 1981 rhizoclonium hieriglyphicum aedes aegypti, culex quinquefasciatus, dhillon et al., 1982 kirchneriella irregularis aedes albopictus marten,1984 kirchneriella irregularis cx. quinquefasciatus marten, 1986a chlorella protothecoides ae. albopictus marten, 1986b ulva lactuca, caulerpa racemosa, sargassum microystum, caulerpa scalpelliformis, gracilaria corticata, turbinaria decurrens, turbinaria conoides and caulerpa toxifolia 4th instar larvae of aedes aegypti, culex quinquefasciatus, anopheles stephensi syed ali et al., 2013 westiellopsis sp. aedes aegypti, culex quinquefasciatus, anopheles stephensi and culex tritaenorhynchus rao et al., 1999 cyanobacteria (blue-green algae) culex sp. marten, 2007 selenastrum capricornatum 3rd instar cx. quinquefasciatus larvae duguma et al., 2017 ulva lactuca (chlorophyta), padina gymnospora, sargassum vulgare (phaeophyta), hypnea musciformis, and digenea simplex (rhodophyta) aedes aegypti guedes et al., 2014 lobophora variegate, spatoglossum asperum, stoechospermum marginatum, sargassum wightii, cx. quinquefasciatus ae. aegypti manilal et al., 2011 rawani a (2023). not sci biol 15(1):11325 9 acrosiphonia orientalis, centroceras clavulatum, padina tetrastromatica larvivorous fishlarvivorous fishlarvivorous fishlarvivorous fish now a day, in developed and developing countries, particularly in urban and peri urban areas, malaria control programs alternatively focus on biological control, which includes a wide range of organisms that helps to control mosquito populations naturally through predation, parasitism, and competition. among them, the use of larvivorous fish was also found to be most effective in the mosquito control program. biological control is the process of the introduction or manipulation of organisms to suppress vector populations. since early 1900, all over the world, larvivorous fish have been used extensively as biological mosquito control agents (chandra et al., 2008a). larvivorous fish means that they feed voraciously on immature stages of mosquitoes. some features are required to meet the criteria of larvivorous fishes, such as must be small, hardy, and capable of getting about quickly in shallow waters amid thick weeds where mosquitoes find suitable breeding grounds. they must have the capacity to live in drinking water tanks and pools, move in deep and shallow waters, and withstand drought (fletcher et al., 1992). they must have the capability to survive through rough handling and transportation for long distances. the fish species must be productive breeders with a short life span that can breed in confined water. another important criterion is that it must be carnivorous and very keen to consume mosquito larvae even in the presence of other food materials, bringing out the outstanding result in regulating the mosquito population. the appearance of larvivorous fish is also a point of selection; it should not be brightly coloured. the fish should not have a nutrition value, so it will not be attractive to fish-eating people (haq and srivastava, 2013). hence, the larvivorous fish should meet the maximum criteria stated above to bring out mosquito control effectively. tables 5 and 6 represent the list of indigenous and exotic fish as biocontrol agents. table 5. table 5. table 5. table 5. potential indigenous larvivorous fishes as biocontrol agent larvivorous fisheslarvivorous fisheslarvivorous fisheslarvivorous fishes mosquito speciesmosquito speciesmosquito speciesmosquito species referencereferencereferencereference aphanius dispar. culex quinquefasciatus. anopheles arabiensis. anopheles gambiae. fletcher 1992; haq and srivastava, 2013; howard et al., 2007; imbahale et al., 2011; ataurrahim, 1981 aplocheilus blockii. anopheles stephensi. aedes albopictus. menon and rajagopalan, 1978; kumar et al., 1998 aplocheilus lineatus. aedes aegypti. mesv, 1988 aplocheilus panchax. an. culicifacies. an. sundaicus. cx. quinquefasciatus. cx. vishnui. mesv, 1988 colisa fasciatus. mansonioides indiana edward, 1930. anopheles annularis van der wulp,1884. mesv, 1988 sharma and ghosh, 1989 colisa lalia. an. annularis. mesv, 1988 colisa sota. an. annularis. mesv, 1988 chanda nama. an. culicifacies. an. balabocensis balabocensis. an. varuna. mesv, 1988 oryzias melastigma. cx. vishnui. sharma and ghosh, 1989 rawani a (2023). not sci biol 15(1):11325 10 anopheles sp. culex sp. macropodus cupanus. cx. fatigans. mathavan, 1980 tilapia mossambicus and aplocheilus latipes an. sinensis kim et al., 2002; yu and lee,1989 oreochromis niloticus cx. quinquefasciatus. ghosh and chandra, 2017 table 6table 6table 6table 6. potential exotic larvivorous fishes as biocontrol agent larvivorous fish mosquito species reference carassius auratus an. subpictus. cx. quinquefasciatus. ar. subalbatus. chatterjee et al., 1997 gambusia affinis an. subpictus. cx. quinquefasciatus. an. subalbatus. ae. aegypti. an. stephensi. an. gambiae. chatterjee and chandra, 1997 rtdc, 2008 zvantsov, 2008 mahmoud, 1985 imbahale et al., 2011 poecilia reticulata (guppy) an. subpictus. an. gambiae. an. subpictus. an. funestus sitaraman et al., 1976; menon and rajagopalan, 1978; sabatinelli et al., 1991 howard et al., 2007; kusumawathie et al., 2008 a, b xenentodon cancila an. subpictus. cx. quinquefasciatus. ar. subalbatus. chatterjee and chandra, 1996 oreochromis mossambica (tilapia) cx. quinquefasciatus. an. culicifacies. sharma and ghosh, 1989 oreochromis niloticus an. gambiae an. funestus howard et al., 2007; ghosh et al., 2006; ghosh and chandra, 2017; ambrose et al., 1993; kim et al., 1994 nothobranchius guentheri anopheles sp. vanderplank, 1941 hyphessobrycon rosaceus cx. vishnui barik et al., 2018 puntius tetrazona cx. vishnui barik et al., 2018 bacteriabacteriabacteriabacteria in biological control, microbial biopesticides also play a significant role in controlling the vector mosquito population. many researchers have reported some bacterial species that are useful to implement in mosquito control programs. bacillus thuringiensis, and bacillus sphericus can be noble alternatives for synthetic pesticides. for the last two decades, their effectiveness has been reported against anopheles, culex, and aedes. however, bacillus sphericus was reported to be very effective against culex sp. bacterial strain is more advantageous than synthetic insecticides as they have long-lasting efficacy without affecting non-target organisms and are cost-effective and safest for the ecosystem. it neither eliminates the pathogen nor the disease but brings them into natural balance (raymond et al., 2010). bacterial strains are isolated from any insect's gut in the biopesticide industry. the soil can be a promising biopesticide agent as it shows long-lasting activity in a polluted environment (lennox et al., 2016). many authors report the mode of action of these bacterial strains. rawani a (2023). not sci biol 15(1):11325 11 the mosquito larvae take up the crystal toxin secreted by the bacterial strain. it solubilizes in the midgut, causes activation of the protoxin by protease into an active toxin, and this toxin binds to specific receptors in the midgut brush border membrane (bravo et al., 2007; silva-filha et al., 2021). then probably, toxins' internalization occurs, and cell lysis occurs (poopathi and tyagi, 2004). however, bacterial pesticides mentioned above, such as bacillus thuringiensis serotype israelensis (bti) and b. sphaericus, are highly efficacious against mosquito larvae and have been used since many decades. another promising bacterial origin, vector mosquito larvicide spinosad, has gained importance in the last decade and is being used in several countries. spinosad is an insecticide product derived via fermentation from a naturally occurring soil actinomycete, saccharopolyspora spinosa mertz and yao. spinosad contains two insecticidal factors, a and d, in an 85:15% ratio within the final product. spinosad is found to be highly active by both contact and ingestion of numerous pests in the orders lepidoptera, diptera, thysanoptera, coleoptera, orthoptera, hymenoptera, and others (hertlein et al., 2010). table 7 shows the list of bacterial strains reported as a mosquito larvicide. table 7. table 7. table 7. table 7. list of bacterial strain reported as a mosquito larvicide sl no.sl no.sl no.sl no. bacterial strainbacterial strainbacterial strainbacterial strain effective against effective against effective against effective against mosquito speciesmosquito speciesmosquito speciesmosquito species effect on effect on effect on effect on pupa/ pupa/ pupa/ pupa/ larvaelarvaelarvaelarvae referencereferencereferencereference 1 bacillus thuringiensis anopheles sp. larvae balakrishnan et al., 2015 rajendran et al., 2018 2 bacillus sphericus culex and anopheles larvae park et al., 2010 balakrishnan et al., 2015 3 bacillus alvei culex fatigans, anopheles stephensi, aedes aegypti. larvae balakrishnan et al., 2015 4 bacillus brevis culex fatigans, anopheles stephensi, aedes aegypti. larvae khyami-horani et al., 1999 5 bacillus circulans cx. quinquefasciatus and anopheles gambiae and aedes aegypti. larvae darriet and hougard, 2002 6 brevibacillus laterosporus cx. quinquefasciatus, aedes aegypti. larvae de oliveira et al., 2004 7 bacillus subtilis cx. quinquefasciatus. larvae balakrishnan et al., 2015 das and mukherjee, 2006 8 clostridium bifermentans anopheles maculates larvae de barjac et al., 1990 9 pseudomonas fluorescens anopheles stephensi, cx. quinquefasciatus, ae. aegypti. toxic to larvae and pupa jenkins, 1964 10 streptococcus species anopheles sp. and culex sp. larvae (l3, l4 stage) kramer, 1964 11 escherichia coli culex mosquito larvae (early inster) jenkins, 1964 12 bacillus cereus anopheles sp. and culex sp. larvae balakrishnan et al., 2015 13 bacillus amyloliquefacien anopheles sp larvae and pupae geetha et al., 2014 14 aneurinibacillus aneurinilyticus anopheles subpictus, cx. quinquefasciatus, aedes aegypti. larva das et al., 2016 rawani a (2023). not sci biol 15(1):11325 12 mosquito control by plant productsmosquito control by plant productsmosquito control by plant productsmosquito control by plant products insecticides of botanical origin can play an essential role in the interruption of the transmission of mosquito-borne diseases. these insecticides act as a larvicide, pupicide, repellent, oviposition deterrent, or fumigants to control the mosquito population. so, insecticides of plant origin are mainly secondary metabolites such as alkaloids, steroids, terpenoids, tannins, and flavonoids deposited in the plant for their defence purposes. earlier shaalan et al. (2005) reported that secondary metabolites from different plant species have insecticidal properties. these secondary metabolites are extracted from the plant that is either built in the body of the herb or various parts of larger plants like fruits, leaves, stems, barks, roots, etc. so, variation in insecticidal properties of these phytochemicals depends on the plant species and the geographical distribution of the plant (chowdhury et al., 2008a). besides that, extraction methodology and the polarity of the solvent used for the extraction process are also supportive forces to define the insecticidal properties of phytochemicals (chowdhury et al., 2008b). after the solvent extraction, phytochemicals containing the active principle for their mosquitocidal activity are concentrated and ready for application in the mosquito control program. here an attempt is made to give an account of the plants that has been reported so far as an insecticidal agent against various life forms of mosquito species (table 8). 15 bacillus sphaericus anopheles subpictus, cx. quinquefasciatus, armigeres subalbatus larva das et al., 2017 16 saccharopolyspora spinosa aedes aegypti an. albimanus an. pseudopunctipennis larva bond et al., 2004 aedes aegypti larva perez et al., 2007 aedes aegypti larva antonio et al., 2009 aedes aegypti an. stephensi cx. pipiens larva romi et al., 2006 aedes aegypti larva darriet and cerbel, 2006 aedes aegypti an. gambiae cx. quinquefasciatus larva darriet et al., 2005 aedes aegypti larva ayesha et al., 2006 ae. albopictus larva liu et al., 2004 a cx. quinquefasciatus larva liu et al., 2004b an. sinensis larva shin et al., 2003 cx. pipiens larva cetin et al., 2005 cx. pipiens larva bahgat et al., 2007 cx. quinquefasciatus ae. aegpti larva jiang and mulla, 2009 cx. quinquefasciatus larva sadanandane et al., 2009 cx. quinquefasciatus larva sadanandane et al., 2018 rawani a (2023). not sci biol 15(1):11325 13 table 8. table 8. table 8. table 8. list of plants and plant parts reported so far as mosquitocidal agent plant plant plant plant sssspeciespeciespeciespecies familyfamilyfamilyfamily plant plant plant plant partpartpartpartssss usedusedusedused target mosquito target mosquito target mosquito target mosquito speciesspeciesspeciesspecies referencesreferencesreferencesreferences artemisia annua asteraceae leaf anopheles stephensi sharma et al., 2006 acacia nilotica fabaceae leaf anopheles stephensi saktivadivel and daniel, 2008 argemone mexicana papaveraceae leaf, seed culex quinquefasciatus karmegan et al., 1997 jatropha curcas euphorbiaceae leaf culex quinquefasciatus rahuman et al., 2007 carica papaya caricaceae seed culex quinquefasciatus rawani et al., 2009 murraya paniculata rutaceae leaf culex quinquefasciatus rawani et al., 2009 aloe barbadensi liliaceae leaf anopheles stephensi maurya et al., 2007 solanum xanthocarpum solanaceae root culex pipiens pollens mohan et al., 2006 cleistanthus collinus euphorbiaceae leaf culex quinquefasciatus rawani et al., 2009 eucalyptus globulus myrtaceae seed, leaf culex pipiens sheeren et al., 2006 thymus capitatus lamiaceae leaf culex pipiens mansour et al., 2000 citrus aurantium rutaceae fruit peel culex quinqefasciatus kassir et al., 1989 myrtus communis myrtaceae flower and leaf culex pipiens molestus traboulsi et al., 2002 alternanthera sessilis amaranthaceae leaf cx. quinquefasciatus rawani et al., 2014 ruellia tuberosa acanthaceae leaf cx. quinquefasciatus rawani et al., 2014 trema orientalis cannabaceae leaf cx. quinquefasciatus rawani et al., 2014 gardenia carinata rubiaceae leaf cx. quinquefasciatus rawani et al., 2014 piper nigram piperaceae seed culex pipiens shaalan et al., 2005 euphorbia hirta euphorbiaceae stem bark culex quinquefasciatus rahuman et al., 2007 polianthes tuberosa agavaceae bud cx. quinquefasciatus an. stephensi rawani et al., 2012 ocimum basilicum lamiaceae leaf anopheles stephesnsi, culex quinquefasciatus maurya et al., 2009 momordica charantia cucurbitaceae fruit anopheles stephensi singh et al., 2006 kaempferia galanga zingiberaceae rhizome culex quinquefasciatus choochote et al., 1999 khaya senegalensis meliaceae leaf culex annulirostris shaalan et al., 2005 rawani a (2023). not sci biol 15(1):11325 14 acacia auriculiformis fabaceae fruit culex vishnui barik et al., 2018 curcuma aromatica zingiberaceae rhizome aedes aegypti choochate et al., 2005 cybistax antisyphilitica bignoniaceae stem wood aedes aegypti rodrigues et al., 2005 eucalyptus citriodora myrtaceae leaf anopheles stephensi, culex quinquefasciatus, aedes aegypti singh et al., 2007 solanum nigram solanaceae dried fruit anopheles culicifacies, anopheles stephensi raghavendra et al., 2009 tridax procumbens compositae leaf anopheles subpictus kamaraj et al., 2011 ageratum conyzoides asteraceae leaf culex quinquefasciatus saxena et al., 1992 cleome icosandra capparaceae leaf culex quinquefasciatus saxena et al., 1992 ageratina adenophora asteraceae twigs culex quinquefasciatus, aedes aegypti raj mahan and ramaswammy, 2007 feonia limonia rutaceae leaf culex quinquefasciatus, aedes aegypti, anopheles stephensi rahuman et al., 2000 millingtonia hortensis bignoniaceae leaf culex quinquefasciatus, anopheles stephensi, aedes aegypti kaushik and saini, 2008 ocimum sanctum labiate leaf aedes aegypti, culex quinquefasciatus anees, 2008 eucalyptu globulus myrtaceae seed and leaf culex pipiens sheeren, 2006 phumbago zeylanica, p. dawei, p. stenophylla plumbaginaceae root anopheles gambiae maniafu et al., 2009 euphorbia tirucalli euphorbiaceae latex and stem bark culex pipiens pallens yadav et al., 2002 nyctanthes arbortristis nyctantheceae flower culex quinquefasciatus khatune et al., 2001 citrus sinensis rutaceae fruit peel anopheles subpictus bagavan et al., 2009 aloe ngongensis asphodelaceae leaf anopheles gambie matasyoh et al., 2008 millettia dura leguminosae seed aedes aegypti yenesew et al., 2003 cassia obtusifolia leguminosae seed aedes aegypti, aedes togoi, culex pipiens pollens yang et al., 2003 atlantia monophylla rutaceae leaf anopheles stephensi sivagnaname and kalyanasundaram, 2004 rawani a (2023). not sci biol 15(1):11325 15 dysoxylum malabaricum meliaceae leaf anopheles stephensi senthil nathan et al., 2006a melia azedarach meliaceae leaf and seed anopheles stephensi senthil nathan et al., 2006b moringa oleifera moringaceae bark culex gelidus kamaraj and rahaman, 2010 ocimum gratissimum lamiaceae leaf culec gelidus kamaraj and rahaman, 2010 solenostemma argel apocynaceae aerial part culex pipiens aldoghaini et al., 2004 chrysanthemam indicum asteraceae leaf culex tritaeniorhynchus kamaraj et al., 2010 mormordica charantia cucurbitaceae leaf culex quinquefasciatus prabhakar and jebanesan, 2004 vitex negundo verbenaceae leaf culex quinquefasciatus krishnan et al., 2007 centella asiatica umbelliferae leaf culex quinquefasciatus rajkumar and jabanesan, 2005 pavonia zeylanica malvaceae leaf culex quinquefasciatus vahitha et al., 2002 coccinia indica cucurbitaceae leaf culex quinquefasciatus, aedes aegypti rahuman et al., 2007 cassia tora caesulpinaceae seed aedes aegypti, culex pipiens pallens jang et al., 2002 annona squamosa annonaceae leaf anopheles sp. das et al., 2007 chamaecyparis obtusa cupressaceae leaf anopheles stephensi jang et al., 2005 acalypha alnifolia euphorbiaceae leaf anopheles stephensi, aedes aegypti, culex quinquefasciatus kovendan et al., 2012 solamum villosum solanaceae leaf, berry anopheles subpictus, aedes aegypti chowdhury et al., 2008a, 2008b, 2009 cestrum diurmum solanaceae leaf anopheles stephensi ghosh and chandra, 2006 solamum nigrum solanaceae leaf, berry culex quinquefasciatus rawani et al., 2010, 2013 cassia obtusifolia leguminosae leaf anpheles stephensi rajkumar and jabanesan, 2009 apium graneolens umbelliferae seed aedes aegypti choochate et al., 2004 rhizophora mucronata rhizophoraceae bark, pith, stem wood aedes aegypti kabaru and gichia, 2009 piper langum piperaceae fruit exocarp aedes aegypti chaithong et al., 2006 rawani a (2023). not sci biol 15(1):11325 16 conclusionsconclusionsconclusionsconclusions for the vector control programme, an abundance of strategies has been developed and are being adopted in different parts of the world. in the present review work, emphasis has been given towards the use of biological control agents as the most effective tool for the control of the vector mosquito population. an effort has been given to control the immature stages of mosquitoes by using biological control agents. in mosquito control programmes, larval forms are a point of attraction because there are confined in water bodies and are easy to locate and control. the development of resistance and adverse effect of synthetic insecticides on the environment and other non-target organisms has shifted the research effort toward an alternative way to reduce the mosquito menace. over the past few years, biological control of mosquitoes has been given more importance to develop safer methods regarding toxicity to man, the environment and other non-target organisms. so biological control of vector mosquitoes includes using aquatic insects, copepods, and larvivorous fish as a predator of mosquito larvae; the use of pathogens like algae, fungi, and bacteria, and insecticides of plant origin. since a single method of control cannot be enough to bring out the desired results, importance should be given to the multiple vector mosquito control approaches, including insecticides, biocontrol agents, and environmental management. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions the author read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, 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entomology 28(4):229‐236. zvantsov ab, kadamov d, fozilov h (2008). experiences and prospects of use of larvivorous fishes for control/prevention of malaria in tajikistan. copenhagen (denmark): world health organization, 2008. the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11490 nsb nath 2023.06.19.docx received: 21 feb 2023. received in revised form: 09 may 2023. accepted: 08 jun 2023. published online: 19 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania nath s et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11490 doi:10.15835/nsb15211490 rererereviewviewviewview articlearticlearticlearticle.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae role of bulking agents, process optimization, and different earthworm role of bulking agents, process optimization, and different earthworm role of bulking agents, process optimization, and different earthworm role of bulking agents, process optimization, and different earthworm species in the species in the species in the species in the vermiremediation process of industrial wastes: a reviewvermiremediation process of industrial wastes: a reviewvermiremediation process of industrial wastes: a reviewvermiremediation process of industrial wastes: a review snigdha nath1, asif qureshi2, subhasish das1* 1mizoram university (a central university), pachhunga university college, department of environmental science, aizawl 796001, mizoram, india; snigdhonath@gmail.com; dassubhasish@pucollege.edu.in (*corresponding author) 2indian institute of technology hyderabad, kandi, department of civil engineering & department of climate change, telangana, india; asif@ce.iith.ac.in abstractabstractabstractabstract rapid industrialization and consumerism have aggravated the generation of industrial waste globally, consequently posing a serious problem related to their treatment, disposal, and management. industrial wastes or sludges are mainly characterised by undesirable levels of heavy metals, toxic chemicals, and other toxic organic compounds. deposition of such wastes in the environmental matrices for prolonged periods may result in serious contamination, and the consequent accumulation of these harmful constituents in the ecological food chain. unavailability of appropriate disposal mechanisms for these sludges is a matter of serious concern that could severely pollute the environment and risk human health. vermicomposting has emerged as a feasible and environmentally friendly bioremediation technology that could provide a solution to this problem. however, the vermicomposting of industrial sludges requires a better understanding of its inextricable factors to make it a viable process. thus, the present study was undertaken to provide insights on the influence of different bulking agents and abiotic factors on the vermicomposting process, as well as, the role of different earthworm species in the successful implementation of this process in the bioremediation of industrial waste. keywords:keywords:keywords:keywords: abiotic factors; bulking agents; earthworms; industrial wastes; vermicomposting abbreviations:abbreviations:abbreviations:abbreviations: aoxs adsorbable organic halides; eds endocrine disruptors; eps -extracellular polymeric substance; mt multifunctional metallothionein; pahs polyaromatic hydrocarbons; phcs petroleum hydrocarbons; tphs total petroleum hydrocarbons introductionintroductionintroductionintroduction the environmentally nonchalant and linear approach to industrialization because of increasing global population and consumerism has resulted in an increased waste production in the 21st century (stoeva and alriksson, 2017; minelgaitė and liobikienė, 2019). recent reports illustrate that industries produce millions of tonnes of wastes every year, and this waste generation is anticipated to increase by three times by 2100 (krishnan et al., 2021). consequent amassment of environmental contaminants from industrial wastes in marine, freshwater, and terrestrial ecosystems has been a matter of serious concern (tornero and hanke, 2016; wani et al., 2022). this has concomitantly aggravated the environmental problems related to industrial waste https://www.notulaebiologicae.ro/index.php/nsb/index nath s et al. (2023). not sci biol 15(2):11490 2 management and disposal (sandberg et al., 2019). in general, industrial wastes are characterized by undesirable levels of heavy metals (pb, cd, cu, cr, as, hg, zn, mn, and ni) and other toxic chemicals or compounds (wang et al., 2015; su et al., 2019). besides heavy metals, organic compounds such as phenols, pyrenes, polyaromatic hydrocarbons (pahs), petroleum hydrocarbons (phcs), adsorbable organic halides (aoxs), endocrine disruptors (eds), etc., constitute other major environmental contaminants (filali-meknassi et al., 2004; da silva et al., 2007; shomar, 2007; nam et al., 2012; oh et al., 2016; johnson and affam, 2019). deposition of large amount of such wastes for longer period may result in percolation of these toxic constituents (toxic metals, phenols, pyrenes, pahs, phcs, aoxs, eds, etc.) into groundwater through soil layers (świerk et al., 2007). besides groundwater contamination (brockway and urie, 1983), other possible consequences attributed to waste deposition include soil contamination and its related plant ecotoxic effects, and spread of harmful pathogens (manzetti and van der spoel, 2015). however, dearth of adequate dehydration and disposal mechanism of these wastes warrants effective mitigation measures of these environmental contaminants (liu et al., 2019; bilal and iqbal, 2020). although several methods have been utilized for industrial wastes treatment, their coverage has been insufficient (raghunandan et al., 2014, 2018; verma and kuila, 2019). techniques such as incineration, oxidation, chemical decomposition, etc., have their own limitations and are largely cost intensive (zouboulis et al., 2019). in addition, the processes of waste incineration, oxidation, chemical decomposition, etc., have detrimental effects on the environment. the major effects on the environment include global warming, smog formation, eutrophication, acidification, formation of other recalcitrant by-products (carboxylic acids, alcohols, aldehydes, etc.), and animal toxicity (kommineni et al., 2000; yang et al., 2012; sharma et al., 2013). conventional management strategies like land-filling does not actually solve the problem of environmental pollution, but rather contribute to further deposition of harmful contaminants in the environment (zouboulis et al., 2019). in this regard, vermicomposting has emerged as a feasible