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  august 2011  vol 3  issue 24 i c o n t e n t s p h c o g j . review articles phytochemistry and pharmacological properties of thunbergia laurifolia: a review 1 eric w.c. chan, suit ying eng, yuen ping tan, zhiew cheng wong kyllinga nemoralis (hutch & dalz) (cyperaceae): ethnobotany, phytochemistry and pharmacology 7 raju s, kavimani s, uma maheshwara rao v, sreeramulu reddy k pharmacognostic studies pharmacognostic studies on the leaves of dyschoriste perottetii nees 11 odoh, u. e., ezugwu, c. o. and ezejiofor, m. medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia 15 jamia azdina jamal, zakiah abd. ghafar & khairana husain biochemical profling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth 25 m. amzad hossain, mohan raj nagooru pharmacognostic standardization of the leaves and root bark of caesalpinia benthamiana 31 dickson ra, annan k, komlaga, g pharmacognostic, physico-chemical and phytochemical evaluation of leaves of a species of ‘paarshva-pipla’: ficus arnottiana 35 kher mn, vaghasiya jv, desai tr, pandya dj pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae 39 k k hullatti, prasenjit bhattacharjee pharmacognostical and phytochemical evaluation of leaves of bauhinia variegata linn. 45 kamal m modh, purvi t parmar, bibhuranjan panigrahi, indermeet singh anand, chhagan n patel profle of elemental composition of oroxylum indicum l.(vent.) collected from different geographical regions of india. 50 k. srilatha srinivas and a.a. saraf pharmacognostical, physicochemical and phytochemical studies of some marketed samples of roots used in ayurvedic medicines 55 amrita mishra, arun k mishra, ashoke k ghosh, shivesh jha pharmacognostic evaluation and phytochemical studies on stem of clitoria ternatea linn. 62 jain r.a., shukla s.h. pharmacological studies mast cell stabilization and membrane protection activity of barleria prionitis l. 67 a.k. maji, s. bhadra, s. mahapatra, p. banerji, d. banerjee in vitro antimicrobial screening of four reputed bangladeshi medicinal plants 72 md. al amin sikder, a. k. m. nawshad hossian, abu bakar siddique , mehreen ahmed, mohammad a. kaisar, and mohammad a. rashid evaluation of in-vitro anthelminthic activity of leucas aspera extracts 77 swati agarwal, simi jacob, nikkita chettri, saloni bisoyi, d. k. badarinath, a. b. vedamurthy, v. krishna and h. joy hoskeri evaluation of laxative activity of oxystelma esculentum 81 pandya dj, anand ispharmacognosy journal  august 2011  vol 3  issue 24 1 r e v i e w a r t i c l e p h c o g j . *address for correspondence: e-mail: erchan@yahoo.com doi: 10.5530/pj.2011.24.1 evening of the same day. [3,4] flowers are not scented. carpenter bees are frequent visitors, creeping into the fowers for the pollen and nectar while black ants are present probably as nectar scavengers. in thai traditional medicine, leaves of t. laurifolia are used as an antidote for poisons and drugs including the treatment of drug addiction. [6,7] the plant has also been reported to have anti-infammatory, anti-diabetic, and antipyretic properties. [8-10] local herbal companies are producing and marketing herbal teas and capsules of t. laurifolia, known as rang jeud in thai. phytochemistry the phytochemistry of t. laurifolia leaves has been studied. [8] two novel iridoid glucosides of 8-epi-grandiforic acid and 3’-o-β-glucopyranosyl-stilbericoside have been isolated, along with seven known compounds of grandiforic acid, benzyl β-glucopyranoside, benzyl β-(2’-o-β-glucopyranosyl)- glucopyranoside, 6-c-glucopyranosyl apigenin, 6,8-di-c-glucopyranosyl apigenin, (e)-2-hexenyl-β-glucopyranoside, and hexanol-β-glucopyranoside. leaves and fowers of t. laurifolia have been found to contain other bioactive phenolic constituents including delphinidin-3, phytochemistry and pharmacological properties of thunbergia laurifolia: a review eric w.c. chan*, suit ying eng,yuen ping tan, zhiew cheng wong faculty of applied sciences, ucsi university, 56000 cheras, kuala lumpur, malaysia a b s t r a c t commonly known as blue trumpet vine or laurel clock vine, thunbergia laurifolia is a popular ornamental vine in the tropics. flowers are attractive with pale purplish-blue petals and a yellow throat. leaves are heart-shaped with a pointed tip and slightly serrated leaf margin. in thailand, leaves of t. laurifolia are believed to have detoxifying effects. they are used as an antidote for poisons and drugs, including the treatment of drug addiction. the plant has also been reported to have antioxidant, anti-diabetic, anti-infammatory, and antipyretic properties. local herbal companies are producing herbal teas and capsules of t. laurifolia, known as rang jeud in thai. compounds isolated from the leaves included iridoid glucosides, grandiforic acid, glucopyranosides, and derivatives of apigenin. other compounds found in leaves and fowers were delphinidin derivatives, and phenolic acids of chlorogenic, caffeic, gallic, and protocatechuic. current knowledge on the pharmacological properties of the species is reviewed. properties reviewed include antioxidant, antimicrobial, antiproliferative, hepatoprotective, and anti-infammatory activities, as well as detoxifying, anti-diabetic, and non-toxic effects. key words: antioxidant, detoxifying, hepatoprotective, anti-diabetic, anti-infammatory, non-toxic introduction thunbergia laurifolia lindl. (thunbergiaceae), or commonly known as blue trumpet vine or laurel clock vine, is native to india. [1,2] the species is grown as an ornamental plant and being a fast-growing vine, it has become an exotic weed in some countries. its leaves are dark green, opposite, heart-shaped, with a pointed tip and slightly serrated leaf margin. [3-5] the leaf blade can grow up to 20 cm in length and 16 cm in width with a petiole up to 6 cm in length. leaves are thin and bright green in colour when young, and tend to be darker green, thicker and slightly variegated as they mature (figure 1). borne on pendulous inforescences, fowers are attractive, trumpet-shaped, with 5-7 rounded and pale purplish-blue petals, and a yellow throat (figure 2). the fower is up to 8 cm long and 6-8 cm across. the plant produces round green stems and a tuberous root system. propagation is from stem or root cuttings. the plant fowers continuously throughout the year with fowers opening early in the morning and aborting in thechan, et al.: phytochemistry and pharmacological properties of thunbergia laurifolia: a review 2 pharmacognosy journal  august 2011  vol 3  issue 24 5-di-o-β-d-glucopyranoside, apigenin, apigenin-7-o-β-d-glucopyranoside, and chlorogenic acid. [6,12] a phenolic profling of water extract of leaves of t. laurifolia showed the presence of apigenin and apigenin glucosides, as well as phenolic acids of caffeic, gallic, and protocatechuic. [12] proximate analysis of the contents of fbre, ash, protein, fat, and carbohydrate in leaves of t. laurifolia (dry weight) were 16.8, 18.8, 16.7, 1.68, and 46.0%, respectively. [13] pharmacology antioxidant activity water, ethanol, and petroleum ether extracts of dried leaf powder of t. laurifolia were evaluated for total phenolic content (tpc), free radical scavenging, and ferric reducing power (frp). [14] based on tpc, it was found that water extraction (2430 mg gae/100 g) was the most effcient compared to ethanol (565 mg gae/100 g) and acetone (142 mg gae/100 g) extraction. the water extract also yielded the highest free radical scavenging with ec 50 value of 0.13 mg gae/ml, whereas ethanol and acetone extracts had ec 50 values of 0.26 and 0.61 mg gae/ml, respectively. figure 1: young (left), developing (middle), and mature (right) leaves of thunbergia laurifolia figure 2: flowers of thunbergia laurifoliapharmacognosy journal  august 2011  vol 3  issue 24 3 chan, et al.: phytochemistry and pharmacological properties of thunbergia laurifolia: a review 38-41 and 50-51%, respectively. microwave-dried leaves remained green with a faint fragrance and when ground, the aromatic green-coloured tea produced a mild tasting green infusion. for the microwave-dried tea, hot water extraction yielded tpc and aeac values that were 1.7-1.9 and 2.0-2.1 times higher than those of methanol extraction. when compared to other commercial teas, tpc, aeac, and frp values of the microwave-dried tea were 6.4, 8.7, and 9.3 times those of the commercial t. laurifolia tea, and were superior to teas of orthosiphon aristatus and aspalathus linearis. antioxidant properties of 13 commercial tropical herbal teas based on screening of their tpc, aeac, and frp have been reported. [16] herbal tea of t. laurifolia was among the low antioxidant category together with herbal teas of alpinia zerumbet, garcinia atroviridis, and cymbopogon citratus. values of herbal tea of t. laurifolia (rang jeud) were 805 50 mg gae/100 g, 591 29 mg aa/100 g, and 43 5 mg gae/100 g, respectively. a study on the effects of various thermal and non-thermal drying methods on the antioxidant properties of leaves and teas of t. laurifolia showed remarkable differences. [17] leaves of t. laurifolia (15 g) were shredded, and microwave-dried (1.5 min), oven-dried (3 h), freeze-dried (overnight), and freeze-withered (2 h). dried leaves were extracted by steeping in hot water (1 h) to obtain the tea infusions. freeze withering and oven drying led to declines in tpc (85 0.6 and 36 2.3%) and aeac (96 2.4 and 25 2.8%), respectively, compared to fresh leaves (table 1). values of freeze-dried leaves remained unchanged i.e. comparable to those of fresh leaves. interestingly, values of microwave-dried leaves were 38 3.2 and 84 6.1% higher than those of fresh leaves, respectively. antioxidant properties of all t. laurifolia teas produced were signifcantly higher than those of the commercial rang jeud tea, with the exception of freeze-withered tea which had comparable properties. [17] freeze-dried, microwave-dried, and oven-dried teas had tpc values the water extract also showed the highest frp (0.93mmol/g), compared to extracts of ethanol (0.18 mmol/g) and acetone (0.04 mmol/g). screening of ethanol extracts from 134 species of edible thai plants for superoxide inhibitive activity, leaf extract t. laurifolia was found to have moderate inhibition rates of 50-69% for total activity and xanthine oxidase inhibition. [15] a study on the optimum time and effciency of methanol extraction for leaves of t. laurifolia demonstrated that 1 h was the optimum extraction time. [3] tpc values for 0.5, 1.0, and 2.0 h were 418 57, 721 105, and 636 71 mg gae/100 g, respectively. based on tpc, the frst extraction extracted about 88% of the phenolic compounds. yields of the second and third extractions were only 10.5 and 3.5%, respectively, suggesting that methanol is effcient in extracting leaves of t. laurifolia. variations in tpc between t. laurifolia leaves of different ages, collection times, and locations were also reported. [3] developing leaves had the highest tpc of 513 8 mg gae/100 g, followed by young and mature leaves with values of 407 11 and 298 9 mg gae/100 g, respectively. tpc values varied from 532 8 to 795 16 mg gae/100 g for four batches of leaves collected from the same source in april and may 2004. leaves collected from plants located in three different locations on the same day had signifcantly different tpc values of 543 15, 734 13, and 892 8 mg gae/100 g, suggesting variation between plants. within plants, leaves and fowers had comparable phenolic content and free radical scavenging ability. the effects of different drying methods on the antioxidant properties of t. laurifolia leaves have been reported. [3,4] antioxidant properties investigated were tpc and ascorbic acid equivalent antioxidant capacity (aeac). leaves (2 g each) were each subjected to three different drying methods. oven drying involved drying for 5.5 hours in an oven set at 50c. sun and microwave drying was for 16 h and 4 min, respectively. for oven and sun drying, tpc and aeac declined 73 and 76%, and 80 and 89%, respectively. for microwave drying, tpc and aeac gained table 1: percentage water loss and gain/loss in total phenolic content (tpc) and ascorbic acid equivalent antioxidant capacity (aeac) of dried leaves in comparison with fresh leaves (fresh weight) drying method water loss (%) gain/loss (%) tpc (mg gae/100 g) aeac (mg aa/100 g) freeze withering 79 0.9 85 0.6 96 2.4 oven drying 78 1.2 36 2.3 25 2.8 freeze drying 80 1.1 0.4 0.7 microwave drying 79 0.6 38 3.2 84 6.1 tpc and aeac are means sd (n 3). abbreviations: gae gallic acid equivalent and aa ascorbic acid.chan, et al.: phytochemistry and pharmacological properties of thunbergia laurifolia: a review 4 pharmacognosy journal  august 2011  vol 3  issue 24 antiproliferative activity ethanolic extracts of nine thai medicinal plants were screened for antiproliferative activity against skbr3 human breast adenocarcinoma cells using the methylthiazoletetrazolium (mtt) assay. [19] leaf extract of t. laurifolia did not show positive antiproliferative activity. similarly, dried leaf powder extract of t. laurifolia showed very weak or no cytotoxic activity against bhk and l929 normal cells, and hepg2 and caco-2 cancer cells using the mtt assay. [14] hepatoprotective activity leaf extract of t. laurifolia protected mice from hepatic injury induced by ethanol. [20] the hepatoprotective activity of aqueous extracts t. laurifolia against ethanol induced liver injury in rats and in primary cultures of rat hepatocytes has also been reported. [21] the extract at appropriate doses increased cell viability of primary cultures of ethanol-treated rat hepatocytes by 2-3 folds and decreased release of alanine transaminase (alt) and aspartate transaminase (ast). it also promoted rat liver recovery after 14 days of ethanol treatment as refected by the decrease in severity of rat liver injury and the normalization in hepatic triglyceride, alt, and ast levels. detoxifying effects the effects of t. laurifolia on endogenous dopamine release from rat striatal slices in comparison with those of amphetamine have been investigated. [6] the effect of hot water extracts of dried t. laurifolia leaves on k stimulated dopamine release from rat striatal slices were compared with amphetamine using hplc with electrochemical detection. results showed that t. laurifolia may stimulate dopamine release in a similar manner to amphetamine. a follow-up study was conducted to determine whether t. laurifolia, which has been used in the treatment of toxicity and addiction, can alter rat brain region activity using in vivo functional nuclear magnetic resonance imaging. [7] it was reported that the methanolic leaf extract of t. laurifolia that were 6.7, 5.3, and 3.1 times, and aeac values that were 11.4, 8.7, and 4.0 times that of the commercial tea (table 2). ranking of the teas based on antioxidant properties was of the order: freeze-dried microwave-dried oven-dried commercial freeze-withered. the various colours of t. laurifolia tea infusions produced from different drying methods are shown in figure 3. tpc and aeac values of 577 39 mg gae/100 g and 398 22 mg aa/100 g of the commercial t. laurifolia (rang jeud) tea were signifcantly lower than values of 805 50 mg gae/100 g and 591 29 mg aa/100 g reported earlier. [16] there is signifcantly difference in antioxidant values between different batches of the commercial t. laurifolia tea, suggesting that manufacturing procedures have not been standardised. antimicrobial activity a total of 41 types of thai medicinal teas were analysed for uv light-activated antimicrobial activities against bacillus subtilis, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, candida albicans, and aspergillus fumigatus. [18] results showed that the ethanolic leaf extract of t. laurifolia did not have any antibacterial or antifungal activities with the exception of uv light-activated activity against b. subtilis. table 2: total phenolic content (tpc) and ascorbic acid equivalent antioxidant capacity (aeac) of tea infusions of thunbergia laurifolia in comparison with the commercial tea (dry weight) tea infusion tpc (mg gae/100 g) aeac (mg aa/100 g) freeze-dried 3850 127 a 4520 100 a microwave-dried 3080 202 b 3450 273 b oven-dried 1800 57 c 1590 55 c commercial tea 577 39 d 398 22 d freeze-withered 488 44 e 219 63 e tpc and aeac are means sd (n 3). abbreviations: gae gallic acid equivalent and aa ascorbic acid. figure 3: infusions of thunbergia laurifolia herbal teas freeze-dried microwave-dried oven-dried commercial freeze-witheredpharmacognosy journal  august 2011  vol 3  issue 24 5 chan, et al.: phytochemistry and pharmacological properties of thunbergia laurifolia: a review non-toxic effects a chronic toxicity study on t. laurifolia aqueous leaf extract on wistar rats showed the extract at doses ranging from 20 to 2,000 mg/kg/day did not affect their body weight, food consumption, behavior, and general health. [9] the extracts did not produce cumulative toxic signs and fatal effects. it was suggested that effects of prolonged oral administration of the extract need to be monitored. in an earlier toxicity study of aqueous leaf extract of t. laurifolia in mice at 1, 2, 4, and 8 g/kg/day, it was reported that no mice died during the frst month, suggesting that the extract is non-toxic, effective, and safe for consumption. [27] anti-inflammatory activity the anti-infammatory effcacy dose of the aqueous leaf extract of t. laurifolia (2.5 g/kg) has been reported to be two-fold that of garcinia mangostana rind extract (5.5 g/kg). [27] alcohol and hexane leaf extracts of t. laurifolia possess anti-infammatory activity against carageenin-induced paw edema in mice. [28] conclusion the growing scientifc interest in t. laurifolia, a medicinal plant in thailand, stems from traditional belief that the species has detoxifying effects, and can used as an antidote for poisons and drugs, including the treatment of drug addiction. local herbal companies are producing and marketing herbal teas and capsules of t. laurifolia, known as rang jeud in thai. in recent years, scientists in thailand have conducted much research on the phytochemistry and pharmacological properties of the species. properties included antioxidant, antimicrobial, antiproliferative, hepatoprotective, and anti-infammatory activities, as well as detoxifying, anti-diabetic, and non-toxic effects. some research interest has also been generated inmalaysia on the antioxidant properties of leaves and herbal teas of t. laurifolia. recently, analyses of the infusion characteristics, sensory attributes, and consumer acceptability of t. laurifolia herbal teas produced by microwave drying, oven drying, freeze drying and freeze withering have been conducted, with comparisons to the commercial rang jeud tea fromthailand. to date, this report represents the frst review of the phytochemistry and pharmacological properties of t. laurifolia. acknowledgements this review forms part of the research project on the effects of different drying methods on the antioxidant properties of leaves and herbal teas of t. laurifolia conducted by three fnal year students of the faculty of applied sciences, ucsi university. the project also included evaluations of the infusion characteristics, sensory increased signal intensity in various parts of the brain, similar to the effects of cocaine or amphetamine. there was a slight decrease in arterial blood pressure. results showed that t. laurifolia can stimulate brain activity in a manner similar to addictive drugs such as amphetamine and cocaine. however, it remains to be confrmed whether t. laurifolia itself can cause addiction or not or whether the effects are indeed relevant to reports that the plant can treat drug addiction. the effects of aqueous leaf extract of t. laurifolia in alleviating lead poisoning in the brain of mice have been studied. [22] results showed that the extract can reduce neuronal cell death and memory loss caused by lead uptake in mice. the extract was found to restore the levels of caspase-3 activity, and maintain total antioxidant capacity and antioxidant enzymes in the brain. the protection of t. laurifolia ethanolic leaf extract against lead toxicity in the nile tilapia (oreochromis niloticus) has been reported. [23,24] when supplemented in fsh food, the extract had the ability to protect the fsh against lead toxicity after 28 days of treatment. the fsh had reduced levels of lead in the liver and muscle, and showed improved growth performance, blood chemistry, hematology, and histology. the detoxifying effects of aqueous t. laurifolia leaf extract on cadmium-induced toxicity in rats have been investigated. [25] two groups of six rats each were injected with cadmium chloride solution at 1.0 mg/kg body weight for 20 days. injected rats fed with drinking water with 0.1 mg/ml of t. laurifolia leaf extract (group 2) had signifcantly higher body weight than those fed with only drinking water (group 1 as control). the rats in group 2 did not show histopathological changes in the kidney that were observed in the control group. the study demonstrated that t. laurifolia leaf extract can protect against cadmium-induced structural damage of rat kidney. another related study investigated the detoxifying effects of aqueous t. laurifolia leaf extract on paraquat-induced toxicity in rats. [26] it was reported that the rats treated with the plant extract had higher survival rates and lower levels of plasma malonaldehyde. anti-diabetic effects the anti-diabetic effects of aqueous leaf extract of t. laurifolia have been studied. [10] results showed that a 15-day treatment with the extract (60 mg/ml/day) decreased levels of blood glucose in diabetic rats. the recovery of some β-cells was found in diabetic rats. whether t. laurifolia leaf contains insulin-like substance(s) which directly act as hypoglycemic agents, or contains substances that induce the regenerative process of β-cells remains to be investigated.chan, et al.: phytochemistry and pharmacological properties of thunbergia laurifolia: a review 6 pharmacognosy journal  august 2011  vol 3  issue 24 attributes, and consumer acceptability of t. laurifolia herbal teas. the support of the faculty and university is gratefully acknowledged. references 1. starr f, starr k, loope l. thunbergia laurifolia. (2003). available from: http://www. hear.org/starr/hiplants/reports/pdf/thunbergia_laurifolia.pdf. 2. nrm. thunbergia: blue trumpet vine. natural resources and mines, queensland. (2003). available from: http://www.nrm.qld.gov.au/pests/ environmental_weeds/weed_ info _series.html. 3. chan ewc.antioxidant activity of flavonoids in thunbergia laurifolia lindl. b.sc.thesis of monash university sunway campus, malaysia. (2004). 4. chan ewc, limyy. antioxidant activity of thunbergia laurifolia tea. j trop forest sci 2006; 18(2):130-6. 5. putiyanan s, chansakaow s, phrutivorapongkul a, charoensup w. standard pharmacognostic characteristic of some thai herbal medicine. chiang mai univ j nat sci 2008; 7(2):239-55. 6. thongsaard w, marsden ca. a herbal medicine used in the treatment of addiction mimics the action of amphetamine on in vitro rat striatal dopamine release. neurosci lett 2002; 329:129-32. 7. thongsaard w, marsden ca, morris p, prior m, shah yb. effect of thunbergia laurifolia, a thai natural product used to treat drug addiction, on cerebral activity detected by functional magnetic resonance imaging in the rat. psychopharmacology 2005; 180:752-60. 8. kanchanapoom t, kasai r, yamasaki k. iridoid glucosides from thunbergia laurifolia. phytochemistry 2002; 60:769-71. 9. chivapat s, chavalittumrong p, attawish a, bansiddhi j, padungpat s. chronic toxicity of thunbergia laurifolia lindl. extract. j thai tradit altern med 2009; 7(1):17-24. 10. aritajat s, wutteerapol s, saenphet k. anti-diabetic effect of thunbergia laurifolia linn. aqueous extract. se asian j trop med public health 2004; 35(2):53-8. 11. purnima m, gupta pc. colouring matters from the flowers of thunbergia laurifolia. j indian chem soc 1978; 55(6):622-3. 12. oonsivilai r, cheng c, bomser j, ferruzzi mg, ningsanond s. phytochemical profiling and phase ii enzyme-inducing properties of thunbergia laurifolia lindl. (rc) extracts. j ethnopharmacol 2007; 114:300-6. 13. jaiboon v, boonyanupahap j, suwansri s, ratanatraiwong p, hansawasdi c. alpha amylase inhibition and roasting time of local vegetables and herbs prepared for diabetes risk reduction chili paste.asian j food ag-ind 2010; 3(1):1-12. 14. oonsivilai r, ferruzzi mg, ningsanond s. antioxidant activity and cytotoxicity of rang chuet (thunbergia laurifolia lindl.) extracts. asian j food ag-ind 2008; 1(2):116-28. 15. jiwajinda s, santisopasri v, murakami a, kim ok, kim hw, ohigashi h. suppressive effects of edible thai plants on superoxide and nitric oxide generation. asian pac j cancer prev 2002; 3:215-23. 16. chan ewc, limyy, chong kl,tan jbl,wong sk.antioxidant properties of tropical and temperate herbal teas. j food compos anal 2010; 23:185-9. 17. eng sy, tan yp, wong zc. antioxidant and sensory properties of herbal teas of thunbergia laurifolia. b.sc. theses, faculty of applied sciences, ucsi university, kuala lumpur, malaysia. (2011). 18. cheeptham n, towers ghn. light-mediated activities of some thai medicinal plant teas. fitoterapia 2002; 73:651-62. 19. moongkarndi p, kosem n, luanratana o, jongsomboonkusol s, pongpan n. anti- proliferative activity of thai medicinal plant extracts on human breast adenocarcinoma cell line. fitoterapia 2004; 75:375-7. 20. chanawirat a. protective effect of thunbergia laurifolia extract on ethanol-induced hepatotoxicity in mice. m.sc. thesis, faculty of graduate studies, mahidol university, bangkok,thailand. (2000). 21. pramyothin p, chirdchupunsare h, rungsipipat a, chaichantipyuth c. hepatoprotective activity of thunbergia laurifolia lindl. extract in rats treated with ethanol: in vitro and in vivo studies.j ethnopharmacol 2005;102:408-11. 22. tangpong j, satarug s. alleviation of lead poisoning in the brain with aqueous leaf extract of thunbergia laurifolia (lindl.). toxicol lett 2010; 198:83-8. 23. palipoch s, jiraungkoorskul w,tansatit t, preyavichyapugdee n, jaikua w, kosai p. effect of thunbergia laurifolia (linn) leaf extract dietary supplement against lead toxicity in nile tilapia (oreochromis niloticus). world j fish mar sci 2011; 3(1):1-9. 24. palipoch s, jiraungkoorskul w,tansatit t, preyavichyapugdee n, jaikua w, kosai p. protective efficiency of thunbergia laurifolia leaf extract against lead (ii) nitrate-induced toxicity in oreochromis niloticus. j med plant res 2011; 5(5):719-28. 25. chattaviriya p, morkmek n, lertprasertsuke n, ruangyuttikarn w. drinking thunbergia laurifolia lindl. leaf extract helps prevent renal toxicity induced by cadmium in rats.thai j toxicol 2010; 25(2):124-32. 26. ussanawarong s, thesiri t, mahakunakorn t, parasupattana s. effect of thunbergia laurifolia linn. on detoxication of paraquat. khon kaen univ res j 2000; 5:11-7. 27. pongphasuk n, khunkitti w, chitcharoenthum m. anti-inflammatory and analgesic activities of the extract from garcinia mangostana linn. acta hortic 2005; 680:125-30. 28. charumanee s, vejabhikul s, taesotikul t, netsingha w, sirisaard p, leelapornpisit p. development of topical anti-inflammatory preparations from thunbergia laurifolia lindl. research report, faculty of pharmacy, chiangmai university, chiangmai,thailand. (1998).pharmacognosy journal  august 2011  vol 3  issue 24 7 r e v i e w a r t i c l e p h c o g j . *address for correspondence: ph: 91 9966164766 email:rajenderreddysama@gmail.com doi: 10.5530/pj.2011.24.2 and it has seen the development of many traditional health care systems. their development was supported by the great biodiversity in fora and fauna due to variations in geography and climate. [2] many weedy plants posses medicinal and therapeutic and therapeutic activities. [3, 4] the cyperaceae family comprising of monocotyledonous fowering plants known as sedges, which superfcially resemble grasses or rushes. the family is large, with some 5,500 species described in about 109 genera. these species are widely distributed, with the centers of diversity for the group occurring in tropical asia and tropical south america. members of the family cyperaceae are called motha as a folkore name in different parts of the country and used as ethno medicinal plants for treatment of diverse ailments. [5] some well-known sedges include the water chestnut (eleocharis dulcis) and the papyrus sedge (cyperus papyrus), fromwhich the ancient egyptian writing material was made. this family also includes cotton-grass (eriophorum), spike-rush (eleocharis), sawgrass (cladium), nutsedge or nutgrass (cyperus rotundus, a common lawn weed), the large genus of carex, and white star sedge (rhynchospora colorata) and whitehead spike sedge (kyllinga nemoralis). this review aims at describing the traditional uses, phytochemical profles kyllinga nemoralis (hutch & dalz) (cyperaceae): ethnobotany, phytochemistry and pharmacology raju s* 1 , kavimani s 2 , uma maheshwara rao v 3 , sreeramulu reddy k 4 1 assistant professor, vijaya college of pharmacy, munaganoor, ranga reddy dist, andhra pradesh, india-505511. 2 mother theresa post graduate institute of health sciences, gorimedu, puducherry, india. 3 nalla narsimha reddy college of pharmacy, korremula, ranga reddy dist, andhra pradesh. 4 assistant manager –clinical r&d, shantha biotechnics limited, hyderabad, andhra pradesh. india- 500004. a b s t r a c t many herbal remedies have so far been employed for the treatment and management of various ailments since the beginning of human civilization. kyllinga nemoralis (hutch & dalz) (cyperaceae) is a plant widely used throughout the world and frequently used for its anti-venom property. the aim of this review was to collect all available scientifc literature published and combine it into this review. the present review comprises the ethanobotanical, phytochemical and pharmacological potential of kyllinga nemoralis. the present review includes 19 references compiled from major databases as chemical abstracts, science direct, scifinder, pubmed, dr. dukes phytochemical and ethnobotany. an exhaustive survey of literature revealed that favonoids, saponins, phenols, terpenes, lipids and glycosides constitute major classes of phytoconstituents of this plant. pharmacological reports revealed that it is having analgesic, antidiabetic, anticancer, antioxidant, antimicrobial, hepatoprotective and antimalarial properties. kyllinga nemoralis seems to hold great potential for in-depth investigation for various biological activities. through this review, the authors hope to attract the attention of natural product researchers throughout the world to focus on the unexplored potential of kyllinga nemoralis, and it may be useful in developing new formulations with more therapeutic value. key words: ethnobotany, phytochemistry, pharmacology, antivenom, kyllinga nemoralis introduction the use of natural products with therapeutic properties is as ancient as human civilization and, for a long time, mineral, plant and animal products were the main sources of drugs. de pasquale, 1984 a. de pasquale, pharmacognosy: the oldest modern science, journal of ethnopharmacology 11 (1984), pp. 1-16. abstract pdf (1361 k) view record in scopus cited by in scopus (15) in recent years, there has been growing interest in alternative therapies and the therapeutic use of natural products, especially those derived from plants. this interest in drugs of plant origin is due to several reasons, namely, conventional medicine can be ineffcient (e.g. side effects and ineffective therapy), abusive and/or incorrect use of synthetic drugs results in side effects and other problems. [1] the indian subcontinent the indian sub-continent comprising of the countries india, pakistan, and bangladesh is the site of one of the oldest civilizations,raju, et al.: kyllinga nemoralis (hutch & dalz) (cyperaceae): ethnobotany, phytochemistry and pharmacology 8 pharmacognosy journal  august 2011  vol 3  issue 24 chiefy in marshy and wet places and is well distributed over all parts of the world. this plant is commonly known as apavisha, nirbishi and velutta nirbasi. it is found in waste places, open grasslands, etc., at low and medium altitudes. it is pantropic in distribution. the plant is more or less glabrous, arising from creeping rootstocks. the stems are usually solitary, 10 to 40 centimeters high. the leaves are up to 15 centimeters in length or longer, 3 to 4 millimeters wide; with the bracts similar. the spikes are ovoid, simple, white, 8 to 13millimeters long. the spikelets are very numerous, 3 to 3.5 millimeters long, the fowering glume distinctly winged along the keel. the fruit is an achene, approximately 1.2-1.5 mm long x 0.5-0.7 mm wide. [7] ethnobotany kyllinga nemoralis leaves and rhizomes containmany biologically active chemicals, and extracts from those tissues have been used in traditional folk medicine to treat many diseases and conditions. the plant leaves are traditionally used for the relief of malarial chills, pruritus of the skin, and thirst due to fever and diabetes. [8] in india plant leaves are used as anti-venom. [9, 10] the rhizomes of the plant are fragrant, sweet, refrigerant, antidiarrhoeal, diuretic, stomachic, and expectorant. [11, 12] the paste of rhizomes mixed with milk is used internally for worm infection. [13] it is also used in fever, hepatopathy, splenopathy, diabetes and tumours. [14] phytochemistry only a few studies have reported on the phytochemistry of k. monocephala. underground parts contain essential oils rich in terpenes α-cyperone, β-selinene, and α-humulene. [15] the methanolic and aqueous extract from the plant leaves were positive for terpenoids, saponins and phenolic compounds. [16] more recently, ethanolic extract of the rhizomes possesses favonoids, triterpenoids and glycosides and the petroleum ether extract was found to possess triterpenoids and glycosides. [17] essential oil from fresh aerial parts by hydrodistillation from cyperus kyllingia endl. was analyzed by a gc, gc-ms. twenty-three compounds were identifed, mainly of oxygenated sesquiterpenes, particularly sesquiterpene hydrocarbons, and carboxylic acid. the most representative compounds were α-cadinol, caryophyllene oxide, α- muurolol, α-humulene, and α-atlantone. [18] pharmacological screening analgesic activity the analgesic activity of the methanol extract of the leaves of kyllinga monocephala rottb. (cyperaceae) was evaluated using and therapeutic potential of various parts of kyllinga nemoralis, which has been used in traditional practice for many years. classification [6] • domain: eukaryota • kingdom plantae – plants • subkingdom tracheobionta – vascular plants • superdivision spermatophyta – seed plants • division magnoliophyta – flowering plants • class liliopsida – monocotyledons • subclass commelinidae • order cyperales • family cyperaceae – sedge family • genus kyllinga rottb. – spike sedge • species kyllinga nemoralis (j.r. forst. & g. forst.) dandy ex hutch. & dalziel – whitehead spike sedge description of kyllinga nemoralis kyllinga nemoralis (hutch & dalz) (family; cyperaceae) is a perennial herb, grass-like in habit, propagated by seed and a creeping rhizome with many synonyms and common names. synonyms include cyperus kyllingia endl, kyllinga monocephala rottb and kyllinga cephalotes (jacq.) and common names include whitehead spike sedge, white kyllinga, white water sedge, white-fowered kyllinga, poverty grass. growpharmacognosy journal  august 2011  vol 3  issue 24 9 raju, et al.: kyllinga nemoralis (hutch & dalz) (cyperaceae): ethnobotany, phytochemistry and pharmacology conclusion kyllinga nemoralis is a wealth of indigenous knowledge and traditional uses have been documented for this species. while this review has attempted to unite the relevant information for this species the data clearly suggests future research priorities. convincing ethnopharmacological evidence is presented alluding to the extensive use of kyllinga nemoralis as antivenom. it is interesting to note that the earlier scientifc investigations of this plant, kyllinga nemoralis, showed the crude extracts exhibited analgesic, antimicrobial, hypoglycemic, anticancer, hepatoprotective and antimalarial properties. this review revealed that favonoids, triterpenoids especially sesquiterpenes, glycosides, saponins, phenolic compounds and lipids constitute major classes of phytoconstituents of this plant. monoterpenes, polyphenols, saponins and favonoids are well known for their biological properties and although a suite of compounds belonging to this class of phytochemicals have been identifed, very few have been subjected to pharmacological assays. this plant can become important sources of novel drugs and lead compounds. references 1. rates smk, plants as source of drugs,toxicon, 2001, 39(5):603-613. 