pharmacognosy journal an ofcial publicaton of pharmacognosy network worldwide [phcog.net] issn : 0975-3575 www.phcogj.com indexed in scopus, scimago journal ranking, chemical abstracts, excerpta medica / embase, google scholar, cabi full text, index coperni-cus, ulrich’s international periodical directory, proquest,index pharmacus, phcogbase, ebscohost, academic search complete, open j-gate, sciaccess.pharmacognosy journal [ phcog j.] (an official publication of pharmacognosy network worldwide) www.phcogj.com editor in chief dr. srisailam keshetti vaagdevi college of pharmacy, ramnagar, hanamkonda, warangal - 506001, andhra pradesh india associate editors dr.m.vijayakumar scientist pharmacognosy & ethnopharmacology division, national botanical research institute, lucknow 226001, india mr. sanjib bhattacharya bengal school of technology delhi road, hooghly calcutta (wb) editorial board dr. vasudeo zambare research scientist centre for bioptocessing r&d south dakota school of mines and technology rapid city, south dakota 57701 usa chinmay rath central council for research in ayurveda & siddha, dept. of ayush, ministry of health & family welfare, janakpuri, new delhi-58, dr. m. k. meghvansi scientist 'c' agro-technology division defence research laboratory, defence r & d organization (min. of defence, govt. of india) post bag no 2, tezpur-784001, assam, india jun-hua wang vice-director of department of cardiology, air force general hospital, chinese people’s liberation army (pla) rajendra m dobriyal cfc-clinicals unilever r&d bangalore-560066 vipra kundoor staff fellow pharmacologist us food and drug administration rockville, maryland, usa yongxu sun department of medicinal chemistry and biomacromolecules qiqihar medical university, qiqihar 161006 heilongjiang province p.r. china anjaneyulu muragundla department of neurology, school of medicine university of maryland, baltimore 655 w. baltimore st rm# 12-044 bressler reserach bldg, baltimore, md-21201, usa shuge tian experimental teaching demonstration center of tcm in xinjiang medical university department of traditional medicine ,tcm xinjiang medical university xinjiang china 830054 fengguo xu dept of epidemiology and public health, yong loo lin school of medicine, national university of singapore, md3, 16 medical drive, singapore 117600. mr. goutam kumar jana asst. professor department of pharmacognosy gayatri college of pharmacy jamadarpali,sason,sambalpur,768200 orissa dr. r.k.asthana sr. scientist medicinal & process chemistry division central drug research institute chttar manzil palace lucknow 226001up india shazia qasim jamshed social and administrative pharmacy school of pharmaceutical sciences universiti sains malaysia penang malaysia dr. ramachandra setty siddamsetty, professor, college of clinical pharmacy, king faisal university, alahsa - 31982 ksa. dr. (mrs.) sayyada khatoon scientist pharmacognosy & ethnopharmacology division national botanical research institute, rana pratap marg, post box 436, lucknow-226001 (u.p.) india sheelendra pratap singh csir-senior research fellow pharmacokinetics and metabolism division central drug research institute lucknow shashi alok asst. professor institute of pharmacy bundelkhand university jhansi (u.p) dr. n. karmegam, professor, department of biotechnology, vmkv engineering college, vinayaka missions university, salem - 636 308, tamil nadu, india. ajay semalty asst. professor department of pharmaceutical sciences h.n.b. garhwal university srinagar garhwal-246174 india dr. vijay d. wagh y.b. chavan college of pharmacy,dr. rafiq zakaria campus, rouza bagh, aurangabad-431001, maharashtra. dr avijit mazumder professor , school of pharmacy & technology management , nmims university v.l mehta road, vile parle(west), mumbai-400056 a.c.tangavelou, dept. of bioethics, biodiversity & drug discovery, bio-sciences techno park, 166/1, gunda salai, moolakulam, pondicherry 605 010, south india sandeep arora professor and director, chitkara college of pharmacy, rajpura campus, chandigarh-patiala highway, punjab 140401 dr. elcey c. daniel, head of department, department of biosciences, kristu jayanti college, k.narayanpura, kothannur post, bangalore 560077. aims and scope of journal pharmacognosy journal [phcog j]- a rapid and swift publication from pharmacognosy network worldwide [phcog.net]. phcog j is not an annual, a quarterly, a monthly, or a weekly. it is being published when the article is ready. highly indexed & abstracted, swift editorial decisions, quality papers, exclusively for pharmacognostic studies. indexed and abstracted in : scopus, scimago journal ranking, chemical abstracts, excerpta medica / embase, google scholar, cabi full text, index copernicus, ulrich’s international periodical directory, proquest,index pharmacus, phcogbase, ebscohost, academic search complete, open j-gate, sciaccess contact publisher emanuscript services, 1713, 41 a cross, 18 main, jayanagar 4 t block, bangalore 41 india email : journals@emanuscript.in submission of manuscript manuscripts could be submitted by email as an attachment submissions@phcogj.com a timely submission, however, is not a guarantee that your work will be accepted for forthcoming publication. all submissions are peer reviewed by the editorial board and a select group of reviewers. please make sure that all guidelines are followed carefully. all the accepted articles will be queued for publication and will appear in the futures issues based on the priorities set by the editorial board. e mail submission format for email submission of manuscript: please place all your documents in one document. it should be titled: first name, last name of corresponding author. e.g. manuscript – sonali_singh.doc ; subsequent files - sonali_singh _1.doc in case of figures, it should be titled: first name, last name of corresponding author followed by the number in chronological order. e.g. figures - sonali_singh_2.jpeg ; subsequent files - sonali_singh_3.xls ; sonali_singh_4.tiff hard copy submission hard copies are not being accepted. check list for submitting a manuscript • covering letter • copyright forms (scanned) • manuscript • illustrations (if any) upon acceptance, non-members are required to pay pre-press charges of rs. 350/page (indian authors) or usd 10/page (foreign authors) is applicable and to be paid to "phcog.net" bangalore.pharmacognosy journal  september 2011  vol 3  issue 25 i c o n t e n t s p h c o g j . review articles a review on phytochemistry of cuminum cyminum seeds and its standards from field to market. 1 ahmad reza gohari and soodabeh saeidnia medicinal plant diversity and their pharmacological aspects of nepal himalayas 6 bhakta prasad gaire and lalita subedi pharmacognostic studies identifcation of a bioactive compound from myrcianthes cysplatensis 18 eugenio damico, stephanie barneche, maria pia cerdeiras and alvaro vázquez comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni from different geographical sources 21 mandal bitasta, madan swati pharmacognostical studies of leaves of lagerstroemia fos-reginae 27 harde pinal a. and shah mamta b. pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) in comparison with market samples 32 rath chinmay, suman kumari, dhar bishnupriya, mohanty rc, dixit renu, padhi mm, babu ramesh pharmacognostic and phytochemical investigation of juglans regia linn. bark 39 thakur nirmladevi, vinita apraj, ashok bhagwat, rashmi mallya, laxman sawant, nancy pandita preliminary phytochemical evaluation of euphorbia fusiformis buch-ham. 44 ashok bk, savitha d bhat, shukla vj, ravishankar b infuence of salt stress on phosphorus metabolism in the roots and leaves of one month old prosopis julifora (sw.) dc seedlings 48 patil, amol v. and chavan, p. d. pharmacognostic and hptlc studies on diospyros montana r. (ebenaceae) 52 shravan kumar, sakshi sehgal, hafsa ahmad, rajiv gupta, shubhini a. saraf pharmacological studies comparative molluscicidal activities of fruit pericarp, leaves, seed and stem bark of blighia unijugata baker 63 agboola o i, ajayi g o, adesegun s a, and adesanya s a. wound healing potential of extract of jatropha curcas l. (stem bark) in rats 67 kamal sachdeva, preeti garg, manmohan singhal, birendra srivastava studies on activity of various extracts of albizia amara against drug induced gastric ulcers 73 t. rajkumar, b.n. sinha acute oral toxicity of abelmoschus manihot and wrightia tinctoria in mice 78 p. s. jain, s. b. bari, s. j. surana. phytochemical screening, antimicrobial and in vitro anti-infammatory activity of endophytic extracts from loranthus sp. 82 govindappa m, channabasava r, sowmya dv, meenakshi j, shreevidya mr, lavanya a and sadananda ts diuretic activity of alcoholic extract of musa sapientum l. flower 91 ashutosh mishra, manas ranjan mishra, dusmanta kumar pradhan, sunil kumar vaishnavpharmacognosy journal  september 2011  vol 3  issue 25 1 r e v i e w a r t i c l e p h c o g j . *address for correspondence: medicinal plants research center, faculty of pharmacy, tehran university of medical sciences, po box 14155-6451,tehran, iran. fax: 98-21-66461178. e-mail: saeidnia_s@sina.tums.ac.ir doi: 10.5530/pj.2011.25.1 are 5-10 cm long, pinnate or bi pinnate, with thread-like leafets and blue green in color and are fnely divided, generally turned back at the ends. the leaves are highly dissected. whitish-red fowers are on a compound umbel (arrangement of fowers looks like an umbrella). the fruit is an elongated, oval shaped schizocarp (an aggregate fruiting body which doesn’t break open naturally and has two single seeded units called mericarps). the fruits are similar to fennel seeds, when chewed has bitter and pungent taste. the fruit are thicker in the middle, compressed laterally about 5 inch long, containing a single seed. [5-6] medicinal plant material dried ripe seeds of c. cyminum are usually used formedicinal or culinary purposes. in iranian traditional medicine, cumin seeds were used for their therapeutic effects on gastrointestinal, gynecological and respiratory disorders, and also for the treatment of toothache, diarrhea and epilepsy. the seeds were also documented as stimulant, carminative and astringent. [2] johri has been recently reported that medicinal usage of cumin seeds has also beenwidespread in diverse ethnomedical systems fromnortherneurope to themediterranean regions, russia, iran, indonesia and north america, where these have remained as an integral part of their folk medicines. [7] a review on phytochemistry of cuminum cyminum seeds and its standards from field to market ahmad reza gohari and soodabeh saeidnia* medicinal plants research center, faculty of pharmacy, tehran university of medical sciences, tehran, iran. a b s t r a c t the small boat shaped seeds of cumin (cuminum cyminum) has been used for many medicinal and culinary purposes from the ancient time in the various countries. cumin is a popular spice in the world from latin america to northern africa and all over the asia and also used as a favoring agent in many products such as cheese, pickle, soup, bean dishes or liqueurs. essential oils of the seeds are also used as a favor or in aromatherapy. many pharmacological effects have been reported from this spicy plant as, anti-diabetic, immunologic, anti-epileptic, anti-tumor and antimicrobial activities. cumin used in the medicinal preparations is supposed to be produced with high quality encompasses all the properties of the fnal product which makes it optimal suitable for use. reproducible quality is a goal, which achieved by the process of standardization. the focus here is rather on harvesting and processing of the cultivated species, because the quality of plant material and processing technology lead to the high quality of the fnal product. the quality of cumin seeds and its essential oil can only be assessed with analytical methods, which include physical, microscopic and chemical analyzing assays. in this paper, the phytochemistry, medicinal properties and the standards from the feld cultivation, harvesting and storage until marketing are reviewed. key words: cuminum cyminum, phytochemistry, adulterants, standards. introduction cuminum cyminum l., belonging to the family apaiaceae, is one of the old cultivated medicinal food herbs in asia, africa and europe. this plant is well-known as cumin and named zireh-sabz or cravieh in persian language. its seeds have been commonly used for culinary and favoring purposes and folklore therapy since antiquity in various countries. [1-3] there are two species of cuminum which growing wildly in iran, c. cyminum l (zireh-sabz means green cumin) and c. setifolium boisskos. pol (zireh-sefd means white cumin). some literature reported that c. setifolium is the sub-species of c. cyminum. [1-4] c. cyminum is an annual herbaceous plant which grows up to 15-50 cm height somewhat angular and tends to droop under its own weight. it has a long, white root. the leavesgohari and saeidnia: phytochemistry and standards of cumin seeds 2 pharmacognosy journal  september 2011  vol 3  issue 25 the cumin oil is reported as a high antioxidant mainly due to the presence of monoterpene alcohols. [11] the presence of phytoestrogens in cumin has been reported which related to its anti-osteoporotic effects. methanol extract of cumin showed a signifcant reduction in urinary calcium excretion and augmentation of calcium content and mechanical strength of bones in animals. [12] furthermore, the aqueous extract of cumin seeds indicated the protective effect against gentamycin-induced nephrotoxicity, which decreased the gentamycin-induced elevated levels of serum urea and enhanced the clearance of the drug. [13] phytochemistry and medicinal propeties antimicrobial activity has been reported from the volatile oils and aqueous extract of cumin. cumin seed oil and alcoholic extract inhibited the growth of klebsiella pneumoniae and its clinical isolates by improvement of cell morphology, capsule expression and decreasing urease activity. cuminaldehyde (1) is the main active compound of cumin for this property. [8-9] limonene (2), eugenol (3), α- and β- pinenes(4, 5) and some other minor constituents have been found in cumin oil and suggested as the active antimicrobial agents. [7,10] o cuminaldehyde (1) limonene (2) or 2 or 1 ho or 3 (10) r 1 glc, r 2 r 3 h (11) r 1 r 3 h, r 2 glc (12) r 1 r 2 h, r 3 glc eugenol (3) o o o o ho oh oh ch 2 oh h h o o o oh h o ho oh oh ch 2 oh oh och 3 a-pinene (4) b-pinene (5) cuminoside a (6) cuminoside b (7) o o hoh 2 c oh oh o or oh oh ho (8) r ch 3 (9) r ch 2 -ch 2 -oh figure 1: chemical structures of the isolated compounds from cumin.pharmacognosy journal  september 2011  vol 3  issue 25 3 gohari and saeidnia: phytochemistry and standards of cumin seeds mediterranean climates. it is grown from seed, sown in spring, and needs fertile, well-drained soil. the plant blooms in june and july. the seeds are normally ripe four months after planting. the plants are threshed when the fruit is ripe and the seeds are dried. [21-22] standards criteria for harvesting, drying and storage appropriate season the cumin seeds are usually ready to harvest during 100-120 days after cultivation. seed harvesting season is different from june to july on the basis of the weather conditions, because the fowering season is infuenced by day long and temperature. [23] literature showed that cumin is better to be in rotation with summer crops such as soybean, millet and sesame. in order to produce satisfed yield in iran, application of 30 kg n, 60 kg p and 30 kg k per hectare has been recommended. cumin crop water requirement is 335 mm/ha. average yield of cumin is reported 1000 kg/ha with percentage of 2.1 to 3.5 for seed volatile oils. [24] appropriate harvesting methods the seeds are harvested about 4 months after planting when the plant begins to wither and the seeds change to brown-yellow color. in traditional method, the whole plants were removed from the soil and collected as sheaves. the sheaves were set up in the felds and sifting and cleaning by winnower. the isolated seeds were then further dried to 10% (moisture content), wither by placing on mats or trays in the sun or using a drier in the humid conditions (like pakistan). the dried seeds are winnowed using a traditional winnowing basket to remove the dirt, dust, leaves and twigs. nowadays, the modern and high capacity combines are used for harvesting, sifting and cleaning of the plants. [23] main physicochemical characteristics the seeds are elongated, oval shaped schizocarp and similar to fennel seeds, when chewed have bitter and pungent taste. the seeds (fruits) are thicker in themiddle, compressed laterally about 5 inch long. five out-standing lines are observed in each parts of mericarp. the seeds are too favored and covered by hairs (sometimes without hair). the fruit pericarp contains high amount of tannins which change color in presence of iron contained compounds. the seeds must contain at least 7%of oil, 13 & resin and 2.5-4% essential oil. the maximum total crude ashes are 9.5% and the maximum acid insoluble ashes are 2% with no more than 9% humidity. [22] qualification and quantification parameters of essential oil different factors may impact on the physicochemical properties of the essential oil of cumin seeds, of which plant anti-epileptic activity of cumin oil was also reported, which decreased the frequency of spontaneous activity induced by pentylene tetrazol (ptz). [14] recently, cumin oil has been found to act as a signifcant analgesic by formalin test in rats and suppress the development and expression of morphine tolerance and also reverse the morphine dependence. [15-17] other important reports consider that dietary cumin can inhibit benzopyrene-induced for stomach tumorigenesis, 3-methylcholanthrene induced uterine cervix tumorigenesis, and 3-methyl-4-dimethyaminoazobenzene induced hepatomas in mice, which was attributed to the ability of cumin in modulating carcinogenmetabolism via carcinogen-xenobiotic metabolizing phase i and phase ii enzymes. [18] literature review on phytochemistry of the cumin seeds revealed the presence of various bioactive compounds, the important secondary metabolites of which are discussed as followed. [19-20] two sesquiterpenoid glucosides, cuminoside a (6) and b (7), and two alkyl glycosides (8, 9) were isolated (figure 1) together with some known compounds from the methanol extract of cumin seeds. their structures were established as (1s,5s,6s,10s)-10-hydroxyguaia-3,7(11)-dien-12,6-olide β-d-glucopyranoside (6), (1r,5r,6s,7s,9s,10r,11r)-1, 9-dihydroxyeudesm-3-en-12, 6-olide 9-o-β-d-glucopyranoside (7), methyl β-d-apiofuranosyl-(16)- β‑d‑glucopyranoside (8) and ethane-1,2-diol 1-o-β‑d‑apiofuranosyl-(16)-β‑d‑glucopyranoside (9). [19] in another report, three glycosides (figure 1), 1-o-β-d-glucopyranoside (10), 3-o-β-d-glucopyranoside (11) and 4-o-β-d-glucopyranoside (12) have been isolated and structural elucidated from the seeds (fruits) of cumin. [20] natural habitat and the land under cultivation cumin is the native species growing in egypt (north africa) and asia. it was originally cultivated in iran and the mediterranean region. in iran, cumin is wildly growing in khorasan. the species, c. setifolium is also found in desert aria such as damghan, sabzevar, tabas and borazjan. cumin (c. cyminum) is mainly cultivated in khorasan followed by east azerbaijan, yazd, semnan, esfahan and some parts of golestanand kerman provinces. ninety percent of the whole products for the export are cultivated in khorasan. iran provides 20-40% of the world production and export of cumin. the cumin seeds are valuable, because the prices of one kilogram seeds are equal to 10 kilogram wheat. cultivation of cumin requires a long, hot summer of 3-4 months, with daytime temperatures around 30 c. this herb is resistant to drought, and is mostly grown ingohari and saeidnia: phytochemistry and standards of cumin seeds 4 pharmacognosy journal  september 2011  vol 3  issue 25 an adulterant in cumin oil. [29] frauds distinguish is very diffcult to detect chemically but it is possible because the synthetic cumin aldehyde may change the refractive index of the oil. [22,30] detection of purity by microscope many of the cumin products contain grinded seeds of cumin. therefore, microscopic analysis is considered for purity determination. grinded cumin is a yellowish-brown powder with a characteristic, aromatic, slightly camphor-like odor and taste. the diagnostic characters are summarized below. [31] the epicarp composed of a layer of colorless cells, with thin, sinuous walls and a faintly and irregularly striated cuticle. stomata are fairly frequent and cicatrices may be present. underlying the epicarp the thin-walled cells of the palisade are sometimes visible. the pale yellowish-brown fragments of the vittae composed of fairly large, thin-walled cells. the fragments are usually wider than the most of the other umbelliferae fruits. the sclereids from the mesocarp are in two main types. one type occurs as a single layer of longitudinally elongated cells with moderately thickened walls and numerous regularly spaced, well-marked pits. second type is found in small groups and composed of considerably elongated cells placed more or less end to end in a longitudinal direction. the endocarp composed of a layer of fairly large, thin-walled cellsand arranged with their long axes parallel. the endosperm composed of moderately thick-walled cells containing aleuronic microrosette crystals of calcium oxalate. [31] thin layer chromatographic analysis in this analysis, the extract of cumin, obtained by percolation of 1 g of dried seeds, is concentrated and dissolved in 0.5 ml toluene. thin layer chromatography (tlc) is carried out on a silica gel tlc plates with the solvent system as toluene: ethyl acetate (7:93) alongside the standards of α- and β-pinenes and α- and β-phellandrenes. the spots are detectable by the anisaldehyde-h 2 so 4 spray reagent followed by heating (105-110 c for 5-10 min). the spots of the above mentioned standards can be visible inside the area of 0.2-0.4 (rf), respectively. the pinene spots show the brown color and the phellandrene spots indicate a yellowish-brown color. [32] conclusion cumin is the second most popular spice in the world, after black pepper, and used as a medicinal plant for aromatherapy and various illnesses. for this reason, the standardization of the plant material from cultivation to storage is too important. to insure the achievement of quality, acceptance criteria for plant material and validating of the technical variety, cultivation area and conditions, date of harvesting and extraction methods are important. quantifcation of the total essential oil of seeds is conducted by hydro-distillation method. in this method, about 20 g of the grind fruits disperse in 500 ml of distilled water (in a 1000 ml fask) and hydro-distilled for 4 h with 3-4 ml/min distillation rate. the oil volume is measured by using xylol. [25-26] physical properties of the essential oil obtained by steam distillation from cumin seeds are summarized in table 1. [22,27] packaging and storage cumin seeds are sensitive to crashing and mechanical damages hence protection of seeds during sifting and cleaning or winnower is too important. sometimes the products lose some parts of humidity and become drier than standards. the quality of seeds has been decrease during the prolonged storage. today, cumin is packed in gunny bags and cleaned by machines in order to remove the stalks, other foreign material, stones and dust in advance. cumin may also pack in tissue, paper or polythene bags depending on the requirements of the buyer. it is preserved at least one to two feet away from the walls in order to save it from moisture in humid countries. the bags should be sealed to prevent moisture entering or exiting. labels should be applied to the products. the label needs to contain all relevant product and legal information such as the name of the product, brand name, names, address and date of manufacture, expiration date, weight of the contents, added ingredients (if relevant) plus any other information that the country of origin and of import may require. [22,28] the essential oils of cumin should be conserved in the amber and tight closed glass or aluminum containers even better to seal by inner epoxy covers. the oil packages should be storage far from the direct sunlight and temperate places. [2] adulterants regarding to this point that the characteristic odor of cumin is caused mainly by aldehydes which are present the essential oil, synthetic cumin aldehyde is sometimes added as table 1: physical properties of the essential oil of cumin seeds obtained by steam distillation physicochemical properties level extraction percentage 2.3-5.7 % color colorless or pale yellow refractive index (20 ºc) 1.47-1.50 density (20 ºc) 0.90-0.94 alcohol solubility (80% v/v) 1:1.3-1:2 aldehyde percentage (on the basis of cuminaldehyde) 35-63% acidity (on the basis of cuminic acid) 0.36-1.8 alcohol percentage (on the basis of cuminol) 3.5 carbonyl index 9.32 steric index 19.24pharmacognosy journal  september 2011  vol 3  issue 25 5 gohari and saeidnia: phytochemistry and standards of cumin seeds 14. janahmadi m, niazi f, danyali s, kamalinejad m. effects of the fruit essential oil of cuminum cyminum linn. (apiaceae) on pentylene tetrazol-induced epileptiform activity in f1 neurones of helix aspersa. j ethnopharmacol. 2006; 104:278-82. 15. koppula s, kopalli sr, sreemantula s. adaptogenic and nootropic activities of aqueous extracts of carum carvi linn (caraway) fruit: an experimental study in wistar rats. aust j med herb. 2009; 21:76-9. 16. haghparast a, shams j, khatibi a, alizaseh am, kamalinejad m. effects of the fruit essential oil of cuminum cyminum linn. (apiaceae) on acquisition and expression of morphine tolerance and dependence in mice. neuroscilett. 2008; 440:134-9. 17. khatibi a, haghparast a, shams j, dianati e, komaki a, kamalinejad m. effects of the fruit essential oil of cuminum cyminum l. on the acquisition and expression of morphine-induced conditioned place preference in mice. neuroscilett. 2008; 448:94-8. 18. gagandeep , dhanalakshmi s, mendiz e, rao ar, kale rk. chemopreventive effects of cuminum cyminum in chemically induced forestomach and uterine cervix tumors in murine model systems. nutr cancer. 2003; 47(2):171-80. 19. takayanagi t, ishikawa t, kitajima j. sesquiterpene lactone glucosides and alkyl glycosides from the fruit of cumin. phytochemistry. 2003; 63:479-84. 20. kitajima j, ishikawa t, fujimatu e, kondho k,takayanagi t. glycosides of 2-c-methyl-d-erythritol from the fruits of anise, coriander and cumin. phytochemistry. 2003; 62:115-20. 21. evans wc. trease and evans pharmacognosy. 14 ed. london: wb saunders company ltd; 1997. p. 267-8. 22. kafi m.cumin (cuminum cyminum) production and processing.mashhad: ferdowsi university of mashhad press; 2002. 23. sadeghi b, rashed-mohassel mh. effects of nitrogen and irrigation regimes on cumin (cuminum cyminum) production. scientific and industrial research center of iran, khorasan. 1990. 24. dehaghi ma, mollafilabi a. production technology for cumin (cuminum cyminum) on the basis of research findings. acta hort. (ishs). 2010; 853:83-92. 25. steinegger e, hansel r. lehrbuch der pharmacognosie auf phytochemischer grundlage. berlin: springer verlag; 1972. p. 348-82. 26. iranian herbal pharmacopoeia commission. iranian herbal pharmacopoeia. ministry of health, treatment and medical education, tehran. 2002:412-8. 27. borges p, pino j. the isolation of volatile oil from cumin seeds by steam distillation. food / nahrung. 1993; 37(2): 123-6. 28. technical information online. cumin processing. available from: http:// practicalaction.org/practicalanswers/product_info.php?products_id86 29. simon je, chadwick af, craker le. 1984. herbs:an indexed bibliography. 1971-1980 the scientific literature on selected herbs, and aromatic and medicinal plants of the temperate zone. 30. rekha s, singhal, pushpa r. kulkarni, dinanath v. rege. handbook of indices of food quality and authenticity. woodhead publishing, india, 1997. 31. jackson bp, snowdon dw. atlas of microscopy of medicinal plants culinary herbs and spices. delhi: cbs publishers; 2000. 32. wagner h, blant s. plant drug analysis. 2 nd ed. berlin: springer verlag; 1996. p. 192-3. process in manufacturing are considered. standardized seeds and essential oils are qualitatively optimized the products or preparations with reproducible content. determination of the physicochemical characteristics of the oil may establish by measurement of extraction yield, refractive index, density, carbonyl and steric indexes together with aldehyde, alcohol and acid contents. microscopic analyzing is very important in the products containing grinded seeds. in addition, thin layer chromatography may help to detect the pinenes and phellandrenes in the seeds as the main and characteristic monoterpenes. cumin aldehyde is not only the active constituent of the cumin seed and its oil but also sometimes added to the oil as a fraud which can diffculty detected by changing the refractive index. references 1. rechinger kh. flora iranica, apiaceae. academische druck-u- verganstalt. graz: austria. 1981; 162:140-2. 2. zargari a. medicinal plants. 5 th ed. vol. 2. tehran: tehran university publications; 2001. 3. mozaffarian v. a dictionary of iranian plant names. tehran: farhang moaser publisher; 1996. p. 168-9. 4. omidbaigi r. production and processing of medicinal plants. 5 th ed. tehran: astan quds publication; 2008. 5. ghahreman a. chromophytes of iran. 2 nd ed. tehran: tehran university publication; 1994. 6. anonymous.the wealth of india:a dictionary of indian raw material and industrial products. new delhi: csir; 1985. 7. johri rk. cuminum cyminum and carum carvi: an update. phcog rev. 2011; 5:63-72. 8. derakhshan s,sattari m,bigdeli m.effect of sub-inhibitory concentrations of cumin (cuminum cyminum l.) seed essential oil and alcoholic extract on the morphology, capsule expression and urease activity of klebsiella pneumoniae. int j antimicrob agents. 2008; 32:432-6. 9. derakhshan s, sattari m, bigdeli m. effect of cumin (cuminum cyminum l.) seed essential oil on biofilm formation and plasmid integrity by klebsiella pneumoniae. pharmacog mag. 2010; 6:57-61. 10. dorman hjd, deans sg. antimicrobial agents from plants: antibacterial activity of plant volatile oils. j appl microbiol. 2000; 88:308-16. 11. de martino l, de feo v, fratianni f, nazzaro f. chemistry, antioxidant, antibacterial and antifungal activities of volatile oils and their components. nat prod commu. 2009; 4:1741-50. 12. shirke ss, jadhav sr, jagtap ag. methanolic extract of cuminum cyminum inhibits ovariectomy-induced bone loss in rats. exp biol med. 2008; 233:1403-10. 13. mahesh cm, gowda kps, gupta ak. protective action of cuminum cyminum against gentamicin- induced nephrotoxicity. j pharmacy res. 2010; 3:753-7.r e v i e w a r t i c l e p h c o g j . 6 pharmacognosy journal  september 2011  vol 3  issue 25 *address of correspondence e-mail: samarpanbp@gmail.com doi: 10.5530/pj.2011.25.2 nepal is blessed with most varied and diverse soil and climate conditions suitable for the growth of veritable plant species. the indigenous people are well acquainted with the properties and uses of plants of their surroundings. until the middle of the 19 th century, plants were the main therapeutic agents used by humans. about 60% of the world population and 60-90% of the population of developing countries rely on traditional medicine [3] and about 85% of the traditional remedies for primary health care are derived from plants. [4] in nepal, at least 1,600 to 1,900 species of plants are commonly used in traditional medicinal practices. [5] traditional medicine in nepal is used extensively by majority of the population, and includes ayurveda, traditional chinese medicine (tcm), unani and various forms of indigenous medicine including tibetan amchi medicine. [6] traditional medicine in nepal comprises those practices based on beliefs that were in existence often for hundreds to thousands of years before the development and spread of modern medicine, and which are still in use today. in the past, many rural areas of nepal, traditional medicinal knowledge and practice were passed down entirely via oral tradition based on a lineage mode of transmission and personal experience. [7] more recently, however, knowledge transfer has also occurred through formally recognized medicinal plant diversity and their pharmacological aspects of nepal himalayas bhakta prasad gaire* 1 and lalita subedi 2 1 department of herbology, college of oriental medicine, kyung hee university, seoul, south korea. (samarpanbp@gmail.com) 2 department of pharmaceutical science, school of health and allied sciences, pokhara university, po box. 427, kaski, nepal (subedilali@gmail.com) a b s t r a c t background: the himalayan range of nepal is affuent with vast diversity of medicinal plants. due to insuffcient supplement of modern allopathic medicine and the traditional believe of ethnomedicinal therapy, still vast majority of nepalese people are dependent on indigenous use of medicinal plant. use of nepalese himalayan medicinal plants is not only limited to erogenous use of nepal himalaya but also regarded as chief ingredients in eastern medicinal system including ayurveda of indian subcontinent, traditional chinese medicine, korean oriental medicine, etc. but due to the lack of effcient pharmacological investigation, himalayan plant diversity is still limited to their ethnomedicinal uses. vigorous pharmacological investigation is mandatory to explore their therapeutic potential. conclusion: here in this review; based on latest published pharmacological research articles, we tried to explore pharmacological aspects of major himalayanmedicinal plant of nepal for thefrst time. there is the current need to investigate further pharmacological potency of these medicinal plants in order to explore their therapeutic potential. key words: ethnomedicine, indigenous use, himalayas background nepal, the kingdom of himalaya, is small, landlocked country situated between india and china. nepal lies on southern slope of central himalaya and occupies a total area of 147181 sq. km between the latitude of 26 22’ and 30 27’ n and the longitude of 80 40’ and 88 12’ e. the average length of the country is 885 km from east to west and width varies 145 km to 241 km from north to south. about 86% of the total land area is covered by hills and high mountains and remaining 14% is covered by fat lands of terai. based on wild altitudal variation (60-8848 m), the climate is broadly classifed into cold arctic/nival (above 3000 m), cold temperate (2000-3000 m), warm temperate (1500-2000 m), subtropical (1000-1500 m) and tropical (below 1000 m). according to the physiological region, nepal is divided into 7 regions including terai, siwaliks, mahabharat lekh, midhills, himalayas, inner himalayas and tibetan marginal mountain range. [1,2]pharmacognosy journal  september 2011  vol 3  issue 25 7 gaire and subedi: medicinal plant diversity of nepal himalayas using these systems was accessed by nepali vaidhyas and kabirajs as early as about 879 ad. [10] therefore, the ayurvedic physicians were incorporating medicinal plants in traditional ayurvedic formulations from early on and the ayurvedic system is reputed all over the indian subcontinent since time immemorial. [11,12] almost of the herbs of nepal himalayas are considered to contain medicinal properties. kunwar and bussmann 2008 reported that 56% of higher plants were ethno botanically important, and 54% were used as ethnomedicine in the nepal himalayas. the topographical characteristics of the himalayas have resulted in a variety of ecological niches that host diverse medicinal plants. it has been estimated that the himalayan region harbors over 10,000 species of medicinal and aromatic plants, supporting the livelihoods of about 600 million people living in the area. [13-15] this reviewwas carried out by dividing nepal himalaya into 3 different region as west nepal (80e to 83e), central nepal (83e to 86e) and east nepal (86e to 88e), according to kunwar and bussmann, 2008. so many researches are carried regarding to the indigenous use and ethnomedicinal potential of nepalese medicinal plants till now. however, according to the knowledge of author, regarding the pharmacological aspects very few investigations have been carried out. here, in this review article we tried to summarize the pharmacological aspects of major nepalese himalayan medicinal plants [table]. school level education. medicinal plants play vital roles in the nepalese livelihood and the use of medicinal plants is frequent in several nepalese regions. the total population of nepal is 23.1 million and about 90% of the nepalese people reside in rural areas where access to government health care facilities is lacking. it is estimated that there is one physician for more than 30,000 people whereas there is one healer for fewer than 100 people in nepal. [8] nepal is a natural storehouse of medicinal plants. each year thousands of tons of raw material are exported, mostly to india, but also to other asian, european and american countries. the government of nepal aims to promote medicinal plant use and conservation programmes for livelihood improvement and poverty alleviation through various policies. [9] the himalayan plant diversity plays pivotal role to fulfll the medicinal demand of nepalese society. the earliest record of medicinal plant use in the himalayas is found in the rigveda (4500 bc and 1600 bc), is supposed to be the oldest repository of human knowledge and describes 67 plants. after the rigveda, ayurveda (the foundation of science of life and the art of healing of hindu culture) describes the medicinal importance of 1200 plants. the charak or charaka samhita (900 bc) and susruta samhita (500 bc) enumerate the art of surgery, therapeutics and medicines in detail on the basis of atharvaveda. the knowledge of table: list of medicinal plants of nepal himalayaas based on their latest pharmacological investigation and indigenous use (w western, c central, e eastern region) s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 1. abies spectabilis (pinaceae) gobre salla (himalayan silver fir) leaves are sniffed for cough and cold pentene of abies leaves is anti-inflammatory and antidepressant. [19] w 2. acacia catechu (mimosaceae) khair (cutch tree) wood is used as local tea for cough and cold cyanidanol, an active ingredient of acacia catechu, is claimed to be effective for treating liver diseases. [20] catechu has hypoglycemic, antipyretic and digestive properties. catechuic acid is valued for expectoration for chest infection. [21] w, c 3. aconitum ferox (ranunculaceae) bikh (himalayan monkshood) root paste is taken for joint pain. alkaloid extract possess anti-inflammatory properties. [22] e, c, w 4. aconitum heterophyllum (ranunculaceae) bish (aconites) rhizome is dried up and taken to relieve body-ache, fever, cold, cough, nose discharge etc. ethanolic root extract of aconitum heterophyllum has anti-inflammatory activity against cotton pellet-induced granuloma in rats. [23] e, c, w 5. aesculus indica (sapindaceae) karu (indian horse chestnut) seed oil is valued for joint pain and skin problems plant is used for delaying hypersensitivity. aescin is cardiostimulant and anti-inflammatory. [24] w continuedgaire and subedi: medicinal plant diversity of nepal himalayas 8 pharmacognosy journal  september 2011  vol 3  issue 25 table: continued s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 6. ageratum conyzoides (asteraceae) gnadhe jhar (ageratum) leaf juice is applied externally to heal wounds. decoction of herb is also given to cure stomach ailments such as diarrhea, dysentery and intestinal colic with flatulence hypoglycemic and antihyperglycemic activity on rat [25] also shows anticancer and antiadrenal activity. [26] e, c, w 7. allium cepa (alliaceae) pyaj/ odal (onion) eating raw bulbs reduces fever acting as cooling agent. it prevents cadmium induced renal dysfunction [27] and has hypoglycaemic effect against type 1 and 2 diabetes mellitus. [28] w, c, e 8. amaranthus spinosus (amaranthaceae) bagani dhap (prickly amaranth) root paste is applied on cuts and wounds. contains several chemical compounds, including tannins (coagulant), steroids (muscle building), flavonoids (antimicrobial), and volatile oils (antiseptic). [29] e, c, w 9. andrographis paniculata (acanthaceae) kalmegh (kariyat) raw plant root juice is considered as antipyretic and effective in infections plant is immunostimulant, anti-inflammatory, antibacterial, analgesic and antiprotozoal. [30] w, c 10. anisomeles indica (lamiacae) ratocharpate (indian catmint) leaf extract is useful for urinary complaints ovatodiolide and pedallitin of anisomeles indica is good anti-inflammatory. pre-flowering plant water extract is analgesic. ethanolic leaf extract is strong antiviral and anti hiv potential. [31] w 11. artemisia indica (asteraceae) titepati (asian mugwort) leaf paste is applied on cuts and wounds. antimicrobial properties and in vitro antimalarial property. [32] e, c, w 12. artemisia vulgaris (asteraceae) tite pati (fleabane) crushed leaves inserted in the nose stop bleeding. water, mixed with crushed leaves, in taking bath prevents and cures allergy. raw leaves chewed are good for mouth ulcer; also find uses in rituals. has antispasmodic and bronchodilator activity in guinea pigs. [33] e, c, w 13. asparagus racemosus (asparagaceae) kurilo (shatavari) tuber paste is used for fever, stomach ache, and diarrhoea ethanol and aqueous extracts from the tubers exhibit significant antidiarrheic activity. [34] e, c, w 14. bauhinia variegate (caesalpiniaceae) koiralo (orchid tree) flower and floral buds are eaten regularly to cure leucorrhoea and mumps. methanol extract of b. variegata bark showed the most remarkable activity as antimicrobial and anticancer. [35] w 15. berberis asiatica (berberidaceae) chutro (barberry) cambium paste is used for rheumatism and pith paste is used for eye problems. widespread use as an extract in eye drops for conjunctivitis. effective as an antipyretic, anesthetic, and antihypertensive. [36] e, c 16. bergenia ciliata (saxifragaceae) pakhanved (frilly bergenia) latex is effective in diarrhea, dysentery, stomachache aqueous and methanolic extract of bergenia ciliata shows the cytoprotective activity. [37] w 17. bischofia javanica (euphorbiaceae) kainjalo (java sedar) chewing raw leaves treat sore throat. drinking bark juices cure diarrhea. betulinic acid derivatives from the bark has the dna