and environmentally clean bioremediation technology which utilizes the biodegradation potential of earthworms (bhat et al., 2017). vermicomposting is generally a non-thermophilic decomposition process in which organic residues or wastes are transformed into a valuable finished clean product called vermicompost with the synergistic action of earthworms and mesophilic microorganisms (bhat et al., 2013; lim et al., 2016). earthworms are capable of expeditious and effectual decomposition as well as remediation of various organic and industrial wastes (hickman and reid, 2008). earthworms secrete an extracellular polymeric substance (eps) through their skin tissues when put under metal stress, which help them to bind with and accumulate heavy metals (khan et al., 2019). these eps (a.k.a. exopolymers) are mainly proteins, polysaccharides, humic acids, nucleic acids, lipids, and enzymes (costa et al., 2018). for instance, a recent study demonstrated that an extracellular polymeric substance (eps) produced by the bacillus licheniformis strain kx657843 isolated from the gut of metaphire posthuma was efficient in the sorption of cu (ii) and zn (ii) (biswas et al., 2020). it can be suggested that eps producing microorganisms are primarily influenced by the earthworm gut environment (biswas et al., 2019). in addition, multifunctional metallothionein (mt) protein production is stimulated in earthworms and these proteins aid in detoxifying various metal ions (gruber et al., 2000). the vermicomposting process is dependent on various factors such as the initial c:n ratio, temperature, ph, moisture, light, and nature of the organic waste. also, these factors influencing the process are inseparably associated with the earthworm species being utilized during the biodegradation process (lim et al., 2016). earthworms can degrade most organic materials with an initial c/n ratio around 30 (ndegwa and thompson, 2000; lim et al., 2016). the bulking agents make the organic wastes more palatable for the survivability of earthworms as well as maintain a conducive milieu for the worms to multiplicate (adhikari et al., 2008). temperature plays an important function in both composting and vermicomposting process as it affects the microbial as well as the earthworms’ activity. as per reports, microbial activity multiplies by two folds per each 10°c increase in temperature and earthworms exhibit efficient activity at mesophilic temperatures ranging nath s et al. (2023). not sci biol 15(2):11490 3 from 15-30°c (rostami et al., 2009; rostami, 2011). the ph of the waste can be a limiting factor affecting the distribution and number of worms as earthworms are very sensitive (ibrahim et al., 2016). also, low moisture content negatively affect the survival and reproductive rates of earthworms (wever et al., 2001). availability of light also influences the vermicomposting process as earthworms have a hostility to bright lights. ultraviolet rays from intense sunlight can cause partial-to-complete paralysis and fatality of earthworms (ibrahim et al., 2016). it is noteworthy to mention that the process of vermicomposting is also dependent on its micro or internal environment (inside the compost pile), for which the process conditions/parameters have to be maintained separately with respect to the above-mentioned factors (bhattacharya and kim, 2016; lim et al., 2016; ganti, 2018). although attempts have been made to understand the efficiency of vermicomposting on the biodegradation of different industrial wastes, the potentiality of different types of earthworms in the management of industrial wastes is yet to be fully explored. it is also necessary to understand the influence of various abiotic factors and bulking agents inextricably associated with the process and its process management for a successful implementation of the overall vermi-remediation process. therefore, the present review was undertaken with the following objectives: i. evaluate the influence of bulking agents, and abiotic factors, and different earthworm species on vermicomposting and its process management. ii. understand the detoxifying mechanism employed by earthworms. vermicomposting: an empirical approach in industrial waste managementvermicomposting: an empirical approach in industrial waste managementvermicomposting: an empirical approach in industrial waste managementvermicomposting: an empirical approach in industrial waste management bioconversion of industrial wastes or sludges by earthworms is an effective method as it decreases the toxicity of these wastes and may act as an effective alternative to the traditional composting process and other inexpensive techniques (ndegwa and thompson, 2001; bhat et al., 2017). importance of bulking agents bulking agents are mainly carbon-based particles which add structure to the compost pile and help in controlling the air supply, moisture content, and other vital composting parameters (e.g., c:n ratio, ph, and temperature) (adhikari et al., 2008; chang and chen, 2010; lim et al., 2016; karwal and kaushik, 2020) (table 1). examples of some popular bulking agents include cow dung, poultry waste, rabbit manure, sawdust, rice bran, rice husk, sugarcane trash, grass clippings, biochar, and fruit and vegetable waste (manish et al., 2013). various bulking agents have mainly shown to induce positive effects on the survivability and reproduction of earthworms during vermicomposting, for instance, domínguez et al. (2000) reported that amendment of paper and carboard mixtures with sewage sludge induced better reproductive rates in eisenia andrei as compared to the control (without bulking agent). kumar badhwar et al. (2020) demonstrated similar positive results, wherein the amendment of cow dung in the vermicomposting of paper mill sludge and tea waste reported an increased reproducibility rate of eisenia fetida. studies have also shown that amendment of cow dung and other bulking agents not only supports earthworm biomass and fecundity, but also enhances the quality of final vermicompost (karmegam et al., 2019). augmentation of bulking materials with industrial wastes/sludges have been reported to accelerate the vermicomposting process mainly because of maceration and mixing of such carbon-rich agents in the earthworm gut (domínguez et al., 1997). the bulking materials are capable of improving or stabilizing the c:n ratio by providing c and reducing the loss of n by ammonia volatilization, which consequently facilitates the bacterial activity during vermicomposting (domínguez et al., 2000; rostami, 2011). bulking agents such as cow dung and sawdust have shown to control the c:n ratio during composting (singh and kalamdhad, 2012; nath s et al. (2023). not sci biol 15(2):11490 4 biruntha et al., 2020). bulking materials such as biogas slurry and wheat straw were observed to decrease the organic c content and increase the available npk content in the final composted product (suthar, 2010). addition of biochar during composting has shown to reduce the loss of nitrogen content (dias et al., 2010). bulking agents help in maintaining ph at an optimum range of 6-8 during composting (chang and chen, 2010). amendment of sugarcane bagasse during the composting of crude oil showed 100% degradation of total petroleum hydrocarbons (tphs) (hamzah et al., 2012). table 1. table 1. table 1. table 1. effects of different bulking agents in the composting/vermicomposting process sl. no.sl. no.sl. no.sl. no. bulking agentsbulking agentsbulking agentsbulking agents process process process process effectseffectseffectseffects referencesreferencesreferencesreferences 1. biochar composting reduction of nitrogen loss (dias et al., 2010) 2. clinoptilolite and saw dust composting high intake of heavy metals and increase in humic substances (zorpas and loizidou, 2008) 3. cotton gin and grape marc composting control ph and temperature, and enhance compost quality (madejón et al., 2001) 4. cotton waste and maize straw composting reduce nitrogen loss, enhance nitrogen fixation, and production of stabilized organic matter (paredes et al., 1996) 5. cow dung vermicomposting increase in worm biomass (gajalakshmi et al., 2002) 6. cow dung vermicomposting reduction in c:n ratio (kaushik and garg, 2003) 7. cow dung composting and vermicomposting increase in enzymatic activity in earthworms (pramanik, 2010) 8. cow dung vermicomposting stabilize ph, reduce heavy metal content, and produce good quality compost (garg and gupta, 2011a) 9. cow dung vermicomposting increase growth and reproductive rate in earthworms (kumar badhwar et al., 2020) 10. cow dung vermicomposting reduction in c:n ratio and increase in npk content (biruntha et al., 2020) 11. cow dung and saw dust composting control ph, bulk density, and carbon content (singh and kalamdhad, 2012) 12. saw dust composting reduction of polyaromatic hydrocarbons (oleszczuk, 2006) 13. grape stalk and olive leaf composting improve aeration, control temperature, regulate c:n ratio, and control ph (alburquerque et al., 2006) 14. onion peel composting reduction in c:n ratio and production of mature compost (abdullah et al., 2013) 15. press mud composting and vermicomposting reduction in ph, total organic carbon, c:n ratio, and favours growth and reproduction of earthworms (karwal and kaushik, 2020) 16. rice husk composting control temperature and moisture (chang and chen, 2010) 17. rice straw composting decrease in total organic carbon and organic matter (zhu, 2007) 18. saw dust composting control ph, moisture, temperature, bulk density, and aeration (adhikari et al., 2008) nath s et al. (2023). not sci biol 15(2):11490 5 19. star grass and sugarcane bagasse composting improve ph, moisture, and total organic carbon (oviedo-ocaña et al., 2015) 20. sugarcane trash composting control ph, moisture, and carbon content (goyal et al., 2005) 21. waste paper and plant residue composting decrease in organic matter, intake of trace metals, decrease in organic c and c:n ratio (tian et al., 2012) composting of industrial sludges with amendment of saw dust reported reduction of polyaromatic hydrocarbons (pahs) (oleszczuk, 2006). studies have shown that adding clinoptilolite (a natural zeolite) in the initial mixture helps in taking up of heavy metals, which warrants its efficiency as a bulking agent in the metal remediation of industrial sludges (zorpas and loizidou, 2008). such favourable conditions induced by bulking agents promote the survival of earthworms (manish et al., 2013; ibrahim et al., 2016). influence of climatic factors apart from the influence of different bulking agents, abiotic factors like temperature, moisture/humidity, and light also play an important role in the quality of compost, as well as, the growth and reproductivity of earthworms (gopal et al., 2004; tang et al., 2007; garg and gupta, 2011b; zhou et al., 2021) (table 2). table 2. table 2. table 2. table 2. influence of abiotic factors on composting/vermicomposting process abiotic factorsabiotic factorsabiotic factorsabiotic factors processprocessprocessprocess effectseffectseffectseffects referencesreferencesreferencesreferences temperaturetemperaturetemperaturetemperature composting increase in microbial population and efficiency of the process at high temperature (chinakwe et al., 2019) composting efficient degradation of tetracyclines and rapid composting at 70 °c (yu et al., 2019) composting higher toc ratio and reduction in c:n ratio at high temperature (46 °c). (kianirad et al., 2010) composting higher decomposition activity of microbes and mass reduction of organic matter at mesophilic temperature (35 °c-37 °c) (tang et al., 2007) composting high protein degradation and high bacterial activity at 54 °c (miyatake and iwabuchi, 2005) composting higher degradation of organic matter and conversion of volatile matter at 60 °c (nakasaki et al., 1985) vermicomposting high enzymatic activity and increase in total nitrogen, total phosphorus, and total potassium content at 30 °c (zhou et al., 2021) vermicomposting decrease in organic matter, high electrical conductivity, and high nitrate content at 25 °c. (zhang et al., 2020) vermicomposting high organic matter degradation, reduction in c:n ratio, increase in total kjeldahl nitrogen in winter (low temperature) (garg and gupta, 2011b) vermicomposting better growth of earthworms and good quality compost in temperature range 15 °c-25 °c (rostami et al., 2009) vermicomposting better vermicompost turnover, number of earthworms (eudrilus sp.) and worm biomass at low temperature (gopal et al., 2004) nath s et al. (2023). not sci biol 15(2):11490 6 vermicomposting efficient life activity of eudrilus eugeniae in temperature range 25 °c-28 °c (shagoti et al., 2001) vermicomposting efficient activity and reproductivity capacity of eudrilus eugeniae at low temperature (amoji et al., 1999) moisture/moisture/moisture/moisture/ humidityhumidityhumidityhumidity composting decrease in total organic matter, increase in total nitrogen and better compost quality at 53% moisture content (li et al., 2021) composting efficient process activity at initial moisture content of 55-70% (yeh et al., 2020) composting high organic matter degradation at 70-75% initial moisture content (makan et al., 2013) composting better stability and maturity of the compost at 6575% moisture content (guo et al., 2012) composting high microbial activity at high moisture content (≥ 50%) (liang et al., 2003) vermicomposting high crude fibre degradation and increase in crude protein at 70% moisture content (hossen et al., 2022) vermicomposting better growth of earthworms and good quality compost in moisture content regime 65-75% (rostami et al., 2009) vermicomposting higher reduction in c:n ratio, decomposition rate, and kinetic reaction rate at 75 ± 5% moisture content (palsania et al., 2008) vermicomposting better vermicompost turnover, number of earthworms (eudrilus sp.) and worm biomass at high relative humidity (gopal et al., 2004) lightlightlightlight vermicomposting increase in earthworms’ photophobic movement with increase in light intensity (lin et al., 2018) vermicomposting higher cast productivity rate of hyperiodrilus africanus in red light colour and least emigration rate in dark light colour (owa et al., 2008) vermicomposting reduced growth rate and reproductive rate of eisenia fetida in exposure to high frequency light (uv rays) (hamman et al., 2003) the investigation on the influence of temperature on vermicomposting showed that earthworms’ gut enzymes exhibited higher activity at an optimum temperature of 30 °c. consequent increment in total npk was also observed at similar temperature (zhou et al., 2021). conversely, zhang et al. (2020) reported that an optimum temperature of 25 °c is essential for better organic matter degradation, whereas increasing the temperature slightly accelerated the rate of decomposition, mineralization, and nitrification. microbes identified in a compost pile (mainly proteo-bacteria and fungi) had greater decomposition activity at mesophilic temperatures (35 °c-37 °c) (tang et al., 2007). the effectiveness of vermicomposting also depends on seasons. it was observed that organic matter degradation by eisenia fetida was higher in the winter as compared to the summer. reports also suggested that eudrilus eugeniae showed efficient activity and reproductive capacity during the colder seasons (amoji et al., 1999). gopal et al. (2004) investigated the influence of prevailing weather conditions on the growth and biomass of eudrilus sp. and the produced vermicompost. it was observed that the vermicompost turnover, number of earthworms, and worm biomass were negatively corelated to atmospheric temperature. rostami et al. (2009) demonstrated that temperatures maintained in a range of 15 °c-25 °c was optimum for the growth of earthworms as well as the decomposition process. shagoti et al. (2001) reported that e. eugeniae demonstrated efficient life activity in optimum temperatures between 25 °c and 28 °c. a pile temperature below 10 °c might induce stress on the earthworms and result in their mortality. nath s et al. (2023). not sci biol 15(2):11490 7 likewise, a significant increase in temperature might reduce the activity of the earthworms, resulting in reduced reproductive rate (ganti, 2018). hence, temperature should be within the earthworms’ tolerance capacity to facilitate biodegradation of sludges (bhattacharya and kim, 2016) (figure 1). liang et al. (2003) investigated the effect of moisture content on the compost microbial activity during the biodeterioration of sludges. hossen et al. (2022) found that an initial moisture content of 70% was suitable for an efficient vermicomposting. another study reported the influence of moisture content variation on the kinetic reaction rate of vermicomposting. it was observed that rate of vermicomposting and kinetic reaction rate was maximum at 75 ± 5% moisture content (palsania et al., 2008). yeh et al. (2020) reported that an initial moisture regime of 55-70% was more suitable for an effective composting of the wastes. guo et al. (2012) reported that moisture content in the regime of 65-75% was optimum for obtaining a stable and mature compost. makan et al. (2013) also reported that an initial moisture content of 70-75% resulted in a high organic matter degradation during composting. li et al. (2021) demonstrated that an initial moisture content of 53% resulted in a considerable decrease in total organic matter and further facilitated in production of good quality compost. the right amount of moisture can be maintained by employing coarse materials in the compost pile which can absorb oxygen (ganti, 2018). an optimum moisture content in the regime of 40-55% inside the pile was suitable for vermicomposting of different wastes (das et al., 2020) (figure 1). composting of organic wastes requires a moisture content of 60-70% for successful results. figure 1. figure 1. figure 1. figure 1. process conditions maintained during vermicomposting process (modified from: (bolong and saad, 2020)) earthworms are generally photo-sensitive to different light intensity and colour (lin et al., 2018). it was reported that exposure of e. fetida to ultra-violet radiations (high frequency light) resulted in a reduced growth rate and reproductive rate in the species and it significantly decreased cocoon fertility rate by 70% (hamman nath s et al. (2023). not sci biol 15(2):11490 8 et al., 2003). additionally, the effect of light colour on the cast productivity as well as the emigration rate of hyperiodrilus africanus was investigated. the results showed that red light colour was the most suitable for cast productivity followed by blue, green, dark, and white. also, the rate of emigration was least in dark light colour indicating the preference of this earthworm species for that particular colour (owa et al., 2008). lin et al. (2018) investigated the effect of different monochromatic lights (white light, yellow light, green light, red light, and incandescent light) and light intensity (10 to 270 lx) on the photophobic reaction of earthworms and found that higher light intensity (270 lx) substantially affected the movement of earthworms away from the light. the results also found that red light exerted the weakest photophobic movement in earthworms. from the results, it can be inferred that exposure to high intensity light and different colours of light can considerably affect the movement of earthworms which consequently might affect the vermicomposting process of sludges and prolong its time period. process management during vermicomposting, the process conditions or control measures inside the compost pile are also to be monitored to achieve efficient earthworm activity (gurav and pathade, 2011; manyuchi and phiri, 2013). some of the process conditions which are maintained or monitored inside the compost pile include c:n ratio, ph, aeration, churning, pre-treatment etc. (raut et al., 2008; getahun et al., 2012; manyuchi and phiri, 2013; das et al., 2020) (figure 1). c:n ratio has to be in an appropriate balance to facilitate better degradation by enhancing the microbial activity during the process (chen et al., 2011). to enhance industrial sludges with low initial c:n ratio, bulking materials with high c:n ratio is often employed (zhang and sun, 2016). ndegwa and thompson (2000) reported that c:n ratio = 25:1 is optimum for vermicomposting of biosolids. similarly, biruntha et al. (2020) tested varied feedstocks (c:n ratio range 23 to 70) in an e. eugenia based vermicomposting system and found that c:n ratio in the range of 23-30 is adequate for earthworm proliferation and waste degradation. in this regard, vermicomposting of sludges is a scalable process as a lot of bulking agents with wide c:n ratios have often been utilized during the process (manish et al., 2013; das et al., 2020). most wastes result in an increase of acidic content of the compost pile during vermicomposting, which consequently affects the survival of earthworms (katiyar et al., 2017). however, earthworms have the ability to maintain the ph by neutralizing both acidic and alkaline feedstocks by producing alkaline exudates and organic acids respectively (goswami et al., 2014). singh et al. (2005) reported that a high acidic initial substrate ph was unfavourable for vermicomposting, whereas initial substrate ph close to neutral favoured the most for waste stabilization. amendment of bulking materials helps in maintaining a near neutral ph inside the compost pile (adhikari et al., 2008; chang and chen, 2010). maintaining proper air circulation in compost piles is a prerequisite for achieving an enhanced biodegradation rate (das et al., 2020). in a bench scale experiment, it was observed that augmenting air for a time period of 4-6 hours at a flow rate of 0.62 l/min per kg was ideal for vermicomposting (palaniappan et al., 2017). earthworms also maintain themselves a proper aeration in vermibeds by regulating their movement through it (kaur, 2020). in this regard, bedding helps in maintaining a proper amount of oxygen during the process (ganti, 2018). selection of bedding materials plays a crucial role in the vermicomposting process. besides maintaining optimum oxygen levels, bedding materials provide protection from extremes in temperature, as well as, a consistency in moisture content (munroe, 2004). they also affect the growth and fecundity of earthworms during vermicomposting, for instance, abd manaf et al. (2009) studied the influence of two bedding materials (saw dust and newspaper) using biological parameters such as growth rate, number of worms, number of cocoons, and worm biomass. the results demonstrated that sawdust bedding was better for cocoon production and number of earthworms, while newspaper bedding was better for earthworm biomass production and growth rate. nath s et al. (2023). not sci biol 15(2):11490 9 for maintaining better aeration and transfer of materials in the vermibeds, churning (turning of compost pile) is a common practice which is to be followed. churning influences the pile thermodynamics and also assist earthworm movement (getahun et al., 2012) (figure 2). abdoli et al. (2019) compared the composting efficiency in a static and frequently churned pile and found good microbial biomass and higher earthworm cocoon count in the latter. it also facilitates good porosity and reduces compaction in the vermibeds which is very essential for earthworm growth and proliferation (bhattacharya and kim, 2016). pre-treatment of sludges in vermicomposting can also influence the duration of the process, survival of earthworms, and nutrient availability. pre-treatment reduces the process duration and eliminates the harmful pathogens (ganti, 2018) (figure 2). the conductivity of the sludges very much affects the process of vermicomposting as earthworms are known to be very sensitive to high conductivity (gunadi et al., 2002). thus, sludges or waste materials containing higher salt content should be leached to reduce the salt content. this is mainly achieved by watering the sludges for some period of time during the pre-treatment phase (kaur, 2020). singh et al. (2021) observed that cellulolytic pre-treatment significantly lessened the earthworm incubation time and reduced c content but increased n availability in the waste. alshehrei and ameen (2021) reviewed the usage of chemical pre-treatment in municipal solid waste vermicomposting. other pre-treatment options like microwave and thermal methods are also possible prior vermicomposting (amiri et al., 2017; tayeh et al., 2020). figure 2. figure 2. figure 2. figure 2. flow chart illustrating the process of vermicomposting of sludges role of different earthworm species in vermiremediationrole of different earthworm species in vermiremediationrole of different earthworm species in vermiremediationrole of different earthworm species in vermiremediation often regarded as the intestines of earth, earthworms play an important role in maintaining soil fertility and in the degradation of wastes (martin, 1976). earthworms are mainly categorized into three types depending on the portion of the soil profile that they inhabit, viz., epigeic, endogeic, and anecic (domínguez, 2018). with regards to vermicomposting, epigeic earthworms are mostly employed in the decomposition process followed by anecic earthworms (table 3). however, the role of endogeic earthworms in soil organic nath s et al. (2023). not sci biol 15(2):11490 10 matter dynamics cannot be overlooked (das et al., 2020). generally, earthworms play two very important roles in vermicomposting, i.e., 1) degradation of wastes and 2) production of quality vermicompost (yadav and, garg 2013). in this regard, the incorporation and influence of monoculture (use of one species) and polyculture (use of more than one species) techniques in vermicomposting have also been studied (khwairakpam and bhargava, 2010). the results of khwairakpam and bhargava (2010) demonstrated that the quality and stability of the produced vermicompost from polyculture technique were better than the vermicompost produced from monoculture technique. also, the overall reduction in ph, total organic carbon, and coliforms was higher in the polyculture reactors. similar results were reported by hussain et al. (2018) who compared the efficiency of kitchen waste vermicomposting by single species (eisenia fetida/eudrilus eugeniae/perionyx excavatus) and three in combination. they found higher compost maturity, compost quality, microbial enrichment, and nutrient contents in the vermicompost prepared by polyculture technique. epigeic earthworms epigeic earthworms are mainly surface dwellers and live in the organic horizon and on or near the soil surface (domínguez, 2018). they mainly feed on fresh organic matter found in forest litter, litter mound, vegetable and animal debris, etc. (aira et al., 2008; domínguez 