2. sarfaraj hussain md, sheeba fareed, mohd ali, hygrophila auriculata (k. schum) heine: ethnobotany, phytochemistry and pharmacology, asian journal of traditional medicines, 2010, 5 (4):122-131. 3. kirtikar k.r and basu bd. indian medicinal plants, vol. iv, bhishan singh mahendra paul singh, dehradun, india, 1935, 2634. 4. jain sk. dictionary of indian folk medicine and ethnobotany, deep publications, new delhi, 1991, p. 68. 5. kumar k, upaddhyay op, and tiwari rk. ethano-medico studies on motha-a folkore name used by tribal community for different species of cyperaceae family, sachitra ayurved, 1996; 49(5):369-372. 6. harold sj. nomenclature of plants, the ronald press company usa, 1958, p. 55. 7. kirtikar kr, basu b.d. indian medicinal plants, third ed. indological and orient publishers, new delhi, 2000, p. 2634. 8. quisumbing e. medicinal plants of the philippines. quezon city, philippines, katha publishing, 1978, pp. 116-117. 9. oudhia p. medicinal weeds in the rice fields of chattisgarh, india, 1999, irrn 24:40. 10. manju panghal, vedpriya arya, sanjay yadav, sunil kumar, jaya parkash yadav. indigenous knowledge of medicinal plants used by saperas community of khetawas, jhajjar district, haryana, india. journal of ethnobiology and ethnomedicine 2010, 6:4:1-11. 11. khory nr and katrak nn. materia medica of india and their therapeutics, bdh printers, new delhi, 1999, 380. 12. sivarajan vv and balachandran i. ayurvedic drugs and their plant sources. oxford and ibh publishing co. pvt. ltd., oxford, 1994, 136. 13. silja vp, samitha verma k, mohanan kv, ethnomedicinal plant knowledge of the mullu kuruma tribe of wayanad district, kerala, indian journal of traditional knowledge, 2008; 7(4):604-612. 14. warrier pk, nambiar vpk, ramankutty c. indian medicinal plants: a compendium of 500 species, orient longman ltd., madras, 1995, 3, 285-286. 15. komai k, tang ch. chemical constituents and inhibitory activities of essential oils from cyperus brevifolius and c. kyllingia, journal chemical ecology, 1989; 15:2171-2176. the acetic acid-induced writhing test on mice and was found to signifcantly reduce the number of writhes in mice by half. following a bioassay-guided fractionation scheme, statistically signifcant analgesic activitywas observedwith both the hexane and ethyl acetate partitions. [19] in another report the methanol extract of k. monocephala was found to signifcantly reduce the number of writhes in mice administered intraperitoneally with acetic acid to induce abdominal constriction. [16] hepatoprotective activity hepatoprotective activity of ethanolic and petroleum ether extracts of rhizomes of kyllinga nemoralis was evaluated against carbon tetrachloride (ccl4)-induced hepatotoxicity in rats at a dose 100 and 200 mg/kg, p.o. both extracts showed signifcant hepatoprotection when compared to control, similar to standard silymarin. histology of liver sections also revealed that the extracts protected liver from injury. ethanolic extract possesses favonoids, triterpenoids and glycosides and the petroleum ether extract was found to possess triterpenoids and lipids. the hepatoprotective effect produced may be probably due to the triterpenoids, which is common in both of these extracts. [17] hypoglycemic activity the hypoglycemic activity of fresh plant infusion of kyllinga nemoralis was monitored using the oral glucose tolerance test. screening of the blood glucose level of the animals was performed by the glucose oxidase method using a commercially available glucometer. kyllinga nemoralis exhibited signifcant hypoglycemic activity when given 15 min after glucose load. [16] antimalarial, anticancer and antimicrobial activities essential oil from fresh aerial parts by hydrodistillation from kyllinga nemoralis was evaluated for antimalarial, anticancer and antimicrobial activities. antimalarial activity against p. falcipalum (k1) was determined by microculture radioisotope techniques. the anticancer activity tested against the nci-h187 cells. the preliminary antimicrobial activities were also evaluated using the agar diffusionmethod. the microorganisms used were: escherichia coli atcc25922, staphylococcus aureus atcc25923, pseudomonas aeruginosa atcc27553, candida albican, aspergillus favus and trichophyton mentagrophyte. the oil showed signifcant activities against p. falcipalum (k1) and nci-h187 (small cell lung cancer) with the ic50 values of 7.52 and 7.72 μg/ml, respectively. the potent activities of the oil might be attributable to its high sesquiterpene content. [18]raju, et al.: kyllinga nemoralis (hutch & dalz) (cyperaceae): ethnobotany, phytochemistry and pharmacology 10 pharmacognosy journal  august 2011  vol 3  issue 24 18. sorachai khamsan, boonsom liawruangrath, saisunee liawruangrath, aphiwatteerawutkulrag,stephen g.pyne andmary j.garson.antimalarial, anticancer,antimicrobial activities and chemical constituents of essential oil from the aerial parts of cyperus kyllingia endl, records of natural products, 2011; 5(4):324-327. 19. amor evangeline c, quanico jusal p, perez grace g. analgesic activity of extracts of kyllinga monocephala, pharmaceutical biology, 2009; 47(7):624-627. 16. jusal p. quanico, evangeline c. amor1, and grace g. perez. analgesic and hypoglycemic activities of bixa orellana, kyllinga monocephala and luffa acutangula, philippine journal of science, 2008; 137 (1):69-76. 17. arumugam s, ramadoss k, vadivel v, balasubramanian d and muthu r. evaluation of hepatoprotective activity of kyllinga nemoralis (hutch & dalz) rhizomes, journal of ethnopharmacology, 2010; 127:555-557.pharmacognosy journal  august 2011  vol 3  issue 24 11 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: phone no. 08063831237 e-mail: email address: estellamaris5@yahoo.com doi: 10.5530/pj.2011.24.3 of phenolic compounds, alkaloids, steroids, saponins and tannin. [3] it was deemed of interest to investigate this plant pharmacognostically such as macroscopical, microscopical and other diagnostic character of the leaves of dyschoriste perrottetii nees, with a view of preparing monograph for its proper identifcation and inclusion in the pharmacopoeia. material and methods plant collection and identification the plant was collected in february 2009 from nsukka, enugu state, nigeria. it was identifed and authenticated by mr. a. ozioko, a taxonomist of the bio-resources and development conservation programme centre (bdcp) nsukka and a voucher specimen (un/ pcog/09/392) deposited in the herbariumof department of pharmacognosy and environmental medicine, university of nigeria, nsukka. macroscopical examination the macroscopical features of the leaves were studied using both the fresh and dried plant collected as described by evans. [7] pharmacognostic studies on the leaves of dyschoriste perottetii nees *odoh, u. e., ezugwu, c. o. and ezejiofor, m. department of pharmacognosy and environmental medicine, faculty of pharmaceutical sciences, university of nigeria, nsukka. a b s t r a c t to ensure reproducible quality of herbal products, proper control of starting material is important. the frst step towards ensuring quality of starting material is authentication. thus, in recent years there has been a rapid increase in the standardization of selected medicinal plants of potential therapeutic signifcance. despite the modern techniques, identifcation of plant drugs by pharmacognostic studies is more reliable. dyschoriste perrottetii nees (family-acanthaceae) is an important medicinal plant used in various ways in the treatment of microbial infections, fever, measles and pains. macroscopic, microscopic and chemo-microscopic studies of powdered and anatomical sections of the leaf were carried out using standard methods. this is necessary, for the purpose of identifcation and monograph preparation. the result shows diacytic stomata on the lower and upper surface, surrounded by wavy walled epidermal cells, unicellular covering trichomes, calcium oxalate crystal, which are mostly single and prismatic, lignifed fbres and a characteristic collenchyma cells below the epidermis. chemo-microscopic examination revealed the presence of starch, tannin, mucilage and cellulose. quantitative evaluation of the powdered leaves gave moisture content of 7.5 %, total ash 12.5 %, water soluble ash 5.3 %, acid insoluble ash of 4.0 %, and alcohol extractive and water soluble extractive of 31.2 and 21 .08 % respectively. these fndings are of importance in the establishing diagnostic indices for the identifcation, result could be used for identifcation and preparation of monograph on the plant. key words: dyschoriste perottetii, macroscopy, microscopy, pharmacognostic evaluation. introduction the plant dyschoriste perrottetii nees (family-acanthaceae) is a shrub of about half a meter high, with branches and square woody stem rooting at lower nodes. [1] it is widely distributed in the tropics frequently in temperate and completely absent in artistic region. [2] in nigeria among the hausas and fulani communities, it is commonly known as fdda hakukuwa the plant is used in traditional medicine for easy labour and in treatment of yellow fever and measles and the seeds used for the removal of foreign material in the eyes. [3] members of the acanthaceae are of used for the relief of pain during child birth. [4] pharmacological and biological study of the family shows that some members exert anticholinestrase activity, histamine antagonist, cardiac depressants, antimicrobial and antifungal effects. [5] recently some were found to exhibit antitumour activity. [6] preliminary phytochemical screening on the herb revealed the presenceodoh, et al.: pharmacognostic studies on the leaves of dyschoriste perottetii nees 12 pharmacognosy journal  august 2011  vol 3  issue 24 ash and water—soluble ash was determined as determined as described. [9] the water and alcohol extractive value were obtained using the method outline. [8] results macroscopical examination the leaves are simple, opposite. the shape is lanceolate with 2.5-5.0 cm wide and 6-12 cm long. the base of the leaf is decurente. the leaves are glabrous dark green in with apex sub acute, the margin shallowly wavy, reticulate venation, smooth and soft texture and petiole about 1.0-2.2 cm . it has characteristic, agreeable odour and slightly bitter. microscopical examination the microscopical features of the fresh and leaves powder were described as follows; diacytic stomata numerous on lower epidermis and moderate on upper epidermis, unicelluler covering trichome 4-12 µm in size, phloem microscopical examination the powdered and transverse section of the leaf was employed for this study; to carry out quantitative and qualitative studies using the method employed. [7] chemo-microscopical examination was carried out to detect the presence or absence of various chemical compounds such as starch, cellulose, tannins, and lignin, fat and oil, mucilage and calcium oxalate crystals. phytochemical studies the preliminary phytochemical screening of the leaf powder was performed following standard qualitative chemical tests [7,8] in other to detect the presence or absence of major secondary plant metabolites of pharmacognostic importance which include; alkaloids, tannins, favonoids, saponins, glycosides, proteins, fats and oils, steroids and carbohydrates. quantitative microscopy themoisture content of the powdered leaves was determined by loss on drying method. [8] the ash value, acid insoluble epidermal cells with diacytic stomata epidermal cells in surface view bundle of fibres with calcium oxalate crystals spiral vessels prisms of calcium oxalate covering trichomes figure 1: macroscopical features of the leaf of dyshoriste perroteti neespharmacognosy journal  august 2011  vol 3  issue 24 13 odoh, et al.: pharmacognostic studies on the leaves of dyschoriste perottetii nees physicochemical standards the water extractive and alcohol extractive, total ash, acid insoluble ash, water soluble ash and moisture content were shown in table 3. discussion the macroscopical features of the plant can be used, as its diagnostic parameters. the microscopical features such as the presence of diacytic stomata on both epidermal surfaces, aggregate of calcium carbonate (cystolith), the parenchymatous cells containing prismatic calcium oxalate crystals conformed with major characteristic features of the family acanthaceace. [10] the chemo-microscopical result indicated the presence of mucilage and tannins. phytochemical screening reveals the presence alkaloids, favonoids, tannins, glycoside, saponnins and sterols. the commonly encountered alkaloid in the acanthaceace family is the trophan alkaloids, quinazoline found to have fbers moderate, 100-200 µm long wih tapering apex, spiral xylem vessels, prisms of calcium oxalate 25-30 µm in size. the transverse section of the lamina through the midrib (figure 2) revealed that the is dorsoventral with epidermis covered externally by a wavy cuticle, mesophyll, diacytic stomata and a characteristic collenchyma cells below the epidermis phytochemical studies phytochemical screening of the leaf powder revealed the presence of alkaloids, favonoids, tannins, glycosides, saponins and sterols. chemo-microscopical examination this revealed the presence of chemical constituents in the cell wall and cell of dyschoriste perrottetii (table 1). quantitative leaf microscopy the results of quantitative microscopy and pharmacognostic standards were presented in table 2. table 1: results of chemomicroscopy of the leaf of dyschoriste perrottetii nees test reagent observation inference chlo-zinc-iodide blue to black colour observed on epidermal cells cellulose () ferric chloride solution greenish leaves in some parenchyma cells tannins () n 50 – iodine blue-black colouration observed on some few grains in parenchyma cells. in transverse section and in powdered leaves. starch () phloroglucinol and conc. hcl no. red colouration observed in the xylem vessels lignin (–) ruthenium red red colouration observed mucilage () 80 % h 2 so 4 crystals of calcium oxalate dissolved calcium oxalate crystals () collenchyma crystal idioblast parenchyma phloem xylem pith cuticle palisade spongy mesophyl collenchyma figure 2: transverse section showing the midrib of the leaf of dyshoriste perrottetii neesodoh, et al.: pharmacognostic studies on the leaves of dyschoriste perottetii nees 14 pharmacognosy journal  august 2011  vol 3  issue 24 conclusion the results presented in this study could serve as diagnostic parameters for proper identifcation as well as preparation of a monograph on dyschoriste perrottetii nees. acknowledgement the authors thank department of pharmacognosy and environmental medicine and department of botany, university of nigeria, nsukka for providing the facilities for the research. references 1. grahams vc, tropical wild flowers: fulton education pub. ltd. london, p. 126, 1963. 2. clerk cb, flora of tropical africa vol. 5, p. 72, 1955. 3. dalziel jm, useful plants of west africa, crown agents for overseas government and administration. millbank, london, pp. 78-91, 1958. 4. harbone jb and herbert b, phytochemical dictionary .a handbook of bioactive compounds from plant,taylor and frances ltd.,pp.270-280,1993. 5. murakami a, possible anti-tumor promoting properties of traditional thai food items and some of their active constituents. asia pacific j. chim.nutr, 1994, 3, 155-191. 6. ciulei 1, methodology for analysis of vegetable drugs, unido romania, pp. 17-25, 1988. 7. hutchison j and dalziel jm, flora of the west tropical africa, 2 nd edition, vol. 2, crown agents for overseas government and administration. millbank, london, pp. 51-54, 1963. 8. evans cw,trease and evans“pharmacognosy”14 th edition,wb saunders ltd., baillere tindal, london, pp. 576, 1996. 9. british pharmacopoeia, vol.2, appendiix x1 her majesty’s stationary office london, a108 - a113, 1988. 10. sharma bb and trivedi bs, introductory taxonomy of angiosperms, 2 nd edition., allah bad kitab, mahd, india, pp. 286-287, 1978. 11. kokate ck, purohit ap and gokhale, sb, pharmacognosy 18 th edition, pub. nirali prakashan, india, p. 520, 2002. utrotonic. [11] this may be responsible for the use of the plant in easing labour. the presence of tannin and other phenolic compounds which are known to have antimicrobial activity were revealed in the phytochemical analysis and chemomicroscopy. this therefore justifes the use of the plant in the traditional treatment venereal diseases, urinary tract infection and diarrohoea. [5] the pharmacognostics standards such as moisture content (7.5 % w/w) of the leaf, which is low, showed that there is less chance for microbial degradation of the drug during storage. table 2: results of quantitative microscopy of the leaf of dyschoriste perrottetii nees standard value palisade ratio  8.4 0.11 stomatal number 23.0 1.32 upper epidermis 19.9 0.50 lower epidermis 37.1 0.16 stomatal index 1.12 1.08 vein islet 15.0 0.84 vein termination 13.0 2.06 values are mean 3 determinations table 3: results of analytical standards of the leaf of dyschoriste perrottetii nees determination values moisture content  7.5 1.50 total ash 17.5 1.92 acid-insoluble ash  4.0 0.21 alcohol extractive 13.2 1.55 water extractive 31.2 0.10 values are mean 3 determinationspharmacognosy journal  august 2011  vol 3  issue 24 15 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: tel: 603-9289 7303; fax: 603-26983271. e-mail: jamia@pharmacy.ukm.my doi: 10.5530/pj.2011.24.4 (b) spiritual. [3,4] the physical characteristic of a person comprises of four elements (fre, earth, wind and water) and humours (damp, cold, dry and hot). [5] often a cold condition, due to either consuming “cold” foods and drinks or being in a cold weather, may result to the person building up excessive wind within the body and consequently this will cause the immediate or precipitating illness. [6] the spiritual aspect, on the other hand, constitutes of the mind and soul substance or vital force (semangat); thus a person with a loss of semangat is said to be vulnerable to the infuence of supernatural or evil spirits. [3] the cause of an ailment is often thought to be due to the imbalance of the above mentioned physical elements and/or loss of semangat. various methods are used in the treatment of illnesses including use of herbal medicines such as spices, medicinal plants and animals; physical treatment such as massage, suction therapy and circumcision; as well as spiritual treatment such as recitation and performing prayers. medication of physical illness is usually prescribed, of which characteristics must be opposite to those of the ailment. medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia jamia azdina jamal 1 *, zakiah abd. ghafar 2 & khairana husain 1 1 drug and herbal research centre, faculty of pharmacy, universiti kebangsaan malaysia, jalan raja muda abdul aziz, 50300 kuala lumpur, malaysia. 2 national pharmaceutical control bureau, ministry of health malaysia, lot 36, jalan universiti, 46200 petaling jaya, selangor darul ehsan, malaysia. a b s t r a c t malay traditional medicine practice concentrates on the primary healthcare including physical and spiritual aspects of human being. most traditional practitioners use medicinal plants in the treatment. hence, the study is aimed to compile preparations and local medicinal plants used traditionally by the women in postnatal care. five malay traditional practitioners based in the district of muar in johor and two in the district of kuala pilah in negeri sembilan were interviewed. from the study, information on 23 preparations, consisting of 128 medicinal plants, was successfully compiled. the preparations were categorised as jamu, fresh herbs, eye drop, poultice, medicated talcum powder and bathing solution. the medicinal plants comprised of 52 species belonging to 42 genera and 27 families. some species were found to occur frequently, such as curcuma longa l., zingiber offcinale roscoe, cinnamomum zeylanicum blume, kaempferia galanga l., piper cubeba bojer, zingiber cassumunar roxb., acorus calamus l., piper nigrum beyr. ex kunth, alyxia stellata roem. & schult., coriandrum sativum l., foeniculum vulgare mill., nigella sativa l. and usnea barbata fries. the part of plants utilised in the preparations include rhizomes, fruits/berries, leaves, seeds, barks, fowers, roots, whole plant, gall and bulb. the study provided useful and important information on the diversity of medicinal plants used by different malay traditional practitioners in postnatal care. key words: medicinal plants, postnatal care, malay traditional medicine introduction the practice of malay traditional medicine has various infuences, for example by the indonesian, chinese and indian traditional medicines, orang asli medicine and including those introduced by the arabs, persians and europeans. [1] however, nowadays the practice is mainly dominated by the arabic unani medicine and galenic philosophy. in the context of socioanthropology, structure of the malay traditional medicine is not fxed and rigid, thus allowing improvements and changes to be made according to suitability and current needs. [2] the malay traditional medicine system believes that a person consists of two aspects: (a) physical, that is the body; andjamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia 16 pharmacognosy journal  august 2011  vol 3  issue 24 preliminary information on plants used traditionally by the women in postnatal care inmalaysia and possible justifcations based on previously published traditional uses and scientifc data for selected medicinal plants. the objective of the study is to compile information on the type and purpose of preparations, as well as the type and part of medicinal plants. this was done by oral interview of randomly selected malay traditional medicine practitioners who are based in the districts of muar in johor and kuala pilah in negeri sembilan, in the peninsular malaysia. materials and methods seven malay traditional medicine practitioners were individually interviewed based on a set of pre-piloted questionnaire. five of them were based in muar, johor and two were in kuala pilah, negeri sembilan (figure 1). the practitioners werehussain, kalsom, painah and salmi from kampung parit medan, muar, johor. zainab was from kampung tiong baru, muar, johor. yah and ujang were fromkampung tanjung jati, kuala pilah, negeri sembilan. information enquired in the questionnaire include: (i) type of preparation, (ii) name of medicinal plant(s), (iii) part of the medicinal plant used, (iv) method of preparation and (v) use of the preparation. the data gathered from the interview was analysed. most of the names of medicinal plants were given in malay, therefore, the scientifc names were cross-checked with several ethnobotanical references such as burkill, [13] zakaria and mohd, [1] and mat-salleh and latiff. [14] the reported traditional uses of these plants in malaysia by burkill [13] were also obtained from dr. duke’s phytochemical and ethnobotanical databases. [15] results and discussion preparations used for postnatal care in malay traditional medicine twenty three preparations consisting of 128 medicinal plants were compiled, as summarised in table 1. the types of preparations included jamu, ulam (fresh herb), eye drops, tapel (poultice), pilis (medicated talcum paste applied onto the forehead), param (medicated talcum paste applied to the whole body) and mandian (herbal bath). in the malay traditional medicine, jamu is traditionally used in post-partum medication to help improve blood fow, warming and refreshing of the body, speed up contraction of the uterus and tightening of the vagina, encourage bowel movement and prevent vaginal discharge. jamu often contains a mixture of various medicinal plants and plant parts that is evidenced from this study (table 1: j1-j8). it is given orally in a form of either herbal pills or hot water for example, a “cold” ailment or that caused by excessive wind in the body will be prescribed with a “hot” medicine. medications containing single or compound medicinal plants may be dispensed in many forms such as powder, capsules, pills, “makjun”, medicated oil, simple distillate, decoction, infusion, paste and poultice. herbal medicines are often used for medicinal purposes and are sometimes self-prescribed for relief of minor illnesses such as colds, fevers, coughs, diarrhea, stomach-aches and headaches. these are also more popularly taken as health supplements for the maintenance of physical ftness and health, restoration of new power and spirit of life, as well as reassuring matrimonial happiness. in malay traditional medicine, traditional midwife (mak bidan) is an important practitioner owing to her role in treating and advising women on health care and health problems. the fundamental function of a traditional midwife is taking care of a pregnant mother before, during and after childbirth. frequently, women seek help from the traditional midwife when they have problems associated with the reproductive system. treatment is often carried out by body massage with either ordinary cooking oil or medicated oil. massage has been found to be benefcial for relaxation of the body, as well as for relieve of joint and muscle pain and stiffness. sometimes a mixture of medicinal plants, commonly known as jamu (unprocessed or dried natural materials used for medicinal or health care purposes), is prescribed to the patient. jamu is traditionally used for the relief of minor illnesses, as health supplements and food supplements, as well in cosmetics. other preparations made by the traditional midwife include air selusuh (medicine for before and during childbirth), ubat periuk (medicine for after childbirth), makjun (spherical semi-solid preparation), bedak sejuk (rice talcum powder), param (medicated talcum paste), liniment and hot compression. [1] most malay communities believe that special attention and care should be given to a new mother during her confnement period in order to help restore energy and vitality. jamu is usually given in the morning, followed by body massage for at least three consecutive days. later, hot compression using a wrapped hot stone is applied onto the abdominal part. a medicated paste is then spread over the stomach and the waist is bound tightly with a long piece of cloth. additionally, a medicated paste may be applied onto the forehead and face. proper documentation of the use of plants in the malay traditional medicine practice is very limited, such as publications by ridley, [7] gimlette, [8,9] gimlette and burkill, [10] gimlette and thomson, [11] and burkill, [12] and these are not regularly updated. no formal training of traditional malay practitioners is currently available. knowledge and traditional prescriptions are passed from generation to generation merely by word of mouth. thus, this paper providespharmacognosy journal  august 2011  vol 3  issue 24 17 jamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia swelling, tonic, urogenital problems and wound healing (table 2). previous studies on c. longa have revealed that the extracts have antidepressant, [16] hypotensive and vasorelaxant [17] effects in vivo. curcumin isolated from c. longa rhizome has been shown to exhibit activity against various pro-infammatory diseases such as cancer, diseases of the heart, lung, liver and skin, neurodegenerative and endocrine disorders, infectious diseases and others, [18] whereas ar-turmerone is a potent inhibitor for collagen-induced platelet aggregation [19] and an immunomodulator. [20] poultice, medicated talcum paste applied onto the forehead, medicated talcum paste applied to the body parts and herbal bath are used as external preparations. in this study, the decoction or hot water mixture. the composition of jamu is found to vary according to the malay traditional practitioners. the fnding seems to agree with the statement of salleh [2] that the practice is not rigid. this is unlike other traditional or complementary practices whereby certain ailment is treated with a specifed medicine. in this study, 61 medicinal plants of 35 species are found to be used in the jamu preparations and the most frequently used include curcuma longa l. (in 5 preparations), coriandrum sativum l. (3), kaempferia galanga l. (3), parkia roxburghii g.don (3), quercus infectoria oliv. (3) and usnea barbata fries (3). the applications of c. longa in the malay traditional medicine have been recorded by burkill [13] for parturition and other ailments related to afterbirth such as amenorrhea, diuretic, lactagogue, figure 1: a map of peninsular malaysia showing the sites of studyjamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia 18 pharmacognosy journal  august 2011  vol 3  issue 24 table 1: plant species, local malay name, parts of plants and applications of medicinal plants used in postnatal care by the malay traditional medicine practitioners in muar, johor and kuala pilah, negeri sembilan no. a species malay name part used applications j1 acorus calamus l. alpinia conchigera griff. alyxia stellata roem. & schult. carum carvi l. cinnamomum zeylanicum blume coriandrum sativum l. curcuma longa l. elaeocarpus grandiflorus sm. foeniculum vulgare mill. illicium tenuifolium (ridl.) a.c.sm. kaempferia galanga l. nigella sativa l. oroxylum indicum (l.) benth. ex kurz parkia roxburghii g.don peucedanum japonicumthunb. piper cubeba bojer piper nigrum beyr. ex kunth piper retrofractum vahl quercus infectoria oliv. rheum officinale baill. saussurea lappa c.b.clarke usnea barbata fries zingiber officinale roscoe jerangau lengkuas padang pulasari jemuju kayu manis ketumbar kunyit anyang-anyang adas pedas bunga lawang bukit cekur jintan hitam bonglai kedaung ganti kemungkus lada putih & hitam cabai sirih manjakani kelembak pucuk kayu angin halia rhizome rhizome bark fruit stem bark fruit rhizome bark fruit flower leaf seed fruit seed rhizome berry fruit fruit gall root root whole plant rhizome hot water mixture of the ground plant materials is given to improve blood circulation, to make the body feel warm, to encourage contraction of the uterus, to expel wind, to prevent fit and as a laxative. j2 coriandrum sativum l. curcuma longa l. parkia roxburghii g.don piper nigrum beyr. ex kunth usnea barbata fries zingiber aromaticum valeton zingiber officinale roscoe ketumbar kunyit kedaung lada putih kayu angin lempoyang halia fruit rhizome seed fruit whole plant rhizome rhizome hot water mixture of ground plant materials is given to improve blood circulation, to regain body strength, to expel wind, to ease muscular and joint pain, as well as to ease abdominal discomfort. j3 alyxia stellata roem. & schult. carum carvi l. cinnamomum zeylanicum blume coriandrum sativum l. eurycoma longifolia jack foeniculum vulgare mill. illicium tenuifolium (ridl.) a.c.sm. kaempferia galanga l. litsea odorifera valeton nigella sativa l. parameria polyneura hook.f. parkia roxburghii g.don piper cubeba bojer quercus infectoria oliv. usnea barbata fries pulasari jemuju kayu manis ketumbar kayu pasak bumi adas pedas bunga lawang bukit cekur terawas jintan hitam kayu rapat kedaung kemungkus manjakani kayu angin bark fruit stem bark fruit root fruit flower leaf flower seed stem bark seed berry gall whole plant hot water mixture of ground plant materials is given to regain body strength, to prevent bad body odour, to expel wind, to encourage contraction of the uterus, to encourage wound healing, to reduce bleeding, to stimulate lactation and as a contraceptive. j4 curcuma longa l. tamarindus indica l. kunyit asam jawa rhizome fruit juice mixture with dark brown sugar and salt is given for slimming. j5 quercus infectoria oliv. manjakani gall ground dried gall is given to encourage contraction of the uterus, to regain body strength,to treat vaginal discharge and to treat abdominal discomfort. j6 rourea humilis blume akar kayu mengecut root its water decoction is given to encourage contraction of the uterus. j7 ananas comosus (l.) merr. curcuma longa l. curcuma heyneana valeton & zijp curcuma mangga valeton & zijp curcuma xanthorrhiza d.dietr. kaempferia galanga l. musa acuminata colla tamarindus indica l. zingiber aromaticum valeton nanas kunyit temu giring temu pauh temu lawak cekur pisang kapas asam jawa lempoyang young leaf rhizome rhizome rhizome rhizome leaf fruit fruit rhizome its juice mixture is given to stimulate lactation.pharmacognosy journal  august 2011  vol 3  issue 24 19 jamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia no. a species malay name part used applications j8 acorus calamus l. curcuma longa l. zingiber cassumunar roxb. jerangau kunyit bongelai rhizome rhizome rhizome hot water mixture of ground plant materials with tamarind and dark brown sugar is given to stimulate lactation and to prevent bad odour of the breasts. u9 centella asiatica (l.) urb. pegaga leaf fresh leaf is eaten to stimulate lactation. u10 zingiber officinale roscoe halia rhizome fried rhizome is eaten to make the body feel warm. e11 piper cubeba bojer kemungkus berry its juice is applied into the eyes to improve eyesight. t12 tamarindus indica l. asam jawa fruit mixture of its juice and lime is applied onto the abdomen for slimming and to encourage contraction of the uterus. t13 zingiber officinale roscoe halia rhizome t14 citrus aurantifolia swingle limau nipis fruit p15 acorus calamus l. alyxia stellata roem. & schult. cinnamomum zeylanicum blume eugenia aromatica kuntze foeniculum vulgare mill. illicium tenuifolium (ridl.) a.c.sm. nigella sativa l. peucedanum japonicumthunb. piper cubeba bojer rheum officinale baill. sesbania grandiflora (l.) pers. zingiber cassumunar roxb. jerangau pulasari kayu manis cengkih adas pedas bunga lawang bukit jintan hitam ganti kemungkus kelembak turi bongelai rhizome bark stem bark flower bud fruit flower seed rhizome berry root leaf rhizome mixture of the ground plant materials and water is applied onto the forehead to help improve eyesight. p16 cinnamomum zeylanicum blume entada phaseoloides (l.) merr. eugenia aromatica kuntze nigella sativa l. piper cubeba bojer piper nigrum beyr. ex kunth kayu manis sintok cengkih jintan hitam kemungkus lada putih stem bark seed flower bud seed berry fruit mixture of the ground plant materials and water is applied onto the forehead to help improve eyesight. p17 allium