topoisomerase ii inhibitory activity. [38] e, c, w 18. cannabis sativa (cannabaceae) ganja (marijuana) plant paste is taken for stomach problems diuretic, anti-emetic, anti-epileptic, painkilling, anti-inflammatory, and antipyretic properties. [39] e, c, wpharmacognosy journal  september 2011  vol 3  issue 25 9 gaire and subedi: medicinal plant diversity of nepal himalayas s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 19. carum carvi (apiaceae) jangali jira (caraway) fruit is stomachic and carminative. seeds are used for their cooling effect. aqueous extract of carum carvi (black zeera) seeds has the renal protective activity in streptozotocin induced diabetic nephropathy in rodents. [40] w, c 20. cedrela toona (meliaceae) tuni (indian mahogany) bark is crushed and the paste is applied to cure ulcers. flower is chewed to promote menstrual discharge in females. has antiproliferative and antitumorogenic activity. [41] e, c 21. celastrus paniculatus (celastraceae) malkauna, kujur (staff tree) seed paste is applied in case of skin irritation/allergy; good for gout. has potent relaxant activity in human ileum. [42] c, e 22. cinnamomum tamala (lauraceae) dalchini,tejpat (malabathrum) leaves are rubbed on the body surface of the scabies affected person. has immunomodulatory activity on rat. [43] c, e, w 23. cissampelos pareira (menispermaceae) batulpate (abuta) plant extract is given to treat diarrhea, dysentery, indigestion and urinary disorders. root is used as antidote. roots are proven to have antineoplastic and antiarthritic activiry. [44] e, c. w 24. citrus medica (rutaceae) bimiro (citrus) chewing dried fruit peel prevents dysentery. fruit is good for indigestion. roots are tied together along with a copper coin and placed in women’s naval during child birth, which is believed to expedite the expulsion of the placenta after child birth. shows good in-vitro inhibitory activity against diabetes mellitus and alzheimer’s disease. [45] e, c, w 25. clematis buchananiana (ranunculaceae) abijalo (clematis) juice extracted by crushing fresh roots is inhaled to treat sinusitis and headache. aquous extracts of clematis buchananiana leaf anti-inflammatory, antinociceptive and antipyretic properties in rats. [46] e, c, w 26. cordyceps sinensis (clavicipitaceae) yarsagumba (cordyceps) whole plant juice is taken as tonic. largely recognized as inducing sexual power and validity. [47] w, c 27. costus speciosus (costaceae) betlauri (wild ginger) rhizome mixed with sugar is used to treat venereal diseases. juice taken before breakfast cures urinary tract infections. eremanthin from costus speciosus shows antidiabetic and antilipidemic effect in stz-induced diabetic rats. [48] e, c 28. curcuma aromatica (zingiberaceae) ban haledo (aromatic turmeric) rhizome powder taken with water relieves nausea, stomachache and expels gas. curcuma aromatica oil has the antineoplastic activity. [49] e 29. curcuma longa (zingiberaceae) besar (turmeric) drinking water boiled with root cures throat pain, cold, cough and fever. more than thousands of researches have been carried out on curcuma longa. recent interests are on anticancer [50] anti-inflammatory [51] and antioxidant [52] activity w, c, e 30. cynodon dactylon (poaceae) dubo (dog’s tooth) crushed root juice is taken to relieve piles. root paste applied heals cuts and wounds. boiled leaf and root juice help in treating diarrhea and dysentery. hydrochloric extract of rhizome shows protective effect against heart failure in rat. [53] e, c 31. dioscorea alata (dioscoreaceae) ghar tarul (winged yam) rhizome is eaten raw to relieve throat pain. this has found to effectively reduced blood pressure of spontaneously hypertensive rat. [54] e, c 32. drymaria cordata (caryophyllaceae) chirbire jhar the plant is warmed while wrapped in a cloth and emanating vapor inhaled in the case of sinusitis, nose blockade and headache.to relive sore throat pain and fever, the plant either eaten raw or cooked. hydroethanolic extract shows anxiolytic effect in animal model. [55] e continuedgaire and subedi: medicinal plant diversity of nepal himalayas 10 pharmacognosy journal  september 2011  vol 3  issue 25 table: continued s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 33. drynaria propinqua (drynariaceae) kammari (dryndria) plant is effective in fever and headache. propinqualin, 4-o-beta-d-glucopyranosyl caffeic acid, beta-sitosterol-3-o-beta-d-glucopyranoside  has been isolated from this plant. [56] c, e 34. engelhardia spicata (juglandaceae) mahuwa (engelhardia) flower juice is drunk for abdominal pain. engelhardtione possesses antituberculer activities. [57] w, c, e 35. entada rheedii (fabaceae) prami (african dream herb) body pain, musculo-skeletal problems. triterpenes isolated from seed of entada rheedii has antiproliferative and antioxidant activity. [58] c 36. ephedra gerardiana (ephedraceae) somlata (ephedra) whole plant is used for respiratory problems. ephedrine from ephedra gerardiana stimulates the respiratory centers, uterus, dilates the bronchi and pupils, contracts the intestines and raises blood sugar. [59] c, e, w 37. equisetum diffusum (equisetaceae) ankhle jhar (horsetail) plant stem juice is given for gonorrhea. methanolic plant extract shows good free radical scavenging activity. [60] w, c, e 38. eupatorium adenophorum (asteraceae) banmara (sticky snakeroot) leaf juice is applied on cuts and wounds. methanolic leaf extracts shows the analgesic effect. [61] e, c, w 39. ficus auriculata (moraceae) timila (roxburgh fig) stem juice is considered effective against diarrhea and fruits are used in dysentery. tannins of the bark extract may reveal anti-inflammatory and analgesic activities. [62] bark extract shows potential antioxidant activity. [63] w, c, e 40. ficus hirta (moraceae) khasreto (ficus) root decoction treats food poisoning. aqueous extracts from ficus hirta have hepatoprotective activity against n, n-dimethylformamide induced acute liver injury in mice. [64] e 41. fraxinus floribunda (oleaceae) lankuree (himalayan ash) bark infusion is used for body pain. anti-inflammatory, anti-oxidative and skin regenerating activities. [65] c 42. fritillaria cirrhosa (liliaceae) kakoli (fertillaria) plant juice is taken for stomach disorders plant contains steroidal alkaloids effective against stomach disorders. [66] e, c 43. helianthus annus (asteraceae) suryamukhi (sunflower) root decoction as a gargle relieves toothache; dried flower chewed cures ulcers, fever, cough and cold. leaves crushed and mixed with water and taken bath cures allergy and skin diseases. terponoids in methanolic and aquous extract of helianthus annus shows anti-inflammatory activity in rat. [67] e, c 44. hibiscus esculentus (malvaceae) ramtoriya (okra) fruit mucilage acts as soothing agent on cuts. methanol extract of hibiscus esculentus seeds shows antihypoxic and antioxidant activity in male mice. [68] e 45. hippophae salicifolia (elaeagnaceae) dale chuk (sea buckthorn) fruit juice is taken for cough, diarrhea, and menstrual disorder. contains high levels of flavonoids (with antimicrobial properties and effectiveness against menopausal symptoms), carotenoids and vitamin c. [69] w, c 46. hippophae tibetana (elaeagnaceae) bhui chuk, (tibetean sea buckthorn) fruit juice is taken for stomach disorders. contains high levels of flavonoids (antimicrobial), carotenoids and vitamin c. [69] c, w 47. hordeum vulgare (poaceae) jau (barley) gruel is made by the powdered grains and given in case of painful indigestion. barley water with honey is prescribed in bronchial coughs. aqueous methanolic extract of this plant shows hepatoprotective activity against acetaminophen induced liver damage in rats. [70] e, cpharmacognosy journal  september 2011  vol 3  issue 25 11 gaire and subedi: medicinal plant diversity of nepal himalayas s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 48. hydrocotyle asiatica (mackinlayaceae) ghortapre (pennywort) fresh plant parts crushed and ingested orally cure sores of throat and lungs. leaf juice is used as eye drops to cure eye infection. dressing with leaf paste reduces swelling or and applied in wounds. juice of shoots treats gastritis and constipation. it has neuroprotective, anti-allergic, anti-pruritic, and anti-inflammatory activities in animal models. [71] e, c, w 49. lantana camara (verbenaceae) masino kada, sitaji phul (spanish flag) the juice of crushed leaves is applied to the fresh cut and wounds to heal. crushed leaves are tied over the sprain to relieve pain. ethanolic extract of leaves and roots shows the antibacterial activity against both gram positive and gram negative bacteria. [72] e 50. lichen species (parmeliaceae) jhau (lichen) lichen extract and decoction is applied to treat moles. parmelia species are antimicrobial [73] and also used to treat warts and cranial diseases. [74] w, c, e 51. lindera neesiana (lauraceae) pahenlo khapate (spicewood) fruit juice taken for diarrhea. essential oil extracted from fruits possess significant antimicrobial activity. [75] e, c 52. lobelia pyramidalis (campanulaceae) aklebir (lobelia) juice of leaves and flowers is rubbed on body parts during body ache. lobeline, the active constituent, may cause nausea, vomiting and diarrhea. [76] w 53. lycopersicon esculentum (solanaceae) rambheda (tomato) raw fruit is taken during indigestion and to prevent bleeding from the gums. it has 5-alpha-reductase enzymatic activity which enhances the formation of testosterone. [77] c, e, w 54. lycopodium clavatum (lycopodiaceae) supari jhar (groundpine) pollen paste is used on cuts and wounds. contains anti-inflammatory alkaloids types of compounds. [78] c, e 55. mentha arvensis (lamiaceae) pudina (mint) raw leaves chewed help to check stomach related disorders: gastritis, acidity, indigestion etc., also used to flavor chutney. various extracts of mentha arvensis clearly shows a protective effect against acid secretion and gastric ulcers in ibuprofen plus pyloric ligation, 0.6 mol/l hcl induced and 90% ethanol-induced ulcer models. [79] w, c, e 56. mucuna macrocarpa (fabaceae) baldengra (mucuna) seed powder taken with water helps remove round worm from stomach. crude methanolic extract of stem have in vitro and in vivo apoptosis-inducing antileukemic effects. [80] e 57. musa paradisiacal (musaceae) kera (banana) person suffering from fever is advised to drink sap released from the plant directly. crude aqueous methanolic extract of leaves shows in vitro anthelmintic effect. [81] e,c,w 58. mussaenda frondosa (rubiaceae) asari (mussaenda) whole plant is boiled and decoction is given to treat fever, asthma and cough. alcoholic and aqueous extract of this plant shows in vitro antioxidant activity. [82] e 59. myrica esculenta (myricaceae) kafal (box myrtle bay berry) fruits are eaten for dysentery and bark decoction is given for bronchitis. crude extract of stem bark shows anti-allergic activity on mice. [83] w, c, e 60. nardostachys grandiflora (valerinaceae) jatamansi (jatamansi) whole plant juice is taken to treat headache and high altitude sickness. ethanol extract from roots showed anticonvulsant activity and are a nervous system stimulant. [84] c, e 61. oroxylum indicum (bignoniaceae) tatelo (indian trumpet) bark and seeds are powdered and mixed with water, and strained; the mixture is fed to patients suffering from high fever or pneumonia, which believed to restore health or brings down fever. unbroken pod is also used in rituals. methanolic extract of root, bark, stem and leaves have the antioxidant activity. [85] e, c continuedgaire and subedi: medicinal plant diversity of nepal himalayas 12 pharmacognosy journal  september 2011  vol 3  issue 25 table: continued s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 62. oxalis corniculata (oxalidaceae) chari amilo (creeping woodsorrell) whole plant is chewed raw and the juice acts as an appetizer; also checks boil. fresh plant decoction taken treats dysentery. fruit is consumed to lessen throat pain. methanol extract of oxalis corniculata shows in-vitro antioxidant and anti-inflammatory activity. [86] e, c, w, 63. paederia scandens (rubiaceae) pat biree (sewer vines) dried fruit is powdered and applied over teeth to relieve tooth ache and prevent tooth decay. several pharmacological activities are reported. most recent are xanthine oxidase inhibitory and uricosuric activity. [87] e, c 64. paris polyphylla (trilliaceae) satuwa (himalayan paris) root paste is taken for fever, vomiting and worms a methanolic extract is gastro protective. also possesses anthelmintic properties. [88] w, c 65. phyllanthus emblica (phyllanthaceae) amala (indian gooseberry) as a tonic to build up lost vitality and vigor and rassayana in ayurveda. also considered as a source of vitamin and amino acid. it has so many action reported included antiviral, antioxidant, etc. recent research shows the antiplasmodic and cytotoxic effect of water extract. [89] c, e, w 66. picrorhiza kurroa (scrophulariaceae) kutki (picrorhiza) dried rhizome is boiled in water and taken to cure fever, cough, etc methanolic and aqueous extract of rhizome has potent antioxidant and antineoplastic activity. [90] e 67. piper longum (piperaceae) pippali (long pepper) dried seed powder paste is applied to reduce sprains; the powdered roots are given to treat cold and cough. it has insecticidal and acaricidal, antifungal, antiameobic, antimicrobial, antiasthmatic, antidiabetic, analgesic, anti- inflammatory, hypocholesteromic, antioxidant, anticancer, immunomodulatory, antidepressant, antiulcer, hepatoprotective effect. [91] e 68. plantago erosa (plantaginaceae) isabgol jhar (greater plantain) leaf paste is applied to heal wounds, cuts, bruises, insect bites, poison-ivy rashes, minor sores and snakebite. seed powder is with water treats diarrhea and dysentery. methanolic extract shows anti inflammatory activity against carageenan induced paw edema in rat and mice. [92] e, c, w 69. podophyllum hexandrum (berberidaceae) laghupatra (himalayan may apple) root juice is taken for liver complaints ethyl acetate extract of podophyllum hexandrum rhizome has antioxidant and protective effect on carbon tetrachloride induced rat liver injury. [93] w, c, e 70. psidium guajava (myrtaceae) amba (guava) young leaves and tender shoots taken raw cure mouth ulcers, sore throat, cough, toothache. drinking bark powder mixed in hot water is best local remedy for dysentery with blood in stool; fruits are edible. ethyl acetate fraction of psidium guajava leaf extract shows antioxidant and antiglycative potential in streptozotocin-induced diabetic rats. [94] e, c, w 71. pteris biaurita (pteridaceae) gulmohar (fern) mashed petiole extract applied on the cuts and wounds stop bleeding and infections alcoholic extracts has the antimicrobial activity. [95] e, c 72. rhododendron arboreum (ericaceae) lali guras (tree rhododendron) dried flowers crushed and mixed with water stop excessive bleeding in female. fresh leaves chewed to cure dysentery. flower juice has the hypolipidemic effect in experimentally induced hypercholestermic rabbits. [96] e 73. rhododendron campanulatum (ericaceae) guras (bell rhododendron) leaves are chewed and the juice from the crushed leaves relieves cough. oleamane, the active triterpenoid, has antibacterial and immunomodulatory activities. [97] e, cpharmacognosy journal  september 2011  vol 3  issue 25 13 gaire and subedi: medicinal plant diversity of nepal himalayas s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 74. rhus semialata (anacardiaceae) arkhar (sweet sumach) sour juice of fruits is boiled with water and raw egg, treats diarrhea and dysentery. it is also used as food preservative. rhus semialata fruit extract has the antidiarrheic activity in rats. [98] e 75. rauvolfia serpentine (apocynaceae) sarpagandha (snake root) use to lower high blood pressure. reserpine, the active alkaloid, produced a dose-dependent depression of the central nervous system. [99] c, e, w 76. rubia cordifolia (rubiaceae) mangito (indian madder) root decoction with water is given to cure urinary infection; paste is used as an ointment to skin diseases. root is also used to make dyes. mollugin, a bioactive phytochemical isolated from rubia cordifolia l, exhibits antimutagenic, antitumor, antiviral, and inhibitory activity in arachidonic acid- and collagen-induced platelet aggregation. it also has neuroprotective and anti-inflammatory effects in mouse hippocampal and microglial cells. [100] e, c, w 77. rubia manjith (rubiaceae) majitho (manjith) root paste is applied over scabies and other skin diseases anti-proliferative against epidermal keratinocytes and also has antiseptic properties. [101] c, e, w 78. rubus ellipticus (rosaceae) ainselu (yellow himalayan raspberry) young shoot is chewed raw to relieve sudden stomach pain. root decoction given to the children to get rid of stomach warm. root paste is applied on forehead during severe headache; fruit is edible. triterpenoid saponins from roots of rubus ellipticus demonstrated inhibitory activities against alpha-glucosidase. [102] e, c 79. rumex nepalensis (polygonaceae) halhale sag (nepal duck) the root is purgative. decoction of the root is applied to dislocated bones. a paste of the root is applied to swollen gums. the leaves are used in the treatment of colic and headaches. root extracts of rumex nepalensis has anti-inflammatory, cycloxygenase (cox)-2, cox-1 inhibitory, and free radical scavenging effects. [103] c, w 80. sapindus mukorossi (sapindaceae) ritho (reetha) scalp is washed with fruit to remove dandruff and lice. saponins from sapindus mukorossi has inhibitory effect on bacterial, fungal and viral genital pathogens. [104] e, c, w 81. schima wallichii (theaceae) sule-chilauni (schima) bark is rubbed on the caterpillar infected portion removes its hair. polyphenolic enriched extract of schima wallichii bark shows anti-inflammatory activity  human peripheral blood mononuclear cells (pbmcs) and in vivo by carrageenan- induced paw edema assay (acute study) and cotton pallet granuloma assay (chronic study). [105] e, c, w 82. schleichera oleosa (sapindaceae) kusum (kusum tree) fruits are eaten as an anthelmintic extracts of bark of schleichera oleosa has cytotoxic and hydroxyl radical-scavenging activities. [106] c, w 83. semecarpus anacardium (anacardiaceae) bhalaayo (marking nut) root paste is applied externally on the affected portion cures skin diseases. decoction of the bark is given to the animals to treat worms. has hypolipidemic activity in streptozocin induced diabetic rats. [107] e, c 84. skimmia anquetilia (rutaceae) narpati (skimmia) leaf infusion is taken for headache and for freshness linalool, from this plant, possess anxiolytic effect. [108] w 85. smilax aspera (smilacaceae) kukurdaino (birdweed) root decoction is used for venereal disease stem juice is used for dropsy and gout. rutinoside has cancer inhibitory effect. [109] w, c continuedgaire and subedi: medicinal plant diversity of nepal himalayas 14 pharmacognosy journal  september 2011  vol 3  issue 25 table: continued s. n. scientific name (family) [16,17] vernacular name (english name) [16,17] indigenous/local use phytochemical/ pharmacological properties (literature review) distribution [16,18] 86. solena heterophylla (cucurbitaceae) bankakri (creeping cucumber) fruits are eaten for common cold and pneumonia of child plant extract is hepatoprotective and plant coumarin and flavonoids inhibit platelet aggregation. [110] w 87. spermadictyon suaveolens (rubiaceae) ban chanp (forest champa) root paste is applied externally to relieve joint pain. ethanolic extract of bark has anti-inflammatory activity on rats. [111] methanol extract of stem bark has hepatoprotective activity on rats. [112] e 88. spondias pinnata (anacardiaceae) amaro (wild mango) plant latex is applied for wounds and cuts. flavonoids of the plant have been known to inhibit intestinal motility and hydro electrolytic secretion, which are known to be altered for diarrheal conditions. [113] w, c, e 89. taxus wallichiana (taxaceae) lauthsalla, barme salla (himalayan yew) respiratory problems. leaf juice is used for cancer and bronchitis. taxol isolated from the bark of this plant shows the in-vitro, in-vivo anticancer activity. it also has antifungal, antiviral anticonvulsant, analgesic, and antipyretic and tumor growth inhibitory activity. [114-116] c 90. terminalia bellirica (combretaceae) barro (baheda) fruit is used as laxative, in headache, leucorrhoea, liver diseases to gastro-intestinal complaints aqueous extract of terminalia bellirica stimulates the secretion and action of insulin and inhibits starch digestion and protein glycation in vitro. [117] c, e, w 91. terminalia chebula (combretaceae) harro (chebulic myrobalan) fruit is used for abdominal problem, headache, bronchitis, and several ayurvedic formulation hydro alcoholic extract of terminalia chebula fruit shows antiulcerogenic activity in rats. [118] c, e , w 92. valeriana jatamansi (valerianaceae) jatamansi (valerian) cuts and wounds, cough and cold dried rhizome extract partially reverses the liver cirrhosis and tissue hyper proliferative response in rats. [119] c, e, w 93. zanthoxyllum alatum (rutaceae) timur (prickly ash, zanthoxylum) branchlet used as toothbrush to relieve toothache. berries taken to cure stomach ache and toothache. berries are crushed and rubbed on the leg which acts as leech guard. crude extract of zanthoxyllum alatum has the spasmolytic activity in gut, airways and cardiovascular diseases. [120] e, c discussion though considerable advances are made in the pharmaceutical sciences, especially in synthetic chemistry, plants and their derivatives continue to maintain their signifcance in medicines. increased interest in natural drugs than synthetic are because of a high degree of adverse side effects caused by the latter. nowadays natural medicines are gaining prominence, because they are economical, easily available and relatively free from side effects. it is evident from the present scenario that herbal cure is gaining world wide acceptance and has emphasized on modern scientifc exploration, extraction and evaluation of foil medicines from plants. these are either used directly as a plant extract or modifed through further synthesis. [121] the himalayas represent the largest mountain chain in the world, and is famous for its rich plant diversity and varied ecosystem, containing large number of plants. the use of plants in curing and healing is as old as man himself. plants containing benefcial and medicinal properties have been known and used in some form or other by primitive people. many plants which are found commonly and are mentioned in above texts are traditional medicine have not been investigated thoroughly. it is necessary to conduct systematic evaluation, standardization, documentation and patenting of these plants. targeted based studies with concentration on mechanism of action, lethal dose/effective dose and bioavailability mechanisms need to be conducted in future to explore scientifcally the hidden potential of these plants so that the ill community gets maximum benefts frompharmacognosy journal  september 2011  vol 3  issue 25 15 gaire and subedi: medicinal plant diversity of nepal himalayas traditional system of medicine. [122] biodiversity of nepal-himalayas is natural wealth and its conservation is important for economic, ecological, scientifc and ethical reasons. 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45:285-90. 96. murty d, rajesh e, raghava d, raghavantv, surulivel mk. hypolipidemic effect of arborium plus in experimentally induced hypercholestermic rabbits.yakugaku zasshi 2010; 130:841-6. 97. tantry ma, khan r, akbar s, dar ar, shawl as, alam ms. an unusual bioactive oleanane triterpenoid from rhododendron campanulatum d. don. chinese chemical letters 2010. article in press. 98. bose sk, dewanjee s, sen gupta a, samanta kc, kundu m, mandal sc. in vivo evaluation of antidiarrhoeal activity of rhus semialata fruit extract in rats. afr j tradit complement altern med 2007; 5:97-102. 99. nammi s, boini km, koppula s, sreemantula s. reserpine-induced central effects: pharmacological evidence for the lack of central effects of reserpine methiodide. can j physiol pharmacol 2005; 83:509-15. 100. jeong gs, lee ds, kim dc, jahng y, son jk, lee sh, kim yc. neuroprotective and anti-inflammatory effects of mollugin via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells. eur j pharmacol 2011; 654:226-34. 101. baral sr, kurmi pp. a compendium of medicinal plants in nepal kathmandu, 2006. 102. li w, fu h, bai h, sasaki t, kato h, koike k. triterpenoid saponins from rubus ellipticus var. obcordatus. j nat prod 2009; 72:1755-60. 103. gautam r, karkhile kv, bhutani kk, jachak sm. anti-inflammatory, cyclooxygenase (cox)-2, cox-1 inhibitory and free radical scavenging effects of rumex nepalensis. planta med 2010; 76:1564-9. 104. talwar gp, dar sa, rai mk, reddy kv, mitra d, kulkarni sv, doncel gf, buck cb, schiller jt, muralidhar s, bala m, agrawal ss, bansal k, verma jk. a novel polyherbal microbicide with inhibitory effect ono r i g i n a l a r t i c l e p h c o g j . 18 pharmacognosy journal  september 2011  vol 3  issue 25 *address for correspondence: dr. alvaro vázquez, cátedra de farmacognosia y productos naturales, facultad de quimica, cc 1157, montevideo 11800, uruguay. e-mail: avazquez@fq.edu.uy doi: 10.5530/pj.2011.25.3 where there is dependence on traditional medicine for a variety of diseases. [4] this wealth of experience and information about medicinal plants as well as the current problems associated with the use of antibiotics has renewed the interest in plants with antimicrobial properties. [5-11] in previous work we undertook the biological and chemical prospection of the gallery forest of the northern uruguay river basin. [12] plants were selected after an exhaustive review of the available literature according at its ethnopharmacological use and submitted to antimicrobial assays and phytochemical characterization. [13] among them, myrcianthes cisplatensis extracts showed striking activity with a broad spectrum of activity that deserves further investigation. many species belonging to the myrtaceae family(that comprises, eucalyptus, psidium and syzygium genus) have been studied for their antimicrobial properties. [14-16] myrcianthes cisplatensis (cambess.) o. berg (myrtaceae) grows freely in uruguay especially in the banks of rivers and streams. it is locally known as guayabo colorado and its fruits are edible and used for the preparation of marmalades. in this work we present the results of the bioguided isolation and structural elucidation of the main active compound as well as its antistaphylococcal activity. identification of a bioactive compound from myrcianthes cysplatensis eugenio damico a# , stephanie barneche a , maria pia cerdeiras b and alvaro vázquez a * a cátedra de farmacognosia y productos naturales, facultad de química, udelar, montevideo, uruguay. b cátedra de microbiología, facultad de química, udelar, montevideo, uruguay. # permanent address: dipartimento di scienze farmaceutiche, universita di bologna, bologna, italy. a b s t r a c t myrcianthes cisplatensis (cambess.) o. berg (myrtaceae) grows freely in uruguay especially in the banks of rivers and streams. it is locally known as guayabo colorado and its fruits are edible and used for the preparation of marmalades. in this work we present the results of the bioguided isolation and structural elucidation of the main active compound as well as its antibacterial activity. through repeated chromatography a pure compound could be obtained. the compound was studied by different spectroscopic techniques and could be unambiguously identifed as α-methyl-1-(2, 4, 6,-trimethoxyphenyl)-1-propanone. when assaying for antistaphylococcal activity, it showed mics of 62.5 µg/ml for the sensible strain (atcc 6538p) and 250 µg/ml for the multirresistant ones (atcc 43300 and atcc 700699). this shows that the bioguided fractionation is appropriate even when not very active compounds are isolated key words: myrcianthes cysplatensis, staphylococcus aureus. introduction in spite of the great advances in chemotherapeutics, infectious diseases are still one of the leading causes of death in the world. the world health organization [1] states that infectious and parasitic diseases account for nearly 11 million among the 57 million total deaths in 2003. although there seems to be a great array of antibacterial and antifungal drugs in clinical use, the appearance of resistant organisms makes them sometimes ineffective or lead to recurrence as stated by theworldhealthorganization. [2] amongst some of the most problematic clinically relevant pathogens at present, methicillin-resistant staphylococcus aureus (mrsa) ranks as one of the most diffcult bacteria to treat. [3] the use of higher plants and preparations made from them to treat infections is an age-old practice in a large part of the world population, especially in developing countries,pharmacognosy journal  september 2011  vol 3  issue 25 19 damico, et al.: identification of a bioactive compound from myrcianthes cysplatensis 1.13 (6h, d, 8hz) α-me and 3-me, 3.03 (1h, m) h-2, 3.78 (6h, s) 2 and 6ome, 3.84 (3h, s) 4ome, 6.12 (2h, s) h-3and h-5. 13 c nmr (cdcl 3 ): 18.0 c-1 and α-me, 41.7 c-2, 55.4 4-ome, 55.8 2and 6-ome, 90.0 c-3and c-5, 113.0 c-1, 158.0 c2 and c-6, 161.0 c4, 208.0 c-1. antibacterial analysis minimum inhibitory concentration (mic) was determined by the microdilution technique according to clinical and laboratory standards institute (clsi, 2006) using sensitive (atcc 6538p) and resistant (atcc 43300 and atcc 700699) staphylococcus aureus strains. results and discussion repeated column chromatography of the dichloromethane extract of m. cisplatensis leaves gave a compound (1) that showed only a spot in tlc and one peak in gc. the esi mass spectrum of 1 showed ions at m/z 239.2879 and 261.2694 ([mh] and [mna] , respectively) indicating a molecular formula c 13 h 18 o 4 (needs 238,2801). in the gc-ms spectra a prominent ion at m/z 195 is shown along with the 238 ion. the uv spectrum showed a maximum absorption at λ 279 nm indicating the presence of an aromatic group. the 1 h nmr spectra showed few signals, with a doublet (6h) at 1.13 ppm, a septuplet at 3.03 ppm and singlets at 3.78, 3.84 and 6.12 ppm. in the 13 c nmr spectra 9 signals could be identifed corresponding to 5 methyl, 3 methine and 5 quaternary carbons according to dept. using a combination of 2d (h,h cosy, hsqc, hmbc) experiments all the signals can be assigned. especially useful were the correlations between the protons at δ h 1.13 (d, 6h) with the signal at 3.03 and carbons at δ c 18.0 (via hsqc) and 41.7 (via hmbc) defning a isopropyl group that in turn is correlated to the carbonyl carbon at 208.2 ppm as can be seen in figure 1. this carbon did not have any other correlation suggesting that is directly linked to the phenyl moiety. this suggestion is further supported by the presence of the peak at m/z 195 in the gc-ms characteristic of a trimethoxyphenyl-carbonyl ion. material and methods plant material m. cisplatensis leaves were collected in the banks of rio uruguay, paysandu and identifed by lic. f. haretche, museo y jardin botanico “atilio lombardo”, montevideo. voucher specimens (n 26349) were kept in the mvjb herbarium, jardín botánico, montevideo. analytical methods gc analysis was performed in a shimadzu gc 14 apparatus with an se-52 column using a temperature program from 100 to 280 with a 5/min gradient. abrucker microtof-q-tof with esi source in positive mode was used for ms spectra and a shimadzu qp 5050 with a se 52 column was used for the gc-ms analysis. tlcwas performed on silicagel or rp c18 plates (macherey nagel, dürin, germany) using chcl 3 /meoh (80:20) or isopropanol/h2 o (50:50) as solvent respectively andh2 so4 / heating or anisaldehyde as detection reagents. 1 h nmr and 13 c nmr spectra were obtained at 400 mhz and 100 mhz reapectively, on a brucker avance dpx 400 spectrometer, using cdcl 3 as solvent and tms (δ h 0.00) and acetone (δ c 31.00) as references. 2d (different h,h-cosy, hmbc, hsqc) experiments were carried out with programs available in the brucker software. bioautography bioautographies were made on developed and dried tlc plates according to the agar overlay method of rahalison et al. [17] using staphylococcus aureus (atcc 6538p). extraction and isolation air dried and coarse milled m. cisplatensis leaves were twice extracted with dichloromethane for one week in the dark. the combined extracts were evaporated under vacuum and used for the following procedures. the extract was dissolved in a minimumvolume of methanol and submitted to column chromatography on polyamide (macherey-nagel, 815600) with meoh and acetone as eluents. the second meoh fraction was submitted to vacuum column chromatography (vlc) on fash silicagel (macherey-nagel, 815380) with ch 2 cl 2 /meoh (100:0 to 90:10) as eluent and the active fraction (95:5) was further purifed a c 18 cartridge to give a single compound (by tlc and gc) conglomerone (1) c 13 h 18 o 4 , dark yellow oil. uv (ch 2 cl 2 ) λ max . 279 nm. ei-ms m/z: 238 [m] , 195 [c 10 h 11 o 4 ] . hr-esi-ms m/z: 239.2879 ([mh] , 261.2694 [mna] . 1 hnmr (cdcl 3 ): figure 1: main correlations in the isopropyl moiety. key to the figure:  cosy   hsqc   hmbcdamico, et al.: identification of a bioactive compound from myrcianthes cysplatensis 20 pharmacognosy journal  september 2011  vol 3  issue 25 acknowledgements e. damico gratefully acknowledges eu for a travel grant through the erasmus programme. references 1. world health organization (2007). the world health report 2007 - a safer future: global public health security in the 21 st century. geneva: world health organization. 2. world health organization (2002). fact sheet 194. antimicrobial resistance available from: http://www.who.int/mediacentre/factsheets/ fs194/en/. accessed on 20 may 2010. 3. michel m, gutmann l. methicillin resistant staphylococcus aureus (mrsa) and vancomycin resistant enterococci:therapeutic realities and possibilities. lancet 1997; 349:1901-1906. 4. cox pa. ethnopharmacology and the search for new drugs. in: chadwick, dj, marsh j, editors. bioactive compunds from plants. chichester: john wiley&sons, 40-48; 1996. 5. potterat o, hamburger m. drug discovery and development with plant-derived compounds. prog drug res 2008; 65:47-118. 6. cherigo l, pereda-miranda r, gibbons s. bacterial resistance modifying tetrasaccharide agents from ipomoea murucoides. phytochem 2009; 70:222-227. 7. chaudhary s, negi a, dahiya v.the study of in vitro antimicrobial activity and phytochemical analysis of some medicinal plants in chamoli garhwal region. phcog j 2010; 2:481-485. 8. sousa ea, silva nf, rodrigues f, campos a, lima s, costa jg. chemical composition and resistance-modifying effect of the essential oil of lantana camara. phcog mag 2010; 22:79-82. 9. al-backri ag, othman g, afifi fa. determination of the antibiofilm, antiadhesive, and anti-mrsa activities of seven salvia species. phcog mag 2010; 24:264-270 10. adu f, gbedema sy, annan k. antimicrobial and resistance modulatory activities of corynanthe pachyceras. phcog res 2009; 1:280-284. 11. vieira a. a comparison of traditional anti-inflammation and anti-infection medicinal plants with current evidence from biomedical research: results from a regional study. phcog res 2010; 2:293-295 12. bertucci a, olivaro c, almeida da silva p, ramos d, cerdeiras mp, vázquez a. initial antimicrobial activity studies of plants of the riverside forests of the southern uruguay river.rev bras farmacog 2009; 19:20‑25. 13. bertucci a, olivaro c, haretche f, vazquez a. prospección química del bosque de galería del río uruguay. rev bras farmacog 2008; 18:21-25. 14. metwally am, omar aa, harraz fm, el sohafy sm. phytochemical investigation and antimicrobial activity of psidium guajava l. leaves. phcog mag 2010; 6:212-218. 15. safarei-ghomi j, abbasia. antimicrobial and antifungal properties of the essential oil and methanol extracts of eucalyptus largiflorens and eucalyptus intertexta. phcog mag 2010; 6:172-175. 16. coppen jjw.the genus eucalyptus. 2002. lavoisier, paris. 17. rahalison l, hamburger m, hostettmann k, monod m, frenk, e. a bioautographic agar overlay method for the detection of antifungal compounds from higher plants. phytochem anal 1991; 2:199-203. 18. clinical and laboratory standards institute m7-a7—methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—seventh edition wayne: clsi; 2006 19. ghisalberti el. bioactive acylphloroglucinol derivatives from eucalyptus species. phytochem 1996; 41:7-22. 20. lahey fn, jones tgh. the constitution and synthesis of conglomerone univ. queensland 21. ricciardi aia, romero-fonseca l, veglia j, pipet n. estructura e identificación de los componentes de agreugenia pungens (berg.) kausel, el guabiyú de corrientes. facena 1990; 4:153-161. papers, dept. chem 1939, chem abs (1940); 34:2346-2. in the same way the correlations between the aromatic protons at δ h 6.12 ppm with carbons at 158.0 and 161.0 ppm and the absence of correlation with carbon at 113.0 ppmdetermined the 2, 4, 6 pattern of substitution in the aromatic group (figure 2). thus the compound could be unambiguously identifed as α-methyl-1-(2, 4, 6-trimethoxyphenyl)-1-propanone. when assaying for antistaphylococcal activity, compound 1 showed . a mic of 62.5 µg/ml for the sensible strain (6538p) and 250 µg/ml for the multirresistant ones (43300 and 700699). this shows that the bioguided fractionation is appropriate even when not very active compounds are isolated. conclusions the bioguided fractionation of m. cisplatensis dichloromethane extract gives a pure compound which using different spectroscopic techniques could be identifed as a propiophenone derivative: α-methyl-1-(2,4,6-trimethoxy-phenyl)-1-propanone. from a biosynthetic point of view the compound could be rationalized as a product of the polyketide pathway with an isobutirylcoa starter and the usual malonylcoa prolonger units through claisen reaction. [19] the compound has been previously isolated by lahey from eucalyptus conglomerata who named it conglomeone. [20] conglomerone was also proposed by ricciardi as a phyletic marker for chemosystematics studies in the myrtaceaee family. [21] however this is the frst complete spectroscopic study of the compound as well as the frst antibacterial activity reported. both the extract and the pure compound showed antibacterial activity against methicillin-sensitive and resistant staphylococcus aureus strains figure 2: main correlations in the aromatic moiety. key to the figure:  cosy   hsqc   hmbcpharmacognosy journal  september 2011  vol 3  issue 25 21 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: mobile: 09891626956 e-mail:swatibalian@yahoo.co.in doi: 10.5530/pj.2011.25.4 brazil for hundreds of years to sweeten local teas, medicines and as a ‘sweet treat’. the plant is also known as sweet herb, honey leaf, or sweet chrysanthemum as it possesses sweet tasting glycosides. [3] it is a storehouse of various bioactive constituents mainly, the ent-kaurene diterpene glycosides (the sweet tasting glycosides) namely- stevioside, steviolbioside, dulcoside a and rebaudioside a, b, c, d and e. [4] these compounds stevioside and rebaudioside are 250-300 times sweeter than sucrose, heat stable, ph stable, and non-fermentable. with reference to its sweetening power, it is estimated that 30ml of stevia extract is equivalent to 3 kg of sucrose. [3] the plant’s leaves, the aqueous extract of the leaves, and purifed steviosides are used as sweeteners. the sweetener extractives have been known to exert benefcial effects on human health- antihypertensive, [5,6] antidiabetic, [7-10] non-carcinogenic, [11,12] antioxidant, [13,14] anti-infammatory activities. [15,16] they are also thought to effect glucose metabolism and renal function. [17] apart from these it also exhibits antimicrobial activities. [18,19] it also plays a benefcial role as a dentifrice as it inhibits the development of plaque and cavities. [20] the current investigation is aimed at a comparative standardization of fve varieties of stevia rebaudiana procured from fve different geographical locations of india viz., comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni from different geographical sources mandal bitasta, madan swati* amity institute of pharmacy, amity university, noida (u.p.) india a b s t r a c t stevia rebaudiana bertoni, a natural non-caloric substitute to conventional sugar, is also popular as the “sweet herb of paraguay”. it is a storehouse of various bioactive constituents mainly, the ent-kaurene diterpene glycosides namely- stevioside, rebaudioside a, b, c, d and e. the plant is known to exhibit a wide range of biological activities like hypoglycemic, anti-oxidant, anticancer, antibacterial activities. the present research is based on a comparative standardization and physicochemical analysis of the dried leaves of fve varieties of stevia rebaudiana procured from fve different geographical locations of india viz., delhi, surat, kangra, bangalore and indore. fluorescence analysis of the powdered leaveswas carried out as ameans for identifcation. the standardization parameters included determination of foreign matter, ash values, loss on drying, extractive values. preliminary phytochemical screening was also performed. the results from the current study can prove to be an indicator to differentiate the fve varieties based on their standardization parameters. key words: stevia rebaudiana, non-caloric substitute, ent-kaurene glycosides, comparative standardization. introduction the modern era faces a number of growing ailments and diseases that are a serious concern to normal sustenance of an individual in this scenario. these include hypertension, diabetes mellitus, premature aging, cancer, dental caries, skin diseases like acne and pruritis, bacterial and fungal infections andmany more. control and cure of these diseases require a source that can overcome these health concerns and that has a minimal potential to cause adverse effects. this situation and need has brought “stevia rebaudiana” (family:-asteraceae) into the picture which is a substitute to conventional sugar existing in nature. it is a non-caloric sweetener which is consumed in many countries. [1] it is a small perennial shrub growing upto 1m tall and with leaves 2-3cm long [2] and native to regions of paraguay and brazil. it is popular as the “sweet herb of paraguay”, as the leaves have been traditionally used by natives of paraguay andbitasta and swati: comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni 22 pharmacognosy journal  september 2011  vol 3  issue 25 krishna herbal (indore) and locally feld grown leaves from chachiyan village (kangra) between the months of september to november, 2010. the identity of the leaves was verifed by dr. h. b. singh, head, raw materials herbarium and museum, niscair, new delhi and a voucher specimen for the leaves was deposited at the herbariumof national institute of science communication and information resources, new delhi respectively. fluorescence analysis 1-2 mg of the dried leaf powder of all the fve varieties of stevia were taken and placed on a microscopic slide and delhi, surat, kangra, bangalore and indore to fnd out which variety best complies with the standardization parameters so that it can be effectively used inmanufacturing of various stevia based products with maximum quality. materials and methods collection dried leaves of stevia rebaudiana were procured fromdifferent suppliers of india: saico healthcare pvt. ltd. (delhi), keshal nursery (surat), deepak trading co.