2018). they exhibit high metabolism and reproductive rates which help them to adapt and survive in changing environmental conditions (domínguez, 2018). these earthworms play a very important role in degradation of organic matter and enhances the rate of decomposition as well as nutrient turnover (gomez-brandon et al., 2011). the influence of epigeic earthworms on soil microbiota is widely varying as they can instigate either an increase or a decrease in microbial biomass (medina-sauza et al., 2019). when it comes to vermicomposting of sludges, epigeic species e. fetida have been employed extensively (domínguez, 2018). apart from this species, e. eugeniae and perionyx excavatus have also been utilized in the vermicomposting process (gajalakshmi et al., 2001; suthar, 2006; ravindran et al., 2015). other lesser utilized epigeic earthworms include species such as dichogaster bolaui (epi-endogeic species) and perionyx simlaensis (bhardwaj and sharma, 2015). vermicomposting of wastes employing e. fetida recorded a significant decrease in ph, organic carbon, c:n ratio, total k and an increase in total n, available p, total ca, and mg. also, heavy metals such as such as fe, mn, zn, cu, cr, and ni in the final vermicompost material were under the permissible levels (ahmed and deka, 2022). reduction of c:n ratio and humification index was reported during the vermicomposting of sludges by e. fetida (boruah et al., 2019). more studies on the vermicomposting potential of e. fetida for different sludges and other organic substances have been reported (garg and kaushik, 2005; singh et al., 2010; hait and tare, 2012; bhat et al., 2013; hanc and chadimova, 2014; bhat et al., 2016; busato et al., 2016; cunha et al., 2016; huang et al., 2016; malińska et al., 2016; mupambwa et al., 2016; xie et al., 2016; ravindran and mnkeni, 2017). reports have shown that eisenia andrei could successfully reduce harmful pathogens such as escherichia coli and salmonella spp. during the vermicomposting of organic wastes (procházková et al., 2018). vermicomposting potential of dairy sludge and paper mill sludge by e. andrei has been investigated (elvira et al. 1998). vermicomposting of lignocellulosic wastes by e. eugeniae showed reduction of c:n ratio in the final vermicompost (pandit et al., 2020). pressmud vermicomposted by e. eugeniae showed a decrease in total organic carbon, c:n ratio, and c:p ratio in the vermicompost (balachandar et al., 2020). various other investigations on the efficiency of e. eugeniae in vermicomposting of sludges/wastes have also been reported (lalander et al., 2015; ravindran et al., 2016; taeporamaysamai and ratanatamskul, 2016; soobhany et al., 2017; biruntha et al., 2020). epigeic species p. excavatus has also shown heavy metal accumulation potential for metals such as cd, cu, pb, and cr during the vermicomposting of sludges. the final produced vermicompost showed a decrease nath s et al. (2023). not sci biol 15(2):11490 11 in total organic carbon, c:n ratio, and c:p ratio and an increase in total npk content (yuvaraj et al., 2018). more studies on the efficiency of p. excavatus in vermicomposting have been reported (suthar, 2006; ananthavalli et al. 2019). the potential of lumbricus rubellus (epigeic) in vermicomposting of sludges has also been reported (azizi et al. 2013; shah et al. 2015) vermicomposting of industrial sludges using consortia of different earthworm species has also been investigated. yuvaraj et al. (2020) investigated the efficiency of vermicomposting of textile sludge using two earthworm species e. eugeniae and p. excavatus and reported good heavy metal accumulation by the earthworms and a significant increase in npk content in the final product. endogeic earthworms endogeic earthworms, characterized by little pigmentation, low reproductive rates and long life cycles, live in deeper section of the soil profile and feed mainly on soils enriched with organic matter (domínguez, 2018). with respect to disturbed soil conditions, they prove themselves to be highly resistant and can survive unfavourable conditions like drought and food shortage (jouquet et al., 2010; domínguez, 2018; das et al., 2020). these earthworms are capable of ingesting a large amount of soil and assimilating greater soil organic matter (bernard et al. 2012). they are highly diverse as they are found in urban soils to temperate soils (schlaghamerský and pižl, 2009; glasstetter, 2012). endogeic earthworms are not extensively used in vermicomposting as compared to the epigeic and anecic earthworms. one possible reason could be that these earthworms are not detrivores for which they do not feed on litter or debris and mostly prefer to live in deeper soil profiles (das et al., 2020). however, a few studies on the vermicomposting potential of endogeic earthworms have been carried out. for instance, das et al. (2016) investigated vermicomposting of sludges by endogeic species metaphire posthuma. the results showed that there was increment in total npk availability, stable humic acid c formation, fulvic acid c, and microbial biomass c in the final vermicompost produced. a decrease in total organic carbon and ph was also recorded. sahariah et al. (2015) also investigated the efficiency of m. posthuma in vermicomposting of municipal sludges. the results showed that this species was potentially capable of accumulating heavy metals such as pb, zn, mn, and cu. moreover, the final vermicompost product recorded an increase in total n and available p, k, and fe content. enhancement in humification rate and fulvic/humic acid c was also reported. reports have also shown the vermicomposting potential of the endogeic species pheretima elongata (munnoli et al., 2000). although other endogeic earthworm species have not been investigated extensively in vermicomposting, but they are equally resourceful when it comes to providing other services such as maintaining soil characteristics and in growth of different plant species (doube et al., 1997; hallam et al., 2021). for instance, doube et al. (1997) investigated the ability of two endogeic species aporrectodea trapezoides and aporrectodea rosea in the growth of three crop plants wheat, barley and faba beans. bernard et al. (2012) reported that pontoscolex corethrurus was capable of inducing a priming effect (stimulation of mineralization of soil organic matter) and subsequently enhancing soil respiration. another species allolobophora chlorotica has shown accumulation potential for the cationic analogue strontium (sr) (morgan et al. 2002). van vliet et al. (2006) reported that a. chlorotica and aporrectodea caliginosa could efficiently accumulate heavy metals such as arsenic, cadmium, and zinc. eck et al. (2015) investigated the priming effect of a. caliginosa on young rhizodeposits and old soil organic matter and found that this species induced strong priming effect on old soil organic matter. anecic earthworms anecic earthworms mainly live in the vertical galleries of the soil profile and feed on soil as well as litter and partially mineralized organic matter (domínguez, 2018; das et al., 2020). they mainly come out to the surface at night to feed on surface litter and other partially decomposed matter (kiyasudeen et al., 2015). anecic earthworms play a crucial role in accelerating the pedological processes by enhancing decomposition of soil organic matter, nutrient cycling, and soil formation (kiyasudeen et al., 2015; gavinelli et al., 2018). bottinelli nath s et al. (2023). not sci biol 15(2):11490 12 et al. (2021) demonstrated that anecic earthworms (amynthas adexilis) could induce soil generation to counteract the effects of soil erosion. the influence of anecic species lampito mauritii on soil enzymatic activity in cadmium-amended soils has also been studied, wherein this species by accumulating cd in their gut reduced the cd-induced stress in microorganisms resulting in an increased microbial enzymatic activity (sivakumar et al., 2015). anecic earthworms, even if not extensively like epigeic earthworms, have been utilized in vermicomposting (das et al., 2020). rajadurai et al. (2022) investigated the vermiremediation efficiency l. mauritii on engine oil contaminated soils. the results showed a total reduction of polyaromatic hydrocarbons (pahs) and total petroleum hydrocarbons (tphs) by 68.6% and 34.3% respectively. also, the vermiremediated soils recorded an elevation in the npk content. prashija et al. (2017) recorded decrease in ph, organic carbon, c:n ratio, c:p ratio, lignin, cellulose, hemicellulose, and phenol content and increase in npk and humic content in the final vermicompost produced from lignocellulosic wastes by l. mauritii. goswami et al. (2014) reported efficient accumulation of heavy metals such as mn, zn, cu, and as by l. mauritii during the vermicomposting of tea factory coal ash. the results also observed reduction of total organic c and ph to neutrality and increase in total n in the final vermicompost. maity et al. (2008) found that l. mauritii was capable of immobilizing pb2+ and zn2+ in metal treated soils. the final vermicast showed reduction of c:n ratio as a result of reduction of organic c and nitrogen fixation. available p and k content also increased as a result of worm activity. banu et al. (2008) investigated the vermicomposting sludges by l. mauritii and found that organic carbon content decreased and total nitrogen increased in the final product. tripathi and bhardwaj (2004) studied the decomposition potential of l. mauritii and reported an increase in organic carbon, nitrogen, phosphorus, and potassium content by 14%, 102%, 33% and 42% respectively and decrease in c:n and c:p ratios by 43% and 14% respectively. gajalakshmi et al. (2001) also investigated the vermicomposting potential of the anecic species drawida willsi. table 3. table 3. table 3. table 3. role of different earthworms in vermicomposting of organic and inorganic wastes categorycategorycategorycategory earthworm earthworm earthworm earthworm speciesspeciesspeciesspecies wastes wastes wastes wastes (organic/inorga(organic/inorga(organic/inorga(organic/inorga nic)nic)nic)nic) effectseffectseffectseffects referencesreferencesreferencesreferences epigeicepigeicepigeicepigeic eisenia fetida patchouli bagasse from oil industry decrease in organic c, c:n ratio, c:p ratio and total k, increase in total p and ca, and heavy metal accumulation (ahmed and deka, 2022) eisenia fetida spent drilling fluid from nature-gas industry decrease in total organic c, c:n ratio, and increase in total npk content (wang et al., 2021) eisenia fetida paper mill sludge + citronella bagasse reduction of c:n ratio and humification index (boruah et al., 2019) eisenia fetida spent grains from brewery decrease in total organic c, and increase in total n and total humic substances (saba et al., 2019) eisenia fetida sewage sludge (malińska et al., 2016) eisenia fetida fruits and vegetable wastes decrease in total organic c and increase in total n (huang et al., 2016) eisenia fetida tannery sludge (cunha et al., 2016) eisenia fetida filter cake decrease in total organic c and total n (busato et al., 2016) nath s et al. (2023). not sci biol 15(2):11490 13 eisenia fetida wastewater sludge decrease in ph value, total organic c, and c:n ratio, and increase in total available p (xie et al., 2016) eisenia fetida fly ash decrease in c:n ratio (mupambwa et al., 2016) eisenia fetida press mud decrease in total organic c, c:n ratio, and k content, and increase in n, p, and na content (bhat et al., 2016) eisenia fetida apple pomace wastes increase in available nutrients such as n, p, k, and mg (hanc and chadimova, 2014) eisenia fetida dyeing sludge decrease in electrical conductivity, c:n ratio, organic c, and k content, and increase in n, p, and na content (bhat et al., 2013) eisenia fetida sewage sludge increase in total n and p content, and decrease in metal content (hait and tare, 2012) eisenia fetida beverage sludge decrease in electrical conductivity, organic c, and k content, and increase in n, p, and na content (singh et al., 2010) eisenia fetida textile sludge reduction in c:n ratio and increase in n and p content. (garg and kaushik, 2005) eisenia andrei apple pomace wastes reduction of pathogenic enterococci and e. coli (procházková et al., 2018) eisenia andrei sewage sludge and kitchen wastes (hanc and dreslova, 2016) eisenia andrei dairy sludge and paper mill sludge increase in n and p content, and low levels of heavy metals (elvira et al., 1998) eudrilus eugeniae coir pith decrease in organic matter, total organic c, c:n ratio, c:p ratio and total phenolic content, and increase in electrical conductivity, total npk and ca content (jayakumar et al., 2022) eudrilus eugeniae lignocellulosic organic wastes stable c:n ratio (15:1) (pandit et al., 2020) eudrilus eugeniae pressmud decrease in ph, total organic carbon, c:n ratio, water-soluble organic c and c:p ratios, and increase in npk content and microbial population (balachandar et al., 2020) eudrilus eugeniae biowastes decrease inorganic matter content, total organic c, lignin, cellulose, c:n ratio and c:p ratio, and increase in npk content (biruntha et al., 2020) eudrilus eugeniae municipal solid waste decrease in c:n ratio (soobhany et al., 2017) eudrilus eugeniae fermented tannery waste reduction in heavy metals, total organic c, and an increase in total kjeldahl n (ravindran et al., 2016) eudrilus eugeniae kitchen waste decrease in organic matter and organic c, and increase in electrical conductivity and total n, p, and k content (taeporamaysa mai and ratanatamskul, 2016) eudrilus eugeniae food waste increase in total n and decrease in total k (lalander et al., 2015) nath s et al. (2023). not sci biol 15(2):11490 14 eudrilus eugeniae tannery waste decrease in total organic c and c:n ratio (ravindran et al., 2015) perionyx excavatus seaweed decrease in organic c and increase in npk content (ananthavalli et al., 2019) perionyx excavatus paper mill sludge decrease in ph, total organic c, c:n ratio and c:p ratio, and increase in electrical conductivity, total n, total p and total k (yuvaraj et al., 2018) perionyx excavatus guar gum industry waste decrease in organic c and increase in total n and p content (suthar, 2006) perionyx simlaensis organic waste decrease in c:n ratio (bhardwaj and sharma, 2015) lumbricus rubellus sugarcane industry waste increase in essential nutrients like n, p, k, ca, and na (shah et al., 2015) lumbricus rubellus sewage sludge decrease in heavy metals cr, cd and pb (azizi et al., 2013) eudrilus eugeniae + perionyx excavatus textile mill wastewater sludge decrease in heavy metal content and increase in npk content (yuvaraj et al., 2020) epiepiepiepi---endogeicendogeicendogeicendogeic endogeicendogeicendogeicendogeic dichogaster bolaui organic waste decrease in c:n ratio (bhardwaj and sharma, 2015) metaphire posthuma jute mill waste decrease in total organic c and ph, and increase in n, p, and k availability (das et al., 2016) metaphire posthuma municipal solid waste reduction in ph and total organic c and increase in total n and availability of p, k, and fe (sahariah et al., 2015) metaphire posthuma organic waste decrease in c:n ratio (bhardwaj and sharma, 2015) pheretima elongata potato peels (munnoli et al., 2000) anecicanecicanecicanecic lampito mauritii lignocellulosic organic waste decrease in ph, organic c, c:n ratio, c:p ratio, lignin, cellulose, hemicellulose and phenol content, and increase in n, p, k content, dehydrogenase and humic acid (prashija et al., 2017) lampito mauritii tea factory coal ash decrease in total organic c and increase in total n content and heavy metal accumulation (goswami et al., 2014) lampito mauritii metal treated soil decrease in c:n ratio and increase in availability of p and k (maity et al., 2008) lampito mauritii sago sludge decrease in organic c and increase in n and p content (banu et al., 2008) lampito mauritii kitchen waste decrease in c:n ratio and c:p ratio and increase in n, p and k content (tripathi and bhardwaj, 2004) underlying mechanism of earthworms in waste detoxification processunderlying mechanism of earthworms in waste detoxification processunderlying mechanism of earthworms in waste detoxification processunderlying mechanism of earthworms in waste detoxification process earthworms are capable of crushing organic materials into smaller fragments with the help of their gut mediated processes. the gut extends from the mouth to the anus and consists of different sections like muscular pharynx, oesophagus, intestines, and associated digestive glands (figure 3). nath s et al. (2023). not sci biol 15(2):11490 15 figure 3. figure 3. figure 3. figure 3. internal mechanism of earthworms in organic material decomposition and metal uptake. the font in red colour designates the three major sections in an earthworm gut (foregut-frontal region; midgutmiddle region, and hindgut-posterior region) (modified from: (lemtiri et al., 2014)) the gut usually consists of mucus and microbes such as bacteria, protozoa, and fungi which contribute to degradation of organic matter at accelerated rates (munnoli et al., 2010). the influential grinding mechanism of the earthworm’s gut is brought about by the strong ligands produced in the gut and peristalsis (contraction and relaxation) which helps in the movement of food (carpenter et al., 2007). it is observed that the gut provides a suitable environment for the incubation of microbial colonies (edwards and lofty, 1977). the gut microbiota degrade the organic matter through secretion of hydrolytic enzymes (swati and hait, 2017). several digestive enzymes such as protease, invertase, amylase, lipase, cellulase, chitinase, etc., present in the alimentary canal help in the process of decomposition (edwards, 1988b). such digestive enzymes decompose organic matter constituents such as cellulose, hemicellulose, lignin, and proteins (garcia et al., 1992; lemtiri et al., 2014). the intestinal mucus of earthworms mainly consists of gluco-proteins and other glucosidic and proteic molecules (morris, 1985). the nitrogenous compounds present in the mucus significantly enhances the gut microbial activity (zhang et al., 2000; lemtiri et al., 2014). the chemical changes of the ingested organic matter are brought about by enzymatic digestion (sharma, 1994). as a consequence of these processes, aromatic protein compounds in the organic matter decrease, whereas humic acid-like and fulvic acid-like substances increase (fernández-gómez et al., 2015). ingestion of organic materials by earthworms increases the microbial count in the gut up to 1000-fold (edwards, 1988a). these microbe species are primarily the n-fixing and decomposer type which are excreted out with nutrients as vermicasts (singleton et al., 2003). reports suggested that the neutral ph and moist conditions of the foregut of earthworms promoted the growth of microbes capable of digesting cellulose (lavelle and gilot, 1994). singleton et al. (2003) reported the presence of hydrocarbon degrading bacteria such as pseudomonas alcaligenes and acidobacterium in the gut of earthworms. hussain et al. (2016) identified and isolated n-fixing and p-solubilizing bacterial strains of the genus bacillus, serratia, burkholderia, and kluyvera from the gut of earthworms. earthworms enhance the process of mineralization by mixing the fragmented organic matter with mineral particles and microorganisms (parmelee, 1998). higher mineralization in the produced vermicasts could be attributed to higher microbial activity and a higher concentration of labile compounds such as soluble carbon and lignin (coq et al., 2007). even in the absence of sufficient gut enzymes, they are capable of digesting nath s et al. (2023). not sci biol 15(2):11490 16 organic matter by stimulating soil microbes (khomyakov et al., 2007; nechitaylo et al., 2010; fujii et al., 2012). primarily, earthworms and microbes stay in a mutualism, wherein earthworms affect the diversity and metabolic activity of microbes and microbes constitute a part of the earthworm diet (lemtiri et al., 2014). earthworms play a crucial role in nutrient cycling processes involving c and n (lemtiri et al., 2014). they play two contrasting roles in regulating organic c dynamics, viz., a) enhancing organic c mineralization by stimulating microbial activity and b) stabilizing organic c by formation of micro and macro aggregates (angst et al., 2019). they are capable of converting organic matter with relatively wide c:n ratios into forms of lower c:n ratios, which resultantly enhances the nutrient cycling of n (syers and springett, 1984). figure 4. figure 4. figure 4. figure 4. bioaccumulation and biotransformation of metals in chloragogenous cells (modified from: (swati and hait, 2017)) earthworms are capable of up-taking potentially toxic elements mainly through two pathways – dermal uptake and dietary uptake (dominguez and edwards, 2011; xiao et al., 2021). a higher content of mobile metal fractions is resultantly produced as metals are unbounded from their ions and carbonates because of the digestion of degradable organic matter. these mobile fractions get accumulated in the cutaneous tissues as a stress response of the earthworms (li et al., 2009; swati and hait, 2017). organic wastes get mineralized and humified to form simpler or short chain organic acids during biodegradation. these newly formed organic acids form stable metal complexes and/or silicate fractions by binding to the available metal content (hait and tare, 2012). the earthworms have adapted to the process of metal accumulation by maintaining a balance between uptake and excretion. the rate of metal excretion increases in earthworms overcoming the metal uptake in tissues which helps them survive the metal induced stress conditions (li et al., 2009; swati and hait, 2017). various metals such as cd, pb, hg, and zn are stored in the chloragogenous tissues of earthworms (figure 4) after their accumulation (song et al., 2014; goswami et al., 2016). different factors like metal type and its exposure level, earthworm species and its physiology and age, production of metal chelating proteins, and substrate characteristics influence the metal accumulation potential and binding mechanism of earthworms (nannoni et al., 2011). the chelation of metals is induced by a low molecular weight protein – metallothionein (mt), wherein this cysteine rich protein chelates metals with the help of their thiol groups and transport them to the chloragogenous tissues (homa et al., 2016). it has been observed that exposure of earthworms to toxic elements like cd and hg up-regulates the expression of mt in the intestine (maity et al., 2009; colacevich et al., 2011). however, the metal accumulation potential of earthworms doesn’t always correlate with the mt nath s et al. (2023). not sci biol 15(2):11490 17 induction in intestines (goswami et al., 2016). interestingly, the induction of other non-mt proteins which are capable of accumulating such toxic elements has been reported, which explains why this incongruity exists (hussain et al., 2021). earthworms also accumulate pahs by dermal absorption and/or intestinal digestion, which are then bio-transformed or biodegraded into more stable harmless compounds (sinha et al., 2008). conclusionsconclusionsconclusionsconclusions the pollution of toxic constituents originating from industrial wastes or sludges has severely affected the environment and human health. even though considerable progress has been made in bioremediation of such pollutants, the scope of vermicomposting in remediation of such pollutants is yet to be fully explored. as discussed in this review, several factors are inextricably associated with vermicomposting which could accelerate the biodegradation process of such toxic constituents and render it for large-scale implementation. empirical studies have shown the positive effects of different bulking agents in vermicomposting which could help in chalking out the suitable bulking materials for vermiremediation of different industrial wastes/sludges. the influence of abiotic factors on vermicomposting and the role of different earthworm species also provides valuable insights for making it a more viable process. it can be suggested that vermiremediation can be made a more scalable process with proper knowledge and understanding of the various co-dependent factors. thus, this study can be taken as an opportunity where further research investigations can be carried out to fill the gaps in developing the vermiremediation process in management of industrial wastes. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions sn: manuscript writing, editing and review; aq: manuscript editing and review; sd: conceptualization, manuscript editing and review. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements the research received funding by university grants commission (ugc) of india (ref: no.30555/2021 (bsr)). conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. nath s et al. 