sativum l. cinnamomum zeylanicum blume bawang putih kayu manis bulb stem bark mixture of the ground plant materials and water is applied onto the forehead to help improve eyesight and to treat headache. p18 acorus calamus l. cinnamomum zeylanicum blume eugenia aromatica kuntze piper cubeba bojer piper nigrum beyr. ex kunth sesbania grandiflora (l.) pers. jerangau kayu manis cengkih kemungkus lada hitam turi rhizome stem bark flower bud berry fruit leaf mixture of the ground plant materials and water is applied onto the forehead to help improve eyesight and to freshen the body. r19 alyxia stellata roem. & schult. foeniculum vulgare mill. kaempferia galanga l. oryza sativa l peucedanum japonicumthunb. zingiber officinale roscoe zingiber cassumunar roxb. pulasari adas pedas cekur beras ganti halia bongelai seed fruit leaf seed rhizome rhizome rhizome mixture of the ground plant materials and a little water is applied onto the face to freshen the body. r20 curcuma longa l. kaempferia galanga l. oryza sativa l usnea barbata fries zingiber officinale roscoe kunyit cekur beras kayu angin halia rhizome leaf seed whole plant rhizome mixture of the ground plant materials and a little water is applied onto the face to freshen the body and to expel wind. r21 curcuma longa l. kaempferia galanga l. oryza sativa l piper nigrum beyr. ex kunth vetiveria zizanioides stapf zingiber cassumunar roxb. zingiber officinale roscoe kunyit cekur beras lada putih larasetu bongelai halia rhizome rhizome seed fruit root rhizome rhizome mixture of the ground plant materials and a little water is used to massage the body and to make the body feel warm. m22 acorus calamus l. alpinia galanga willd. carica papaya l. citrus aurantifolia swingle coriandrum sativum l. curcuma longa l. jerangau lengkuas betik limau nipis ketumbar kunyit leaf leaf leaf leaf leaf leaf mixture of the water decoction and plenty of water is used for bathing in order to remove bad body odour and to freshen the body. (continued)jamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia 20 pharmacognosy journal  august 2011  vol 3  issue 24 table 1: continued no. a species malay name part used applications curcuma xanthorrhiza d.dietr. cymbopogon citratus stapf piper betle l. psidium guajava l. zingiber cassumunar roxb. temu lawak serai makan sireh jambu batu bongelai leaf leaf leaf leaf leaf m23 alpinia galanga willd. coleus blumei benth. cymbopogon nardus (l.) rendle datura fastuosa l. pandanus odorus ridl. zingiber cassumunar roxb. lengkuas ati-ati serai wangi kecubung pandan bongelai leaf leaf leaf leaf leaf leaf mixture of the water decoction and plenty of water is used for bathing in order to remove bad body odour and to make the body feel warm. a indicates the type of preparation, that is, jjamu, uulam (fresh herbs), etitisan mata (eye drops), ttapel (poultice), ppilis (medicated talcum powder applied onto the forehead), rparam (medicated talcum powder applied to the body), mmandian (bathing solution). defnition of each preparation is described in the text. table 2: families of the medicinal plant species used medicinal plants used after childbirth by the malay traditional medicine practitioners in muar, johor and kuala pilah, negeri sembilan and the reported traditional uses family species reported traditional uses by burkill (1966) acoraceae acorus calamus l. diarrhea, odontosis, parturition, pediculifuge, splenomegaly, venereal, vermifuge. apiaceae carum carvi l. – centella asiatica (l.) urb. – coriandrum sativum l. cough, fever, nausea, ophthalmia, rheumatism. foeniculum vulgare mill. abdomen, dermatosis, gastralgia, hepatosis, rheumatism. peucedanum japonicumthunb. constipation, fever, giddiness, miscarriage, parturition, sapraemia, smallpox. apocynaceae alyxia stellata roem. & schult. – parameria polyneura hook.f. parturition, tonic, toothblack, uteromegaly. asteraceae saussurea lappa c.b.clarke – bignonaceae oroxylum indicum (l.) benth. ex kurz ache (stomach and tooth), cholera, diarrhea, dysentery, enterosis, fever, gastrosis, parturition, rheumatism, splenomegaly, swelling, vertigo. bromeliaceae ananas comosus (l.) merr. abortifacient, diphtheria, diuretic, emmenagogue, gonorrhoea, vermifuge. caricaceae carica papaya l. abortifacient, arthritis, asthma, boil, colic, dysuria, fever, fumitory, gravel, vermifuge. connaraceae rourea humilis blume – elaeocarpaceae elaeocarpus grandiflorus sm. sore, tonic. fabaceae entada phaseoloides (l.) merr. abdomen, cosmetic, enterosis, hematochezia, parturition, piscicide, shampoo, watervine, wound. parkia roxburghii g.don – sesbania grandiflora (l.) pers. aperient, bruise, cosmetic, diarrhea, dysentery, edema, emetic, enterosis, gastrosis, glossitis, leucorrhea, ophthalmia, scabies, sore (throat), sprain, sprue, stomatitis, thrush, tonic, tonsillitis. tamarindus indica l. abortive, boil, conjunctivitis, cosmetic, dermatosis, fever, itch, mucusitis, pimple, rheumatism, sore, sting (caterpillar), thrush, wound. fagaceae quercus infectoria oliv. – illiciaceae illicium tenuifolium (ridl.) a.c.sm. – lamiaceae coleus blumei benth. cachexia, dyspepsia, ophthalmia lauraceae cinnamomum zeylanicum blume – litsea odorifera valeton lactagogue liliaceae allium sativum l. vermifuge musaceae musa acuminata colla – myrtaceae eugenia aromatica kuntze ache (head and tooth), colic, parturition, tonic, vaginamegaly. psidium guajava l. ache (stomach), dermatosis, diarrhea, emmenagogue, epilepsy, hysteria, leucorrhea, swelling, vermifuge. pandanaceae pandanus odorus ridl. anemia, cosmetic, gonorrhoea, measles, parturition, sapraemia, syphilis. parmeliaceae usnea barbata fries –pharmacognosy journal  august 2011  vol 3  issue 24 21 jamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia poultice is made up of a mixture of lime (citrus aurantifolia swingle) and juice of either tamarindus indica l. or zingiber offcinale roscoe and is applied onto a mother’s abdomen. this is said to encourage contraction of the uterus and slimming of the abdominal part (table 1: t12-t14). the latter traditional use could be associated with the fndings that methanol and ethyl acetate extracts of z. offcinale reduce abdominal fat deposition in vivo. [21] additionally, the use of z. offcinale in this preparation is also in accordance to the reported traditional use of z. offcinale in parturition and during puerperium [13] (table 2). furthermore, the sundaneses in west java, indonesia have also used t. indica fruit in their post-partum remedies. [22] the medicated talcum paste, pilis, applied onto a mother’s forehead is traditionally believed to help improve poor vision and to treat headache after childbirth. fifteen species in 4 preparations have been compiled in this study. cinnamomum zeylanicum blume (in 4 preparations), eugenia aromatica kuntze (3) and piper cubeba bojer (3) are three most commonly utilised in the pilis (table 1: p15-p18). only e. aromatica has been reported to be used traditionally in the treatment of headache [13] (table 2). eugenol, which is the active ingredient of e. aromatica, is found to inhibit monoamine oxidase a in vitro and has antidepressant activity in vivo. [23] traditionally, medicated talcum paste param used for massaging or applied to the entire body is considered to help regain body fgure, expel wind, eliminate stretch marks and smoothen the skin. in param, k. galanga (in 3 preparations), oryza sativa l. (3) and z. offcinale (3) are most popular ingredients (table 1: r19-21). it has been revealed that methanolic extract of k. galanga has antinociceptive effect in vivo [24] and the hexane extract has sedative property. [25] in addition, o. sativa has been used in the traditional family species reported traditional uses by burkill (1966) piperaceae piper betle l. abscess, ache (tooth), antiseptic, asthma, cough, earache, epistaxis, gingivitis, gonorrhoea, halitosis, hoarseness, itch, lactafuge, leucorrhea, mucositis, otosis, parturition, pimple, sore, stimulant. piper cubeba bojer aphrodisiac, diuretic, dysentery, dyspepsia, enterosis, gonorrhoea, panacea, rheumatism, stimulant. piper nigrum beyr. ex kunth abortifacient, ache (head), cholera, colic, gonorrhoea. piper retrofractum vahl cramps, dyspepsia, hepatosis, osteosis, weakness. poaceae cymbopogon citratus stapf ache (tooth), diaphoretic, diuretic, dyspepsia, emmenagogue, gingivitis, neuritis, rheumatism, sprain. cymbopogon nardus (l.) rendle dyspepsia, emmenagogue. oryza sativa l. – vetiveria zizanioides stapf cosmetic, parturition. polygonaceae rheum officinale baill. cosmetic, freckles, fumitory, purgative, tonic. ranunculaceae nigella sativa l. – rutaceae citrus aurantifolia swingle ache (head and stomach), cough, dermatosis, dysentery, gonorrhoea, neuralgia, yaws. simaroubaceae eurycoma longifolia jack ache (head), fever, malaria, parturition, smallpox, sore, syphilis, wound. solanaceae datura fastuosa l. – zingiberaceae alpinia conchigera griff. – alpinia galanga willd. – curcuma heyneana valeton & zijp deodorant, obesity, wound. curcuma longa l. abscess, amenorrhea, cold, conjunctivitis, cosmetic, diarrhea, diuretic, dysentery, gonorrhoea, gravel, hepatosis, jaundice, lactagogue, parturition, pyuria, scabies, sore, swelling, tonic, urogenital, wound. curcuma mangga valeton & zijp fever, stomachic. curcuma xanthorrhiza d.dietr. amenorrhea, choleretic, constipation, dyspepsia, emmenagogue, gallstones, hepatosis, parturition, rheumatism. kaempferia galanga l. abdomen, cosmetic, cough, fever, mastitis, ophthalmia, otosis, rheumatism, sore (throat), swelling. zingiber aromaticum valeton bilious, chlorosis, cholecystosis, gout, parturition, pertussis, tonic. zingiber cassumunar roxb. abdomen, ache (head), ache (stomach), anodyne, colic, constipation, cosmetic, cramps, fever, flatulence, gonorrhoea, jaundice, malaria, numbness, parturition, vermifuge. zingiber officinale roscoe abortive, ache (back, head and stomach), ague, colic, congestion, cosmetic, cough, dyspepsia, fever, gingivosis, gynecology, hepatosis, infection, panacea, parturition, puerperium, rheumatism, rhinosis, sore, swelling, syphilis, tonic. (–) information not available in burkill (1966).jamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia 22 pharmacognosy journal  august 2011  vol 3  issue 24 preparation of the sundaneses in west java, indonesia in the treatment of dermatitis. [22] o. sativa could also be used as an excipient in the powdered mixture due to the high content of starchy materials for easy and uniform application of the paste. in the malay traditional medicine, new mothers are encouraged to bathe with water-boiled leaves or roots. in this study, 17 medicinal plants in 2 preparations were compiled and alpinia galanga willd. (in 2 preparations), zingiber cassumunar roxb. (2) and cymbopogon spp. (2) are found to be common ingredients of the solutions (table 1: m22-m23). the use of aromatic herbs is believed to help remove bad body odour, to freshen the body, to make the body feel warm, to help expel wind from the body and to smoothen the skin. the applications on the skin could be supported by the fact that essential oil of z. cassumunar is active against dermatological infections caused by bacteria, dermatophytes and yeasts [26] and c. citratus oil has potent in vitro antifungal effect against candida spp. [27] in addition, a. galanga extract has protective effects on uva-dependent melanogenesis. [28] in this study, it is found that fresh herbs (table 1: u9-u10) are consumed, for example, leaf of centella asiatica (l.) urb. for stimulation of breast milk and rhizome of z. offcinale for warming of the body. although there is no scientifc evidence that c. asiatica is galactagogue, interestingly its water extract has been shown to enhance learning and memory of mice during the postnatal developmental stage. [29] additionally, the methanolic extract of c. asiatica has antioxidant activity [30] and the asiaticoside has wound healing effects. [31] one of the practitioners also mentioned the use of p. cubeba juice eye drop to improve poor eyesight of a mother after childbirth. the use of herbal eye drop in postnatal treatment is not a common practice. however, methanolic extract of p. cubeba has been shown to exhibit anti-infammatory activity. [32] most of the medicinal plants used in the preparations obtained from the study are believed to be “heaty” components so as to complement the “cold” condition after childbirth. this probably explains why most preparations are meant to improve blood circulation, to make the body feels warm, to expel wind, to ease abdominal discomfort and cramps, to ease muscular and joint pain, to stimulate lactation and to act as a laxative and a contraceptive. in fact, the external preparations utilise mainly aromatic medicinal plants, especially the zingiberaceae species. in aromatherapy, essential oils of c. zeylanicum, e. aromatica and z. offcinale have been used to ease emotional and mental fatigue. thus, the use of these aromatic species could help the new mothers to reduce anxiety, reduce stress and improve mood after childbirth. [33] medicinal plants used for postnatal care in malay traditional medicine in this study, as many as 52 species of medicinal plants from 42 genera are found to be used in the 23 post-partum traditional preparations. these species belong to 27 families, that is, zingiberaceae (10 species), apiaceae (5), fabaceae (4), piperaceae (4), poaceae (4), apocynaceae (2), lauraceae (2), myrtaceae (2), acoraceae (1), asteraceae (1), bignonaceae (1), bromeliaceae (1), caricaceae (1), connaraceae (1), elaeocarpaceae (1), fagaceae (1) illiciaceae (1), lamiaceae (1), liliaceae (1), musacea (1) pandanaceae (1), parmeliaceae (1) polygonaceae (1), ranunculaceae (1), rutaceae (1), simaroubaceae (1) and solanaceae (1) (table 2). some medicinal plants are found to be commonly utilised in the preparations such as curcuma longa l. (7 preparations), z. offcinale (7), c. zeylanicum (6), k. galanga (6), p. cubeba (6), z. cassumunar (6), acorus calamus l. (5), piper nigrum beyr. ex kunth (5), alyxia stellata roem. & schult. (4), c. sativum (4), foeniculum vulgare mill. (4), nigella sativa l. (4) and u. barbata (4). it is anticipated that malay traditional medicine practitioners make use of the easily accessible and inexpensive herbs and spices such as turmeric (c. longa), ginger (z. offcinale), aromatic ginger (k. galanga), cassumunar (z. cassumunar), cinnamon (c. zeylanicum), white and black pepper (p. nigrum), coriander (c. sativum) and sweet fennel (f. vulgare) in their preparations. these can be either collected from their home gardens or the neighbouring forests, or purchased from retail stores or local herbal suppliers. however, the use of black seed or black cumin (n. sativa) that is not grown locally is rather surprising but could suggest the infuence of prophetic medicine in the malay traditional medicine because of the belief that black cumin is a remedy for every disease except death. [34] besides, several pharmacological properties of n. sativa have been reported including cns-depressant, [35] anti-infammatory and analgesic, [36] hypotensive, anti-nociceptive, uricosuric, choleretic, anti-fertility, anti-diabetic and anti-histaminic, [37] as well as anti-oxidant, anti-microbial, antitumor and immunomodulatory properties. [38] parts of the medicinal plants frequently utilised in the preparations are rhizomes (25%), fruits/berries (22%) and leaves (19%); followed by seeds (9%), barks (9%), fowers (5%), roots (5%), whole plant (3%), gall (2%) and bulb (1%) (figure 2). most of the preparations use rhizomes of the zingiberaceae species; whereas the leaves are mainly used in the bathing preparations. it is also interesting to learn that different parts of a plant are used for different preparations and purposes. for example, the z. cassumunar rhizome is used as internal jamu preparation whereas the leaves are used in the external herbal bath. frequency of the use of plant parts found in this study is different from the previously reported ethnobotanical studies, wherebypharmacognosy journal  august 2011  vol 3  issue 24 23 jamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia roots and leaves are the most commonly utilised plant parts in the remedies. [39,40] this may exemplify the diversity of traditional practices based on cultural differences. conclusion in conclusion, the study has successfully gathered information on the 23 preparations containing 128 medicinal plants used for postnatal care in the malay traditional medicine at muar, johor and kuala pilah, negeri sembilan. diverse types of species and parts of plants were used by different practitioners. these are traditionally prepared in various forms, either freshly prepared or dried; powdered or extracted; in a single or compound composition and either taken internally or used externally. similar studies should be carried out elsewhere in order to compile more data on the use of medicinal plants in the malay traditional medicine. acknowledgements the authors would like to thank the malay traditional practitioners who were willing to contribute to the study and to the development of knowledge on medicinal plants of malaysia. references 1. zakaria m, mohd ma. traditional malay medicinal plants. kuala lumpur: penerbit fajar bakti sdn. bhd.; 1994. 2. salleh h. perubatan tradisi malaysia dari aspek sosio-antropology. in: soepadmo e, goh sh, wong wh, din l, chuah ch, editors. malaysian traditional medicine, kuala lumpur. kuala lumpur: institute of advanced studies, university of malaya and malaysian institute of chemistry; 1988; p. 281. 3. kasimin a.asas dan falsafah perubatan melayu tradisional dari perspektif islam. in: din l, sulaiman na, hussein fn, mohd-riji h, mohd-anuar h, editors. proceedings: national workshop on the integration of traditional medicine in primary health care. kuala lumpur: (publisher unknown); 1990; p. 20-23. 4. rahman haa. asas dan falsafah perubatan melayu. in: din l, sulaiman na, hussein fn, mohd-riji h, mohd-anuar h, editors. proceedings: national workshop on the integration of traditional medicine in primary health care. kuala lumpur: (publisher unknown); 1990; p. 13-16. 5. jamal ja. malay traditional medicine: an overview of scientific and technological advancement. asia pacific tech monitor. 2006; 23(6):37-49. 6. supathan r. the role of the malay traditional medicine. in: shaari k,­ abd-kadir a, mohd-ali, ar, editors. medicinal products fromtropical rain forest. kuala lumpur: forest research institute of malaysia; 1992; p. 57-62. 7. ridley hn. materials for a flora of the malayan peninsula. singapore: methodist publishing house; 1907. 8. gimlette jd. some superstitious beliefs occurring in the theory and practice of malay medicine. (publisher unknown); 1913. 9. gimlette jd.malay poisons and charm cures.newyork:oxford university press; 1971. 10. gimlette jd, burkill ih.the medical book of malayan medicine. singapore: botanic gardens; 1930. 11. gimlette jd, thomson hw. a dictionary of malay medicine. london: oxford university press; 1939. 12. burkill ih. a dictionary of the economic products of the malay peninsula. london: crown agents; 1935. 13. burkill i h. a dictionary of the economic products of the malay peninsula. kuala lumpur: ministry of agriculture and co-operatives; 1966. 14. mat-salleh k, latiff a. tumbuhan ubatan malaysia. selangor: pusat pengurusan penyelidikan, universiti kebangsaan malaysia; 2002. 15. dr. duke’s phytochemical and ethnobotanical databases. (http://www.ars-grin.gov/duke) 16. xia x, cheng g, pana y, xia zh, kong ld. behavioral, neurochemical and neuroendocrine effects of the ethanolic extract from curcuma longa l. in the mouse forced swimming test. journal of ethnopharmacology. 2007; 110:356-363. 17. adaramoye oa, anjos rm, almeida mm, veras rc, silvia df, oliveira fa, et al. hypotensive and endothelium-independent vasorelaxant effects of methanolic extract from curcuma longa l. in rats. journal of ethnopharmacology. 2009; 124:457-462. figure 2: frequency of plant parts used in the preparations of the malay traditional medicine for postnatal carejamal, et al.: medicinal plants used for postnatal care in malay traditional medicine in the peninsular malaysia 24 pharmacognosy journal  august 2011  vol 3  issue 24 18. aggarwal bb, harikumar kb. potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. the international journal of biochemistry and cell biology. 2009; 41:40-59. 19. lee hs. antiplatelet property of curcuma longa l. rhizome-derived ar‑turmerone. bioresource technology. 2006; 97:1372-1376. 20. yue ggl, chan bcl, hon pm, lee myh, fung kp, leung pc, et al. evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from curcuma longa. food chemistry andtoxicology. 2010; 48:2011-2020. 21. goyal rk, kadnur sv. beneficial effects of zingiber officinale on goldthioglucose induced obesity. fitoterapia. 2006; 77:160-163. 22. roosita k, kusharto cm, sekiyama m, fachrurozi y, ohtsuka r. medicinal plants used by the villagers of a sundanese community in west java, indonesia. journal of ethnopharmacology. 2008; 115:72-81. 23. tao g, irie y, li dj, keung wm. eugenol and its structural analogs inhibit monoamine oxidase a and exhibit antidepressant-like activity. bioorganic and medicinal chemistry. 2005; 13:4777-4788. 24. ridtitid w, sae-wonga c, reanmongkol w, wongnawa m. antinociceptive activity of the methanolic extract of kaempferia galanga linn. in experimental animals. journal of ethnopharmacology. 2008; 118:225-230. 25. huang l, yagura t, chen s. sedative activity of hexane extract of keampferia galanga l. and its active compounds. journal of ethnopharmacology. 2008; 120:123-125. 26. pithayanukul p, tubprasert j, wuthi-udomlert m. in vitro antimicrobial activity of zingiber cassumunar (plai) oil and a 5%plai oil gel.phytotherapy research. 2007; 21(2):164-169. 27. silva cde b, guterres ss,weisheimer v, schapoval ee. antifungal activity of the lemongrass oil and citral against candida spp. brazilian journal of infectious diseases. 2008; 12(1):63-66. 28. panich u, kongtaphan k, onkoksoong t, jaemsak k, phadungrakwittaya r,thaworn a, et al. modulation of antioxidant defense by alpinia galanga and curcuma aromatica extracts correlates with their inhibition of uva-induced melanogenesis. cell biology andtoxicology. 2009; 26(2):103-116. 29. rao sb, chetana m, uma devi p. centella asiatica treatment during postnatal period enhances learning and memory in mice. physiology and behaviour 2005; 86:449-457. 30. zainol mk., abd-hamid a,yusof s, muse r. antioxidative activity and total phenolic compounds of leaf, root and petiole of four accessions of centella asiatica (l.) urban. food chemistry. 2003; 81:575-581. 31. shukla a, rasik am, jain gk, shankar r, kulshrestha dk, dhawan bn. in vitro and in vivo wound healing activity of asiaticoside isolated from centella asiatica. journal of ethnopharmacology. 1999; 65:1-11. 32. choi em, hwang jk. investigations of anti-inflammatory and antinociceptive activities of piper cubeba, physalis angulata and rosa hybrid. journal of ethnopharmacology. 2003; 89:171-175. 33. louis m, kowalski sd. use of aromatherapy with hospice patients to decrease pain, anxiety, and depression and to promote an increased sense of well-being. american journal of hospital palliative care. 2002; 19:381-386. 34. sahih bukhari.translation of sahih bukhari. volume 7, book 71, number 591. (http://www.iium.edu.my/deed/hadith/bukhari/071_sbt.html) 35. al-naggar tb, gómez-serranillos mp, carretero me, villar am. neuropharmacological activity of nigella sativa l. extracts. journal of ethnopharmacology. 2003; 88:63-68. 36. al-ghamdi ms.the anti-inflammatory, analgesic and antipyretic activity of nigella sativa. journal of ethnopharmacology. 2001; 76:45-48. 37. ali bh, blunden g. pharmacological and toxicological properties of nigella sativa. phytotherapy research. 2003; 17:299-305. 38. salem ml. immunomodulatory and therapeutic properties of the nigella sativa l. seed. international immunopharmacology. 2005; 5:1749-1770. 39. ragunathan m, solomon m. the study of spiritual remedies in orthodox rural churches and traditional medicinal practice in gondar zuria district, northwestern ethiopia. pharmacognosy journal. 2009; 1(3): 178-183. 40. afolayan aj, mbaebie bo. ethnobotanical study of medicinal plants used as anti-obesity remedies in nkonkobe municipality of south africa. pharmacognosy journal. 2010; 2(11):368-373.pharmacognosy journal  august 2011  vol 3  issue 24 25 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: dr. m. amzad hossain, natural product chemistry laboratory, biotechnology research institute, universiti malaysia sabah, locked bag no. 2073, 88400, kotakinabalu, sabah, malaysia. tele: 610109571462; fax: 61088320993 23 e-mail: dramzadh@gmail.com doi: 10.5530/pj.2011.24.5 peoples’ traditional medicine and a common element in ayurvedic, homeopathic, naturopathic, traditional oriental, and native american indian medicine. who had noted that of 119 plant-derived pharmaceutical medicines, about 74% are used in modern medicine in ways that correlated directly with their traditional uses as plant medicines by native cultures. the major pharmaceutical companies in the world are presently conducting extensive research on plant materials which are gathered from the rain forests and other places for their potential medicinal value. today, the u.s. pharmacopoeia, with its reliance on herbal compounds, has been all but forgotten. the physician’s desk reference, an extensive listing of chemically manufactured drugs is where most of the modern physicians rely on. it is important to note that each entry in this enormous volume, in addition to specifying the chemical compound and actions of a particular drug, also includes an extensive list of contraindications and possible side effects. rather than using a whole plant as different types of organic extracts, pharmacologists identify, isolate, extract, and synthesize individual components, thus capturing the active properties. biochemical profiling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth m. amzad hossain* a , mohan raj nagooru b a natural product chemistry laboratory, biotechnology research institute, universiti malaysia sabah, locked bag no. 2073, 88400 kota kinabalu, sabah, malaysia. b biotechnology, school of science and technology, universiti malaysia sabah, locked bag no. 2073, 88999 kota kinabalu, sabah, malaysia a b s t r a c t now a day the importance of medicinal plants has been increasing both for pharmaceutical industry and traditional users. most of the countries either it is developing and under developing mostly rely on traditional medicines. this herbal or traditional medicine involves the use of different type organic extracts or the bioactive pure chemical constituents. this type of biochemical investigation provides the health application at minimum cost. this survey such as ethnomedicine keenly represents one of the best avenues in searching new economic plants for medicine. keeping this view in mind, the present study is carried out in corydyline terminalis l. kunth leaves collected from rainforest area at sabah state, malaysia. this plant has several benefcial properties. the powder leaves of corydyline terminalis l. kunth was extracted with organic solvent such as hexane, ethyl acetate, chloroform, butanol and methanol. the total phenolic contents of the extracts was determined by willet method with modifcation. the results for total favonoids content of the extracts as caffeic acid equivalents were found to be highest in hexane extract (68.02%) followed by ethyl acetate (61.50%), methanol (39.27%), butanol (19.08%) and chloroform (15.75%). based on these results it can be suggested that the biochemical properties of the leaves for curing various ailments. key words: biochemical screening, flavonoids content, soxhlet extraction, organic extracts, corydyline terminalis l. kunth. introduction ancient man is well known for their knowledge of utilizing vast variety of drugs for millennia. the crude form of many traditionally used herbs and plants are known to be very useful based on current knowledge. there are at least 121 chemical substances of known structure are still extracted from plants that are useful as drugs around the globe. [1] the world health organization (who) estimates that more than 4 billion people, 80% of the total world population, presently medication herbal medicine for some aspect of primary health care. in the third world countries, herbal medicine is a major component or ingredient in all indigenoushossain, et al.: biochemical profiling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth 26 pharmacognosy journal  august 2011  vol 3  issue 24 the plants samples such as favonoids, isofavones, favones, anthocyanins, catechins and other phenolics. [9-11] various curable and incurable diseases has been linked to the oxidative stress, [9-11] but the food industry has been concerned with issues such as rancidity and oxidative spoilage of foodstuffs. [12] the oxidation for enzyme as well as auto oxidation of amino acid or lipids during storage and processing is the major reaction responsible for the deterioration in food quality affecting the colour, favour, texture and nutritive value of the foods. they are oftenly added antioxidants to the foods to prevent the radical chain reactions of oxidation by inhibiting the initiation and propagation step leading to the termination of the reaction and a delay in the oxidation process. corydyline terminalis l.kunth, asparagaceae (monocotyledonous fowering plant) is one of the popular traditional plant used extensively as remedy in southeast asia for the treatment of wide range of diseases. cordylines are known to the world by many names and are crowned as “king of tropical foliage”. corydyline terminalis l. kunth is a shrub plant, with woody stem that can reach a height of 6 metres. this plant which is native to tropical asia, australia and new zealand does not have many branches and its roots can swell to form starch extract, usually occurs in root plants. the name cordyline comes from the greek word kordyle, meaning “club,” a reference to the enlarged underground stems or rhizomes. [13] the leaves are simple and in an arrangement of twisted leaves, long, oval or in shape of spear as long as 45 cm and between 7.5-15 cm in width. the stalk consists of long and fat leaves. consisting of leaves of various colour; red, pink, purplish strips and also with borders of whitish leaves it is reaching high popularity among gardeners, landscapers and collectors alike. the fowers grow in a bunch which is in medium size up to 70 cm long, yellow or purple in colour. meanwhile the fruits form in ball shapes, reddish in colour with diameter of 2 cm. in malaysia, especially sabah, kadazandusun’s ethnic use corydyline terminalis l. kunth as medicinal use for curing cough, bloody cough, dysentery, high fever, diffculties in urine, bloody urine, kidney diseases, tibiae, headache and infammation in the digestive tract. [14] furthermore, it is also can also be used to heal scurf and joint pain. [14] people also used corydyline terminalis l. kunth as popular ornamental plant, with numerous cultivars available, many of them selected for green or reddish or purple foliage despite its variety of medicinal uses. this species is usually planted by the stem or leafy shoots. some of the villagers plant it by a local method name ’tut’. it is easily planted where the stem part easily grows into a new plant. it is convenient to be planted in pots and on the soil individually, in rows or in groups. this is the frst time a research work is being carried out on this tropical traditional medicinal plant. during this can create problems, however, in addition to active ingredients, plants contain minerals, vitamins, volatile oils, glycosides, alkaloids, biofavanoids, and other substances that are important in supporting a particular herb’s medicinal properties. these elements also provide an important natural safeguard isolated or synthesized active compounds can become toxic in relatively small doses; it usually takes a much greater amount of a whole herb, with all of its components, to reach a toxic level. herbs as traditional medicine can have powerful effects. they should not be taken lightly. now a day the suggestions to practitioner for herbal treatments in this book are not intended to substitute for consultation with a qualifed health care, but rather to support and assist you in understanding and working with your physician’s advice. chemical substances derived from the plants remain the basis for a large proportion of the commercial medications used today for the treatment of heart disease, high blood pressure, pain, asthma, and other problems. some medicinal herb used in ‘traditional chinese medicine’ for more than two thousand years to treat asthma and other respiratory problems. ephedrine, the active ingredient in ephedra, is used in the commercial pharmaceutical preparations for the relief of asthma symptoms and other respiratory problems. it helps the patient to breathe more easily. there are so many groups or families of phytochemicals and they help or accelerate to the human body in several ways. chemical constituents inside the plants may be protecting human body from a host of diseases. chemical constituents are non-nutritive plant bioactive chemicals that have protective or disease preventive properties. plant produces itself these bioactive chemicals to protect itself but recent research demonstrates that many chemical constituents can protect humans against diseases. there are so many groups of bioactive chemicals in fruits, vegetables and herbs and each works differently. so many possible ways to fght or kill these diseases is to improve our body’s antioxidant defenses. comparatively high consumption of vegetables and fruits has been associated with a lowered incidence of such degenerative and incurable diseases. [2] fruits constituents also help to improve health in other ways. due to active chemical constituent of fruit juice, can also be taken to alleviate sore throat and seasickness. the functional bioactivity of a plant organic extract, in general, depends upon the presence of compounds such as polyphenols, carotenoids, terpenoids and chlorophyll. [3] plants also can contribute in this area primarily due to the antioxidant activity of phenolic and favonoids compounds. [4-8] by the researcher several studies have done and revealed that the antioxidant capacity may be from compounds insidepharmacognosy journal  august 2011  vol 3  issue 24 27 hossain, et al.: biochemical profiling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth methanol fractions (7 g), respectively. the extract was ltered through whatman no. 41 lter paper to obtained particle free crude extract. the residue was re extracted twice follow the same procedure and ltered. the combined extracts were concentrated and dried under vacuum. determination of total flavonoids the total favonoids content of the crude extracts of corydyline terminalis l. kunth were determined by using colorimetric method as described by willet, [15-17] with some modifcations. methanol extracts (0.5 ml), 10% aluminium chloride (0.1ml), 1mpotassium acetate (0.1ml) and distilled water (4.3 ml) were mixed. at room temperature the mixture samples was incubation for 30 min. the absorbance of the crude extracts was measured at 415 nm using spectro (thermo fisher scientifc, model 4001/4) spectrophotometer. quercetin was used to make the calibration curve. the calculation of total favonoids content in the extracts was carried out in