(bangalore), shri table 1: fluorescence analysis of powdered leaves of stevia rebaudiana from delhi treatment stevia rebaudiana (delhi) day light uv light 254 nm 366 nm powder as such light green greyish brown dark brown powder 1n naoh (aq.) brownish yellow bright green blackish green powder 1n naoh (alc.) yellowish green yellowish green brownish green powder 1n hcl yellowish green bright green greenish black powder nh 3 dark green blackish green purplish black powder 5% iodine greyish green silvery green blackish green powder 5% fecl 3 blackish green greenish black dark green powder acetic acid light brown blackish green blackish brown powder 1n h 2 so 4 yellowish green bright green blackish green powder 1n hno 3 yellowish green light green blackish green table 2: fluorescence analysis of powdered leaves of stevia rebaudiana from surat treatment stevia rebaudiana (surat) day light uv light 254 nm 366 nm powder as such dark green dark green brownish green powder 1n naoh (aq.) dark brown light green dark brown powder 1n naoh (alc.) dark green dark green brownish green powder 1n hcl brownish green light green purplish black powder nh 3 blackish green blackish green black powder 5% iodine blackish green dark green purplish green powder 5% fecl 3 dark green blackish green brownish green powder acetic acid dark green light green brownish green powder 1n h 2 so 4 dark green blackish green blackish green powder 1n hno 3 orange green dark green black table 3: fluorescence analysis of powdered leaves of stevia rebaudiana from bangalore treatment stevia rebaudiana (bangalore) day light uv light 254 nm 366 nm powder as such yellowish green yellowish green brownish green powder 1n naoh (aq.) brownish green dark green purplish black powder 1n naoh (alc.) brownish green bright green brown powder 1n hcl brownish yellow light green grey powder nh 3 blackish green dark green black powder 5% iodine greyish green bright green purplish grey powder 5% fecl 3 yellowish green dark green purplish black powder acetic acid dark brown dark green purplish brown powder 1n h 2 so 4 yellowish green light green black powder 1n hno 3 orange brown blackish green blackpharmacognosy journal  september 2011  vol 3  issue 25 23 bitasta and swati: comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni table 4: fluorescence analysis of powdered leaves of stevia rebaudiana from kangra treatment stevia rebaudiana (kangra) day light uv light 254 nm 366 nm powder as such dark green light green blackish green powder 1n naoh (aq.) blackish green blackish green black powder 1n naoh (alc.) dark green dark green black powder 1n hcl light green light green blackish green powder nh 3 blackish green greenish black brownish green powder 5% iodine dark green dark green black powder 5% fecl 3 blackish green brownish green blackish green powder acetic acid dark green dark green purplish green powder 1n h 2 so 4 light green light green blackish green powder 1n hno 3 dark brown dark green blackish green table 5: fluorescence analysis of powdered leaves of stevia rebaudiana from indore treatment stevia rebaudiana (indore) day light uv light 254 nm 366 nm powder as such light green yellowish green dark green powder 1n naoh (aq.) brownish green blackish green dark green powder 1n naoh (alc.) light brown bright green dark brown powder 1n hcl yellowish green dark green brownish green powder nh 3 dark green bright green blackish green powder 5% iodine brownish green light green blackish green powder 5% fecl 3 yellowish green blackish green black powder acetic acid brownish green dark green blackish green powder 1n h 2 so 4 yellowish green bright green blackish green powder 1n hno 3 dark brown light green black table 6: foreign matter of the different varieties of stevia rebaudiana stevia rebaudiana weight of sample taken (g) foreign matter (%) delhi 100 2.35 surat 100 0.58 bangalore 100 1.65 kangra 100 1.80 indore 100 0.85 table 7: total ash, acid insoluble ash and water soluble ash of the different varieties of stevia rebaudiana stevia rebaudiana total ash (%w/w) acid insoluble ash (%w/w) water soluble ash (%w/w) delhi  9.00 1.25 6.25 surat 13.50 2.25 7.25 bangalore 12.75 1.25 7.75 kangra  7.75 0.75 4.25 indore 11.50 1.75 6.75 table 8: loss on drying of the different varieties of stevia rebaudiana stevia rebaudiana weight of sample taken (g) loss on drying (%w/w) delhi 10 5.75 surat 10 7.90 bangalore 10 5.35 kangra 10 8.15 indore 10 7.25 using petroleum ether (b.p. 40-60), chloroform, methanol, methanol:water(1:1) and chloroform:water (1:99) as solvents. the different extracts were concentrated using rota vapor. extractive values in different solvents (petroleum ether soluble, chloroform soluble, methanol soluble, diluted methanol soluble and water soluble) were then determined observed in day light as well as in short wave uv light (254nm) and long wave uv light (366 nm). the powdered drugs were then treated with different reagents as 1n sodium hydroxide (aqueous), 1 n sodium hydroxide (alcoholic), 1 n hydrochloric acid, ammonia, 5% iodine, 5% ferric chloride, acetic acid, 1 n sulphuric acid, 1 n nitric acid [21,22,23] and the results were noted. (table 1,2,3,4,5) standardization and physicochemical parameters physicochemical parameters of the leaves which included determination of foreign matter, ash values (total ash, water soluble ash and acid insoluble ash) and loss on drying [24,25,26] and the results were taken. (table 6,7,8) extraction the fve varieties of the leaves collected were taken and subjected to both hot soxhlation as well as cold macerationbitasta and swati: comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni 24 pharmacognosy journal  september 2011  vol 3  issue 25 hot air oven at temperatures below 50 c. [29] the different successive solvent extractive values were then recorded. (table 11) preliminary phytochemical screening the methanolic extracts of all the fve varieties were subjected to preliminary phytochemical screening to judge the presence of various classes of phytoconstituents as per the method. [17,30] the different chemical tests included the tests for alkaloids, saponins, carbohydrates, glycosides (general), anthraquinone glycosides, cardiac glycosides, according to the method [24,27] and noted. (table: 9 and table 10) successive solvent extraction successive solvent extraction of the air-dried drug powdered leaves was carried out using the same solvents as earlier successively in increasing order of polarity starting with petroleum ether (b.p. 40-60), chloroform, methanol, methanol:water(1:1) and fnally with chloroform: water (1:99) by cold maceration. [28] before extracting with a new solvent, the powdered material was dried in table 9: extractive values in different solvents by hot soxhlation stevia rebaudiana petroleum ether (%w/w) methanol (%w/w) methanol-water (%w/w) chloroform-water (%w/w) delhi 2.40 31.15 29.50 21.85 surat 2.55 38.10 33.80 26.50 bangalore 3.60 35.25 32.00 24.20 kangra 6.00 28.55 35.40 24.90 indore 3.25 29.00 32.65 23.40 table 10: extractive values in different solvents by cold maceration stevia rebaudiana petroleum ether (%w/w) chloroform (%w/w) methanol (%w/w) methanol-water (%w/w) chloroform-water (%w/w) delhi 3.15 11.95 39.95 14.95 18.60 surat 3.85 10.55 45.30 20.95 25.15 bangalore 4.90 13.80 47.00 17.05 24.50 kangra 2.55 10.10 41.95 19.55 20.25 indore 5.45 14.25 44.30 15.50 21.20 table 11: successive solvent extractive values of the different varieties of stevia rebaudiana stevia rebaudiana petroleum ether (% w/w) chloroform (% w/w) methanol (% w/w) methanol-water (% w/w) chloroform-water (% w/w) delhi 2.00 4.90 19.20 11.20 10.60 surat 2.90 4.00 24.50 10.40 12.70 bangalore 3.00 6.40 22.40  7.20 15.40 kangra 3.20 6.30 27.20 12.00 11.30 indore 2.40 5.80 21.20  9.60 12.20 table 12: preliminary phytochemical screening of the methanolic extracts of the different varieties of stevia rebaudiana test stevia rebaudiana delhi surat bangalore kangra indore alkaloids ve ve ve ve ve saponins ve ve ve ve ve carbohydrates ve ve ve ve ve glycosides (general) ve ve ve ve ve anthraquinone glycosides ve ve ve ve ve cardiac glycosides ve ve ve ve ve coumarin glycosides ve ve ve ve ve cyanogenetic glycosides –ve –ve –ve –ve –ve tannins ve ve ve ve ve proteins –ve –ve –ve –ve –ve steroids ve ve ve ve ve waxes ve ve ve ve ve flavonoids ve ve ve ve ve amino acids ve ve ve ve ve acidic compounds –ve –ve –ve –ve –vepharmacognosy journal  september 2011  vol 3  issue 25 25 bitasta and swati: comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni presence of a wide range of phytoconstituents including alkaloids, glycosides (anthraquinone, cardiac, coumarin), saponins, carbohydrates, favonoids, tannins, amino acids, steroids, waxes supporting the reason for its wide range of biological activities. conclusion hence, the current research assists to differentiate the fve varieties of stevia rebaudiana based on their standardization and physicochemical parameters. the fuorescence analysis of the powder, various physicochemical parameters like foreign matter, loss on drying, ash values, extractive values as well as phytochemical studies including preliminary phytochemical screening supported the identifcation and authentifcation of the fve varieties for the present study. the results may thus, be helpful in obtaining the variety of best quality to be used in manufacturing of various stevia based products. references 1. saenphet k, aritajat s, saenphet s, manosroi j, manosroi a. safety evaluation of aqueous extracts from aegle marmelos and stevia rebaudiana on reproduction of female rats. southeast asian jtrop med public health 2006; 37(3):203-205. 2. mishra pk, singh r, kumar u, prakash v. stevia rebaudiana- a magical sweetener. global journal of biotechnology & biochemistry 2010; 5(1):62-74. 3. sharma m, thakral nk, thakral s. chemistry and in vivo profile of ent-kaurene glycosides of stevia rebaudiana bertoni –an overview. natural product radiance 2009; 8(2):181-189. 4. pasquel a, meireles maa, marques mom, petenate aj. extraction of glycosides with carbon dioxide water and carbon dioxide ethanol. braz j chem eng 2000; 17:3. 5. melis ms.chronic administration of aqueous extract of stevia rebaudiana in rats: renal effects. journal of ethnopharmacology 1995; 47(3):129-134. 6. chan p, xu dy, liu jc, chen yj, tomlinson b, huang wp, et al. the effect of stevioside on blood pressure and plasma catecholamines in spontaneously hypertensive rats. life sciences 1998; 63(19):1679-1684. 7. jeppensen pb, gregersen s, alstrup kk, hermansen k. stevioside induces antihyperglycaemic, insulinotropic and glucagonostatic effects in vivo: studies in the diabetic goto-kakizaki (gk) rats. phytomedicine 2002; 9(1):9-14. 8. jeppensen pb,gregersen s, rolfsen se, jepsen m, colombo m,agger a, et al.antihyperglycemic and blood pressure-reducing effects of stevioside in the diabetic goto-kakizaki rat. metabolism 2003; 52(3):372-378. 9. chen th, chen sc, chan p, chu yl, yang hw, cheng jt. mechanism of the hypoglycemic effect of stevioside, a glycoside of stevia rebaudiana. planta med 2005; 71(2):108-113. 10. kujur rs,singh v,ramm,yadava h,singh kk,kumari s,et al.antidiabetic activity and phytochemical screening of crude extract of stevia rebaudiana in alloxan-induced diabetic rats. pharmacognosy journal 2010; 2(14):27-32. 11. konoshima s, takasaki m. cancer-chemopreventive effects of natural sweeteners and related compounds. pure appl chem 2002; 74(7):1309-1316. 12. jayaraman s, manoharan ms, illanchezian s. in-vitro antimicrobial and antitumor activities of stevia reabudiana (asteraceae) leaf extracts. tropical journal of pharmaceutical research 2008; 7(4):1143-1149. coumarin glycosides, cyanogenetic glycosides, tannins, proteins, steroids, waxes, favonoids, amino acids and acidic compounds and the results were taken. (table 12) results and discussion the current investigation assessed in a detailed and comparative standardization and physicochemical analysis of the dried leaves of fve varieties of stevia rebaudiana procured from fve different geographical locations of india viz., delhi, surat, kangra, bangalore and indore. from the current study, it was possible to differentiate the fve varieties based on their standardization parameters. the results may prove to be a valuable indicator in fnding out a suitable variety that best matches in accordance with the standardization parameters so that it can be effectively used in manufacturing of various stevia based products with reasonable and fair quality. the various physicochemical parameters carried out for the purpose of standardization and authentication included determination of foreign matter, loss on drying, ash values (total ash, acid insoluble ash, water soluble ash), extractive values in different solvents as petroleum ether (b.p. 40-60), chloroform, methanol, methanol-water (1:1), chloroform-water (1:99). both hot soxhlation, coldmaceration and successive solvent extraction were carried out for all the fve varieties in all the fve solvents and it was found that successive solvent extraction led to lower extractive values compared to hot soxhlation and cold maceration. the foreign matter was found to be the highest in the leaves from delhi with a value of 2.35% w/w and lowest in the leaves from surat with a value of 0.58% w/w. presence of moisture which was determined through loss on drying (lod) was found to be the maximum in kangra variety i.e., 8.15% w/w and the minimum in the bangalore variety i.e., 5.35% w/w. ash values were mainly determined with the purpose of estimating the inorganic salts naturally occurring in the drug and adhering to it as well as the inorganic matter added for the purpose of adulteration and it was found that the total ash and acid insoluble ash was found to be the maximum in the surat variety with a value of 13.50%w/w and 2.25%w/w respectively and minimum in the kangra variety with a value of 7.75%w/w and 0.75% w/w respectively. however, the water soluble ash was found to be the highest in the bangalore variety i.e., 7.75% w/w and the lowest in the kangra variety i.e., 4.25% w/w. additionally, fuorescence analysis for the powdered leaves was carried out using various reagents in day light and uv light (254 nm and 366 nm) which served as a parameter for identifcation of the plant material. preliminary phytochemical screening was carried out on the methanolic extracts of all the varieties and revealed thebitasta and swati: comparative standardization and physicochemical evaluation of the leaves of stevia rebaudiana bertoni 26 pharmacognosy journal  september 2011  vol 3  issue 25 21. kokoski j, kokoski r and slama fj. fluorescence of powdered vegetable drugs under ultraviolet radiation. j am pharmacol assoc 1958; 47:75-78. 22. kalidass c, mohan vr, amish ad. pharmacognostic studies on capparis sepiaria (l.) r.br. pharmacognosy journal 2009; 1(2):121-125. 23. kumar v, yadav ps, pratap su, bhat hr, rana a, zaman mk. pharmacognostical evaluation of cuscuta reflexa roxb. pharmacognosy journal 2010; 2(6)74-82. 24. the ayurvedic pharmacopoeia of india. part-i, vol. iv. 1 st edition. new delhi: government of india, ministry of health and family welfare, department of ayush. 2004; 159-160. 25. who. quality control methods for medicinal plant materials, geneva, 1998. 26. radhika b, nasreen b, srisailam k. pharmacognostic and preliminary phytochemical evaluation of the leaves of bixa orellana. pharmacognosy journal 2010; 2(7):132-136. 27. mukherjee pk. quality control of herbal drugs. business horizons, new delhi, india. 1 st ed. (reprint) 2005; 187-188. 28. agrawal ss. herbal drug technology. universities press (india) private limited. 2007; 326. 29. lawania rd, prasad r, mishra a, gupta r. pharmacognostic and phytochemical studies of bark of oroxylum indicum. pharmacognosy journal 2010; 2(9):297-303. 30. kokate ck, purohit ap, gokhale sb. pharmacognosy. nirali prakashan, pune, india. 13 th ed. 2005; 593-597. 13. vignais pv, duee ed, vignais pm, huet j. effects of atractyligenin and its structural analogues on oxidative phosphorylation and on the translocation of adenine nucleotides in mitochondria. biochim biophys acta 1966; 118:465-483. 14. ghanta s, banerjee a, poddar a, chattopadhyay s. oxidative dna damage preventive activity and antioxidant potential of stevia rebaudiana (bertoni) bertoni, a natural sweetener. j agric food chem 2007; 55(26):10962-10967. 15. boonkaewwan c, toskulkao c, vongsakul m. anti-inflammatory and immunomodulatory activities of stevioside and its metabolite steviol on thp-1 cells. j agric food chem 2006; 54(3):785-789. 16. jeong iy, lee hj, jin ch, park yd, choi ds, kang ma. anti-inflammatory activity of stevia rebaudiana in lps-induced raw 264.7 cells. j food sci nutr 2010; 15:14-18. 17. tadhani m, subhash r. preliminary studies on stevia rebaudiana leaves: proximal composition, mineral analysis and phytochemical screening. j med sci 2006; 6(3):321-326. 18. debnath m. clonal propagation and antimicrobial activity of an endemic medicinal plant stevia rebaudiana. journal of medicinal plants research 2008; 2(2):45-51. 19. ghosh s, subudhi e, nayak s. antimicrobial assay of stevia rebaudiana bertoni leaf extracts against ten pathogens. international journal of integrative biology 2008; 2(1):27-31. 20. wu cd,johnson sa,sriakantha r,kinghorn ad. intense natural sweetener and their effect on cariogenic bacteria. j dental res 1998; 77:283.pharmacognosy journal  september 2011  vol 3  issue 25 27 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: e-mail: pinalharde@gmail.com doi: 10.5530/pj.2011.25.5 phytochemical studies showed presence of triterpene acids like corosolic acid, ursolic acid, oleanolic acid, maslinic acid, asiatic acid and arjunolic acid. [4,5] leaves also revealed presence of tannin derivatives like lagertanin, lageracetal, lagerstroemin, fosin b, reginin a, reginin c and reginin d. [6,7,8] besides it also showed presence of sterols like daucosterol, β-sitosterol, phytol, sitosterol acetate. [9] a corosolic acid from alcoholic extract of leaves was reported to have antidiabetic activity when administered in vivo as well as in vitro. [10,11] tannins isolated from leaves showed signifcant hypoglycemic activities in different models as well. [6] however, there are no reports on the systematic pharmacognostical studies of the leaves of l. fos-reginae. hence, the present investigation is an attempt in this direction and includes evaluation of the leaves of l. fos-reginae by its macroscopical, microscopical, physicochemical parameters and preliminary phytochemical screening of different extracts. materials and methods plant material the fresh leaves were collected from a healthy and well developed tree of lagerstroemia fos-reginae (l.) pers from pharmacognostical studies of leaves of lagerstroemia flos-reginae harde pinal a. 1* and shah mamta b. 2 1 department of pharmacognosy, c. k. pithawalla institute of pharmaceutical science and research, dumas road, surat-395007, gujarat, india. 2 department of pharmacognosy, l. m. college of pharmacy, navrangpura, ahmedabad-380009, gujarat, india. a b s t r a c t introduction: lagerstroemia fos-reginae (l) pers. (hindi-jarul) belonging to lythraceae is found throughout india, especially in assam, bengal and deccan peninsula. a decoction of the leaves of l. fos-reginae in form of tea is widely used for diabetes mellitus in philippines. methods: the pharmacognostical investigations of leaves of l fos-reginae was done by evaluating its morphological, microscopical studies, leaf constants, phytochemical screening and various physicochemical parameters. results: the microscopical studies revealed presence of epidermis with striated cuticle, bilayered palisade, rannanculaceous stomata, abundant calcium oxalate rosettes and prisms, fragments of bordered pitted xylem vessels and lignifed pericyclic fbres in groups. physical constants of leaf powder showed 14.23% alcohol soluble extractive value, 15.75% water soluble extractive value, 8.94% total ash, 8.65%water soluble ash, 1.94% acid insoluble ash, phytochemical analysis revealed presence of triterpenoid saponins, tannins, alkaloids, steroids, sugars and proteins. conclusion: the above pharmacognostical and preliminary phytochemical studies will be benefcial for proper identifcation and authentifcation of leaves of l. fos-reginae. key words: lagerstroemia fos-reginae, leaf constants, microscopy, physicochemical parameters introduction lagerstroemia fos-reginae l. (syn. lagerstroemia speciosa) belonging to family lythraceae is popularly known as banaba, jarul in hindi and queen’s crape myrtle in english. it grows widely in the philippines, india, south east asian countries including vietanam, malaysia and south china. in india, it is found especially in assam, bengal and deccan peninsula. [1,2] it is one of the well known ornamental trees and is cultivated widely in gardens as an avenue tree. it is a medium sized to large deciduous tree about 9-18 m high with a rounded crown. bark smooth, grayish, exfoliating in irregular fakes. flowers are 5-7.5 cm across in large panicles, sometimes reaching to 30 cm. long; mauve to purple in colour. capsule ellipsoid or subglobose. seeds are pale brown in colour. [3] the tea from the leaves of banaba has traditionally been used in philippines, as a folk medicine for the treatment of diabetes. the leaves have also been used as purgative, deobstruent and diuretic. [1]pinal and mamta: pharmacognostical studies of leaves of lagerstroemia flos-reginae 28 pharmacognosy journal  september 2011  vol 3  issue 25 fluorescence analysis fluorescence analysis of powdered leaves was carried out by standard methods. [19,20] preliminary phytochemical screening for the preliminary phytochemical analysis, 5 g powdered drug was extracted in soxhlet extractor with petroleum ether (60-80 c), ethyl acetate, n-butanol, methanol and water successively. the presence or absence of different phytoconstituents viz. triterpenoids, steroids, alkaloids, sugars, tannins, coumarins and favanoids, etc. were detected by usual prescribed methods. [21,22] results and disscusion macroscopical characters the leaves are simple, pinnate and opposite. they are elliptical to oblong, 8-22 cm 3.5-7.2 cm in size. the apex of leaves is subacute. leaves have entire margin and coriaceous texture. the leaves show fne reticulations both surfaces. main nerves are in 10-13 pairs, prominent and curving upwards. the odour is characteristic and taste is bitter (figure 1). the bapalal vaidya botanical garden of veer narmad south gujarat university, surat, india in june 2007, when the fowering was in bloom. they were identifed as leaves of lagerstroemia fos-reginae (l). pers. by comparing the morphological characters described in the literature .[1,12,13] the authenticity of the plant was further confrmed by dr. minooh. parabia, head of department and botanist, bapalal vaidya botanical researchcentre (department of bioscience) the veer narmad south gujarat university, surat and voucher specimen number was given as pah/23082007/01 and deposited at bioscience department of the veer narmad south gujarat university, surat, india for future reference. macroscopic evaluation the leaves of l. fos-reginae were evaluated macroscopically to photographed to view its extra features. microscopic evaluation i) sectioning transverse sections of fresh leaves of l. fos-reginae were taken by microtome and free hand sectioning. numerous temporary mounts of transverse sections were prepared using lactophenol as a mounting agent and examined microscopically. histochemical reactions were applied with hydrochloric acid-phloroglucinol to reveal lignifed elements, iodine-iodide for starch, sudan iv for lipophilic substances, dragendorff ’s reagent for alkaloidal substances, ruthenium red formucilage and ferric chloride for phenolic compounds. [14] photomicrographs of the microscopical sections were taken with the help of magnus mlx-dx photomicroscope provided with honestech software. ii) powder characteristics microscopical examination of powder of leaves were carried out. photomicrographs were taken preliminary examination, behavior of powder with different chemical reagents were performed. [15,16] iii) leaf constants the leaf constants of l. fos-reginae were determined by standard methods. [17] photomicrographs of important microscopical structures were taken with the help of magnus mlx-dx photomicroscope provided with honestech software. iv) micrometry the measurements of different cells and cell contents were done with the help of calibrated ocular micrometer. physico-chemical parameters percentage of total ash, acid-insoluble ash and water soluble ash were calculated. water soluble and alcohol soluble extractive values of the leaves were determined. [18] figure 1: a leaf of lagerstroemia flos-reginaepharmacognosy journal  september 2011  vol 3  issue 25 29 pinal and mamta: pharmacognostical studies of leaves of lagerstroemia flos-reginae walled parenchymatous cells called pith in which sclerenchymatous cells are found in groups. the vascular bundle is encircled by continuous band of the pericyclic sclerenchymatous lignifed, thick walled fbres. pericycle is surrounded by parenchymatous cells of ground tissue. prismatic crystals and rosettes of calcium oxalate are abundant in midrib and mesophyll. microscopical powder studies (figure 3) stomata: rannanculaceous types of stomata epidermal cells: straight walled epidermal cells with striated cuticle palisade: compactly arranged bilayer palisade cells calcium oxalates crystals in form of rossetts and prism xylem vessels: spiral and annular different leaf constants and micrometric analysis are tabulated in table 1 and 2 respectively. results of physico-chemical parameters for leaf powder of l. fos-reginae is shown in table-3. quantitative standards revealed that the ash content was 3.56 0.15%. water soluble ash and acid insoluble ash was 2.51 0.11 and 0.33  0.05% respectively. the water soluble extractive value was 15.75 0.35 indicating the presence of sugar, microscopical characters transverse section of leaf (figure 2) lamina the leaf of l. fos-reginae is dorsiventral with distinct adaxial and abaxial faces. epidermis: it consists of single layered rectangular cells, covered with thin and striated cuticle. some epidermal cells contain mucilage. the adaxial epidermal cells are about twice as large as those of the abaxial epidermis. mesophyll: mesophyll is well differentiated and composed of double layered, compact, radially elongated palisade tissue followed by spongy mesophyll composed of 3-4 layers of loosely arranged parenchymatous cells with scattered calcium oxalate cluster crystals. midrib transverse section passing through midrib represented concavity on abaxial surface and a convex or rounded adaxial surface. vascular bundle: midrib consists of two bicollateral vascular bundles in which secondary xylem vessels are arranged in form of cup and lid shape. xylem shows presence of tracheids, xylem parenchyma and xylem vessels. distinct band of phloem tissue can be seen on either sides of xylem. the central region of vascular bundle is made up of thin figure 2: transverse section of leaf of l. flos-reginae c-collenchyma, cr-calcium oxalate crystals, f-pericyclic fiber, i.ph‑interxylary phloem, l.ep-lower epidermis, u.ep-upper epidermis, ph-phloem, p-palisade, sp-spongy parenchyma, vt-veinpinal and mamta: pharmacognostical studies of leaves of lagerstroemia flos-reginae 30 pharmacognosy journal  september 2011  vol 3  issue 25 figure 3: microscopical powder characteristics of leaf of l. flos-reginae a- bilayered palisade b- striated cuticle c-anomocytic stomata d-xylem vessel with annular and spiral thickening e- prisms in veins f calcium oxalate rosettes crystals table 1: leaf constants of l. flos-reginae leaf constants values stomatal number upper surface: nil lower surface: 400-450 stomatal index 18.8-21.35 vein -islet number 9-12 vein-termination number 10-12 table 2: measurement of cells in t.s. of l. flos-reginae type of cells size in μ upper epidermis 13.6 40.8 collenchyma 20.4-27.2 palisade cells 40.8-54.4 parenchyma 40.8-108.8 xylem parenchyma 7.8-20.4 xylem vessels 20.4-108.8 sclerenchymatous fibres 13.6-149.6 phloem 13.6-26.4 calcium oxalate rosettes 13.6-54.4 table 3: physicochemical constants for powder of leaf of l. flos-reginae physicochemical constants (% w/w) total ash  3.56 0.15 water soluble ash  2.51 0.11 acid insoluble ash  0.33 0.05 water soluble extractive value 15.75 0.35 alcohol soluble extractive value (%w/w) 14.23 0.18 loss on drying (lod)  4.55 0.03 table 4: fluorescence analysis of powdered leaves of l. flos-reginae treatment with chemical reagent ordinary light long uv light powder 1n naoh in methanol greenish brown greenish brown powder 1n naoh in water reddish brown reddish brown powder 1n hcl light green light green powder 50% hno 3 reddish brown orange red powder 50% h 2 so 4 green brownpharmacognosy journal  september 2011  vol 3  issue 25 31 pinal and mamta: pharmacognostical studies of leaves of lagerstroemia flos-reginae 2. satyavati gv, gupta ak, tandon n. medicinal plants of india-ii, indian council of medical research, new delhi. 1987; 124. 3. rajpal v. standardization of botanicals(testing and extraction methods of medicinal herbs, vol.2), eastern publishers,the house of pharmaceutical books, new delhi-110048, india, 2008; 206-15 4. wenli h, yanfang l, qiang z, xin w, aihua p, lijuan c. et al. triterpene acids isolated from lagerstroemia speciosa leaves as α-glucosidase inhibitors. phytother res, 2009; 23(5): 614-18. 5. okaday,omaea.okuyamat.a new triterpenoid isolated fromlagerstroema speciosa (l.) pers. chem pharm bull. 2003; 51 (4):452-54. 6. hayashi t, maruyama h, kasai r, hattori k, takasuga s, hazeki o. et al. elllagitannins from lagerstroemia speciosa as activators of glucose transport in fat cells. planta med. 2002; 68(2):173-75. 7. takahashi m, osawa k, ueda j, yamamoto f, tsai ct. the components of the plants of lagerstroemia genus. iii on the structure of the new tannin“lagertannin” from the leaves of lagerstroemia speciosa, yakugaku zasshi. 1976; 96:984-87. 8. xu ym, tanaka t, nanaka g and nishioka i. tannins and related compounds. cvii: structure elucidation of three new monomeric and dimeric ellagitannins, flosin b and reginins c and d isolated from lagerstroemia speciosa retz., chem pharm bull. 1991; 39(3): 647-50. 9. ragasa cy,ngo ht,rideout ja.terpenoids and sterols fromlagerstroemia speciosa, j asian nat prod res. 2005; 7(1):7-12 10. murakami c, myoga k, kasai r, ohtani k, kurokawa t, ishibashi s. screening of plant constituents for effect on glucose transport activity in ehrlich ascites tumor cells. chem pharm bull. 1993; 41(12):2129-31. 11. miura t, itoh y, kaneko t, ueda n, ishida t, fukushima m. corosolic acid induces glut4 translocation in genetically type 2 diabetic mice. biological & pharmaceutical bulletin, 2004; 27(7):1103-05. 12. kirtikar kr, basu bd. indian medicinal plants. reprinted edition, vol.2, l.m. basu, allahabad, 1933; 1079-81. 13. shah gl. flora of gujarat. vol.1, sardar patel university, anand, 1978, 122. 14. kay la. the microscopic studies of drugs. bailliere tindall and cox, london, 1938, 18-21. 15. reddy ysr, venkatesh s, ravichandra t. pharmacognostical studies on wrightia tinctoria bark. pharm biol 1999; 37:291-95. 16. wahi ak, geetha m. pharmacognostical studies on leaves of barleria prionitis linn. indian j nat prod. 2002; 16; 16-19. 17. wallis te. practical pharmacognosy. j. and a. churchill ltd., london, 1953, 139. 18. anonymous. indian pharmacopoeia. vol.2. 4 th ed. controller of publications, govt. of india, ministry of health and family welfare, delhi. 1996. 19. pratt rj, chase cr. fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. j am pharm. ass. 1949; 38:324-33. 20. kokoski j, kokoski r, salma fj. fluorescence of powdered vegetable drugs under ultraviolet radiation. j am pharm ass. 1958; 47:715-17. acid and inorganic components. the alcohol soluble extractive value was 14.23 0.18 which shows the presence of polar and non polar secondary metabolites present in the plant materials. loss and drying at 105 c was revealing 4.55 0.03 the moisture content in the plant. the results of fuorescence analysis were tabulated in table 4. the results of phytochemical screening of powder of leaves of l.fos-reginae is mentioned in table 5. conclusion the present study deals with pharmacognostical study of leaves of l. fos-reginae, which include striated cuticle, bilayered palisade, typical vascular bundles arranged in form of cup and lid surrounded by sclerenchymatous fbers on inner as well as outer side, abundant calcium oxalate rosettes, spiral as well as annular xylem vessels. the physicochemical parameters and leaf constants would help in the authentication of this plant. the preliminary qualitative phytochemical screening shows presence of saponins, steroids, tannins, alkaloids, carbohydrates and proteins. the microscopic features, leaf constants and physicochemical parameters would be useful for laying down pharmacopoeial standards. further studies are in progress in our laboratory to isolate the active constituents. acknowledgement authors are greatful to dr. minoo parbia, ex. dean of bioscience dept., the veer narmad southgujarat university for proper authentication of plant. authors are also thankful to c. k. pithawalla institute of science and research for providing the facility to carry out research work. references 1. anonymous.wealth of india raw materials. sp-w.vol 6. publications and information directorate, new delhi; csir. 1959; 26-27. table 5: qualitative phytochemical analysis of various extracts of leaves of l. flos-reginae constituents pet. ether extract ethyl acetate extract n-butanol extract methanolic extract aqueous extract tannins – – saponins – – steroids – – – flavonoids – – coumarins – – – – carbohydrate – – – – alkaloids – – – proteins – – – – ‘’ indicates presence and ‘–’ indicates absenceo r i g i n a l a r t i c l e p h c o g j . 32 pharmacognosy journal  september 2011  vol 3  issue 25 *address for correspondence: senior research fellow, central council for research in ayurveda & siddha, dept. of ayush, ministry of health & family welfare, janakpuri, new delhi-58, ph: 919968532138 (m) e-mail: rath.chinmay@gmail.com doi: 10.5530/pj.2011.25.6 where botanical identity of plant source has not been established and these are subjected to adulterations/ substitutions. so the aim of the present study was focussed on the pharmacognosy, and phytochemical properties, which would like to facilitate quick identifcation and selection of the drug from various substitutes and adulterants and its comparison with market samples collected in the name of kakoli and ksheerkakoli to standardize and maintain the ‘quality control’ the drug. material and methods fresh tubers of botanically identifed plants of roscea procera wall. and lilium polyphyllum d. don were collected from medicinal plants garden of regional research institute of himalayan flora, tarikhet and indian medicines pharmaceutical corporation limited, mohan (almora). tubers were washed, cut into pieces and preserved in formalo-acetyl-alcohol (faa) and labelled rp4 and lp4 for pharmacognostical study. microtome section were taken, stained and mounted following the usual plant microtechniques [9,10] and representative diagram sketched through camera lucida and some were shade dried and coarse (20 to 30 #) powdered for qualitative tests and physico-chemical study as per ip/api / who guidelines. the physicochemical parameters like total ash, acid insoluble ash, water and alcohol soluble extractives pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) in comparison with market samples rath chinmay*, suman kumari # , dhar bishnupriya # , mohanty rc ## , dixit renu # , padhi mm # , babu ramesh # # central council for research in ayurveda & siddha, dept. of ayush, ministry of health & family welfare, janakpuri, new delhi-58. ## department of botany, utkal university, vani bihar, bhubaneswar, orissa a b s t r a c t kakoli (roscea procera wall.) of family zingiberaceae and ksheerkakoli (lilium polyphyllum d. don) of family liliaceae, are mentioned as drugs of astavarga, but the botanical identity of both the species are controversial and different drugs are sold/ used in the name of kakoli and ksheerkakoli. this has created frightening problem with regards to the quality, safety and stability of raw materials and their desired therapeutic effcacy. so efforts have been made to lay down pharmacopoeial characters and to identify botanical identity of market samples by comparision with genuine drugs. key words: macroscopy, microscopy, thin layer chromatography, physico chemical evaluation. introduction the identity of astavarga drugs suffer a lot of confusion in ayurvedic literature. according to various ayurvedic nighantu books composed or commented by different vaidyas, it constitutes a group of eight drugs, which form an important constituent of a number of ayurvedic preparations. these are known in sanskrit as jivaka, rishibhak, mahameda, meda, kakoli, ksheerkakoli, riddhi and vriddhi. [1-3] bhav mishra in his bhav prakash nighantu has further mentioned that none of astavarga drugs are true. [4] kakoli and ksheerkakoli have been described under “brhneeya” (the drugs which promote the formation of mansadhatu that is fesh formation) in dhanvantri nighantu, [5] caraka samhita, [6] sushruta samhita [7] and astanghridaya. [8] till date none has tried to work out on their pharmacognosy and chemistry evaluation therefore they need further investigation. there is however a number of crude drugspharmacognosy journal  september 2011  vol 3  issue 25 33 chinmay, et al.: pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) covered with hard membranous scales arranged in a concentric manner and breaking readily with a short fracture; cut surface white to creamish-yellow and starchy; scars of adventitious roots seen; odour, pleasant; taste, bitter. microscopic transverse section of bulb shows concentric layers of scale leaves; axis of bulb show three concentric layers of scale leaves. t.s of scale shows with an outer and inner epidermis consisting of single layered parenchymatous, pentagonal cells with mucilage; cuticle of both epidermis of all the samples were carried out adopting standard procedures. [11-13] the thin layer chromatography of 90 per cent ethanolic extract of all samples were performed on pre-coated silica gel 60 f 254 aluminium plates and the plates were developed in solvent system toluene: ethyl acetate: methanol (8.0:1.6:0.4). the developed plates were observed under uv 254 nm and uv 366 nm and after derivatization visualisation under uv 366 nm. market samples rp1, rp2, rp3 and lp1, lp2, lp3 were collected in the name of kakoli and ksheerkakoli from new delhi, jaipur (rajasthan), mandi (himachal pradesh), respectively and compared pharmacognostically as well as phytochemically with authentic drugs. results macro – and microscopical studies of roscea procera wall. macroscopic roots occur in bunches of 4-15; straight or curved, dark brown; each root about 3-8 (10) cm long, upto 0.9 cm thick; external surface rough due to presence of longitudinal wrinkles; odour, slightly aromatic; taste acrid. microscopic tuberous root shows circular in outline; cork 10-12 layered, consisting of thin-walled, tangentially elongated, almost radially arranged cells, upper cells flled with reddish-brown content; below the cork phellogen layer is present; cortex consisting of oval to elongated, thinwalled, parenchymatous cells flled with abundant, simple, ovoid to ellipsoidal starch grains, measuring 5-11μm in dia.; vascular bundles composed of usual elements, xylem vessels arranged alternatively with phloempatches, vessels mostly solitary with spiral thickening and separated by multiseriate medullary rays; xylem fbres and xylem parenchyma are associated with xylem vessels; phloem consists of sieve tubes, companion cells, phloem parenchyma; pith composed of oval to polygonal, thin-walled, parenchymatous cells. powder greenish-yellow; slightly aromatic in smell; shows cork cells in surface view and section view; in sectional view cork cells with underlying cortex cells, cortex cells flled with crystalline material, simple, ovoid to ellipsoidal starch grains, measuring 5-11 μm in dia., simple pitted vessel and tracheids. macro - and microscopical studies of lilium polyphyllum d. don macroscopic whole bulbs (tuber), conical 1.4 to 3.0 in width and 2.5 to 4.0 cm in length, transluscent with slight longitudinal ridges, figure 1a: t.s. root of roscea procera wall. (diagrammatic); figure 1b: t.s. root of roscea procera wall. (showing cork and cortex region); figure 1c: t.s. root of roscea procera wall. (showing phloem and xylem woody region); figure 1d: powder characteristics of roscea procera wall. (diagrammatic)chinmay, et al.: pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) 34 pharmacognosy journal  september 2011  vol 3  issue 25 comparative pharmacognostic characters of market samples the detail macro- and microscopic characters of all samples rp1-rp3 and lp1-lp3 were studied carefully and compared with authentic samples of roscea procera wall. and it was found that only rp1 are genuine samples and the macro- and microscopical characters of rp2 resembles with withania somnifera (l.) dunal. the details of pharmacognostical characters are given below macroscopic root tuberous attaining diameter of 1 to 2.5 cm, occasionally branched, the distal ends taper slightly woody, outer surface is yellowish brown in colour, smooth but sometimes shallow short longitudinal fssures are seen. the cut end of the tuberous root reveals