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(2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11585 doi:10.15835/nsb15211585 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae iiiin vitron vitron vitron vitro regeneration ofregeneration ofregeneration ofregeneration of haloxylonhaloxylonhaloxylonhaloxylon ammodendronammodendronammodendronammodendron ping wang, lingjuan man, li ma, jiaxin qi, yanping ren, zhengpei yao, bo wang, cong cheng, hua zhang* xinjiang agricultural university, college of life sciences, urumqi, xinjiang, china; 1827278246@qq.com; 990559779@qq.com; 125667729@qq.com; 874531321@qq.com; renyanping923@163.com; yaozp@xjau.edu.cn; wangbo@xjau.edu.cn; 2017216030@njau.edu.cn; hazelzhang@163.com (*corresponding author) abstractabstractabstractabstract haloxylon ammodendron (c.a.mey) bunge is one of the important species of arid desert vegetation in china, and it is also an ideal material for studying plant stress resistance, which plays an extremely important role in protecting desert ecosystems and preventing land desertification. however, there are relatively few studies on the regeneration of the fusiform, and the genetic transformation system has not been successfully reported, which restricts the in-depth study of the molecular mechanism of shuttle propagation and stress resistance. in the present study, the seeds, hypocotyls, cotyledons, cotyledon nodes, terminal buds and fixed buds were used as explants, and a set of tissue culture and plant regeneration system was established by inducing adventitious buds, adventitious bud rooting and transplanting. the results showed that amongst different h. ammodendron explants, i.e., seeds, hypocotyls, cotyledons and cotyledon nodes, the last induced budding effect was better. the optimal medium for inducing clandine buds by cotyledon segment differentiation is 0.5 mg·l1 naa+0.5 mg·l-1 6-ba induced budding rate was high, reaching 61.90%, rooting medium was 1/2 ms+1 mg·l-1 naa +1 mg·l-1 iba + 1 mg·l-1 iaa with a rooting rate of 50%. the results of this study will provide a theoretical basis for the genetic transformation of h. ammodendron. keywords:keywords:keywords:keywords: adventitious buds; root induction; organogenesis; ex vivo regeneration; h. ammodendron introductionintroductionintroductionintroduction haloxylon ammodendron is a small perennial tree of the genus haloxylon in the family amaranthaceae, widely distributed in desert areas of asia and africa, and is a major building block of desert vegetation. in china, h. ammodendron is mainly distributed in xinjiang, inner mongolia, qinghai, gansu and ningxia provinces and regions; h. ammodendron is a small perennial tree, sometimes shrub-like, which is unique to desert areas drought tolerance (when the soil moisture content is 1.3~2.6%, it can still grow normally), high temperature resistance, salinity resistance (salt resistance critical range can reach 4% ~ 6%), wind erosion resistant plants (kung et al., 1979). it is a typical c4 species of high drought tolerance and salt tolerance (casati et al., 1999), having the morphological and physiological characteristics of a super-arid plant. to adapt to the environment of extreme soil water scarcity and intense transpiration at high temperatures, h. https://www.notulaebiologicae.ro/index.php/nsb/index wang p et al. (2023). not sci biol 15(2):11585 2 ammodendron degenerates into scale-like, juicy green shoots (‘assimilated shoots’) with high salt content, which can be used as preferred fodder for livestock and is a woody fodder in desert arid areas (kazuo et al., 2000; xu et al., 2007; wu et al., 2019). h. ammodendron forests play an important role in protecting the security of the oasis by fixing a large amount of drifting sand around the oasis and have a high ecological value in combating desertification (su et al., 2007; maina et al., 2015). the natural desert vegetation (known as "desert forest") formed by h. ammodendron as the dominant species provides a suitable breeding environment for nearly 200 species of desert plants, and also provides a habitat and breeding place for many desert animals and insects, which is an important place for biodiversity conservation in desert plain areas, and has important ecological and economic value in mitigating desertification, maintaining regional ecological balance and protecting the ecological environment. under natural conditions, h. ammodendron is reproducing extensively by seeds, which have a short germination retention period and are prone to interspecific variation and intergenerational degeneration. the heterogeneity of its germplasm and limited germination shelf life, limit the natural renewal and development of h. ammodendron. the high degree of lignification and thin bast of assimilated branches of h. ammodendron is one of the reasons why it is difficult to form adventitious roots. using relatively young and tender branches for cuttings, the spike rot is also serious, resulting in cuttings rooting cannot continue, and using histogenic seedling technology can overcome these shortcomings. the use of in vitro culture technology allows not only to obtain new plantings and varieties, but also to obtain the metabolic substances required by humans. still, the plant tissue culture technology has a high reproduction factor and can provide a large number of seedlings in a short period of time. tissue culture of woody plants has always had problems such as low reproduction coefficient, difficulty in rooting and serious pollution. however, with the development of time, a variety of regeneration systems for fruit trees and forest trees have been successfully established, for example, zhang et al. (2014) used apple leaves as explants to obtain regenerated plants. bertsouklis et al. (2023) studied in vitro propagation of juniperus oxycedrus adult native plants, examining the effects of seasonal explant collection (spring, summer, winter), different culture media, and cytokinin types. there are also citrus (jardak et al., 2020), fir (hu et al., 2017) and pine (kim et al., 2014) that have successfully established regeneration systems. some economically important species have been widely used in plantation production, such as poplar (chan et al., 2016) and eucalyptus (fernando et al., 2016). yu-qing et al. (2018) used leaves of zenia insignis chun as explants to obtain regenerated fertile plants through guaired tissue induction, adventitious shoot induction, and rooting. in view of this, the present study was conducted to investigate the shoot induction and adventitious shoot rooting of h. ammodendron weeds, hypocotyls, cotyledons, cotyledon nodes, terminal buds and fixed buds as experimental materials, with a view to providing a theoretical basis for genetic transformation of h. ammodendron. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods seed treatment and acquisition of sterile seedlings picked uniform and full seeds of h. ammodendron weeds (harvested from gurbantunggut desert in northern jimsar county, changji prefecture, xinjiang. -20 °c for storage), add 75% (v/v) ethanol, shake for 3 min, discard the liquid; rinse with sterile water 3 times, shake for 3 min; finally soak the seeds with sodium hypochlorite for 10 min, rinse with sterile water 6 times; soak in sterile water overnight, cultured on ms solid medium the next day to germinate, and obtain sterile seedlings of h. ammodendron after 35 d (figure 1). wang p et al. (2023). not sci biol 15(2):11585 3 figure 1.figure 1.figure 1.figure 1. h. ammodendron sterile seedlings (note: a: seed b: hypocotyl c: cotyledon d: cotyledon nodes e: fixed bud f: terminal bud) induction of indeterminate buds when the sterile seedlings grew to 3-5 cm, the hypocotyl, cotyledons, cotyledon nodes, staple buds and terminal buds of about 0.5-1 cm in size were rapidly cultured into the germination induction medium, three bottles of each combination of different hormone concentrations, and seven explants were inoculated in each bottle. germination was observed and recorded 10 days after inoculation. germination percentage = (number of explants forming shoots/number of inoculated explants) × 100%. (see results and analysis section for details of hormone combinations) three hormones iba (indole-3-butyric acid), naa (1-naphthal acetic acid), and iaa (indole acetic acid) were added to the basic medium of 1/2 ms, and four hormone concentration gradients of 1, 2, 3, and 4 mg l-1 were orthogonally designed for the induction of adventitious roots in 16 treatments. after the adventitious shoots induced by the h. ammodendron nodes grew about 0.5~1 cm in size, they were put into the induction rooting culture flasks; three replicate controls were made for each treatment, and about 7 explants were placed in each culture flask. after 35 d of incubation, the growth status was observed and the rooting rate was counted. rooting percentage (%) = number of rooted plants/number of inoculated explants × 100%. the data were processed with microsoft excel 2010 software. (see results and analysis section for details of hormone fractions). refining seedlings for transplanting the regenerated seedlings with well-developed root systems and more robust growth were opened in the artificial climate chamber, cling film was applied, small holes were poked in the cling film, and the seedlings were refined for about one week, then the regenerated seedlings were removed from the culture bottles, the medium attached to the root surface was rinsed with sterile water, transplanted into sterilized vermiculite: charcoal: sand = 1:1:1, and placed in the culture room for cultivation. wang p et al. (2023). not sci biol 15(2):11585 4 results results results results effect of different hormone concentration ratios on the induction of adventitious shoots from the results of the experiment, it can be seen that h. ammodendron seeds, cotyledon nodes, fixed buds and terminal buds can be induced to germinate, but hypocotyl and cotyledons did not germinate (table 1). during the experimental observation, it was found that seeds and cotyledons emerged preferentially within 14 d, while fixed and terminal buds emerged one after another after 14 d. since the material of fixed and terminal buds was difficult to obtain, although the seeds could also induce buds, it could be seen from the pictures that the quality of the buds was not as good as that of the indeterminate buds induced by the cotyledon nodes, and for the consideration of subsequent rooting, the most suitable explants for the induction of buds were determined to be the cotyledon nodes (figure 2). the cotyledonary nodes were induced in treatment 11 with a higher rate of 61.90% at ms + 0.5 mgl-1 naa + 0.5 mgl-1 6-ba. the induced shoots had vigorouw growth and dark green color. in treatment 3, cotyledonary nodes did not induce buds when no phytohormones were added, thus showing that phytohormones play a very important role in cotyledonary node induction of buds. the germination rate of cotyledon nodes in treatment 8 and 13, ms+1 mg·l-1 iba+1 mg·l-1 kt was 14.5%, ms+1 mg·l-1 naa+1 mg·l-1 6-ba was 23.81%, which indicated that the combination of naa and 6-ba and iba and kt hormones was at a concentration of 1 mgl-1, it is not suitable as a medium for cotyledon nodes, inducing adventitious buds. the budding rate of cotyledonary node in treatment 5 and 15 was the same, second only to treatment 11, and the budding rate reached 57.14%. in the combination with high germination rate, 6-ba was found to be involved, indicating that 6-ba played a role in the process of inducing adventitious budding in the h. ammodendron cotyledonary node. germination of cotyledon nodes started within 14 d. adventitious shoots were vigorous, had normal leaf color, usually appeared in compact clusters, and it was difficult to quantify the number of regenerated shoots, with a few vitrification seedlings, but no deformed shoots. figurefigurefigurefigure 2222.... induction of germination from six exosomes of h. ammodendron (35d) (note: a: seed b: hypocotyl c: cotyledon d: cotyledon nodes e: fixed bud f: terminal bud) wang p et al. (2023). not sci biol 15(2):11585 5 tabletabletabletable 1111.... effect of adding different hormone concentration ratios on germination induction of six explants (% germination induction rate) treatment code media type a seed b hypocotyl c cotyledon d cotyledonary nodes e fixed bud f terminal bud 1 ms+0.5 mg·l-1 6-ba+1 mg·l-1 kt 76.01ab 0 0 33.33bcde 47.62ab 28.57ab 2 ms+1 mg·l-1 6-ba+0.5 mg·l-1 kt 61.9abc 0 0 42.86abcde 33.33ab 23.81ab 3 ms 42.86bc 0 0 0f 0b 0b 4 ms+0.5 mg·l-1 iba+1 mg·l-1 6-ba+1 mg·l-1 kt 47.62bc 0 0 47.62abcd 23.81ab 33.33ab 5 ms + 0.5 mg·l-1 iba + 0.5 mg·l-1 6-ba 61.91abc 0 0 57.14ab 38.1ab 23.81ab 6 ms + 0.5 mg·l-1 iba + 0.5 mg·l-1 kt 47.62bc 0 0 52.38abc 0b 19.05ab 7 ms+1 mg·l-1 iba+1 mg·l-1 6ba 71.43abc 0 0 28.57cde 4.76ab 23.81ab 8 ms+1 mg·l-1 iba+1 mg·l-1 kt 47.62bc 0 0 14.5ef 0b 0b 9 ms+1 mg·l-1 iba+0.5 mg·l-1 6-ba+0.5 mg·l-1 kt 38.1c 0 0 47.62abcd 33.33ab 14.29ab 10 ms+0.5 mg·l-1 naa+1 mg·l1 6-ba+1 mg·l-1 kt 71.43abc 0 0 42.86abcde 33.33ab 52.38a 11 ms+0.5 mg·l-1 naa+0.5 mg·l-1 6-ba 71.43abc 0 0 61.9a 52.38a 33.33ab 12 ms + 0.5 mg·l-1 naa + 0.5 mg·l-1 kt 61.9abc 0 0 52.38abc 23.81ab 9.52b 13 ms+1 mg·l-1 naa+1 mg·l-1 6-ba 66.67abc 0 0 23.81de 0b 0b 14 ms+1 mg·l-1 naa+1 mg·l-1 kt 66.67abc 0 0 33.34bcde 47.62ab 23.81ab 15 ms+1 mg·l-1 naa+0.5 mg·l1 6-ba+0.5 mg·l-1 kt 85.71a 0 0 57.14ab 28.57ab 33.33ab note: different letters indicate significant differences at p ≤ 0.05 effects of different concentrations of iba, naa and iaa on the rooting of regenerated plants the results of 1/2 ms as the basal medium with different concentrations of iaa, iba, and naa were used to induce rooting of adventitious shoots in the culture, and the results are shown in table 2 below. from the test results, it can be seen that the highest rooting rate of adventitious shoots in 1/2 ms + 1 mg·l-1 iba + 1 mg·l-1 naa + 1 mg·l-1 iaa medium was 50%, and the average root length was 6.96 mm. in the course of the experiment, it was found that the overall rooting was better when 1/2 ms was used as the basal medium than when ms was used as the basal medium, so 1/2 ms was used as the basal medium for subsequent adventitious shoot induction rooting. although the rooting rate was higher when 1/2 ms was used as the basal medium, the effective number of roots was lower, and during the rooting process, most of the adventitious shoots in the medium would gradually disappear, leaving only a large number of healed and adventitious roots produced. after the main roots had lignified and a certain number of fibrous roots had grown, the caps of the histoponic bottles were left open for a small period of time; after 1~2 d, the caps were left open for another period of time to allow the regenerated seedlings to gradually adapt to the natural environment. after one week of refinement, the root medium of the regenerated seedlings was rinsed with sterile water and transferred into sterilized vermiculite: charcoal: sand = 1:1:1. to maintain a certain temperature and humidity, the nursery pots for transplanting regenerated seedlings were covered in multiple layers using cling film. wang p et al. (2023). not sci biol 15(2):11585 6 based on the above results and the observation during the experiment, it is concluded that the seeds, cotyledons, fixed buds and top buds can induce budding, and the cotyledons have the best bud-inducing effect, and the most suitable medium for cotyledons to induce budding is ms+0.5 mg·l-1 naa+0.5 mg·l-1 6-ba, the germination rate was 61.9%, and the budding was successively after 14 days. the optimal medium for inducing adventitious bud rooting is 1/2 ms+1 mg·l-1 naa +1 mg·l-1 iba + 1 mg·l-1 iaa, with a rooting rate of 50%, took root after 14 days, and the h. ammodendron regeneration system of organogenesis was successfully established. figurefigurefigurefigure 3333.... flow chart of organogenesis regeneration system of h. ammodendron (note: a: h. ammodendron weed; b: sterile seedling; c: cotyledon nodes; d~g: cotyledon node induced shoot; h~k: adventitious shoot induced rooting; l: h. ammodendron regenerated seedling; m~o: transplant) tabletabletabletable 2.2.2.2. effects of adding different concentrations of hormones on the rooting of regenerated plants with 1/2 ms as the base medium treatment code iba (mg·l )-1 naa (mg·l )-1 iaa (mg·l )-1 rooting rate (%) average root length(mm) growth 1 2 2 4 8.5b 0.75b after the formation of healing wounds, the buds fade away, with roots but no buds 2 2 1 2 0b 0b after the formation of healing wounds, the buds fade away, with roots but no buds 3 4 1 4 0b 0b after the healing wound is formed, the buds fade away. 4 3 2 1 18.5ab 3.5ab after the healing wound is formed, the buds fade away. 5 3 1 3 8.5ab 1.9ab more roots, shorter, more healing, fewer shoots, slower growth 6 1 3 4 14.5ab 4.17ab more roots, slender, fewer shoots, more healing tissue 7 1 1 1 50a 6.96a many roots, good growth condition, more healing tissue, forming a complete plant 8 1 4 2 36.5ab 1.52b many roots, slender, with roots and shoots, slow-growing 9 3 4 4 33.5ab 1.3b after the formation of healing wounds, the buds fade away, with roots but no buds 10 4 2 3 47a 1.36b few slender roots, no buds, many healing wounds, slow growth 11 4 2 2 36ab 1.87ab after the formation of healing wounds, the buds fade away, with roots but no buds 12 4 4 3 0b 1.23b after the formation of healing wounds, the buds fade away, with roots but no buds 13 2 4 1 40.5ab 2.39ab after the formation of healing wounds, the buds fade away, with roots but no buds wang p et al. (2023). not sci biol 15(2):11585 7 14 3 3 2 12.5ab 1.56b after the formation of healing wounds, the buds fade away, with roots but no buds 15 4 3 1 8.5ab 1.97ab after the healing wound is formed, the buds fade away. 16 2 3 3 24.5ab 1.9ab buds disappeared, only healing wounds remain, roots few, slender discussiondiscussiondiscussiondiscussion the use of plant tissue culture techniques to obtain regenerated plants directly or indirectly through the somatic embryo pathway or organogenesis pathway is the basis for biotechnological breeding, protoplast culture, somatic cell hybridization and gene transformation research. in this study, the induction of adventitious buds was carried out using h. ammodendron seeds, hypocotyls, cotyledons, cotyledon nodes, fixed buds and terminal buds as explants, and it was found that h. ammodendron seeds, cotyledon nodes, fixed buds and terminal buds could be induced to produce buds, but hypocotyls and cotyledons did not produce buds. the cotyledonary nodes showed fast and high germination rate when germination was induced. due to the long time for the formation of definitive and terminal buds in h. ammodendron sterile seedlings, the source of material was limited and time-consuming; the cotyledon nodes induced better quality of germination, and the cotyledon nodes were finally identified as the best explants for germination induction. yasmin et al. (2003) showed that the types and proportions of different exogenous plant hormones have a significant effect on the regeneration of the same plant. 6-ba favours cell division and affects organ differentiation, and in agreement with chen and gao reported (dabauza et al. 1977), a mixed fraction of 6-ba and growth hormone was found to increase the rate of germination induction in plumbago auriculata. higher 6-ba concentrations strongly inhibited shoot elongation, resulting in little or no production of large shoots. this finding is consistent with the cotyledon explants of kale type oilseed rape (xiaolan et al. 2001). this may be because the plant has reached a specific balance between internal and external hormones and the hormone receptors are saturated (miedema, 1984; pasqualetto et al., 1986; leshem et al., 1988; liu et al., 2008) found that high levels of cytokinins can cause vitrification in in vitro cultures of phytoplankton with high water content, respectively. however, there was no significant vitrification of adventitious shoots in the present study. the specific cause of this problem deserves further investigation. in addition, it was found in the pre-test pre-experiment that the h. ammodendron regeneration seedlings could be rooted when ms and 1/2 ms were used as the base medium, but it was found that the growth of explants on 1/2 ms medium was better than ms during the test observation, so in this experiment, we chose 1/2 ms as the base medium for rooting, and the rooting rate could reach when iba, iaa & naa were added to 1/2 ms 50%. during the rooting phase, iba, iaa, and naa are the three most used hormones. the effects of iba on rooting have been shown to be more effective than other plant growth regulators (amiri and elahinia, 2011). in addition, the addition of naa had some promotion effect on the growth of adventitious roots. this may be due to the appropriate concentration of growth hormone acting on the cells, which facilitates the binding to atpase on the plasma membrane. this acidifies the cell wall environment and some unstable hydrogen bonds are easily broken, allowing the molecular structures of cell wall polysaccharides to interweave. the cell wall tends to relax, making it easy for the cells forming adventitious roots to break through (li et al., 2009). therefore, it is necessary to investigate the optimal concentration for rooting. in addition, many studies have shown that iaa can promote root formation and growth, such as banana and bergamot (miilion et al., 2015; jose et al., 2015). although the root rate of this graduate student is high, most adventitious shoots will disappear in the later culture, and only calluses and adventitious roots will appear, which is guessed to be related to the concentration of plant hormones, and subsequent experiments also need to adjust the concentration of plant hormones to induce rooting. browning, vitrification, and contamination are relatively common and poorly controlled phenomena in in vitro plant culture. vitrification is a physiological lesion in wang p et al. (2023). not sci biol 15(2):11585 8 which the regenerated shoots are translucent and have an abnormal morphological appearance. the majority of vitrification seedlings are adventitious shoots from stem tips or stem cut cultures, and usually the recovery percentage of vitrification seedlings is very low, and vitrification seedlings still form in succession cultures. in this experiment, the occurrence of vitrification seedlings was less frequent at the induction stage of shoot emergence, and it was observed that the occurrence of vitrification seedlings was reduced accordingly by decanting the residual water in the culture flasks. in summary, this study establishes a system for h. ammodendron regeneration by organogenesis. the method will provide a basis for the application of h. ammodendron ex vivo regeneration and genetic transformation. conclusionsconclusionsconclusionsconclusions it plays an extremely important role in protecting desert ecosystems and preventing desertification. ex vivo reproduction is an effective method for species conservation and restoration. in this study, six explants of the h. ammodendron were used to establish an ex vivo regeneration system of the shuttle by inducing adventitious buds, adventitious bud rooting and transplanting. the optimal medium for inducing clandine buds in cotyledon nodes differentiation is 0.5 mg·l-1 naa+0.5 mg·l-1 6-ba induced budding rate was high, reaching 61.90%, rooting medium was 1/2 ms+1 mg·l-1 naa +1 mg·l-1 iba + 1 mg·l-1 iaa with a rooting rate of 50%. this study will lay a solid technical foundation for in-depth study of the molecular mechanism of drought resistance and cultivation of excellent germplasm. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization: hua zhang and bo wang; data curation: ping wang, lingjuan man and li ma; formal analysis: ping wang, lingjuan man and li ma; investigation: ping wang, lingjuan man and li ma, jiaxin qi; methodology: yanping ren and cong chen; supervision: ping wang, lingjuan man and li ma; visualization: lingjuan man and ping wang; writing original draft: ping wang; writing review and editing: hua zhang and zhengpei yao. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this work was supported by natural science fondation of xinjiang uygur autonomous rigion 2022d01d35. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. wang p et al. 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(microsoft word 11564 nsb galv\341n-moreno 2023.06.28.docx) received: 10 may 2023. received in revised form: 26 jun 2023. accepted: 27 jun 2023. published online: 28 jun 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania galván-moreno vs et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 2, article number 11564 doi:10.15835/nsb15211564 research articleresearch articleresearch articleresearch article.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae evaluation of two sampling methods for evenevaluation of two sampling methods for evenevaluation of two sampling methods for evenevaluation of two sampling methods for even----age age age age pinuspinuspinuspinus fffforestorestorestorest in northern mexicoin northern mexicoin northern mexicoin northern mexico viridiana s. galván-moreno1,2, jesús m. olivas-garcía2*, joel rascón-solano1,2, javier hernández-salas2 1autonomous university of nuevo león, faculty of forestry sciences, doctoral program in science with a focus on natural resource management, national highway #85, km. 145, linares, postal code 67700, nuevo león, mexico; viridiana2910@outlook.com; forestal_rascon@hotmail.com 2autonomous university of chihuahua, faculty of agricultural and forestry sciences, 2.5 km on delicias-rosales road, postcode 33000, delicias, chihuahua, mexico; jolivas@uach.mx (*corresponding author); jhernans@uach.mx abstractabstractabstractabstract the objective was to evaluate and compare the precision of estimation of dasometric variables, survey times and costs of two sampling methods versus a census, applied to a natural even-age mass of pinus arizonica engelm. in the municipality of guachochi, chihuahua, mexico. nine fixed-dimension and nine variabledimension sites were performed with bitterlich's angular sampling at coincident sample points. times from start to finish were taken by site and technicians from the state were surveyed to determine sampling costs. anova tests were developed at a significance level of 0.05, comparing both methods with the census. the results indicate that, in both samplings, the number of trees per hectare does not present statistical differences with respect to the census. the basal area by both methods was not statistically different, however, fixed dimensions present an error greater than 5%. finally, the volume per hectare estimated was similar to that of the census, according to the rates carried out, greater precision was found by the variable area method (error=0.03%). the execution time showed significant differences (p=0.0001), the fixed dimension site required a mean time of 44 minutes and the variable dimension one 10 minutes 26 seconds. the total costs are 679.90 and 654.33 euro to sample nine fixed-dimension and nine variable-dimension sites respectively, showing significant differences (p=0.0008). it is concluded that both methods are statistically acceptable for the variables evaluated in the type of forest mass studied. keywords:keywords:keywords:keywords: angular sampling; forest sampling; fixed area sampling; sampling costs; sampling times; variable area sampling introductionintroductionintroductionintroduction forests are the largest terrestrial ecosystem, occupying 30% of the planet's surface area (food and agriculture organization of the united nations (fao), 2007). mexico occupies the twelfth place in forested areas worldwide (fao, 2010). it has an area under timber harvesting of 6,290,610 hectares (secretaría del medio ambiente y recursos naturales (semarnat), 2020). https://www.notulaebiologicae.ro/index.php/nsb/index galván-moreno vs et al. (2023). not sci biol 15(2):11564 2 ortiz-reyes et al. (2015) and martín-garcía et al. (2017) indicate that the most common practice to estimate the population parameters of the dasometric stocks is the forest inventory, and it is supported by sampling tools based on a design and estimators based on models (ruíz, 1982). the precision of an inventory estimate is determined by the sampling method used (paula-neto, 1990). in sampling, a part of a population is selected and used to obtain estimates of the characteristics of that population (moscovich and brena, 2006; köhl and magnussen, 2016). in this way, and depending on the type of data obtained in the field, it is possible to calculate different levels of precision (schroeder, 1992). some of the objectives, when carrying out a forest inventory through sampling, are to obtain reliable results to know the tree diversity and the density condition of the stands (aguirre and jiménez, 1995), calculate the volume (kramer and akça, 1987) and plan forestry activities (schreuder et al., 2006). these parameters are necessary to obtain dasometric estimates of the population of interest (schreuder et al., 2006; köhl, magnussen and marcheti, 2006; aguirre-salado et al., 2009). prodan et al. (1997) indicate that there are various sampling methods to estimate the variables of a tree population. sampling through the use of sites of fixed dimensions of 0.1 hectares (ha) is the most used in forest inventories (aguirre et al., 1995). in this method, the selection of individuals is carried out proportionally to the surface area of the minimum management or sampling unit and, in turn, by the frequency of individuals found within it (pellico-netto and brenna, 1997). on the other hand, the bitterlich or variable dimensions method (schreuder et al., 1993) is used only to a limited extent. finger (1992) mentions that the number of trees in a stand, whose normal diameter (dn1.30), from a fixed point, appears greater than a constant alpha angular value, is proportional to the basal area in square meters per hectare (g-1). regarding the levels of precision, schroeder (1992) and machado and león (2005) mention that in forest inventories it is feasible to obtain different levels of precision. on the other hand, the estimation accuracy of the variables has a direct effect on the quality of the planning, management and evaluation of existing possibilities in the forests (bettinger et al., 2009). it is important to highlight that, given the importance of the inventory for the development of sustainable forest management strategies, it must be carried out according to the established precision levels, optimizing time, costs and quality of the data collected (aguirre et al., 1997). additionally, lara and espinosa (1994) mention that the conservation and use of forest resources in a continuous and sustained manner will only be possible when greater precision is achieved in their distribution and quantification. in this sense, the objective of this research was to evaluate and compare the precision of estimation of dasometric variables, sampling times and costs of two temporal sampling methods based on a census applied to a natural even-age mass of pinus arizonica engelm. in the municipality of guachochi, chihuahua, mexico. the objective of this study is based on the hypothesis that the sampling methods do not present statistical differences in the precision of estimations of dasometric variables in even-age forests the and the execution costs are statistically different between sampling methods. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods study location it was carried out in the municipality of guachochi, located in the southwest of the state of chihuahua, mexico, in the physiographic province of sierra madre occidental. in the area there is a cold temperate forest of pinus arizonica engelm (figure 1). the type of soil is eutric regosol, of medium to fine texture (inegi, 2014), and the predominant climate is c(e)(w2)(x'), humid semi-cold with an average annual temperature between 5 and 12 °c, and an average annual rainfall of 621.3 mm (inegi, 2008). galván-moreno vs et al. (2023). not sci biol 15(2):11564 3 figure 1.figure 1.figure 1.figure 1. location of the uniform-age pine forest studied in northern mexico materials and equipment used the dasometric data for the study were taken from each of the individuals. the database was made up of trees whose normal diameter (dn1.30) ≥ 7.6 cm was measured with a 90 cm haglöf® caliper and the total height with a suunto® pm5-15-20 hypsometer. additionally, the species to which each individual belongs was recorded. the simple bitterlich relascope was used to estimate the basal area in sites of variable dimensions. methods a standard hectare was delimited in a 40-year-old even-aged forest, with flat topography, without a logging program, with coordinates 26° 50' 43.28" n and 107° 04' 55.54" w (figure 2). the dasometric data of the total trees, species, normal diameter and total height of each of the individuals were recorded. figure 2.figure 2.figure 2.figure 2. individuals identified numerically in an even-aged mass of pinus arizonica within the established type hectare galván-moreno vs et al. (2023). not sci biol 15(2):11564 4 within the census area, nine sites of 1,000 m2 and nine sites of variable dimensions of bitterlich were established with a systematic sampling method. these data were collected to determine the statistical precision of each sampling method based on actual stocks. for this, the number of trees per hectare (n arb ha-1) was calculated with the following relationship: n trees ha¯ = ∑ ������ m ∗ 10 where: n trees ha-1 = number of trees per hectare. n tressi = number of trees in the site i. m = number of sites. the estimation of basal area per hectare (ab m2 ha-1) was carried out with the following procedure: ab m² ha¯ = ∑ g� (m �) m ∗ 10 where: ab m2 ha-1 = basal area in square meters per hectare. gi (m2) = basal area of site i expressed in square meters. m = number of sites. the volume (vol m3 ha-1) was determined using the equation developed by the umafor-0807 biometric model (conafor chihuahua government umafor 0807, 2014), presented below: vta = 0.000033 ∗ (dn^1.957172) ∗ (h^1.103479) + 0.000132 ∗ (dn^2) where: vta = total tree volume. dn = normal diameter (1.30 m). h = total height. the times from the installation of each of the methods, until the end of data collection, and transfers from site to site were recorded. the comparison of the methods in the efficiency in the estimation of times, was made through the calculation of the relative efficiency (re) by means of the following equation (silva, 1998). re = 1 t/ ∙ cv � where: re = relative efficiency. tx = installation and measurement time. cv = coefficient of variation. to determine fixed costs, variable costs and total costs, the application of 20 structured interviews was carried out with providers of technical forestry services that use or have used sampling both through sites of fixed dimensions and variable dimensions in the municipalities of madera, guachochi and bocoyna, chihuahua, and the costs of equipment, personnel training, salaries, food, fuel and medical service were considered, mainly. statistical analysis in order to detect if there are statistical differences in the precision of estimates of dasometric variables between methods and in comparison, with the census, data recording times and costs involved in the development of both methods, the homogeneity of the variances of the sampling methods was evaluated employed by levene's tests at a significance value of p ≤ 0.05. analysis of variance (anova) tests were applied at a significance level of p ≤ 0.05 to determine the statistical differences between methods with respect to the census in the variables of number of trees per hectare, basal area per hectare and total tree volume per hectare. the statistical package ibm-spss® version 25 was used to carry out the analysis of the variables. galván-moreno vs et al. (2023). not sci biol 15(2):11564 5 results results results results analysis of the number of trees per hectare the difference in the number of trees per hectare estimated by the fixed dimension method is not significant (p = 0.8975) with respect to the census, the same as the variable dimension method (p = 0.8607). figure 3 shows that the sites of fixed dimensions presented absolute ranges from 310 to 540 individuals per hectare, according to the results of the nine established sample units. for its part, the bitterlich method shows absolute ranges from 291 to 629 trees per hectare. these values indicate that despite the fact that the mass is coetaneous and the estimates are similar in terms of the actual number of trees per hectare, the irregular distribution of the trees may have an effect on the precise calculation of density. figure 3.figure 3.figure 3.figure 3. comparison of ranges to estimate the number of trees per hectare by dasometric sampling method *common letter indicates equality in anova test (p ≤ 0.05). the analysis of the type or census of the hectare indicates the presence of 398 individuals of pinus arizonica. on the other hand, the estimate product of the sampling of fixed dimensions (nine sites of 0.1 ha) resulted with 388 trees per hectare, which represents an underestimation of -2.57 %. the sampling by variable dimensions method estimates 418 trees per hectare, representing a 5.14% surplus with respect to the result of the census. according to the analysis, this estimation error would have an effect on the sustainable management of the stand; however, the statistical analysis indicates that the difference is not significant as previously mentioned. this indicates that the fixed dimension method is more efficient; but, as mentioned above, the difference between the two methods is not significant. analysis of basal area per hectare the nine sites with fixed dimensions presented ranges from 20.85 to 32.54 m2 ha-1 of basal area (figure 4), the site that presented the highest value represents outlier data. this result indicates the presence of a greater number of individuals, or diametric dimensions greater than the average for the study area. the variable area method shows basal area levels between 22.00 and 38.00 m2 ha-1 depending on the nine measurement points. the results found indicate that the distribution of the trees, their diameter, and the density of the mass, generate variability in the estimation of basal area per hectare between sampling sites. however, they do not galván-moreno vs et al. (2023). not sci biol 15(2):11564 6 represent a significant effect on the estimated mean values. this assertion is supported by a p value = 0.7483 in the comparison of census means and sites of one tenth of a hectare. the variable area sampling method showed similarity with the results obtained from the total count, with a significance of p = 0.8582. figure figure figure figure 4444.... comparison of ranges for estimation of basal area per hectare by dasometric sampling method * common letter indicates equality in anova test (p ≤ 0.05). according to the values obtained by the different types of sampling, in basal area estimation (ba), the census or total count recorded 27.13 m2 ha-1, and the fixed dimensions method resulted in a basal area of 26.01 m2 ha-1, therefore, underestimated with -4.12%; the bitterlich method resulted in a basal area of 28.22 m2 ha1, with an overestimation of 4.02 % with respect to that estimated by the census of the typical hectare. the bitterlich method was the one that came closest to the average, which is due to the regular mass structure, which is the type of forest where the study was carried out. analysis of total tree volume per hectare the difference in total tree volume per hectare estimated in the fixed dimension method is not significant (p = 0.7382) with respect to that the census. likewise, the bitterlich method does not show differences in the estimation of volumetric stocks compared to the total survey (p = 0.9992). the sites of fixed dimensions presented volumetric ranges from 179.71 to 366.58 m3 ha-1 in the results of the nine sample units established. for its part, the variable area method shows ranges from 201.42 to 459.43 m3 ha-1. the estimation of the volume of the sites presents variability in the ranges, due in part to the fact that in the sites of variable dimensions a reference tree is taken with representative dasometric characteristics of the sampling point; instead, fixed-size sites take into account the individual volume of trees in the sample area. according to the results obtained by the different types of sampling, in estimation of total tree volume, the census or total count has 287.40 m3 ha-1, the fixed dimensions method resulted in a volume of 265.75 m3 ha-1, therefore, it underestimated the volumetric stocks with -7.53 %, in this case, the volume calculated by this method exceeds the expected error of 5.00 %, which implies a problem in decision making in the sustainable management of the mass. using the bitterlich method, a total tree volume of 287.31 m3 ha-1 was calculated, having an underestimation of -0.03 % with respect to that estimated by the census of the typical hectare. the bitterlich method was the one that came closest to the average (figure 5), which indicates that it is an adequate method to estimate the volumetric stocks in forests with characteristics similar to those shown in this study. galván-moreno vs et al. (2023). not sci biol 15(2):11564 7 figure figure figure figure 5555.... comparison of ranges for estimation of total tree volume per hectare by dasometric sampling method * common letter indicates equality in anova test (p ≤ 0.05). the estimates made indicate that both sampling methods are adequate to determine the stocks present in pine forests with regular and even-aged stands. however, the absolute differences presented by the fixed dimension method in the estimation of the number of trees per hectare (5.12 % error) and total tree volume per hectare (7.53 % error) based on the total count, indicate the need for perform deeper analyzes in the sampling methods, because the spatial distribution of the individuals (location and diameter) may affect the values obtained, or even the size of the site may not be the most adequate to obtain a sample accurate in this forest condition. inventory data collection time in the present study, with the fixed sampling method, ranges from 31 to 54 trees per site were obtained. the general data recording and quantification of trees represents an estimated time of 28 to 67 minutes. with the variable area sampling method, under the basal area factor parameter used (factor 4), ranges from 6 to 12 individuals per sampling point were obtained. the general record of forest inventory data and the estimation of basal area present survey times of 9 to 12 minutes. according to the information shown in figure 6, the sites with fixed dimensions show a significant increase in the time taken to carry out the sampling as a function of the density of trees within the plot. on the other hand, the regression obtained through the relationship of survey time and number of trees registered in the bitterlich angular count sites, indicates that the density of the trees represents an increase to a lesser degree in the registration time than the sites of fixed dimensions. galván-moreno vs et al. (2023). not sci biol 15(2):11564 8 figure figure figure figure 6666.... relation between the number of trees registered and the time involved in the survey of forest sampling sites (fd = fixed dimensions; vd = variable dimensions) contrast of data collection times the applied methods presented significant differences (p = 0.0001) in the comparison of times in the survey of the sites. the fixed-size sampling method presented an average of 44 minutes, and the bitterlich method showed greater efficiency, spending less time in the sampling data collection with an average of 10 minutes 26 seconds (figure 7). based on the results, it is estimated that according to the average time to execute a site of fixed dimensions, it is possible to make at least four sites of variable dimensions in a forest mass such as the one studied. according to the maximum range of time for establishing a fixed dimension site, it is possible to make between five and seven variable dimension sites based on their upper and lower limits. likewise, it is possible to measure two to three bitterlich sites in the time required to record the trees of a fixed dimension site. figure figure figure figure 7777.... graphical comparison of anova test results for run time by sampling method (fd = fixed dimensions; vd = variable dimensions). * common letter indicates equality in statistical test (p = 0.05). galván-moreno vs et al. (2023). not sci biol 15(2):11564 9 estimated cost of sampling according to the survey applied to providers of technical forestry services in the municipalities of guachochi, madera and bocoyna, chihuahua, average fixed costs of 48.62 euro were generated in the concept of medical service, 200.35 euro is the average cost of training personnel for the two inventory methods, and finally, the cost of forestry inventory equipment, which in the case of fixed dimensions has an average of 394.03 euro, which includes a pocket diameter tape with an estimated cost of 19.71 euro; a meter of height by tangent of 23.24 euro; a bounded cable to delimit the surface of the site at a cost of 24.96 euro; a satellite geopositioning device (gps) for 117.48 euro and an auger for the extraction of increment samples with a value of 211.03 euro. for its part, bitterlich sampling requires an investment in equipment of at least 381.09 euro. in general, the same type of equipment is used as in the previous method, except that in this case the bounded cable is replaced by a simple bitterlich relascope or a “jalometro” with an approximate cost of 12.02 euro. a mean salary of 2.80 euro per fixed-size site and 1.40 euro per variable-size site was estimated, with a p value = 0.0001; in food, kitchen equipment and food for the brigade were taken into account, obtaining an average of 1.00 euro. regarding the material for data recording, paper, pencil and eraser, an average of 0.08 euro was obtained. based on the maximum and minimum distances to the areas where an inventory has been carried out, an average of 0.19 euro in fuels per site was determined. according to the analysis of the estimates of the costs of the methods of fixed and variable dimensions through the anova test, it was found that the fixed and total costs present significant differences (p = 0.0001 and p = 0.0008 respectively) (figure 8), while for variable costs the difference is not significant (p = 0.1615). figure figure figure figure 8888.... estimated differences in sampling cost accounting (fd = fixed dimensions; vd = variable dimensions) discussiondiscussiondiscussiondiscussion moscovich and brena (2006) mention that by analysis of variance it was verified that the methods they applied (fixed area, strand, prodan, bitterlich and quadrantes), did not show significant differences in the estimation of volume, number of trees per hectare and basal area. this comparison coincides with the results in the present study, since the fixed area and variable area (bitterlich) methods did not present significant galván-moreno vs et al. (2023). not sci biol 15(2):11564 10 differences in the estimation of the number of trees per hectare, basal area per hectare and total tree volume per hectare. roldán-cortes et al. (2014) determined that in the case of the basal area/volume ratio, it turned out that for each m2 of ba in a site, there are approximately 8.05 m3 of standing volume. like the results obtained in the comparison of stocks in the census and estimates from the bitterlich method, this relationship can be used to carry out rapid inventories when only the relascope is used to determine the basal area. on the other hand, ríos et al. (2000) indicate that the sampling error, as expected, increases with the increase in the basal area factor, or also with the decrease in the plot area. defined for each factor of basal area and mean diameter. corvalán (2019) refers to the fact that the survey time of the sites depends on the area that will be inventoried and the speed of movement within the plots. similarly, the results of this research indicate that the greater the number of individuals to be recorded in the sampling, the greater the time required to carry out the forest inventory. on the other hand, sites of variable dimensions do not require a large sample of individuals, which means a decrease in the time for collecting dasometric data. manzanero and pinelo (2004) determined the variable costs of a silvicultural, remanence, and diagnostic sampling for the petén forest in guatemala, where 500 sites were surveyed (50 sites per day), for ten days, with a budget of $831.17 dollars (usd), equivalent to 762.34 euro for salary. according to the information obtained through interviews, the registration of field data requires an investment for the concept of salary of 2.80 euro per site of fixed dimensions and 1.40 euro per site of variable dimensions, these amounts being higher and lower respectively compared to the costs registered in petén, guatemala. finally, lara and espinosa (1994) indicate that, in a census carried out in the state of michoacán, mexico, it was found that sites with variable dimensions have greater precision in estimating ba than sites with fixed dimensions (1,000 m2). regarding the time required to register the individuals in the sampling, the bitterlich angular site requires 3.8 times less time than the fixed dimension sampling. lastly, the equipment used for measuring sites of variable dimensions tends to be less expensive than that used to survey fixeddimension sites. in the present study it was found that the variable area method turns out to be more precise (error = 4.02%), however, the precision differences turn out to be non-significant also for the fixed dimension site. regarding the time taken to record the inventory data, it is estimated that the fixed dimensions site requires 4.19 times more time than the bitterlich angular sampling. conclusionsconclusionsconclusionsconclusions the methods studied present acceptable statistical precision when estimating the number of trees per hectare, basal area and volume. the time involved in collecting inventory data is more efficient in the application of the bitterlich method, since it requires only 23.72% of the time used in the fixed dimensions method. the cost per execution of both samplings presents significant differences, being the method of variable dimensions more favorable when taking field data in even-aged forests with absolute presence or dominance of the genus pinus. although variable-size sampling tends to be cheaper than fixed-size sampling, there is no statistically significant difference. the differences observed in the fixed dimension method for estimating the number of trees per hectare and total tree volume per hectare compared to the total count, indicate the need to conduct further analyses on sampling methods, due to the random spatial distribution of trees, which can result in biases in the estimations made. the spatial characteristics of the individuals can affect the values obtained, or even the dimension of the site, is not the most adequate to obtain an accurate sample in this forest condition. the proposed hypothesis is accepted because the sampling methods did not present statistical differences in the precision of the estimates of dasometric variables in natural even-age forests. likewise, the execution costs of the forest sampling are statistically different between methods, because the variable galván-moreno vs et al. (2023). not sci biol 15(2):11564 11 dimension method turned out to be less expensive. these results can be applied to carry out larger-scale forest sampling in even-age forests in order to obtain accurate information quickly and at a lower cost. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions conceptualization of the research idea, designing the experiment, and writing-original draft preparation, v.s.g.m.; formal analysis, validation and discussion, j.m.o.g., j.h.s. and j.r.s.; resources, data curation and writing-review and editing, c.m.k.d. and h.t.v.; supervision, v.s.g.m. and j.m.o.g. all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. acknowledgementsacknowledgementsacknowledgementsacknowledgements this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. the authors would like to express our gratitude to the anonymous reviewers who, with their comments, have improved the content of this manuscript. conflict of interestsconflict of interestsconflict of interestsconflict of interests the authors declare that there are no conflicts of interest related to this article. referencesreferencesreferencesreferences aguirre oa, jiménez j (1995). guía de densidad para pinus teocote schl. et cham. en nuevo león [density guide for pinus teocote schl. et cham. in nuevo leon]. memoria del ii congreso mexicano sobre recursos forestales. pp 22 montecillo, estado de méxico. aguirre oa, jiménez j, meráz b (1995). optimización de inventarios para manejo forestal: un caso de estudio en durango, méxico [inventory optimization for forest management: a case study in durango, mexico]. investigación agraria: sistemas y recursos forestales 4(1):107-118. https://doi.org/10.5424/540 aguirre o, jiménez j, treviño e, meráz b (1997). evaluación de diversos tamaños de sitio de muestreo en inventarios forestales [evaluation of various sample site sizes in forest inventories]. madera y bosques 3(1):71-79. https://doi.org/10.21829/myb.1997.311380 aguirre-salado ca, valdez-lazalde jr, ángeles-pérez g, de los santos-posadas hm, haapanen r, aguirre-salado ai (2009). mapeo de carbono arbóreo aéreo en bosques manejados de pino patula en hidalgo, méxico [aerial tree carbon mapping in managed patula pine forests in hidalgo, mexico]. agrociencia 43:209-220. bettinger p, boston k, siry jp, grebner dl (2009). forest management and planning. new york: academic press, pp 360. conafor-gobierno de chihuahua-umafor 0807 [conafor-gch-umafor 0807] (2014). sistéma biométrico umafor 0807, guachochi, chihuahua [biometric system umafor 0807, guachochi, chihuahua]. chihuahua, méxico: comisión nacional forestal, pp 54. galván-moreno vs et al. 