triplicate and the results were averaged. preliminary phytochemicals screening one gram of hexane, ethyl acetate, chloroform, butanol and aqueous methanol crude plant extracts of the powder leaves of corydyline terminalis l. kunth were dissolved in 100 ml of its own mother solvents to obtain a stock of concentration 1% (v/v). the obtained crude extracts were subjected to preliminary biochemical screening following the methodology of hatano [18] and birt. [19] screening procedure test for alkaloids the stock crude extract (5 ml) was added hydrochloric acid (2 ml). one milliliter of dragendroff ’s reagent was added to this acidic medium. an orange or red precipitation immediately produced that indicates the presence of alkaloids. test for amino acids the crude stock extract solution (1 ml) was added few drops of ninhydrin reagent. the purple colour appearance shows the presence of amino acids. test for anthraquinones the crude stock extract solution (5 ml) was hydrolysed by diluted concentrated sulphuric acid extracted with benzene. finally dilute ammonia solution was added to it. the rose pink coloration obtained. it is suggested that the positive response for anthraquinones. test for flavonoids the crude stock extract solution (1 ml) and a few drops of dilute sodium hydroxide were added. an intense yellow colour was appearance in the plant crude extract, which our study on the biologically active constituents of this plant, we examined the biochemical screening of the leaves of corydyline terminalis l. kunth widely used in sabah community, malaysia. hence, the aim of this present study has been made to investigate the biochemical screening and total favonoids contents of the leaves crude extracts of corydyline terminalis l. kunth. materials and methods general all the solvent used in this experiment were of analytical grade or gc grade. uv-visible spectra measurements were done using spectro (thermo fisher scientifc, model 4001/4) spectrophotometer ultrospeck inmethanol (λmax in nm). the water was purifed from water distillation plants in our laboratory. all solvents were analytical reagent grade. plant material the fresh green leaves of corydyline terminalis l. kunth were collected from the rainforest hilly area of southern part of sabah, malaysia. the leaves of this plant were harvested during the month of may, 2011. the leaves were collected at afternoon on may 21, 2011 and packed in polyethylene bags and bring to the laboratory and stored at 4c until required. plant material grinding the samples were washed thoroughly with fresh running water, dried under shade room temperature (25 1 c) for 3 days. the plant initially identifed by morphological features and data base present in the library, institute for tropical biology and conservation, universiti malaysia sabah, sabah, malaysia and a voucher specimen has been deposited at the borneo herbarium, sabah, malaysia with voucher number 031. approximately 50 g of leaves were ground using a grinder (blender 80115) for 20 s. the unfermented corydyline terminalis l. kunth leaves were kept in the oven at 40 c and put in a desiccator for at least 24 h prior to analysis. the small pieces of the samples were homogenised in a grinder for 3 min to 40-mesh size. the air-dried leaves and stems of corydyline terminalis l. kunth were pulverized into powdered form. extraction coarse powders (500 g) were separately soxhlet extracted with methanol solvent (1 l, 72 h) at the temperature between 45-65 c. the viscous semi solid masses were dried at 40 c in rotary evaporator frozen and lyophilized (buchi labortech ag, model 1, r-215). the methanol extract was (30 g) suspended in water and then defatted and fnally extracted successively with hexane, chloroform, ethyl acetate and butanol to give hexane (5.03 g), ethyl acetate (4.67 g), chloroform (3.39 g), and butanol (2.58 g) and residualhossain, et al.: biochemical profiling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth 28 pharmacognosy journal  august 2011  vol 3  issue 24 the correlation is obtained in bivariate correlations. p values 0.05 were regarded signifcant. results and discussion the percentage yields of extraction of the leaves powder of corydyline terminalis l. kunth were hexane (3.19 g), ethyl acetate (16.31 g), chloroform (1.59 g), and butanol (9.50 g) and residual methanol fractions (6.34 g), respectively. the total favonoids contents of the different organic crude plant extracts were determined bywillet method are reported as quercetin equivalents (table 1). among the extracts, hexane extract was containing highest (68.02%) amount of favonoids content compounds followed by ethyl acetate (61.50%), methanol (39.27%), butanol (19.08%) and chloroform (15.75%) (table 1). in our previous several studies, it has been reported that the yield of extractable compounds was highest in methanol extract from the leaves, peel and seeds of pomegranate in comparison with the solvents such as chloroform, butanol, ethyl acetate and hexane. furthermore, the extraction of favonoids contents from the leaves, peel and seeds is commonly achieved with methanol or aqueous ethanol. the result obtained in the present investigation (table 2), the ethyl acetate, chloroform, butanol, aqueous ethanol and methanol extracts of the leaves powder of corydyline terminalis l. kunth showed the presence of alkaloids, amino acids, favonoids, glycosides, saponins, steroids, tannins and triterpenoids. further, all the organic extracts of the leaves showed the absence of anthraquinones. in the hexane extract, all the group alkaloids, amino acids, favonoids, glycosides, saponins and triterpenoids was absence except triterpenoids and steroids. . all the herbs and herbal extracts contain different chemical constituents with biological activity that can be show of valuable therapeutic and medicinal index. most of the protective effect of medicinal plants, fruits and vegetables has been attributed by active biochemicals, which are the non-nutrient plant compounds. different type’s active biochemicals have been found to possess a wide range of activities, which may help in protection against uncureable become colourless on addition of a few drops of dilute acid indicates the presence of favonoids. test for glycosides the crude extract was hydrolysed by hydrochloric acid for few hours on a water bath. pyridine (1 ml) was added to the hydrolysate and a few drops of sodium nitroprusside solutions were added to it and then it was made alkaline with sodium hydroxide solution. the pink to red colour obtained shows the presence of glycosides. test for phytosterol the plant crude extract solution was refuxed with solution of alcoholic potassium hydroxide till to complete saponifcation takes place. the whole mixture was diluted with water and then extracted with ether. the ether layer was evaporated by water bath and the residue was tested for the presence of phytosterol. the residue was dissolved with few drops of diluted acetic acid then 3 ml of acetic anhydride was added followed by few drops of conc. h 2 so4 . the bluish green colour was appearance showed the presence of phytosterol. test for saponins the crude extract stock solution (1 ml) was diluted with 20 ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. the formation of 1 cm foam layer showed the presence of saponins. test for steroids the crude plant extracts solution (1 ml) was dissolved in chloroform (10 ml) and added equal volume of concentrated sulphuric acid by sides of the test tube. the upper layer turns into red and sulphuric acid layer showed yellow with green fuorescence. this indicated the presence of steroids. test for tannins the crude extract solution (3 ml) and a few drops of 1% lead acetate were added. a yellow precipitate was formed, indicates the presence of tannins. test for triterpenoids the dry crude plant extract (5 mg) was dissolved in chloroform (2 ml) and then acetic anhydride (1 ml) was added following the addition of 1 ml of conc. h 2 so 4 . formation of reddish violet colour indicates the presence of triterpenoids. statistical analyses experimental results were mean s.d. of three parallel measurements and analyzed by spss 10 (spss inc. chicago, il). differences between means were determined using tukey multiple comparisons and least signifcant difference (lsd). by using the pearson correlation coeffcient table 1: total flavonoids content extracts of the leaves of corydyline terminalis extract total flavonoids (% w/w) hexane extract 68.02 2.15 ethyl extract chloroform extract butanol extract 61.50 1.51 15.75 0.06 19.08 1.92 methanol extract 39.27 1.72 the values are means sd of three replicates.pharmacognosy journal  august 2011  vol 3  issue 24 29 hossain, et al.: biochemical profiling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth acknowledgements the authors are grateful to associate prof. dr. vijay kumar, actingdirector, biotechnology research institute, university malaysia sabah, malaysia for their continuous encouragement during the work and the use of all laboratory facilities. we are also thanks to biotechnology research institute, university malaysia sabah, malaysia for providing the seed money. thanks are also due to mr. emran raga, laboratory assistant, biotechnology research institute, university malaysia sabah, malaysia for his help to assist our entire work. references 1. alothman m., bhat r., karim a. a. antioxidant capacity and phenolic content of selected tropical fruits from malaysia, extracted with different solvents. food chemistry. 2003. 115:785-788. 2. bajpai v.k., yoon j.i., kang s.c. antioxidant and antidermatophytic activities of essential oil and extracts of magnolia liliflora desr. food chemical toxicology. 2009. 47:1355-1361. 3. negi p.s., jayaprakasha g.k., jena b.s. antioxidant and antimutagenic activities of pomegranate peel extracts. food chemistry. 2002. 80:293-297. 4. allan a.r., blowers a., earle e.d. expression of a magainin-type antimicrobial peptide gene (msi-99) in tomato enhances resistance to bacterial speck disease plant cell reports. 2004. 22:388-396. 5. higdon j.v., frei b. terpenoids- chemistry, metabolism, cardioprotective effects and dietary sources. critical review food science and nutrition. 2002. 43:89-143. 6. li h.b.,wong c.c., cheng k.w., chen f.antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. lwt – food science technology. 2008. 41:385-390. 7. mhatre m., tilak-jain j., de s., devasagayam t.p.a. evalution of the antioxidant activity and non transfored and transformed pine apple. food chemical toxicology. 2009. 37:2696-2702. 8. bhattacharjee p., kshirsagar a., singhal r.s. supercritical carbon dioxide extraction of 2-acetyl-1-pyrroline from pandanus amaryllifolius roxb,food chemistry. 2005. 91:255-259. 9. abdille m.h., singh r.p., jayaprakasha g.k., jena b.s. antioxidant activity of the extracts from dillenia indica fruits. food chemistry. 2005. 90(4):891-896. 10. isabelle m., lee b.l., lim m.t., koh m.t., huang d., nam c. flavonoids- chemistry, metabolism effects and dietary sources food chemistry. 2010. 123:77-84. diseases. biochemicals and phytochemicals such as saponins, terpenoids, favonoids, tannins, steroids and alkaloids have antiinfammatory effects. [20-24] some complex glycosides, favonoids, tannins and alkaloids have also very high hypoglycemic activities. [25-26] recently by allan [4] have reported that saponins possess hypocholesterolemic and antidiabetic properties. the chemical constituents such as mono di and triterpenoids have also been shown to decrease or reduce the blood sugar level in animal studies. [27-30] almost all high molecular weight such as glycosides, steroids and triterpenoids showed the analgesic properties. [31-35] the steroids and saponins are also responsible for central nervous system activities. [9] the biochemical and phytochemicals screening of the hexane, ethyl acetate, chloroform, butanol, aqueous ethanol and methanol extracts of corydyline terminalis l. kunth powder leaves used in this present study revealed that the crude extracts contained alkaloids, amino acids, favonoids, glycosides, phytosterols, saponins, steroids, tannins and triterpenoids (table 2). this study is only a preliminary study of the occurrence of certain properties of corydyline terminalis l. kunth leaves an in-depth study will provide a good concrete base of all the biochemicals and phytochemicals functions mention above. conclusion in this present study, we have found that biologically active biochemical and phytochemicals were present in the ethyl acetate, chloroform, butanol and methanol extracts of corydyline terminalis l. kunth. the high favonoids contents of corydyline terminalis l. kunth leaves crude extracts may be due to the presence of bioactive phytochemicals. further studies are in progress in our laboratory is to isolate the bioactive components from the leaves of corydyline terminalis l. kunth. table 2: the analysis of biochemical in the hexane, ethyl acetate, chloroform, butanol and methanol crude extract of corydyline terminalis biochemicals inference hexane ethyl acetate chloroform butanol methanol alkaloids amino acids – – – anthraquinones – – – – – flavonoids glycosides – – phytosterol – – – – saponins – tannins – triterpenoids steroids presence; - absencehossain, et al.: biochemical profiling and total flavonoids contents of leaves crude extract of endemic medicinal plant corydyline terminalis l. kunth 30 pharmacognosy journal  august 2011  vol 3  issue 24 24. chung k.t., wong t.y., wei c.i., huang y.w., lin y. tannins and human health: a review. critical review food science nutrition. 1998. 38:421-433. 25. deighton n., brennan r., finn c., davies h.v. antioxidant properties of domesticated and wild rubus species. journal of science food agriculture. 2000.80:1307-131. 26. greenberg e.r., baron j.a.,tosteson t.d. cholesterol and coronary heart disease. engineering journal medicine. 1994. 331:141-150. 27. jena s., sahoo p., mohanty s., das a.b., das p. karyotype variation and cytophotometric estimation of in situ dna content in some minor and associate mangroves of india. cytologia. 2002. 67:15-24. 28. das n.p., pereira t.a. effects of flavonoids on thermal autooxidation of palm oil: structure- activity relationship. journal of americal oil chemistry society. 1990. 67:255-258. 29. halliwell b., gutteridge j.m.c. free radicals in biology and medicine. oxford university press, oxford. 2007. 30. halliwell b. antioxidant characterization methodology and mechanism. biochemical pharmacology. 1995. 49:1341-1348. 31. pandhair v., sekhon b.s. reactive oxygen species and antioxidants in plants: an overview. journal of plant biochemistry and biotechnology. 2006.15:71-77. 32. cowan m.m. plant products as antimicrobial agents. clinical microbiology review. 1999. 12(4):564-571. 33. kessler m., ubeaud g., jung l. anti- and pro-oxidant activity of rutin and quercetin derivatives. journal of pharmceutical. 2003. 55:131-142. 34. miller n.j., rice-evans c.a.the relative contributions of ascorbic acid and phenolic antioxidants to the total antioxidant activity of orange and apple. food chemistry. 1997. 60:331-337. 35. moore j., hao z., zhou k., luther m., costa j.y.l. carotenoid, tocopherol, phenolic acid, and antioxidant properties of maryland-grown soft wheat. journal of agriculture food chemistry. 2005. 53:6649-6657. 11. kahkonen m.p., hopia a. i., vuorela h.j. antioxidant activity of some medicinal plants. journal of agriculture chemistry. 1999. 47:3954-3962. 12. shahidi f., wanasundara p.k.j.p.d.) phenolic antioxidants. critical review food science and nutrition. 1992. 32:67-103. 13. bok-mun h. cordyline obtecta. australian national botanic gardens. ht tp: / /www.cpbr.gov.au/gnp/ interns-2006/cordyl ine-obtecta.html . retrieved, 2006. 2008-04-12. 14. kulip j.. an ethnobotanical servey of medicinal and other useful plants of muruts in sabah, malaysia.telopea. 2003. 10(1):81-98. 15. velioglu y.s., mazza g., gao l., oomah b.d. antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. journal of agricultural food chemistry. 1998. 46:4113-4117. 16. vinson j.a., hao y., zubic s.k. food antioxidant quantity and quality in foods: vegetables. journal of agricultural food chemistry. 1998. 46:3630-3634. 17. willett w.c. balancing life-style and genomics research for disease prevention. science. 2002. 296:695-698. 18. hatano t., kagawa h.,yasuhara t., okuda t.two new flavonoids and other constituents in licorice root: their relative astringency and radical scavenging effects. chem pharma bull. 1999. 36:1090-2097. 19. birt d.f., hendrich s., wang w.q. dietary agents in cancer prevention: flavonoids and isoflavonoids. pharmacological theraphy. 2001. 90:157-177. 20. manach c., regerat f., texier o. metabolism effects and dietary sources. nutrition research. 1996. 16:517-544. 21. liu c.s.,tsai c.s., kuo c.l.,chen h.w., lii c.k.,may.s.,wei y.h.oxidative stress-related alteration of the copy number of mitochondrial dna in human leukocytes. free radical research.2003. 37(12):1307-1317. 22. blois m.s. antioxidants determination by the use of a stable free radical. nature. 1958. 4617:1199-1200. 23. choi y., lee j. anti- and pro-oxidant activity of rutin and quercetin derivatives. food chemistry. 2009. 114:1386-1390.pharmacognosy journal  august 2011  vol 3  issue 24 31 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: ra. dickson, department of pharmacognosy, faculty of pharmacy and pharmaceutical sciences, college of health sciences, kwame nkrumah university of science and technology, kumasi-ghana. tel.: 233-(0)3220-60366 e-mail: radickson.pharm@knust.edu.gh doi: 10.5530/pj.2011.24.6 an infusion of the dried root is drunk or used as a bath for general malaise in senegal. [4] the aqueous decoction of the roots is used in traditional medicine as aphrodisiacs and the vasorelaxant properties have been reported. [5] the anti-diarrhoeal effects of the aqueous extract of the plant have also been studied. the results obtained showed that the plant possessed anti-diarrhoeal activity due to its inhibitory effects on gastrointestinal propulsion and intestinal fuid accumulation. [6] the analgesic, antipyretic and anti-infammatory effects of the aqueous extract of the plant have been also been evaluated in mice, rats and rabbits. the data obtained show that c. benthamiana root bark extract possesses analgesic and antipyretic activities but lacked anti-infammatory properties. [6] in a phytochemical investigation conducted by dickson et al., [7] the bioactivity-guided fractionation of the light petroleum extract of the root bark of the plant led to the isolation of two novel cassane diterpenoids, designated as benthaminin 1 and 2. a third compound, a deoxy form of caesaldekarin c (also pharmacognostic standardization of the leaves and root bark of caesalpinia benthamiana dickson ra 1 *, annan k 2 , komlaga, g 1 1 department of pharmacognosy, 2 department of herbal medicine, faculty of pharmacy and pharmaceutical sciences, kwame nkrumah university science & technology, kumasi a b s t r a c t caesalpinia benthamiana (baill.) herend. and zarucchi (mezoneuron benthamianum baill.) (caesalpiniaceae) has been traditionally used in management of erectile dysfunction, dysentery, urethral discharges, skin diseases and wounds. despite a long tradition of use in the treatment of various ailments, no systematic pharmacognostic standardization work has been carried out on c. benthamiana. one major obstacle in the systematic exploration of the plant may be the non-availability of authentic plant material. in the present investigation, various pharmacognostic standards for the plant have been generated including the macro and micro morphological studies, one of the who accepted parameters for identifcation of medicinal plants by way of establishing the salient diagnostic characters and constants. this was carried out on the leaves and root bark of c. benthamiana. the leaves were bipinnate, oblong, with entire margin. the apex was obtuse, and possessed a symmetric base and reticulates venation. the terminal leaves were however obovate in shape. actinocytic and paracytic types of stomata were observed. trichomes were uniseriate and unicellular and epidermal cells were found to be wavy. surface data analysis revealed the stomatal index to be 1.69% to 11.11% for the upper surface and 16.94% to 28.52% for the lower surface. vein-islet number was recorded as 12.5 to 16.5 whilst the veinlet termination number was 22.25 to 35 with palisade ratio ranging between 11.25 and 13.75. the water- soluble extractive value was 9.2% and 3.7% for the leaves and root bark respectively. whilst the alcohol-soluble extractive value for the leaves was found to be 6.7% and 2.6% for the root bark. the total ash value determinations were observed to be 5.6% for the leaves and 7.9% for the root bark. the result of this study may be useful in setting diagnostic indices for the identifcation and preparation of a monograph for the plant. key words: caesalpinia benthamiana, pharmacognostic studies, quantitative microscopy and extractive values introduction caesalpinia benthamiana (baill.)herend. andzarucchi (mezoneuron benthamianum baill.) (caesalpiniaceae), [1] is an african tropical shrub found mostly in the secondary forest zones. the roots of c. benthamiana are considered to be an effective dysentery remedy in ghana. [2] the powdered roots are used mixed with shea butter or palm kernel oil to treat skin diseases and wounds in ghana. [3] a decoction of the root, bark, and leaves is used in guinea for urethral discharges. [2]dickson, et al.: pharmacognostic standardization of the leaves and root bark of caesalpinia benthamiana 32 pharmacognosy journal  august 2011  vol 3  issue 24 microscopic evaluation leaves were cut into smaller sizes and cleared in chloral hydrate, mounted with glycerin and observed under a compound microscope. the presence /absence of the following were observed: epidermal cells, stomata (type and distribution) and epidermal hairs (types of trichomes and distribution). the transverse section through the fresh petiole of the leaf was also examined. preliminary phytochemical investigation the leaf and root bark powders of the plant were separately subjected to soxhlet extraction using 70% ethanol. chemical tests were employed in the preliminary phytochemical screening for various secondary metabolites such as tannins (ferric chloride test), cardiac glycosides (keller-killiani and kedde tests), alkaloids (mayer’s; dragendorff ’s; wagner’s and 1% picric acid reagents), saponin glycosides (frothing and haemolysis tests), anthracene derivatives (borntrager’s test for combined and free anthraquinones) and cyanogenetic glycosides (sodium picrate paper test). [10,11,12,13] quantitative investigations quantitative leaf microscopy to determine palisade ratio, stomata number, stomata index, vein- islet number and veinlet termination number were carried out on cleared sections of the leave. other parameters determined for the powdered leaves were moisture content, total ash, acid - insoluble ash, water - soluble ash, alcohol (90% ethanol) and water soluble extractive values. [14] the moisture content of the powdered leaves and root bark of the plant was also determined using the dean-stark apparatus. [9] determination of total ash 2 g of the powdered leaves and root bark of c. benthamiana were weighed separately in a pre-weighed ash-less flter paper and incinerated at 400c for about 3-4 min or until the vapours completely ceased. the temperature was gradually reduced to come to normal and then the ash was collected and weighed. determination of alcohol soluble extractive accurately weighed powder (10 g) of both leaves and root bark were taken separately and macerated with 100 ml of 95% alcohol for 24 hours. the contents were frequently shaken during the frst 6 hours and allowed to remain for another 18 hours. after 24 hours, the extract was fltered and 20 ml of the fltrate was evaporated to dryness. the extract was dried at 105c to a constant weight. determination of water soluble extractive water soluble extractive value was determined using the procedure described for alcohol soluble extractive, except that chloroform water was used for maceration in this instance. referred to as methyl vouacapenate) which had previously been isolated from caesalpinia major, caesalpinia bonducella, vouacapoua americana and vouacapoua macropetala, was also isolated, together with beta-sitosterol and stigmastenone. the antibacterial and antioxidant activities of these cassane diterpenoids have also been assessed. [7] the resistance modifying activities of extracts from this plant on standard antibiotics against staphylococcus aureus have also been assessed by the same group of researchers. a 4-fold potentiation of the activity of norfoxacin was observed for the ethanol extract, whilst the petroleum spirit extract resulted in a 2-fold potentiation. [8] despite a long tradition of use in the treatment of various ailments, c. benthamiana has not been explored properly by way of establishment of standards in the identifcation and quality control of this plant. the cassane-type diterpenoids possessing antimicrobial, antioxidant and wound healing properties isolated from the plant could serve as leads in the search for new biomolecules as drugs. the need to standardize this plant can therefore not be overemphasized. it is also worthwhile to note that some drugs of plant origin in conventional medical practice are not pure compounds but direct extracts or plant materials that have been prepared appropriately and standardized. the use of artemisia annua, digitalis and senna leaves are a few examples. the establishment of the pharmacognostic profle of the leaves and root bark of c. benthamiana will assist in standardization, which can guarantee quality, purity and identifcation of samples to ensure that only the authentic plant is explored properly for its traditional claims. materials and methods the fresh leaves and roots of the plant was collected from the ayeduase in the ashanti region of ghana and authenticated by mr. henry sam of the department of herbal medicine, kwame nkrumah university of science and technology, kumasi, ghana, where voucher specimens were deposited with the numbers radcb 01 and radcb11 respectively for the leaves and roots of the plant. organoleptic evaluation in the organoleptic evaluation, various sensory parameters of the plant material such as colour, odour, taste and texture were investigated. [9] macroscopic evaluation the following macroscopic characters for the fresh leaves were noted: the type of leaves, its arrangement, colour, shape, length and width of the leaves, size and surfaces, venation, presence or absence of petiole, the apex, margin, base, lamina, texture. [9,10]pharmacognosy journal  august 2011  vol 3  issue 24 33 dickson, et al.: pharmacognostic standardization of the leaves and root bark of caesalpinia benthamiana discussion the standardization of a crude drug is an integral part of establishing its correct identity. [17] caesalpinia benthamiana is employed in ethnomedicine in the management of various disease states without standardization. the quantitative determination of some pharmacognostic parameters is useful for setting standards for crude drugs. [18] the vein islet, and vein termination numbers and the other parameters determined in the quantitative microscopy, are relatively constant for plants and can be used to differentiate between closely related species. [19] also, the physical constant evaluation of the crude drugs is an important parameter which is a valuable tool in detecting adulteration or improper handling of crude drugs. the moisture content of the powered drugs may be said to be high since the bp stipulates an allowable value of not more than 10%. any value beyond this encourages microbial growth and subsequent deterioration of the stored powdered drugs. equally important in the evaluation of crude drugs, is the ash value and acid-insoluble ash value determination. the total ash is particularly signifcant in the evaluation of purity of the crude drugs, i.e. the presence or absence of foreign inorganic matter such as metallic salts and/ or silica. [20] the macro - and micro – morphological features of the leaf described, distinguishes it from other members of the genera. for example whereas the leaves of caesalpinia spinosa lack petioles those of caesalpinia benthamiana are petiolated. [15] the length of the leaves of caesalpinia benthamiana falls between 33 to 38 cm which is within the range for those of caesalpinia pulcherrima which are also between 20 cm and 40 cm but the leaves of caesalpinia gilliesii are shorter, falling within the range of 10 cm and 15 cm. [16] by and large, the pharmacognostic constants including extractive values, ash values and the phytochemical profle of caesalpinea benthamiana obtained for the leaves and root bark of this plant, the diagnostic microscopic features and the numerical standards reported in this work could be useful for the compilation of a suitable monograph for the proper identifcation and use of this plant. conclusion these parameters which are being reported for the frst time, could serve as useful information in preparing a monograph of the plant which can be locally incorporated into the ghana herbal pharmacopoeia which may ultimately serve as a signifcant addition to international herbal pharmacopoeias. any crude drug which is claimed to be results macroscopically, the plant possess compound leaves which are bipinnate, alternate in arrangement, apex and base are obtuse, margin is entire, venation is reticulate, shape oblong with terminal leaves being obovate. full length of leaves 33-38 cm, six (6) pairs of pinnae, with length of each pinnae being 6.5-7.5 cm and four (4) pairs of leafets on each pinnae. the average size of individual leaf is 2.0-5.0 cm 0.5 (length) and 1.5-3 cm 0.3 (width). fresh leaves are green in colour, odourless with a gritty texture. it is petiolated with the length of petiole between 4-5 cm, shape of petiole cylindrical and brownish-green in colour. the leave arrangement may be similar to that of c. spinosa but whereas the leaves of c. spinosa lack petioles those of c. benthamiana are petiolated. [15] the length of the leaves of c. benthamiana falls between 33 to 38 cm which is within those of c. pulcherrima which are also between 20 cm and 40 cm but are longer than those of c. gilliesii which falls between 10 cm and 15 cm long. [16] the leaves of c. benthamiana have two types of stomatal arrangements and these are the actinocytic and paracytic types. the types of trichomes observed were uniseriate clothing hairs and unicellular hairs. the epidermal cells were found to be wavy. micromorphological features revealed that anticlinal walls are thin and wavy. the type of stomata revealed actinocytic arrangement and these were few as compared to the paracytic ones which were more. uniseriate covering trichomes are present on both surfaces. it has isobilateral leaf arrangement. the midrib bundle is surrounded by a zone of pericyclic fbres possessing double layered parenchymatous cells. the roots are brown in colour and the texture is gritty. vein islet number was determined to be 12.5 to 16.5, veinlet termination number ranging from 22.5 to 35, stomatal index of 1.69% to 11.11% for the upper surface and 16.94% to 28.52% for the lower surface and palisade ratio of 11.25 to 13.75. water- soluble extractive values were 9.2% and 3.7% for the leaves and roots respectively. the alcohol-soluble extractive value of 6.7%was obtained for the leaves and 2.6% for the roots. also, the total ash values for both leaves and roots were 5.6% and 7.9%. phytochemical evaluation revealed the presence of true tannins, pseudotannins and terpenoids mainly for both leaves and roots. these secondary plant metabolites are known to possess various pharmacological effects and may be responsible for the various actions of c. benthamiana. see tables 1 to 6 numerical and quantitative values as well as morphological descriptions.dickson, et al.: pharmacognostic standardization of the leaves and root bark of caesalpinia benthamiana 34 pharmacognosy journal  august 2011  vol 3  issue 24 9. evans wc. trease and evans pharmacognosy. wb saunders ltd. london. 2006; pp 32, 33, 95-99, 512, 547. 10. wallis, t. e. textbook of pharmacognosy. published by sk jain. 1985; 572-575. 11. brain kr, turner to. practical evaluation of phytopharmaceuticals. wright - scientechnica, bristol. 1 st ed. 1975; 144. 12. ciulei i. methodology for analysis of vegetable drugs. unido romania. 1981; 17-25. 13. harborne jb. phytochemical methods. a guide to modern technique of plant analysis. chapman and hill, london. 1992; 279. 14. british pharmacopoeia 11: ash values, acid insoluble ash, water soluble, extractive and alcohol soluble extractive. appendix xi. her majesty’s stationery office, london. 1980; a108, a113. 15. mcclintock e.the jepson manual:higher plants of california,caesalpinia. in: hickman j c. (ed.). university of california press. 1996; 57-59. 16. counter sa. “amazon mystery: a medicine man understood the secrets of this plant long before we did. how?” caesalpinia pulcherrima. the boston globe 2006; 29-30. 17. kalidass c, mohan vr. amish ad. pharmacognostic studies on capparis sepiaria (l.) r. br. pharmacognosy journal. 2009; 1(2), 121-125. 18. sikarwar ms, patil mb, kokate ck, sharma s, bhat v. pharmacognostical, phytochemical screening and acute toxicity study of crateva nurvala stem bark. pharmacognosy journal. 2009; 1 (2), 116-120. 19. soni s, kondalkar avinash,tailang,m., pathak,a. k. pharmacognostic and phytochemical investigation of stevia rebaudiana. pharmacognosy magazine, 4 (13), 89-93 (2008). 20. musa, k.y., katsayal., a.u., ahmed, a., mohammed, z. and danmalam, u h. pharmacognostic investigation of the leaves of gisekia phamacioides. african j of biotechnology, 5 (10), 956-957, (2006). caesalpinea benthamiana but whose characters signifcantly deviate from the accepted standards above may be considered to be contaminated, adulterated, substandard or fake. references 1. herendeen ps, zarucchi jl. validation of caesalpinia subgenus mezoneuron (desf.) vidal and new combinations in caesalpinia subgenus species from africa. ann. miss. bot.garden. 1990; 77:854-5. 2. irvine fr. woody plants of ghana. 1961; pp. 312-313, oxford university press, london. 3. attah y. personal communcation 2003. 4. burkill hm. the useful plants of west tropical africa. 1994; pp. 755, the trustees of the royal botanic gardens, kew, uk. 5. zamble a, martin-nizard f, sahpaz s, hennebelle t, staels b, bordet r, duriez p, brunet c, bailleul f. vasoactivity, antioxidant and aphrodisiac properties of caesalpinia benthamiana roots., j. ethnopharmacol. 2008; 116:112-119. 6. mbagwu ho, anene ra, adeyemi oo. analgesic, antipyretic and anti-inflammatory properties of mezoneuron benthamianum baill (caesalpiniaceae). nig. q. j hosp med. 2007; 17:35-41. 7. dickson ra, houghton pj, hylands pj. antibacterial and antioxidant cassane diterpenoids from caesalpinia benthamiana. phytochemistry. 2007; 68:1436-1441. 