an inner smooth white starchy tissue surrounded by an outer narrow brown ring. microscopic transverse section of root shows narrow cork, a moderate cortex and a large wood. cork shows 6 to 8 rows of thin walled cubical to slightly tangentially elongated cells. the inner portion of cork tissue. cortex is 3 to 4 mm in thickness, composed of 14-18 thin walled slightly tangentially elongated cells. simple to compound type of starch grains are abundantly embedded in cortex and vary in size, compound starch grains having 2-5 components and almost rounded with a dia. of 15-30 μm and small is slightly wavy and horny, mesophyll consists of 6 to 9 layered hexagonal parenchymatous cells; starch grains gelatinized which are eccentric type (hilium present on one side), deposited in parenchymatous cells and are of various shape i.e. polygonal, oval and truncated and 6-10 in one parenchymatous cell. raphides ranging from 100 to 230 μm in length are also present in the mesophyll; surface view of upper epidermis show compactly arranged rectangular, elongated thin walled cells. longitudinal section of bulb scale shows tracheids, which is composed of narrow, elongated and tubular cells. the lignifed secondary cell wall is scalariform i.e. secondary cell wall material is deposited on primary cell wall forming a ladder like pattern. transverse section of root shows single layered epidermis, the cells of epidermis are pentagonal in shape. cortex is made up of parenchymatous cells which are hexagonal in shape, without intercellular spaces. below the cortex is a thick walled single layered endodermis. pericycle is present which is composed of thin and single layered cells. primary xylem is distributed towards the pith zone and surrounded by phloem. the main part of pith area is occupied by metaxylem. pith is parenchymatous. powder powder creamish with pleasant smell; raphides present; powder treated with ruthenium red, mucilage turns bright pink. figure 2a: bulb peel of lilium pophyllum d.don; figure 2b: t.s. scale of lilium pophyllum d.don; figure 2c: l.s. scale of lilium pophyllum d.don; figure 2d: t.s. root of lilium pophyllum d.donpharmacognosy journal  september 2011  vol 3  issue 25 35 chinmay, et al.: pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) vessels; radially cut medullary rays crossing the vessels and fbres; thin-walled xylem fbres. the physico-chemical analysis (table 1) and tlc of 90 percent ethanolic extract of powders of all samples are carried out using solvent system toluene: ethyl acetate: methanol (8.0:1.6:0.4). and observation are compared. the pharmacognostic studies of lp1-lp3 were also carried out and observed that the macro-and microscopic characters of sample lp3 was found similar to chlorophytum arundinaceum. the details of pharmacognostical characters are given below:- macroscopic peeled dried tuberous root shriveled and sharply tapering at both ends cylindrical and arcuate or 5-7 cm long, 0.4 (0.7) to 1 (1.2) cm thick, longitudinally ridged and furrowed, transversely cracked at places; unpeeled tubers more rough, exhibit root scars. fracture short and brittle; root brownish starch grains of diameter 9 μm. a narrow ring of phloem passing inner to centre is enclosing the wood and intercepted by medullary rays. phloem composed of small sized thin walled polygonal cells. thin walled 2-3 layered broadly rectangular cambium tissue is also present. xylem is a wide consisting of isolated or rarely groups of 2 to 3 vessels embedded in thin-walled fbres occupying the major area of the wood, parenchyma are vesicentric and paratracheal and medullary rays are bent at places expecially when run adjacent to the vessel. powder shows abundant, simple and compound, spherical, oval or cup shaped starch grains with slit like or stellate hilum, scattered as such throughout or embedded in the parenchymatous cells of the cortex; microsphenoidal crystals of calciumoxalate embedded in the parenchymatous cells of the cortex; fragments of suberised cork in transverse and surface view; fragments of longitudinally cut and horse-shoe shaped pitted table 1: observations of physicochemical parameters of powdered samples of kakoli s. no. parameters % rp1 rp2 rp3 rp4 1. total ash (% w/w) 4.5 5.2 4.6 4.0 2. acid insoluble ash (% w/w) 2.0 1.3 1.8 1.5 3. ethanol soluble extractive (% w/w) 6.0 7.0 6.5 5.0 4. water soluble extractive (% w/w) 8.5 8.0 9.2 9.0 5. tlc (figure 3) under uv 254 nm (r f values) 0.15, 0.29 0.20 – – under uv 366 nm (r f values) 0.08, 0.19, 0.23, 0.52, 0.87 0.11, 0.25, 0.39, 0.86 0.12, 0.26, 0.40, 0.88 0.11, 0.26, 0.40, 0.52, 0.68, 0.89 after derivation visualisation under uv 366 nm (r f values) 0.10, 0.40, 0.68, 0.90 0.08, 0.11, 0.46 0.09, 0.14, 0.40, 0.59, 0.11, 0.25, 0.38, 0.49, 0.66 iwhughulydwl]dwlrqylvxdolvdwlrq xqghu89qp 5 i ydoxhv solvent system : toluene : ethyl acetate : methanol (8.0 : 1.6 : 0.4) figure 3: tlc fingerprint of 90% ethanolic extract of samples samples of roscoea procera from rp 1: new delhi, rp 2: jaipur (rajasthan), rp 3: mandi (himachal pradesh), rp 4: tarikhet (uttarakhand)chinmay, et al.: pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) 36 pharmacognosy journal  september 2011  vol 3  issue 25 the apical and lower parts of the root. an endodermis and pericyclic layer are distinct. groups of phloem tissue alternating with exarch xylem bundles in a ring encircled by lignifed xylem parenchyma, lie underneath the pericyclic fbre. pith is very narrow. ts of the peeled root are devoid of epiblema and exodermis tissue. powder powder shows bundles or free acicular crystals of calcium oxalate scattered as such throughout and emebedded in the parenchyma cells; fragments of lignifed, thick-walled radially arranged cells of epiblema in surface view; fragments of three sided thickened lignifed cells of the endodermis in surface view; cortical parenchyma flled with mucilage and acicular crystals of calcium oxalate; pitted and reticulate xylem vessels and pitted lignifed xylem parenchyma. the physico-chemical analysis (table 2) and tlc of 90 percent ethanolic extract of powders of all samples are externally, whitish internally. taste slightly sweet and mucilaginous and devoid of any odour. microscopic diagrammatic transverse section of the root is somewhat circular in outline exhibiting epiblema, an outermost layer, followed by exodermis and wide parenchymatous cortex containing bundles of acicular crystals of calcium oxalate and central very narrow pith encircled by alternately arranged phloem and xylem bundles. detailed transverse section of unpeeled root shows a layer of epiblema with outer lignifed and radially thickened dome shaped cells bearing long hairs which usually get detached in the dry samples. underneath this lie 3 to 5 rows of tangentially elongated cells, exodermis which are oval in shape and radially arranged followed by wide parenchymatous cortical zone containing mucilage and bundles of acicular crystals of calcium oxalate, more in after derivatization visualisation under uv 366 nm (r f values) solvent system : toluene : ethyl acetate : methanol (8.0 : 1.6 : 0.4) figure 4: tlc fingerprint of 90% ethanolic extract of samples samples of lilium polyphyllum from lp 1: new delhi, lp 2: jaipur (rajasthan), lp 3: mandi (himachal pradesh), lp 4: tarikhet (uttarakhand) table 2: observations of physicochemical parameters of powdered samples of ksheerkakoli s. no. parameters % lp1 lp2 lp3 lp4 1. total ash (% w/w) 5.5 6.5 6.7 6.0 2. acid insoluble ash (% w/w) 1.0 1.5 2.0 1.2 3. water soluble ash (% w/w) 22.0 24.0 26.5 25.0 4. water soluble extractive (% w/w) 27.5 26.5 29.0 28.0 5. tlc (figure 4) under uv 254 nm (r f values) – – – – under uv 366 nm (r f values) 0.08, 0.20, 0.34 0.07, 0.23, 0.44, 0.64 0.06, 0.36, 0.45, 0.68 0.09, 0.21, 0.25, 0.36, 0.51, 0.64, 0.83 after derivatization visualisation under uv 366 nm (r f values) 0.02, 0.35, 0.60 0.05, 0.29, 0.65 0.02, 0.32, 0.51, 0.65, 0.82 0.04, 0.35, 0.51, 0.64, 0.83pharmacognosy journal  september 2011  vol 3  issue 25 37 chinmay, et al.: pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) carried out using solvent system toluene: ethyl acetate: methanol (8.0:1.6:0.4), and result compared to the authentic samples. discussions and conclusion macroscopically the roots are straight and curved and dark brown in roscea procera and in withania somnifera roots are occasionally branched, buff to yellow brown while longitudinal wrinkles are present at outer surface in both species. microscopically the tuberous root of roscea procera shows circular in outline, cork consisting of thin walled cells with reddish brown content, cortex thin walled with oval to ellipsoidal starch grains, 5-11 dia. and vascular bundles composed of usually elements, xylem vessels arranged alternatively with phloem parenchyma. the roots of withania somnifera shows narrow cork, moderate cortex and a large wood. in cortical region starch grains are abundant. starch grains are of 2 types, compound with 2-5 components and 15-30 μm and small with 9 μm, a narrow ring of phloem passing inner to centre, enclosing the wood and intercepted by medullary rays are present. cortex cell flled with starch grains and crystalline material and simple pitted vessel and tracheids in powder of roscea procera and in withania somnifera microspheroidal crystals of calcium oxalate present in cortical region and fragments of suberised cork in transverse and surface view and longitudinally cut and horse shoe shaped pitted vessels. in lilium polyphyllum bulbs are conical, translucent with slight longitudinal ridges covered with hard membranous scales. while peeled tuberous roots of shriveled, cyclindrical, sharply tapering at both ends. transverse section of bulb of lilium polyphyllum shows a concentric rings of scale leaves. ts of root shows a single layered epidermis followed by parenchymatous cortexwithout intercellular spaces and the main part of pith is occupied by metaxylem. however in chlorophytum arundinaceum outer layer of epiblema with long hairs which usually get detached in dry samples, followed by compact layers of exodermis and a wide parenchyma cortical zone with mucilage and bundles of acicular crystals of calcium oxalate. the observation of physico-chemical evaluation indicates that the most drugs available in market are not genuine and adulterated with other plant drugs which are easily available in the market. in this dimension pharmacognostic anjd phytochemical studies onmarket samples is a substantial figure 5a: t.s. root of withania somnifera (l.) dunal (diagrammatic); figure 5b: t.s. root of withania somnifera (l.) dunal (showing cellular details); figure 5c: powder characteristics of withania somnifera (l.) dunalchinmay, et al.: pharmacognostical & phytochemical studies of roscea procera (kakoli) and lilium polyphyllum (ksheerkakoli) 38 pharmacognosy journal  september 2011  vol 3  issue 25 5. sharma pv. dhanwantri nighantu. varanasi, india: chaukambha orientalia; 1998. 6. charaka samhita. part i (hindi commentary by kashi nath shastri) varanasi, india: chaukhamba sanskrit series office; 1969. 7. susruta samhita (hindi commentary by ambika dutt shastri). varanasi, india: chaukhamba sanskrit sansthan; 1953. 8. astangahridayam of vagbhata (padradakar bh (vaidya)). varanasi, india: chaukambha orientalia; 1969. 9. kay al. microscopically studies on drugs. london; balliere tindall and cox: 1938. 10. trease ge, evans wc. pharmacognosy. balliere,tindall: 1983. 11. kokate ck, purohit ap, gokhale sb. pharmacognosy. pune, india; nirali prakashan: 2001. 12. chase cr, pratt fj. fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. j am pharm assoc1949; 38:324-3. 13. anonymous. indian pharmacopoeia, 2 nd ed., delhi; 1966. step and it further requires a long term study and this type of work is benefal for scientists for identifcation of raw material and for industries also. references 1. puri hs. traditional herbal medicine for modern times, rasayana – ayurvedic herbs for longevity and rejuvenation. london: taylor & francis; 2003 2. pandey g. dravyaguna vijnana. ii nd edition: varanasi india. krishnadas academy; 2002. 3. gogte vaidya v m. ayurvedic pharmacology and therapeutic uses of medicinal plants – dravyagunavignyan. (english translation). mumbai: bharatiya vidya bhavan; 2000. 4. chunekar kc. bhav prakash nighantu. varanasi, india: chaukhambha bharti academy; 1982. figure 6a: t.s. root of chlorophytum arundinaceum baker (diagrammatic); figure 6b: t.s. root of chlorophytum arundinaceum baker (showing tissues of outer region); figure 6c: t.s. root of chlorophytum arundinaceum baker (showing tissues of central region); figure 6d: powder characteristics of chlorophytum arundinaceum bakerpharmacognosy journal  september 2011  vol 3  issue 25 39 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: thakur nirmladevi ph: 919987046862 e-mail: nirmlaanywhere@gmail.com doi: 10.5530/pj.2011.25.7 world has been using plants from thousands of years for treating various ailments of humans & animals. [3] herbal medicines are promising choice over modern synthetic drugs. they show minimum/no side effects and are considered to be safe. generally herbal formulations involve use of fresh or dried plant parts. correct knowledge of such crude drugs is very important aspect in preparation, safety and effcacy of the herbal product. pharmacognosy is a simple and reliable tool, by which complete information of the crude drug can be obtained. [4- 8] though the traditional indian system of medicine has a long history of use, they lack adequate scientifc documentation, particularly in the light of modern scientifc knowledge. [9] to ensure reproducible quality of herbal medicines, proper control of starting material is utmost essential. the frst step towards ensuring quality of starting material is authentication followed by creating numerical values of standards for comparison. pharmacognostical parameters for easy identifcation like leaf constants, microscopy & physic chemical analyses are few of the basic protocol for standardization of herbals. [10-11] the numbers of reports of patients experiencing negative health consequences caused by the use of herbal medicine has increased in recent year. analysis & studies have revealed a variety of reasons for such problem. one of the major pharmacognostic and phytochemical investigation of juglans regia linn. bark thakur nirmladevi 1* ; vinita apraj 1 ; ashok bhagwat 2 ;rashmi mallya 3 ; laxman sawant 4 ; nancy pandita 4 1 school of science, svkm’s nmims, vile parle (west), mumbai -56. 2 svkm’s c.b. patel research centre, vile parle (west), mumbai -56. 3 svkm’s dr. bhanuben nanavati college of pharmacy, vile parle (west), mumbai -56. 4 school of pharmacy & technology management, svkm’s nmims, vile parle (west), mumbai -56 a b s t r a c t juglans regia linn belongs to family juglandaceae. it is commonly known as walnut tree. juglans regia bark has been claimed to possess anti-infammatory, blood purifying, anticancer, depurative, diuretic and laxative activities. the bark is fnely powdered and used to prevent bleeding gums and as a mouth rinse. the present investigation deals with microscopic evaluation of bark and establishment of its quality parameters, including physicochemical, phytochemical evaluation, hptlc analysis & microbial load. chief microscopic characters include cork, phloem fbres with stone cells & calcium oxalate crystals. phytochemical screening revealed presence of reducing sugars; alkaloids; tannins & phenols; steroids & saponins. the bark powder was found to be free from pathogenic organisms. the study will provide referential information for the correct identifcation of the crude drugs. key words: juglans regia linn, pharmacognostic study, phytochemical analysis, hptlc analysis. introduction juglans regia linn known as akhort in india, a native of eastern europe to north asia i.e. china, iraq, mexico, spain, turkey, nepal, india (forests in himalayas) is a member of juglandaceae family. it is a woody, deciduous and frost-tender tree growing to 20m height. the wood is heavy, durable and polishes well. the bark is resinous and scented. this valuable tree has a long history of medicinal use to treat a wide range of health complaints. almost all parts of the plant are medicinally important. the root and stem bark are anti-helmentic, astringent and detergent. the stem bark is dried and used as a tooth cleaner. the decoction of leaves and bark is used with alum for staining wool brown. [1] herbal medicine is a triumph of popular therapeutic diversity. plants above all other agents have been used for medicine from time immemorial because they have ftted the immediate personal need are easily accessible and inexpensive. [2] human population in countries around thenirmladevi, et al.: pharmacognostic and phytochemical investigation of juglans regia linn. bark 40 pharmacognosy journal  september 2011  vol 3  issue 25 physiochemical analysis physicochemical properties such as the percentage of total ash, acid insoluble ash, water soluble ash, alcohol soluble extractive &water soluble extractive values were determined as per the standard procedure. [14] percentage of ash value is indicative of the purity of the drug and extractive values represent the presence of polar and non polar compounds in the extract. fluorescence analysis fluorescence study is an essential parameter for frst line standardization of crude drug. the crude powders were subjected to these studies & their fuorescence patterns were noted. the powder material were treated separately with different reagents & exposed to visible, ultraviolet light to study their fuorescence behaviour. [18] the colors obtained by application of different reagents in different radiations were recorded hptlc analysis chromatographic fnger-printing of phytoconstituents can be used for the assessment of quality consistency and stability of herbal extracts or products by visible observation and comparison of the standardized fngerprint pattern. the fngerprint has potential to determine authenticity and reliability of chemical constituents of herbal drug and formulations. chromatographic separation of hot & cold methanolic extracts of juglans regia bark were performed on 10 cm 10 cm aluminum-backed hptlc plates coated with 200 μm layers of silica gel 60gf254 (merck, darmstadt, germany). standard solution of gallic acid & methanolic extracts (10 μl each) were applied on to hptlc plate as 8 mm wide bands and 12 mm apart from middle of bands by spray-on technique along with nitrogen gas supply for simultaneous drying of bands, by means of a camag automatic tlc sampler 4 (ats4). a constant spot application rate of 10 μl/sec was used. plates were developed to a distance of 80 mm at room temperature (28 2 c) with chcl 3 : ethylacetate: formic acid (7.5:6:0.5) (v/v) as mobile phase in a camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. chromatography was performed in camag’s twin-trough chamber. after development, the plates were dried in air & then scanned at 340nm with camag tlc scanner with camag wincats planar chromatography manager software (version 1.4.2). the plate was later on derivatized with anisaldehyde sulphuric acid & heated at 105 c till bands develop. determination of microorganisms medicinal plant materials normally carry a great number of bacteria & moulds, often of soil origin. while a large cases of reported adverse events are directly linked to the poor quality of herbal drug and raw medicinal plant materials. [12] this traditional knowledge about the plants can be transferred to several generations only by proper documentation of their botanical, physicochemical, phytochemical characters and along with their medicinal uses in the form of monographs. the monograph of these plants are prepared according to thewhoguidelines and presented as herbal pharmacopoeia. these guidelines enable to identify, authenticate, detect adulterants and standardize the plant material. [3] this present work, thus aims to standardize juglans regia linn bark by pharmacognostic and preliminary phytochemical analysis. materials and methods collection and authentication juglans regia linn dried bark was procured from the local market in mumbai. it was identifed & authenticated by prof. bindu of botany department of svkm’s mithibai college of science &commerce, vile parle (west), mumbai. the dried bark was used for section cutting & the bark powder was used for phytochemical analysis. the morphological studies such as colour, odour and taste of juglans regia bark were studied. microscopic sections were cut by free hand sectioning method. the sections of bark were cleared with chloral hydrate solution & then stained with phloroglucinol & hcl & mounted in glycerine. numerous mounts of the microscopical sections of the bark specimens were made and examined microscopically. photomicrographs of the microscopical sections were taken with the help of motic photomicroscope provide with motic image plus 2.0 software. phytochemical analysis thepowderedbarkwas subjected topreliminaryphytochemical screening for qualitative detection of phytoconstituents. the dried and coarsely powder (50 g) was extracted in (300ml) methanol by soxhlet method (hot methanolic extraction) & for cold maceration (cold methanolic extraction) 25g powder in 150 ml methanol. the concentrated extracts were evaporated to dryness and the extracts were then weighed. their percentages were calculated in terms of initial air dried plant material. the colors of the extracts were observed. the extracts as mentioned above, were subjected to various qualitative phytochemical tests for the identifcation of chemical constituents present in the plant material as per standard procedure. [13-17]pharmacognosy journal  september 2011  vol 3  issue 25 41 nirmladevi, et al.: pharmacognostic and phytochemical investigation of juglans regia linn. bark powder microscopy juglans regia powder was brown in colour & showed presence of stone cells, fbers & calcium oxalate crystals (figure 4). preliminary phytochemical test preliminary phytochemical test for hot & cold methanolic extract of the drug was carried out. both the extracts showed the presence of reducing sugars; alkaloids; tannins & phenols; steroids & saponins (table 1). physico-chemical constants the powdered bark of juglans regia was studied for their physico-chemical constant which included percentage of range of bacteria & fungi form the naturally occurring microfora of herbs, aerobic spore forming bacteria frequently predominate. current practices of harvesting, handling & production often cause additional contamination & microbial growth. determination of total viable count & detection of pathogens was performed as per the method in who guideline on “quality control methods for medicinal plant materials”. [19] results and discussion morphology bark of juglans regia was dull blackish brown in colour. it was thin with whitish epidermal layer tough and fbrous and somewhat mealy. inner fbers were tough and fattened; the outer ones were white and silky. the taste of bark slightly bitter and astringent microscopy the transverse section of juglans regia showed one cell layer thick cork on the outermost side of the bark. it also showed presence of phloem fbres with stone cells present in them. crystals of calcium oxalate were found to be scattered amongst the stone cells (figure 1-3). figure 1: transverse section of dried bark of juglans regia linn figure 2: transverse section of dried bark of juglans regia linn showing calcium oxalate crystals figure 3: powder microscopy of dried bark of juglans regia linn figure 4: hptlc profile of methanolic extracts of juglans regia linn. bark 1- jugalns regia cold methanolic extract (10 mg/ml); 2- jugalns regia hot methanolic extract (10 mg/ml) & 3, 4- standard gallic acid (0.5 mg/ml).nirmladevi, et al.: pharmacognostic and phytochemical investigation of juglans regia linn. bark 42 pharmacognosy journal  september 2011  vol 3  issue 25 quantifcation of gallic acid in the extract was carried out. hot & cold methanolic extract of juglans regia showed 1.4% & 1.08% of gallic acid respectively. determination of microorganisms total aerobic plate count of juglans regia bark powder was found to be 2.41 10 5 cfu/ml & no fungal propogules were observed in total fungal count (table 4). the bark was also found to be free from objectnable pathogens. discussion the information obtained from preliminary phyto-chemical screening will be useful in fnding out the genuity of the drug. ash values; extractive values & fuorescence analysis are few parameters, which normally are adopted to get the qualitative information about the purity & standard of the crude drug. the percent extractives indicate the quantity and nature of constituents in the extracts. morphological and anatomical studies discussed can be considered as a distinguishing parameter to identify & decide the authenticity of this drug. these simple but reliable standards will be useful to a lay person in using the drug as a home remedy. conclusion the data produced in the present investigation is also helpful in the preparation of the crude drug’s monograph and inclusion in various pharmacopoeias. also the manufacturers total ash, acid-insoluble ash, water-soluble ash, alcohol soluble extractives (table 2). fluorescence analysis of extract and drug powder the fuorescence analysis of the powdered drug of juglans regia in various solvents and chemical reagents were performed under normal and uv light. there was no fuorescence observed under uv long (365nm) with any of the chemicals (table 3). hptlc analysis hptlc analysis of methanolic extracts was carried out using chcl 3 : ethylacetate: formic acid (7.5:6:0.5) (v/v) as a mobile phase. hptlc screening of the extracts was established to substantiate the standardization data on juglans regia linn (figure 5). as gallic acid was used as standard, table 1: results of phytochemical screenings of extract of juglans regia linn test juglans regia bark reducing sugars amino acids – flavonoid – alkaloid tannins and phenols steroids – saponins key: present, – not present table 2: results of physicochemial properties of dried bark powder of juglans regia linn physicochemial properties result (% w/w) total ash 9.51% acid insoluble ash 0.125% water soluble ash 1.035% alcohol soluble extractive 6.03% water soluble extractive 4.02% table 3: fluorescence analysis of dried bark powder of juglans regia linn treatment day light uv (254 nm) powder 1n naoh (aq.) light brown black powder 1n naoh (alc.) chocolate brown black powder conc. hcl chocolate brown black powder conc. h 2 so 4 yellowish brown black powder conc. hno 3 yellowish black black powder chloroform orange black powder glacial acetic acid yellow yellow powder 5% naoh brown black powder 5% koh chocolate brown black powder 5% fecl 3 chocolate brown black powder ammonia greenish black black table 4: total viable count of dried bark powder of juglans regia linn microbial method medium for plating microbial counts total aerobic plate count letheen agar 2.41 10 5 cfu/ml total fungal count sabourauds dextrose agar no fungal propagules were observed figure 5: transverse section of dried bark of juglans regia linn showing phloem fiberspharmacognosy journal  september 2011  vol 3  issue 25 43 nirmladevi, et al.: pharmacognostic and phytochemical investigation of juglans regia linn. bark 7. trease ge and evans wc. pharmacognosy, 15 th ed. harcourt brace & co. asia, pvt. ltd., w.b. saunders company ltd, 2002. 8. biren n. shah and avinash k. seth. pharmacognostic studies of the lagenaria siceraria (molina) standley. international journal of pharmtech research 2010; 2 (1):121-24. 9. k. raveendra retnam & a john de britto. pharmacognostical study of hybanthus enneaspermus (linn.) f. muell. natural product radiance 2007; 6 (5):386-90. 10. d. sathis kumar, n. srisutherson, b. pradeep kumar reddy, s. vinitha, t. yadhagiri rao, david banji. pharmacognostic studies on boswellia ovalifoliolata. journal of pharmacy research 2011; 4 (5): 1374-75. 11. d. sathis kumar,veena mandarapu, david banji, rao knv, chandrashekar, sudhakar. k, et al. pharmacognostical study on piper trioicum roxb. int j pharm pharm sci 2011; 3(3):129­32. 12. richardo rmf.medicinal plants into drugs (ed.) by ahemad i,aquil f. and owais m willey ‐ vch bioactive phytocompound: new approaches in the phytosciences in 13. mordern phytomedicine, gmbh & co. weinheim, 2006; 1 ‐ 24. 14. kokate ck. practical pharmacognosy, 4 th edition. new delhi, vallabh prakashan, 2004; p.123. 15. khandelwal kr. practical pharmacognosy,techniques and experiments, 12 th edition. nirali prakashan, 2004; p.157. 16. the ayurvedic pharmacopoeia of indian ministry of health and family welfare new delhi, 1996; part-1, 1:24. 17. harborne jb. method of extraction and isolation in. phytochemical methods. chapman & hall, london. 1998; 60-66. 18. ali m. text book of pharmacognosy, 2 nd edition. cbs publishers and distributers, new delhi, 2003; 64-65, 96-97, 139-140, 272, 283. 19. shanta, t.r., j.k.p. shetty, i. ammal & t. bikshapathi. pharmacognostical studies on vata shrung, (ficus bengalensis linn, leaf primordium). indian j.trad. knowledge 2006; 5:388-93. 20. who/pharm/92.559/rev.1. quality control method of medicinal plant materials (organization mondaile de la santé, geneva), 1992; 48-54. can utilize them for identifcation and selection of the raw material for drug production. acknowledgement the author is grateful to dr. aparna khanna, dean, school of science, nmims university; dr. bhanuben nanavati college of pharmacy for extending laboratory competence & providing necessary amenities to carry out this work. author is also highly obliged to anchromtest lab for allowing to carry out hptlc analysis at their premises. references 1. kale a., sapana shah, sucheta gaikwad, kavita mundhe, nirmala deshpande, jyoti salvekar. elements from stem bark of orchard tree - juglans regia. international journal of chemtech research 2010; 2 (1):548-50. 2. pullaih t. flora of andhra pradesh, anantapur, scientific publishers, 1997; 1:206. 3. k.ashokkumar,sramachandrasetty and laxmi narsu.pharmacognostic and phytochemical investigations of roots of hibiscus micranthus linn. rjpbcs 2010; 1(4):324-37. 4. gokhale sb.textbook of pharmacognosy, nirali prakashan, 1979. 5. mukherjee pk.quality control of herbal drugs-an approach to evaluation of botanicals, business horizons pharmaceutical publishers, 2002. 6. raghunathan k and mitra r. pharmacognosy of indigenous plants, central council for research in ayurveda and siddha, 1982.o r i g i n a l a r t i c l e p h c o g j . 44 pharmacognosy journal  september 2011  vol 3  issue 25 *address for correspondence: research assistant, pharmacology laboratory, institute for post graduate teaching & research in ayurveda, gujarat ayurved university, jamnagar - 361 008, gujarat, india ph: 09429333816 e-mail: drashokbeekay@yahoo.co.in doi: 10.5530/pj.2011.25.8 healers extensively use this plant to treat abdominal tumors. further, the ethnobotanical value of the tuberous root of this plant refers to its recognized action as a remedy for several diseases like rheumatism, gout, paralysis and arthritis [6,7] with proven anti-infammatory [8] and anti-bacterial activities. [9] previously we have explored analgesic activity of tuberous roots of this plant. [10] regarding the phytochemical profle only constituents like diterpene lactone caudicifolin, methylellagic acid and euphol were reported. [11,12] however no reports are available regarding physicochemical and chromatographic profles of this drug till date. hence the present study was undertaken to evaluate preliminary phytochemical and chromatographic profle of tuberous roots of e. fusiformis. materials and methods plant materials: the tuberous roots of e. fusiformis were collected fromwaghai forest, dang, gujarat, india in fully matured condition in the month of november and the material was authenticated by the taxonomist of our institute. the tuberous roots were made into slices and shade dried for 12 days. the dried root slices were pulverized to fne powder and utilized for phytochemical analysis. analysis of physicochemical parameters: physicochemical parameters like loss on drying at 105 c, total ash value, preliminary phytochemical evaluation of euphorbia fusiformis buch-ham. ashok bk 1 *, savitha d bhat 2 , shukla vj 3 , ravishankar b 4 1* research assistant, pharmacology laboratory, ipgt & ra, dhanvantri mandir, gujarat ayurved university, jamnagar. 2 ph.d scholar, department of dravyaguna. i.p.g.t. & r.a. (mail: anudivas@yahoo.co.in) 3 head, pharmaceutical chemistry lab, i.p.g.t. & r.a. 4 director, sdm research centre for ayurveda and allied sciences, kuthpady, udupi. karnataka – 574118 (mail: gravishankar2000@yahoo.com) a b s t r a c t the present investigation was undertaken to analyze the physicochemical and chromatographic profle of dried tuberous roots of euphorbia fusiformis. physicochemical parameters like loss on drying, total ash value, acid insoluble ash, water insoluble ash, various extractive values, ph etc., were carried out. further, qualitative tests for various functional groups like alkaloids, glycosides etc., were carried out in methanol and water extracts. the results of the preliminary phytochemical screening indicated the presence of carbohydrates, starch, favanoids and steroids. thin layer chromatography was carried out using different solvent systems which revealed two common spots indicating the presence of some common phyto-constituents. the parameters of present study can be used as a reference for further scientifc investigations. key words: euphorbia fusiformis, euphorbiaceae, chromatography, total ash, caudicifolin introduction the use of medicinal plants still plays a vital role to cover the basic health needs in both developed and developing countries. [1,2] the medicinal value of these plants lies in some chemical active substances that produce a defnite physiological action on the human body. each and every plant has got its own chemical characteristics which help in separating it from other closely related species. [3] to explore the hidden secrets of the plant kingdom such as their complex compounds or active principles which are thought to be responsible for their effectiveness, it is necessary to undertake the analytical evaluation. euphorbia fusiformis buch.-ham. (family: euphorbiaceae) is a rare medicinal plant found in tropical himalaya up to 1500 ft. fromgarhwal to nepal. it is also found in konkan and deccan hills. [4] in gujarat state it is found in dang, rajpippala and chotaudaipur regions, [5] where traditionalpharmacognosy journal  september 2011  vol 3  issue 25 45 ashok, et al.: phytochemical evaluation of euphorbia fusiformis results the results of physico-chemical parameters have been depicted in table-1. the results of the preliminary phytochemical screening for various functional groups indicated the presence of carbohydrates, starch, favanoids and steroids (table -2). the rf values of different extracts have been tabulated in table 3, where rf values 0.95 and 0.97 were common to all the three extracts. acid insoluble ash, water insoluble ash, water soluble extractive value, alcohol soluble extractive value and ph value were carried out by referring standard procedure. [13,14] qualitative test for various functional groups: qualitative tests for various functional groups like alkaloids, glycosides etc., were carried out by using the aqueous and methanol soluble extracts of the sample . [15-17] chromatographic evaluation: the chromatographic studies were performed using various solvent systems to confrm the phytochemical studies. silica gel gf 254 (precoated plates) were used for the chromatographic evaluation. [18,19] sample preparation methanol extract: about 5 g of accurately weighed powder sample was taken in a conical fask and 100 ml methanol was added to it, shaken and kept overnight. next day it was fltered. then it was concentrated to 5 ml and sample was used for spotting (a). petroleum ether extract: about 5 g of accurately weighed powder sample was taken in a conical fask and 100 ml petroleum ether was added to it, shaken and kept overnight. next day it was fltered. then it was concentrated to 5 ml and sample was used for spotting (b). chloroform extract: about 5 g of accurately weighed powder sample was taken in a conical fask and 100 ml chloroform was added to it, shaken and kept overnight. next day it was fltered. then it was concentrated to 5 ml and sample was used for spotting (c). chromatographic conditions: a. for steroids:  mobile phase: tolune: ethyl acetate (9:1)   stationary phase: silica gel gf 254 (precoated plates)  detection: a) short u-v (254 nm): figure 1,  b) long u-v (366 nm): figure 2,   c) spraying with vanillin sulphuric acid followed by heating at 110 c for 10 min. figure 5 b. for favonoids  mobile phase: ethyl acetate: formic acid: water (6.7:1.5:2.6)   stationary phase: silica gel gf 254 (precoated plates)  detection: a) short u-v (254 nm): figure 3  b) spraying with vanillin sulphuric acid followed by heating at 110 c for 10 min. figure 4 figure 1: tlc profile of e. fusiformis root table 1: physico-chemical parameters of roots of e. fusiformis parameters results loss on drying at 105 c 11.50% w/w ash value 7% w/w acid insoluble ash 0.6% w/w water insoluble ash 4% w/w water soluble extractive 8.38% w/w methanol soluble extractive 5.56% w/w ph 6.9ashok, et al.: phytochemical evaluation of euphorbia fusiformis 46 pharmacognosy journal  september 2011  vol 3  issue 25 to ensure the absence of an undue proportion of extraneous mineral matters introduced accidentally or mixed at the time of collection or in subsequent treatment. in present study test drug have shown total ash content of 7% w/w. treatment of ash with hydrochloric acid leaves virtually only silica. hence it is done to detect the silica in the drug. the ash obtained was further analyzed for acid insoluble particles in ash. in present study values of acid insoluble ash and water insoluble ash were 0.6% w/w and 4% w/w respectively. the information obtained from preliminary phytochemical screening will be useful in fnding out the genuity of the drug and also to fnd out the phytoconstituent present in the test drug. the results indicated the presence of carbohydrates, starch, favanoids and steroids which may be responsible for various biological expressions. the preliminary phytochemical test results were rationalized by the thin layer chromatographic studies, which revealed only two common spots in three different extracts, indicating the presence of some common components. conclusions at our best knowledge this is the frst preliminary physico-chemical and chromatographic study on dried tuberous roots of e. fusiformis and this will be helpful for the identifcation of this drug in powder form. references 1. world health organization (who): the promotion and development of traditional medicine.technical report series, p. 622; 1978. 2. siddiqui hh. safety of herbal drugs-an overview. drugs news and views. 1993; 1(2):7-10. 3. ahmad m, khan ma, zafar m, arshad m, sultana s, abbasi bh, din su. use of chemotaxonomic markers for misidentified medicinal plants used in traditional medicines. j med plant res. 2010; 4(13):1244-52 4. gamble js. flora of presidency of madras. published under authority of the secretary of state of london, vol. ii, p.1272; 1921. 5. bedi sj. ethno botany of the ratan mahal hills gujarat. economic botany. 1978; 32:278-84. discussion the plants of euphorbiaceae family are known for their therapeutical interest in both organized (such as ayurveda and unani) and un-organized (folklore) system of medicine. [20-21] they exhibit great chemical diversity and several of them have been listed as source of valuable drugs. [22] one of the genus of this family euphorbia comprises a large and diverse group of plants, which are characterized by the presence of white milky latex and reported to have a number of interesting biological agents. [20,23-25] many substances absorb moisture on storage, presence of moisture may affect the preservation quality of the drug. loss on drying in a sample corresponds to moisture content and volatile matter content in it. the loss on drying at 105 c was 11.50% w/w, indicative of some moisture content in drug. total ash content of crude drug is the inorganic residue remaining after incineration. it represents the inorganic salts occurring naturally in the drug and also inorganic matter from external sources. the ash value is determined table 2: qualitative tests for various functional groups functional groups tests performed results carbohydrates molish’s test reducing sugar test keller kiliani test test for starch alkaloid dragendorff ’s test – mayer’s reagent test – flavonoid shinoda test steroid lb reagent sarkowski reaction: tannin neutral fecl 3 – gelatin test – resin acetic anhydride – glycoside molisch’s test – saponin distilled water – lead acetate – coumarin ammonia test – table 3: tlc profile of roots of e. fusiformis conditions rf values of samples figures steroids: a b c short u-v (254 nm): 0.13, 0.19, 0.97 [3] 0.10, 0.15, 0.93 [3] 0.10, 0.16, 0.88 [3] figure 1 long u-v (366 nm) 0.26 [1] 0.17, 0.99 [2] 0.99 [1] figure 2 derivatization: vanillin sulphuric acid 0.16, 0.24, 0.42, 0.66 [4] 0.13, 0.55, 0.97 [3] 0.33, 0.61 [2] figure 5 flavonoids: short u-v (254 nm): 0.38, 0.63, 0.97 [3] 0.97 [1] 0.97 [1] figure 3 derivatization: vanillin sulphuric acid 0.89, 0.95 [2] 0.95 [1] 0.95 [1] figure 4 total numbers of spots are provided in parenthesis next to rf values.pharmacognosy journal  september 2011  vol 3  issue 25 47 ashok, et al.: phytochemical evaluation of euphorbia fusiformis 15. harborne jb. phytochemical methods- a guide to modern techniques of plant analysis. 3 rd edition, springer india pvt. ltd, p. 49-129; 1998. 16. aj baxi, vj shukla, ub bhat. methods of qualitative testing of some ayurvedic formulations, gujarat ayurved university, jamnagar, india, p. 05‑12; 2001. 17. kr khandelwal. practical pharmacognosy techniques and experiments. nirali prakashan, pune, india, p.149-56; 2000. 18. wagner h, bladt s. plant drug analysis- a thin layer chromatography atlas, ii edition,thomson press ltd. india, p. 275-78; 1996. 19. stahl e. thin- layer chromatography- a laboratory handbook, i reprint, springer-verlag- berlin-heidelberg, p.241-247, p.427-431; 2005. 20. julius t, mwine, patrick van damme. why do euphorbiaceae tick as medicinal plants? a review of euphorbiaceae family and its medicinal features. journal of medicinal plants research. 2011; 5(5):652-62. 21. seigler ds. phytochemistry and systematics of the euphorbiaceae. ann. mol. bot. gard. 1994; 81:380-401. 