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(2020). anuario estadístico de la producción forestal 2017 [statistical yearbook of forest production 2017]. ciudad de méxico: secretaría de medio ambiente y recursos naturales (semarnat), pp 284. silva i (1998). aplicação do método de amostragem de strand para estimação da densidade na regeneração natural de espécies arbóreas e arbustivas tropicais na amazônia occidental. tesis de maestría en ciencias [application of strand's sampling method for density estimation in the natural regeneration of tropical tree and shrub species in the western amazon. master's thesis in science]. curitiba, brasil: universidade federal do paraná, pp 113. the journal offers free, immediate, and unrestricted access to peer-reviewed research and scholarly work. users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. license license license license ---articles published in notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae are open-access, distributed under the terms and conditions of the creative commons attribution (cc by 4.0) license. © articles by the authors; licensee smtct, cluj-napoca, romania. the journal allows the author(s) to hold the copyright/to retain publishing rights without restriction. notes:notes:notes:notes:  material disclaimer: the authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  maps and affiliations: the publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  responsibilities: the editors, editorial board and publisher do not assume any responsibility for the article’s contents and for the authors’ views expressed in their contributions. the statements and opinions published represent the views of the authors or persons to whom they are credited. publication of research information does not constitute a recommendation or endorsement of products involved. microsoft word 11414 nsb zouhri 2023.03.22.docx received: 17 dec 2022. received in revised form: 30 jan 2023. accepted: 06 mar 2023. published online: 22 mar 2023. from volume 13, issue 1, 2021, notulae scientia biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. the journal will continue to appear quarterly, as before, with four annual numbers. shstshstshstshst horticulture and forestryhorticulture and forestryhorticulture and forestryhorticulture and forestry society of transylvaniasociety of transylvaniasociety of transylvaniasociety of transylvania zouhri a et al. (2023) notulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicaenotulae scientia biologicae volume 15, issue 1, article number 11414 doi:10.15835/nsb15111414 rererereviewviewviewview articlearticlearticlearticle.... nsbnsbnsbnsb notulae scientia notulae scientia notulae scientia notulae scientia biologicaebiologicaebiologicaebiologicae a review of moroccan medicinal plants used a review of moroccan medicinal plants used a review of moroccan medicinal plants used a review of moroccan medicinal plants used in the treatment of inflammationin the treatment of inflammationin the treatment of inflammationin the treatment of inflammation aziz zouhri1,3*, yahya el-mernissi2, naoual el menyiy3, toufik bouddine1, nora mir1, kamal el aissaoui4, hassan amhamdi2, abdelhay elharrak1, lhoussain hajji1 1moulay ismail university, faculty of sciences, bioactives and environmental health laboratory, meknes b.p. 11201, morocco; az.zouhri@edu.umi.ac.ma; t.bouddine@edu.umi.ac.ma; nora120@hotmail.fr; a.elharrak@umi.ac.ma; l.hajji@umi.ac.ma 2abdelmalek essaadi university, applied chemistry team, faculty of science and techniques, al hoceima 32003, morocco; yahya.elmernissi@etu.uae.ac.ma; h.amhamdi@uae.ac.ma 3national agency for medicinal and aromatic plants, laboratory of pharmacology, taounate 34025, morocco; nawal.elmenyiy@usmba.ac.ma 4translation, dialog of civilisations and arbitration, school of arts and humanities, meknes n13, morocco; kamal1elaissaoui@gmail.com abstractabstractabstractabstract inflammation is a mechanism that occurs against aggressive agents such as infections, wounds, pollens, autoimmune diseases, and tissue damage. morocco has traditionally used herbal remedies to treat many illnesses, including inflammation. the objective of this study was to describe the moroccan medicinal plants used in inflammation treatment. this systematic review examines ethnobotanical studies published in english and french in the literature as well as the pharmacological studies that assess anti-inflammatory activity in vitro and in vivo, from databases (scopus, web of science, science direct, and springer). the research was carried out until august 2021. in ethnobotanical surveys, 100 medicinal plants were identified, which belonged to 52 families of which the most often cited are lamiaceae with 18 species, followed by apiaceae (13 species) and asteraceae (10 species). the most used plants to treat inflammation in morocco are: coriandrum sativum l, caralluma europaea (guss.) nebr., opuntia ficus-indica (l.) mill., capparis spinosa l., anacyclus pyrethrum (l.) lag, ajuga iva (l.) schreb., thymus atlanticus (ball) pau, thymus saturejoides coss, thymus zygis l., lawsonia isermis l. and rosmarinus officinalis l. this review highlighted moroccan medicinal plants used in traditional medicine to treat inflammation. many of this plant species require additional pharmacological, and clinical studies to verify which plants are effective in treating inflammation. keywords:keywords:keywords:keywords: anti-inflammatory; inflammation; moroccan; medicinal plants; systematic review; traditional medicine https://www.notulaebiologicae.ro/index.php/nsb/index zouhri a et al. (2023). not sci biol 15(1):11414 2 iiiintroductionntroductionntroductionntroduction inflammation is a response to harmful stimuli, such as infections, injuries, allergens, autoimmune conditions, and tissue damage (konstantopoulos, 2005; ribaldone et al., 2018). inflammation is characterized by warmth, edema, pain, redness, and impaired function of affected tissues (chen et al., 2018; ferrero-miliani et al., 2007). according to its course, inflammation is divided into acute, and chronic. acute inflammation is an innate, primary reaction that occurs mainly over a short time due to tissue damage (suzuki, 2019). if not stopped in time, the inflammation will develop into a chronic phase leading to cancer (abdalla et al., 2020), alzheimer's disease, cardiovascular disease, and type 2 diabetes (nishimura et al., 2009; pahwa et al., 2021; saltiel and olefsky, 2017). to treat these conditions, nonsteroidal anti-inflammatory drugs are the most widely used drugs to alleviate pain, and inflammatory symptoms (bribi et al., 2015). secondary metabolites like flavonoids, terpenoids, alkaloids, tannins, coumarins, and essential oils are considered alternative traditional herbal remedies (guimarães et al., 2012; lima et al., 2013; srinivasan et al., 2001). morocco (figure 1) is known for its climate diversity, and richness in medicinal plants, many of which are used to treat inflammation (amrati et al., 2021; benayad et al., 2014; bouhlali et al., 2016; bouyahya et al., 2021; derouich et al., 2020; el azhary et al., 2017; el kharraf et al., 2021; hmidani et al., 2019; jawhari et al., 2020; kebbou et al., 2019; khouya et al., 2015, 2020; manouze et al., 2017; mechchate et al., 2021; moutia et al., 2016; zouhri et al., 2017) not witstanding its importance, there are no reviews on anti-inflammatory activity in morocco. in this context, the purpose of this first research is to identify ethnobotanical studies that can reveal plants used to treat inflammation in different regions of morocco. on the other hand, to identify articles referring to the pharmacological evaluation of these plants in experimental inflammation. materials and methodsmaterials and methodsmaterials and methodsmaterials and methods web of science, scopus, science direct, and springer search engines were explored using terms related to the following subject areas: ethnobotanical/ethnomedicinal studies reporting on medicinal plants used for traditional inflammation treatment in morocco, moroccan medicinal plants, inflammation, and medicinal plants in morocco, inflammation, and treatment with medicinal plants in morocco, the anti-inflammatory activity of plants of morocco (figure 1). the electronic databases were assessed between june/2021 and august/2021. this systematic research was carried out according to prisma (moher et al., 2016). two investigators identified and examined the resulting papers for relevance based on their titles, and abstract. plant names, and families are confirmed through data available on site (www.theplantlist.org). microsoft excel is used for statistical data analysis. zouhri a et al. (2023). not sci biol 15(1):11414 3 figure 1.figure 1.figure 1.figure 1. map of morocco results results results results and discussionand discussionand discussionand discussion study selection a total of 8964 relevant articles are identified by two researchers from an electronic database search. of these, 4637 are from web of science, 3528 springer, 494 from scopus, 305 from science direct. after removing duplicates by consulting the associated titles, and abstracts, a total of 171 articles are retrieved for full text review. after a detailed review of each article, 97 articles are excluded, and 74 articles are retrieved including 23 ethnobotanical articles, 51 pharmacology papers (figure 2). figure 2.figure 2.figure 2.figure 2. flowchart for systematic literature search zouhri a et al. (2023). not sci biol 15(1):11414 4 ethnobotanical studies 100 plant species belonging to 52 botanical families are inventoried in this study (table 1). the most represented families are lamiaceae with (18 species), apiaceae (13 species), asteraceae (10 species), leguminoseae, oleaceae, rutaceae (4 species each), amaranthaceae, brassicaceae, compositae, and solanaceae (3 species each), amaryllidaceae, anacardiaceae, asparagaceae, cucurbitaceae, euphorbiaceae, fabaceae, fagaceae, lauraceae, papaveraceae, rosaceae, salicaceae, urticaceae, verbenaceae, zingiberaceae (2 species), and apocynaceae, araceae, aristolochiaceae, asclepiadaceae, boraginaceae, cactaceae, capparaceae, caryophyllaceae, combretaceae, cupressaceae, ericaceae, gentianceae, geraniaceae, jungladaceae, liliaceae, linaceae, malvaceae, myristicaceae, myrtaceae, nitrariaceae, plantaginaceae, pinaceae, piperaceae, poaceae, pteridaceae, ranunculaceae, rhamnaceae, zygophyllaceae (1 species each) (figure 3). the prevalence of these families in medicinal plants is likely due to their abundance in morocco's flora. with regards to the most frequently used plants are, salvia officinalis l. was mostly reported (7 mentions), opuntia ficusindica (l.) mill. (6), rosmarinus officinalis l. (6), ajuga iva (l.) schreb. (5), lavandula stoechas l. (5), lawsonia inermis l. (5), nerium oleander l. (4), artemisia herba-alba asso (4), calendula officinalis l. (4), allium cepa l. (3), origanum vulgare l. (3), mentha pulegium l. (3), peganum hamala l. (3), olea europaea l. (3), plantago coronopus l. (3), nigella sativa l. (3), the species recorded are shown in table 1. figure 3.figure 3.figure 3.figure 3. moroccan medicinal plants families used to treat inflammation plant parts the leaves, flowers, roots, seeds, whole plant, and aerial parts are commonly used to treat inflammation. the leaves are the most used part of the plant (39.33%), which are followed by flowers (13.33%), root (10.67%), seed (9.33%), and the whole plant (8.67%). fruits, stem, bulb, and rhizome are also presented with a low percentage (figure 4). the researchers suggest that the leaves are frequently used in herbal medicine as they are easily obtainable (bouyahya et al., 2017). zouhri a et al. (2023). not sci biol 15(1):11414 5 figure 4.figure 4.figure 4.figure 4. percentage use of different plant parts against inflammation preparation methods and route of administration the most commonly used methods traditionally for the treatment of inflammation are decoction (n = 71), infusion (n = 25), powder (n = 22), and cataplasm (n = 12). other less reported preparation methods include maceration (n = 5), and raw (n = 1). the percentage of use of the different preparation methods is presented in figure 5. the majority of anti-inflammatory preparations are taken orally (60%), followed by local application (24.44%), mouthwash (10.37%), and much less by another external route, and inhalation (5.18%) (figure 6). however, the oral route of administration allows a rapid physiological effect to promote the effective action of the herbal remedy. figure 5.figure 5.figure 5.figure 5. percentage use of various preparations methods of plants against inflammation zouhri a et al. (2023). not sci biol 15(1):11414 6 figure 6.figure 6.figure 6.figure 6. percentage various modes of administration of plants against inflammation moroccan medicinal plants used traditionally to treat inflammation diseases a survey was conducted between march and april 2018 in rabat, morocco to gather information on plants with anti-inflammatory properties. the survey results showed that there are three types of plants from two families, asteraceae, and lamiaceae. the most commonly used plants are artemisia herba-alba asso (chih), salvia officinalis l. (sâlmiya) and rosmarinus officinalis l. (azir) (skalli et al., 2019).... another study which was carried out also in same region by salhi et al. (2019) demonstrates the importance of medicinal plants use by local populations in the skin burns treatment. the results identified 36 species belonging to 23 families, and 35 genera used in the skin burns treatment in the region. according to the authors, the most frequently used in the inflammation treatment are: linum usitatissimum l., lawsonia inermis l and plantago coronopus l. in casablanca city (morocco), an ethnobotanical study identified 9 medicinal plants, divided into 8 families. they are reported for the traditional treatment of inflammation in morocco. these are: pistacia lentiscus l., calamintha officinali, mentha suaveolens ehrh., cinnamomum verum j.presl, eucalyptus globulus labill., adiantum capillus-veneris l., nigella sativa l., populus nigra l and urtica urens l (zougagh et al., 2019). an ethnobotanical study was conducted among 785 people from the region of the ksar lakbir (located at the southwestern limit of the rif area, morocco) identified 19 species belonging to 12 botanical families. the species used are anabasis aretioides moq. & coss. ex bunge, cachrys libanotis l., kundmannia sicula (l.) dc., thapsia garganica l., brassica nigra (l.) koch., saponaria officinalis l., artemisia herba-alba asso, carlina gummifera (l.) less. , citrullus colocynthis (l.) schrad., euphorbia falcata l., juglans regia l., ajuga iva (l.) schreb., lavandula stoechas l., origanum majorana l., mentha rotundifolia (l.) huds., salvia verbenaca l., melilotus indicus (l.) all., fraxinus angustifolia vahl, and cynodon dactylon (l.) pers. (merzouki et al., 2000). a survey was conducted in collaboration with herbalists in the province of marrakech to identify medicinal plants used to treat inflammation. the most commonly used plant species identified was quercus ilex l.(ouarghidi et al., 2013). in imouzzer ida outanane region (south-western morocco), a survey was carried out to take stock of the main medicinal plants used in traditional medicine to treat inflammation. the results obtained made it possible to identify 15 medicinal plants used against inflammation. the most cited herbs for treating inflammation are: thapsia transtagana brot., periploca angustifolia labill., artemisia herbaalba asso, dittrichia viscosa (l.) greuter, warionia saharae benth. coss., lavandula stoechas l., mentha pulegium l., rosmarinus officinalis l., salvia officinalis, myristica fragrans houtt., phillyrea angustifolia l., nigella sativa l., ruta montana l., alpinia officinarum hance, zingiber officinale rosc. the leaves of plants are zouhri a et al. (2023). not sci biol 15(1):11414 7 the most commonly used parts of plants. decoctions, and infusions are the most common preparation methods in traditional medicine. (saadi et al., 2013).... according a survey conducted among herbalists and users of medicinal plants to treat inflammation in the zaer region (western morocco), seven plant species belonging to six botanical families have been identified. the species were coriandrum sativum l., asparagus officinalis l., opuntia ficusindica (l.) mill., ajuga iva (l.) schreb., origanum vulgare l., peganum hamala l., olea europaea l (lahsissene and kahouadji, 2010).... in the same vein, another ethnobotanical survey of the local population of the taounate province (northern morocco) indicated that a total of 11 plant species from 8 families in the region are used to treat inflammation. among these listed species, four types are the most cited: lavandula stoechas l., rosmarinus officinalis l., salvia officinalis, plantago coronopus l. (el-hilaly et al., 2003).... in the eastern region of morocco, an ethnobotanical study identified 148 medicinal plants, divided into 60 families. eleven are reported for the traditional treatment of inflammation in morocco. these are: nerium oleander l., borago officinalis l., brassica napus l., saponaria officinalis l., ricinus communis l., laurus nobilis l., cassia senna l., peganum hamala l., citrus sp., citrus sinensis (l.) osbeck, salix alba l. (jamila & mostafa, 2014). also an ethnobotanical survey conducted by ziyyat et al (ziyyat et al., 1997) which reports ten plants used as anti-inflammatory remedies in different regions oriental of morocco, among which peganum hamala l., urtica dioica l. and arbutus unedo l. are the most used. a study by zouhri et al (zouhri and aarab, 2018) in targuist (north morocco) reports that 90 plant species were cited in 3,500 questionnaires for the treatment of inflammation. five plants are most frequently used: calendula officinalis l, opuntia ficus-indica (l.) mill, malva sylvestris l., plantago coronopus l. and datura stramonium l. investigation shows that the seeds, and leaves are the part of the plant most commonly used in herbal preparations. also, in the northern region of morocco (region of bouhachem natural regional park), an ethnobotanical study identifies 101 medicinal plants used in the treatment of diseases. the most cited herbal remedies used for their anti-inflammatory effects are hammada scoparia (pomel) iljin, allium cepa l., allium sativum l., ammi visnaga (l.) lam., nerium oleander l., aristolochia baetica l., artemisia herba-alba asso, cynara humilis l., scolymus hispanicus l., brassica oleracea l., opuntia ficusindica (l.) mill., ajuga iva (l.) schreb., cinnamomum verum j. presl, anthyllis cytisoides l ., lawsonia inermis l., fraxinus excelsior l., triticum durum desf., urtica dioica l. (bachar et al., 2021). similarly, another ethnobotanical survey was conducted from july 1st, 2016 to july 30th, 2018 on the population of the rif, in the northern morocco indicates that a total of 13 species belonging to 11 families of plants are used to treat inflammation. namely: dysphania ambrosioides (l.) mosyakin & clemants, rhus pentaphylla (jacq.) desf, arisarum vulgare o.targ.tozz, agave americana l., carlina gummifera (l.) less., dittrichia viscosa (l.) greuter, lactuca virosa habl., euphorbia falcata l., quercus ilex l., lawsonia inermis l., glaucium flavum crantz, cedrus atlantica (endl.), capsicum frutescens l. the results show that decoction, and infusion are the most common preparation methods (chaachouay et al., 2022). in the rif region of morocco, another ethnobotanical study conducted by chaachouay et al. has identified five herbal remedies used in inflammation treatment. the plants used are arisarum vulgare o.targ.tozz, carlina gummifera (l.) less., dittrichia viscosa (l.) greuter, lactuca virosa habl and cedrus atlantica (endl.) (chaachouay et al., 2021). an analogous study by bouyahya et al (bouyahya et al., 2017) in ouezzane (north-west of morocco) reports that eight plant species are cited for the management of inflammation. three plants, ajuga iva (l.) schreb., lavandula stoechas l. and salvia officinalis are the most used. in the province of errachidia (southeastern morocco), a survey was carried out to classify the plants traditionally used to treat inflammation. the authors have identified 194 species belonging to 69 families, of which 17 species were cited for the first time. the plants most commonly cited are nerium oleander l., calendula officinalis l. and cassia senna l. (eddouks et al., 2017). similarly, an ethnobotanical study in the zouhri a et al. (2023). not sci biol 15(1):11414 8 same region identified 64 medicinal plants belonging to 33 families used in the treatment of diseases. the plants most cited for their anti-inflammatory effects are opuntia ficus-indica (l.) mill., lavandula stoechas l., salvia officinalis l. (lahsissene and kahouadji, 2010). another ethnobotanical survey of the local population of the zagora region (southeastern morocco) indicates that a total of 7 species belonging to 7 plant families were used to treat inflammation. among these identified species, three are the most cited as anti-inflammatory plants in this region, namely: carum carvi l., terminalia chebula retz., allium sativum (l.), lawsonia inermis l., olea europaea l., nigella sativa l., zygophyllum gaetulum (emb. and maire) (boufous et al., 2017). while in the region of meknès-tafilalet (north-central morocco), an ethnobotanical study was conducted to identify plants often used to treat inflammation include: cuminum cymimum l., calendula officinalis l., santolina rosmarinifolia l., medicago sativa l., trigonella foenumgraecum l., pelargonium graveolens l'hér., lavandula angustifolia mill., origanum vulgare l., mentha pulegium l., rosmarinus officinalis l., lawsonia inermis l., papaver rhoeas l., rosa centifolia mill., citrus aurantium l. and verbena officinalis l. (fadil et al., 2015). likewise, in the north-central region of morocco (fez), an ethnobotanical study reported that 75 species are divided into 41 families for the treatment of 34 diseases. among these species, eight plants have antiinflammatory effects: ajuga iva (l.) schreb., lavandula angustifolia mill., origanum vulgare l., melissa officinalis, mentha pulegium l., rosmarinus officinalis l., salvia officinalis, thymus vulgaris l. (mikou et al., 2015). the medicinal plants used to treat inflammation in el hajeb province, according to a survey was carried out to show that the most important species is pimpinella anisum l. (ajebli et al., 2017). a field study in the central middle atlas (morocco), 8 plant species belonging to 4 families were reported for the treatment of inflammation in the region. the most important are ammi visnaga (l.) lam., artemisia arborescens (vaill.) l., artemisia herba-alba asso., carlina gummifera (l.) less, matricaria chamomilla l., origanum compactum benth, rubus ulmifolius schott and urtica urens l (najem et al., 2020). ultimately an ethnobotanical survey by mouhaddach et al (lahsissene and kahouadji, 2010) in different regions of morocco during march, april, and june 2014 reported that opuntia ficusindica (l.) mill. used for the inflammation treatment. pharmacological studies according to the selection criteria that characterise the research direction, 51 studies on the antiinflammatory activity of moroccan medicinal plants are identified and classified in table 2. these articles cover six different experimental models. among them, 38 articles use in vivo models, and 13 articles use in vitro models. the plant extracts tested in vitro and in vivo have been successful in reducing the inflammation induced experimentally in animals. 56 plants are explored experimentally, namely: allium subvillosum salzm. ex schult. & schult.f., aphloia theiformis (vahl) benn., ammodaucus leucotrichus coss., apium graveolens, coriandrum sativum, cuminum cyminum l., petroselinum crispum (mill.) fuss, petroselinum sativum hoffm., aphloia theiformis (vahl) benn., caralluma europaea (guss.) n.e.br., phoenix dactylifera l., dipcadi serotinum (l.) medik., moricandia sinaica (boiss.) boiss., opuntia ficus-indica (l.) mill., capparis spinosa l., cistus ladanifer l., cistus monspeliensis l., cistus salviifolius l., androcymbium gramineum (cav.) j.f. macbr., anacyclus pyrethrum (l), dittrichia viscosa (l.) greuter, lactuca sativa l., kleinia anteuphorbium (l.) haw., allanblackia gabonensis (pellegr.) bamps, garcinia mangostana l., coriaria myrtifolia l., tetraclinis articulata (vahl) mast., erica arborea l., euphorbia granulata forssk., albizia anthelmintica, pelargonium graveolens l'hér., ajuga iva (l.) schreb., melissa officinalis l., mentha pulegium l., origanum compactum benth., rosmarinus officinalis l., thymus atlanticus (ball), thymus broussonetii boiss., thymus maroccanus ball, thymus saturejoides coss, thymus vulgaris l., thymus willdenowii boiss., thymus zygis l., bauhinia reticulata dc., bauhinia thonningii schum., lawsonia inermis l., punica granatum l., eugenia uniflora l., syzygium aromaticum (l.) merr. & l.m. perry, ziziphus lotus (l.) lam., rosa × damascena herrm., argania spinosa (l.) skeels, withania frutescens (l.) zouhri a et al. (2023). not sci biol 15(1):11414 9 pauquy, thymelaea hirsuta (l.) endl., thymelaea lythroides barratte & murb. and tetraena gaetula (emb. & maire) beier & thulin. the effects of untapped moroccan plant species on inflammation need to be further investigated, in particular the mechanism of action of these plant extracts, in order to obtain additional data on the pharmacological effects of these plants. coriandrum sativum l, caralluma europaea (guss.) nebr., opuntia ficus-indica (l.) moulin., capparis spinosa l., anacyclus pyrethrum l, ajuga iva (l.) schreb., thymus atlanticus (ball) pau, thymus saturesioides coss, thymus zygis l., lawsonia inermis l. and rosmarinus officinalis l. are plants most commonly used to treat inflammation. these plants will be discussed in detail below. plants used most frequently for the inflammation treatment in morocco coriandrum sativum l. coriandrum sativum l. (kassbour) is a plant of the apiaceae family, widely cultivated for its seeds (nadeem et al., 2013). kassbour seeds are a common species, and have a large number of documented traditional medicinal uses. they can be used to treat diabetes, kidney and heart problems, and gastrointestinal diseases (el-hilaly et al., 2003; es-safi et al., 2020; lahsissene and kahouadji, 2010; mechchate et al., 2021) and to fight worms, rheumatism and joint pain (nadeem et al., 2013). the lipid-lowering effect of whole kassbour seed has been studied (chithra and leelamma, 1999), mainly its effect on lowering blood sugar and increasing insulin release (eidi and eidi, 2009), and the effect of scavenging free radicals (de almeida melo et al., 2005) and anti-inflammatory activity (shraddha and anuradha, 2019).... this activity has been studied in vivo (mechchate et al., 2021), and in vitro (derouich et al., 2020). using carrageenan-induced paw edema model, mechchate et al (mechchate et al., 2021) reported that methanolic extract of coriandrum sativum whole plant exhibited potent anti-inflammatory activity. at a dose of 25 mg / kg this extract exhibited a potent effect, revealing a higher inhibitory effect when compared with vehicle control in mice. morever, hydromethanolic extracts of coriandrum sativums show nitric oxide inhibition with ic50 vaue of 218,63 ± 6.41 μg/ml (derouich et al., 2020). caralluma europaea (guss.) n.e.br. caralluma europaea (guss.) n.e.br. : (eddaghmouss) is a wild medicinal plant belonging to the apocynaceae family, widespread in many mediterranean countries including morocco, algeria, tunisia, egypt, jordan, libya, spain, and italy (dra et al., 2019). in traditional medicine, it is recommended to use the aerial parts of eddaghmouss in the form of juice or powder mixed with honey or milk to treat inflammation, ulcers, diabetes, bacterial