8. dickson ra,houghton pj,hylands pj.antimicrobial, resistance-modifying effects, antioxidant and free radical scavenging activities of mezoneuron benthamianum baill., securinega virosa roxb. &wlld. and microglossa pyrifolia lam. s., phytother res. 2006; 20:41-45.pharmacognosy journal  august 2011  vol 3  issue 24 35 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: pandyadevang82@yahoo.com doi: 10.5530/pj.2011.24.7 pharmacognostic, physico-chemical and phytochemical evaluation of leaves of a species of ‘paarshva-pipla’: ficus arnottiana kher mn, vaghasiya jv, desai tr, pandya dj* r. k. college of pharmacy, rajkot – 360020, gujarat, india. a b s t r a c t introduction: ‘paarshva-pipla’ is an important drugmentioned in the traditional medicinal texts. however, two completely different species are used as ‘paarshva-pipla’: thespesia populnea (malvaceae) and ficus arnottiana (moraceae). recent pharmacological fndings indicate that leaves of ficus arnottiana possess signifcant anti-ulcer activity which complies with the claims made in the traditional medicinal texts regarding ‘paarshva-pipla’. however, no conclusive pharmacognostic study of the leaves has been performed yet. methods: the present investigation deals with the qualitative and quantitative microscopic evaluation of the leaf material and establishment of its quality parameters, including physicochemical and phytochemical evaluation. results: main characters of the transverse section include presence of bi-layered palisade, cystoliths, pericyclic fbers and perimedullary phloem. diagnostic characters of powder include pericyclic fbers, cystoliths, anomocytic stomata, horse-shoe shaped xylem vessels and xylem vessels with annular thickening. phytochemical analysis showed the presence of many important classes of phytoconstituents like alkaloids, cardiac glycosides, saponins, favonoids, phenolics and carbohydrates. conclusion: such a study would help in distinguishing ficus arnottiana from thespesia populnea and make way for isolation of phytoconstituents, therapeutic investigations and standardization of formulations containing its leaf material. most importantly, it may throw light on the true botanical identity of ‘paarshva-pipla’. key words: ficus arnottiana, indian rock fg, moraceae, paarshva-pipla, urostigma arnottiana introduction ficus arnottiana miq. syn. urostigma arnottiana (family - moraceae) is also known as (english) indian rock fg, (hindi) paras pipal and (sanskrit) paarshva-pipla, plaksha. [1] its leaves are used in stomach ulcers. [2] the present investigation deals with the qualitative and quantitative microscopic evaluation of the leaf material and establishment of its quality parameters, including physicochemical and phytochemical evaluation. materials and methods collection and authentication of leaves leaves of ficus arnottiana were collected from the garden of primary health center, gadhaka near rajkot, gujarat, in july 2010. herbariums and voucher sample were prepared and deposited in department of pharmacognosy, r. k. college of pharmacy (voucher no. rkcp/cog/06/2010). authentication was done by dr. a. n. pandey, department of biosciences, saurashtra university. pharmacognostic studies morphology of fresh leaves of ficus arnottiana was studied. photomicrography of stained and unstained transverse sections of fresh leaves was performed using usb camera and software. leaf constants were established using camera lucida. the leaves were dried under shade, powdered to 60#, stored in airtight containers and used for powder study and quantitative microscopy (table 1). [3] physico-chemical evaluation various physico-chemical parameters like loss on drying, ash values (total ash, water soluble ash and acid-insoluble ash) and extractive values (water soluble, alcohol soluble and petroleum ether soluble extractives) were established using the powdered drug (table 2). [4]kher, et al.: pharmacognostic, physico-chemical and phytochemical evaluation of leaves of a species of ‘paarshva-pipla’: ficus arnottiana 36 pharmacognosy journal  august 2011  vol 3  issue 24 phytochemical study the powder was extracted separately with 500 ml each of distilled water and alcohol at 70 c for two hours each. various phytoconstituents present in the leaves were detected by their respective chemical tests using the appropriate extracts (table 3). [5-12] results and discussion pharmacognostic study macroscopical characteristics leaves are simple, 7.5-15 cm 3.2-6.3 cm, ovate-oblong or elliptic-lanceolate, apex acuminate, margin wavy, surface figure 1: leaves of ficus arnottiana (a- upper surface, b-lower surface) figure 2: a – schematic diagram (t.s.), b- detailed t. s. of leaf (u co, upper collenchyma; u ep, upper epidermis; l co, lower collenchyma; l epi, lower epidermis; pal, palisade; xyl, xylem; phl, phloem; pe fib, pericyclic fiber; sp pa, spongy parenchyma; pph, perimedullary phloem; cys, cystolith; va bun, vascular bundle) table 3: phytochemical screening phytoconstituent test result alkaloids dragendorff ’s test hager’s test wagner’s test mayer’s test ve ve ve ve flavonoids shinoda test lead acetate test ve ve phenolics ferric chloride test folin ciocalteu test ve ve sterols and triterpenoids salkowski test libermann-buchardt test ve ve cardiac glycosides legal test baljet test keller killiani test ve ve ve saponins foam test lead acetate test ve ve anthraquinone glycosides borntrager test modified borntrager test –ve –ve carbohydrates fehling’s test molisch test ve ve gums ruthenium red test ve glabrous and glaucous, texture membranous, base symmetric, venation reticulate. color of upper surface is dark green and lower surface is light green. three prominent veins seem to be arising from the base of the lamina. petiole is 8-10 cm long, angular and reddish-brown (figure 1). table 1: quantitative microscopy leaf constant mean value sd stomatal index upper surface lower surface  4.9 0.4 15.9 0.5 palisade ratio  5 1 vein islet number 12 1 vein termination number 15 1 number of observations 10 sd standard deviation table 2: physicochemical evaluation parameter mean sd loss on drying 11.5 0.3%w/w ash values total ash acid insoluble ash water soluble ash 13.1 0.3%w/w  0.9 0.1%w/w  3.8 0.3%w/w extractive values water soluble extractive alcohol soluble extractive 18.4 0.3%w/w  6.3 0.2%w/w number of observations 3 sd standard deviationpharmacognosy journal  august 2011  vol 3  issue 24 37 kher, et al.: pharmacognostic, physico-chemical and phytochemical evaluation of leaves of a species of ‘paarshva-pipla’: ficus arnottiana microscopy: transverse section lamina of the transverse section shows large, tabular epidermal cells covered by thick cuticle. underlying the upper epidermis are bi-layered, compact, radially elongated palisade cells followed by spongy mesophyll composed of 3-4 layers of loosely arranged parenchymatous cells containing calcium carbonate crystals which dissolve in acetic acid (cysoliths) and a cavity at the lower epidermis. a well-developed collenchyma is seen beneath each epidermis in the mid-rib. ground tissue of the mid-rib consists of loosely arranged polygonal parenchymatous cells having calcium oxalate prism crystals and orange color pigment. vascular bundles are bicollateral, crescent shaped; with patches of perimedullary phloem and presence of secondary vascular bundles. a continuous layer of pericyclic fbers is present surrounding vascular bundles. trichomes are rare. (figure 2, 3) microscopy: powder characteristics it is dark green with no distinct odor or taste. the important diagnostic features of the powder include parts of epidermis in surface view showing straight walled epidermal cells and anomocytic stomata, xylem vessels with annular thickening, fragments of horse-shoe shaped xylem vessels, cystoliths, orange color matter and bundles of pericyclic fbers. (figure 4) figure 3: t. s. of leaf of ficus arnottiana showing single enlarged portions (x400) (u co,upper collenchyma; u ep, upper epidermis; l co, lower collenchyma; l epi, lower epidermis; pal, palisade; xyl, xylem; phl, phloem; pe fib, pericyclic fiber; sp pa, spongy parenchyma; pph, perimedullary phloem) figure 4: powder study (x400) (a, horse-shoe shaped xylem vessel; b, cystoliths and anomocytic stomata in surface view; c, orange matter; d, xylem vessels with annular thickening; e, bundle of pericyclic fibers ; f, epidermis in surface view; g, calcium oxalate prism crystals.)kher, et al.: pharmacognostic, physico-chemical and phytochemical evaluation of leaves of a species of ‘paarshva-pipla’: ficus arnottiana 38 pharmacognosy journal  august 2011  vol 3  issue 24 references 1. kirtikar kr, basu bd. indian medicinal plants. vol. i and vol. iii. 2 nd ed. dehradun: international book distributors; 2005. 2. gregory marslin et al, anti-ulcer (ulcer-preventive) activity of ficus arnottiana miq. (moraceae) leaf methanolic extract, american journal of pharmacology and toxicology 2009 vol. 4 issue 3 3. khandelwal kr, kokate ck, gokhale sb. practical pharmacognosy techniques and experiments. pune: nirali prakashan; 1996. 4. world health organization. quality control methods for medicinal plant materials. geneva (switzerland); 2002. 5. feigl f. identification of individual organic compound. in: spot tests in organic analysis. 4 th ed. london: elsevier, p. 237-45; 1956. 6. fishcher r. praktikum der pharmakognosic. 3 rd ed. berlin: springer verlag; 1952. 7. geissman a. modern methods of plant analysis. vol. iii. berlin: springer verlag; 1955. 8. harborne jb. phytochemical methods. 2 nd ed. london: chapman & hall; 1973. 9. hodge je, hofreiter bt. determinations of reducing sugars and carbohydrates analysis. in: roy lw, wolfrom ml, editors. methods in carbohydrate chemistry. vol. i. london: academic press, p. 388-405; 1962. 10. list ph, horhammer l. hager hand buch der pharmazeutischem praxis. vol. i. berlin: springer verlag; 1967. 11. robinson t.the organic constituents of higher plants, their chemistry and interrelationships. minneapolis: burgers; 1964. conclusion the present work deals with the macroscopic, microscopic, physicochemical and phytochemical evaluation of the leaves of ficus arnottiana. diagnostic microscopic characters include pericyclic fbers, cystoliths, anomocytic stomata, horse-shoe shaped xylem vessels and xylem vessels with annular thickening. various physicochemical parameters were established which can be important in detecting adulteration and mishandling of the crude drug. phytochemical analysis showed the presence of many important classes of phytoconstituents like alkaloids, cardiac glycosides, saponins, favonoids, phenolics, carbohydrates, sterols and triterpenoids. this indicates that these both the plant can be useful for treating different diseases because the therapeutic activity of a plant is due to the presence of particular class of compounds. development of such a monograph would help in distinguishing ficus arnottiana fromthespesia populnea, thereby providing evidence on the true botanical identity of ‘paarshva-pipla’ and paving the way for isolation of phytoconstituents, therapeutic investigations and standardization of formulations containing its leaf material.pharmacognosy journal  august 2011  vol 3  issue 24 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: e-mail: drhullatti@klepharm.edu doi: 10.5530/pj.2011.24.8 genkwanin and volatile oils in the leaves. [11] ethanolic and aqueous leaf extracts of the plant has been found possess signifcant diuretic activity. [12] the plant is also known to contain the constituents responsible for cytotoxicity and anti bacterial activity. [13] meterials and methods plant material coleus amboinicus was collected from the surrounding shimoga city, karnataka and authenticated by prof. d. rudrappa, dept. of botany, s.r.n.m. college, shimoga. the collected plants are washed thoroughly with water, all the three parts i.e. leaves, stems and roots were separated and dried in shade at room temperature and powdered using hand mill to make a coarse powder. then they are stored in well-closed light resistant container until further use. morpho-anatomical studies of coleus amboinicus the macroscopy and microscopy of the plant was studied according to the methods of brain & turner, [14] the cross sections were prepared and stained. physico-chemical analysis air dried plant material was used for the quantitative determination of ash and extractive values were determined as per the who guidelines. [15] fluorescence analysis of the extract(s) was carried out by the method of chase & pratt. [16] pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae k k hullatti* 1 , prasenjit bhattacharjee 2 1 department of pharmacognosy, kle university college of pharmacy, belgaum, india - 010. 2 department of pharmacognosy, national college of pharmacy, india, shimoga – 201 a b s t r a c t coleus amboinicus lour. belonging to the family lamiaceae commonly known as karpuravalli, omavalli in tamil, patta ajavayin, patharcur in hindi, country borage in english is a large succulent aromatic perennial herb. the leaves of this plant have been used in malarial fever, hepatopathy, renal and vesicle calculi, cough, chronic asthma, hiccough, bronchitis, anthelmintic, colic and convulsions. this paper deals with the pharmacognostical evaluation of leaves, stems and roots of coleus amboinicus by usingdifferent parameterswhich includemorpho-anatomical studies, physicochemical properties and fuorescence analysis to set the quality control parameters for the raw material. key words: quality control, microscopy, physico-chemical, morpho-anatomical introduction the plant coleus amboinicus (synonym: plectranthus amboinicus, coleus aromaticus) commonly known as country borage, indian borage, is a dicotyledonous plant belonging to the family lamiacea. [1,2] it is a large succulent aromatic perennial herb. much branched, feshy highly aromatic pubescent herb with distinctive smelling leaves. the plant is distributed through out india, cultivated in the gardens. it is a folkloric medicinal plant used to treat malarial fever, hepatopathy, [3] renal and vesical calculi, cough, chronic asthma, [4] hiccough, bronchitis, helminthiasis, colic, convulsions and epilepsy. [5] it is used to treat colds and cough as well as arthritic infammations. [6] its insect repellent properties have been tested [7] and another member of the coleus genus, c. aromaticus, has been found to cause reduction in egg laying capacity, retard in adult emergence and weight loss in the pulse beetle callosobruchus maculatus. [8] a moderate allelopathic effect of the powdered leaves of c.amboinicus against the water hyacinth is also on record. [9] studies performed in india demonstrated the “fungistatic” properties of the essential oil of this plant. [10] the phytochemical study reveals the presence of various favonoids like quercetin, apigenin, luteolin, salvigenin,hullatti, et al.: pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae 40 pharmacognosy journal  august 2011  vol 3  issue 24 chromatograms were developed and were examined under uv and daylight as well as after spraying with anisaldehyde-sulphuric acid reagent to detect the presence of different phytoconstituents. results and discussions macroscopic characters leaf, stem and root parts of coleus amboinicus were evaluated on morphological parameters. leaves are feshy, thick, acute apex, pinnate, dentate margin and symmetrical base. stems are fexible, smooth, small, and feshy and having fbrous structured with large number of nodes and inter-nodes (figure 1a). roots are cylindrical to torus and sharp edges at the base with large numbers of rootlets. the details are given in table-1. preliminary phytochemical screening preliminary phytochemical screening was carried out by using standard procedure described by kokate et al. [17] and harbone. [18] total phenol content of the extracts was determined by using the folin-ciocalteu method. [19] total favonoid content was determined as described by singleton & rossi. [20] the amount of total alkaloids and total saponins was determined according to method given by rajpal. [21, 22] thin layer chromatography all the three extracts were subjected for thin layer chromatography. various mobile phases were tried and the one with maximum number of spots was selected. the most suitable mobile phase was found to be toluene: dioxan: glacial acetic acid (90:25:4). all the three plant extracts were dissolved in methanol and applied to pre-coated tlc silica gel plates (silica gel 60 f 254 , alugram, germany). figure 1: morpho-anatomical features of coleus amboinicus a. photograph from the natural habitat showing leaves and stem, b. transverse section of lamina of the leaf, c. transverse section of midrib of the leaf, d. transverse section of stem, e. transverse section of root. gl tr, glandular trichome; pal, palisade cells; cov tr, covering trichome; vb, vascular bundle; tr, trichome; cor, cortex; ph, phloem; xy, xylem; par, parenchyma; crk, cork; phd, phelloderm; phl, phellogen; scl, sclerenchyma; mr, medullary rayspharmacognosy journal  august 2011  vol 3  issue 24 41 hullatti, et al.: pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae endodermis separates the cortex region from the stellar region. around 7-9 collateral, conjoint, opened type of vascular bundle are arranged in ring. phloem is towards the periphery and containing sieve tubes, companion cells, phloem-parenchyma, and phloem fbers. cambium is made up of thin-walled radially arranged cells. xylem is made up of meta-xylem, proto-xylem and contains xylem-parenchyma and xylem fbers. below the endodermis, patches of pericyclic fbers are present in the form of semi-lunar shape which forms the bundle-cap. pith region is large andmade up of thin-walled polygonal parenchymatous cells with inter-cellular spaces (figure 1d). microscopic characters of root the transverse section of the matured root presents a circular outline with following important tissues- from periphery to the centre. cork is stratifed and consists of smaller, suberised unlignifed cells up to 5-6 rows and larger lignifed cells up to 6-8 rows in radial depth. phellogen is distinct and seen as thin-walled cells. phelloderm is 6-8 layered immediately below the phellem. cells are parenchymatous and with inter-cellular spaces. secondary phloem lies towards outside, consists of sea-tubes, companion cells, and phloem parenchyma. secondary xylem lies towards inside and which forms main bulk of the root. it is made up of meta-xylem and proto-xylem arranged alternatively in a row. xylem consists of vessels, wood-fbers and lignifed parenchyma. medullary rays runs radially from centre to the cortex through phloem. medullary rays are lignifed in nature in 2-3 cells wide (figure 1e). determination of physicochemical parameters: various physicochemical parameters were evaluated for the leaf, stem and root parts as per who guidelines. signifcant amount of acid insoluble ash had been detected which indicates presence of various silicacious substances. cellulosic substances also contributed signifcantly in total ash as indicated by water soluble ash. the plant contains fewer amounts of non-polar substances in comparison to polar substances, as extractive value microscopic characters of leaf the transverse section of leaf shows the dorsiventral characters. the important tissues in the lamina and midrib regions are as follows-upper epidermis is made up of single-layered rectangular cells with cuticle. both covering and glandular trichomes are seen. covering trichomes are uniseriate, multicellular with acute apex. the basal cells of the trichomes are comparatively wider. covering trichomes are made up of 2-3 cells. glandular trichomes made up of 2-3 celled base and unicellular head (figure 1b). mesophylls is differentiated into 2 regions, towards upper epidermis single-layered compact and radically elongated palisade cells and towards lower epidermis more or less spherical parenchymatous cells without intercellular space containing chloroplast. lower epidermis is identical to upper epidermis and is discontinuous due to the presence of stomata and numerous covering and glandular trichomes. midrib appears as plano-convex with fat dorsal side and hemispherical ventral side. in the epidermal layer, lamina is continuous with the midrib region. vascular bundles are small, single and less prominent and consist of 4-6 rows of xylem and thin arc of phloem (figure 1c). surface preparation shows diacytic type of stomata. microscopic characters of stem the transverse section of stem is almost circular in nature. margin is silky due to presence of numerous multicellular covering trichomes. some of the important tissues from the periphery to the centre as follows-epidermis contains single row of fattened closely arranged cells with cuticles. some of the cells of epidermis developed into multicellular covering trichomes. base is 2-3 celled, uniceriate and head is unicellular in nature. 3 distinct portions can be clearly observed in the cortex region. the outer most 3-4 layers of cholenchymatous cells are closely arranged where the cells are thickened due to deposition of pectin. it is followed by 4-5 layers of loosely arranged parenchymatous cells with inter cellular space. the cells are more or less spherical in nature. the inner most layer of the cortex (endodermis) is made up of closely arranged radially elongated parenchymatous cells. the table 1: organoleptic evaluation of leaves, stem and root parts parameters leaf stem root colour light green green to pink brown odour highly aromatic aromatic aromatic taste slightly bitter to acrid slightly bitter to acrid aromatic size max. 6.5 cm in length, and max. 6 cm in width 70 to 80 cm in length extended up to 15 to 20 cmhullatti, et al.: pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae 42 pharmacognosy journal  august 2011  vol 3  issue 24 increases with the increase in the polarity of solvents. however, alcoholic and aqueous extractives showed signifcant yields. signifcant amount of moisture had been also found in air-dried materials (table-2). preliminary phytochemical screening the preliminary phytochemical screening revealed the presence of alkaloids, favonoids, tannins, triterpenoids, saponins in all the three extracts and carotenoids were detected in leaf and stem extracts. the total polyphenols, total favanoids, total alkaloids and total saponis in the different parts of the plant are given in the table 3. the fuorescence analysis is a tool for the determination of constituents in the plant that gives a defnite idea of the chemical nature. thus the fuorescence analysis of the drug powder was carried out and data is presented in the table 4, 5 and 6. thin layer chromatography the solvent system which showed maximum separation of constituents for all the extracts was- toluene: dioxan: glacial acetic acid (90:25:4). in visible light and in uv both short and long wavelengths, the rf value of quercetin matches with the rf values of the frst spots of all the three extracts. this indicates the presence of quercetin in all the three parts of the plant. in visible light and in uv both short and long wavelengths leaf extract showed presence of about 10 components, whereas stem and root extract showed less components. but when sprayed with anisaldehyde-sulphuric acid reagent, about 14 spots have been detected with both leaf and stem extracts, whereas about 16 spots detected with root extract. this indicates presence of more numbers of triterpenoids and saponins in the root extract (figure 2). table 3: amount of various active constituents in different part of coleus amboinicus extracts amount of major active constituents (% w/w) total phenolics flavonoids alkaloids saponins cale 19.62 0.83 4.21 0.39  4.3 0.74 2.09 0.33 case 14.30 0.49 3.59 0.41 1.28 0.50 0.84 0.15 care 12.91 0.60 1.54 0.25 0.60 0.19 0.52 0.16 values represent the mean sd of fve readings cale - coleus amboinicus leaf extract, case - coleus amboinicus stem extract, care - coleus amboinicus root extract table 4: fluorescence characters of powdered leaf particulars of treatment under visible light under uv light short wavelength longwavelength water pale brown light-black brownish-black 5% picric acid pale brown black brownish-black nh 3 solution greenish brown black greenish-black methanol brown black brownish-black acetic acid purple light bluish-black greenish-black conc.h 2 so 4 dark brown deep bluish-black green conc.hno 3 yellowish brown black light-green conc.hcl greenish yellow light-blue yellowish-green 5% iodine yellowish brown deep blue deep green naoh soln. brownish black black greenish-black table 2: physicochemical parameters of leaves stem and root parts parameters leaf stem root extractive values water soluble extractive value 30.38 0.30% 14.93 0.16% 11.13 0.21% alcohol soluble extractive value 13.10 0.33%  7.00 0.20%  4.40 0.17% ether soluble extractive value  3.50 0.19%  1.08 0.09%  0.88 0.11% ash values total ash 21.03 0.03% 11.43 0.02%  9.23 0.02% water soluble ash value  7.90 0.12%  5.85 0.22%  8.05 0.31% acid insoluble ash value  2.05 0.21%  0.95 0.18%  1.75 0.13% loss on drying 11.96 0.15%  9.30 0.10%  8.29 0.51% *values are in mean standard deviation, where as n6.pharmacognosy journal  august 2011  vol 3  issue 24 43 hullatti, et al.: pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae table 5: fluorescence characters of powdered stem particulars of treatment under visible light under uv light short wavelength long wavelength water brownish-black light black greenish-black 5% picric acid yellowish-black bluish-black yellowish brown nh 3 solution greenish-black black greenish-black methanol brown black greenish-black acetic acid yellowish-brown bluish-black light yellowish-black conc.h 2 so 4 dark-brown dark-black greenish-black conc.hno 3 yellowish-brown light-black yellowish-green conc.hcl purple light-black brownish-green 5% iodine blackish-brown light-black light greenish-black naoh soln brownish-black dark-black greenish-black table 6: fluorescence characters of powdered root particulars of treatment under visible light under uv light short wavelength long wavelength water light-brown light bluish-black brown 5% picric acid light-brown deep blue yellowish brown nh 3 solution dark-brown bluish-black greenish-black methanol light-brown black light brownish-black acetic acid yellowish brown black greenish-black conc.h 2 so 4 dark brown black greenish-black conc.hno 3 brown black dark-green conc.hcl brownish-black black greenish-black 5% iodine brownish-black black greenish-black naoh soln dark brown black greenish-black conclusion the data generated from the present studies would help in the authentication of various parts of coleus amboinicus. the microscopic features and the quantitative standards would be useful for laying down pharmacopoeial standards. the different spots observed in tlc would be defnitely useful in deciding the purity and quality of the drug. morphology as well as various pharmacognostic aspects of different parts of the plant were studied and described along with phytochemical, physicochemical and tlc studies in authentication for quality control. references 1. warrier ps. indian medicinal plants, arya vaidya sala, kottakkal. hyderabad: orient longmann limited. 1994; 315-7. 2. chopra rn. nayar sl. chopra ic. the glossary of indian medicinal plants. new delhi: csir. 1956; 74. 3. kirtikar kr. basu bd. indian medicinal plants (2 nd edition). dehradun: international book distributors. 1975; 1971. figure 2: tlc profile of the alcoholic extract of various parts of coleus amboinicus. a. visible light, b. uv 366, c. uv 254, d. after spraying with anisaldehyde- sulphuric acid reagent. cale- coleus amboinicus leaf extract, case- coleus amboinicus stem extract, and care- coleus amboinicus root extract.hullatti, et al.: pharmacognostical evaluation of different parts of coleus amboinicus lour., lamiaceae 44 pharmacognosy journal  august 2011  vol 3  issue 24 13. da costa jgm. campos ar. brito sa. pereira ckb. erlânio o souza. rodrigues ffg. biological screening of araripe basin medicinal plants using artemia salina leach and pathogenic bacteria. ph cog mag. 2010; 6(24):331-334. 14. brain kr,turner td. in: the practical evaluation of phytopharmaceuticals, wright-sciencetechnica, bristol, 1975; 4-9. 15. anonymous. quality control methods for medicinal plant materials. world health organization (who), geneva. 1998; 22-34. 16. chase cr, pratt r. fluorescence of powdered vegetable drugs with particular reference to development of a system of identification, j am pharmacol assoc 1949; 38:324-331. 17. kokate ck. practical pharmacognosy, (1 st edition). vallabh prakashan, new delhi. 1986; 111. 18. harbone jb. method of extraction and isolation, in: phytochemical methods, chapman & hall, london. 1998; 60-66. 19. sadasivam s.manikam a. biochemical methods for agricultural sciences. new delhi, wiley eastern limited. 1992; 187. 20. singleton vl. rossi jr ja. colorimetry of total phenolics with phosphomolybdic acid-phosphotungtic acid reagents. am j enol viticult. 1965; 16:44-158. 21. rajpal v. standardization of botanicals, eastern publishers, new delhi. 2002; 256. 22. rajpal v. standardization of botanicals, eastern publishers, new delhi. 2002; 41. 4. nadkarni ak. indian materia medica (2 nd edition). mumbai: popular prakashan. 1996; 371. 5. gil-otaiza r. plantas usuales en la medicina popular venezolana edición c.d.c.h.t-ula (mérida). 1997; 52-54. 6. gomez-rodriguez a. margarita. medicina popular, ediciones de la federación farmacéutica venezolana, series 3 (caracas), (vol. 1). 1982; 119. 7. kathiresan r.m. allelopathic potential of native plants against water hyacinth. crop protection. 2000; 19 (8-10):705-708. 8. babu a. raja n. albert s. ignacimuthu s. dorn s. comparative efficacy of some indigenous plant extracts against the pulse beetle callosobruchus maculatus f. (coleoptera: bruchidae). biol agric hort. 1999; 17(2):145-150. 9. samuel co, srivastava l,tripathi s. protection of dry-fruits from fungal infestation by essential oil of coleus amboinicus. indian journal plant prot. 1995; 23:174-179. 10. rastogi rp, mehrotra bn. compendium of indian medicinal plants. lucknow: cdri; and new delhi: publication and information directorate. 1979; 201. 11. buznego mt. perez-saad h. anti-epileptic effect of plectranthus amboinicus (lour.) spreng. (french marjoram), rev neurol. 1999; 29(4):388-389. 12. patel r. mahobia nk. gendle r. kaushik b. singh sk. diuretic activity of leaves of plectranthus amboinicus (lour) spreng in male albino rats. pharmacog res. 2010; 2(2):86-88.pharmacognosy journal  august 2011  vol 3  issue 24 45 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 m modh, department of pharmacology, shri sarvajanik pharmacy college, nr. arvind baug, mehsana - 384 001 gujarat, india. tel. no.: 91-2762-247711 tele-fax no.: 91-2762-247712 e-mail: siddhkamal@yahoo.co.in doi: 10.5530/pj.2011.24.9 leprosy, piles, snake bite and liver complaints [3] and its extracts have been found to have antibacterial and antifungal ativity. [4] it is also used in fever, diarrhoea, dysentery, hemorrhoids, piles, edema, skin diseases, wound healing, obesity, stomatitis, dyspepsia, fatulence and as tonic, astringent, laxative, anthelmintic, antileprotic, antigoitrogenic, antitumor, and carminative. [5] the leaves of other bauhinia species are reported to have antiophidian, [6] antidiabetic, [7] antimalarial, [8] and antioxidant potential. [9] previously reported phytochemical constituents from the plant are lupeol, β-sitosterol, tannins, kaempferol-3-glucoside, [10] amides, carbohydrates, reducing sugars, crude protein, vitamin c, fbers, [11] calcium, phosphorus, [12] rutin, quercetin, quercitrin, apigenin, apigenin -7-o-glucoside, [13] dotetracont-15-en-9-ol and heptatriacontan-12,13-diol. [14] inspite of its abundant uses, the phytochemical standards of bauhinia variegata leaves have not been reported. the present investigation deals with the qualitative and quantitative pharmacognostical and phytochemical evaluation of the leaf material. pharmacognostical and phytochemical evaluation of leaves of bauhinia variegata linn. kamal m modh 1 *, purvi t parmar 2 , bibhuranjan panigrahi 3 , indermeet singh anand 1 , chhagan n patel 3 1 department of pharmacology, shri sarvajanik pharmacy college, nr. arvind baug, mehsana – 384 001, gujarat, india. siddhkamal@yahoo.co.in; inderlilly@yahoo.com 2 department of pharmacognosy, shri sarvajanik pharmacy college, nr. arvind baug, mehsana – 384 001, gujarat, india. ptparmar@sspcmsn.org 3 department of pharmaceutical chemistry, shri sarvajanik pharmacy college, nr. arvind baug, mehsana – 384 001, gujarat, india. bibhu_lmcp@yahoo.co.in; drcnpatel2000@yahoo.co.in a b s t r a c t bauhinia variegata linn. (caesalpiniaceae) syn: kovidara and kachnar is a medium sized deciduous tree generally found in sub himalayan region of india. almost all parts of this plant are used in traditional medicine for the treatment of various ailments. the present work was undertaken to establish the pharmacognostic and phytochemical standards along with hptlc densitometric analysis of leaves for evaluating the plant material. the macro and microscopical studies indicated presence of pulvinus base with grooved petiolate leaf, emarginated apex, rough surface with 11-13 reticulate and palmate-divergent venation with scattered prismatic calcium oxalate crystals throughout the transverse section. physiochemical studies revealed that total ash is 9.42%, acid insoluble ash is 5.72%, water-soluble extractive value is 3.30% and loss on drying at 105 c is 6.27%. preliminary phytochemical analysis revealed the presence of alkaloid, tannin, favonoid, steroid, triterpenoid and saponin in different extracts. hptlc fngerprinting for favonoids revealed presence of two favonoids rutin and kaempferol. the results of the study could be useful in setting some diagnostic indices for the identifcation and preparation of a monograph of the plant. key words: bauhinia variegata, leaf microscopy, physico-chemical constants, phytochemical, hptlc fnger print introduction about 250 species of bauhinia grow in the tropical regions of the world. it includes shrubs, trees and vines that are frequently planted for their showy fowers and ornamental foliage. [1] bauhinia variegata linn. is native to south eastern asia and grows throughout india and china. it is most commonly cultivated in india. [2] bauhinia variegata linn. (caesalpiniaceae) is a medium sized deciduous tree, known as (sanskrit) kanchanara, (hindi) kovidara and (marathi) raktakanchan. almost all parts are used in traditional medicine for the treatment of various ailments like asthma, ulcer,modh, et al.: pharmacognostical and phytochemical evaluation of leaves of bauhinia variegata linn. 46 pharmacognosy journal  august 2011  vol 3  issue 24 solution test) were detected by usual method prescribed in standard text. [19,20] densitometric hptlc analysis for favonoids a densitometric hptlc analysis was performed for the development of characteristic fnger printing profle. 50% hydroalcoholic and methanolic extract of bauhinia variegata linn. leaves were dissolved with hplc grade methanol 10 mg/ml. the solutions were sonicated for 10 min and used for hptlc analysis. then, 10 µl of the samples were loaded as 5 mm band length in the 10 10 silica gel 60f 254 tlc plates using hamilton 254 syringe and desaga sarstedt-gruppe as 30 instrument. the sample loaded plate were kept in tlc twin trough developing chamber (after saturation with solvent vapor) with respective mobile phase along with 2000 µg/ml rutin and 200 µg/ml kaempferol standard solutions. the plates were developed in the ethyl acetate-butanol-formic acid-water (10:6:2:2 v/v/v/v) for rutin (rf 0.48) and toluene-ethyl acetate-methanol-formic acid (6:3:0.2:0.4 v/v/v/v) (rf 0. 