22. webster g.the genera of euphorbiaceae in the southern united states. j. arnold arb harvard university. 1967; 48:303-430. 23. kawadikar sr, kazmi sm, trivedi vb. effect of extracts of some euphorbias on fungi. bull bot soc.1976; 23-24. 24. reeta k, vijaya rani, eugine leo s, prakash, geetha k, dhandapani b, dhanabal k. evaluation of pharmacognostic, phytochemical and antimicrobial activity of euphorbia rothiana phcog j. 2009; 1(1), 22-24. 25. fred-jaiyesimi aa, abo ka. phytochemical and antimicrobial analysis of the crude extract, petroleum ether and chloroform fractions of euphorbia heterophylla linn whole plant. pharmacognosy journal. 2010;(2)16:1-4. 6. prakash a, singh kk. use of medicinal plants by certain tribal people in north india. journal of tropical medicinal plants. 2001; 2:225-9. 7. singh gb, kaur s, satti nk, atal ck, maheswari jk. anti-inflammatory activity of euphorbia aqualis roxb. journal of ethnopharmacology. 1984; 10:225-33. 8. natarajan d, britto sj, srinivasan k, nagamurugan n, mohanasundari c, perumal g, antibactrial activity of euphorbia fusiformis - a rare medicinal herb. journal of ethnopharmacology. 2005; 102:123-6. 9. andimuthu ramachandran, devarajan natarajan, chokkalingam mohanasundari, kesavan srinivasan, sebastian soosairaj, ekambaram natarajan, screening for antibacterial activity of combined extracts of euphorbia fusiformis fromtamil nadu, india,a new application. journal of biological research. 2005;(9):97-102. 10. ashok bk, savitha bhat, ravi rao s, ravishankar b. screening of antinociceptive activity of euphorbia fusiformis buch-ham. ijrap. 2011; 2(1):27-9. 11. khanna nm. chemical examination of e. acaulis roxb. ind j pharma. 1954; 16:110-11. 12. ram rastogi, mehotra bn. compendium of indian medicinal plants. central drug research institute lucknow and national institute of science communication, new delhi, vol. 4:308; vol. 5:351; 1999. 13. karnick cr. pharmacopoeial standards of herbal plants. sri saguru publication, p.124; 1994. 14. anonymous.the ayurvedic pharmacopoeia of india, part i,vol. i, edition i, (the controller of publications,civil lines,delhi on behalf of government of india, department of indian systems of medicine and homeopathy), p.143; 2000.o r i g i n a l a r t i c l e p h c o g j . 48 pharmacognosy journal  september 2011  vol 3  issue 25 *address for correspondence: e-mail: prosopis.1912@gmail.com doi: 10.5530/pj.2011.25.9 tree species, as the major problems of such soils are high water table, high salinity impeded drainage and less soil aeration for tree growth, singh . [3] phosphorus metabolism occupies a key position in cellular-biochemistry as it is related with energy relation in respiration and photosynthesis. hence, an attempt has been made to study the phosphorus metabolism in the roots and leaves of prosopis julifora seedlings grown under salinity stress in laboratory conditions. materials and methods for the experiment, seeds were obtained from the pod of prosopis julifora plants growing in the salt affected agriculture feld in sangli district in themonth of april-may. mechanically scarifed seeds were used to raise the seedlings. after the establishment of seedlings for 5 days, they were treated with increasing concentration of salt (100, 200, and 300mmnacl) mix with half strength hoagland solution. the seedling were grown for one month and then analysed for phosphorus metabolism. the method of sekine et al, [4] was employed for estimation of phosphorus from the root and leaves. fresh leaves and roots were used for the assay of enzymes of phosphorus metabolism. for enzyme acid phosphatase crude enzyme was prepared in 0.1 m acetate buffer (ph 5) and assayed according to the method of mclachlan . [5] the activity of enzyme atpase was determined following the method influence of salt stress on phosphorus metabolism in the roots and leaves of one month old prosopis juliflora (sw.) dc seedlings *patil, amol v. and chavan, p. d. department of botany, shivaji university, kollhapur-416 004 (ms) india. a b s t r a c t a sand culture experiment was designed to study the effect of sodium chloride salinity on phosphorus metabolism in the roots and leaves of one month old prosopis julifora (sw.) dc seedlings. it was found that the p level in the roots as well as leaves was decreased with increasing level of salinity in rooting medium. however, the activities of enzymes acid phosphatase and atpase were increased in both the parts of seedlings grown in saline conditions. the activities of alkaline phosphatase and inorganic pyrophosphatase were found to be decreased in the root and leaves of seedlings grown under saline conditions. key words: salinity, phosphorus, enzymes, prosopis julifora. introduction the selection and breeding of salt tolerant crops is regarded as one of the main approaches to deal with a serious problem of salt affected soils throughout the world. in order to achieve this strategy it is necessary to identify the mechanisms of salt tolerance in the plant species well adapted to such problem soils. prosopis julifora is one such plant species which can successfully grow and complete its life cycle in a variety of problem soils. it is noticed that the plant has successfully established in farmlands of digraj (dist. sangli) which are heavily affected by secondary salinization. prosopis julifora is a multipurpose plant of great economic potential. the ability of this species to grow on the poorest soil, under arid conditions and on saline soil is well known pasiecznik et al,. [1] according to dagar and tomar , [2] in india about 8.53 million ha land is waterlogged, 5.50 million ha land is saline and 3.88 million ha land is alkaline and more and more land is becoming water logged due to several factors. according to cssri these soils can be judiciously utilized for raising forestry, agriculture and horticulture crops. afforestration programme for saline soil requires the proper selection ofpharmacognosy journal  september 2011  vol 3  issue 25 49 patil, et al.: influence of salt stress on phosphorus metabolism in the roots and leaves of one month old prosopis juliflora (sw.) dc seedlings which appears to be quite stable during salt stress in this species, the phosphorus nutrition in -prosopis julifora seems to be sensitive to salt stress. the disturbance in p nutrition can have signifcant effects on overall plant metabolism in view of a key role of this element in cellular biochemistry. effect of sodium chloride salinity on the activity of enzyme acid phosphatase in the leaves and roots of prosopis julifora is recorded in fgure 2(a). it is evident that the activity of this enzyme in both root and leaves is stimulated at all salinity levels except 300 mmnacl, at which it has decreased in the roots. enhancement in the activity of acid phosphatase in the leaves of spinach grown under saline condition has been reported by pan. [18] similar observations have been made by karadge and chavan [19] in sesbania. lila arab and ehsanpour [20] measured acid phosphatase activity in the leaf and stem of in vitro grown medicago sativa under saline conditions and found that the activity was increased due to increasing salt concentration. chakrabarti andmukharji [21] have also found that the salt stress caused to increase the activity of acid phosphatase in the leaf and roots of mung bean. parida and das [22] studied effect of various levels of salinity (0, 100, 200, 400mm nacl) on the activity of acid phosphatase in bruguiera parvifora growing under hydrophonic culture. their experiments also revealed that the salinity causes stimulation of activity of this enzyme. effect of nacl salinity on the activity of enzyme alkaline phosphatase in the leaves and roots of prosopis julifora is depicted in the fgure 2 (b). it is evident that the activity of this enzyme is decreased in the root and leaves with increasing level of salt in the medium. weimberg [23] noticed a decrease in the level of alkaline phosphatase in pea seedlings due to nacl salinity. a contrasting behavior of acid and alkaline phosphatases under saline conditions was noticed by ahmad and huq [24] in halophytic spinach. in the case of horsegram only lower concentration of salt (25 mm of nacl) caused the real increase in alkaline phosphatase activity. [25] parida and das [22] noticed that the activity of this enzyme in a mangrove, bruguiera parvifora was increased under varying levels of salinity (0, 100, 200, 300 mmnacl). the effect of salt stress on alkaline phosphatase was studied by pan [26] in spinach. he found that the enzyme alkaline phosphatase was inhibited by salinity ( 150mmnacl). in case of prosopis julifora a trend more or less similar to that in spinach and pea is evident in both root and leaf tissues. acid phosphatase and alkaline phosphatase in the root and leaves of this plant, however have shown an opposite trend. a difference in ionic balance resulting in a shift in cellular ph might be a reason for such alterations. effect of nacl salinity on enzyme atpase in the leaves and roots of prosopis julifora is shown in fgure 2(c). it is evident that the activity of enzyme atpase in the root was described by todd and yoo , [6] and liberated phosphorus was estimated by the standard method . [7] the method described by weimberg [8] was employed for the study of activity of enzyme alkaline phosphatase. amethod by kar andmishra [9] was employed for the determination of the activity of enzyme alkaline inorganic pyrophosphatase. the soluble proteins in the enzyme preparations were determined according to the method of lowry et al,. [10] results phosphorus is an important macronutrient essential for all living organisms. it plays a major role in energy transfer during plant metabolism like respiration, photosynthesis in the form of atp, nadp and also in cell division and cell expansion. phosphorus is involved in the formation of cell membrane lipids, which play a vital role in ionic regulation . [11] there are many reports indicating suppression of p uptake due to salt stress. [12,13] nieman and clark [14] also found depression of total p in the corn leaves due to salinity at low level of inorganic phosphorus in the nutrient solution. in case of prosopis cineraria seedlings ramoliya et al, [15] noticed that phosphorus content was signifcantly decreased in the leaves with increase in soil salinity while that was gradually decreased in the stem and root tissues. a decrease in p content of root tissue and that increase in the leaf tissue of salt grown poncirus trifoliata was evident in the experiments by tozly et al. [16] prosopis julifora seedlings have shown a pattern similar to that in prosopis cineraria since in both root and leaves a decline in p content was evident in the seedlings exposed to salt stress (figure 1) according to gibson [17] phosphorus defciency induced by salinity could reduce the cellular ability to accumulate optimum concentration of ion without reduced growth. thus in contrast to calcium and potassiumnutrition 0 0.05 0.1 0.15 0.2 0.25 0.3 g 100 g –1 dry weight control 100 200 300 nacl treatment ( mm) root leaves figure 1: effect of sodium chloride salinity on phosphorus content in the roots and leaves of prosopis juliflora (sw.).patil, et al.: influence of salt stress on phosphorus metabolism in the roots and leaves of one month old prosopis juliflora (sw.) dc seedlings 50 pharmacognosy journal  september 2011  vol 3  issue 25 treated aster plant while f-atpase activity was increased with increase in nacl concentration. thus it is clear that this enzyme plays an important role in salt tolerance process. this increase may help in regulation of ion uptake as well as contribute energy to growth processes. effect of nacl salinity on the activity of enzyme alkaline inorganic pyrophosphatase in the leaves and roots of prosopis julifora is shown in the fgure. 2 (d). it is evident that the activity of this enzyme decreases in the root and leaf tissue with increasing salt concentration. this trend is quite prominent upto 300mmnacl treatment. this enzyme plays important role in regulating the level of pyrophosphate and supplying pi for various reactions requiring pi in the cell. rea and sander [33] reported that inorganic pyrophosphatase can also acts as proton pump across the tonoplast membrane. vianello and macri [34] noted that in higher plants, cell membrane bound proton pumping pyrophosphatase and three moitochondrial h ppiase present in the inner surface of inner mitochondrial increases with increasing nacl treatment upto 200 mm and later decreased signifcantly at 300 mm nacl. while, in the leaf tissue its activity was increased with increasing level of salt. weimberg [8] found that in the seedlings of pea grown under highly saline media, the activity of atpase was slightly reduced. kuiper et al. [27] noticed that the activities of mg 2 dependent atpase was increased due to increased mineral level in the root of wheat seedlings and juvenile plants of plantago major. lin et al., [28] noted that the activity of h atpase was increased due to 75 mm nacl in the seedlings of cotton. horovitz and waisel [29] reported that this enzyme is associated with salt tolerance with many halophytes. they also observed a stimulation of this enzyme in glycophytic bean and carrot root and inhibition of the same in atriplex and suaeda roots after exposure to salt. under salinity stress its expression is down regulated in root and upregulated in shoot of pearl millet. [30] leaf of maize plant treated with 125 mm nacl showed slight increase in h atpase. [31] balasubbramaniam et al., [32] reported a decrease in the activity of atpase in 3 % nacl 0 10 20 30 40 50 60 enzyme activity µ moles p.nitrophenol h –1 mg –1 protein control 50 100 200 300 control 50 100 200 300 nacl treatment (mm) enzyme acid phosphatse root leaves 0 0.2 0.4 0.6 0.8 1 1.2 enzyme activity µ moles phosphorus h –1 mg –1 protein nacl treatment ( mm) enzyme atpase root leaves 0 5 10 15 20 25 enzyme activity µ moles p.nitrophenol h –1 mg –1 protein control 50 100 200 300 nacl treatment (mm) enzyme alkaline phosphatse root leaves 0 2 4 6 8 10 12 14 16 18 20 enzyme activity µ moles phosphorus h –1 mg –1 protein control 50 100 200 300 nacl treatments in mm enzyme alkaline inorganic pyrophosphatase root leaves a b c d figure 2: effect of sodium chloride salinity on the activity of (a) enzyme acid phosphatase (b) enzyme alkaline phosphatase (c) enzyme atpase and (d) enzyme alkaline inorganic pyrophosphatase in the roots and leaves of prosopis juliflora (sw.).pharmacognosy journal  september 2011  vol 3  issue 25 51 patil, et al.: influence of salt stress on phosphorus metabolism in the roots and leaves of one month old prosopis juliflora (sw.) dc seedlings encyclopedia of plant physiology, new series. 1983; 15. pp. 422-449. sringer-verlag new york. 12. fageria n.k. salt tolerance of rice cultivars. plant and soil, 1985; 88:237‑243. 13. indulkar b.s. and more s.d. interactive effect of nature of salinity and nitrogen on growth and nutrient composition of sorghum. j. indian soc. soil sc. 1985; 33 (8):641-645. 14. nieman r.h. and clark r.a. interacive effect of salinity and phosphorus nutrition on the concentration of phosphste and phosphsate esters in mature photosynthesizing corn leaves. plant physiol. 1976; 57:157-161. 15. ramoliya p.j., patel h.m., joshi j.b. and pandey a.n. effect of salinization of soil on growth and nurtien accumulation in seedlings of prosopis cineraria . j. of plant nutrition. 2006; 29:283-303. 16. tozly i., moore g.a. and gey c. effect of nacl concentration on stem elongation dry mass production and micronutrient of r. trifoliata. aust. j. of plant physiol. 2000; 27 (1):35-42. 17. gibsont.s.carbohydrate metabolism and phosphorus /salinity interaction in wheat (triticum aestivum l.). plant and soil. 1988; 111:25-35. 18. pan s.m. characterization of multiple acid phosphates in salt stressed spinach leaves. aust j. plant physiol. 1987; 14:117-124. 19. karadage b.a. and chavan p.d. physiological studies in salinity tolerance of sesbania aculeata poir. biol. plant. 1983; 25 :412-418. 20. lila a. and ehsanpour a. the effect of ascorbic acid on salt induced alfalfa (medicago sativa l.) in in vitro culture. nigerian society for experimental biology. 2006; 18(2):63-69. 21. chakrabarti n. and mukherji s. growth regulator mediated changes in leaf area and metabolic activity in mungbean under salt stress condition. indian j. of plant physiol. 2003; 7(3):256-263. 22. parida a.k. and das a.b. effect of nacl stress on nitrogen phosphorus metabolism in a true mangrove bruguiera parviflora grown under hydrophonic culture. j. of plant physiol. 2004; 161 (8):921-928. 23. weimberg r. effect of growth in highly salinized media on the enzymes of the photosynthetic apparatus in pea seedlilngs. plant physiol. 1975; 56:8-12. 24. ahmad r. and huq z. some physiological and biochemical studies on spinach growing on saline soil. pak. j. bot. 1974; 6:49-52. 25. nigwekar a.s. physiological studies in horse-gram (dolichos biflorus l.). a ph.d. thesis submitted to shivaji university, kolhapur. india :1988. 26. pan s.m.the effect of salt stress on the betain aldehyde dehydrogenase in spinach. taiwania. 1983; 28:128-137. 27. kuiper d., sommarin m. and kylin a. the effect of mineral nutrition and benzyl adenine on the plasmlemma. atpase activity from roots of wheat and plantago major ssp pleiosperma. physiol. plant. 1991; 81:169-174. 28. lin h., salus s.s. and schumakar k.s. salt sensitivity and the activities of h atpase in cotton seedling. crop sci. 1997 ; 37:190-197. 29. horovitz c.t. and waisel y. different atpase system in glycophytic and halophytic plant species. experientia. 1970; 26:941-42. 30. tyagi w., singla p., nair s., reddy m.k. and sopory s.k. a novel isoform of atpase subunit from pearl millet that is differentially regulated in response to salinity and calcium. plant cell reports. 2006; 25(2):156-163. 31. zoerb c., stacke b.,trumitz b. and denter d. does h pumping by plasma membrane limit leaf growth atpase in maize during 1 st phase of salt stress. journal of plant nutrition and soil science. 2005; 168 (5):550-557. 32. balasubbramaniam r.,thilo r., ahmed b., ralf s., burnhard h., ahlert s. and jatta p. aster tripolium l. and sesuvium portulacastrum l.: two halophytes two strategies to survive in habitat. plant physiology and biochemistry. 2006; 44(5-6):395-404. 33. rea p.a. and sander d. tonoplast energization: two h -pump, one membrane. physiol. plant. 1987; 71:131-141. 34. vianello a and macri a. proton pyrophosphatase from higher plant mitochondria. physiol. plant. 1999; 105:763-768. 35. simmons s. and butter l.g. alkaline inorganic pyrophosphatase of maize leaves. biochim. biophys. acta. 1969; 172:150-157. 36. murumkar c.v. and chavan p.d. influence of salt stress on phosphorus metabolism in leaves of chickpea cicer arietinum l. indian bot. reprt. 1990; 9(2):56-60. membrane involved in the specifc hydrolysis of ppi coupled to proton transport. simmons and butter [35] indicated that high activity of this enzyme in certain plants is directly related to high photosynthetic effciency. murumkar and chavan [36] reported that in the leaves of salt sensitive legume cicer arietinum, a stimulation of inorganic pyrophosphatase was evident under saline conditions. in salt sensitive plants such an increase may play same role in energy dependent processes because atp level is affected due to salt stress. but in salt tolerant prosopis julifora such situation perhaps may not occur which demands greater breakdown of ppi when atp level becomes limiting conclusion in conclusion it can be stated that due to salinity, there is defnite changes in phosphorus metabolism in the salt tolerant species prosopis julifora. some of these changes are probably related to mechanisms underlying salt tolerance in this species. acknowledgement authors are highly thankful to dr. b. a. karadge the ex. head, department of botany, shivaji university, kolhapur (ms, india) for his valuable suggestions. references 1. pasiecznik, n., felker, p., harris, p.j.c., harsh, l.n., cruz, g., tewari, j.c. cadoret, k. and maldonado, l.j. 2001. the prosopis juliflora, prosopis pallida complex: a monograph. hdra, coventry, uk. 2. dagar j.c. and tomar o.s. utilization of salt affected soil and poor quality water for sustainable biosaline agriculture in arid and semiarid regions of india. 12 th isco conference beijing; 2002. 3. singh g.the role of prosopis in reclaiming high ph soils and in meeting firewood and forage need of small farmers. pp.1.3-1.27. in: prosopis: semiarid fuelwood and forage tree; building consensus for the disenfranchised. (eds.) p. felker and j. moss. center for semi-arid forest resources, kingsville,texas, usa, 1996. 4. sekine t, sasakawa t, morita s, kimura t and kuratom k. cf. labrotory manual for physiological studies of rice (eds.) yoshida, s., forno, d., cook, j.b. and gomez, k.a. pub. international rice research institute, manila, india: 1972. 5. mclachlan k.d. acid phosphatase of intact roots and phosphorus nutrition in plants. aust. j. agric. res. 1980; 31:441-448. 6. todd g.w. and yoo b.y. enzymatic changes in detached wheat leaves as affected by water stress. phyton (buenos aires), 1964; 21:61. 7. fiske c.h.and subbaraoy.the calorimetric determination of phosphorus. j. biol. chem. 1925; 66:375-400. 8. weimberg r. enzyme levels in pea seedlings grown in highly salinized media . plant physiol. 1970; 46:466-470. 9. kar m. and mishra d. inorganic pyrophosphatase activity during rice leaf senescence. can. j. bot. 1976; 53:503-511. 10. lowry o.h, rosenbrough n.j., furr a.l. and randall r.j. protein measurement with folin phenol reagent. j. biol. chem. 1951; 193:262‑263. 11. bieleski r.l. and ferguson i.b. physiology and metabolism of phosphate compounds (eds.a. lauchi and r.l. bieleski). in: inorganic plant nutrition.o r i g i n a l a r t i c l e p h c o g j . 52 pharmacognosy journal  september 2011  vol 3  issue 25 address for correspondence: prof. rajiv gupta, head, dept. of pharmacognosy, faculty of pharmacy, babu banarasi das national institute of technology and management, lucknow. mobile no.: 91 9839278227; fax. no. 0522-2815187 e-mail: rajiv961@rediffmail.com doi: 10.5530/pj.2011.25.10 diospyrin (a bioactive bis-naphthoquinonoid). [5-7] free radical scavenging activity, anthelmintic, anti cancer and leishmanicidal activity has been reported in the plant. [8-11] pharmacognostic studies have not been reported for this plant. therefore the main aim of the present investigation is to study the macro, microscopic and physicochemical standards of leaves, stem bark, root and seeds of diospyros montana r. as per who guidelines which could be of use in preparing a monograph of the plant. material and method collection and authentication: the specimens of the plant diospyros montana r. for the present study were collected from local areas of district sultanpur, uttar pradesh, authenticated by national botanical research institute (csir) lucknow, also a voucher specimen was submitted for future reference (ref no. nbri/cif/180/2010). morphological profile the morphological characteristics of the specimens like leaves, root, stem bark and seeds were studied and the pharmacognostic and hptlc studies on diospyros montana r. (ebenaceae) shravan kumar, sakshi sehgal, hafsa ahmad, rajiv gupta*, shubhini a. saraf faculty of pharmacy, babu banarasi das national institute of technology and management, lucknow, uttar pradesh. a b s t r a c t diospyros montana r. commonly known as bistendu, is one of the medicinally important plant, widely distributed throughout india. almost all parts of diospyros montana r. possess good therapeutic value in traditional system of medicine. method: the present investigation deals with the pharmacognostic investigation of the fresh leaves, stem bark, root and seeds of diospyros montana r. which were used to determine its morphological, macroscopical, microscopical (including quantitativemicroscopy) physicochemical and chromatographic profle. result: themicroscopy of leaf revealed the presence of anomocytic stomata, multicellular glandular trichomes; prism shaped and clustered crystals of calcium oxalate. the microscopy of bark reveals the presence of sclerides or stone cells, pericyclic fbers, medullary rays. the root shows the presence of cork cells, cortex, medullary rays and xylem parenchyma. the total ash, acid insoluble ash, water ‐ soluble ash values of leaves were 14.535% w/w, 0.85% w/w, 12.60% w/w respectively. for roots it was 16.25% w/w, 0.25% w/w, 1.25% w/w respectively and for stem bark and seeds it was 7.85% w/w, 1.1% w/w, 6.6% w/w and 2.03% w/w, 0.10% w/w and 0.50% w/w respectively. hptlc studies were also carried out for detection and quantifcation of β-sitosterol in leaves and roots of diospyros montana r. conclusion: the fndings would be useful towards establishing pharmacognostic standards on identity, purity and quality and also helpful in preparing the monograph for future references. key words: diospyros montana, bistendu, hptlc, standardization introduction diospyros montana r.(ebenaceae) called as bistendu (hindi), mottled ebony (english), tumala (sanskrit). [1,2] it is distributed throughout india, malaya, australia. the parts used are leaves, fruit, seed, stem bark and heart wood. ethnomedicinally used in boils, fever, dysuria, diarrhea, spider bite, jaundice, deep wound etc. mundas of chota nagpur use the crushed leaves to poison fsh. the fruit is supposed to be poisonous. the ‘bhistis’ apply it to boils which generally appears on their hands. [3-4] the anatomical features of the plant can be utilized to determine the identity. recent investigations and preliminary phytochemical studies fnd reports on separation of quinonoids andpharmacognosy journal  september 2011  vol 3  issue 25 53 kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. reddish brown when ripe. [1] seeds usually reddish brown in color and it is about 1.0-1.2 cm by 6-7mm and oval in nature with acute apex. root is also reddish brown in color and internally wooden part of root is yellowish in nature with longitudinal striations are present on the root bark. morphologically the outer surface of stem bark light grey photographs were taken with the help of canon digital ixus 200 is, 12.1 mega pixel camera. diospyros montana r. (ebenaceae) is a small tree with rather slender stem and smooth grey bark. leaves alternate 3.8-14 by 2.5-7.5 cm; ovate-oblong or elliptic, apex acute or sub acuminate, base usually rounded. fruit upto 2.5 cm in diameter, globose, figure 1a: leaf figure 1c: root figure 1d: seeds figure 1b: stem barkkumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. 54 pharmacognosy journal  september 2011  vol 3  issue 25 such as stomatal number, stomatal index, vein-islet, vein termination number and palisade ratio were determined by using fresh leaves of the plant [14,15] (table 1). physico-chemical evaluations were done including ash values (total ash, acid-insoluble ash, and water-soluble ash), extractive values (alcohol soluble and water soluble extractive values), moisture content and saponin content [16,17] (table 2). hptlc studies on leaves and roots of diospyros montana r. solvents: all the solvents used were of ar grade. reference standard: the reference standard (ß-sitosterol) was procured from sigma aldrich, usa. chromatographic conditions instrument: hptlc system equipped with a sample applicator device camag linomat 5. camag twin trough chamber, camag tlc scanner and integration software (wincats) hptlc plate: silica gel g f 254 (merck) 15 10 cm mobile phase: toluene: ethyl acetate (9:1) wavelength: 273 nm standard preparation a stock solution of β-sitosterol (1000 μg ml -1 ) was prepared by dissolving 10.0 mg of accurately weighed β-sitosterol in methanol and diluting it to 10.0 ml with methanol. further dilutions were made with methanol to obtain working standards 200, 400, 600, 800 and 1000 µg ml -1 . [18] sample preparation 100 mg of air dried powdered plant material (leaves and root) was defatted with n-hexane and then soxhlet extracted with methanol for 16 hours. the methanolic extract was vaccum evaporated and concentrated and 10 mg of the concentrated methanolic extract was redissolved in 10 ml methanol to yield a test sample (1000 μg ml -1 ) and inner surface is yellowish grey in color. the stem bark is curved in shape and on the outer surface longitudinal striations were observed. (figure 1a, 1b, 1c, 1d) microscopic profile for microscopic studies t.s. of midrib and lamina, fresh stem bark, fresh root and ripe seeds were used. the fne section of leaves, root, bark and seeds were cut by free hand. the chlorophyll and the other pigments of the above mentioned part of plant were removed by treating the sections with 5% potassium hydroxide (koh) and 20% chloral hydrate as required. photomicrographs: microscopic descriptions of tissues were supplemented with microphotograph wherever necessary. photographs of different magnifcations were taken with olympus microscope, model olympus (india) and it was attached with yoko ccd camera. pharmacognostic investigation: morphological studies were carried by using simple microscope and visual examination. microscopic studies were done by free hand cutting thin section of leaves, root, stem bark and seeds of diospyros montana r. with platinum coated razor blade. [12,13] the sections were cleared with 20% chloral hydrate solution and 5% potassium hydroxide wherever required, stained with phloroglucinol-hydrochloric acid (1:1), mounted in glycerin and observed under above mentioned microscope attached with camera. quantitative microscopy of leaf table 1: quantitative microscopy or (leaf constant) of leaves of diospyros montana r. parameters range stomatal number adaxial epidermis abaxial epidermis 11-14 8-10 stomatal index 2.78 vein islets 16.67 vein termination no 19.44 palisade ratio 6.31 table 2: physicochemical analysis of leaves, root, stem bark and diospyros montana r. physicochemical constants parts of plant used ash value (%) leaves root stem bark seeds total ash 14.55 16.25 7.85 2.03 acid insoluble ash 0.85 0.25 1.1 0.10 water soluble ash 12.60 1.25 6.6 0.50 extractive values (%) water soluble extractive 14.05 6.8 13.9 8.6 ethanol soluble extractive 11.75 3.9 34.1 6.6 loss on drying (lod)% 7.3 7.9 7.8 7.6 foaming index 100 100 100 100pharmacognosy journal  september 2011  vol 3  issue 25 55 kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. procedure the tlc plate was activated by placing in an oven at the temperature of 110 c for 20 min. the spots were applied on the plate with test extract and standard solution maintaining a distance of 15mm from the edge of tlc plate. it was developed upto 75mm in the twin trough chamber using mobile phase toluene: ethyl acetate (9:1), dried in an oven and subjected for tlc scanning at 273nm. result and discussion microscopic features of the leaves t.s. of midrib and lamina: the abaxial part of leaf has more prominent convex shape than adaxial part of the midrib with thick lamina. the midrib has thin layer of epidermis comprising of small, circular thin walled cells. the ground tissue around the vascular structure is spongy parenchymatous; the cells are circular in nature. the xylem is thick and broad. the phloem is thin and less prominent. it has very thick sclerenchyma bundle sheath. the mesophyll has double layer cylindrical palisade cells which are closely arranged. the palisade cells are more prominent in abaxial side than adaxial side. (figure 2a, 2b) epidermal cells and stomata: the epidermal cells are small, tetragonal in outline with fairly thick, straight walls. the abaxial epidermis having more stomata than the adaxial side. the stomata are anomocytic in nature which is surrounded by three subsidiary cells. the guard cells are elliptic, wide and possess slit like aperture. the epidermal cells are wider, tetragonal and have thick, straight walls. (figure 2c) venation pattern and crystals in lamina: the lamina has dense reticulate venation. the vein islets are wide and distinct and they are polyhedral in outline. the vein terminations are long, thick and well developed. most of them are branched repeatedly forming a dendroid (free-like) outline. the calcium oxalate crystals are abundant in the lamina. the crystals are prismatic type; they are rhomboidal, cuboidal or polyhedral in shape. (figure 2d) and (table 1). microscopic features of the stem bark t.s. of stem bark: the parenchyma layer consist of sclerenchymatous cells which are also known as parenchymatous cells and it is in polyhedral in shape. they have thick lignifed walls, the lumen are slit like hollow to a fairly large sub rectangular cavity. medullary rays composed of parenchymatous cells which tend to be enlarged horizontally. pericyclic fbers are lignifed and embedded in stone cells layer like bunch of grapes (figure 3a, 3b). figure 2b: t.s. of midrib with lamina showing single head multicellular glandular trichome and double layer palisade cells (10x) figure 2a: t.s. of midrib showing spongy parenchyma and xylem vessels (40x)kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. 56 pharmacognosy journal  september 2011  vol 3  issue 25 figure 3b: t.s. of stem bark showing medullary rays figure 2d: venation pattern in lamina with prismatic calcium oxalate crystals (10x) figure 3a: t.s. of stem bark showing stone cells. figure 2c: stomatal morphology showing anomocytic stomata embedded in epidermal cells (10x) microscopic features of the root t.s. of root: cork has stratifed isodiametric lignifed cells usually orange brown color. there are many layers of thin walled cellulosic parenchyma with very small intercellular space. xylem vessels are yellow pitted or reticulately thickened walls which are surrounded by xylem parenchyma cells. pith is absent in root. medullary rays composed ofpharmacognosy journal  september 2011  vol 3  issue 25 57 kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. palisade cells. the epidermis consists of polygonal cells. the inner epidermis shows the yellow pigmented layer. the inner most circular part shows the embryo of the seed. (figure 5a, 5b) hptlc studies under the chromatographic conditions described above, the rf value of ß-sitosterol was about 0.76. the chromatograms of standard ß-sitosterol are exhibited in figure 6 (a-e) and that of ß-sitosterol in leaves and roots of diospyros montana are shown in figure 7 (a, b). the area under the parenchymatous cells which tend to be elongated vertically. (figure 4a, 4b) microscopic features of the seed t.s. of seed: cotyledon in sectional view showing the lower epidermis and cells of mesophyll as well as underlying figure 5b: t.s of seed showing polygonal cells of epidermis figure 5a: t.s. of seed showing embryo figure 4b:t.s of root showing xylemvessels and xylemparenchyma cells figure 4a: t.s. of root showing cork and cortexkumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. 58 pharmacognosy journal  september 2011  vol 3  issue 25 figure 6a: a typical hptlc chromatogram of ß-sitosterol working standard track 1 (200 μg ml -1 ) figure 6b: a typical hptlc chromatogram of ß-sitosterol working standard track 2 (400 μg ml -1 )pharmacognosy journal  september 2011  vol 3  issue 25 59 kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. figure 6d: a typical hptlc chromatogram of ß-sitosterol working standard track 4 (800 μg ml -1 ) figure 6c: a typical hptlc chromatogram of ß-sitosterol working standard track 3 (600 μg ml -1 )kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. 60 pharmacognosy journal  september 2011  vol 3  issue 25 curve (auc) obtained for various tracks are enumerated in table 3 . the calibration curve was linear in the range of 200 to 1000 μg ml -1 , as illustrated in figure 8. from the regression equation, y5.574x1436, the concentrations of the test samples i.e. leaves and roots was estimated to be about 651.99 and 447.14 μg ml -1 respectively. figure 7a: a typical hptlc chromatogram of ß-sitosterol in diospyros montana (leaves) figure 6e: a typical hptlc chromatogram of ß-sitosterol working standard track 5 (1000 μg ml -1 )pharmacognosy journal  september 2011  vol 3  issue 25 61 kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. table 3: area under curve values for different concentrations of working standards of ß-sitosterol for linear calibration tracks concentrations of working standard of ß-sitosterol (μg ml -1 ) area under curve (au) track1 200 2660.3 track2 400 3307.3 track3 600 4870.0 track4 800 6356.4 track5 1000 6710.0 conclusion as per w.h.o. guidelines, developing countries like india must try to fully explore their treasure of medicinal plants so as to reduce their dependency on imports and thus make them economically independent. diospyros montana is a commonly available plant of rural india which is not yet fully explored (villagers apply it to boils and for deworming purpose) as per literature survey and as revealed by its phytochemical profle. standards for herbal drugs figure 8: standard curve (line of best fit) for ß-sitosterol figure 7b: a typical hptlc chromatogram of ß-sitosterol in diospyros montana (roots)kumar, et al.: pharmacognostic and hptlc studies on diospyros montana r. 62 pharmacognosy journal  september 2011  vol 3  issue 25 6. hazra b, sarma md, sanyal u. separation methods of quinonoid constituents of plants in oriental traditional medicines. j chromatogr b. 2004; 812:259-275. 7. sanyal u, bhattacharyya s, patra a, hazra b. liquid chromatographic separation of diospyrin, a bioactive bis-naphthoquinonoid plant-product, and analogous naphthyl compounds. j chromatogr a. 2003; 1017:225-232. 8. pathak av, itankar pr, singhai ak. free radical scavenging activity of heartwood of diospyros montana roxburg. journal of pharmacy research. 2009; 2(3):483-86. 9. rajarajeshwari n, ganapaty s, kumar dh. in vitro anthelmintic activity of five rare species of diospyros. international journal of pharmaceutical sciences. 2010; 1:445-7. 10. sagar s, kaur m, minneman kp, bajic vb. anticancer activity of diospyrin, its derivatives and analogues .european journal of medicinal chemistry. 2010; 45:3519-30. 11. mukharjee p, majee sb, ghosh s, hazra b. apoptosis like death in leishmania donovani promastigotes induced by diospyrin and its ethanolamine derivative. int j. antimic ag. 2009; 34:596-601. 12. khandelwal kr. practical pharmacognosy.techniques and experiments. 4 th ed. pune: nirali prakashan; 1998. 13. kokate ck. practical pharmacognosy. 4 th ed. new delhi: vallabh prakashan; 1994. 14. the ayurvedic pharmacopoeia of india. 1 st ed. part 1, vol 1. new delhi: government of india, ministry of health and family welfare; 2001. 15. wallis te, editor.textbook of pharmacognosy. new delhi: cbs publisher and distributors; 2005. 16. evans wc, trease ge. text book of pharmacognosy. 15 th ed. saunders wb; 2002. 17. quality control methods for medicinal plant materials; world health organization, geneva [serial online] 1998 [cited 2010, dec 07; 2; 33 pm]. available from: url: http://apps.who.int/medicinedocs/pdf/h1791e/ h1791e.pdf. accessed on 05 august 2011 18. kumar a, lakshman k, jayaveera kn,tripathi snm, satish kv. estimation of gallic acid, rutin and quercetin in terminalia chebula by hptlc. jordan journal of pharmaceutical sciences 2010; 3(1):63-7. are being developed worldwide, which is also helpful in developing the monograph of the medicinal plant, and to explore it commercially if found promising. standardization and quality control for herbal drugs are need of the hour. the present study attempts to outline basic requirements necessary to develop technical standards for making the medicinal plant worth exploring for further research work. acknowledgement the authors are thankful to the a.i.c.t.e for the generous grant under modrobs project (file no. 8024/rid/ bor/mod-458/2009-10), for making the present research work possible. references 1. kirtikar kr, basu bd, editors. indian medicinal plants. 2 nd ed. dehradun, uttaranchal (india): oriental enterprises; 2001. 2. rastogi rp, mehrotra bn. compendium of indian medicinal plants. new delhi: csir; 2006. 3. tandon n, sharma m. review on indian medicinal plants. new delhi: icmr; 2009. 4. chopra rn, nayar sl, chopra ic. glossary of indian medicinal plants. new delhi: csir; 2002. 5. maridass m,ghanthikumar s, raju g. preliminary phytochemical analysis of diospyros species. ethnobotanical leaflets. 2008; 12:868-72.pharmacognosy journal  september 2011  vol 3  issue 25 63 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: e-mail: bunmiagboola2000@yahoo.com or bunmi.agboola@ndu.edu.ng doi: 10.5530/pj.2011.25.11 temporary abatement of human parasites burden because of rapid re-infection rates subsequent to drug intervention and the fact that the drug is ineffective to immature stage of the parasite. [5,6,7] experience has shown that in high risk setting, cessation of drug treatment for even a few years can result in recurrence of high level of schistosoma infection among adults and children as if the community had never been treated. [8] evidence suggest that countries such as china and philippines controlled their schstosomiasis by combining the destruction of amphibian snails control with treatment of infected humans [9] and without changes in schistosomiasis transmission potential even multiple years of annual drug treatment will not be adequate to prevent schistosoma infection in many high risk areas. and this may lead to onset of both community and donor fatigue in large scale drug treatment projects if disease control is not fully effective and durable over the long term. [8] this insight couple with the indication that resistance to praziquantel might develop in future and the fact that some side effects associated with praziquantel may reduce drug compliance in primary health care [10] has buttressed the view that molluscciding schistosomes transmitting snails still has a useful part to play in integrated control schemes for this important disease and a pressing need for more selective and effcient molluscicide for the control of snail vector. [11,12] comparative molluscicidal activities of fruit pericarp, leaves, seed and stem bark of blighia unijugata baker * 1 agboola o i, 2 ajayi g o, 2 adesegun s a, and 3 adesanya s a. 1 department of pharmacognosy and herbal medicine, faculty of pharmacy, niger delta university wilberforce island, bayelsa state. 2 department of pharmacognosy, faculty of pharmacy university of lagos. 