infections, antiulcer, antinociceptive, antihyperglycemic, antioxidant, and cytotoxic activities (adnan et al., 2014; amrati et al., 2021; bellakhdar, 1997). the anti-inflammatory effects of hydroethanol, n-butanol and fractions rich in polyphenols of this plant have been studied through paw edema induced by carrageenin. after 6 hours of treatment, the part rich in polyphenols is the best edema inhibitor, with an inhibition rate of 75.68% (amrati et al., 2021). furthermore, caralluma europaea ethanol extract reduced xylene-induced edema by 62.04% at 100 mg / kg (kebbou et al., 2019).... opuntia ficus-indica (l.) mill. opuntia ficus-indica (l.) mill. (lhindia) or cactus belongs to the cactaceae family, this plant is native to mexico, cultivated in arid and semi-arid regions of south, and central america, and was introduced in north africa (d’aquino et al., 2017). the fig tree grows quickly, adapts well to poor soils, and requires low water consumption. in traditional folk medicine it’s cladodes, and its fruits are also used as a source of nutrients, and for the treatment of inflammatory, diabetes, stomach ulcers, and kidney disease, and antioxidant activity (alimi et al., 2010; feugang et al., 2006; kaur, 2012). the anti-inflammatory effects of aqueous acetone zouhri a et al. (2023). not sci biol 15(1):11414 10 extract of this plant were studied through the inhibition of nitric oxide. opuntia ficus-indica extract demonstrated potent no inhibitory activity (90%) with ic50 value of 0.19 mg / ml (benayad et al., 2014). capparis spinosa l. capparis spinosa l. (kabâr) is a medicinal plant of the capparaceae family, native to the mediterranean region. this plant is found in many parts of the world in north africa, italy, central asia and greece (zarei et al., 2021). in traditional medicine, the different parts of this plant are used to treat rheumatism, digestive diseases, headaches, toothaches, as well as diuretics, antihypertensives, and tonics (tlili et al., 2010). kabâr can be used as an antioxidant, antifungal, anti-hepatotoxic, and anti-inflammatory (gadgoli and mishra, 1999; germanò et al., 2002). it also treats many illnesses, such as bronchitis, bronchial asthma, liver disease and tuberculosis. the anti-inflammatory effect of capparis spinosa was confirmed by ethanolic extracts of the leaves in a model of paw edema in mice. capparis at 1.07 g / kg reduced inflammation by 73.44% (el azhary et al., 2017).... aqueous extracts of capparis spinosa have also showed an anti-inflammatory effect by the inhibition of the proinflammatory cytokine, il-17 at 500 µg / ml (moutia et al., 2016).... this activity attributed to the presence of alkaloids, biologically active lipids, polyphenols, flavonoids, and glucosinolates in capparis spinosa (rodrigo et al., 1992), which are known for its bioactive properties. anacyclus pyrethrum l. anacylcus pyrethrum l., commonly known as oud al attass, botanically classified in the compositae family, is a plant native to north africa (mohamed fennane and oualidi, 2014). according to experts in traditional moroccan medicine, oud al attass is one of the medicinal plants used to relieve toothache, angina, salivary secretions (abbas zaidi et al., 2013; doudach et al., 2012), neuralgia, paralysis, and the common cold (bendjeddou et al., 2003). other pharmacological, and biological properties are antimicrobial (jalayer-naderi et al., 2016), antidiabetic (tyagi et al., 2011), antioxidant (pahuja et al., 2013) and anti-inflammatory (manouze et al., 2017; rimbau et al., 1999).the authors attributed those differents activities to many chemical constituents in anacylcus pyrethrum l., including alkaloids, reducing compounds, tannins, flavonoids, coumarins, and also saponins, and sesamin (elazzouzi et al., 2014). in a recent study, jawhari et al (jawhari et al., 2020) have reported the anti-inflammatory effect of hydroalcoholic extract of anacylcus pyrethrum l., in rats using carrageenin-induced paw edema test. the hydroalcoholic extract of the roots at a dose of 300 mg / ml significantly reduced inflammation by 96%. the active compounds of hydroalcoholic extract of the seeds are able to reduce inflammation by 96% at a dose of 500 mg / ml. a similar study shows a significant antiinflammatory effect (62%) of aqueous extract of anacylcus pyrethrum l. at a dose of 500 mg / kg by employing xylene-induced ear edema test on rats (manouze et al., 2017). ajuga iva (l.) schreb. in morocco ajuga iva (l.,) schreb is called "chendgoura" and is a medicinal plant of the lamiaceae (bouyahya et al., 2020). this species is traditionally used in morocco to treat assorted diseases, such as diabetes (bouyahya et al., 2017; lahsissene and kahouadji, 2010). biological studies on extracts, and volatile compounds of chendgoura essential oil have shown that this plant has antiparasitic (bellakhdar et al., 1991), antibacterial (makni et al., 2013), antidiabetic (fettach et al., 2019), anti-inflammatory (taleb-senouci et al., 2012), anticancer effects, antioxidants, and antihypertensives (el-hilaly et al., 2004). while the dermatoprotective effect is studied by inhibiting the activity of elastase in vitro. the essential oils of the aerial parts have shown an ic50 value of 192.21 μg/ml (bouyahya et al., 2021).... zouhri a et al. (2023). not sci biol 15(1):11414 11 thyme varieties thymus l, also known as "ziitra or azukni," is an aromatic plant from the lamiaceae family that is native to north africa, europe, and asia. it has been used for centuries in morocco to treat various illnesses, including inflammatory diseases. traditional medicine recognizes its many benefits, such as treating infections caused by bacteria, fungi, and parasites (hosseinzadeh et al., 2015), relieving coughs, and increasing appetite (jarić et al., 2015). thymus saturejoides coss, thymus atlanticus (ball), and thymus zygis l. are species of morocco (ramchoun et al., 2012) that are known for their anti-inflammatory properties and are used in folk medicine (bellakhdar et al., 1991). several studies have demonstrated the anti-inflammatory effect of aqueous thyme extracts using three models: mouse ear edema induced by croton oil, paw edema induced by carrageenin in rats, and albumin denaturation inhibition test. after 5 hours of treatment, at a dose of 50 mg / kg of thymus atlanticus (ball) the paw edema induced by carrageenin has been reduced by 3.74% ± 0.01% and 9.52% ± 0.04 % for thymus zygis l. and thymus atlanticus (ball), respectively (p <0.001) (khouya et al., 2015). while after eight hours treatment, the aqueous extract significantly reduced the volume of croton oil-induced ear edema by 70.47%, 84.62% and 29.66% at 900 mg / ear for thymus zygis l., thymus atlanticus (ball) and thymus saturejoides coss respectively (khouya et al., 2015). according to hmidani et al. (2019), the order of albumin denaturation inhibition of the aqueous extracts was: thymus atlanticus (ball) (ic50 = 122.90 μg / ml), followed by thymus zygis l. (ic50 = 133.25 μg / ml), and thymus saturejoides coss (ic50 = 181.42 μg / ml). in a recent study, khouya and collaborators (khouya et al., 2020) reported that aerial parts of thymus atlanticus (ball) have shown a 70% inhibition of paw edema induced by carrageenan at a concentration of 100 mg / kg of aqueous extract of thymus atlanticus (ball). lawsonia inermis l. lawsonia inermis l. (family lythraceae), commonly known as lhena, is a medicinal plant widely cultivated around the world, and its leaves have many medicinal, and cosmetic uses (rostkowska et al., 1998). pharmacological studies have revealed that it can be used in the treatment and prevention of inflammation, diarrhea, diabetes and ulcers and other diseases. in addition, it has antioxidant, anticancer, antibacterial, antiparasitic, antifungal, analgesic and antipyretic effects (rostkowska et al., 1998). ineed, zouhri et al. (2017) reported the anti-inflammatory effect of lawsonia inermis l fixed oils on paw edema induced by carrageenan in rats. the results showed that lawsonia inermis reduced inflammation by 90.30% at 0.1 mg / ml after five hours. the anti-inflammatory activity of the methanolic extract of the leaves of lawsonia inermis was also studied by bouhlali et al. (2016). they found that when the extract was administered at a dose of 200 μg/ml, it significantly reduced protein denaturation with an ic50 of 103.21 μg/ml. rosmarinus officinalis l. rosmarinus officinalis l. (azir) is a medicinal, and aromatic plant of the lamiaceae family, which is widely distributed in the mediterranean region, but today it is cultivated all over the globe (bendif et al., 2017; karadağ et al., 2019). the leaves are used as a spice to flavor cooking in traditional medicine, and because of its healing properties (prior et al., 1998), such as stomach diseases, respiratory diseases, diabetes and inflammatory diseases (bakırel et al., 2008). azir is also used to reduce anxiety and depression (nematolahi et al., 2018), and for its antioxidant, and antibacterial properties (de melo et al., 2011; gonzález-trujano et al., 2007). the anti-inflammatory effect of rosmarinus officinalis l. has been confirmed by the essential oil from the leaves in a lipoxygenase assay model. this plant reduced inflammation at a concentration of 4.21 mg/ml with an ic50 value of 0.548 ± 0.005 mg/ml (el kharraf et al., 2021). zouhri a et al. (2023). not sci biol 15(1):11414 12 table 1.table 1.table 1.table 1. plants used in the inflammation treatment in morocco, cited in ethnobotanical studies familyfamilyfamilyfamily scientific namescientific namescientific namescientific name common orcommon orcommon orcommon or local namelocal namelocal namelocal name part usedpart usedpart usedpart used preparation preparation preparation preparation methodsmethodsmethodsmethods modes of modes of modes of modes of administradministradministradministr ationsationsationsations number number number number of of of of citationscitationscitationscitations referencesreferencesreferencesreferences amaranthaamaranthaamaranthaamarantha ceaeceaeceaeceae anabasis aretioides moq. & coss. ex bunge chajra limayhazha rrih aerial parts decoction oral 1 (merzouki et al., 2000) dysphania ambrosioides (l.) mosyakin & clemants mkhinza leaves decoction oral 1 (chaachouay et al., 2022) haloxylon scoparium pomel tirguelt seeds, whole plant decoction local applicatio n 1 (bachar et al., 2021) amaryllidaamaryllidaamaryllidaamaryllida ceaeceaeceaeceae allium cepa l. l’bssale bulb, whole plant decoction and cataplasm oral or local applicatio n 3 (bachar et al., 2021; bouyahya et al., 2017; salhi et al., 2019) allium sativum l. toum bulb, whole plant decoction, cataplasm oral or local applicatio n 1 (bachar et al., 2021) anacardiacanacardiacanacardiacanacardiac eaeeaeeaeeae pistacia lentiscus l. drou leaves, flowers decoction mouthwa sh 1 (zougagh et al., 2019) searsia pentaphylla (jacq.) f.a barkley tizgha leaves decoction oral 1 (chaachouay et al., 2022) apiaceaeapiaceaeapiaceaeapiaceae ammi visnaga (l.) lam. bouchnikha, khala seeds, whole plant, flowers decoction oral or mouthwa sh 2 (bachar et al., 2021; najem et al., 2020) artemisia arborescens (vaill.) l. chiba leaves decoction mouthwa sh 1 (najem et al., 2020) carlina gummifera (l.) less dad root decoction mouthwa sh 1 (najem et al., 2020) cachrys libanotis l. kollikh root decoction oral 1 (merzouki et al., 2000) carum carvi l. ni seeds decoction oral 1 (boufous et al., 2017) coriandrum sativum l. qasbor leaves decoction oral 1 (lahsissene and kahouadji, 2010) cuminum cymimum l. kamoun seeds maceration oral 1 (fadil et al., 2015) matricaria chamomilla l. babounj lahmir leaves, flowers ni ni 1 (najem et al., 2020) pimpinella anisum l. ni seeds decoction oral 1 (ajebli et al., 2017) kundmannia sicula (l.) dc. ziyata root decoction oral 1 (merzouki et al., 2000) thapsia garganica l. addaryas aerial parts infusion oral 1 (merzouki et al., 2000) thapsia transtagana brot. deriass root decoction oral 1 (saadi et al., 2013) apocynaceapocynaceapocynaceapocynace aeaeaeae nerium oleander l. dafla, ariri , alili leaves decoction oral 4 (bachar et al., 2021; eddouks et al., 2017; jamila and mostafa, 2014; salhi et al., 2019) araceaearaceaearaceaearaceae arisarum vulgare o.targ.tozz irni whole plant decoction oral 2 (chaachouay et al., 2022) aristolochiaristolochiaristolochiaristolochi aceaeaceaeaceaeaceae aristolochia baetica l. berez’tem aerial parts, whole plant powder oral 1 (bachar et al., 2021) zouhri a et al. (2023). not sci biol 15(1):11414 13 asclepiadaasclepiadaasclepiadaasclepiada ceaeceaeceaeceae periploca angustifolia labill. asellif leaves, root decoction and powder local applicatio n 1 (saadi et al., 2013) asparagaceasparagaceasparagaceasparagace aeaeaeae agave americana l. sabra whole plant cataplasm local applicatio n 1 (chaachouay et al., 2022) asparagus officinalis l. sekoum root cataplasm local applicatio n 1 (lahsissene and kahouadji, 2010) asteraceaeasteraceaeasteraceaeasteraceae artemisia herba-alba asso chih leaves, whole plant infusion and decoction oral 5 (bachar et al., 2021; merzouki et al., 2000; najem et al., 2020; saadi et al., 2013; salhi et al., 2019; skalli et al., 2019) carlina gummifera (l.) less. addad whole plant decoction oral 3 (chaachouay et al., 2022; merzouki et al., 2000) calendula officinalis l. jemra flowers powder local applicatio n 4 (eddouks et al., 2017; fadil et al., 2015; salhi et al., 2019; zouhri and aarab, 2018) cynara humilis l timta, taymeth root, whole plant powder local applicatio n 2 (bachar et al., 2021; salhi et al., 2019) dittrichia viscosa (l.) greuter magraman, terrahla leaves decoction, infusion local applicatio n, oral 3 (chaachouay et al., 2022; saadi et al., 2013) lactuca virosa habl. ahchlaf nssem whole plant decoction oral 1 (chaachouay et al., 2021) santolina rosmarinifolia l. oue-zouaza flower, leaves infusion oral 1 (fadil et al., 2015) scolymus hispanicus l. guernina rhizome infusion oral 1 (bachar et al., 2021) warionia saharae benth. coss. afessas leaves decoction local applicatio n 1 (saadi et al., 2013) boraginaceboraginaceboraginaceboraginace aeaeaeae borago officinalis l. lahricha, lisan attur leaves decoction oral 2 (el-hilaly et al., 2003; jamila and mostafa, 2014) brassicaceabrassicaceabrassicaceabrassicacea eeee brassica napus l. laft root, leaves decoction and maceration oral 1 (jamila and mostafa, 2014) brassica nigra (l.) koch. al khardal lak’hal seeds cataplasm local applicatio n 1 (merzouki et al., 2000) brassica oleracea l. kroumb leaves cataplasm local applicatio n 1 (bachar et al., 2021) cactaceaecactaceaecactaceaecactaceae opuntia ficusindica (l.) mill. el hendia whole plant, rackets cataplasm oral and local applicatio n 6 (bachar et al., 2021; lahsissene and kahouadji, 2010; salhi et al., 2019; zouhri and aarab, 2018) capparacecapparacecapparacecapparace aeaeaeae capparis spinosa l. kebbar fruits decoction oral 2 (el-hilaly et al., 2003; ziyyat et al., 1997) caryophyllcaryophyllcaryophyllcaryophyll aceaeaceaeaceaeaceae saponaria officinalis l. sarghina, tighighacht root, seeds powder and decoction oral or external applicatio n 2 (jamila and mostafa, 2014; merzouki et al., 2000) zouhri a et al. (2023). not sci biol 15(1):11414 14 combretaccombretaccombretaccombretac eaeeaeeaeeae terminalia chebula retz. hlilaj khal fruits powder local applicatio n 1 (boufous et al., 2017) compositacompositacompositacomposita eeee anthemis tomentosa boiss. nouar el jenna flowers maceration external applicatio n 1 (el-hilaly et al., 2003) cucurbitaccucurbitaccucurbitaccucurbitac eaeeaeeaeeae bryonia dioica l. enab dib root decoction oral 1 (el-hilaly et al., 2003) citrullus colocynthis (l.) schrad. handal, hdejja fruits decoction oral 2 (merzouki et al., 2000; ziyyat et al., 1997) cupressacecupressacecupressacecupressace aeaeaeae juniperus phoenicea l. ar’ar stem powder oral 1 (bouyahya et al., 2017) ericaceaeericaceaeericaceaeericaceae arbutus unedo l. sasnou root powder local applicatio n 1 (ziyyat et al., 1997) euphorbiaeuphorbiaeuphorbiaeuphorbia ceaeceaeceaeceae euphorbia falcata l. hayat annofos whole plant infusion oral 2 (chaachouay et al., 2022; merzouki et al., 2000) ricinus communis l. lkharouaa leaves decoction inhalatio n 1 (jamila and mostafa, 2014) fabaceaefabaceaefabaceaefabaceae medicago sativa l. fassa seeds infusion oral 1 (fadil et al., 2015) trigonella foenum graecum l. halba seeds infusion oral 1 (fadil et al., 2015) fagaceaefagaceaefagaceaefagaceae quercus ilex l. d’bagh ou fernân root powder local applicatio n 2 (chaachouay et al., 2022; ouarghidi et al., 2013; salhi et al., 2019) vicia faba l. fûl leaves powder local applicatio n 1 (salhi et al., 2019) gentianceagentianceagentianceagentiancea eeee centaurium erythraea rafn. korsat lhaya flowers maceration local applicatio n 1 (bouyahya et al., 2017) geraniaceageraniaceageraniaceageraniacea eeee pelargonium graveolens l'hér. ifer laâtar flowers, leaves infusion oral 1 (fadil et al., 2015) jungladacejungladacejungladacejungladace aeaeaeae juglans regia l. asswak leaves infusion oral 1 (merzouki et al., 2000) lamiaceaelamiaceaelamiaceaelamiaceae ajuga iva (l.) schreb. chendgoura leaves decoction oral 5 (bachar et al., 2021; bouyahya et al., 2017; lahsissene and kahouadji, 2010; merzouki et al., 2000; mikou et al., 2015) calamintha officinalis moench manta leaves decoction mouthwa sh 1 (zougagh et al., 2019) lavandula angustifolia mill. khzama flowers, leaves decoction oral 2 (fadil et al., 2015; mikou et al., 2015) lavandula multifida l. hlihla stem decoction oral 1 (el-hilaly et al., 2003) lavandula stoechas l. halhal flowers decoction oral 5 (bouyahya et al., 2017; el-hilaly et al., 2003; lahsissene and kahouadji, 2010; merzouki et al., 2000; saadi et al., 2013) origanum compactum benth zaatar tadlaoui leaves, flowers decoction mouthwa sh 1 (najem et al., 2020) zouhri a et al. (2023). not sci biol 15(1):11414 15 origanum majorana l. merdeddouch aerial parts infusion oral 1 (merzouki et al., 2000) origanum vulgare l. zâtar flowers, leaves decoction oral 3 (fadil et al., 2015; lahsissene and kahouadji, 2010; mikou et al., 2015) marrubium vulgare l. merrîwut leaves powder local applicatio n 1 (salhi et al., 2019) melissa officinalis l. naanae souf leaves infusion oral 1 (mikou et al., 2015) mentha pulegium l. fliou flowers, leaves infusion oral or inhalation 3 (fadil et al., 2015; mikou et al., 2015; saadi et al., 2013) mentha rotundifolia (l.) huds. mchichtro arial parts infusion oral 1 (merzouki et al., 2000) mentha suaveolens ehrh. timija leaves decoction oral 1 (zougagh et al., 2019) mentha spicata l. naenae leaves cataplasm local applicatio n 1 (salhi et al., 2019) rosmarinus officinalis l. azir, yazir flowers, leaves infusion oral 6 (el-hilaly et al., 2003; fadil et al., 2015; mikou et al., 2015; saadi et al., 2013; skalli et al., 2019) salvia officinalis l. sâlmiya leaves maceration and infusion oral and local applicatio n 7 (bouyahya et al., 2017; el-hilaly et al., 2003; lahsissene and kahouadji, 2010; mikou et al., 2015; saadi et al., 2013; salhi et al., 2019; skalli et al., 2019) salvia verbenaca l. al’khiata aerial part powder local applicatio n 1 (merzouki et al., 2000) thymus vulgaris l. ziitra flowers infusion ni 1 (mikou et al., 2015) lauraceaelauraceaelauraceaelauraceae cinnamomum verum j.presl l’karfa bark infusion and decoction oral or mouthwas h 2 (bachar et al., 2021; zougagh et al., 2019) laurus nobilis l. rend, ouarkat sidna moussa leaves decoction oral 2 (jamila and mostafa, 2014; ziyyat et al., 1997) leguminosleguminosleguminosleguminos eaeeaeeaeeae anthyllis cytisoides l. chtappa root decoction oral 1 (bachar et al., 2021) cassia senna l. lsana leaves decoction oral 2 (eddouks et al., 2017; jamila and mostafa, 2014) ceratonia siliqua l. kharoub seeds decoction oral 1 (bouyahya et al., 2017) melilotus indicus (l.) all. azroud leaves, flowers infusion oral 1 (merzouki et al., 2000) liliaceaeliliaceaeliliaceaeliliaceae allium sativum (l.) ni bulb decoction oral 1 (boufous et al., 2017) linaceaelinaceaelinaceaelinaceae linum usitatissimum l. zariat al kettan seeds powder local applicatio n 1 (salhi et al., 2019) zouhri a et al. (2023). not sci biol 15(1):11414 16 lythraceaelythraceaelythraceaelythraceae lawsonia inermis l. l’hanna leaves powder and decoction local applicatio n or oral 5 (bachar et al., 2021; boufous et al., 2017; chaachouay et al., 2022; fadil et al., 2015; salhi et al., 2019) malvaceaemalvaceaemalvaceaemalvaceae malva sylvestris l. khobbeza leaves cataplasm local applicatio n 1 (zouhri and aarab, 2018) myristicacemyristicacemyristicacemyristicace aeaeaeae myristica fragrans houtt. lgouza seeds powder oral 1 (saadi et al., 2013) myrtaceaemyrtaceaemyrtaceaemyrtaceae eucalyptus globulus labill. kalitus flowers, leaves infusion and decoction oral or mouthwas h 2 (ziyyat et al., 1997; zougagh et al., 2019) nitrariaceanitrariaceanitrariaceanitrariacea eeee peganum hamala l. lharmal stem, seeds decoction oral 3 (jamila and mostafa, 2014; lahsissene and kahouadji, 2010; ziyyat et al., 1997) oleaceaeoleaceaeoleaceaeoleaceae fraxinus angustifolia vahl touzalt, alssan attir, addardar leaves, fruits powder local applicatio n 1 (merzouki et al., 2000; ziyyat et al., 1997) fraxinus excelsior l. dardar leaves decoction oral 1 (bachar et al., 2021) olea europaea l. zebbûj, zitoun leaves, fruits powder and decoction oral and local applicatio n 3 (boufous et al., 2017; bouyahya et al., 2017; lahsissene and kahouadji, 2010) phillyrea angustifolia l. benzemmour leaves decoction local applicatio n 1 (saadi et al., 2013) papaveracepapaveracepapaveracepapaverace aeaeaeae glaucium flavum crantz merzak halabi flowers decoction oral 1 (chaachouay et al., 2022) papaver rhoeas l. bellamane flowers infusion oral 1 (fadil et al., 2015) plantaginaplantaginaplantaginaplantagina ceaeceaeceaeceae plantago coronopus l. l-messâssa, rjal laghrab leaves powder local applicatio n 3 (el-hilaly et al., 2003; salhi et al., 2019; zouhri and aarab, 2018) pinaceaepinaceaepinaceaepinaceae cedrus atlantica (endl.) arz el atlas leaves decoction oral 1 (chaachouay et al., 2021) piperaceaepiperaceaepiperaceaepiperaceae triticum durum desf. kam’h salb bran decoction oral 1 (bachar et al., 2021) poaceaepoaceaepoaceaepoaceae cynodon dactylon (l.) pers. anjem rhizome decoction oral 1 (merzouki et al., 2000) pteridaceaepteridaceaepteridaceaepteridaceae adiantum capillusveneris l. ziata leaves decoction mouthwa sh 1 (zougagh et al., 2019) ranuncularanuncularanuncularanuncula ceaeceaeceaeceae nigella sativa l. habba sawda, sanouj seeds powder and decoction oral or mouthwas h 3 (boufous et al., 2017; saadi et al., 2013; zougagh et al., 2019) rhamnacearhamnacearhamnacearhamnacea eeee ziziphus lotus (l.) lamk. sadra leaves decoction oral 1 (ziyyat et al., 1997) rosaceaerosaceaerosaceaerosaceae rosa centifolia l. lward lbeldi flowers infusion oral 1 (fadil et al., 2015) rubus ulmifolius schott. tût azzarb, serrmû leaves decoction mouthwa sh 1 (najem et al., 2020) rutaceaerutaceaerutaceaerutaceae citrus aurantium l. range leaves decoction oral 1 (fadil et al., 2015) citrus sp. ouadmi root powder oral 1 (jamila and mostafa, 2014) citrus sinensis (l.) osbeck litchin leaves decoction oral 1 (jamila and mostafa, 2014) zouhri a et al. (2023). not sci biol 15(1):11414 17 ruta montana l. fidjel aerial part, leaves decoction and cataplasm oral or local applicatio n 2 (saadi et al., 2013; ziyyat et al., 1997) salicaceaesalicaceaesalicaceaesalicaceae populus nigra l. safsaf leaves decoction mouthwa sh 1 (zougagh et al., 2019) salix alba l. oud lma leaves decoction inhalatio n 1 (jamila and mostafa, 2014) solanaceaesolanaceaesolanaceaesolanaceae capsicum frutescens l. sudaniya, flfel har fruits cataplasm local applicatio n 1 (chaachouay et al., 2022) datura stramonium l. chdak jmal leaves cataplasm local applicatio n 1 (zouhri and aarab, 2018) mandragora automnalis bertol. bid el gul fruits raw oral 1 (el-hilaly et al., 2003) urticaceaeurticaceaeurticaceaeurticaceae urtica dioica l. horriga leaves decoction local applicatio n 2 (bachar et al., 2021; ziyyat et al., 1997) urtica urens l. horiga elmelsa leaves decoction mouthwa sh 2 (najem et al., 2020; zougagh et al., 2019) verbenaceaverbenaceaverbenaceaverbenacea eeee verbena officinalis l. lwiza leaves infusion oral 1 (fadil et al., 2015) vitex agnus-castus l. el kherwae leaves infusion oral 1 (el-hilaly et al., 2003) zingiberaczingiberaczingiberaczingiberac eaeeaeeaeeae alpinia officinarum hance khudenjal root decoction oral 1 (saadi et al., 2013) zingiber officinale rosc. skinjbir rhizome decoction and infusion oral 1 (saadi et al., 2013) zygophyllazygophyllazygophyllazygophylla ceaeceaeceaeceae zygophyllum gaetulum (emb. and maire) ni leaves, stem powder and decoction oral or external applicatio n 1 (boufous et al., 2017) zouhri a et al. (2023). not sci biol 15(1):11414 18 table 2.table 2.table 2.table 2. anti-inflammatory activities of moroccan medicinal plants used traditionally to treat inflammation familyfamilyfamilyfamily plant speciesplant speciesplant speciesplant species local local local local namenamenamename parts parts parts parts usedusedusedused form ofform ofform ofform of extractextractextractextract dosedosedosedose methods usedmethods usedmethods usedmethods used therapeutictherapeutictherapeutictherapeutic activityactivityactivityactivity mechanism of mechanism of mechanism of mechanism of aaaa ntintintinti---inflammatory inflammatory inflammatory inflammatory activityactivityactivityactivity referencesreferencesreferencesreferences amaryllidaamaryllidaamaryllidaamaryllida ceaeceaeceaeceae allium subvillosum salzm. ex schult. & schult.f. basilah bulbs hydroethan olic extract 300 µg/cm2 carrageenaninduced ear edema in mice antioxidant, antiinflammator y effect 22 % inhibition at 300 µg/cm2 (moussaid et al., 2011) aphloiacaphloiacaphloiacaphloiaceaeaeaea eeee aphloia theiformis (vahl) benn. mfandrab o leaves methanol extract 100-200 mg/kg carrageenaninduced paw edema in rats immunomo dulatory and antiinflammator y activities 93 % inhibition at 200 mg/kg after 5h (hsoidrou et al., 2014) apiaceaeapiaceaeapiaceaeapiaceae ammodaucus leucotrichus coss. kamoun n es-sofi seeds hydroethan olic extract 100-200 mg/kg carrageenaninduced paw edema in rats antidiabetic and antiinflammator y activities 84 % inhibition at 200 mg/kg after 6h (es-safi, mechchate, amaghnouje, jawhari, et al., 2020) apium graveolens l. karafs stems and leaves hydrometh anolic extract 20 1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 270,38 ± 5.25 μg/ml) (derouich et al., 2020) coriandrum sativum l. kasbour stems and leaves hydrometh anolic extract 20 1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 218,63 ± 6.41 μg/ml) (derouich et al., 2020) coriandrum sativum l. kasbour seeds methanol extract 25-50 mg/kg carrageenaninduced paw edema in rats antioxidant, antiinflammator y and antidiabetic