56) for kaempferol up to 90 mm. the developed plate was dried using hot air to evaporate solvents from the plate and kept in photo-documentation chamber (desaga) and captured the images at uv254. finally, the plates were fxed in scanner stage and scanned at 380 nm and 254 nm for rutin and kaempferol respectively. materials and methods plant material the plant material was collected from the natural park of the mehsana district, gujarat, india in the month of february 2009. the plant was authenticated by pro. y. b. dabgar, head , department of botany, shri c. l. parikh & r. r. mehta science college, palanpur, gujarat, india and a voucher specimen (no. sspc/cog/05/2009) was deposited indepartment of pharmacognosy, shri sarvajanik pharmacy college, mehsana, gujarat, india. pharmacognostical studies morphology morphological studies such as shape, size, apex, surface, base, margin, venation, taste and odour of leaves were carried out. microscopy microscopical studies were carried out using nicon labphot-2 instrument (japan). the transverse sections with average thickness of 10-12 µm were taken with the help of rotary microtome. dewaxing of the sections was performed by customary procedure. [15] the section was stained with toluidine blue as per the method. [16] since, toluidine blue is a polychromatic stain, it rendered pink colour to the cellulose walls, blue to the lignifed cells, dark green to suberin, violet to the mucilage and blue to the protein bodies. wherever necessary, sections were also stained with saffranin, fast-green and iodine potassium iodide reagents (for starch). as a part of quantitative microscopy, stomatal number, stomatal index, vein islet number and vein termination number were studied by taking paradermal sections as well as clearing of leaf with 5% sodium hydroxide and epidermal peeling off by partial maceration employing jeffrey’s maceration fuid [17] as shown in table 1. physico-chemical constants physicochemical constants of the leaf such as the total ash, acid insoluble ash, water soluble ash and loss on drying were calculated based upon standard procedures. [18] phytochemical analysis for preliminary phytochemical studies, 300 g of powdered material was extracted in soxhlet apparatus with petroleum ether, chloroform, methanol and water. extracts were dried in rotary evaporator and weighed. the extractive values for each extract is shown in table 2. the presence of various phytoconstituents like steroids and triterpenoids (liberman and burchard test), alkaloids (dragandroff ’s test), tannin (ferric chloride test), favonoid (shinoda test), sugar (fehling table 1: leaf constants of the bauhinia variegata linn. leaf leaf constant values palisade ratio 4.8 stomatal index 5.6 vein-islet number 5.8 vein termination number 3.7 table 2: extractive values of the bauhinia variegata linn. leaf extract colour % w/w petroleum ether yellowish brown 3.41 chloroform brownish black 1.72 methanol brown 8.76 aqueous greenish brown 7.25 figure 1: leaf of bauhinia variegata linn.pharmacognosy journal  august 2011  vol 3  issue 24 47 modh, et al.: pharmacognostical and phytochemical evaluation of leaves of bauhinia variegata linn. midrib ventral side of the midrib is slightly concave. shape of the middle vein portion in the ts is oblong but elongated tapered at ventral side and also shows the irregularities on its lower epidermis. midrib contains ‘u’- shape well developed vascular bundle at the centre surrounded by sclerenchyma (pericyclic lignifed fbrous tissue in a band). vascular bundle shows the presence of the xylem at the upper side and phloem at the lower side and well developed collenchyma below the upper epidermis and above the lower epidermis with scattered prismatic calcium oxalate crystals. ts of the leaf also show the presence of unicellular, 3-5 celledmulticellular uniseriate- cuticlerised covering trichomes as well as unicellular sessile and unicellular head with unicellular stalcked glandular trichomes. trichomes are more prominent on the lower epidermis than upper epidermis. powder microscopy the powder microscopy showed the presence of 142.8-199.92 µm long multicellular uniseriate trichomes, 57.12 µm in diameter prismatic calcium oxalate crystals, lignifed annular and spiral xylem vessels, portion of epidermal cells with anomocytic stomata as shown in fgure 3. results and discussion organoleptic characters colour of bauhinia variegata linn. leaves is green on both side when fresh and brown in dry state. size and shape is 13-15 12-14 cm, long as broad as or rather broader than long, cleft 1/4 to 1/3 of the way down into 2 obtuse lobes, pulvinus base with grooved petiolate, linear-lanceolate with entire margin with soft stipules. apex of the leaf is broad and emarginated. the surface of leaf is rough surface with 11-13 reticulate, palmate-divergent venation as shown in fgure 1. taste is slightly bitter and having weak odor. microscopy transverse section of the leaf (figure 2) lamina it shows dorsi-ventral nature; more densely covered upper epidermis with cuticle than lower epidermis and made up of thin walled tangentially elongated rectangular cells. mesophyll in the lamina shows the presence of 2-3 layers of palisade parenchyma below the upper epidermis and spongy parenchyma above the lower epidermis as shown in fgure 2 (b). scattered prismatic calcium oxalate crystals are present throughout the mesophyll. figure 2: leaf structure of bauhinia variegata linn. figure 3: powder microscopy of leaves of bauhinia variegata linn.modh, et al.: pharmacognostical and phytochemical evaluation of leaves of bauhinia variegata linn. 48 pharmacognosy journal  august 2011  vol 3  issue 24 quantifcation of rutin and kaempferol in both extracts shows that the sample of 50% hydroalcoholic extract (1.114 mg/100 g) contains more rutin than the sample from the methanolic extract (0.075 mg/100 g) whereas the sample of 50% hydroalcoholic extract (0.193 mg/100 g) contains more kaempferol than the sample from the methanolic extract (0.014 mg/100 g). conclusion a variety of standardization parameters like morphological, microscopical, physico-chemical, phytochemical and chromatographic characterization were studied and generated physico-chemical parameters the percentage of total ash, acid-insoluble ash, water soluble ash and loss on drying are tabulated in table 3. qualitative phytochemical analysis the results demonstrated presence of alkaloids, favonoids, saponin, tannins, steroids and sterol, triterpenoids and sugar in the leaf of bauhinia variegata linn. the presences of various phytoconstituents in various organic and aqueous extracts are summarized in table 4. quantifcation of rutin and kaempferol both extracts of the plant developed a characteristic hptlc fngerprinting for rutin and kaempferol as shown in fgure 4. it gave a distinct spot at rf value 0.48 and 0.56 for rutin and kaempferol respectively as shown in table 5. the peak purity was assessed by comparison of overlay spectra of standards and both the extracts as shown in fgure 5. the linear regression equation was y 1060 151.5*x and the correlation coeffcient (r 2 ) was 0.993 for rutin and y 1576 895.2*x and the correlation coeffcient (r 2 ) was 0.992 for kaempferol. table 3: physicochemical constants of the bauhinia variegata linn. leaf sr. no. parameter % w/w 1 total ash 9.42 2 acid insoluble ash 5.72 3 water soluble ash 3.30 4 loss on drying 6.27 table 4: qualitative phytochemical analysis of the bauhinia variegata linn. leaf constituents pet.ether chloroform methanol aqueous alkaloids – – – coumarins – – – – flavonoids – – – anthraquinone – – – – saponin – – – tannins – – steroids and sterol – triterpennoids – – sugar – fixed oils – – – – presence; - absence figure 4: tlc plate at 254 nm for (a) rutin and (b) kaempferolpharmacognosy journal  august 2011  vol 3  issue 24 49 modh, et al.: pharmacognostical and phytochemical evaluation of leaves of bauhinia variegata linn. 5. mali rg, mahajan sg, mehta aa. raktachandan (bauhinia variegate): chemistry, traditional and medicinal uses- a review. phcog rev. 2007; 1:314-319. 6. oliveira cz,maiorano va,marcussi s,santana cd, januario ah, lourenco mv, et al. anticoagulant and antifibrinogenolytic properties of the aqueous extract from bauhinia forticata against snake venoms. j ethanopharmacol. 2005; 98:213-216. 7. silva fr, szpoganicz b, pizzolatti mg, willrich ma, desousa e. acute effect of bauhinia forticata on serum glucose levels in normal and alloxan induced diabetic rats. j ethanopharmacol. 2002; 83:33-37. 8. kittakoop p, kirtikara k, tanticharoen m, thebtaranonth y. antimalarial preracemosols a and b, possible biogenetic precursors of racemosol from bauhinia malabarica roxb. phytochem. 2000; 55:349-352. 9. argolo ac, santana ae, pletsch m, coelho lc. antioxidant activity of leaf extracts from bauhinia monandra. biores techno. 2004; 95:229-233. 10. amonymous.the wealth of india; a dictionary of indian raw materials and industrial products, vol. 2(b). new delhi, csir, 1998; 49-52. 11. sharma dd, gill rs, chander s, negi ss. chemical composition of some fodder tree leaves in the kangra district. j res. 1966; 3:438-442. 12. sharma dd, chawla ms, negi ss. chemical composition and nutritive value of bamboo and kanchar tree leaves. j res. 1968; 5:253-258. 13. spilkova j, hubik j. biologische wirkungen von flavonoiden ii. pharmazie in unserer zeit. 1992; 21(4):174-182. 14. singh rs, pandey hs, ghanshyam.two new long chain compounds from bauhinia variegate linn. ind j chem. 2006; 45b(9):2151-2153. 15. johanson da. plant microtechniques. 1 st edition. new york, mc graw hill, 1940; 126-154. 16. obrien tp, feder n, mccull me. polychromatic staining of plant cell walls by toluidine-o. protoplasma. 1964; 59:364-373. 17. anonymous. the pharmacopoeia of india. vol. i. 4 th ed. new delhi: the controller of publication; 1996; a-74. 18. kaliappan nd,viswanathan pk. pharmacognostical studies on the leaves of plectranthus amboinicus (lour) spreng. int j green pharm. 2008; 2(3):182-184. 19. khandelwal kr. practical pharmacognosy. 19 th ed. pune:nirali prakashan; 2008:149-160. 20. kokate ck, purohit ap, gokhale sb. pharmacognosy. 39 th ed. pune: nirali prakashan; 2007:607-611. data could be useful for the assessment of quality of plant material, and also to check the adulteration and substitution etc., for future reference. the pharmacognostic study of bauhinia variegata linn., have furnished a set of qualitative and quantitative standards that may substantiate to ascertain it’s identity and to establish the quality and purity of this plant material in closely related species. densitometric hptlc analysis may serve a supplementary data for the standardization of the drug, particularly of different batches. this could also serve in the establishing data for preparation of monograph of this plant. acknowledgement authors are thankful to dr. yogesh b. dabgar, head, department of botany, shri c. l. parikh & r. r. mehta science college, palanpur, gujarat, india for authentication of plant sample. references 1. larsonss.pollenmorphologyof thai speciesof bauhinia (caesalpiniaceae). grana. 1974; 14:114-31. 2. anonymous.the ayurvedic pharmacopoeia of india.vol. i. new delhi:the controller of publication; 2001:321-322. 3. kirtikar kr, basu bd. indian medicinal plants. vol. ii. 2 nd ed. dehradun: international book distributors; 1999:891-902. 4. rajkapoor b, jayakar b, murugesh n. antitumor activity of bauhinia variegata on dalton’s ascetic lymphoma. j. ethanopharmacol. 2003; 89; 107-109. table 5: densitometric hptlc analysis of the bauhinia variegata linn. leaf extract rutin kaempferol rf value area concentration (mg/100 g) rf value area concentration (mg/100 g) 50% hydroalcoholic 0.48 2048 1.114 0.56 3379.36 0.193 methanolic 0.47 1907.05 0.075 0.56 3314.10 0.014 figure 5: overlay spectra of 50% hydroalcoholic and methanolic extracts of leaves of bauhinia variegata linn. over standard (a) rutin and (b) kaempferolo r i g i n a l a r t i c l e p h c o g j . 50 pharmacognosy journal  august 2011  vol 3  issue 24 *address for correspondence: e-mail: draparnasaraf@yahoo.co.in doi: 10.5530/pj.2011.24.10 over the past decades have focused on the clinical signifcance of trace elements in human health and disease. trace quantities of these elements are essential for enzyme catalyzed biological processes in plants. consequently, the presence of these elements attributes medicinal properties to the plant. it is often observed that these elements are present at varying levels of concentration in different parts of the plants such as roots, stem, leaves etc. it is also observed that these elements concentration vary depending upon the geographical location of the plant. it has been reported that whatever is consumed as medicine could cause metabolic disturbances subject to the allowed upper and lower limits of trace elements. [1] both the defciency and excess of essential micronutrients and trace elements of toxic metals may cause serious effects on human health. [2,3] according to who, medicinal plants should be checked for the presence of heavy metals. it is an established fact that the overdose or prolonged ingestion of the medicinal plant leads to the chronic accumulation of different elements which cause various health problems. [4,5] in this context elemental contents of the medicinal plants are very important and need to be screened for their quality control. [6] for the present study, oroxylum indicum l.(vent.) a medicinal plant, belonging to the family bignoniaceae was selected. it is profile of elemental composition of oroxylum indicum l.(vent.) collected from different geographical regions of india k. srilatha srinivas and a.a. saraf* department of botany, the institute of science, mumbai-32, india. a b s t r a c t mineral content was quantifed in oroxylum indicum l.(vent.) collected from two different geographic regions of india viz. western ghats (maharashtra) and northern uttar pradesh. the main aimof this study was determination of elemental composition in different parts of the plant oroxylum indicum l.(vent.), which is extensively used in ayurvedic preparations. specifc parts (leaves, stem and root) often used in indian ayurvedic system were analyzed for 10 elements viz cu,na,ca,cr,mn,fe,ni,cd,zn and pb by employing icpes techniques. all of the detected values for metallic elements in plant studied here were found to be below the who permissible levels. the elemental concentration in different part of medicinal plants and their biological effects on human beings are discussed. key words: oroxylum indicum l.(vent.), icpes, elemental composition. introduction the curative properties of plants have been well documented in ancient indian literature. the different parts of the plant are used as ingredients in several medicinal preparations in different systems of medicine including ayurveda. the importance of herbal medicines in the health care system of the larger section of the world’s population, the developing countries, is also an undeniable fact. they form and inseparable part of the traditional systems of medicine and in many cases bridge the gap between the availability of and demand for modern medicine. world health organization estimates that about 80% of the population living in rural areas use or depend on plant based medicinal preparations for preventive and curative health care. epidemiological studies over the past decades have documented the importance of trace elements in human health and disease. prompted by this development the pharmaceutical companies have been marketing as general tonics a variety of formulations containing combinations of different trace element contents .various medical studiespharmacognosy journal  august 2011  vol 3  issue 24 51 srinivas and saraf: profile of elemental composition of oroxylum indicum l.(vent.) collected from different geographical regions of india perchloric acid was then added to the above solution and heated for 5 min. this was followed by addition of aqua regia and heated .the volume was then made up to 250 ml in a standard fask by adding de ionized water. estimation of elements was carried out using inductively coupled plasma - atomic emission spectrometer (model: arcos from m/s. spectro, germany) results and discussion the results presented in table 1 exhibit that the various plant parts of oroxylum indicum l.(vent.) are a good source of trace and major elements. copper: copper content was found to be variable among the plant parts in the two plants, ranging from 19.16 ppm to 55.69 ppm, with oroxylum indicum l.(vent.) stem sample from western ghats recording the highest level. this is in accordance with reports [10] which state that range of copper content in 50 medicinally important leafy materials growing in india is in the range of17.66 ppm to 56.3 ppm. copper (cu) is an essential redox ‐ active transition element that play vital role in various metabolic processes. being toxic, its quantity should be mentioned very low. it is well known that the high content of transition metal like cu catalyzes the formation of hydroxyl (oh) radicals, hence their excess quantity can cause oxidative stress in plants and consequently increase the antioxidant response. [11] it is essential to the human body since it forms a component of many enzyme systems, such as cytochrome oxidase, lysyl oxidase and an iron-oxidizing enzyme in blood. the observation of anemia in copper defciency is probably related to its role in facilitating iron absorption and in the incorporation of iron in hemoglobin. however copper defciency in humans is a rare occurrence. copper could be toxic depending on the dose and duration of exposure. [12] the permissible limit set by fao/who [13] in edible plants a medium sized, deciduous tree found in india in eastern andwesternghats andnorth east regions. it is an important herb in ayurvedic medicine and indigenous medical system for over thousands of year. [7] as the plant is used in many medicinal formulations likechyawanprash, dashamoolarishtha [8] etc, a detailed study is carried out to analyze the concentration levels of elements. the stem bark and root bark possess anti allergic properties and are used in treating allergic diseases urticaria, jaundice, asthma, sore throat, laryngitis andmeasles. [9] as the quantitative profle of a plant medicine may vary depending upon geological and ecological factors, the same species is collected from two different locations viz., western ghats and northern region of india to compare the various elemental concentrations. the elemental concentration in different part of medicinal plant and their biological effects on human beings are discussed. materials and methods sample collection whole plant parts of oroxylum indicum l.(vent.) were collected during the fowering season from two different geographical regions viz., western ghats (village – pophali, kumbharli ghat near chiplun, dist raigarh , maharashtra) and khiri village, dist lakhimpur, northern u.p. the plants were identifed and authenticated at blatter’s herbarium, st. xavier’s college, mumbai, (accession no 54436). the plants collected from different regions were sorted out and individual plant parts were separated. sample preparation plant parts were washed with de ionized water and oven dried at 40 ºc for four days and then subjected to grinding for powder formation. the powder was stored in air tight glass containers and used for further analysis. digestion two gram powder of each plant part was dissolved in nitric acid and heated until the reddish brown fumes disappear. table 1: concentration of elements in oroxylum indicum l.(vent.) collected from different regions of india elements analysed western ghats uttar pradesh root stem leaves root stem leaves cu 45.45 55.69 19.16 33.37 21.47 23.93 na 279.22 272.98 840.84 152.27 310.16 1685.32 ca 4513.49 7708.04 9311.72 3275.90 7017.47 10657.53 cr nd nd nd nd nd nd mn 46.70 12.49 26.26 28.26 14.73 86.62 fe 2288.34 233.77 293.31 1347.61 402.90 1129.11 ni nd nd nd nd nd nd cd nd nd nd nd nd nd zn 27.85 59.94 28.00 21.29 17.60 27.79 pd nd nd nd nd nd nd (all values in ppm -- microgram per gram; nd—values less than 0.01 ppm )srinivas and saraf: profile of elemental composition of oroxylum indicum l.(vent.) collected from different geographical regions of india 52 pharmacognosy journal  august 2011  vol 3  issue 24 figure 3: concentration of calcium in different plant parts collected from different regions of india (wg –western ghats; up –uttar pradesh) figure 4: concentration of manganese in different plant parts collected from different regions of india (wg –western ghats; up –uttar pradesh) figure 5: concentration of iron in different plant parts collected from different regions of india (wg –western ghats; up –uttar pradesh) figure 6: concentration of zinc in different plant parts collected from different regions of india (wg –western ghats; up –uttar pradesh) figure 1: concentration of copper in different plant parts collected from different regions of india (wg –western ghats;up –uttar pradesh) figure 2: concentration of sodium in different plant parts collected from different regions of india (wg –western ghats; up – uttar pradesh) figures: elemental concentration in oroxylum indicum l.(vent.) collected from different regions of india.pharmacognosy journal  august 2011  vol 3  issue 24 53 srinivas and saraf: profile of elemental composition of oroxylum indicum l.(vent.) collected from different geographical regions of india nickel : nickel is considered to be highly mobile element within a plant. accumulation of ni takes place only in the leaves. [20] ni toxicity in human is not very common occurrence as its absorption by the body is very low. [21] the permissible limit for nickel set by who in edible plants was 1.63 ppm and the amount of nickel concentration in all the samples analyzed was below the permissible level. the permissible limits for medicinal plants have yet not been set. cadmium: cadmium is toxic metal having functions neither in human body nor plants. [22] accumulation of cd in kidney leads to high blood pressure and renal diseases. its accumulation also leads in damaging the nerve cells, inhibition of release of acetylcholine and activation of choline esterase enzyme, resulting in a tendency for hyperactivity of the nervous system. [23] the permissible level (who) for cadmium in edible plants was 0.21 ppm and for medicinal plants is 0.3 ppm. [14] the amount of cadmium concentration in all the samples analyzed was found to be within the permissible limits. this may be due to low level of cadmium present in the available soil for plant growth. lead: exposure to increased concentrations of lead is a health hazard. the permissible limit for lead set by fao/ who in edible plants was 0.43 ppm. the amount of lead concentration in all the samples analyzed was in minimal amount and well below the permissible level. zinc: zinc is essential to all organisms and has an important role in metabolism, growth, development and general wellbeing. it is an essential co-factor for a large number of enzymes in the body. zinc defciency leads to coronary heart diseases and various metabolic disorders. highest amount found inw. ghats plant stem 59.94 ppm and lowest in17.60 ppm in u.p. stem. the results above indicated that the plants contain large amounts of nutrients and are rich in fe, copper, ca and na. the abundance of fe, ca and cu, in the result of this analysis, was in agreement with previous fndings that these three elements represent the most abundant metal constituents in plants. [24,25] high contents of ca are important, because of its role in human and studied plants show satisfactory level of ca accumulation conclusions in view of above facts, the medicinal plant, oroxylum indicum l.(vent.) studied is a source of biologically important elements, which may play a part in the observed therapeutic use of this plant. ayurvedic formulations do demonstrate is 3.00 ppm. however for medicinal plants the who limits have yet not been established for cu. [14] sodium: sodium is essential to all living organisms. na is an important element for the maintenance of acid–base equilibrium and of osmotic pressure of body fuids. [15] concentration of sodium element was observed to be much higher in leaves from both plants than the concentration in other plant parts. the lowest amount, 152.27 ppm was found in roots of oroxylum indicum l.(vent.) from u.p and highest amount was found in leaf of oroxylum indicum l. (vent.) from u.p. calcium: calcium is an important trace element because of its role in bones, teeth, muscular system and heart functions. [16] it is required for absorption of dietary vit. b, for synthesis of neurotransmitter acetylcholine and is also required for activation of enzyme pancreatic lipase. [17] it is observed that amongst all the metals studied in the analyzed samples, calcium accumulation is highest in all parts than the concentration of other metals. maximum concentration is found in leaves than the other plant parts. u. p root sample showed the lowest 3275.90 ppm while u.p. leaf has the highest amount i.e. 106.57 ppm. chromium: chromium is known to regulate carbohydrate, nucleic acid and lipoproteinmetabolism and it also potentiates insulin action. [18] chronic exposure to cr may result in liver, kidney and lung damage. [19] chromium also acts as an activator of several enzymes. defciency of chromium decreases the effciency of insulin and increases sugar and cholesterol in the blood. chromium defciency can cause an insulin resistance, impair in glucose tolerance and may be a risk factor in atherosclerotic disease. the permissible limit for chromium as set by fao/who [13] in edible plants is 0.2 ppm. however the permissible limit for medicinal plants has yet not been set. the chromium concentration in all the samples studied was below the permissible levels. manganese: the highest concentration of manganese found in the sample, up leaf was 10657.53 ppm and the least was found in w. ghats stem i.e. 12.49 ppm. the permissible limit for mn is 2 ppm in edible plants. the permissible limits for mn in medicinal plants have yet not been set. iron: the permissible level set by who for iron in edible plants was 20 ppm. iron is important for the formation of haemoglobin and also plays an important role in oxygen and electron transfer in human body. in all the samples studied, the amount of iron accumulated is much higher than the permissible levels. studies suggest that the intake of iron in higher concentration is hazardous to health. w.ghat root sample showed highest concentration 2288.34 ppm and lowest concentration was found inw.ghat stem, 233.77 ppm.srinivas and saraf: profile of elemental composition of oroxylum indicum l.(vent.) collected from different geographical regions of india 54 pharmacognosy journal  august 2011  vol 3  issue 24 7. joshi kc, prakash l, shah rk. chemical examination of the roots of tabebuia rosea and heart wood of oroxylon indicum. plant med. 1977; 31:257-58. 8. kirtikar kr, basu bd. indian medicinal plant. oriental enterprises, dehradun, vol. 4, 2001; 1105-1107. 9. lawania rd, mishra a, gupta r. oroxylum indicum: a review. phcog j. 2010; vol. 2, issue 9:304-310. 10. reddy pr, reddy sj. elemental concentrations in medicinally important leafy materials. chemosphere, 1997; 34:2193-2212. 11. joya m.s.,alist.k.,kazi g.h.,detection of trace elements in heliotropium europeum l. var. hamdard. food chem 1998; 10(4):50 ‐ 52. 12. obi e, akunyili d, ekpo b, orisakwe o. heavy metal hazards of nigerian herbal remedies. sci total environ 2006; 369:35-41. 13. fao/who contaminants. in codex alimentarius, vol. xvii, edition 1. fao/ who, codex alimentarius commision, rome. 1984. 14. jabeen s, shah mt, khan s & hayat mq .determination of major and trace elements in ten important folk therapeutic plants of haripur basin,pakistan journal of medicinal plants research, 2010, 4 april; vol. 4(7), 559-566. 15. martin jr., d.w., mayers p.a., rodwell, v.w., granner, d.k. harper’s review of biochemistry, 20 th ed. lange medical publications, california, 1985; 651-660. 16. brody t . nutritional biochemistry. san diego, ca: academic press. 1994. 17. lokhande r, singare p, andhele m & acharya r. study of some indian medicinal plants by application of inaa and aas techniques. natural science, 2010; vol. 2, no.1, 26-32. 18. kaplan, l.a., pesce, a.j., kazmierczak, s.c., clincal chemistry—theory, analysis, correlation, fourth ed. mosby, london; 2003. 19. zayed am, terry n. chromium in the environment: factors affecting biological remediation. plant soil. 2003; 249:139-156 20. mc grath sp, chromium and nickel. in: alloway b.j. (ed.) heavy metals in soils. blackie academic & professional, london, 1995; 152-179. 21. onianwa pc, lawal ja, ogunkeye aa, orejimi bm . cadmium and nickel composition of nigerian foods. j. food compos. anal. 2000; 13:961-969. 22. iqbal hussain, lajber khan. international journal of pharmacognosy and phytochemical research 2010; 2(1):15-18. 23. shumacher m, bosque m a & domingo jl, carbella j . bull. environ. toxicol, 1991; 46, 320. 24. maiga, a., d. diallo, r. bye and b.s. paulsen, determination of some toxic and essential metal ions in medicinal and edible plants from mali. j agric food chem., 2005; 53:2316-2321. 25. özcan, m.m. and m. akbulut,. estimation of minerals, nitrate and nitrite contents of medicinal and aromatic plants used as apices, condiments and herbal tea food chem., 2007; 106:852-858. signifcant success in treatment of many diseases. these medicines contain trace elements whose activity has an impact on its overall pharmacological action. there is no direct link that has been established between elemental content and curative capability of the plant. but such studies will help us to understand the pharmacological action of the herb and thus provide the vital link between the two. the data obtained in the present work will be helpful in the synthesis of new ayurvedic drugs which can be used for the control and cure of various diseases. however, in order to develop a stronger basis for appreciating the curative effects of medicinal plant, oroxylum indicum l.(vent.) there is a need to study the effect of soil and climatic conditions on the elemental contents of this medicinal plant. medicinal plant oroxylum indicum l.(vent.) is rich in metals fe, copper, ca and na and it is expected that plants with high contents of the above-mentioned macro and micronutrients, might play an important role in maintenance of human health. also, all of the detected values for metallic elements in plant studied here are below the who permissible levels and may not constitute a health hazard for consumers. references 1. shad ali khan et al. profile of heavy metals in selected medicinal plants.,pak. j. weed sci. res. 2008; 14(1-2):101-110, . 2. underwood, e.j.trace element in human and animal nutrition. 4 th edition, academic press inc. new york; 1997. 3. reilly, c. metal contamination of food, 1 st ed. chapter 5 and 6. applied science publishers london; 1980. 4. who, evaluation of certain food additives and contaminants. who technical report series 776, geneva: world health organization; 1989. 5. sharma kr, agrawal m, marshall mf. heavy metals in vegetables collected from production and market sites of a tropical urban area of india. food chem.toxicol. 2009; 47:583-591. 6. arceusz a,radecka i,wesolowski m. identification of diversity in elements content in medicinal plants belonging to different plant families. food chem. 2010; 120:52-58.pharmacognosy journal  august 2011  vol 3  issue 24 55 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: amrita mishra, department of pharmacognosy, college of pharmacy, iftm, lodipur-rajput, moradabad-244001 india. mobile: 91-9452072531 e-mail: amrita_azam@rediffmail.com doi: 10.5530/pj.2011.24.11 pharmacognostical, physicochemical and phytochemical studies of some marketed samples of roots used in ayurvedic medicines amrita mishra 1 *, arun k mishra 1 , ashoke k ghosh 1 , shivesh jha 2 1 department of pharmacognosy, college of pharmacy, iftm, moradabad india-244001. 