3 department of pharmacognosy, faculty of pharmacy obafemi awolowo university ile-ife nigeria. a b s t r a c t introduction: the plant is used in ethno-medicine as a fsh poison and belongs to family known to contain saponins which are toxic to cold-blooded animals including snails acting as vectors of organisms responsible for many human diseases including schistosomiasis. methods: the mollusccidal activities of 50% ethanolic extracts of the seeds, fruit pericarp, leaves and stem bark as well as the fractions of the fruit pericarp of b unijugata baker were evaluated on biomphalaria glabrata.snails results: the crude extract of fruit pericarp was the most active among the morphological parts tested with lc 50 of 15 μg/ml while ethyl- acetate fractions showed the highest activity of the 3 fractions with a lc 50 of 7.6 μg/ml and satisfed the condition set by world health organization for a potential plant molluscicides either as a crude extract or as a fractions conclusions: the results confrmed the ethno-medicinal uses of the plant and can be so regarded as a potential molluscicides in the snail vector of schistsomiasis key words: blighia unijugata, bak, fruit pericarp, biomphalaria glabrata, fruit pericarp, schistosomiasis introduction schistosomiasis is a debilitating disease affecting close to 4-5% of the world population [1] and approximately 90% of these estimated cases of human schistosomiasis lives in sub-saharan africa. within the sub-saharan africa, nigeria is the country with the most cases of human schstosomiass which is widespread in both the urban and rural communities. [2] epidemiological studies showed the prevalence rates to be high, for example 26% of school children were found to be infected in anambra state, south eastern nigeria. [3] while 21% of school children, 18.4% of local dry cleaners and 15 .8% of vehicles washers were found to be infected in studied population in ibadan south western nigeria while prevalence rates of 26.6-36.8%were found in some localities in kano, north eastern nigeria. [4] chemotherapy is one of the most valuable methods in the cure of schistosomiasis but chemotherapy provides onlyagboola, et al.: comparative molluscicidal activities of fruit pericarp, leaves, seed and stem bark of blighia unijugata baker 64 pharmacognosy journal  september 2011  vol 3  issue 25 dryness under vacuum to yield 10.30 g of the dried extract and out of this 9.80 g was dissolved in water and partitioned between ethyl acetate, butanol and water to give 2.77 g of ethyl acetate fraction, 2.81 of butanol and 3.35 of water fraction respectively. molluscicidal screening snails for the experiment were collected from streams that has not been subjected to either synthetic or plant molluscicides. the snails were identifed by dr. olorunmola of drug research and production unit of obafemi awolowo university ile-ife osun state. they were allowed to acclimatize in the laboratory for two weeks before use. the molluscicidal test was divided to two stages; rapid screening test the methods described by various authors [16,17,18] were used with slight modifcation such that concentrations of 1000 and 500 ppmwere used. extract which show 100% activities at concentration 500 ppm were then used for the fnal screening test. the extract with the activity within the who recommended guidelines was fractionated into, ethyl acetate, butanol and water fractions and each fraction subjected to further screening to determine where activity resides final screening test a different method [19] was used for the fnal screening test but copper sulphate was used as positive control at a concentration of 1 ppm and was set up in duplicate which gave 100% mortality and 500 ml de-chlorinated water was used as negative control. the same method used for the crude extracts was also used for the screening of the various fractions. the lethal concentration that kills 50% of the snails was determined with the use of probit analysis table with value plotted on graph paper to determine the lc 50 . result and discusson results of rapid screening test data in table 1 below from rapid screening show that the leaves and the seed gave 100% mortality at 1000 ppm the plant material the plant blighia unijugata baker family sapindaceae is a small to medium-sized tree up to30 m tall widespread in tropical africa. ethno-medicinal uses like all other plants used in ethno-medicine the uses varies from place to place but the traditional used related to the present work is the use of macerated twigs, leaves, fowers and fruit as a fsh poison and the coastal people in nigeria. the leaves are eaten as vegetable and various part of the tree are considered to have sedative and analgesic properties and are used in traditional medicine for the treatment of rheumatism, kidney pain and stiffness. fruits have also been used for the treatment of nausea and vomiting. [13] like all other members of the sapindaceae family saponins are believed to be present. saponins are naturally occurring plant glycosides, which form a soapy latter with water. there is a high correlation between plants employed as fsh poison or soap substances and their molluscicidal activity [14] and many potent molluscicides of plant origin were triterpenoids saponins and some triterpenods have actually been isolated from this particular plant and they include the following triterpenoids friedelin and epifriedelinol. [15] materials and methods plant collection the various parts of the plants were collected along ologuneru in iddo local government of oyo state in march 2010 with the assistance of mr. benjamin daramola and mr. odewo both member of staff of university of lagos herbariumwho identifed it and a voucher specimen was deposited at the university of lagos herbarium with voucher number luhn 3325. each plant part was allowed to dry at room temperature and 50 g of leaves 10 g each of seed, stem bark and fruit pericarp were macerated with 50% ethanol for 72 hours fltered and concentrated to dryness at 30 c under vacuum using a rotary evaporator. the extraction was carried out thrice and the yield for each plant part was calculated. for the preparation of the fractions from the fruit pericarp, 71 g of the powdered peicarp was macerated for 72 hours with 50% ethanol, fltered and the fltrate concentrated to table 1: results of rapid screening plant parts concentration ppm %mortality leaves 1000 100  500   0 seed 1000 100  500   0 stem bark 1000 100  500 100 fruit pericarp 1000 100  500 100pharmacognosy journal  september 2011  vol 3  issue 25 65 agboola, et al.: comparative molluscicidal activities of fruit pericarp, leaves, seed and stem bark of blighia unijugata baker powdered dried fruit were found to be lower as carried out by [20] where the lc 95 values were 98.7 for adult bulinus globules and 98.5 for bulinus truncates. the seed has been shown to be a good source of protein, carbohydrate, minerals and crude fber and can serve as feed supplement and the oil from the seed can be used in the production of soap and lather shaving cream. [21] this fact should serve as impetus to the local people who can be encouraged to exploit the commercial benefts as well as the health benefts of this plant since schistosomiasis has been shown to be both a cause and an effect of continuing rural poverty in endemic areas. [22] in the case of the 3 fractions of the fruit pericarp activity increases from the water fraction to butanol fraction with ethyl-acetate fraction having the highest activity with lc 50 of 7.6 ppm respectively. for a plant to be considered as a potential mollusiccide according to the world health originations (who) guidelines a methanolic or lipopholic extracts should be active at equal to or less than 20 μg/ml to kill 90% of snails exposed for 24 hours. [12] it must also be freely solubility in water since the medium of fnal usage will be water. it is only the fruit pericarp of bl. unijugata baker that met this condition and may be a candidate for further studies while the criteria for solubility and concentration were met, other consideration like the effect on non target organisms such as fshes and other amphibian inside the river has to be investigated before it can be declare a candidate molluscicide and this together with the isolation of the compounds responsible for activity will be the focus of future research. it is interesting to note that in all cases of death, death occurs within 6 hours of application of the extracts to the snails with a reddish fuid around the snails which may suggest heamolysis of the blood and this gives noxious odor the following day. it was also observed that both the extracts and the various fractions produced what can be call all or none response as efforts to get a concentration that will not give 100%mortality was not successful. this action of the extracts and fractions would be of immense utility if the plant is to be used in the control of the snails as it will be certain that if the appropriate concentration is used total eradication of the snails in the affected community will be accomplished and this will ensure that there will not be residual snails that can serve as intermediate hosts for further infection of humans. conclusions this study has confrmed the ethno botanical use of the plant as a fsh poison and as a potential mollluscicidal agent which the people living in endemic areas can be encourage but no activity at 500 while the stem bark and fruit pericarp show 100%mortality at both 1000 and 500 ppm and both are then used for the molluscicidal screening result from table ii above showed that the fruit pericarp has the highest activity and was subsequently fractionated to ethyl-acetate, butanol and water the result of the molluscicdal bioassay showed that of the four morphological parts tested, only the stem bark and the fruit pericarp were active at concentration of 500 ppm and below with the fruit pericarp having the highest activity with lc 95 and lc 50 of 15 and 7.6 ppm. the result of the fruit pericarp alone without the seed suggest that the concentration of the active compounds are more in the fruit pericarp and that may be the reason why molluscicidal activity of the table 2: results of mollucsicdal screening of stem bark and fruit pericarp plant parts concentration (ppm) % mortality lc 50 ppm stem bark 1000  500  250 100 100   0 fruit pericarp 1000  500  250  125  100   75   50   40   30   20   10    5 100 100 100 100 100 100 100 100 100   0   0   0 15 positive control copper sulphate 1 ppm 100 negative control (500 ml) de-chlorinated water 0 table 3: results of molluscicidal activity of the fractions of fruit pericarp fractions conentration ppm %mortality lc 50 ethyl-acetate 30 100 20 100 7.6 10 100  5   0 butanol 30 100 20 100 15 10   0  5   0 water 30 100 20   0 25 10   0  5   0 positive and negative control were set up as in above.agboola, et al.: comparative molluscicidal activities of fruit pericarp, leaves, seed and stem bark of blighia unijugata baker 66 pharmacognosy journal  september 2011  vol 3  issue 25 macchakos district, kenya, measured by long term snail sampling and cercaririomrtyry. parasitology. 1994; 109, 443-453. 8. king ch, sturocrf, kariuki hc, hambuger j. transmission control for schistosomiasis-why it matters now trends in parasitology. 2010; 22, 575‑582. 9. fenwick a, webster jp. schistosomiasis; challenges for control, treatment and drug resistance. current opinion in infectious diseases. 2006; 19, 577-582. 10. berhe n, gunderson gs, abebe f, birrie h, medhim g, gemetchu t. praziquantel side effects and efficacy related to schistosoma mansoni egg loads and morbidity in primary school children in north-east ethiopia. acta tropcal, 1998; 72, 53-63. 11. whitfield pj. medicinal plants and the control of parasites, novel anthelmintic compounds and molluscicides from medicinal plants. transactions of the royal society of tropical medicine and hygiene 1996; 90, 596-600. 12. hostetmann k. plant derived molluscicides of current importance. economic and medicinal plant research 1989; 3, 72-103. 13. burkill hm . the useful plants of west tropical africa second edition royal botanical garden, kew2000. 14. singh sk, ram py, singh a. molluscicides from some common medicinal plants of eastern uttar pradesh, india. journal of applied toxicology 2010. 30, 1-7. 15. ongarora dsb,thoithi gn, kmau fn, abuga ko, mwangi jw kibwage o. triterpenoids from the stem bark of blighia unjugata bak ethiopian pharmaceutical journal 2009; 27, 71-74. 16. nick a. ralt, sticher o. biological screening of traditional medicinal plants from papua new guinea. journal of ethnopharmacology 1995; 49, 147-156. 17. al-zanbagi na, barret j, abdul-elah ab. laboratory evaluation of molluscicidal properties of some saudi arabian euphorbiales against biomphalaria pfeifferi. acta ttopical 2001; 78, 23-28. 18. bilia an, braca a,mendez j,morelli i.molluscicdal and piscicidalactivities of venezuelan chrysobalanaceae plants. life sciences 2000; 66, 53-59. 19. truiti mct, ferreira icp, zamuner ml, mnakamura cv, sarragioto mh, souza mc. antiprotozoal and molluscicidal activities of five brazilian plants brazilian journal medical and biological research 2005; 38, 1873-1878. 20. anto f, aryeetey me, anyorigiyat, asoala v, kpikpi j. the relative susceptibilities of juvenile and adult bulinus globosus and bulinus truncatus to the molluscicidal activities in the fruit of ghanaian blighia sapida, blighia unijugata and balanites aegptiaca ann trop med parasitol 2005; 99, 211-7. 21. oderinde r, ajayi ia, adewuyi a. evaluation of the mineral nutrient’s, characterization and some possible uses of blighia unijugata bak seed and seed oil. electronic journal of environmental, agriculture and food chemistry, 2009; 8, 120-129. 22. king ch, parasites and poverty; the case of schistosomiasis acta tropica 2010; 113, 95-104. to use the fruit pericarp after extracting the oil from the seed which can serve as additional source of income but such usage is subject to further research to determine its effect on other aquatic organisms and to isolate the agents responsible for actvity from the various fractions. effort is on to isolate the active compounds responsible for activity from various fractions. acknowldgment dr felix olorunmola of drug research and production unit, obafemi awolowo university ile-ife for the identifcation of snails and mr julius solomon and ms susan umoh for the collection and care of the snails. references 1. clark te, appleton cc, drewers se. a semi- quantitative approach to the selection of appropriate candidate plant molluscicides – a south african application journal of ethnopharmacology 1997; 56, 1-13. 2. ugbomoiko us, ofoezie ie, okoye ic, heukelabach j. factors associated with urinary schistosomiasis in two peri-urban communities in south-western nigeria. annals of tropical medicine and parasitology 2010; 104, 409-419. 3. amazigo uo, anago-amanze ci, okeibunor jc. urinary schistomiasis among school children in nigeria; consequences of indigenous beliefs and water contact activities. journal of biosocial science 1997; 29; 9-18. 4. betterton c, ndiffon gt, bassey se, tan rm, oyeyi t. schistosomiasis in kano state,nigeria 1.human infection near dam sites and the distribution and habitat preferences of potential snails intermediate hosts. ann trop med parasitol 1988; 62, 561-70. 5. ernould jc, ba k, sellin b. increase in intestinal schistosomiasis after praziquantel treatment in a schistosoma heamatobium and schistosoma mansonii mixed focus acta tropical 1999; 73, 143-152. 6. sturrock rf, kinyanju h,thiongo fw,tosha s, ouma jh, kng ch, koech d, siongok ta mamoud aaf. chemotheraphy-based control of schistosomiasis heamatobium 3 snails studies monitoring the effect of chemotherapy on snail transmissions in msanbweni area, kenya, transactions of the royal society of tropical medicine and hygiene 1990; 84, 257-261. 7. sturrock rf, klumpp rk, ouma jh, butterworth ae, fulford jc kariuk hc, thiongo fw, koech d. observation on the effects of different chemotherapy strategies on the transmission of schistisoma mansoni inpharmacognosy journal  september 2011  vol 3  issue 25 67 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: kamal sachdeva, m. pharm student, jaipur national university, jaipur, 302025, india email: kamal.pharmaresearch@gmail.com doi: 10.5530/pj.2011.25.12 thick glorious branch lets. the tree has a straight trunk and grey or reddish bark, masked by large white patches. it has green leaves with a length and width of 6 to 15 cm, with 5 to 7 shallow lobes. the branches contain whitish latex, which causes brown stains. inforescences are formed terminally on branches. the plant is monoecious and fowers are unisexual. [4-5] after pollination, a trilocular ellipsoidal fruit is formed. the seeds are black and in the average 18 mm long and 10 mm wide ripe jatropha fruits. [6] it is a multipurpose species with many attributes and considerable potential. the wood and fruit of jatropha can be used for numerous purposes including fuel. it is used against dermatomucosal diseases, arthritis, gout, jaundice, toothache, gum infammation, gum bleeding, diarrhoea and pyorrhea. [7] plant extract used to treat allergies, burns, cuts and wounds, infammation, leprosy, leucoderma, scabies and small pox. water extract of branches used in hiv, tumor andwound healing. the plant contains organic acids, cyclic triterpenes stigmasterol, [8] curcacycline a, curcin, [9] a lectin phorbolesters esterases, sitosterol and its d-glucoside. [10] the leaf and bark have been shown to contain glycosides, tannins, phytosterols, favanoids and steroidal sapogenins. [7] the plant is reported to have properties against diseases. in view of these cited activities, observations and traditional uses of plant, the present study was undertaken to explore the wound healing potential of extract of this plant in excision and incision experimental models. wound healing potential of extract of jatropha curcas l. (stem bark) in rats kamal sachdeva*, preeti garg, 1 manmohan singhal, 2 birendra srivastava 2 1 school of pharmacy, suresh gyan vihar university, jaipur, india. 2 school of pharmaceutical sciences, jaipur national university, jaipur, india a b s t r a c t introduction: the present study provides a scientifc evaluation for the wound healing potential of extract of jatropha curcas l. stem bark. jatropha curcas l. or physic nut, is a bush or small tree (up to 5 m height) and belongs to the euphorbiaceae family. methods: excision and incision wounds were inficted upon four groups of six rats each. group i was assigned as control (ointment base), group ii was treated with standard silver sulfadiazine (0.01%) cream, group iii and group iv was treated with 5% and 10% extract ointment respectively. the parameters observed were percentage of wound contraction, hydroxyproline content and tensile strength including histopathological studies. results: it was noted that the effect produced by the extract ointment showed signifcant (p 0.01) healing in both the wound models when compared with control group. all parameters such as wound contraction, hydroxyproline content, tensile strength and histopathological studies showed signifcant changes when compared to control. conclusion: the result shows that extract ointment demonstrates wound healing potential in both excision and incision models. key words: histopathological, hydroxyproline, euphorbiaceae, sulfadiazine, tensile introduction herbal medicines have been enjoying revitalization among the clients all over the world. there are hundreds of medicinal plants that have a long history of curative properties against various diseases and ailments. however, screening of plants for their activity is very crucial and needs imperative attention in order to know the value of the plant. the assessment of the plants for their therapeutic activity is done on the basis of either their chemotaxonomic examination or ethnobotanical information for a particular disease. [1] jatropha curcas l. or physic nut, is a bush or small tree (up to 5 m height) and belongs to the euphorbiaceae family and contains approximately 170 known species. [2] jatropha, a drought-resistant shrub or tree, which is widely distributed in the wild or semi-cultivated areas in central and south america, africa, india and south east asia. [3] it is a multipurpose, drought resistant, perennial plant gaining lot of importance for the production of biodiesel. it hassachdeva, et al.: wound healing potential of extract of jatropha curcas l. (stem bark) in rats 68 pharmacognosy journal  september 2011  vol 3  issue 25 shaved 1 day prior to the experiment. an excision wound was inficted by cutting away a 300 mm 2 full thickness of skin from a predetermined shaved area. [13] the wounds were left undressed to the open environment. the ointment base, standard drug ointment (0.1% silver sulfadiazine) and extract of plant ointment (5%, w/w) & (10%, w/w) were applied topically to the control group, standard group and treated group respectively, till the wound was completely healed. in this model, wound contraction was monitored. wound contraction was measured as percent contraction in each 2 days after wound formation. from the healed wound, a specimen sample of tissue was collected from each rat for histopathological examination. [14-15] incision wound model in incision wound model, [16] all the animals of each group were anaesthetized under light ether anesthesia. two full thickness paravertebral long incisions were made through the skin at the distance of about 1 cm from midline on each side of the depilated back of rat. after the incision was made the both edges of skin kept together and stitched with black silk surgical thread (no. 000) and a curved needle (no. 11) was used for stitching. the continuous threads on both wound edges were tightened for good closure of the wound. after stitching, wound was left undressed then ointment base, standard ointment and extracts ointment were applied daily up to 10 days; when wounds were cured thoroughly the sutures were removed on the day 10 and tensile strength of cured wound skin was measured using tensiometer. [17] wound healing evaluation parameters measurement of wound contraction an excision wound margin was traced by following the progressive changes in wound area planimetrically, excluding the day of wounding. the size of wounds was traced on a transparent paper in every 2 days, throughout the monitoring period. the tracing was then shifted to graph paper, from which the wound surface area was evaluated. [18] the evaluated surface area was then employed to calculate the percentage of wound contraction, taking initial size of wound, 300 mm 2 , as 100%, by using the following formula as: % wound contraction initial wound size specifc day wound size initial wound size 100 measurement of tensile strength the force required to open the healing action is known as tensile strength. it is used to measure the completeness of healing. it also indicates how much the repaired tissue resists to breaking under tension and may indicate in part the quality of repaired tissue. the sutures were removed material and methods plant material fresh stembark of jatropha curcas l. was collected from a local area of jaipur were identifed in the department of botany, rajasthan university, jaipur. a voucher specimen number rubl20844 was deposited in the department. the fresh stem bark was air-dried to constant weight, pulverized and stored in an air-tight container for further use. extraction of plant drug powder of dried stem bark was subjected to soxhlet extraction with methanol: acetone: water (70:20:10). the extract was then fltered and the fltrate was concentrated to dryness. preliminary phytochemical screening the extract was subjected to phytochemical tests for tannins, steroids, alkaloids and glycosides, favanoids, carbohydrates, proteins and amino acid using reported methods. [11-12] preparation of formulation and standard used 5% (w/w) & 10% (w/w) simple ointment containing the extract of plant was prepared by trituration method in a ceramic mortar and pestle using white soft paraffn base. for this, 5 g & 10 g extract was incorporated in 100 g of the base. silver sulfadiazine (0.01%) obtained from rexin pharmaceutical pvt. ltd. was used as standard drug for comparing the wound healing potential of extract in different animal models. animals albino rats of either sex (150-200 g) were used for the experimental study. the animals obtained from shree dhanvantary pharmaceutical analysis and research centre, kim, surat were maintained under standard husbandry conditions in polypropylene cages and provided with food and water ad libitum. the animals were kept on fasting overnight prior to the experimentation and all the procedures used in these studies were approved by the institutional animal ethics committee. grouping of animals four groups of animals containing six in each were used for excision and incision wound models. the animals of groups i, ii, iii and iv were considered as the control, reference standard, (5%) & (10%) extract ointment respectively. in vivo studies excision wound model the animals were divided into three groups with six in each were anaesthetized by open mask method with anesthetic ether before wound creation. the particular skin area waspharmacognosy journal  september 2011  vol 3  issue 25 69 sachdeva, et al.: wound healing potential of extract of jatropha curcas l. (stem bark) in rats statistical analysis results obtained from both wound models have been expressed as mean sem and the treated group was compared with control group. the results were analyzed statistically using dunnet test followed by one-way anova, to analyze the differences between the treated and control. the data were considered signifcant at p 0.01. results wound contraction a better healing pattern with complete wound closure was observed in standard and treated group (10%, w/w), (5%, w/w) within 18, 19 & 22 days respectively while it was about 26 days in control rats as shown in (table i). tensile strength of incision wound model tensile strength for the treated group on 10 th day was found to be signifcant (p 0.01) than control group as shown in (table ii). hydroxyproline content treated group showed signifcant increase in hydroxyproline content when compared to control group (p 0.01) as depicted in (table ii). histopathological examinations in standard and treated albino rats with extract (5%) & (10%), excision and incision type of wounds have shown signifcant healing as in fbroblasts cells (f), collagen fbres (cf) and new blood vesicles (bv) in (figure i, ii and iii) respectively while in control rats wounds shown incomplete healing in (figure iv). control group has shown to slightest wound healing ability when compared to extract treated and reference ointment group. fibroblast cells, on the 9 th day after wounding and the tensile strength was measured on 10 th day. for this purpose, the newly formed tissue including scar was excised and tensile strength was measured with the help of tensiometer. [19] in this method, wound-breaking strength was measured as the weight of water at the time of wound breaking per area of the specimen. hydroxyproline estimation hydroxyproline is an uncommon amino acid present in the collagen fbres of granulation tissues. its estimation helps clinically to understand progress rate at which the healing process is going on in the connective tissue of the wound. for the determination of hydroxyproline content, the wound tissues were excised and dried in a hot air oven at 60-70 c to constant weight and were hydrolysed in 6nhcl at 130 c for 4 h in sealed glass tubes. the hydrolysate was neutralized to ph 7.0 and was subjected to chloramine-t oxidation for 20 min. the reaction was terminated by addition of 0.4m perchloric acid and color was developed with the help of ehrlich reagent at 60 c. the absorbance was measured at 557 nm using a spectrophotometer. the amount of hydroxyproline in the samples was calculated using a standard curve prepared with pure l-hydroxyproline. [20] histopathological examinations a specimen sample of skin tissues from control, standard and treated groups was taken out from the healed wounds of the animals in excision and incision wound models for histopathological examinations. the thin sections were cut and stained with haematoxylin and eosin [21] and observed under microscope for the histopathological changes such as fbroblast proliferation, collagen formation and angiogenesis. table 1: effect of methanolic extract of jatropha curcas l. and standard ointment on % of wound contraction of excision wound models in rats post wounding days % of wound contraction control standard extract ointment (5%) extract ointment (10%) 2 8.72 1.791% 13.31 1.229% 12.11 1.538% 9.03 3.02% 4 19.13 1.528% 30.59 2.492% 22.45 1.748% 29.14 2.688% 6 30.55 3.055% 51.74 0.564% 45.34 3.332% 49.95 2.164% 8 40.47 2.107% 63032 2.538% 57.56 3.396% 59.05 2.816% 10 47.79 1.51% 81.04 3.016% 65.65 2.068% 67.11 2.729% 12 55.21 2.473% 87.90 2.488% 74.98 1.469% 81.56 2.791% 14 66.70 2.91% 93.49 1.412% 81.38 1.790% 88.11 2.049% 16 74.12 3.276% 97.82 0.311% 86.64 1.331% 93.39 0.723% 18 83.41 3.602% 99.29 0.113% 92.54 1.086% 97.63 0.345% 20 89.95 1.67% – 96.13 2.49% 99.89 1.452% 22 93.64 4.71% – 99.58 3.49% – 24 97.38 1.82% – – – 26 99.91 5.98% – – –sachdeva, et al.: wound healing potential of extract of jatropha curcas l. (stem bark) in rats 70 pharmacognosy journal  september 2011  vol 3  issue 25 discussion and conclusion wound healing is stepwise process, which consists of different phases such as hemostasis, infammation, proliferative and remodeling or maturation. the genetic response regulating the body’s own cellular resistance mechanisms contributes to the wound and its repair. [22] hence in this study, excision and incision wound models were used to evaluate the effect of extract ointment on various phases. in incision wound, the increase in tensile strength of treated wounds may be due to the increase in collagen concentration and stabilization of the fbres. [23] increase in blood vessels and role of antioxidants were experimentally proved. [24] in excision wound, the extract showed faster healing with earlier wound contraction compared with control groups. collagen fbres and blood vessels are prominently present in standard and extract treated group as compared to control. table 2: effect of jatropha curcas l. extract and standard ointment on various wound parameters of incision wound model in rats groups hydroxyproline (mg/g tissue) tensile strength (g/mm 2 ) control 25.76 0.003 413.80 3.665 standard 61.52 0.004* 607.22 3.717* extract ointment (5%) 43.39 0.002* 493.75 4.136* extract ointment (10%) 55.76 0.003* 582.80 3.665* n 6 albino rats per group; values represents mean sem. *p 0.01 (comparison of control with standard& extracts). figure 1: hepatological characteristics of healed tissue on 18 th day by treatment with standard ointment figure 2: histopathological characteristics of rat skin on 18 th day by treatment with 5% extract ointment figure 3: histopathological characteristics of rat skin on 18 th day by treatment with 10% extract ointment figure 4: histopathological characteristics of rat skin on 18 th day treatment with ointment basepharmacognosy journal  september 2011  vol 3  issue 25 71 sachdeva, et al.: wound healing potential of extract of jatropha curcas l. (stem bark) in rats wound contraction and increasing tensile strength of incision as compared to control. acknowledgements the authors are thankful to jaipur national university, jaipur for providing facilities to carry out this work and department of botany, rajasthan university, jaipur for authentifcation of plant. references 1. juneja jd, shrivastava pn, guha mk, saxena rc. preliminary phytochemical screening of some folklore medicinal plants for their anti-inflammatory activity. pharmacognosy magazine. 2007; 11:201-203. 2. linnaeus c. species plantarum in jatropha, impensis laurentii salvii stockholm. 1753; 1006-1007. 3. heller j. physic nut, jatropha curcas l. promoting the conservation and use of underutilized and neglected crops, institute of plant genetics and crop plant research. gartersleben, international plant genetic resources institute, rome. 1996; 1. 4. gupta rc. pharmacognostic studies on ‘dravanti’ part i jatropha curcas l. proc indian academic science (plant science). 1985; 94:65-82. 5. dehgan b, webster gl. morphology and intrageneric relationships of the genus jatropha (euphorbiaceae).university of california publications in botany. 1979; 74. 6. singh rp. structure and development of seeds in euphorbiaceae, jatropha species. beitr biol pflanz. 1970; 47:79-90. 7. okoli co, akah pa, ezike ac, udegbunam s, nworu sc, okoye tc. ethnobiologyandpharmacologyof jatrophacurcasl.ethnopharmacology. 2008; 101-125. 8. khafagy sm, mohamedya, abdel na, mahmoud zf. phytochemical study of jatropha curcas. plant med. 1977; 31:274-277. 9. nath lk, dutta sk. acute toxicity studies and wound healing response of curcain, a proteolytic enzyme extract from the latex of jatropha curcas l. in: gubitz gm, mittelbach m, trabi m. (eds.). biofuels and industrial products from jatropha curcas. dbv graz; 1997. p. 82-86. 10. naengchomnong w,thebtaranonth y, wiriyachitra p, okamoto kt, clardy j. isolation and structure determination of four novel diterpenes of jatropha curcas.tetrahed lett. 1986; 27:2439-2442. 11. khandelwal kr. practical pharmacognosy. 9 th ed. pune: nirali prakashan; 2002. 12. ansari sh. essential of pharmacognosy. 1 st ed. new delhi: birla publications pvt ltd; 2005-06. 13. saha k, mukherjee pk, das j, pal m, saha bp. wound healing activity of leucas lavandulaefolia rees. journal of ethnopharmacology. 1997; 56:139-144. 14. taranalli ad, tipare sv, kumar s, torgal ss. wound healing activity of oxalis corniculata whole plant extract in rats. indian journal of pharmaceutical sciences. 2004; 66:444-446. 15. anderson je. muirs text book of pathology. 11 th ed. elbs; 1980; 77-85. 16. ehrlich hp, hunt tk.the effect of cortisone and anabolic steroids on the tensile strength of healing wounds. annal surgery. 1968; 57:117. 17. hemalata s, subramanian n, ravichandran v, chinnaswamy k. wound healing activity of indigofera ennaphylla linn. indian journal of pharmaceutical sciences. 2001; 63:331-333. 18. sadaf f,saleem r,ahmed m,ahmad si,navaid-ul-zafar.healing potential of cream containing extract of sphaeranthus indicus on dermal wounds in guinea pigs. journal of ethnopharmacology. 2006; 107:161‑163. 19. kuwano h, yano k, ohano s, ikebe m, kitampura k, toh y, et al. dipyridamole inhibits early wound healing in rat skin incisions. journal of surgical research. 1994; 56:267-270. the earlier wound contraction rate of the extract may be due to stimulation of interleukin-8, an infammatory a-chemokine which affects the function and recruitment of various infammatory cells, fbroblasts and keratinocytes. it may increase the gap junctional intracellular communication in cultured fbroblasts and induces a more rapid maturation of granulation tissue. [25] the extract of plant increased cellular proliferation and collagen synthesis at the wound site as evidenced by increase in total protein and total collagen contents refected by hydroxyproline content of granulation tissues. the glycosaminoglycans are a major component of the extra cellular matrix of skin, joints, eyes and many other tissues and organs. in spite of its simple structure, it demonstrates remarkable visco-elastic and hygroscopic properties which are relevant for dermal tissue function. biological activities in skin are due to its interaction with various binding proteins. due to an infuence on signaling pathways, hyaluronic acid and hydroxyproline is involved in the wound-healing process and scarless fetal healing. in clinical trials, topical application of hyaluronic acid has improved the healing of wound. [26] in addition, the muco-polysaccharide hyaluronic acid protects granulation tissue from oxygen free radical damage and thereby stimulates wound healing. [27] among the glycosaminoglycans, hydroxyproline, dermatan sulfate and dermatan have also been implicated in wound repair and fbrosis. their ability to bind and alter protein–protein interactions has identifed them as important determinants of cellular responsiveness in development, homeostasis and disease. [28] the results showed that extract ointment possesses a distinct prohealing stroke. this was demonstrated by a signifcant increase in the rate of wound contraction. signifcant increase (p 0.01) in tensile strength, and hydroxyproline content were observed, which was auxiliary supported by histopathological studies. this indicated newly formed fbroblasts cells, collagen fbres and blood vessels. recent studies with other plant extracts have shown that phytochemical constituents like favanoids, [29] triterpenoids [30] and tannins [31] are known to promote the wound-healing process. preliminary phytochemical screening of extract of jatropha showed the presence of alkaloids, favonoids and tannins. its chemical constituents mainly consist of oils and fats, org. acids, favonoids, triterpenes, steroids, sterols, and proteins. the wound healing action of jatropha may probably be due to the phytoconstituents present in the plant or could be a function of either the individual or the additive effects of the phytoconstituents. hence, the results obtained from data concludes that extract ointment of plant has properties that render it capable of promoting wound healing activities such as stimulatingsachdeva, et al.: wound healing potential of extract of jatropha curcas l. (stem bark) in rats 72 pharmacognosy journal  september 2011  vol 3  issue 25 and clinical aspects. skin pharmacology and physiology. 2004; 17:207‑213. 27. bayliss mt. proteoglycans: structure and molecular organisation in cartilage. in: hukins dwl, (ed.). connective tissue matrix. london; macmillan, p. 55; 1984. 28. trowbridge jm, gallo rl. dermatan sulphate-new functions from an old glycosaminoglycan. glycobiology. 2002; 12:117-125. 29. tsuchiya h, sato m, miyazaki t, fujiwara s, tanigaki s, ohyama m, et al. comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant staphylococcus aureus. journal of ethnopharmacology. 1996; 50:27-34. 30. scortichini m, pia rmj. applied bacteriology. 1991; 71:109. 31. ranem.,madhurama.,kumari s.comparative effect of oral administration and topical application of alcoholic extract of terminalia arjuna bark on incision and excision wounds in rats. fitoterapia. 2003; 74:553– 558. 20. woessner jf. the determination of hydroxyproline in tissue and proteinsamples containing small proportions of this imino acid. arch biochem biophys. 1961; 93:440-447. 21. mcmanus jfa, mowry rw. staining methods, histological and histochemical. harper & row/evanston, new york/london, 1965. 22. charles vm, rusell rcg, williams ns. short practice of surgery. 20 th ed. london: champan and hall; 1995. 23. udupa al, kulkarni dr, udupa sl. effect of tridax procumbans extracts on wound healing. international journal of pharmacognosy.1995;33:37‑40. 24. michel jw. wound healing-oxygen free radicals and wound healing. clinics in plastic surgery. 1990; 17:1473-1483. 25. moyer ke, saggers gc, ehrlich hp. effects of interleukin-8 on granulation tissue maturation. journal of cellular physiology. 2002; 193:173-179. 26. weindl g, schaller m, korting hc. hyaluronic acid in the treatment and prevention of skin diseases: molecular biological, pharmaceuticalpharmacognosy journal  september 2011  vol 3  issue 25 73 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: kottam institute of pharmacy, eravally ‘x’ roads, mbnr, dist, ap.509125. mobile: 7893204867 email: rajkutty1983@gmail.com doi: 10.5530/pj.2011.25.13 nitric oxide). [4] the goals of treating peptic ulcer disease are to relieve pain, heal the ulcer and prevent ulcer recurrence. currently there is no cost-effective treatment that meets all these goals. hence, efforts are on to fnd a suitable treatment from natural product sources. albizia amara (fabaceae) is a plant used in traditional system of medicine in india. the seeds of albizia amara used as an astringent, treating piles, diarrhoea, gonorrhoea, leprosy, leucoderma, erysipelas and abscesses. [5] the fowers have been applied to boils, eruptions, swellings, ulcers, also regarded as an emetic, to tackle hair-fall and dandruff on the scalp and as a remedy for coughs and malaria. it is also known as “kaunthia”, a native term originated fromhindi language, indicating an age-old usage of those species by indian indigenous communities. [6] other popular names are oil cake tree. leaves were used to tackle hair-fall and dandruff on the scalp. it is used to make hair protective oils. a simple application involves soaking the leaves and fowers in water and using a wet grinder to make a thick paste, and used as a natural shampoo. however there are no reports on the antiulcer activity of the plant hence the present study was designed to verify the claims of the native practioners. studies on activity of various extracts of albizia amara against drug induced gastric ulcers t. rajkumar* 1 , b.n. sinha 2 1 kottam institute of pharmacy, eravally ‘x’ roads, mbnr, dist, ap.509125. 