proprieties 87 % inhibition at 25 mg/kg after 6h (mechchate et al., 2021) cuminum cyminum l. kemmûn seeds methanol extract 50 800 μg/ml inhibition of protein denaturation antioxidant and antiinflammator y properties 57,66 % inhibition at 200 μg/ml (ic50 = 134.87 μg/ml) (bouhlali et al., 2016) petroselinum crispum (mill.) fuss maadnou s stems and leaves hydrometh anolic extract 20 1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 142.45 ± 5.59 μg/ml) (derouich et al., 2020) petroselinum sativum hoffm. maadnou s aerial parts hydroethanolic extract 500-1000 mg/kg carrageenaninduced paw edema in rats estrogenic and antiinflammator y activities 63,33 % inhibition at 500 mg/kg after 6h (slighoua et al., 2021) petroselinum sativum hoffm. maadnou s aerial parts polyphenol fraction 500-1000 mg/kg carrageenaninduced paw edema in rats estrogenic and antiinflammator y activities 81,33 % inhibition at 220 mg/kg after 6h (slighoua et al., 2021) aphloia theiformis (vahl) benn. mfandrab o leaves methanol extract 100-200 mg/kg carrageenaninduced paw edema in rats mmunomod ulatory and antiinflammator y activities 93 % inhibition at 200 mg/kg after 5h (hsoidrou et al., 2014) apocynaceapocynaceapocynaceapocynace aeaeaeae caralluma europaea (guss.) n.e.br. eddaghm ouss aerial parts ethanol extract 100–200 mg/kg xylene induced mouse ear edema antioxidant activity, antiinflammator y and analgesic effects 62,04 % inhibition at 100 mg/kg (kebbou et al., 2019) caralluma europaea (guss.) n.e.br. eddaghm ouss aerial parts ethyl acetate extract 100–200 mg/kg xylene induced mouse ear edema antioxidant activity, antiinflammator y and analgesic effects 57,05 % inhibition at 100 mg/kg (kebbou et al., 2019) zouhri a et al. (2023). not sci biol 15(1):11414 19 caralluma europaea (guss.) n.e.br. eddaghm ouss aerial parts hydroethan ol 50–100 mg/kg carrageenaninduced paw edema in rats antiinflammator y, antifungal, and antibacteria l activities 69,50 % inhibition at 100 mg/kg after 4h (amrati et al., 2021) caralluma europaea (guss.) n.e.br. eddaghm ouss aerial parts n-butanol 50–100 mg/kg carrageenaninduced paw edema in rats antiinflammator y, antifungal, and antibacteria l activities 76,32 % inhibition at 100 mg/kg after 5h (amrati et al., 2021) caralluma europaea (guss.) n.e.br. eddaghm ouss aerial parts polyphenol rich fractions 50–100 mg/kg carrageenaninduced paw edema in rats antiinflammator y, antifungal, and antibacteria l activities 76,70 % inhibition at 100 mg/kg after 6h (amrati et al., 2021) arecaceaearecaceaearecaceaearecaceae phoenix dactylifera l. jihl fruit aqueous extract 2 mg/kg carrageenaninduced paw edema in rats antiinflammator y activity 68,2 % inhibition at 2 mg/kg (bouhlali et al., 2018) phoenix dactylifera l. boufgous seeds methanol extract 20–1000 μg/ml croton oilinduced ear edema in mice antiinflammator y activity 58,37 % inhibition at 500 μg after 4h (bouhlali et al., 2020) phoenix dactylifera l. boufgous seeds methanol extract 20–1000 μg/ml carrageenaninduced paw edema in rats antiinflammator y activity 58,37 % inhibition at 30 mg/kg after 6h (bouhlali et al., 2020) phoenix dactylifera l. boufgous seeds methanol extract 20–1000 μg/ml inhibition of protein denaturation antiinflammator y activity inhibition of albumin denaturation (ic50 = 241.65 ± 6.69 μg/ml) (bouhlali et al., 2020) phoenix dactylifera l. boufgous seeds methanol extract 20–1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 114,45 ± 7.63 μg/ml) (bouhlali et al., 2020) phoenix dactylifera l. boustha mmi seeds methanol extract 20–1000 μg/ml croton oilinduced ear edema in mice antiinflammator y activity 77,17 % inhibition at 500 μg after 4h (bouhlali et al., 2020) phoenix dactylifera l. boustha mmi seeds methanol extract 20–1000 μg/ml carrageenaninduced paw edema in rats antiinflammator y activity 58,37 % inhibition at 30 mg/kg after 6h (bouhlali et al., 2020) phoenix dactylifera l. boustha mmi seeds methanol extract 20–1000 μg/ml inhibition of protein denaturation antiinflammator y activity inhibition of albumin denaturation (ic50 = 138.04 ± 7.83 μg/ml) (bouhlali et al., 2020) phoenix dactylifera l. boustha mmi seeds methanol extract 20–1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 118,36 ± 5.92 μg/ml) (bouhlali et al., 2020) phoenix dactylifera l. jihl seeds methanol extract 20–1000 μg/ml croton oilinduced ear edema in mice antiinflammator y activity 71,69 % inhibition at 500 μg after 4h (bouhlali et al., 2020) phoenix dactylifera l. jihl seeds methanol extract 20–1000 μg/ml carrageenaninduced paw edema in rats antiinflammator y activity 58,37 % inhibition at 30 mg/kg after 6h (bouhlali et al., 2020) phoenix dactylifera l. jihl seeds methanol extract 20–1000 μg/ml inhibition of protein denaturation antiinflammator y activity inhibition of albumin denaturation (ic50 = 116.63 ± 6.75 μg/ml) (bouhlali et al., 2020) zouhri a et al. (2023). not sci biol 15(1):11414 20 phoenix dactylifera l. jihl seeds methanol extract 20–1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 108,57 ± 5.15 μg/ml) (bouhlali et al., 2020) phoenix dactylifera l. majhoul seeds methanol extract 20–1000 μg/ml croton oilinduced ear edema in mice antiinflammator y activity 50,64 % inhibition at 500 μg after 4h (bouhlali et al., 2020) phoenix dactylifera l. majhoul seeds methanol extract 20–1000 μg/ml carrageenaninduced paw edema in rats antiinflammator y activity 58,37 % inhibition at 30 mg/kg after 6h (bouhlali et al., 2020) phoenix dactylifera l. majhoul seeds methanol extract 20–1000 μg/ml inhibition of protein denaturation antiinflammator y activity inhibition of albumin denaturation (ic50 = 209.38 ± 9.01 μg/ml) (bouhlali et al., 2020) phoenix dactylifera l. majhoul seeds methanol extract 20–1000 μg/ml nitric oxide inhibition assay antiinflammator y activity no inhibition (ic50 of 163,63 ± 6.39 μg/ml) (bouhlali et al., 2020) asparagaceasparagaceasparagaceasparagace aeaeaeae dipcadi serotinum (l.) medik. bssal eddib bulbs hydroethan olic extract 300 µg/cm2 carrageenaninduced ear edema in mice antioxidant, antiinflammator y effect 22 % inhibition at 300 µg/cm2 (moussaid et al., 2011) brassicaceabrassicaceabrassicaceabrassicacea eeee moricandia sinaica (boiss.) boiss. krombe aerial parts hydrometh anolic extract 250–500 mg/kg carrageenaninduced paw edema in rats analgesic, antiinflammator y and antipyretic activities 52,4 % inhibition at 500 mg/ml (el-mekkawy et al., 2020) cactaceaecactaceaecactaceaecactaceae opuntia ficusindica (l.) mill. lhindia flowers aqueous acetone extract 0,18–2.25 mg/ml nitric oxide inhibition assay antioxidant and antiinflammator y activities 90% no inhibition (ic50 of 0,19 mg/ml) (benayad et al., 2014) capparacecapparacecapparacecapparace aeaeaeae capparis spinosa l. kabâr leaves ethanol extract 1.07 g/kg paw oedema assay in mice antiinflammator y activity 73,44 % inhibition at 1,07 g/kg (el azhary et al., 2017) capparis spinosa l. kabâr leaves aqueous extract 100-500 µg/ml unknown antiinflammator y activity inhibition of another pro inflammatory cytokine, il-17 at 500 μg/ml (moutia et al., 2016) cistaceaecistaceaecistaceaecistaceae cistus ladanifer l. arguil, bouchikh leaves aqueous extract 150-200 mg/kg carrageenaninduced paw edema in rats antiinflammator y and analgesic effects 93,77 % inhibition at 200 mg/kg after 3h (el hamsas el youbi et al., 2016) cistus monspeliensis l. arguil, bouchikh aerial parts aqueous extract 500 mg/kg carrageenaninduced paw edema in rats antiinflammator y and analgesic activities 85,78 % inhibition at 500 mg/kg after 6h (sayah et al., 2017) cistus salviifolius l. arguil, bouchikh aerial parts aqueous extract 500 mg/kg carrageenaninduced paw edema in rats antiinflammator y and analgesic activities 91,57 % inhibition at 500 mg/kg after 6h (sayah et al., 2017) colchicacecolchicacecolchicacecolchicace aeaeaeae androcymbiu m gramineum (cav.) j.f.macbr. ssgëm lerneb bulbs hydroethan olic extract 300 µg/cm2 carrageenaninduced ear edema in mice antioxidant, antiinflammator y effect 25 % inhibition at 300 µg/cm2 (moussaid et al., 2011) compositacompositacompositacomposita eeee anacyclus pyrethrum (l) oud al attass roots hydroalcoh olic extract 300 mg/kg carrageenaninduced paw edema in rats analgesic, antiinflammator y, and wound healing properties 96 % inhibition at 300 mg/ml (jawhari et al., 2020) zouhri a et al. (2023). not sci biol 15(1):11414 21 anacyclus pyrethrum (l) oud al attass seeds hydroalcoh olic extract 500 mg/kg carrageenaninduced paw edema in rats analgesic, antiinflammator y, and wound healing properties 96% inhibition at 500 mg/ml (jawhari et al., 2020) anacyclus pyrethrum (l) oud al attass capitul a hydroalcoh olic extract 500 mg/kg carrageenaninduced paw edema in rats analgesic, antiinflammator y, and wound healing properties 98% inhibition at 500mg/ml (jawhari et al., 2020) anacyclus pyrethrum (l) oud al attass roots aqueous extract 125-500 mg/kg xylene–induced ear edema antiinflammator y, antinocicep tive and antioxidant activities 62 % inhibition at 500 mg/kg (manouze et al., 2017) anacyclus pyrethrum (l) oud al attass roots methanol extract 125-500 mg/kg xylene–induced ear edema antiinflammator y, antinocicep tive and antioxidant activities 65 % inhibition at 500 mg/ml (manouze et al., 2017) dittrichia viscosa (l.) greuter amakarm an leaves aqueous extract 200 mg/kg carrageenaninduced paw edema in rats anti-infam matory and antioxidant activities 54 % inhibition at 200 mg/kg (lounis et al., 2018) lactuca sativa l. mesiouka , harouka leaves hydroalcoh olic extract 200–400 mg/kg formaldehydeinduced pedal edema test in rats antioxidant and antiinflammator y activities 50,1 % inhibition at 400 mg/ml (zekkori et al., 2018) kleinia anteuphorbiu m (l.) haw. cigar leaves aqueous extract 200 mg/kg carrageenaninduced paw edema in rats anti-infam matory and antioxidant activities 43% inhibition at 200 mg/kg (lounis et al., 2018) clusiaceaeclusiaceaeclusiaceaeclusiaceae allanblackia gabonensis (pellegr.) bamps ntia stem bark methylene chloride fraction 37,5 300 mg/kg carrageenaninduced paw edema in rats antiinflammator y and antinociceptive activities 83,33 % inhibition by carrageenan at 150mg/ml (nguemfo et al., 2007) allanblackia gabonensis (pellegr.) bamps ntia stem bark methylene chloride fraction 37,5 300 mg/kg carrageenaninduced paw edema in rats antiinflammator y and antinociceptive activities 42,10 % inhibition by histamine at 150 mg/ml (nguemfo et al., 2007) allanblackia gabonensis (pellegr.) bamps ntia stem bark methylene chloride fraction 37,5 300 mg/kg carrageenaninduced paw edema in rats antiinflammator y and antinociceptive activities 64,20 % inhibition by arachidonic acid at 150 mg/ml (nguemfo et al., 2007) allanblackia gabonensis (pellegr.) bamps ntia stem bark methylene chloride fraction 37,5 300 mg/kg carrageenaninduced paw edema in rats antiinflammator y and antinociceptive activities 40,29 % inhibition by dextran at 150 mg/ml (nguemfo et al., 2007) garcinia mangostana l. tamoul leaves dichlorome thane extract 31.25 1000 µg/ml inhibition of protein denaturation antioxidant, antiinflammator y and cytotoxicol ogical properties inhibition of protein denaturation (ic50 = 152.79±3.34 μg/m) (chadon alphonsine assemian et al., 2019) zouhri a et al. (2023). not sci biol 15(1):11414 22 garcinia mangostana l. tamoul leaves ethanol extract 31.25 1000 µg/ml inhibition of protein denaturation antioxidant, antiinflammator y and cytotoxicol ogical properties inhibition of protein denaturation (ic50 = 652.33 ± 12.23 μg/ml) (chadon alphonsine assemian et al., 2019) coriariacecoriariacecoriariacecoriariace aeaeaeae coriaria myrtifolia l. arwaz or rewiza leaves ethyl acetate extract 0,005 0,013 mg/kg the plantar edema model induced in rabbits by carrageenan. antioxidant and antiinflammator y activities 68,2 % inhibition at 0,005 mg/kg dose after 3h (hafsé et al., 2017) cupressacecupressacecupressacecupressace aeaeaeae tetraclinis articulata (vahl) mast. al'araar or azouka leaves essential oil 100–200 mg/kg carrageenaninduced paw edema in rats antioxidant and antiinflammator y activities 68,48 % inhibition at 200 mg/kg dose after 3h (el jemli et al., 2017) tetraclinis articulata (vahl) mast. al'araar or azouka leaves essential oil 100–200 mg/kg trauma induced paw edema in rats antioxidant and antiinflammator y activities 84,51 % inhibition at 200 mg/kg dose after 3h (el jemli et al., 2017) ericaceaeericaceaeericaceaeericaceae erica arborea l. khlendj aerial parts ethanol extract 200–400 mg/kg carrageenaninduced paw edema in rats antioxidant and antiinflammator y activities 59 % inhibition at 400 mg/kg dose after 3h (amezouar et al., 2013) euphorbiaeuphorbiaeuphorbiaeuphorbia ceaeceaeceaeceae euphorbia granulata forssk. lubaina whole plants methanol extract 20–200 mg/kg carrageenaninduced paw edema in rats antiinflammator y, antipyretic, analgesic activity 59,12 % inhibition at 200 mg/kg after 3h (ghauri et al., 2021) fabaceaefabaceaefabaceaefabaceae albizia anthelmintica brongn. _ leaves methanol extract 200–400 mg/kg carrageenaninduced paw edema in rats antiinflammator y, pain killing and antipyretic activities 34 % inhibition at 400mg/ml (sobeh, rezq, et al., 2019) albizia anthelmintica brongn. _ leaves methanol extract 200–400 mg/kg cyclooxygenase assay antiinflammator y, pain killing and antipyretic activities cox-1 inhibition (ic50 of 4,11 μg/ml) cox-2 inhibition (ic50 of 0,054 μg/ml (sobeh, rezq, et al., 2019) albizia anthelmintica brongn. _ leaves methanol extract 200–400 mg/kg lipoxygenase assay antiinflammator y, pain killing and antipyretic activities 5-lox inhibition (ic50 of ≥ 1,74 μg/ml) (sobeh, rezq, et al., 2019) geraniaceageraniaceageraniaceageraniacea eeee pelargonium graveolens l'hér. el aatricha whole plants essential oil 250 mg/kg carrageenaninduced paw edema in rats oxidative and sodium nitroprussid e stress and infammatio n 86,76 % inhibition at 250 mg/kg dose after 6h (marmouzi et al., 2019) lamiaceaelamiaceaelamiaceaelamiaceae ajuga iva (l.) schreb. chendgo ura aerial parts essential oil 0,5 –3 mg/ml inhibition of elastase activity antiinflammator y activity elastase inhibition (ic50 of 192.21 ± 7.80 μg/ml) (bouyahya et al., 2021) ajuga iva (l.) schreb. chendgo ura aerial parts essential oil 40–160 µg/ml inhibition of tyrosinase activity antiinflammator y activity tyrosinase inhibition (ic50 of 87.49 ± 0.98 μg/ml) (bouyahya et al., 2021) melissa officinalis l. trandjan e leaves essential oil 200–400 mg/kg trauma induced paw edema in rats antiinflammator y activity 94,44 % inhibition at 400 mg/kg after 6h (bounihi et al., 2013) melissa officinalis l. trandjan e leaves essential oil 200–400 mg/kg carrageenaninduced paw edema test in rats antiinflammator y activity 70,58 % inhibition at 400 mg/kg after 6h (bounihi et al., 2013) zouhri a et al. (2023). not sci biol 15(1):11414 23 mentha pulegium l. fliou aerial parts hydroethan olic extract 300 µg/cm2 carrageenaninduced ear edema in mice antioxidant, antiinflamm atory effect 28 % inhibition at 300 µg/cm2 (moussaid et al., 2011) origanum compactum benth. zaâtar leaves essential oil 4.21 mg/ml lipoxygenase assays antioxidant, antiinflamm atory, and antibacterial effect lox inhibition (ic50 of 0.129 ± 0.004 mg/ml) (el kharraf et al., 2021) origanum compactum benth zaâtar leaves essential oil 4.21 mg/ml acetylcholineste rase assays antioxidant, antiinflamm atory, and antibacterial effect inhibition (ic50 of 0.13 ± 0.01 mg/ml) (el kharraf et al., 2021) rosmarinus officinalis l. azir leaves essential oil 4.21 mg/ml lipoxygenase assays antioxidant, antiinflammator y, and antibacterial effect lox inhibition (ic50 of 0.548 ± 0.005 mg/ml) (el kharraf et al., 2021) rosmarinus officinalis l. azir leaves essential oil 4.21 mg/ml acetylcholineste rase assays antioxidant, antiinflammator y, and antibacterial effect inhibition (ic50 of 1.74 ± 0.17 mg/ml) (el kharraf et al., 2021) thymus atlanticus (ball) ziitra or azukni leaves aqueous extract 50–150 mg/kg carrageenaninduced paw edema in rats antiinflammator y and anticoagulan t effects 70 % inhibition at 100 mg/ml (khouya et al., 2020) thymus atlanticus (ball) ziitra aerial parts aqueous extract 900 µg/ear croton oilinduced ear edema in mice antiinflammator y, anticoagulan t and antioxidant effects 55,15 % inhibition at 900 µg/ear (khouya et al., 2015) thymus atlanticus (ball) ziitra aerial parts aqueous extract 50 mg/kg carrageenaninduced paw edema in rats antiinflammator y, anticoagulan t and antioxidant effects 62,15 % inhibition at 50 mg/kg (khouya et al., 2015) thymus atlanticus (ball) ziitra aerial parts aqueous extract 200– 1500 μg/ml inhibition of albumin denaturation antioxidant, antiinflammator y and anticoagulan t activities inhibition of albumin denaturation (ic50 = 122.90 μg/ml) (hmidani et al., 2019) thymus broussonetii boiss. z’iitra leaves chloroform extract 300 μg cm 2 croton oilinduced ear edema in mice antiinflammator y activity 83,36 % inhibition at 1000 μg cm 2 dose after 6h (ismaili et al., 2002) thymus maroccanus ball z’iitra aerial parts ethanol extracts 300–600 mg/kg carrageenaninduced paw edema in rats antioxidant activity, antiinflammator y potential 55,36 % inhibition at 300 mg/kg dose after 6h (oubihi et al., 2020) thymus maroccanus ball z’iitra aerial parts methanol extract 300–600 mg/kg carrageenaninduced paw edema in rats antioxidant activity, antiinflammator y potential 57,51 % inhibition at 300 mg/kg dose after 6h (oubihi et al., 2020) thymus saturejoides coss azukni leaves hexane extract 300 µg cm−2 croton oilinduced ear edema in mice antiinflammator y and in vitro 24 % inhibition at 300 µg cm−2 dose after 6h (ismaili et al., 2004) zouhri a et al. (2023). not sci biol 15(1):11414 24 antioxidant activities thymus saturejoides coss azukni leaves methanol extract 300 µg cm−2 croton oilinduced ear edema in mice antiinflammator y and in vitro antioxidant activities 29 % inhibition at 300 µg cm−2 dose after 6h (ismaili et al., 2004) thymus saturejoides coss azukni leaves chloroform extract 300 µg cm−2 croton oilinduced ear edema in mice antiinflammator y and in vitro antioxidant activities 65 % inhibition at 300 µg cm−2 dose after 6h (ismaili et al., 2004) thymus saturejoides coss. zaiitra aerial parts aqueous extract 900 µg/ear croton oilinduced ear edema in mice antiinflammator y, anticoagulan t and antioxidant effects 2,83 % inhibition at 900 µg/ear (khouya et al., 2015) thymus saturejoides coss zaiitra aerial parts aqueous extract 50 mg/kg carrageenaninduced paw edema in rats antiinflammator y, anticoagulan t and antioxidant effects 8,93 % inhibition at 50 mg/kg (khouya et al., 2015) thymus saturejoides coss zaiitra aerial parts aqueous extract 200– 1500 μg/ml inhibition of albumin denaturation antioxidant, antiinflammator y and anticoagulan t activities inhibition of albumin denaturation (ic50 = 181.42 μg/ml) (hmidani et al., 2019) thymus vulgaris l. z’itra aerial parts essential oil 100–400 mg/kg carrageenaninduced paw edema in rats antiinflammator y activity 58,4 % inhibition at 400 mg/ml (abdelli et al., 2017) thymus willdenowii boiss. z’itra leaves chloroform extract 300 μg cm 2 croton oilinduced ear edema in mice antiinflammator y activity 92 % inhibition at 300 μg cm 2 dose after 6h (ismaili et al., 2001) thymus zygis l. z’itra plant aqueous extract 900 µg/ear croton oilinduced ear edema in mice antiinflammator y, anticoagulan t and antioxidant effects 31,52 % inhibition at 900 µg/ear (khouya et al., 2015) thymus zygis l. z’itra plant aqueous extract 50 mg/kg carrageenaninduced paw edema in rats antiinflammator y, anticoagulan t and antioxidant effects 87,20 % inhibition at 50 mg/kg (khouya et al., 2015) thymus zygis l. z’itra aerial parts aqueous extract 900 µg/ear croton oilinduced ear edema in mice antioxidant, antiinflammator y and anticoagulan t activities 39,83 % inhibition at 900 µg/ear after 3h (khouya et al., 2016) thymus zygis l. z’itra aerial parts dichlorome thane fraction 900 µg/ear croton oilinduced ear edema in mice antioxidant, antiinflammator y and anticoagulan t activities 28,32 % inhibition at 900 µg/ear after 3h (khouya et al., 2016) thymus zygis l. z’itra aerial parts aqueous extract 50 mg/kg carrageenaninduced paw edema in rats antioxidant, antiinflammator y and anticoagulan t activities 86,61 % inhibition at 50 mg/kg (khouya et al., 2016) zouhri a et al. (2023). not sci biol 15(1):11414 25 thymus zygis l. z’itra aerial parts dichlorome thane fraction 50 mg/kg carrageenaninduced paw edema in rats antioxidant, antiinflammator y and anticoagulan t activities 12,66 % inhibition at 50 mg/kg (khouya et al., 2016) thymus zygis l. z’itra aerial parts aqueous extract 200– 1500 μg/ml inhibition of albumin denaturation antioxidant, antiinflammator y and anticoagulan t activities inhibition of albumin denaturation (ic50 = 133.25 μg/ml) (hmidani et al., 2019) leguminosleguminosleguminosleguminos aeaeaeae bauhinia reticulata dc. nguiguis leaves methanol extract 40 μg/ml to 1000 μg/ml inhibition of heat induced protein denaturation antioxidant and the antiinflammator y effects 58,69 % inhibition of albumin denaturation at 1000 μg/ml (ic50 = 121.43 ± 1.55 μg/ml) (boualam et al., 2021) bauhinia thonningii schum. nguiguis leaves methanol extract 40 1000 μg/ml inhibition of heat induced protein denaturation antioxidant and the antiinflammator y effects 67,39 % inhibition of albumin denaturation at 1000 μg/ml (ic50 = 116.4 ± 0.73 μg/ml) (boualam et al., 2021) lythraceaelythraceaelythraceaelythraceae lawsonia inermis l. lhana leaves methanol extract 50 800 μg/ml inhibition of protein denaturation antioxidant and antiinflammator y properties 82,42 % inhibition at 200 μg/ml (ic50 = 103.21 μg/ml) (bouhlali et al., 2016) lawsonia inermis l. lhena leaves oil 0,1 mg/ml carrageenan induced pedal edema test in rabbits antioxidant, antiinflammator y and photoprotec tive effects 90,30 % inhibition at 0,5 mg/ml after 5h (zouhri et al., 2017) punica granatum l. ounk hmam fruit methanol extract 50-150 mg/kg albumin induced hind paw edema antiinflammator y and antinocicept ive effects 51 % inhibition at 150 mg/kg after 2h (ouachrif et al., 2012) myrtaceae eugenia uniflora l. _ leaves methanol extract 100 mg/kg carrageenaninduced hind paw edema antioxidant, antiinflammator y activities 32 % inhibition at 100 mg/kg after 1h (sobeh, el-raey, et al., 2019) eugenia uniflora l. _ leaves methanol extract 100 mg/kg cyclooxygenase assay antioxidant, antiinflammator y activities cox-1 inhibition (ic50 of 5,63 μg/ml) cox-2 inhibition (ic50 of 0,18 μg/ml) (sobeh, el-raey, et al., 2019) eugenia uniflora l. _ leaves methanol extract 100 mg/kg lipoxygenase assay antioxidant, antiinflammator y activities 5-lox inhibition (ic50 of 2.14 μg/ml) (sobeh, el-raey, et al., 2019) syzygium aromaticum (l.) merr. & l.m.perry kranfal whole plants essential oil 250 mg/kg carrageenaninduced paw edema in rats oxidative and sodium nitroprussid e stress and infammatio n 93,83 % inhibition at 250mg/kg after 6h (marmouzi et al., 2019) rhamnacerhamnacerhamnacerhamnace aeaeaeae ziziphus lotus (l.) lam. sedra seeds oil 200-300 mg/kg carrageenaninduced paw edema in rats antiinflammator y activity 64,33 % inhibition at 200mg/ml (el hachimi et al., 2017) rosaceaerosaceaerosaceaerosaceae rosa × damascena herrm. _ flowers methanol extract 50800 μg/ml inhibition of protein denaturation antioxidant and antiinflammator y properties 79,5 % inhibition at 200 μg/ml (ic50 = 129.04 μg/ml) (bouhlali et al., 2016) sapotaceaesapotaceaesapotaceaesapotaceae argania spinosa (l.) skeels argan seeds argan oil 300 -500 mg/kg carrageenaninduced paw edema in rats antiinflammator y activity 70,68 % inhibition at 500 mg/kg after 6h (kamal et al., 2019) zouhri a et al. (2023). not sci biol 15(1):11414 26 conclusionsconclusionsconclusionsconclusions the present review reported the anti-inflammatory effects of moroccan medicinal plants from their traditional use to the pharmacological studies, which linked the ethnopharmacological uses to the biological activities. it was noticed that many moroccan herbal remedies could be used as anti-inflammatory remedies. the study found that 100 plant species belonging to 52 families are used to treat inflammation. the dominant family in this study concerning species number was lamiaceae, and the most cited plant species are salvia officinalis l, opuntia ficus-indica l, lavandula stoechas l, lawsonia isermis l, and rosmarinus officinalis l. in terms of efficiency, the aqueous extract of cistus ladanifer, the hydroalcoholic extract of anacyclus pyrethrum, essential oil of melissa officinalis and syzygium aromaticum are the most effective anti-inflammatory agents in vivo models. likewise, methanolic extracts of cuminum cyminum, lawsonia inermis, rosa damascena and acetone extract of opuntia ficus-indica are the most effective plant extracts for regulating inflammatory, and pro-inflammatory cells in the in vitro model. however, numerous plants used in moroccan traditional medicinal have not been yet tested for their anti-inflammatory effects in laboratory. therefore, further investigations are required regarding the in vitro and in vivo models and their bioactive compounds as well as their clinical investigations. in this way, it will also be essential to determine the toxicity of these plants in order to be able to apply them in clinical terms and contribute to develop now anti-inflammatory drugs. authors’ contributionsauthors’ contributionsauthors’ contributionsauthors’ contributions all authors read and approved the final manuscript. ethical approvalethical approvalethical approvalethical approval (for researches involving animals or humans) not applicable. argania spinosa (l.) skeels argan seeds argan oil 300 -500 mg/kg trauma induced paw edema in rats antiinflammator y activity 49,63 % inhibition at 500 mg/kg after 6h (kamal et al., 2019) solanaceaesolanaceaesolanaceaesolanaceae withania frutescens (l.) pauquy irram foliar parts ethanol extract 350 -450 mg/kg carrageenaninduced paw edema in rats analgesic, antiinflammator y, and healing activities 82,20 % inhibition at 450 mg/kg after 6h (el moussaoui et al., 2020) thymelaeathymelaeathymelaeathymelaea ceaeceaeceaeceae thymelaea hirsuta (l.) endl. _ aerial parts aqueous extract 500 mg/kg carrageenaninduced hind paw edema anti-inflam matory and antiarthritic activities 68 % inhibition at 500 mg/ml after 5h (oudghiri and azza, 2015) thymelaea lythroides barratte & murb. _ aerial parts methanol extract 200 mg/kg lps induced microglial activation antiinflammator y activity thymelaea lythroides reduces lps-induced microglial activation (berkiks et al., 2018) zygophyllazygophyllazygophyllazygophylla ceaeceaeceaeceae tetraena gaetula (emb. & maire) beier & thulin _ aerial parts aqueous extract 5oo mg/kg carrageenaninduced paw edema in rats antiinflammator y activity 47 % inhibition at 500mg/ml (khabbal et al., 2006) zouhri a et al. 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