2 department of pharmaceutical sciences, birla institute of technology, mesra, ranchi, india-835215 a b s t r a c t background: kuth (saussurea lappa), nisotha (operculina turpethum), akarakara (anacyclus pyrethrin) and chitrak (plumbago zeylanicum) are some common plants used in ayurvedic system of medicines and herbal drugs. objective: the objective of the present study is to evaluate the quality of the samples of same plant marketed into different area, on the standardization parameters given in ayurvedic pharmacopoeia. methods: three different marketed samples of these roots were subjected to pharmacognostic, physicochemical and phytochemical analysis and results were compared to the standards given in ayurvedic pharmacopoeia. results: variations were found in physicochemical and phytochemical parameters of two samples in each case of saussurea lappa, operculina turpethum, anacyclus pyrethrin and of one sample in case of plumbago zeylanicum. pharmacognostical parameters were found same as given in ayurvedic pharmacopoeia for all the samples. conclusions: the outcome of the study suggested that there is a lot of difference in the quality of the same drug marketed in different parts of country which may also cause variation in products prepared from them. these fndings may be very useful for the identifcation of the species which may be useful to pharmaceutical industries for the quality control of the commercial samples. keywords: ayurvedic pharmacopoeia, commercial sample, quality control. introduction kuth (saussurea lappa), nisotha (operculina turpethum), akarakara (anacyclus pyrethrin) and chitrak (plumbago zeylanicum) are some common plants used in ayurvedic system of medicines and herbal drugs. [1,2,3,4] kuth is used for anti-infammatory, anti-ulcer, anticancer and hepatoprotective activities; nisotha is an important ingredient of ayurvedic formulation viz. avipattikara churna used for the treatment of gastric ulcer, gastrointestinal related disturbances and also used in treatment of piles, tumors and jaundice. [5,6,7] it also reported to have hepatoprotective and antimicrobial activities. [8,9] akarakara plant is used in traditional system of medicine as a tonic to the nervous system and also reported to have antibacterial, antidepressant and anti-infammatory activities. [10] chitrak is used against a number of ailments including skin diseases, diarrhea and leprosy. it also possesses antibacterial, antifungal, anti-carcinogenic, antitumor properties. [11] these plant drugs are available in local market of many cities in uttar pradesh, fromwhere they are utilized by local people as home remedies and by small scale ayurvedic drug manufacturers. these plant materials may vary in their quality and therefore in its therapeutic effect according to different places of collection, with different times in a year for collection, with collection at the same time and places but in different years and with different environmental factors surrounding the cultivation of a particular medicinal plant. [12] this difference may cause batch to batch variation or also may cause city to city variation in quality, safety and effcacy of same formulation. the objective of the present study is to evaluate the quality of the samples of same plant marketed into different area, on the standardization parameters given in ayurvedic pharmacopoeia.mishra, et al.: pharmacognostical, physicochemical and phytochemical studies 56 pharmacognosy journal  august 2011  vol 3  issue 24 materials and methods sample collection the samples of all four drugs were purchased from three different locations and labeled properly as per [table 1]. authantication samples were authenticated by dr. n. k. dubey, professor, department of botany, bhu, varanasi and a voucher specimen is preserved in herbarium section for future reference. organoleptic properties organoleptic properties were evaluated including appearance, size, color, taste and odour following the method described by wallis et. al, 1989. [13] for determining the odor of an innocuous material, small portion of the sample was placed in the beaker of suitable size, and examined by slow and repeated inhalation of the air over the material. if no distinct odor was perceptible, the sample was crushed between the thumb and index fnger, between the palms of the hands, using gentle pressure or if the material was known to be dangerous, by other suitable means such as pouring a small quantity of boiling water onto the crushed sample placed in a beaker. first, the strength of the odor was determined (none, weak, distinct, strong) and then the odor sensation (aromatic, fruity, musty, moldy, rancid, etc.) was studied. taste was distinctively classifed as aromatic, pungent, sweet, sour, astringent, mucilaginous, or bitter. microscopic study all samples were cleaned and boiled separately. their transverse sections were cut, stained, mounted and observed under microscope. [14] physicochemical analysis water soluble extractive value five gm. of the air-dried, coarsely powdered drug was macerated with 100 ml of chloroform water in a closed fask for 24 hours, shaked frequently during the frst 6 hours and allowed to stand for 18 hours. there often fltered rapidly evaporated 25 ml of the fltrate to dryness in a tarred fat bottomed shallow dish and was dried at 105 c and weighed. percentage of water soluble extractive was calculated with reference to the air dried drugs. ethanol soluble extractive value five gm. of the air dried coarsely powdered drug was macerated with 100 ml of ethanol of the specifed strength in a closed fask for 24 hours, shaked frequently during the frst 6 hours and allowed to stand for 18 hours. thereafter rapidly taking precautions against loss of ethanol, evaporated 25 ml of the fltrate to dryness in a tarred fat-bottomed shallow dish and was dried at 105c, and weighed. percentage of ethanol soluble extractive was calculated with reference to the air dried drug. total ash value accurately 2 g of the air dried crude drug was weighed in a tarred platinumor silica dish and incinerate at a temperature not exceeding 450 until free from carbon and then cold and weighed again. percentage of ash was calculated with reference to the air-dried drug. acid insoluble ash value accurately 2 g of air dried crude drug was weighed in a tarred platinum or silica dish and incinerated at temperature not exceeding 450c until free from carbon and then cold and weighed again. then the ash was boiled with 25 ml of 2m hydrochloric acid for 5 minutes. the insoluble matter was collected in a gooch crucible or on an ash less flter paper, washed with hot water, ignited, cold in desiccators and weighed. percentage of acid insoluble ash was calculated with reference to the air-dried drug. [15] qualitative phytochemical analysis to detect the presence of various phytoconstituents in these samples, phytochemical investigation was performed. [16,17] thin layer chromatography tlc of the alcoholic extract of ot1, ot2 and ot3 was developed on silica gel ‘g’ plate using toluene: ethylacetate (9:1) as mobile phase. vanillin-sulphuric acid reagent was table 1: location of sample procurement and coding sample name location of collection code for labeling kuth (saussurea lappa) dinanath gola market, varanasi sl1 buddha bazaar, moradabad sl2 chauk market, jhansi sl3 nisotha (operculina turpethum) dinanath gola market, varanasi ot1 buddha bazaar, moradabad ot2 chauk market, jhansi ot3 akarakara (anacyclus pyrethrin) dinanath gola market, varanasi ap1 buddha bazaar, moradabad ap2 chauk market, jhansi ap3 chitrak (plumbago zeylanicum) dinanath gola market, varanasi pz1 buddha bazaar, moradabad pz2 chauk market, jhansi pz3pharmacognosy journal  august 2011  vol 3  issue 24 57 mishra, et al.: pharmacognostical, physicochemical and phytochemical studies transverse section of root samples pz1, pz2 and pz3 showed outer most layer cork with 5-6 rows of light brown cells, rectangular in shape; starch grains compactly packed in the cortex region, phloem well developed with phloem fbers. groups of phloem fbers were present near the phloem. cambium single layered, xylemwas well developed with xylem vessels .medullary ray was single to multilayered and loaded with simple to compound starch grains [figure 2]. used as detecting agent .color, number and rf values of spots were compared with the standards given in ayurvedic pharmacopoeia. [18] results organoleptic properties the results of organoleptic evaluations are presented in [table 2] and [figure 1]. microscopic study transverse section of root samples sl1, sl2 and sl3 showed the presence of cork, 3-5 layered wide, secondary phloem consisting of mostly storage parenchyma, modularly rays multi seriate, resin canals throughout as cavities, xylem, fbers, vessels and xylem parenchyma groups were found scattered in the center and inulin was observed in storage parenchyma. transverse section of root samples ot1, ot2 and ot showed thin cork, consisting of 3-5 rows of brown cells, broad cortex consisted of clusters of parenchyma cells and resin canals. it was consisting of continuous circular zone of secondary phloem and dense secondary xylem, cleared radially in to wide four or fve fan shaped segments by narrow xylem rays. the wide vessels, calcium oxalate crystals in prisms and rosettes shape were also observed. the starch grains were found scattered in cortex, phloem parenchyma, xylem parenchyma and medullary ray cells. transverse section of root samples of ap1, ap2 and ap3 showed cork consisting of tabular cells, many of which developed as sclerenchyma, a few sclerenchymatous cells also found scattered in secondary cortex; developed secondary phloem; cambium 2-5 layered; secondary xylem very wide consisting of xylem vessels, tracheids and xylem parenchyma; vessels pitted; medullary rays numerous, running straight, bi to tri and multi seriate; oleo-resinous schizogenous glands found scattered in secondary cortex, secondary phloem and medullary rays. table 2: results of organoleptic properties test sample colour odour taste size and shape s l1 brown none blunt 10-15 cm. long, 1.5 cm broad, thick , cylindrical sl2 brown none blunt 5-8.5 cm. long, 1 cm broad, thick, cylindrical, hard sl3 grayish none blunt 5-10 cm. long, 1.5 cm broad, thick, cylindrical hard. ot1 dull grey none acrid 1-7 cm long, 1 cm diameter, cylindrical, longitudinal wrinkles. ot2 brown none acrid 1-10 cm long, 1 cm diameter, cylindrical, longitudinal wrinkles, thin rootlets. ot3 dull grey none acrid 1-15 cm long, 1 cm diameter, cylindrical, longitudinal wrinkles, ap1 dark brown none pungent 8-10 cm long, tapering, hairy rootlets. ap2 dark brown slightly pungent 5-10 cm long, tapering, hairy rootlets. aromatic ap3 grayish brown none blunt 5-10 cm long, tapering, hairy rootlets. pz1 brown none acrid 10-15 cm long, 1.2 cm dia. cylindrical pz2 brown disagreeable acrid 15-25 cm long, 1 cm dia., cylindrical pz3 brown none acrid 15-20 cm long, 1 cm dia. cylindrical. figure 1: photographs of sl1, ot1, ap1 and pz1mishra, et al.: pharmacognostical, physicochemical and phytochemical studies 58 pharmacognosy journal  august 2011  vol 3  issue 24 figure 2: microscopy of kuth (saussurea lappa), nisotha (operculina turpethum), akarakara (anacyclus pyrethrin), chitrak (plumbago zeylanicum)pharmacognosy journal  august 2011  vol 3  issue 24 59 mishra, et al.: pharmacognostical, physicochemical and phytochemical studies at rf 0.21, 0.41, 0.48, 0.58, 0.60, 0.62 and 0.96. ot3 showed seven spots appearing at rf 0.21, 0.41, 0.49, 0.58, 0.60, 0.92 and 0.97. the reported numbers of spots are seven with rf values 0.21, 0.41, 0.49 (all light violet), 0.58, 0.70, 0.90 and 0.97 (all violet) as per pharmacopoeia [figure 3]. discussion kuth (saussurea lappa), nisotha (operculina turpethum), akarakara (anacyclus pyrethrin) and chitrak (plumbago zeylanicum) are some of the very common plants used in ayurvedic system of medicine. evaluation of qualitative pharmacognostical parameters, physicochemical parameters and qualitative phytochemical screening can be useful in physicochemical analysis upon physicochemical analysis for all the roots samples on several parameters, outcome was matched with standard and presented in [table 3][table 4]. all the tests were performed in triplicate and result is presented in mean sem. qualitative phytochemical analysis upon phytochemical investigation of all the samples, different constituents were reported for all the samples. [table 5] thin layer chromatography analysis ot 1 showed fve spots of violet color appearing at rf. 0.20, 0.40, 0.49, 0.57 and 0.97. ot2 showed seven spots appearing table 4: results of ash value analysis total ash value standard value acid insoluble ash value standard value sl1  5.23 0.06% not more than 4% 2.82 0.40% not more than 1% sl2  3.61 0.12% 0.97 0.14% sl3  4.80 0.31% 1.06 0.23% ot1 12.58 0.45% not more than 10% 2.13 0.04% not more than 1.5% ot2 11.36 0.21% 2.53 0.16% ot3  8.31 0.19% 1.38 0.23% ap1  8.43 0.42% not more than 10% 2.05 0.05% not more than 2% ap2 10.54 0.31% 1.94 0.13% ap3  9.37 0.07% 1.28 0.06% pz1  2.60 0.14% not more than 3% 0.78 0.12% not more than 1% pz2  1.96 0.07% 0.54 0.15% pz3  3.81 0.27% 1.30 0.20% table 3: results of extractive value analysis water soluble extractive value standard value ethanol soluble extractive value standard value sl1 14.73 0.26% not less than 20 % 10.89 0.12% not less than 12% sl2 20.65 0.32% 13.06 0.29% sl3 18.87 0.14% 12.78 0.11% ot1  5.80 0.52% not less than 8% 10.45 0.26% not less than 10% ot2  8.11 0.17%  9.88 0.56% ot3  9.86 0.36% 10.67 0.12% ap1 12.87 0.08% not more than 22%  5.72 0.12% not less than 8% ap2 11.46 0.65%  7.56 0.32% ap3 15.73 0.06%  9.40 0.45% pz1 13.47 0.32% not less than 12% 16.10 0.14% not less than 12% pz2 15.20 0.22% 12.66 0.24% pz3 10.75 0.18%  8.42 0.33% table 5: results of qualitative phytochemical analysis sl1 sl2 sl3 ot1 ot2 ot3 ap1 ap2 ap3 pz1 pz2 pz3 alkaloid – – – – – – carbohydrates glycosides – – – tannins – – – – – – – – – – – – phenolic com. – – – – – – – – – flavnoids – – – – – – fixed oil – – – – – – – – – – – – saponins – – – – – – proteins – – – – – – – – – – – – steroids – – – – – – – – –mishra, et al.: pharmacognostical, physicochemical and phytochemical studies 60 pharmacognosy journal  august 2011  vol 3  issue 24 difference in storage conditions, collection process and age of plant. the qualitative pharmacognostical parameters were found uniform in all the samples. the variation was observed in physicochemical properties; sl1, sl3, ot1, ot2, ap1, ap2 and pz3 showed variation in all physicochemical parameters, from the standard value given in ayurvedic pharmacopoeia. the physicochemical parameters like extractive value, ash value indicates the quality and purity of drugs. extractive values are representative of the presence of the polar or nonpolar extractable compounds in a plant material. the total ash usually consists of carbonates, phosphates, silicates, and silica, which include both physiologic ash and nonphysiologic ash. the variation in these parameters from standard value indicates the low quality of the samples .in qualitative phytochemical analysis the sample were found uniform but in the thin layer chromatography results, absence of some spots in sample ot1 and ot2 was observed which indicates the absence of a particular group of compound in these samples. conclusion by the above study, it can concluded that there is absence of uniformity in the quality of same plant material marketed in different area, which can result into variation in quality, safety and effcacy of same formulation manufactured in different area. therefore emphasis is to be laid on the collection process, sources, storage conditions and standardization of raw materials in order to maintain the quality aspects of the product throughout worldwide. acknowledgement the authors are thankful to managing director, iftm, moradabad for providing all the laboratory facilities and chemicals to carry out this work. references 1. pandey mm, rastogi s, rawat aks. evaluation of pharmacognostical characters and comparative morphoanatomical study of saussurea costus (falc.) lipchitz and arctium lappa l. roots.nat prod sci 2007;13(4):304-310. 2. kumar abs, prabhakaran v, lakshman k , nandeesh r, khan s, tripathi m, et al. histological and physico-chemical evaluation of operculina turpethum linn. root. ethnobot leaflets 2009; 13:215-20. 3. sharma pv.dravyaguna vigyan-i. 4 th ed.varanasi.chaukhambha sanskrit sansthan; 1978, p-55-58. 4. gupta s, ahirwar d, jhade d, sharma nk, ahirwar b, muthal n. pharmacognostic standardization, physico and phytochemical evaluation of plumbago zeylanica linn. root. drug inv today 2010; 2(9):408-410. 5. pandey mm, rastogi s, rawat aks. saussurea costus: botanical, chemical and pharmacological review of an ayurvedic medicinal plant. j ethnopharmacol 2007; 110(3):379-390. 6. bhande rm, laakshmayya, kumar p, mahurkar nk, setty sr. pharmacological screening of root of operculina turpethum and its formulations. acta pharm sci 2006; 48:11-17. standardization of the marketed samples of these drugs. in the present study, all samples were collected fromdifferent parts of uttar pradesh, india to evaluate the uniformity of the quality of rawmaterials used by small scale ayurvedic drug manufacturers. the samples were found almost uniform in there organoleptic properties. the variation observed may be due to the figure 3: tlc image of nisotha (operculina turpethumpharmacognosy journal  august 2011  vol 3  issue 24 61 mishra, et al.: pharmacognostical, physicochemical and phytochemical studies 12. ekka nr, namdeo kp, samal pk. standardization strategies for herbal drugs-an overview. research j pharm and tech 2008; 1(4):310-312. 13. wallis te.text book of pharmacognosy. cbs publishers and distributors, delhi. 1989, p-356-549. 14. evans wc.trease and evans- pharmacognosy, 14 th edn: wb sacenders company ltd. london; 1996, p-194. 15. who-quality control methods for medicinal plants material.world health organization, geneva, ait us publisher and distributor delhi; 1998. p 10. 16. khandelwal kr. practical pharmacognosy. 12 th ed: pune:nirali prakashan; 2004. 149-160. 17. kokate ck, purohit ap, gokhale sb. pharmacognosy. 39 th ed: pune nirali prakashan; 2007. p 108-109. 18. anonymous, the ayurvedic pharmacopoeia of india, part i, vol. iii, govt. of india, m.h & f.w, dept. of ism & h; 2001. 7. kirtikar k, basu bd. indian medicinal plant 2 nd ed. vol. 1 international book distributors, rajpur road, dehradun india 1987, p-153. 8. suresh kumar sv, sujatha c, syamala j, nagasudha b, mishra sh. protective effect of root extract of operculina turpethum linn. against paracetamol-induced hepatotoxicity in rats. indian j pharm sci 2006; 68(1):32-35. 9. hussain t, arshad m, khan s, sattar h, qureshi ms. in vitro screening of methanol plant extracts for their antibacterial activity. pak j bot 2001; 43(1):531-538. 10. badhe sr, badhe rv, ghaisas mm, chopade vv, deshpande ad. evaluations of antidepressant activity of anacyclus pyrethrum root extract. int j green pharm. 2010; 4(2):79-82. 11. vishnukanta, rana ac. evaluation of anticonvulasant activity of plumbago zeylanica linn leaf extract. asian j pharm clinical res 2010; 3(1):76-78.o r i g i n a l a r t i c l e p h c o g j . 62 pharmacognosy journal  august 2011  vol 3  issue 24 *address for correspondence: e-mail: reshmajain_pharmacy@yahoo.com doi: 10.5530/pj.2011.24.12 for treatment of snake bite. [7] the fatty acid content of clitoria ternatea seeds includes palmitic, stearic, oleic, linoleic, and linolenic acids. [8,9,10] the seeds also contain a water-soluble mucilage, delphinidin 3, 3’, 5’-triglucoside useful as a food dye; beta-sitosterol. [11,12] c. ternatea have number of pharmacological activities such as possessing nootropic, anxiolytic, antidepressant, anticonvulsant, sedative, antipyretic, anti-infammatory and analgesic activities. [13,14,15] hypoglycemic activity of methanolic extract of c.ternatea roots in steptozotocin induced diabetic rat. enhance memory, and increase acetylcholine content and acetylcholinesterase activity in rats. [16,17,18] ethanol and benzene extract of clitoria ternatea seeds at doses 75 mg/ kg and 100 mg/kg inhibit clonidine induced catalepsy, milk induced eosinophili and leucocytosis in mice. [19,20] the main objective of present study was to perform pharmacognostic investigation and preliminary phytochemical screening of the stem of clitoria ternatea linn. material and methods collection and authentication the stem of clitoria ternatea l. was collected in bulk from local areas of vallabh vidyanagar in august 2006, identifed pharmacognostic evaluation and phytochemical studies on stem of clitoria ternatea linn. jain r.a. 1 *, shukla s.h. 2 1 research scholar, pharmaceutical quality assurance laboratory, centre of relevance and excellence in novel drug delivery system, pharmacy department, g. h. patel building, donor’s plaza, the maharaja sayajirao university of baroda, fatehgunj, vadodara, gujarat, india – 390 002. 2 associate professor, dept. of pharmacognosy, indukaka ipcowala college of pharmacy, new vallabh vidyanagar.dist. anand a b s t r a c t pharmacognostic evaluation is the frst and foremost step to determine identity and to assess the quality and purity of the crude drug. the genus “clitoria” includes about 48 species under the family “fabaceae”. clitoria ternatea a very common garden fowering plant is found all over india especially in southern india. c. ternatea have reported to possess a number of pharmacological activities such as nootropic, anxiolytic, anticonvulsant, sedative, antipyretic, anti-infammatory and analgesic. the roots, stems and fowers are recommended for treatment of snake bite. the current study describes some pharmacognostical and preliminary phytochemical investigations undertaken on the stem of one of those species namely clitoria ternatea. transverse section of clitoria ternatea linn. stem shows cork, cortex, pericyclic fbers, xylem parenchyma, xylem fbers and medullary rays. quantitative pharmacognostic analysis of the powder of the stem revealed moisture content of 5.92 0.23%, total ash of 3.76 0.32%, acid-insoluble ash of 11.6 0.09%, alcohol extractive value of 9.84 0.19%, and water extractive value of 24.8 0.22%. key words: clitoria ternatea., fabaceae , pharmacognosy, preliminary phytochemical screening introduction clitoria ternatea l. (family: fabaceae), commonly known as “aparajita” is widely used as a substitute of shankhpushpi. it is a perennial twinning herb, steams are terete, more or less pubescent and leaves are imparipinnate, elliptic oblong, obtuse, glabrous or with a few short appressed hairs, fowers are axillary and solitary with pedicel. [1,2,3] there are two varieties of clitoria ternatea white-fower and blue fower varieties. the leaves of both varieties contain an ester and resin glycosides. seeds contain a fxed oil, a bitter acrid resin, tannic acid, glucose. root bark contains starch, tannins and resins. [4,5] the roots have a sharp bitter taste and cooling, laxative, diuretic, anthelmintic, anti-infammatory properties and useful in severe bronchitis, asthma, hectic fever and also used in ascites and abdominal enlargement (ayurveda). [6] the infusion of root bark is used in gonorrhea and irritation of the bladder and urethra. the seeds are powerful cathartic, laxative and aperients actions in combinations with ginger powder. the roots, stems and fowers are recommendedpharmacognosy journal  august 2011  vol 3  issue 24 63 jain and shukla: pharmacognostic evaluation and phytochemical studies on stem of clitoria ternatea linn. results morphological evaluation the stems were feshy, long, slender and fexible with 0.5‑3 m long, hairy or bald, sometimes somewhat erect stem. (figure 1) fracture of the dried stem was fbrous and 15-20 cm in length, 5-10 mm in width. and authenticated by taxonomist, bioscience department, sardar patel university, vallabh vidyanagar. remaining material was cut into small pieces and shade dried. the dried plant material was powdered using mechanical grinder, and sieved by using sieve no. 60. then the fnal uniform powder was used for the extraction of active constituents of the plant. morphological and microscopical studies the morphology of the stem was studied according to standard methods. transverse sections of the stem were taken manually, cleared, stained and mounted and representative photographs of sections were taken with the help of digital microscope. the powder characteristics were studied according to standard methods. [21,22,23] determination of physicochemical parameters physico-chemical parameters i.e. percentage of moisture content, percentage of ash values and extractive values were performed according to the offcial methods. [23,24] preliminary phytochemical screening the powdered plant was extracted with petroleum ether (60-80 c), toluene, chloroform, acetone, methanol and distilled water using soxhlet apparatus. all the extracts were screened qualitatively for the presence of various groups of phytoconstituents using different chemical tests. [23,24,25] figure 1: entire plant of clitorea ternatea figure 2: transverse section of clitoria ternatea stem (p.f. pericyclic fibres, m.r. medullary rays, ph. phloem, x.v. xylem vessels)jain and shukla: pharmacognostic evaluation and phytochemical studies on stem of clitoria ternatea linn. 64 pharmacognosy journal  august 2011  vol 3  issue 24 microscopical studies of the powder are shown in figure 3 and contained xylem fbers (a), xylem vessels (b), cork (c), cork with cortex (d) and pericyclic fbers (e). starch and crystals of calcium oxalate were not observed. physicochemical parameters the proximate analysis result showed that the moisture content, total ash value, acid insoluble ash value, alcohol soluble extractive value and water soluble extractive value were 5.92 0.23%, 3.67 0.32%, 0.12 0.09%, 9.84 0.19%, and 24.8 0.22% respectively (table 1). successive solvent extractions were shown in percentage of yield along with physical appearance. the percentage yield values for petroleum ether, toluene, chloroform, acetone and methanol were 1.62%, 1.35%, 6.07%, 3.02%, and 6.01% respectively (table 2). all extracts were then subjected to qualitative examination to detect the presence of phytoconstituents. the preliminary phytochemical studies revealed that petroleum ether extract showed the presence of fxed oil and fats, toluene microscopical evaluation transverse section of clitoria ternatea linn. stem (figure 2) shows 1-2 layers of thin walled rectangular, tangentially elongated cork cells. the cortex followed by the cork was composed of 8-9 rows of thin walled parenchymatous cells, some of which contained chlorophyll. the cortex was followed by a continuous layer of lignifed pericyclic fbre, the cells of which were polygonal, lignifed and thick walled. the pericyclic fbres were followed by phloemwhich consisted of phloem parenchyma. xylem as a continuous layer and wedge shaped was seen which consisted of large xylem vessels, xylem parenchyma and xylem fbres. xylem parenchymatous cells were pitted and form the 1-5 serriate medullary rays. the centre of the stem was composed of the pith in which the cells were thin walled parenchymatous in nature. starch and calcium oxalate were not observed. powder characters the powder was green in colour and odour and taste are characteristic. texture is fbrous. figure 3: powder characteristics of clitoria ternatea stempharmacognosy journal  august 2011  vol 3  issue 24 65 jain and shukla: pharmacognostic evaluation and phytochemical studies on stem of clitoria ternatea linn. acids, saponin, phenolics (table 3). the extracts which showed the presence of phytoconstituents of interest were further subjected to tlc and results are tabulated in table 4. conclusion the various morphological, microscopical, physicochemical standards developed in this study will help for botanical identifcation and standardization of clitoria ternatea. further, the authentic plant material can be explored for its pharmacological and phytochemical potential. references 1. shah gl, flora of gujarat state, sardar patel university, v.v.nagar, 1978;(pt 1):189-190. 2. maheshwari jk, flora of delhi, csir, new delhi, 1963:131-132. 3. verma sk, flora of bhagalpur, 1969;(pt 1): 201. 4. rastogi rp, mehroltra bn, compendium of indian medicinal plants, central drug research institute, lucknow, 1993; vol. i, p. 114-115. 5. chatterjee a, chandra ps. the treatise on indian medicinal plants, national institute of science communication, csir, new delhi, 1992; vol. 2, p. 79. 6. terahara, n, saito n, honda t, toki k, osajima y, phytochemistry, volume 29(3):, p. 949-953; 1990. 7. kirtikar kr, basu bd. indian medicinal plants. vol. i, 2 nd ed., dehradun: international book distributor; 1995; page no. 802-804. 8. debnath nb, chakravarti d, ghosh a, chakravarti rn. fatty acids of clitoria ternatea seed oils. j of the institution of chemists (india). 1975; 47:253-5. 9. husain s, devi ks. fatty acid composition of three plant species: clitorea ternatea, mandulea suberosa and ruta chalapensis. j of the oil technologists association of india. 1998; 30:162-4. 10. joshi ss, shrivastava rk, shrivastava dk. chemical examination of clitoria ternatea seeds. j of american oil and chemical society. 1981; 58:714-5. 11. macedo mlr, xavier-filho j. purification and partial characterization of trypsin inhibitors from seeds of clitoria ternatea. j of the science of food and agriculture. 1992; 58:55-8. 12. sinha, a. β-sitosterol from the seeds of clitoria ternatea. current science, 1960; 29:180-1. 13. jain nn, ohal cc, shroff sk, bhutada rh, somani rs, kasture vs, kasture sb. clitoria ternatea and the cns. pharmac biochem behav. 2003; 75:529-36. 14. kulkarni c, pattanshetty jr, amruthraj g. effect of alcoholic extract of clitoria ternatea linn. on central nervous system in rodents. indian j exp biol. 1988; 26:957-60. extract showed the presence of fxed oils, chloroform extract contained carbohydrates and saponins, acetone extract contained carbohydrates, saponins and phenolics, while methanolic extract contained carbohydrate, proteins and amino table 1: evaluation of stems of clitoria ternatea linn. parameters value obtained on dry weight basis (% w/w)* moisture content 5.92 0.23 total ash value 3.67 0.32 acid insoluble ash 0.12 0.09 alcohol soluble extract 9.84 0.19 water soluble extract 24.8 0.22 *average of three reading sem on of stems of clitoria ternatea linn. table 2: successive solvent extraction of stems of clitoria ternatea linn. solvent used color & consistency average extractive values on dry weight basis (% w/w) petroleum ether green solid mass 1.62 toluene dark green solid mass 1.35 chloroform blackish green residue 6.07 acetone dark brown semisolid mass 3.02 methanol reddish brown solid mass 6.1 table 3: chemical examination of various extracts of stems of clitoria ternatea linn. constituents extract p t c a m alkaloids – – – – – carbohydrates – – proteins & amino acids – – – – saponin – – fixed oil/ fat – – – gums/ mucilage – – – – – flavonoids – – – phenolic – – – p petroleum ether extract; t toluene extract; c chloroform extract; a acetone extract; m methanol extract. table 4: tlc screening of various crude drug extract of clitoria ternatea linn. solvent system used detection reagent observation inference p t c a m toluene : ethyl acetate (93:7) vs reagent red/yellow/brown/blue-green essential oil – – – as reagent pink/green essential oil – – – ethyl acetate : methanol : water (100:13.5:10) as reagent red/yellow/brown/blue-green bitter principle – – – vs reagent blue saponin – – np/peg/ and uv yellow/green/orange flavonoid – – – 10% koh yellow colour anthrone – chloroform : methanol : formic acid (10:0.3:0.1) lb reagent dark green phytosterol – – – – (p petroleum ether extract; t toluene extract; c chloroform extract; a acetone extract; m methanol extract, vs vanillin sulphuric acid, as anisaldehyde sulphuric acid, lb libermann burchard, indicates presence of constituents. – indicates absence of constituents)jain and shukla: pharmacognostic evaluation and phytochemical studies on stem of clitoria ternatea linn. 66 pharmacognosy journal  august 2011  vol 3  issue 24 20. taur dj, patil ry. effect of clitoria ternatea seeds extract on milk-induced leucocytosis and eosinophilia in mice. journal of pharmacy research. 2009; 2(12):1839-41. 21. mukherjee pk, quality control of herbal drugs, business horizon’s pharmaceutical publishers, new delhi) p. 138-141, 2002. 22. wallis t.e., practical pharmacognosy, 6 th edition, (j. & a. churchill ltd., london) 115-116, 179-182, 1953. 23. khandelwal kr. practical pharmacognosy technique and experiments. 13 th ed. nirali prakashan, pune, 146-159, 2005. 24. anonymous. the indian pharmacopoeia. vol. ii. the controller of publications, new delhi, a-53, a-54; a-89, 1996. 25. evans wc. trease and evans pharmaconosy. 15 th ed. w.b. sounders company ltd, london. 224, 230, 336, 541-545, 2005. 15. parimaladevi b, boominathan r, mandal sc.anti-inflammatory, analgesic and antipyretic properties of clitoria ternatea root. fitoterapia. 2003; 74:345-9. 16. rai ks, murthy kd, karanth ks, rao ms. clitoria ternatea linn. root extract treatment during growth spurt period enhances learning and memory in rats. indian j physiol pharmac. 2001; 45:305-13. 17. rai ks, murthy kd, karanth ks, nalini k, rao ms, srinivasan kk. clitoria ternatea root extract enhances acetylcholine content in rat hippocampus. fitoterapia. 2002; 73:685-9. 18. taranalli ad, cheeramkuczhi tc. influence of clitoria ternatea on memory and central cholinergic activity in rats. pharm biol. 2000; 38:51-6. 19. taur dj, patil ry, khalate ah. phytochemical investigation and evaluation of clitoria ternatea seeds on clonidine induced catalepsy in mice. pharmacology online, 2009; 3:215-220.pharmacognosy journal  august 2011  vol 3  issue 24 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: dr. debdulal banerjee, department of botany and forestry, vidyasagar university, midnapore(w)-721 102, india. telefax: 91-3222-275329 e-mail: debu33@gmail.com doi: 10.5530/pj.2011.24.13 reaction frequently associatedwith painwhich leads to increase of vascular permeability, protein denaturation and alteration of membrane integrity. [11] the mast cells plays important role in infammatory events. [12] hence, the present work has been highlighted the effect of hydroalcoholic extract of b. prionitis on infammatory events such as membrane stabilization and mast cell protection. materials and methods plant material b. prionitis l. (acanthaceae) whole plant was procured from local vendor and sample was authenticated from the department of botany and forestry, vidyasagar university, india. voucher specimen (no: vbdb/10/17) was preserved in the department of botany and forestry, vidyasagar university, india. preparation of plant extract the air dried plant material (300 g) was powdered in grinder and extracted with hydro-methanol (70%) by coldmaceration process for 15 days to obtain crude extract. the crude extract was fltered through whatman no. 1 flter paper and fltrate was evaporated and dried in a rotary evaporator followed by lyophilization. the percentage yield of the extract was calculated. the dried extract was dissolved in distilled water before use. mast cell stabilization and membrane protection activity of barleria prionitis l. a.k. maji 1 , s. bhadra 2 , s. mahapatra 1 , p. banerji 3 , d. banerjee 1 * 1 department of botany and forestry, vidyasagar university, midnapore (w)-721 102, india; 2 school of natural product studies, jadavpur university, kolkata, india; 3 ulysses research foundation, kolkata-700 029, india. a b s t r a c t barleria prionitis is a well-known medicinal plant, traditionally used for the treatment of various infammatory diseases because of its anti-infammatory activity. but the effect of the extract on infammatory mediators and cell membrane in response to toxic chemicals was not studied before. here, we evaluated the membrane stabilization and mast cell protection activity of hydroalcoholic extract of b. prionitis whole plant. the hydroalcoholic extract signifcantly inhibited the hypo-saline induced erythrocyte membrane hemolysis and the compound 48/80 induced mast cells degranulation in a dose dependent manner. the extract at dose concentration of 10 µg/ml, reduced the rat mesenteric mast cells degranulation up to 64.91% and prevented hypotonic solution induced hemolysis of rat erythrocytes by 27.10%. these fndings clearly validate the anti-infammatory activity of b. prionitis whole plant extract and provide support for traditional usage for infammatory disorders. key words: barleria prionitis, anti-infammatory, phytochemical studies introduction barleria prionitis l. (family: acanthaceae) is a well-known medicinal plant traditionally used inayurveda for the treatment of bronchial asthma, rheumatic affections, infammation, glandular swelling and this plant is found throughout the humid climatic zone of india. [1] the plant reported to contain barlerin, acetylbarlerin, scutellarein-7-neohesperidosideetc, balarenone, pipataline, lupeol, prionisides, barlerinoside, shanzhisidemethyl ester, lupulinoside verbascoside, 6-o-trans-p-coumaroyl-8-o-acetylshanzhiside methyl ester and its cis‑isomer. [1-3] the plant was reported to show antibacterial, anti-arthritic, anti-infammatory, [4-5] hepatoprotective, [6] antioxidant, [7] antidiabetic, [8] anthelmintic, [9] antiviral, [2] and antifertility activities. [10] with this scope we have studied the erythrocytes membrane stabilization and mast cell protection activity of hydroalcoholic extract of b. prionitis whole plant against toxicants. infammation is a complex immunologicmaji, et al.: mast cell stabilization by barleria prionitis l. 68 pharmacognosy journal  august 2011  vol 3  issue 24 animals male wistar rats (200-225 g) were obtained from the authenticated vendor and the animal were housed in the animal facilities at vidyasagar university. the animals were maintained in a laminar air-fow room at a temperature of 22 1 ºc under the relative humidity of 55 10% with standard food and water ad libitum throughout the study. the animal studies were done in accordance to the institutional guidelines and the protocols were approved by the institutional ethical committee for animal care and use at vidyasagar university before study. reagents compound 48/80 (c 48/80) was purchased from sigma-aldrich (usa), o-toludine blue was obtained from spectrochem pvt. ltd, india, disodium cromoglycate (dscg) from cipla, india. indomethacin (indo) was obtained from genpharma international pvt. ltd. india. all other analytical grade solvents and reagents utilized in this study were purchased from the local vendors. phytochemical screening the dried extract was subjected for the colour reactions to screen the nature of chemical components present in the extract. small portion of dried extract was dissolved in purifed water. the screening of phytochemical classes was performed according to the standard phytochemical procedures. [13-15] tests for alkaloids the aqueous extract (2 ml) incubated with few drops of dilute hydrochloric acid in four different test tubes and then fltered. each fltrate was tested with alkaloidal reagents either of the mayer’s (cream precipitate) or dragendorffs (orange brown precipitate) or hager’s (yellow precipitate) or wagner’s (reddish-brown precipitate) reagents. development of colour precipitates was observed in each test tube. tests for glycosides the dried extract (50 mg) incubated with concentrated hydrochloric acid for 2 h in a water bath and fltered. filtrate was subjected for borntrager’s test (pink colour) and legal’s test (pink colour). development of pink colour in both tests indicated the presence of glycosides. tests for saponins the dried extract (50 mg) dissolved in 20 ml of distilled water and shaken in graduated cylinder for 15 minutes. the formation of a 1-2 cm layer of foam indicated the presence of saponins. tests for favonoids the dried extract dissolved in distilled water and fltered. 