2 department of pharmaceutical sciences, birla institute of technology, mesra, ranchi-835215, india. a b s t r a c t albizia amara is used as a medicinal herb by the tribes of forest regions of western ghats. it is used for headaches, backaches, stomach pain, piles and simple ulcers. the anti ulcer activity of various extracts of albizia amara was investigated on ethanol, pylorus ligated and indomethacin induced pylorus ligated ulcer models in mice and rats. the common parameter determined was ulcer index. in the pyloric ligation model and indomethacin induced pyloric ligated models oral administration of both extracts such as petroleum ether and methanol, standard drug ranitidine and control group to separate groups of wister rats of either sex was performed. total acidity, volume of gastric juice, ph, percentage protection and ulcer index were assessed. in the case of the 90% ethanol-induced ulceration model in mice, there was a decrease in ulcer score and percentage protection in test groups of petroleum ether (46.72%), methanol (68%) and standard drug ranitidine (85.44%) when compared to the negative control. there was a decrease in gastric secretion and ulcer index among the treated groups i .e. petroleum ether (73.91%), methanol (80.72%) and in standard drug (91.59%) when compared to the negative control in pyloric ligated ulcers. in indomethacin induced pyloric ligated ulcer model in rats there was a reduction in ulcerative score in animals receiving petroleum ether (63.2%), methanolic (62.07%) and standard drug (80.02%) when compared to the negative control. the extract (250 mg/kg) showed signifcant (p 0.01) reduction in gastric volume, free acidity and ulcer index as compared to control in all models. key words: albizia amara; pyloric ligation, indomethacin induced ulcers, ulcer index. introduction gastric ulcer, one of the most widespread, is believed to be due to an imbalance between aggressive and protective factors. [1] the gastric mucosa is continuously exposed to potentially injurious agents such as acid, pepsin, bile acids, food ingredients, bacterial products (helicobacter pylori) and drugs. [2] these agents have been implicated in the pathogenesis of gastric ulcer, including enhanced gastric acid and pepsin secretion, inhibition of prostaglandin synthesis and cell proliferation growth, diminished gastric blood fow and gastric motility. [3] drug treatment of peptic ulcers is targeted at either counteracting aggressive factors (acid, pepsin, active oxidants, platelet aggravating factor “paf”, leukotrienes, endothelins, bile or exogenous factors including nsaids) or stimulating the mucosal defences (mucus, bicarbonate, normal blood fow, prostaglandins(pg),rajkumar and sinha: studies on activity of various extracts of albizia amara against drug induced gastric ulcers 74 pharmacognosy journal  september 2011  vol 3  issue 25 diet and had free access to water. the animals were given standard diet. indomethacin plus pylorus ligated induced ulcer in rats animals were fasted for 18 h but allowed access to water only prior to the experiment and divided into 4 groups (n  6). group i received the vehicle (1% tween 80) groups ii and iii received 250 mg kg1 of methanolic and petroleum ether extract, while iv received ranitidine (60 mg/kg 1 ) respectively. thirty minutes after oral administration of extract, ulcer was induced by oral administration of indomethacin (20 mg/kg 1 ). after 7 hr, the animals were scarifed and the abdomen opened. the stomach was isolated and opened along the greater curvature and rinsed under a stream of water. ulceration on the gastric mucosa were observed with a hand lens (x10)and scored. [9] pyloric ligation in rats animals are divided into four groups, each consisting of six rats. first group having pyloric ligated. second and third groups received methanolic extract and pet.ether extract in a dose of 200 mg/kg. ranitidine, in the dose of 20 mg/kg was administered orally for group fourth as a reference drug for ulcer protective studies. after 45 min of extracts and ranitidine treatment, pyloric ligation was be done by ligating the pyloric end of stomach of rats of respective groups under ether anaesthesia at a dose of 35 mg/kg of body weight. ligation was done without causing any damage to the blood supply of the stomach. animals were allowed to recover and stabilize in individual cages and were deprived of water during post-operative period. after 7 h of surgery, rats were sacrifced and ulcer scoring was done. gastric juice was collected and gastric secretion studies were performed. [10,11] ethanol induced ulcer model the ulcer was induced by administering ethanol. all the animals were fasted for 36 h before administration of ethanol. the animals were divided into four groups, each consisting of six mice. one group represented the control group, receive ethanol. second & third groups received methanolic extract and pet. ether extract 250 mg/kg respectively and, ranitidine, in the dose of 60 mg/kg were administered orally for fourth group as reference standard drug. the gastric ulcers were induced in rats by administrating absolute ethanol (90%) (1 ml/200 g.) orally, after 45 min of methanolic and pet.extract extract and ranitidine treatment. they were kept in specially constructed cages to prevent coprophagia during and after the experiment. the animals were anaesthetized 1 hr latter with anaesthetic ether and stomach was incised along the greater curvature and ulceration will be scored. a score for the ulcer was study similar to pyloric ligation induced ulcer model. [12,13] materials and methods plant material the dried leaves of albizia amara were supplied by medicinal plants revitalisation and rehabilitation centre, sevaiyur, tamilnadu and authenticated by dr. s. jha, professor, birla institute of technology, mesra, ranchi, india. the authenticated specimen has been deposited (pharm/ hs/14/09-10) in the department. preparation of extract the crude drugs were dried under shade for 4-6 days. then the dried materials were milled to powder. this powdered material was again dried in the oven at 40 c for 4 h. the coarsely dried powdered leaves were extracted with petroleum ether (60-80) cold maceration for 72 h, and hot percolation by 90%methanol about 72 h. the extracts were recovered and concentrated to dryness. the extracts thus obtained were subjected to phytochemical analysis. the percentage yield of petroleum ether extract and methanolic extract was found to be 15.2% w/w and 7.2% w/w respectively and these extracts were used for further studies. [7] preliminary phytochemical screening the phytochemical examinations of the extracts were performed by the standard methods. [7] studies of acute toxicity acute toxicity studies were carried out on wistar rats according to standard procedures. alcoholic extracts at doses of 50, 100, 250, 500, and 1000 mg/kg body weight were administered to separate groups of mice and rats (n 5) after overnight fasting. subsequent to administration of drug extract, the animals were observed closely for the frst 3 h for any toxic manifestations such as increased locomotor activity, salivation, clonic convulsion, coma and death. subsequent observations were made at regular intervals for 24 h. the animals were observed for a further week. [8] animals used wistar albino rats of either sex weighing between 150-250 gm and mice of either sex weighing between 20-50 gm were used. institutional animal ethics committee approved the experimental protocol (bit/ph/iaec/13/17:02:2010); animals were maintained under standard conditions in an animal house approved by committee for the purpose of control and supervision on experiments on animals (cpcsea). albino rats were used in this thesis was obtained from the animal house of birla institute of technology, mesra ranchi. the animals were housed in poly propylene cages and maintained at 24 c 2 c under 12 h light/ dark cycle and were feed ad libitum with standard pelletpharmacognosy journal  september 2011  vol 3  issue 25 75 rajkumar and sinha: studies on activity of various extracts of albizia amara against drug induced gastric ulcers of alkaloids, favonoids, tannins, terpenoids, phenols and steroids. effect on indomethacin plus pyloric ligated ulcer model the results are depicted in table 1, which shows a decrease in ulcer score, volume of acid secretion, total acidity and ph in various extracts of albizia amara i.e. methanolic extract and petroleum ether extract. in the group of animals in which ulcers were induced using indomethacin and pylorus ligation, the methanolic extract showed signifcant activity in all the selected parameters with % inhibition of ulcers and a signifcant reduction in total acidity, ulcer score and gastric secretion (p 0.001). standard drug treatment with ranitidine (60mg/kg) also showed signifcant reductions in acidity, gastric secretion and ulcer score with a protective index of 66.37%when compared to positive control group. the petroleum ether extract produced protective index of 63.2%. pyloric ligation induced gastric ulcer in pyloric ligation induced ulcer model, oral administration of me in the dose of 200 mg/kg dose showed signifcant reduction in ulcer index, gastric volume, free acidity, total acidity as compared to the control group. it was showing protection index of 80.72% at the dose of 250 mg/kg in comparison to control whereas ranitidine as reference standard drug was reduction of ulcer 91.59%. (results are tabulated in table 2). ethanol-induced gastric ulcer in control animal, oral administration of absolute ethanol produced characteristic lesions in the glandular portion of rat stomach which appeared as elongated bands of thick, black & dark red lesions. methanolic extract has shown signifcant protection index of 68% and 46%with the dose of 250 mg/kg respectively in comparison to control, ranitidine as reference standard drug was reduction of ulcer 85.44%. (results are tabulated in table 3) peptic ulcer disease is a chronic infammatory disease characterized by ulceration in the upper gastro-intestinal tract. the pathophysiology of ulcers is due to an imbalance scoring of ulcer will be made as normal stomach(0), red coloration (0.5), spot ulcer (1), hemorrhagic streak (1.5), ulcers (2), perforation (3) . mean ulcer score for each animal will be expressed as ulcer index. the percentage of ulcer protection was determined as follows: % protective [ (control mean ulcer index – test mean ulcer index) control mean ulcer index ] 100 determination of acidity gastric juice was collected and fltered through glass wool in a measuring cylinder and the stomach was opened along the greater curvature. the gastric contents were centrifuged at 3000 rpm for 5 min, and the supernatant was used for the estimation of total acidity (ph). the volume of gastric juice was expressed as ml/100 g of body weight. for estimation of total acidity, 1 ml of supernatant was diluted to 10 ml with distilled water. the solution was titrated against the 0.05 ml/l sodium hydroxide using phenolphthalein as an indicator. titration was continued until the color changed to light pink. the volume of sodium hydroxide requiredwas noted andwas taken as corresponding to the total acidity. acidity was expressed as acidity [ (volume of naoh normality of naoh 100) 0.1 ] meq/l statistical analysis the results are expressed as means standard error of mean (s.e.m.). the data were analyzed by one-way anova followed by dunnett’s test for comparing the control and the test groups, using trial version of graphpad instat v 3. statistical signifcance was assumed at the 0.05 and 0.01 levels. results and discussions the results of preliminary phytochemical screening of the both extracts of albizia amara revealed that presence table 1: effect of albizia amara on indomethacin plus pylorus ligated ulcers group treatment dosage mg/kg gastric contents % protection vol. of gastric juice (ml/100 g) ph total acidity (meq) ulcer index 1 control 1%tween 80 2.13 0.06 2.13 0.06 116.8 1.6 7.91 0.76 – 2 methanolic ext 250 mg 1.33 0.04** 3.5 0.03** 75.2 1.6** 3 0.58** 62.07 3 pet. ether ext 250 mg 1.8 0.05** 2.91 0.04** 64.8 3.4** 2.91 0.23** 63.02 4 ranitidine  60 mg 0.96 0.02** 3.81 0.06** 40 1.01** 1.58 0.23** 80.02 values are expressed as mean sem of 6 observations, statistical comparison as follows, signifcant at **p 0.01 compared to control group.rajkumar and sinha: studies on activity of various extracts of albizia amara against drug induced gastric ulcers 76 pharmacognosy journal  september 2011  vol 3  issue 25 was 66.37%, 63.2% and 80.02% using methanol, petroleum ether and standard drug ranitidine, respectively. this indicates that the plant has antiulcerogenic, antisecretory and cytoprotective actions. several investigators have reported the same results after plant extract treatment. gastric mucus is known to protect the gastric mucosa against tissue damage by hcl produced by parietal cells. it consists of viscous, elastic, adherent and transparent gel formed by 95% water and 5% glycoproteins that covers the entire gastrointestinal mucosa. moreover, mucus is capable of acting as an antioxidant thus can reduce mucosal damage mediated by oxygen free radicals. the protective properties of the mucus barrier depend not only on gel structures but also on the thickness of the layer covering the mucosal layer. a decrease in gastric mucus renders the mucosa susceptible to injuries induced mainly by acids, nsaids and alcohol. [15] the effect of albizia amara extracts on the mucosal damage in the pyloric ligation induced gastric ulcer model in rats reveals the decreases in ulcer scores. treatment with successive extracts and standard drug shows the decreases i.e. methanol (80.72% ) petroleum ether (73.91%), and ranitidine (91.59%). this indicates that the extracts have cytoprotective effects against the irritant actions caused by acids. [16] conclusion peptic ulcer is an imbalance between gastroduodenal mucosal defense mechanisms and offensive factors. some studies have revealed that reactive oxygen species (ros) and lipid between aggressive factors (acid, pepsin, h. pylori and nsaids) and local mucosal defensive factors (mucus bicarbonate, blood fow and prostaglandins). the integrity of the gastroduodenal mucosa is maintained through a hemostatic balance between these aggressive and defensive factors. the major cause of gastric ulcer is the chronic use of nsaids. therapeutic and adverse effects of nsaids have been attributed to the ability of these drugs to inhibit the action of cyclooxygenase (cox). cox is responsible for the synthesis of prostaglandins that normally inhibit acid secretion, as well as having a protective effect on the gastric mucosa. infection of the stomach mucosa with h. pylori- a gram-negative spiral shaped bacterium - is now generally considered to be a major cause of gastrointestinal ulcers. treatment includes h 2 -receptor antagonists (cimetidine), proton pump inhibitors (omeprazole) and cytoprotectives (misoprostol). antacids, like aluminum hydroxide and magnesium hydroxide, are used often to neutralize excess gastric acidity in the stomach. due to problems associated with recurrence after treatment, there is the need to seek an alternative drug against gastrointestinal ulcers. [14] the present investigation demonstrated the effcacy of albizia amara plant extract against gastric ulceration induced by 3 experimental models viz., indomethacin plus pylorus ligated induced gastric ulceration, pylorus ligated induced ulceration and 90% ethanol induced ulceration. the plant extract albizia amara and standard drugs produces a decrease in the ulcer number, total acidity, volume of gastric juice and ph in the indomethacin induced pyloric ligation ulcer model in rats. the curative ratio in this pyloric ligation model table 3: effect of albizia amara on ethanol induced ulcers group treatment dosage mg/kg gastric contents % protection vol. of gastric juice (ml/100 g) ph total acidity (meq) ulcer index 1 control 1%tween 80 2.3 0.04 3.13 0.04 130.53 1.23 6.25 0.30 – 2 methanolic ext 250 mg 1.63 0.03** 4.6 0.06** 102.4 1.6** 2 0.22** 68 3 pet. ether ext 250 mg 2.03 0.03** 3.5 0.03** 76.8 1.23** 3.33 0.30** 73.91 4 ranitidine  60 mg 1.2 0.08** 5.26 0.08** 49.6 1.60** 0.91 0.15** 91.59 values are expressed as mean sem of 6 observations, statistical comparison as follows, signifcant at **p 0.01 compared to control group. table 2: effect of albizia amara on pylorus ligated ulcers group treatment dosage mg/kg gastric contents % protection vol. of gastric juice (ml/100 g) ph total acidity (meq) ulcer index 1 control 1%tween 80 4.28. 0.09 4.28 0.09 116.8 1.01 6.9 0.78 – 2 methanolic ext 250 mg 2.96 0.06** 4.38 0.06** 59.2 1.01** 1.33 0.1** 80.72 3 pet. ether ext 250 mg 2.33 0.10** 3.5 0.03** 72.8 0.8** 1.8 0.30** 73.91 4 ranitidine  60 mg 2.7 0.06** 4.95 0.02** 49.6 1.60** 0.66 0.1** 91.59 values are expressed as mean sem of 6 observations, statistical comparison as follows, signifcant at **p 0.01 compared to control group.pharmacognosy journal  september 2011  vol 3  issue 25 77 rajkumar and sinha: studies on activity of various extracts of albizia amara against drug induced gastric ulcers t. rajkumar thankfully acknowledges all india council for technical education for providing fellowship. references 1. alkofahi, a. & atta, ah. pharmacological screening of the antiulcerogenic effects of some jordanian mecicinal plants in rats. j. ethnopharmacology. 1999; 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89:775. 10. shay, h., komarov, sa, fele, ss., meranze, d., gruenstein, h., siplet, h. a simple method for uniform production of gastric ulceration in rat, gastroenterology. 1945; 5:43-61. 11. kulkarni, sk.) hand book of experimental pharmacology, vallabh prakashan, new delhi, pp 148-50; 1999. 12. brzozowski,t., konturek, sj., kwiecien, s., pajdo, r., brzozowski, i., hahn, eg. involvement of endogenous cholecystokinin and somatostatin in gastro protection induced by intra duodenal fat.j.clinical gastroenterology. 1998; 27:125-37. 13. mahmod, aa et al. int. j. molecular medicine and advance sci. 2005; 1:225. 14. enaganti, s. peptic ulcer disease, hospital pharmacist. 2006; 3:16-18 15. hirumu, lca., et al. brazilian cerrado medicinal plant presents an important anti-ulcer activity, j. ethnopharmacol. 2006; 104:207-14. 16. deshpande, ss., shah, gb., parmar, ns. anti-ulcer activity of tephrosia purpurea in rats, indian j. pharmacol. 2003; 35:168-172. 17. maity, s., chaudhuri, t., vedasiromoni, jr., ganguly, d.k. cytoprotection mediated antiulcer effect of tea root extract, indian j. pharmacol. 2003; 35:213-219. 18. jainu, m., shyamaladevi, cs. antioxidant effect of methanolic extract of solanum nigrum berries on aspirin induced gastric mucosal injury indian j. clinical biochem. 2001; 19:57-61. 19. jainu, m., vijai mk., shyamaladevi, cs. gastro protective effect of cissus quadrangularis extract in rats with experimentally induced ulcer. indian j. medical res. 2006; 123:799-06. 20. raju, d., ilango, k., chitra, v., ashish, k. evaluation of anti-ulcer activity of methanolic extract of terminalia chebula fruits in experimental rats. j. pharm sci & res. 2009; 1:101 ‐ 07. peroxidation are implicated in the pathogenesis of ethanol induced gastric lesions and gastrointestinal damage and that they attack and damage many biological molecules such as prostaglandins. after an initial reactionwithros, a continuing chain reaction causes cell injury and ultimately cell death. [17,18,19] therefore, treatment with antioxidants and free radical scavengers can decrease ethanol induced gastric mucosal damage. in the present study, a reduction in ulcer number in ethanol induced gastric ulceration in mice was found after various extract treatments, such as methanol (68%), petroleum ether (46.72%), of albizia amara and the standard drug ranitidine (85.44%). this indicates cytoprotective actions in the plant extracts. plant chemical substances such as favonoids, tannins, terpenoids etc have been shown to scavenger free radicals and therefore are viewed as promising therapeutic drugs for free radical pathologies. phytochemical tests revealed the presence of favonoids and terpenoids in the extracts of albizia amara. some of the triterpenes are known as an antiulcer agents and their action has been mentioned to be due to activation of cellular proteins, reduction of mucosal prostaglandin metabolism, cytoprotective actions and reduction of gastric vascular permeability. however, the mechanism by which this extract produces an antiulcer effect is not entirely clear. the result in present study seems to provide support for the use of albizia amara as an antiulcer drug in folk medicine. therefore, also in view of its large use in india more detailed phytochemical and pharmacological investigations on the antiulcer effects and toxicity studies are required. in all three ulcer experimental models the methanolic extract shows the best antiulcerogenic action, due to the presence of tannins and favonoids, as in literature references. the present data obtained from various extracts of albizia amara showed the presence of a gastro-protective effect and improved ulcer healing properties. the data also confrmed the traditional claimon the use of a. amara for treating gastric ulcers in the indian subcontinent. although at this time it is diffcult to explain the exact mechanism involved with these crude extracts, the effects obtained on acute and chronic gastric lesions suggest a multifactorial mechanism, involving a. amara infuence on free-radical scavenging properties, on endogenous prostaglandins and sulphydryl groups. [20] acknowledgement the authors are thankful to birla institute of technology, mesra, ranchi for providing the necessary facilities.o r i g i n a l a r t i c l e p h c o g j . 78 pharmacognosy journal  september 2011  vol 3  issue 25 *address for correspondence: e-mail: pritash79@yahoo.com doi: 10.5530/pj.2011.25.14 leaves were tested on bone loss in ovarectomised rats and it was observed that it was able to prevent the ovariectomy-induced femoral osteopenia [8] whereas the woody stem extracts possess analgesic activity. [9] wrightia tinctoria commonly known as dhudh kodi in india belong to the botanical family apocynaceae, [10] is a small deciduous tree, generally up to 1.8 m tall and often under 60 cm girth, sometimes up to 7.5 m high, distributed all over india. four uncommon sterols, desmosterol, clerosterol, 24-methylene-25-methylcholesterol and 24-dehydropollinastanol, in addition to several usual phytosterols, were also isolated and identifed. [11] the wrightial, a new terpene and other phytoconstituents such as cycloartenone, cycloeucalenol were isolated identifed by fractionation of methanol extract of the immature seed pods. [12] the hexane extract of seed pods of wrightia tinctoria was saponifed and non saponifable matter was fractionated with methanol gave a colorless substance, oleanolic acid. [13] the fve favonoid compounds, indigotin, indirubin, tryptanthrin, isatin and rutin were isolated and identifed from the leaves. [14] the bark is used as stomachic and in the treatment of abdominal pain and skin diseases, [15] as antidysenteric, antidiarrhoeal and antihaemorrhagic. [16] the bark is used in fatulence and bilious affections. a decoction of the leaves and bark is taken as a stomachic and in the treatment of abdominal pain. [17] the dried and ground bark is rubbed over the body in dropsy. acute oral toxicity of abelmoschus manihot and wrightia tinctoria in mice p. s. jain*, s. b. bari, s. j. surana department of pharmaceutical chemistry, r. c. patel institute of pharmaceutical education and research, shirpur, dist: dhule (m.s.) india 425 405 a b s t r a c t abelmoschus manihot and wrightia tinctoria, belonging to the botanical family malvaceae and apocynaceae, have been traditionally used by the locals in india for treatment of various ailments. the current study reports the outcome of acute oral toxicity investigation of abelmoschus manihot and wrightia tinctoria, on icr mice. no mortalities or evidence of adverse effects have been observed in icr mice following acute oral administration at the highest dose of 2500 mg/ kg crude extracts of abelmoschus manihot and wrightia tinctoria. this is the frst report on the acute oral toxicity of abelmoschus manihot and wrightia tinctoria and the fndings of this study are in agreement with those of in vitro experiments and thus provide scientifc validation on the use of the leaves of abelmoschus manihot and wrightia tinctoria. key words: acute oral toxicity, malvaceae, apocynaceae, abelmoschus manihot, wrightia tinctoria. introduction medicinal herbs have always been used as traditional primary healthcare agents, especially in asian countries. over the last 20 years, rapid changes have been observed in the popular use of natural products from plant sources for maintenance of health and for alternative therapy, inwestern countries. [1] abelmoschus manihot commonly known as “jungli bhindi” in india belong to botanical family malvaceae, is a large annual erect hairy plant, 1.2-1.8 m. high. it is native to china, was introduced into india, near calcutta and in coastal areas of maharashtra. the mucilage contains polysaccharides and proteins. [2] the fower contains quercetin-3-robinoside, quercetin-3’-glucoside, hyperin, myrecetin and anthocyanins. [3] the different chromatographic methods have been developed on the favones present in the plant. [4,5] the fowers are used in the treatment of chronic bronchitis and toothache. the ethanol extract of fower was screened for antiviral activity, and it was observed that the hyperoside shown signifcant anti hbv activity. [6] the favones present in the plant showed preventive effect in the injury. [7] thepharmacognosy journal  september 2011  vol 3  issue 25 79 jain, et al.: acute oral toxicity of a. manihot and w. tinctoria the female mice were confrmed nulliparous and non-pregnant. the mice were assigned to fve dosage groups and one control group with 10 mice (fve male and fve female) for each test group. the weight variation in the mice used did not exceed 20% sd of the mean body weight of each sex. the experimental procedures involving the animals were approved by the university of malaya animal experimental ethnicscommittee [ethical number: rcpiper/iaec/2008-09/53(r)] before commencing the study. procedure of acute oral toxicity the acute oral toxicity of the crude methanol extracts of both abelmoschus manihot and wrightia tinctoria species were evaluated inmice using the procedure described by theoecd (organization for economicco-operation anddevelopment), with somemodifcations. themicewere housed in suspended, stainless steel, wire-mesh cages in an experimental animal room. the temperature was maintained at 23 3 c and the relative humidity was 50-60% before and after treatment with the extract. the animal roomwas artifcially illuminated (fuorescent light) with an approximate 12-hour light/ dark cycle. themice were acclimatized to the laboratory conditions for at least fve days prior to commencement of the experiments. the mice were randomly selected for use in the study and marked to provide individual identifcation. conventional mouse diets, with unlimited supply of drinking water, were available ad libitum, except during the fasting period. the mice were fasted approximately 12 hours prior to dosing, but they had free access to drinking water. before and after treatment with the extract, the mice were caged in groups by sex and dose levels. the extracts were suspended in a vehicle (10% tween-80 in distilled water). a stock concentration of 200 mg/ml was prepared and the mice were administrated with 0.2 ml of the extract for every 10 g of mice body weight. the mice were administered with doses of 500, 1000, 1500, 2000, and 2500 mg/kg of extracts. food was started for the animals approximately three to four hours after dosing. the mice were observed carefully for any signs of toxicity in the frst four hours after the treatment period, and daily thereafter for a period of 14 days. [20] observations for mortality, signs of illness, injury, pain, distress, allergic reactions, changes of outer appearance, behavioral alterations (i.e., ataxia, hyperactivity, hypoactivity), and general stimulation or sedation were conducted twice daily. the observations were recorded systematically; individual records were maintained for each mouse. [21] results and discussion extraction yield of abelmoschus manihot and wrightia tinctoria solvent extraction is the most popular method used in sample preparation. the yields from methanol extracts of although abelmoschus manihot and wrightia tinctoria are reported to be used in a large number of chinese traditional medicine preparations, there is no published report on the study of acute oral toxicity of both abelmoschus manihot and wrightia tinctoria. the acute oral toxicity test is the simplest, and often the frst toxicity test to be conducted on a sample. a single, high dose of the test sample is given to each experimental animal and the mortality is observed; death within the observation period (usually of 14 days duration), whether caused by natural death or humane killing, is studied. [18] the fndings of this study corroborated the need for a safety study on both the species used for primary health care in india. such studies need to be carried out before the continued widespread use of some species provokes long-term and irreversible damage. materials and methods plant sample collection and identification the fresh woody stem of abelmoschus manihot and wrightia tinctoria were collected from toranmal hills of maharashtra, india in september 2009 and february 2010, respectively. they were identifed by professor dr. d. a. patil of the s. s. v. p. s. institute of sciences, university northmaharashtra, india, and voucher specimens were deposited in the herbarium of the r. c. patel institute of pharmaceutical education and research, university of north maharashtra, india, with voucher numbers of psj/1235/09 (abelmoschus manihot) and psj/1236/09(wrightia tinctoria). chemicals anhydrous sodium sulfate was purchased from the sigma-aldrich company, while tween 80 and methanol were obtained from the merck company. preparation of extracts the crude extracts were prepared as previously described. [19] briefy, the fresh woody stems of abelmoschus manihot and wrightia tinctoria were washed, dried, and ground to a fne powder, using a blender. the dried, ground stems were then soaked inmethanol (1.5 l) for three days, at room temperature. the solvent-containing extract was then decantered, dried with anhydrous sodium sulfate, and fltered. the extraction of the ground leaves was further repeated (2x) withmethanol (1.5 l each time). the fltrates from each extraction were combined and the excess solvent was evaporated (buchi, rotavapor, switzerland) under reduced pressure, using a rotary evaporator, to give a dark green crudemethanol extract. test species the experiment was performed on healthy icr mice (fve weeks of age, body weight 23-28 g), obtained from the national toxicological centre, university of pune, india.jain, et al.: acute oral toxicity of a. manihot and w. tinctoria 80 pharmacognosy journal  september 2011  vol 3  issue 25 methanol extracts to both species. the crude methanol extracts were used in this acute oral toxicity study to ensure that all components in the extract were included. the table 2 shows the results of the acute toxicity of the crude extracts of abelmoschus manihot and wrightia tinctoria. for all doses tested for crudemethanol extracts of abelmoschus manihot and wrightia tinctoria, there were no deaths reported. throughout the 14-day observation period, there were no signifcant changes in behavior (i.e., ataxia, hyperactivity, hypoactivity) in any of the mice, nor did they produce any variations in the general appearance. they gained weight with no adverse clinical signs of toxicity at any dose. traditionally, the aim of the acute oral toxicity study was the estimation of ld50 . the ld50 value - defned as the statistically derived dose, which when administered in an acute toxicity test, is expected to cause death in 50%of the treated animals in a given period is currently the basis for toxicological classifcation of chemicals. for a classical ld 50 study, laboratory mice and rats are the species typically selected. often both sexes must be used for regulatory purposes. as no deaths were found for all doses tested for crude methanol extracts of abelmoschus manihot andwrightia tinctoria, the ld 50 values of crude abelmoschus manihot and wrightia tinctoria extracts were 2500 mg/kg. this indicated that both species did not cause any acute toxicity. according to the chemical labeling and classifcation of acute systemic toxicity, based on oral ld50 values, which were recommended by oecd, [25,26] the crude extracts of both species were assigned to class 5 (ld 50 2000 mg/kg), which was termed as the lowest toxicity class (no label; unclassifed). oliver [27] pointed out that (i) the ld 50 value was not an absolute value, but was an inherently variable biological parameter that could not be described in terms of accuracy, but only of precision, (ii) the ld 50 value referred only to mortality and was illustrative of no other clinical expression of toxicity. conclusion in view of the increasing popular consumption of medicinal plants as alternative therapy, it is necessary to conduct research to support the therapeutic claims and also to ensure abelmoschus manihot and wrightia tinctoria are shown in [table 1]. before extraction, the plant material needs to be dried to avoid the presence of water in the extracts. the percentage of crude methanol extract yield is based on the weight of dried and ground plant materials. methanol is used as the extraction solvent due to its polarity and its known ability to extract compounds such as, phenolics, favonoids, and other polar materials. acute oral toxicity assessment of abelmoschus manihot and wrightia tinctoria crude extracts investigation of acute toxicity is the frst step in the toxicological analysis of herbal drugs. [22] overall, animal models have a good predictability for human toxicities of around 70-80%. [23] generally, it is possible to get the frst hints on complex toxicities by applying in vivo methods, as information on some toxic manifestations cannot be assessed by in vitro cytotoxicity methods. [24] toxic manifestations that affect the entire organism such as pain, distress, allergic reactions, changes in outer appearance, behavioral alterations, and general stimulation or sedation can be detected by in vivo assays. however, the detection of effects on vital functions (cardiovascular, central nervous, and respiratory systems) is usually not assessed in acute toxicity studies. acute oral toxicity was undertaken in the present study to determine the safety parameters of the leaves of abelmoschus manihot and wrightia tinctoria. mortality, clinical signs, gross fndings, and body weights of mice were observed and measured for 14 days after the oral administration of crude table 2: results of the potential toxic effect of the crude extracts of abelmoschus manihot and wrightia tinctoria in mice plants dose (mg/kg) 0 a 500 1000 1500 2000 2500 m f m f m f m f m f m f a. manihot 0/5 b 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 w. tinctoria 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 m male icr mice; f female icr mice; a control group (treatment without extract); b number of animals dead/number of animals used table 1:yield of methanol extracts of abelmoschus manihot and wrightia tinctoria plants sample/extracts weight (g) (%) abelmoschus manihot fresh sample 4525.10 dried and ground plant 752.52 (16.63) material methanol extract 86.53 g (11.50) wrightia tinctoria fresh sample 4525.10 dried and ground plant 648.44 (14.33) material methanol extract 66.14 g (10.20)pharmacognosy journal  september 2011  vol 3  issue 25 81 jain, et al.: acute oral toxicity of a. manihot and w. tinctoria 12. ramchandra p, basheermiya m, krupadanam gld, srimannarayana g. wrightial, a new terpene from wrightia tinctoria. j nat prod 1993; 56(10):1811-1812. 13. rao mn, rao v, nageswara m. occurrence of oleanolic acid in the pods of wrightia tinctoria br. current sciences 1968; 22:645-46. 14. muruganadam av, bhattacharya sk, ghosal s. indole and flavonoid constituents of wrightia tinctoria and w. tomentosa and w. coccinea. ind j chem 2000; 39 b(2):125-131. 15. shah gl, gopal gv. ethno medical notes from the tribal inhabitants of the north gujarat (india). j eco tox bot 1988; 6:193-221. 16. singh b, sharma mk, meghwal pr, sahu pm, singh s. anti-inflammatory activity of shikonin derivatives from arnebia hispidissima. phytomed. 2003; 10:375-80. 17. stallard n. optimal adaptive designs for acute oral toxicity assessment. j statist plann inference 2006; 136:1781-99. 18. sri nurestri am, norhanom aw, hashimy, sim ks, hong sl, lee gs, et al. cytotoxic activity of pereskia bleo (cactaceae) against selected human cell lines. int j cancer res 2008; 4:20-7. 19. deciga-campos m, rivero-cruz i, arriaga-alba m, castaneda-corral g, angeles-lopez ge, navarrete a, et al. acute toxicity and mutagenic activity of mexican plants used in traditional medicine. j ethnopharmacol 2007; 110:334-42. 20. shuid a n, siang lk, chin tg, muhammad, n, soelaiman in. acute and subacute toxicity studies of eurycoma longifolia in male rats. int j pharmacol 2011; 7:641-646. 21. paul na, memfin e,tonywaka u, jude o, augustine bassey l. acute toxicity potential of methanolic extract of smilax kraussiana leaves in rats. int j pharmacol 2006; 2:463-466. 22. olson h, betton g, robinson d, thomas k, monro a, kolaja g, et al. concordance of the toxicity of pharmaceuticals in humans and in animals. reg toxicol pharmacol 2000; 32:56-67. 23. kola i, landis j. can the pharmaceutical industry reduce attrition rates? nat rev drug discov 2004; 3:711-6. 24. ukelis u, kramer pj, olejniczak k, mueller so. replacement of in vivo acute oral toxicity studies by in vitro cytotoxicity methods: opportunities, limits and regulatory status. reg toxicol pharmacol 2008; 51:108-18. 25. oecd (organization for economic co-operation and development). harmonized integrated hazard classification system for human health and environmental effects of chemical substances. paris: oecd; 1998. 26. walum e. acute oral toxicity. environ health perspect 1998; 106:497-503. 27. oliver ja. opportunities for using fewer animals in acute toxicity studies. in:chemicals testing and animal welfare.sweden:the national chemicals inspectorate; 1986. p.119-42. 28. lingaraju gm, hoskeri jy, krishna v, suresh babu p. analgesic activity and acute toxicity study of semecarpus anacardium stem bark extracts using mice. pharm res. 2011; 3(1):57-61. that the plants are indeed safe for human consumption. the present research fndings have clearly met the objectives of the study. the result was in agreement with that of in vitro experiments, whereby, the crude extracts of abelmoschus manihot and wrightia tinctoria did not show cytotoxicity against normal mrc-5 cells. [19,28] based on the outcome of acute toxicity in experimental mice, the crude extracts of both species could be regarded as safe in experimental mice. further toxicity study over a longer period of time involving detection of effects on vital organ functions would ensure that the plants are safe for human consumption. references 1. wills rb, bone k, morgan m. herbal products: active constituents, modes of action and quality control. nutr res rev 2000; 13:47-7. 2. kiritikar kr, basu bd. indian medcinal plants. 2 nd ed. india: allahabad; 1994. p.1606-09, 1783- 92. 3. lai xy, zhao yy, liang h. studies on chemical constituents in flower of abelmoschus manihot. china j chin mat med 2006; 31(19):1597-600. 4. liang h, lai x, zhao y, bai y, wang b, guo d. spe-hplc method for the determination of four flavonols in rat plasma and urine after oral administration of abelmoschus manihot extract. j chromatogr b analyt tech biomed life sci 2007; 852(1-2):108-14. 5. lai x, liang h, zhao y, wang b. simultaneous determination of seven active flavonols in the flowers of abelmoschus manihot by hplc. j chromatogr sci 2009; 47(3):206-10. 6. linlin wu, xin-bo y, zhengming h, hezhi l, guangxia wu. in vivo and in vitro antiviral activity of hyperoside extracted from abelmoschus manihot (l) medic. acta pharmacol sin 2007; 28(3):404-409. 7. wen jy,chen zw. protective effect of pharmacological preconditioning of total flavones of abelmoschus manihot on cerebral ischemic reperfusion injury in rats. am j chin med 2007; 35(4):653-61. 8. puel c, mathey j, davicco mj, lebecque p, chanteranne b, horcajada mn, coxam v. preventive effect of abelmoschus manihot (l) medik on bone loss in the overiectomised rats. j. ethnopharmacol 2005; 99:55-60. 9. jain ps, bari sb. analgesic activity of abelmoschus manihot extracts. int j. pharmacol 2011; 1-5. 10. anonymous: the wealth of india: raw materials. india, new delhi: publication and information directorate csir; 1976. 11. akihisa t, ishtiaque a, singh s, tamura t, matsumoto m. 14α-methylzymosterol and other sterols from wrightia tinctoria seeds. phytochem 1988; 27(10): 3231-3234.o r i g i n a l a r t i c l e p h c o g j . 82 pharmacognosy journal  september 2011  vol 3  issue 25 *address for correspondence: department of biotechnology, shridevi institute of engineering & technology, sira road,tumkur-572 106, karnataka, india, phone: 91-9686114847, 91-816-2212626, fax: 91-816-2212629 email: dravidateja07@yahoo.co.in doi: 10.5530/pj.2011.25.15 potential of flling this need because of structures are different from those of the more studied and their those of the more action may too very likely differ. [1] in this growing interest, many of the phytochemical bioactive compounds from a medicinal plants have shown many pharmacological activities. [2,3] screening of various bioactive compounds from plants has lead to the discovery of new medicinal drug which have effcient protection and treatment roles in against various diseases. [4] the rapid emergence of multiple drug resistance strains of pathogens to current antimicrobial agents has generated an urgent intensive for new antibiotics from medicinal plants. many medicinal plants have been screened extensively for their antimicrobial potential worldwide. [5,6] endophytic fungi are relatively unexplored producers of metabolites useful to pharmaceutical and agricultural industries. [7] endophytes are the microorganisms that grow inside the plants; both phytochemical screening, antimicrobial and in vitro anti-inflammatory activity of endophytic extracts from loranthus sp. govindappa m 1 *, channabasava r 1 , sowmya dv 2 , meenakshi j 2 , shreevidya mr 2 , lavanya a 2 , gustavo santoyo 3 and sadananda ts 1 1 department of biotechnology, shridevi institute of engineering & technology, sira road, tumkur-572 106, karnataka, india. 2 department of biotechnology, shridevi pg center, sira road, tumkur-572 106, karnataka, india. 3 instituto de investigaciones quimico-biologicas, universidad michoancana de san nicolas de hidalago, edificio b-5 ciudad universitaria, morelia, michocan, mexico, 58060. a b s t r a c t four different endophytes were isolated from different parts of loranthus sp. methanol and water extracts of all the endophytes was assessed for its antimicrobial and anti-infammatory activity and phytochemical screening. phytochemical analysis revealed the presence of tannins, favonoids, terpenoids, steroids, alkaloids, phenols and saponins. the antimicrobial effcacy was determined using paper disc diffusion method against different fungi and bacteria. sensitivity in terms of zones of inhibition and phytochemical composition of the all endophytic extracts were also determined. the results show that, a. niger, penicillium sp. and alternaria alternata extracts effective against all the bacteria and fungi tested, whereas a. favus extract was failure in inhibiting the growth of all bacteria and bacteria. in vitro anti-infammatory activity was evaluated using albumin denaturation, membrane stabilization assay and proteinase inhibitory assay. aspirin was used as a standard drug for the study of anti-infammatory activity. a. niger, penicillium sp. and alternaria alternata methanol fractions showed in vitro anti-infammatory activity by inhibiting the heat induced albumin denaturation (87.88, 86.89, 87.03 g/ml) and red blood cells membrane stabilization with 78.42, 77.61, 77.98 g/ml respectively. proteinase activity was also signifcantly inhibited by the a. niger (85.21 g/ml), alternaria alternata (84.09 g/ml) and penicillium sp. (79.17 g/ml). bsa anti-denaturation and hrbc membrane stabilization assay indicated that the methanol extracts of a. niger, penicillium sp. and alternaria alternata possess constituents with anti-infammatory properties. from the result, it is concluded that phytochemicals (tannins, favonoids, terpenoids, phenols, steroids, alkaloids and saponins) present in the a. niger, penicillium sp. and alternaria alternata extract may be responsible for the antimicrobial and anti-infammatory activity. key words: endophytes, antimicrobial, anti-infammatory, phytochemicals, loranthus sp. introduction the increase in prevalence of multiple drug resistance has showed down the development of new synthetic antimicrobial, anti-infammatory drugs and the new drug is necessary to search for new antimicrobial, antioxidant and anti-infammatory from alternative sources. phytochemicals frommedicinal plants showing antimicrobial, antioxidant and anti-infammatory activities have thepharmacognosy journal  september 2011  vol 3  issue 25 83 govindappa, et al.: endophytic extracts from loranthus sp. identifed by their vernacular names and later it was compared with the herbarium of department of studies in botany, manasa gangothri, university of mysore, mysore and government ayurvedic college, mysore, india. isolation and identification of endophytic fungi the protocol for isolation follow methods used in other endophyte study [23] but adjusted for the specifc plant tissues used here following pilot experiments. the plant tissues were washed in running tapwater for one hour. fifty segments of leaves from each plant were cut into 5 mm 2 pieces, including a vein (25 samples) and intervein (25 samples). 25 segments of branches were then cut randomly to a length of 5 mm. endophytic fungi were isolated from the bark of the plant (25 segments). twenty fve segments (5 mm long) were cut from the stems and the roots. the total 150 segments of plant material were treated by triple surface sterilization technique. [24] each piece was then placed on malt extract agar (malt extract (20 g/l), rose bengal (0.033 g/l), chloromphenicol (50 mg/l, agar (15 g/l). all plates were incubated at 26 2 c until mycelium grew out hyphal tips were cut and transferred to potato dextrose agar (pda). half strength pdawas used for subculture and stock culture. identifcation was based on colony and hyphal morphology of the fungal cultures, characteristics of the spores. [25,26] fungal cultivation and extraction of metabolites the fungal endophytes were cultivated on potato dextrose broth (himedia, germany) by placing agar blocks of actively growing pure culture (3 mm diameter) in 250 ml erlenmeyer fasks containing 100 ml of the medium. the fasks were incubated at 26 2 c for 1 week with periodical shaking at 150 rpm. after the incubation period, the cultures were taken out and fltered through sterile cheesecloth to remove the mycelia mats. solvents identifcation of the phytochemical active substances carried out using methanol solvent at 5 g/15 ml (w/v). phytochemical analysis chemical analysis was carried out in the methanol and water extracts of the all endophytes of loratnthus sp. using standard procedures to identify constituents, as described by harborne (1984), trease and evans (1979) and sofowara (1993). [27,28,29] determination of antimicrobial activity antimicrobial assay bacillus subtilis, pseudomonas fuorescens, clavibacter michiganensis sub sp. michiganensis, xanthomonas oryzae pv. oryzae, xanthomonas axanopodis pv. malvacearum and strains of staphylococcus aureus, e. coli, pseudomonas aeruginosa and klebsiella pneumonia bacteria were obtained from stock cultures presented at –80 c at (plant and endophytes) will be benefcial. fungal endophytes residing within these plants could also produce metabolites similar to or with more activity than that of their respective hosts. [8] microorganisms are a rich source of biologically active metabolites that fnd wide-ranging exploitation in medicine, agriculture and industry. [9] many of the anticancer agents are explored from endophytes rather than host (taxol from pestalotiopsis microspora). [10] various research groups have identifed more than hundreds of endophytic isolates from south indian medicinal plants that showed promising activity against antitumour and antimicrobial agents. [11,12] the development of drug resistance in human and pathogenic bacteria and fungi has prompted a search for more and better antibiotics, especially as disease caused by pathogenic microorganisms, now represents a clear and growing threat to world health. [13,14] many of the endophytic fungal strains have attracted special attention because they have the capability of producing different colored pigments with high chemical stability. globally, there are at least one million species of endophytic fungi in all plants, [15] which can potentially provide a wide variety of structurally unique, bioactive, natural products. [16,17] increasing evidence indicate that reactive oxygen species (ros), (example, o 2 - and oh-) and free radical mediated reactions can cause oxidative damage to biomolecules (for example, lipids, proteins and dna), eventually contributing to; aging, cancer, atherosclerosis, coronary heart ailment, diabetes, alzhemier’s disease and other neurodegenerative disorders. [18,19] loranthus is a genus of parasitic plants that grow on the branches of woody trees. it belongs to the family loranthaceae (the showy mistletoe family). loranthus micranthus exhibited various degree of antimicrobial [20] and antidiabetic activity. [21] l. europaeus have showed hematopoietic activity. [22] the literature survey indicates that no reports are available from india regarding antimicrobial and anti-infammatory activity of loranthus sp. endophytic extract. the present study was aimed to examine the total phenolic content and phytochemical analysis of water and methanol extract endophytes of loranthus sp. were screened for antimicrobial and anti-infammatory properties using standard methods. the fndings from this work may add to the overall value of the medicinal potential of the plant. materials and methods the plant was collected in november 2010 from our college campus (shridevi institute of engineering & technology, sira road, tumkur, karnataka, india). the plant wasgovindappa, et al.: endophytic extracts from loranthus sp. 84 pharmacognosy journal  september 2011  vol 3  issue 25 where abs control is the absorbance of the dpph radical solvent, abs sample is the absorbance of dpph radical sample extract/standard. membrane stabilization test preparation of red blood cells (rbcs) suspension fresh whole human blood (10 ml) was collected and transferred to the centrifuge tubes. the tubes were centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of normal saline. the volume of blood was measured and re constituted as 10% v/v suspension with normal saline. [33] heat induced hemolytic the reaction mixture (2 ml) consisted of 1 ml of test sample solution and 1 ml of 10%rbcs suspension, instead of test sample only saline was added to the control test tube. aspirin was taken as a standard drug. all the centrifuge tubes containing reaction mixture were incubated in water bath at 56 c for 30 min. at the end of the incubation the tubes were cooled under running tap water. the reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm. the experiment was performed in triplicates for all the test samples. percent membrane stabilization activity was calculated by the formula mentioned above. [33] protein inhibitory action the test was performed according to the modifed method of oyedepo and femurewas (1995) and sakat et al. (2010). [34,33] the reaction mixture (2 ml) was containing 0.06 mg trypsin, 1 ml of 20 mm tris hcl buffer (ph7.4) and 1 ml test sample of different concentrations. the reaction mixture was incubated at 37 c for 5 min and then 1 ml of 0.8% (w/v) casein was added. the mixture was inhibited for an additional 20 min, 2 ml of 70% perchloric acid was added to terminate the reaction. cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. the experiment was performed in triplicate. the percentage of inhibition of proteinase inhibitory activity was calculated. bsa anti-denaturation assay five ml of each extract was dried in vacuum oven and redissolved in 5 ml of isosaline. then, 1 mg/ml of all extracts were made from the abovementioned stock solution. to 1.8 ml of 1% of bsa solution, 0.2 ml of extract solution in isosaline was added. the ph was adjusted to 6.5 using 1nhcl. this solution was incubated at 37 ºc for 20 minutes and then heated to 57 ºc for 10 to 15 minutes. after cooling, turbidity was measured at 660 nm. control was taken without the extracts. [35] department of studies in applied botany, seed pathology and biotechnology, university of mysore, manasagangothri, mysore, karnataka, india and department of studies in biotechnology and microbiology, bangalore university, gnana bharathi, bangalore, india respectively. three gram positive bacteria tested were bacillus subtilis, clavibacter michiganensis sub sp. michiganensis, staphylococcus aureus and six gram negative bacterias tested were pseudomonas fuorescens, xanthomonas oryzae pv. oryzae, xanthomonas axanopodis pv. malvacearum, e. coli, pseudomonas aeruginosa and klebsiella pneumonia. all bacteria were grown on nutrient agar media. fungi (aspergillus favus, aspergillus niger, aspergillus nidulans, aspergillus faviceps, alternaria carthami, alternaria helianthi, cercospora carthami, fusarium solani, fusarium oxysporum, fusarium verticilloides and nigrospora oryzae were obtained from department of studies in applied botany, seed pathology and biotechnology, university of mysore, manasa gangothri, mysore, karnataka, india and department of studies in microbiology, bangalore university, gnana bharathi, bangalore, india respectively. all fungi were grown on potato dextrose agar medium. paper disc method diameter of zone of inhibition was determined using the paper disc diffusion method as described by lai et al. (2009) and adedapo et al. (2008). [30,31] a swab of the bacteria suspension containing 1 10 8 cfu/ml was spread on to petri plates containing nutrient agar media. each extracts were dissolved in ethanol to fnal concentration of 10 mg/ml. sterilized flter paper discs (6 mm in diameter) impregnated with 1 mg of plant extracts were placed on culture plates. the plates were incubated at 37 c for 24 h. the methanol served as negative control while the standard streptomycin (10 μg) discs were used as positive controls. antimicrobial activity was indicated by the presence of clear inhibition zone around the discs. the assay was repeated thrice and mean of three experiments was recorded. in vitro anti-inflammatory activity inhibition of albumin denaturation methods of mizushima and kobayashi (1968) and sakat et al. (2010) [32,33] followed with minor modifcations. the reaction mixture was consisting of test extracts and 1% aqueous solution of bovine albumin fraction, ph of the reaction mixture was adjusted using small amount at 37 c hcl. the sample extracts were incubated at 37 c for 20 min and then heated to 51 c for 20min. after cooling the samples the turbidity was measured spectrophotometrically at 660 nm. the experiment was performed in triplicate. percent inhibition of protein denaturation was calculated as fallows, % inhibition [ {abs control –abs sample } abs control ] 100,pharmacognosy journal  september 2011  vol 3  issue 25 85 govindappa, et al.: endophytic extracts from loranthus sp. and cardio glycosides in methanol extracts whereas no phytochemicals was observed in water extracts. other three endophytic extracts yielded all the phytochemicals in both methanol and water extracts viz., carbohydrates, tannin, steroids, cardiac glycosides, favonoids, terpenoids, alkaloids, phenol, saponins and anthraquinones (table 2). antimicrobial assay the antimicrobial activities of methanol and water extracts of endophytes of loranthus sp. gave different zones of inhibition on the organisms tested (table 3). the ethanol aspergillus niger, penicillium sp. and alternaria alternata extract inhibited the growth of all most all the bacteria and fungal species signifcantly. e. coli, pseudomonas fuorescens, xanthomonas oryzae pv. oryzae, a. helianthi and cercospora carthami are inhibited by methanol extract of aspergillus favus minimally, in water extracts there is no activity against all the bacteria and fungi (table 3). anti inflammatory properties inhibition of albumin denaturation denaturation of proteins is a well documented cause of infammation. as part of the investigation on the mechanism of the anti infammation activity, ability of extract protein denaturation was studied. it was effective in inhibiting heat induced albumin denaturation (table 4). maximum inhibition 87.88% was observed from methanol a. niger extract hrbc membrane stabilization assay blood was collected freshly and mixed with equal volume of alsever solution. it was then centrifuged at 3000 rpm for 15 minutes. the packed cells were washed with isosaline and a 10% suspension was made with isosaline. to 0.5 ml of extract, 1 ml phosphate buffer, 2 ml hyposaline and 0.5 ml hrbc suspension were added. this was incubated for 30 minutes at 37 ºc and then centrifuged at 3000 rpm for 20 minutes. absorbance was measured at 560 nm. control was taken without the extract. [36] statistical analysis analysis of variance (anova) was used to determine the signifcance of difference between treatment groups (p 0.05). means between treatment groups were compared for signifcance using duncan’s new multiple range post test. results phytochemical analysis loranthus sp. was collected from neem plants (figure 1). all the incubated parts exhibited the presence of four different endophytic fungal species viz., aspegillus niger, aspergillus favus, penicillium sp. and alternaria alternata (table 1). in the phytochemical screening of endophytes, aspergillus favus has showed only presence of carbohydrates figure 1: a: loranthus sp on neem plant, b: flowers of loranthus sp. c: different endophytes from different parts of loranthus sp. table 1: list of endophytes from different parts of loratnthus sp on pda media types of endophytes leaves bark stem root petiole vein inter-vein aspergillus niger aspergillus flavus penicillium sp. alternaria alternata experiments were repeated for thrice for each sample, presencegovindappa, et al.: endophytic extracts from loranthus sp. 86 pharmacognosy journal  september 2011  vol 3  issue 25 followed by penicillium sp. (86.89%) and alternaria alternata (87.03%). aspirin, a standard anti-infammation drug showed the maximum inhibition 76.69% at the concentration of 200 μg/ml. in water endophytic extract, maximum inhibition 77.33% was observed from a. niger followed by penicillium sp. (76.54%) and alternaria alternata (77.21%)(table 4). membrane stabilization test stabilization of rbcs membrane was studied for further establishes the mechanism of anti-infammatory action of different methanol and water extracts of different endophytes. all the extracts were effectively inhibiting the heat induced hemolysis. these results provide evidence for membrane stabilization as an additional mechanism of their anti infammatory effect. this effect may possibly table 2: phytochemical analysis of ethanol extract of different plant parts tests methanol extract water extract 1 2 3 4 1 2 3 4 carbohydrates – tannin – – steroids – – cardiac glycosides – flavonoids – – terpenoids – – alkaloids – – phenol – – saponins – – anthraquinones – – experiments were repeated for thrice for each sample, ve: positive, –ve: negative, 1-aspergillus favus, 2- a. niger, 3-penicillium sp., 4-alternaria alternata table 3: in vitro inhibition assay from methanol and water extracts of endophytes species methanol extract water extract 1 2 3 4 1 2 3 4 bacterial pathogens e. coli – pseudomonas aeruginosa – – staphylococcus aureus – – klebsiella pneumonia – – pseudomonas fluorescens – clavibacter michiganensis sub sp. michiganensis – – xanthomonas oryzae pv. oryzae – xanthomonas axanopodis pv. malvacearum – – fungal pathogens aspergillus flavus – – a. niger – – a. nidulans – – a. flaviceps – – alternaria carthami – – a. helianthi – cercospora carthami – fusarium solani – – f. oxysporum – – f. verticilloides – – nigrospora oryzae – – average, minimum activity, – no activity, 1-aspergillus favus, 2- a. niger, 3-penicillium sp., 4-alternaria alternate, repeated the experiments three times for each replicates table 4: effect of methanol and water extracts of different endophytes on albumin denaturation, membrane stabilization and proteinase inhibitory activity percentage inhibition test sample albumin denaturation membrane stabilization proteinase inhibition methanol extract aspergillus niger 87.88 0.006 a 78.42 0.03 a 85.21 0.03 a aspergillus flavus 44.76 0.006 a 54.29 0.03 a 53.34 0.03 a penicillium sp. 86.89 0.006 c 77.61 0.03 b 79.17 0.03 b alternaria alternata 87.03 0.006 a 77.98 0.03 a 84.09 0.03 a water extract aspergillus niger 77.33 0.006 a 72.54 0.03 a 82.04 0.03 a aspergillus flavus 39.41 0.006 a 54.81 0.03 a 50.19 0.03 a penicillium sp. 76.54 0.006 c 71.87 0.03 b 77.89 0.03 b alternaria alternata 77.21 0.006 a 71.95 0.03 a 81.86 0.03 a aspirin (200μg/ml) 75.89 0.006 b 85.92 0.03 a 92.87 0.05 a repeated the experiments three times for each replicates, according to duncan’s multiple rangetest (dmrt), values followed by diferent subscripts are signifcantly diferent at p 0.05, se-standard error of the mean.pharmacognosy journal  september 2011  vol 3  issue 25 87 govindappa, et al.: endophytic extracts from loranthus sp. bsa anti-denaturation assay the inhibitory effect on protein (bsa) denaturation by the water and methanol extracts of endophytes is shown in figure 2. all the extracts were tested at 200 μg/ml concentration. the a. niger, penicillium sp. and alternaria alternata water and methanol fractions showed good activity, whereas the a. favus extract showed comparatively lower activity. at 200 μg/ml concentration, a. niger methanol extract showed 79% inhibition of denaturation followed by alternaria alternata (78.6%) and penicillium sp. (65.84%). the water endophytic extracts also showed signifcant inhibition of denaturation by a. niger (76%), alternaria alternata (76.1%) followed by (75.3%) (figure 3). denaturation of proteins is well documented cause of infammation and rheumatoid arthritis. several anti-infammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation. [37] when bsa is heated, it undergoes denaturation and expresses antigens associated with type iii hypersensitive reaction and which are related to diseases such as serum sickness, glomerulonephritis, rheumatoid arthritis and inhibit the release of lysosomal content of neutrophils at the site of infammation. the extracts inhibited the heat induced hemolysis of rbcs to varying degree (table 4). the maximum inhibitions 78.42% frommethanol aspergillus niger extract followed by penicillium sp. (77.61%) andalternaria alternata (77.98%). the aspirin standard drug standard drug showed the maximum inhibition 85.92%. in water endophytic extract, maximum inhibition 72.54% was observed from a. niger followed by penicillium sp. (71.87%) and alternaria alternata (71.95%)(table 4). proteinase inhibitory activity the different endophytic ethanol extract exhibited signifcant antiproteinase activity. the maximum inhibition was observed from methanol a. niger extract (85.21%), in decreasing order was penicillium sp. (79.17%) and alternaria alternata (84.09%). the standard aspirin (92.87%) drug showed the maximum proteinase inhibitory action. in water endophytic extract, maximum inhibition 82.04% was observed from a. niger followed by penicillium sp. (77.89%) and alternaria alternata (81.86%)(table 4). figure 2: per cent inhibition of bsa denaturation from methanol extract of endophytes figure 3: % inhibition of bsa denaturation from water extract of endophytesgovindappa, et al.: endophytic extracts from loranthus sp. 88 pharmacognosy journal  september 2011  vol 3  issue 25 the favonoids from extracts have been found to possess antimicrobial and anti infammatory properties in various studies. [44,45] the presence of terpenoids have shown as antimicrobial [46] and anti-infammatory properties. [47] strong presence of tannins in all extracts may explain its potent bioactivities are known to possess potent antimicrobial activities [41] and anti-infammatory properties. [48] the saponins have already shown as antimicrobial activity [49] and anti-infammatory activity. [50] in vitro anti inflammatory properties denaturation of proteins is a well documented cause of infammation. the infammatory drugs (salicylic acid, phenylbutazone etc) have shown dose dependent ability to thermally induced protein denaturation. [42] similar results were observed from many reports from plant extract. [33] the extracts may possibly inhibit the release of lysosomal content of neutrophils at the site of infammation. these neutraphils lysosomal constituents include bactericidal enzymes and proteinases, which upon extracellular release cause further tissue infammation and damage. [51] the precise mechanism of this membrane stabilization is yet to be elucidated, it is possible that the endophytes, a. niger, penicillium sp. and alternaria alternate of loranthus sp. produced this effect surface area/volume ratio of the cells, which could be brought about by an expansion of membrane or the shrinkage of cells and an interaction with membrane proteins. [52] proteinases have been implicated in arthritic reactions. neutrophils are known to be a source of proteinase which carries in their lysosomal granules many serine proteinases. it was previously reported that leukocytes proteinase play important role in the development of tissue damage during in infammatory reactions and signifcant level of protection was provided by proteinase inhibitors. [53] recent studies have shown that many favonoids and related polyphenols contributed signifcantly to the antioxidant and anti-infammatory activities of many plants. hence, the presence of bioactive compounds in the methanol extracts of different endophytes, a. niger, penicillium sp. and alternaria systemic lupus erythematosus. thus, this assay was applied for the detecting compounds, which can stabilize the protein from denaturation process. several nonsteroidal anti-infammatory drugs such as indomethacin, ibufenac, diclofenac sodium, salicylic acid and fufenamic acid prevent denaturation of bsa at pathological ph (6.2-6.5). [38] hrbc membrane stabilization assay after the initial screening of endophytes, it was found that the methanol extract showed activity similar to diclofenac, the standard anti-infammatory drug used, for treating infammation. various endophytic methanol extracts in isosaline were tested and it was observed at 250 μg/ml both diclofenac and the endophytic extracts showed similar effects (table 5). the analogous activity makes the extract a potential candidate for further studies. discussion in recent years, the search for phytochemicals possessing antimicrobial and anti infammatory properties have been on the rise due to their potential use in the therapy of various chronic and infectious diseases. epidemiology and experimental studies have implicated oxidative cellular damage arising from an imbalance between free radical generating and scavenging systems as the primary cause of cardiovascular, diseases, cancer, aging etc. [39] due to risk of adverse effects encountered with the use of synthetic antibiotics, medicinal plants may offer an alternative source for antimicrobial agent with signifcant activity against pathogenic and infective microorganisms. in addition, a number of antibiotics have lost their effectiveness due to the development of resistant strains, mostly through the expression of resistance genes. [40] results of our fndings confrmed the use of endophytes, a. niger, penicillium sp. and alternaria alternata as traditional medicine.we found strong antimicrobial and anti-infammatory activities specifcally in the methanol extract of a. niger, penicillium sp. andalternaria alternata. plant phenolic compounds have been found to possess potent antimicrobial [41,42] and anti infammatory activity. [33,43] table 5: hrbc membrane stabilization of endophytic extracts, standard drug concentration % stabilization by by diclofenac methanolic extract water extract 1 2 3 4 1 2 3 4  50 μg/m 79.6 36.1 78.9 79.1 77.4 32.4 76.6 77.1 68.09 100 μg/ml 80.4 47.4 79.6 80.2 79.8 45.7 78.8 79.5 80.48 250 μg/ml 81.6 49.3 80.7 81.1 80.7 46.8 80.2 80.3 82.74 500 μg/ml 68.7 34.3 67.2 69.4 68.8 33.1 65.9 68.1 88.21 repeated the experiments three times for each replicates,pharmacognosy journal  september 2011  vol 3  issue 25 89 govindappa, et al.: endophytic extracts from loranthus sp. 8. strobel ga. endophytes as sources of bioactive products. microbes and infection 2003; 5:535-544. 9. strobel ga, daisy b. bioprospecting for microbial endophytes and their natural products. microbiol mol biol rev 2003; 67:491-502. 10. strobel g, yang xs, sears j, kramer r, sidhu rs, hess wm. taxol from pestalotiopsis microspora, an endophytic fungus of taxus wallachiana. microbiology 1996; 142:435-440. 11. gangadevi v, muthumary j. isolation of colletotrichum gloeosporioides, a novel endophytic taxol-producing fungus from the leaves of a medicinal plant, justicia gendarussa. mycologia balcanica 2008; 5:1-4. 12. gangadevi v, muthumary j. 2007. preliminary studies on cytotoxic effect of fungal taxol on cancer cell lines. african journal of biotechnology 007; 6(12):1382-1386. 13. raviglione mc, snider de, kochi a. global epidemiology of tuberculosis. morbidity and mortality of a worldwide epidemic. journal of the american medical association, 1995; 273:220-226. 14. pablos-mendez, mayeux a, ngai r, shea c, lars b. association of apo e polymorphism with plasma lipid levels in a multiethnic elderly population. arterioscler thromb vasc biol 1997; 17:3534-3541. 15. ganley rj, brunsfeld sj, newcombe g. a community of unknown, endophytic fungi in western white pine. proc. natl. acad. sci 2004; 101:10107-10112. 16. tan rx, zou wx. endophytes: a rich source of functional metabolites. natural product reports 2001; 18:448-459. 17. huang wy, cai yz, hyde kd, corke h, sun m. endophytic fungi from nerium oleander l (apocynaceae): main constituents and antioxidant activity. world journal of microbiology and biotechnology 2007; 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20:169-173. alternata of w. trilobata may contribute to its, antimicrobial and anti-infammatory activity. the present investigation has shown that the a. niger, penicillium sp. and alternaria alternata extracts have active phytochemicals which are able to inhibit plant and animal pathogenic bacteria and fungi. the methanol extract fractions showed signifcantly antimicrobial activity against all gram-positive and gram-negative bacteria and different fungi tested. strong anti-infammatory properties were confrmed in the methanol endophytic extract fractions. these activities may be due to strong occurrence of polyphenolic compounds such as favonoids, tannins, terpenoids, phenols and saponins. the anti-infammatory activity was comparable with standard ascorbic acid, bht and aspirin. these fndings provide scientifc evidence to support traditional medicinal uses and indicate a promising potential for the development of an antimicrobial and anti-infammatory agent from a. niger, penicillium sp. and alternaria alternata. these endophytes by in vitro results appear as interesting and promising and may be effective as potential sources of novel antimicrobial and anti-infammatory drugs. acknowledgements we thank dr mr hulinaykar, managing trustee, sri shridevi charitable trust (r.) and dr ma venkatesh, principal, siet, tumkur, india for encouragement, dr s lokesh, dos in applied botany and biotechnology, university of mysore, mysore, india for assistance in identifying endophytic fungi. references 1. fabricant ds, fansworth nr. the value of plants used in traditional medicine for drug discovery. environment health perspective 2001; 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2(1):146-155. 34. oyedepo oo, femurewas aj. anti-protease and membrane stabilizing activities of extracts of fagra zanthoxiloides, olax subscorpioides and tetrapleura tetraptera. in j of pharmacong 1995; 33:65-69. 35. grant nh, album he, kryzanauskas c. stabilization of serum albumin by anti-inflammatory drugs. biochem. pharmacol. 1970; 19:715-722. 36. mizushima y. screening test for anti-rheumatic drugs. lancet. 1966; 2:443-448. 37. sadique j, al- rqobahs wa, bughait mf, el gindi ar. fitoterapia. 1989; 60:525-532. 38. williams l,connar ao, latore l,dennis o,ringer s,whittaker ja,conard j, vogler b, rosner h, kraus w. west indian med j. 2008; 57(4):327-331. 39. halliwell b. antioxidants in human health and disease. annu rev nutr 1996; 6:33-50. 40. berahou aa, auhmani a, fdil n, benharref a, jana m, gadhi ca. antibacterial activity of quercus ilex bark’s extracts. j. ethnopharmacol 2007; 112:426-429. 41. kaur gj, arora ds. antibacterial and phytochemical screening of anethum graveolens, foeniculum vulgare and trachyspermum ammi. bmc complement and alternate medicine 2009; 9:30. 42. lai hy,yau yy, kim kh. blechnum orientale linn - a fern with potential as antioxidant, anticancer and antibacterial agent. bmc complem. altern. med 2010; 10:15. 43. garg vkr, jain m, sharma pkr, garg g. anti inflammatory activity of spinacia oleracea. international journal of pharma professional’s research 2010; 1(1): 1-4. 44. lopez-lazaro m. distribution and biological activities of the flavonoid luteolin. mini rev med chem 2009; 9:31-59.pharmacognosy journal  september 2011  vol 3  issue 25 91 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: dusmanta kumar pradhan, asst. professor, gayatri college of pharmacy, sambalpur, odisha. 768200 email: dkpradhan74@gmail.com doi: 10.5530/pj.2011.25.16 the inforescence is big with a reddish brown bract and is eaten as vegetables. the ripe fruits are sweet, juicy and full of seeds and the peel is thicker than other banana. [2] the fruit m. sapientum is traditionally used in diarrhoea (unripe), dysentery, intestinal lesions in ulcerative colitis, diabetes (unripe), in sprue, uremia, nephritis, gout, hypertension, cardiac disease. [3,4] it is also used in the treatment of excess menstruation with canna indica l. var. speciosa. [5] banana leaves (ashes) are used in eczema, [6] as cool dressings for blister and burns. [3] flowers are used in dysentery and menorrhagia. stem juice of fruited plant is used for treating diarrhoea, dysentery, cholera, otalgia, haemoptysis, dysentery, diabetes and menorrhagia. [3] the root is used as anthelmintic, [4] blood disorders, venereal diseases. [3] the plant is also used in infammation, pain and snakebite. [7] banana has played interesting and important roles in the history of human civilizations. banana is very rich in carbohydrates, vitamin c, a, b and several important minerals, including potassium, copper, magnesium, calcium, and iron. the banana “tree” grows in humid lowland to upland tropical areas; these plants die if they are exposed to cold temperatures. [8] carbohydrates have been isolated fromm. sapientum. [9] catecholamines such as norepinephrine, serotonin, dopamine, [10,11] tryptophan, indole compounds, [12] pectin have been found in the pulp. several favonoids and related compounds (leucocyanidin, quercetin and its 3-ogalactoside, 3-o-glucoside, and 3-o-rhamnosyl glucoside) were isolated from the unripe pulp of diuretic activity of alcoholic extract of musa sapientum l. flower ashutosh mishra 1 , manas ranjan mishra 1 , dusmanta kumar pradhan 1 *, sunil kumar vaishnav 2 1 gayatri college of pharmacy, jamadarpali, sambalpur, odisha-768200. 2 jk college of pharmacy, bilaspur, cg. a b s t r a c t the present study was designed to investigate the diuretic activity of musa sapientum l. (family- musaceae) fowers. the dried powder of the fower was subjected to soxhlet extraction with alcohol and this extract was used for diuretic activity in wistar albino rats using lipschitz method. the diuretic activity was assessed in terms of urine output and, concentration of sodium, potassium and chloride ions in urine. the result obtained revealed that the alcoholic extract showed signifcant diuretic activity at a dose of 250 and 500 mg/kg body weight by increasing the total volume of urine and, concentration of sodium, potassium and chloride ions with respect to standard drug furosemide. key words: diuretic, sodium, potassium, lipschitz method, furosemide. introduction the modern era of diuretic therapy began in 1949 when sulphanilamide was discovered to possess diuretic and natriuretic properties. a diuretic is an agent that increases the rate of urination thereby decreasing body fuid, especially the extracellular fuid. diuretics play an important role in situations of fuid overload, like acute and chronic renal failure, hypercalciuria, cirrhosis of liver and also act as an antihypertensive agent. [1] a number of diuretics like mannitol, thiazides, furosemide, ethacrynic acid are used in practice. still there is a need for more effective and less toxic diuretic. that’s why there is a great need to search of safer and less toxic diuretic drug from natural resources. there are so many natural diuretic herbs like aadraka (zingiber offcinale), brahmi (centella asiatica), gokshura (tribulus terrestris), ikshuraka (saccharum offcinarum), kantakaari (solanum xanthocarpum), punarnava (boerhavia diffusa), sariba (ichnocarpus frutescens), satavari (asparagus racemosus), vacha (acorus calamus), banana (musa sapientum) etc. reported in different traditional literature and practices by natural healers. musa sapientum is a tree like perennial herb that grows 5-9 m in height, with tuberous rhizome, hard, long pseudo stem.mishra, et al.: diuretic activity of alcoholic extract of musa sapientum l. flower 92 pharmacognosy journal  september 2011  vol 3  issue 25 ad libitum. the animals were housed in polypropylene cages maintained under environmental conditions (12 h light and 12 h dark cycle; 25 3c). the animals were treated strictly according to the cpcsea guidelines. acute toxicity the rats were fasted overnight, divided into groups (n 3) and were orally fed with increasing doses (250, 500, 750 and 1000mg/kg body weight) of alcoholic extract suspended in tween 80. after administration of the extracts, the animals were observed during frst 3 h for their gross behavioral changes and once in 30 min for next 5 h, then once in 24 h for next 72 h to fnd out percentage mortality. [16,17,18] diuretic activity in rats the diuretic activity of the extract was assessed by the method previously described by lipschitz et al. for the assessment of diuretic activity, the urine output, sodium, potassium and chloride ion concentration in urine were measured. the animals were divided into four groups each group containing three animals. the animals were deprived of food and water for 12 h prior to the experiment. [16] before the oral administration of test drugs, the animals were dosed with 25 ml/kg body weight of normal saline. among the four groups of animals, group i received tween 80 (control, vehicle for the extracts) and group ii received the standard diuretic drug furosemide at 20 mg/kg body weight. alcoholic extract was studied at two concentrations. group iii received 250 mg/kg and group iv received 500 mg/kg body weight of alcoholic extract in tween 80. [19] immediately after administration, the animals were placed in fabricated metabolic cages individually to allow separation of urine and faeces. the bottom of the metabolic cage was fxed with a glass funnel inserted into a measuring cylinder containing mineral oil. the presence of mineral oil in the measuring cylinders prevents loss of urine through evaporation. the urine was collected for six hours after administration of control, standard and extract. the bladder was emptied by pulling the base of tail of each rat. [20] diuretic assay parameters were observed for each rat. the observed parameters were total urine volume, sodium, potassium and chloride ions concentrations. the concentrations of plantain. [13,14,15] serotonin, nor-epinephrine, tryptophan, indole compounds, tannin, starch, iron, crystallisable and non-crystallisable sugars, vitamin c, b-vitamins, albuminoids, fats, mineral salts have been found in the fruit pulp of m. paradisiaca and m. sapientum. [3] the review of the scientifc literature did not expose any data on the diuretic activity of banana fower. in this study, an attempt was made to assess the effcacy of this indigenous plant for its diuretic activity in terms of urine output and, sodium, potassium and chloride ions concentration in experimental animals with respect to standard. materials and methods materials wistar albino rats 150-170 gms, 36; standard furosemide (20 mg/kg); control normal saline (5 ml/kg); test solution alcoholic extract of m. sapientum fower (500 mg/kg). collection and authentication of plant the fower was identifed and authenticated as a fower of musa sapientum l. by dr. netrabhanu pradhan botanist, prof. and h.o.d. dept. of botany, panchayat college, bargarh, orissa and a specimen of fower was deposited in the herbarium museum of college. musa sapientum l. fowers were collected in the month of nov.-dec. 2008 from the cultivar of dularpali, mahasamund chhattisgarh. care was taken to obtain best condition of m. sapientum fowers and it was subjected to dry under shade, powdered with laboratory mixer and sieved. methods extraction the dried fower powder was soxhlet extracted with alcohol. the obtained solvent extract was concentrated using rotary vacuum evaporator and dried in desiccators. animal healthy wistar albino rats of either sex approximately of same age and weighed about 150-170 g were used for the study. they were fed with standard indian diet and water table 1: diuretic activity of m. sapientum flower name of the drug/extract dose (mg/kg) urine volume (ml) concentration of ions (meq/l) sodium potassium chloride tween 80 5 ml/kg 0.74 0.47 51.75 1.67 10.84 0.47 52 1.45 furosemide  20 2.80 0.60* 71.33 2.31 12.87 0.19 92 2.38 alcoholic extract 250 1.40 0.10* 56.69 0.92 11.42 0.09 51.42 1.26 alcoholic extract 500 1.93 0.49* 64.32 096 11.93 0.25 53.02 2.40 *p 0.05, values are mean sem, n 3pharmacognosy journal  september 2011  vol 3  issue 25 93 mishra, et al.: diuretic activity of alcoholic extract of musa sapientum l. flower references 1. hussain md s, ahmed kfh n, ansari md zh. preliminary studies on diuretic effect of hygrophila auriculata (schum) heine in rats. international journal of health research 2009; 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6(6):263-266. 10. vettorazz g. 5-hydroxytryptamine content of bananas and banana products. food cosmet toxicol. 1974; 12:107-113. 11. waalkes tp, sjoerdsma a, creveling cr, weissbach h, udenfriend s. serotonin, norepinephrine, and related compounds in bananas. science 1958; 127(3299):648-650. 12. shanmugavelu kg, rangaswami g. tryptophan and indole compounds in banana ovaries. nature 1962; 194(4830):775-776. 13. ragasa cy, martinez a, chua jey, rideout ja. a triterpene from musa errans. philippine j sci. 2007; 136(2):167-171. 14. lewis dl, field wd, shaw gp. a natural flavonoid present in unripe plantain banana pulp (musa sapientum l. var. paradisiaca) protects the gastric mucosa from aspirin-induced erosions. j ethnopharmacol. 1999; 65:283‑288. 15. lewis da, shaw gp. a natural flavonoid and synthetic analogues protect the gastric mucosa from aspirin-induced erosions. j nutr biochem. 2001; 12:95-100. 16. rathi bb, jain bs, bodhankar sl. evaluation of diuretic activity of citrullus vulgaris rind in rats. int j pharmacol biol sci. 2007; (3):93‑96. 17. ghosh mn. fundamentals of experimental pharmacology, 2 nd edition, scientific book agency, kolkata, 1984, pp 156-157. 18. kulkarni sk. hand book of experimental pharmacology, 3 rd edition, vallabh prakasan, pune, 1999, pp 117-171. 19. lipschitz wl, haddin z, kerpscar a. bioassay of diuretics. j pharmacol exp ther. 1943; 79:97-110. 20. radhika b, begum nasreen, srisailam k, reddy vm. diuretic activity of bixa orellana linn. leaf extracts. indian j of natural products and resources 2010; 1(3):353-355. 21. mishra a, pradhan dk, mishra mr, sen bk, alok k, vaishnaw sk. in vitro anthelmintic activity of musa sapientum linn. flower. international j of pharmagenesis 2010; 1(2):161-163. sodium and potassium ions were measured by fame photometry and chloride ion concentration was estimated by titration with silver nitrate solution (n/50) using 5% potassium chromate as indicator. statistical analysis the results were presented as mean sem. “one-way anova with dunnett’s post t-test was performed using graph pad prism version 3.00 for windows. graph pad software, san diego california usa, p 0.05 were considered signifcance. results and discussions in acute toxicity study, all the animals were found to be surviving after 72 h. this indicates that the extract was found to be safe up to the dose level studied. since, all the animals survived at a dose of 1000 mg/kg body weight, the ld 50 of the extract will be 1000 mg/kg body weight. no major behavioral changes were observed during this period of study. the result of diuretic activity of m. sapientum fower showed signifcant as compared to the standard drug furosemide and control. the higher dose of extract (500 mg/kg) showed more signifcant activity as compared to the lower dose of extract (250 mg/kg). the diuretic activity of the m. sapientum fower can be attributed to its presence of amino acids and proteins [21] that plays an important role in the human body urea cycle, which removes nitrogen from the blood and help it to convert into urine. [16] determination of urinary electrolyte concentration revealed that alcoholic extract 500 mg/kg body weight was effective in increasing urinary electrolyte concentration for all the ions tested (sodium, potassium and chloride) in comparison to 250 mg/kg dose and control. conclusion on the basis of above results it can be concluded that the alcoholic extract produce dose dependent diuretic effect. the present data support the ethanomedical application of m. sapientum fower as diuretic.