5 ml of dilute ammonia solution was added with some portion fltrate followed by the addition of concentrated h 2 so 4 . formation yellow colouration indicated the presence of favonoids and colouration disappeared on standing. tests for terpenoids salkowski test was performed to determine the presence of terpenoids. five ml of aqueous extract was mixed with 2 ml of chloroform followed by the careful addition of 3 ml concentrated h 2 so 4 to form a layer. a reddish brown colour was formed in the inter face indicated the presence of terpenoids. test for tannins the dried extract (50 mg) was dissolved in 20 ml of distilled water and fltered. with the fltrate few drops of 0.1% ferric chloride was added and the formation brownish-green or a blue-black colouration indicated the presence of tannins. test for steroids the dried extract (50 mg) was dissolved in 2 ml of acetic anhydride. with this, 1-2 drops of concentrated h 2 so 4 was added slowly. the colour of reaction mixture was changed from violet to blue or green colour indicated the presence of phytosteroids. membrane stabilization activity in vitro membrane stabilization was performed using the method of hypotonicity induced rat erythrocyte hemolysis described by shinde and his co-workers (1999). [16] whole blood of rats was collected by retro-orbital puncture using heparinized syringe. the whole blood samples were centrifuged at 3000 rpm for 10 minutes. the erythrocytes pellet was collected and washed by re-suspending with isotonic buffered solution (154 mnacl in 10m phosphate buffer ph 7.4). the procedure repeated three times and each time erythrocytes were centrifuged at 3000 rpm for 10 minutes. the test sample consisted of varying concentrations of extract (10, 100 and 1000 µg/ml) or indo (10 µg/ml) and 0.50 ml of stock erythrocyte suspension in 4.0 ml of hyposaline solution. the control sample consisted of 0.5 ml of stock erythrocyte suspension with hypotonic buffered saline solution. the reaction mixtures were incubated at 56 1 c for 30 min and centrifuged at 3000 rpm for 10 minutes. the absorbance of the supernatant was measured at 540 nm. the inhibition percentage of erythrocyte haemolysis was calculated according to the method of shinde et al (1999). [16] inhibition of erythrocyte heamolysis (%) [ od1 od2 od1 ] 100 where: od1 optical density of control od2 optical density of test samplespharmacognosy journal  august 2011  vol 3  issue 24 69 maji, et al.: mast cell stabilization by barleria prionitis l. statistical analysis all the results were expressed as mean sem of the number of the experiments. statistical signifcance was performed by one-way anova followed by bonferroni’s multiple comparison or dunnett’s multiple comparison test wherever applicable. the p values 0.05 were considered as statistically signifcant. the data analysis was performed using graph pad prism software. results phytochemical screening the vacuum-dried extracts gave 4.15% yield of whole plant extract. the results of preliminary phytochemical analysis of hydroalcoholic extract of whole plant are shown in table-1. the extract gave positive results for the presence of glycosides, saponins, favonoids, steriods and tannins. effect on protection of mast cells the c 48/80 at dose concentration of 0.8 μg/ml signifcantly degranulated rat mesenteric mast cells (93.08 4.59%, p 0.001) in compared to the control (2.65 0.84%). the hydroalcoholic extract showed dose dependent inhibition against c 48/80 induced mast cells degranulation as shown in figure-1a and 1b. at a dose concentration of 10 μg/ml mast cells protecting activity the protection of mast degranulation induced c 48/80 was performed using the method describe by norton (1954). [17] the overnight fasted male wister rats were anesthetize with excess ether and cut the whole abdomen to expose the intestine. the intestinal mesenteries were collected in ringer-locke solution and the mesenteries were cut into small pieces. the pieces of mesentery were placed in different petri dishes consisting of different concentrations of extract (10, 100 and 1000 µg/ml) or dscg (10 µg/ml) prepared in ringer locke solution. the petri-dishes were incubated with toxicant c 48/80 (0.8 μg/ml) at 37 ºc for 30 minutes. two sets of control were prepared by incubating pieces mesentery with or without toxicant c 48/80 in ringer-locke solution. the tissues were placed on a clean microscopic slide and remove the fatty layer carefully. the trimmed tissues were stained with 4% formaldehyde solution containing 0.1%o-toludine blue for 30 minutes. the tissues were then de-stained by successive washing with acetone and xylene for 5 minutes. the stained mesentery pieces were examined under digital light microscope at 100x magnifcation. the mast cell was considered as degranulated if 4-5 granules were present around the cells. the percentage of degranulated and intact mast cells was calculated on the basis of counting 100 mast cells for each sample. for each set of test sample including control 6-8 pieces of mesentery were observed. table 1. phytochemical constituents of hydroalcoholic extract of b. prionitis whole plant test observation inference tests for alkaloids no colour formation with dragendorffs, hager’s, mayer’s, wagner’s alkaloidal reagents. absence of alkaloids tests for glycosides pink colour with borntrager’s and legal’s test presence of glycosides tests for saponins formation of foam upon shaking. presence of saponins tests for flavonoids yellow colouration up on the addition of dilute ammonia and concentrated h 2 so 4 & colouration disappeared on standing. presence of flavonoids tests for terpenoids (salkowski test) no reddish brown colour formation in inter face of chcl 3 & h 2 so 4. absence of terpenoids test for tannins formation of blue-black colour with fecl 3 solution. presence of tannin test for steroids change of violet to blue or green colour with acetic anhydride & h 2 so 4 . presence of steroids figure 1: mast cells protection by b. prionitis hydroalcoholic extract a. degranulated mast cells in control; b. intact mast cells in hydroalcoholic extract treated sample.maji, et al.: mast cell stabilization by barleria prionitis l. 70 pharmacognosy journal  august 2011  vol 3  issue 24 release histamine, heparin, proteases and other mediators to produce infammatory effects. [20] these effects can be manipulated therapeutically by regulating the function of these mediators. constituents from natural resources including plant origin may able to modulate such effects. in this experiment it was found that hydroalcoholic extract of b. prionitis whole plant provide signifcant mast cells stabilization and membrane protection. conclussion in conclusion, the result of the present investigation suggested that hydroalcoholic extract of b. prionitis whole plant has signifcant mast cell stabilizing and membrane protection activities. these effects validate the earlier reported anti-infammatory activity and the traditional usage in infammatory disorders. these effects could be the mechanism of action of anti-infammatory action of this plant and this may due to the presence of glycosides, saponins, favonoids, steroids, tannins in the extract. thus, this plant may offer benefcial effects in the management of infammatory conditions. however, further studies should be performed on isolated chemical compounds to establish the chemical responsible for the activity. acknowledgements authors are thankful to the ulysses research foundation, india for providing fnancial assistance to carry out this research work. extract reduced the degranulation of mast cells up to 64.91 2.64% which was comparable with the standard dscg (19.32 6.92%, 10 μg/ml) with signifcance of p 0.001, whereas 100 μg/ml of extract reduced the degranulation up to 31.42 6.79% (p 0.05) (figure 2). effect on membrane stabilization the extract at dose concentration of 10 μg/ml provided signifcant membrane protection (27.10 3.18%) and the results were comparable with known standard drug, indo (61.29 6.37%, 10 μg/ml) with signifcance of p 0.01, whereas 100 μg/ml extract showed membrane protection by 32.48 5.26% (p 0.01) (figure 3). discussion the cell membrane integrity is essential for the normal growth, development and function of the cells. exposure of erythrocytes to injurious medium, hyposaline solution, leads to rupture of its membrane followed by haemolysis and oxidation of haemoglobin. [18] the lysis of such bio-membranes leads to the generation of free radicals which enhanced the secondary cellular damage. [19] thus, the compound with membrane-stabilizing property could offer signifcant protection of cellular membrane from toxic substances and interfere in early phase infammatory reactions via inhibiting the formation of infammatory mediators. [16] the mast cells have an important role in the development of infammatory anaphylactic and allergic reactions. during anaphylactic reaction ige degranulated the mast cells to control c48 / 80 4 (µg / ml) extract 10 (µg / ml) extract 100 (µg / ml) extract 1000 (µg / ml) 0 20 40 60 80 100 a a a b b % of degranulated mast cells dscg 10 (µg / ml) figure 2: mast cell protection activity of b. prionitis extract the bars of the graph represents mean sem of three observations (n 3). statistical analysis was done through one-way analysis of variance (anova) followed by bonferroni’s multiple comparison test. control vs c 48/80, a p 0.001; c 48/80 vs dscg (a p 0.001) and extract treated groups (a p 0.001; b p 0.01). extract 1000 (µg/ml) indo 10 (µg/ml) extract 10 (µg/ml) extract 100 (µg/ml) 0 20 40 60 80 b b c % of erythrocyte heamolysis figure 3: membrane stabilization activity of b. prionitis extract the bars of the graph represents mean sem of three observations (n 3). statistical analysis was done through one-way analysis of variance (anova) followed by dunnett’s multiple comparison test. indo vs extract treated groups (b p 0.01; c p 0.05).pharmacognosy journal  august 2011  vol 3  issue 24 71 maji, et al.: mast cell stabilization by barleria prionitis l. 11. umapathy e, ndebia ej, meeme a, adam b, menziwa p, nkeh-chungag bn, et al. an experimental evaluation of albuca setosa aqueous extract on membrane stabilization, protein denaturation and white blood cell migration during acute inflammation. j med plants res. 2010 may 4; 4(9):789-95. 12. galli sj, grimbaldeston m, tsai m. immunomodulatory mast cells: negative, as well as positive, regulators of immunity. nat rev immunol. 2008 jun; 8(6):478-86. 13. evans wc. trease and evans’ pharmacognosy. 13 th ed. bailliere tindall: london, 1989. 14. harborne jb. phytochemical methods. 1 st ed. chapman and hall ltd: london, 1973. 15. mukherjee pk. quality control of herbal drugs - an approach to evaluation of botanicals. 1 st ed. business horizons: india, 2002. 16. shinde ua, phadke as, nair am, mungantiwar aa, dikshit vj, saraf vo. membrane stabilizing activity–a possible mechanism of action for the anti-inflammatory activity of cedrus deodara wood oil. fitoterapia. 1999 june 1; 70(3):251-7. 17. norton s. quantitative determination of mast cell fragmentation by compound 48/80. br j pharmacol chemother. 1954 dec; 9(4):494-7. 18. ferrali m, signorini c, ciccoli l, comporti m. iron release and membrane damage in erythrocytes exposed to oxidizing agents, phenylhydrazine, divicine and isouramil. biochem j. 1992 jul 1; 285 (pt1):295-301. 19. halliwell b, hoult jr, blake dr. oxidants, inflammation and anti-inflammatory drugs. faseb j. 1988 oct; 2(13):2867-73. 20. subramoniam a, evans da,valsaraj r, rajasekharan s, pushpangadan p. inhibition of antigen-induced degranulation of sensitized mast cells by trichopuszeylanicus in mice and rats. j ethnopharmacol. 1999 dec 15; 68(1-3):137-43. references 1. khare cp. indian medicinal plants: an illustrated dictionary. 1 st ed. springer: usa, 2007. 2. chen jl, blance p, stoddart ca, bogan m, rozhon ej, parkinson n, et al. new iridoids from the medicinal plant barleria prionitis with potent activity against respiratory syncytial virus. j nat prod. 1998 oct; 61(10):1295-7. 3. ata a, kalhari ks, samarasekera r. chemical constituents of barleria prionitis and their enzyme inhibitory and free radical scavenging activities. phytochem lett. 2009 feb 19; 2(1):37-40. 4. singh b, bani s, gupta dk, chandan bk, kaul a. anti-inflammatory activity of ‘taf’ an active fraction from the plant barleria prionitis linn. j ethnopharmacol. 2003 apr; 85(2-3):187-93. 5. amoo so, finnie jf, van staden j. in vitro pharmacological evaluation of three barleria species. j ethnopharmacol. 2009 jan 21; 121(2):274-7. 6. singh b,chandan bk,prabhakar a,taneja sc,singh j,qazi gn.chemistry and hepatoprotective activity of an active fraction from barleria prionitis linn. in experimental animals. phytother res. 2005 may; 19(5):391-404. 7. jaiswal sk, dubey mk, das s,verma ar, rao cv. a comparative study on total phenolic content, reducing power and free radical scavenging activity of aerial parts of barleria prionitis. int j phytomed. 2010; 2:155-9. 8. dheer r, bhatnagar pa. study of the antidiabetic activity of barleria prionitis linn. indian j pharmacol. 2010 apr; 42(2):70-3. 9. chavan cb, hogade mg, bhinge sd, kumbhar m, tamboli a. in vitro anthelmintic activity of fruit extract of barleeia prionitis linn. against pheretima posthuma. int j pharmacy pharm sci. 2010 feb 28; 2(3):49-50. 10. gupta rs,kumar p,dixit vp,dobhal mp.antifertility studies of the root extract of the barleria prionitis linn in male albino rats with special reference to testicular cell population dynamics.j ethnopharmacol.2000may;70(2):111‑7.o r i g i n a l a r t i c l e p h c o g j . 72 pharmacognosy journal  august 2011  vol 3  issue 24 *address for correspondence: mohammad a. rashid; tel.: 880-2-9661900, extn. - 8137; 8131; 8030 fax: 880-2-8615583 e-mail: rashidma@univdhaka.edu doi: 10.5530/pj.2011.24.14 commonly known as snake plant or mother in-law’s tongue is an evergreen herbaceous perennial plant which is found throughout malaysia [4] where it has been traditionally used for the treatment of ear pain, swellings, boils and fever. phytochemical screening with this plant has shown to contain carbohydrates, saponins, glycosides [5] and steroids. [6] hydnocarpus kurzii (king warb) (family: achariaceae also placed in: flacourtiaceae [7-8] ) is well known for its chaulmoogra oil, which is expressed from the dried ripe seeds [9] and is also known as hydnocarpus oil, kalaw tree oil, leprosy oil. [10] the oil and the crushed seeds have long been used in southeast asia to treat various skin diseases like scabies, eczema, psoriasis, scrofula, ringworm, and intestinal worms and it has been shown that the active principles of the oil (hydnocarpic and chaulmoogric acids) exhibited strong antibacterial activity. for this reason h. kurzii is employed in hindu medicine to treat leprosy. the bark contains principles capable of reducing fevers. seeds are usually applied externally as a dressing for skin diseases; combined with walnut oil and pork lard for ringworm; with calomel in vitro antimicrobial screening of four reputed bangladeshi medicinal plants md. al amin sikder a , a. k. m. nawshad hossian a , abu bakar siddique a , mehreen ahmed b , mohammad a. kaisar c , and mohammad a. rashid c, * a department of pharmacy, state university of bangladesh, dhaka-1205, bangladesh b department of pharmacy, the university of asia pacific, dhaka-1209, bangladesh c department of pharmaceutical chemistry, faculty of pharmacy, university of dhaka, dhaka-1000, bangladesh a b s t r a c t the antimicrobial activity of extractives of different plants has been recognized for many years. in present study the crude methanolic extracts and their kupchan partitioning fractions of four medicinal plants of bangladesh namely, sansevieria trifasciata (fam: asparagaceae), justicia gendarussa (fam: acanthaceae), hydnocarpus kurzii (fam: achariaceae) and kigelia pinnata (fam: bignoniaceae) were investigated for their in vitro antimicrobial properties. all fractions were tested against 11 different gram positive and gram negative bacteria by the disc diffusion technique for bacteria, where kanamycin (30 μg/disc) disk used as standard. among the extractives, the methanol extract and their pet-ether, carbon tetrachloride and chloroform soluble kupchan fractions of leaf extract of h. kurzii, and the aerial part extract of s. trifasciata showed signifcant antibacterial activity, where as chloroform soluble extracts of leaves of h. kurzii revealed highest activity against vibrio mimicus (15.00 mm) and the methnol extract of whole plant of s. trifasciata demonstrated highest activity against v. mimicus (14.67 mm). the chloroform soluble fractions of j. gendarussa also showed mild to moderate antimicrobial activity with zone of inhibition ranging from 8.33-13.00 mm, in which highest activity was seen against shigella boydii (13.00 mm). all the extractives of k. pinnata, on the other hand, demonstrated mild antimicrobial activity, where highest zone of inhibition was displayed by the chloroform soluble extract against s. boydii (11.00 mm) and pseudomonas aeruginosa (11.00 mm). key words: antimicrobial activity, disc diffusion, hydnocarpus kurzii, justicia gendarussa, kigelia pinnata and sansevieria trifasciata. introduction the shade-loving, quick-growing, evergreen plant justicia gendarussa (burm f.) (family: acanthaceae), is mostly found in moist areas. it is believed to be native to china and is distributed widely across india, sri lanka, and malaysia. [1] it is an erect, branched, smooth undershrub, 0.8-1.5 meters in height with long leaves (7 to 14 cm). [2] the plant is used in traditional medicinal practice for chronic rheumatism, infammations, bronchitis, vaginal discharges, dyspepsia, eye diseases and fever. the plants of this genous are known to contain lignans, naturally occurring phenolic dimers and triterpenoids. [3] sansevieria trifasciata (prain) (family: ruscaceae),pharmacognosy journal  august 2011  vol 3  issue 24 73 sikder, et al.: in vitro antimicrobial screening of four reputed bangladeshi medicinal plants specimens (dacb 35489, 35490, 35491 and 34998, respectively) have been deposited in bangladesh national herbarium for future reference. leaves and bark of j. gendarussa, whole plant of s. trifasciata and leaves of h. kurzii and k. pinnaata were sun dried for several days after washing. the plant materials were then oven dried for 24 hours at 40 c and then ground to a coarse powder. the powdered materials (300 gmeach) were then soaked inmethanol (1.5 liter each) and kept for 10 days at room temperaturewith occasional shaking. the crude extracts were then fltered through cotton plug followed by whatman no. 1 flter paper individually and the extracts were concentrated with rotary evaporator. extraction and fractionation a portion (5 g) of each of the concentrated methanol extract (me) was fractionated by the modifed kupchan partitioning method [21] into pet-ether (pesf), carbon tetrachloride (ctsf), chloroform (cfsf), and aqueous (aq) soluble fractions. evaporation of solvents afforded various organic soluble extractives and aqueous soluble materials as shown in table 1. test organisms both gram positive (bacillus cereus, b. subtilis, sarcina lutea, staphylococcus aureus) and gramnegative (escherichia coli, salmonella paratyphi, shigella boydii, s. dysenteriae, pseudomonas aeruginosa, vibrio mimicus, v. parahemolyticus), bacterial strains used for the experiment were collected as pure cultures from the institute of nutrition and food science (infs), university of dhaka, bangladesh. and sesame oil for leprosy; and with sulfur and camphor for scabies. in india, the seeds are considered to be an alternative tonic. [11] kigelia pinnata (lam. benth) belonging to the family of bignoniaceae and has a wide geographical distribution in the west and central africa. the tree grows on river banks, wet areas along streams and on foodplains of nigeria, cameroon, kenya, guinea and senegal. [12] the kigelia plant have medicinal properties not only because of its perceived characteristics such as bitterness, astringent taste or smell but also because of forces that it seems to emit in connection with its location, orientation and association with other plants. [13] k. pinnata is widely used for antidiarrhoeal, [14] antileprotic, [15] antimalarial, [16] anti-infammatory, [12] anticancer, [17] gynecological disorders, [18] anti-microbial [19] and rheumatism. [20] we, here in, report the results of preliminary antimicrobial screening of the methanolic crude extracts and the corresponding kupchan partitioning fractions of four bangladeshi medicinal plants having folklore reputation for the frst time. material and methods collection and preparation of the plant materials j. gendarussa, s. trifasciata, and h. kurzii were collected from dhaka botanical garden in february 2011 while k. pinnata was obtained from rangpur in february 2010, voucher table 1: amount of different partitionates of j. gendarussa, s. trifasciata, h. kurzii, k. pinnaata partitionates j. gendarussa s. trifasciata h. kurzii k. pinnaata pet-ether (pesf)  730 mg  750 mg  720 mg  720 mg carbon tetrachloride (ctsf)  500 mg  520 mg  550 mg  600 mg chloroform (cfsf)  480 mg  470 mg  410 mg  580 mg aqueous (aq) soluble fractions 1200 mg 1100 mg 1350 mg 1420 mg table 2: antimicrobial activity of crude extracts and kupchan fractions of sansevieria trifasciata test microorganisms diameter of zone of inhibition (mm) me pesf ctsf cfsf aq kanamycin gram positive bacteria bacillus cereus 10.00 1.00 9.33 2.31 12.67 1.15 14.00 1.00 – 32.00 1.00 b. subtilis 12.67 2.08 10.33 1.53 14.00 1.00 13.67 1.53 – 34.00 1.00 staphylococcus aureus 10.67 1.15 11.67 0.58 11.67 2.08 11.67 0.58 – 34.33 1.15 sarcina lutea 11.33 2.08 12.00 2.00 10.00 1.00 12.33 2.08 – 33.67 2.31 gram negative bacteria escherichia coli 11.33 1.53 11.00 2.00 13.67 1.53 12.67 1.15 8.33 1.53 32.67 1.15 pseudomonas aeruginosa 12.00 3.00 11.67 0.58 11.33 1.15  9.00 1.00 – 33.00 1.00 salmonella paratyphi 11.67 2.08 11.33 2.31 12.33 1.53 11.67 1.53 – 32.00 2.65 shigella boydii 12.33 2.52 11.67 2.08 12.00 2.00 12.00 2.65 – 33.67 0.58 s. dysenteriae 11.67 1.15 10.33 0.58 10.33 0.58 11.67 2.08 – 32.67 2.52 vibrio mimicus 14.67 2.00 11.67 1.53 13.67 1.53 13.33 1.15 – 33.33 2.08 vibrio parahemolyticus 12.00 2.00 12.00 1.73 12.67 2.08 12.67 1.15 – 33.00 1.00sikder, et al.: in vitro antimicrobial screening of four reputed bangladeshi medicinal plants 74 pharmacognosy journal  august 2011  vol 3  issue 24 activity with zone of inhibition ranging from10.00-14.67mm with the highest being seen against vibrio mimicus (14.67mm). moderate activity was seen against bacillus subtilis (12.67 mm), vibrio parahemolyticus and pseudomonas aeruginosa (12.00 mm each). the chloroform soluble fraction of this palnt also revealed mild to moderate antimicrobial activity (9.00-14.00 mm), where highest activity was found against bacillus cereus (14.00 mm). different extractives of leaves of j. gendarussa also demonstratedmild tomoderate antimicrobial activity and its chloroform soluble kupchan partitionate showed antimicrobial activity with zone of inhibition (table 3) ranging from 8.33-13.00 mm. this fraction revealed strong activity against b. cereus (13.33 mm) and moderate inhibitory activity agent salmonella paratyphi (12.67 mm). on the other hand, the chloroform soluble partitionate of the methanol extract of leaves of h. kurzii exerted signifcant antimicrobial activity having inhibitory zone (table 4) ranging from 9.00-16.00 mm. the chloroform soluble fraction demonstrated antimicrobial activity against gram positive bacteria like b. cereus (11.67 mm) b. subtilis (11.33 mm), sarcina lutea (11.33 mm) and gram negative bacteria like escherichia coli (11.67 mm), p. aeruginosa (14.00 mm), s. paratyphi (14.00 mm), experimental procedure the antimicrobial study was carried out by disc diffusion technique for bacteria. [22] standard kanamycin disc (kan.) (30 μg/disc) and discs containing the test materials (400 μg/disc) and respective solvents were used as positive and negative controls, respectively. according to this method, the antimicrobial potency of the test samples was measured by determining the diameter of the zones of inhibition in millimeter. results and discussion in our preliminary antimicrobial screening of four local medicinal plants, it was observed that, among different fractions the methanol extract and its pet-ether, carbon tetrachloride, chloroform soluble fractions of leaves of h. kurzii showed signifcant activity against most of the grampositive and gramnegative bacteria at a dose of 400 μg/ disc (tables 2-5). all the extractives of s. trifasciata exhibited moderate antimicrobial activity, (table 2) especially the methnol extract revealed mild to moderate antimicrobial table 4: antimicrobial activity of crude extracts and kupchan fractions of hydnocarpus kurzii test microorganisms diameter of zone of inhibition (mm) me pesf ctsf cfsf aq kanamycin gram positive bacteria bacillus cereus 12.67 1.53  9.00 1.00 11.67 1.53 11.67 1.15 – 31.67 0.58 b. subtilis 11.00 1.00  9.67 0.58 10.33 0.58 11.33 1.53 – 32.33 1.53 staphylococcus aureus 14.67 0.58 10.67 0.58 11.67 2.08 10.33 1.15 – 33.67 1.15 sarcina lutea 14.67 1.15 12.33 0.58 11.33 1.53 11.33 1.15 – 32.00 2.65 gram negative bacteria escherichia coli 11.33 1.53 11.33 1.53 12.67 1.15 11.67 1.53 12.00 2.00 32.00 1.00 pseudomonas aeruginosa 10.67 0.58 10.33 0.58 10.33 1.53 14.00 1.00 – 32.67 0.58 salmonella paratyphi 12.67 0.58 11.00 1.73  9.67 1.15 14.00 2.00 – 32.67 2.08 shigella boydii 10.33 1.15 11.00 1.00 12.00 1.00 11.67 1.53 – 31.67 0.58 s. dysenteriae 10.67 0.58 12.00 2.00 10.67 2.08 11.67 0.58 – 33.67 1.15 vibrio mimicus 10.33 0.58 11.67 0.58 11.00 1.00 15.00 1.00 – 31.67 2.08 vibrio parahemolyticus 11.00 1.00  9.67 0.58 11.33 1.15 12.33 1.53 – 30.67 0.58 table 3: antimicrobial activity of crude extracts and kupchan fractions of justicia gendarussa test microorganisms diameter of zone of inhibition (mm) me pesf ctsf cfsf aq kanamycin gram positive bacteria bacillus cereus 11.67 1.53 11.33 0.58 – 13.33 1.53 – 32.33 1.53 b. subtilis  9.00 1.00 11.67 2.08 –  8.33 1.53 – 34.00 1.00 staphylococcus aureus  9.33 0.58 11.00 1.00 – 11.33 2.08 – 32.00 1.00 sarcina lutea  9.67 1.53  8.67 1.15 –  8.33 1.53 12.33 2.52 33.33 1.53 gram negative bacteria escherichia coli 10.00 1.00 11.00 1.00 – 12.33 0.58 – 34.33 1.53 pseudomonas aeruginosa 10.33 0.58  8.33 0.58 –  8.33 1.53 – 33.67 2.31 salmonella paratyphi 10.00 1.00 11.00 1.73 – 12.67 1.15 – 32.67 2.08 shigella boydii 11.00 1.73 13.00 1.73 – 11.67 0.58 – 32.00 1.00 s. dysenteriae 12.00 1.00 10.00 1.00 – 12.33 2.08 – 33.67 1.15 vibrio mimicus  9.67 0.58 11.33 1.15 – 11.00 1.73 – 33.00 2.00 vibrio parahemolyticus 11.00 2.65 11.67 2.00 – 11.67 0.58 – 31.67 0.58pharmacognosy journal  august 2011  vol 3  issue 24 75 sikder, et al.: in vitro antimicrobial screening of four reputed bangladeshi medicinal plants 2. sumathi e, janarthanam b. in vitro regeneration of justicia gendarussa burm. f. libyan agriculture research center journal internation 2010; 1(5):284-7. 3. periyanayagam k, umamaheswari b, suseela l, padmini m, ismail m. evaluation of antiangiogenic effect of the leaves of justicia gendarussa (burm. f) (acanthaceae) by chrio allontoic membrane method. american journal of infectious diseases 2009; 5(3):180-2. 4. anbu jeba sunilson j, jayaraj p, varatharajan r, thomas j, james j, muthappan m. analgesic and antipyretic effects of sansevieria trifasciata leaves. african journal of traditional, complementary and alternative medicines 2009; 6(4):529-33. 5. yoshihrio m, toshihiro i, minpei k, yutaka s. steroidal saponins from sansevieria trifasciata. phytochemistry 1996; 43:1325-31. 6. yoshihrio m, toshihiro i, minpei k, yutaka s. pregnane glycosides from sansevieria trifasciata. phytochemistry. 1997; 44:107-11. 7. aubréville a, leroy jf. flore du cambodge du laos et du viet-nam. ed muséum national d’histoire naturelle. paris: eds. 1960-1987; 1-13. 8. council of scientific and industrial research, india.the wealth of india: a dictionary of indian raw materials and industrial products. raw materials. delhi. (wealth india rm) 1959; 5:141-142. 9. dean al, wrenshal r. preparation of chaulmoogra oil derivatives for the treatment of leprosy. public health rep. 1922; 37(23):1395-456. 10. tun uk, than up. myanmar medicinal plant database. [updated 2006 august 11] available from: http://www.tuninst.net/myanmedplants/til/ famf/flacourtiacae.htm#hydnocarpus-kurzii 11. herbnet. medicinal herb facts c, chaulmoogra (hydnocarpus kurzii). available from: http://www.herbnet.com/herb%20uses_c.htm 12. owolabi oj, omogbai eki. analgesic and anti-inflammatory activities of the ethanolic stem bark extract of kigelia africana (bignoniaceae). afr. j. biotechnol. 2007; 6:582-5. 13. saini s, kaur h, verma b, ripudaman, singh sk. kigelia africana (lam.) benth. — an overview. natural product radiance. 2009; 8(2):190-7. 14. akah pa. antidiarrhoeal activity of the aqueous leaf extract of kigelia africana experimental animal. j. herbs spices med. plants. 1996; 4(2):31‑8. 15. lal sd,yadar bk. folk medicines of kurukshetra district (haryana). india econ. bot. 1983; 37:299-305. 16. weenen h, nkunya mhh, bray dh, mwasumbi lb, kinabo ls, kilimali vaeb. antimalaria activity of tanzanian medicinal plants. planta medica. 1990; 56:368-70. 17. msouthi jd, mangombo d. medicinal herbs in malawi and their uses. hamdard 1983; 26:94-100. 18. grace om, light me, lindsey kl, moholland da, staden jv, jager ak. antibacterial activity and isolation of antibacterial compounds from fruit of v. mimicus (15.00 mm) and v. parahemolyticus (12.33 mm). among the extractives of leaves of k. pinnaata, the chloroform soluble extractive showed weak antimicrobial activity (8-11mmzone of inhibition) with better antimicrobial activity against gram negative bacteria, especially p. aeruginosa and shigella boydii (11.00 mm each) (table 5). conclusion the methanolic extracts of leaf of s. trifasciata, j. gendarussa, h. kurzii and k. pinnata and their kupchan fractions (pet-ether, carbon tetrachloride and chloroform) demonstrated mild to moderate antibacterial activity against gram ve and gram –ve bacteria. the antibacterial activity demonstrated by the extractives of s. trifasciata, j. gendarussa, h. kurzii and k. pinnata are in agreement with the folk uses of the plants against chronic rheumatism, infammations, bronchitis, vaginal discharges, dyspepsia, eye diseases and fever. however, further studies are warranted to isolate and characterize the compounds reported for the antimicrobial property. acknowledgement we are grateful to the department of pharmacy, state university of bangladesh, for materialistic supports and to the institute of food and nutrition, university of dhaka, for supplying the pure standard microorganisms. references 1. paval j, kaitheri sk, potu bk, govindan s, kumar rs, narayanan sn, et al. anti-arthritic potential of the plant justicia gendarussa burm f. clinics 2009; 64(4):357-62. table 5: antimicrobial activity of crude extracts and kupchan fractions of kigelia pinnaata test microorganisms diameter of zone of inhibition (mm) me pesf ctsf cfsf aq kanamycin gram positive bacteria bacillus cereus 9.67 1.53 10.33 1.53  7.67 0.58  8.33 0.58 – 30.67 0.58 b. subtilis 8.00 1.73  8.67 2.08  9.00 1.53  9.67 1.53 – 31.67 1.53 staphylococcus aureus 7.67 1.15 10.33 0.58  9.33 0.58  9.67 0.58 – 33.00 1.73 sarcina lutea 7.33 0.58  9.00 1.00 10.67 1.53 10.33 1.53 – 33.67 1.15 gram negative bacteria escherichia coli 8.00 1.00  7.33 0.58  8.67 1.53  9.33 1.53 – 32.67 2.08 pseudomonas aeruginosa 9.67 1.15  9.33 0.58  8.33 1.00 11.00 1.00 – 33.00 1.00 salmonella paratyphi 7.33 0.58 10.33 1.53  8.33 2.00 10.00 2.00 – 33.33 2.08 shigella boydii 9.33 1.53  8.67 1.15  9.00 1.00 11.00 1.00 – 32.00 1.00 s. dysenteriae 7.33 1.15  8.33 1.53  9.00 1.53  8.33 1.53 – 33.33 0.58 vibrio mimicus 8.00 1.00  9.33 2.52  9.00 2.08  9.67 2.08 – 32.00 1.00 vibrio parahemolyticus 8.67 1.53 10.33 0.58  8.00 1.00 10.00 1.00 – 34.00 1.73 the average values of three calculations are presented as mean s.d. (standard deviation); me methanolic extract; pesf pet-ether soluble fraction; ctsf carbon tetrachloride soluble fraction; cfsf chloroform soluble fraction; aq aqueous soluble fraction of the methanolic extract of the plants.sikder, et al.: in vitro antimicrobial screening of four reputed bangladeshi medicinal plants 76 pharmacognosy journal  august 2011  vol 3  issue 24 21. wagenen bc, larsen r, cardellina jh, dazzo dr, lidert zc, swithenbank c. ulosantoin. a potent insecticide from the sponge ulosa ruetzleri. j org chem. 1993; 58:335-7. 22. barry al. principle & practice of microbiology. 3 rd ed. philadelphia: lea & fabager; 1976. the traditional african medicinal plant, kigelia africana. s. afr. j. bot. 2002; 68:220-2. 19. kela sl,ogunsusi ra,ogbogu n,nwude vc.screening of some nigerian plants for molluscidal activity. revue. elev. med. vet. pays trop. 1989; 42:20‑195. 20. gill ls. ethomedical uses of plants in nigeria. benin city: uniben press. 1992; 143.pharmacognosy journal  august 2011  vol 3  issue 24 77 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: joybioinfo@gmail.com doi: 10.5530/pj.2011.24.15 evaluation of in vitro anthelminthic activity of leucas aspera extracts swati agarwal 1 , simi jacob 1 , nikkita chettri 1 , saloni bisoyi 1 , d. k. badarinath 1 , a. b. vedamurthy 1 , v. krishna 2 and h. joy hoskeri* ,1,2 1 department of biotechnology, the oxford college of science, bangalore – 560 102, karnataka, india. 2 department of p.g. studies and research in biotechnology and bioinformatics, kuvempu university, shankarghatta – 577 451, shimoga, karnataka, india. a b s t r a c t helminths infections are also among the most common infections in human, affecting a large proportion of the world’s population in developing countries and produce a global burden of disease. pherithema posthuma a helminthes is commonly known as earth-worms, leucas aspera herb is distributed throughout india. the anthelminthic property of leucas aspera was evaluated using pherithema posthuma as an experimental model. piperazine citrate was used as the standard reference. earthworm belonging to control group showed paralysis time as 64.33 min and death time as 200 min. among the various concentrations of aqueous extract tested, concenration at 250 mg/ml showed effcient anthelminthic activity and among all the concentrations ethanol extract tested, concentration at 250 mg/ml gave signifcant results. this investigation revealed that ethanol extract of leucas aspera showed signifcant anthelminthic activity against pheretima posthuma when compared aqueous extract. ethanol extract also proved to be effcient than the standard drug. this investigation supported the ethnomedical claims of leucas aspera as anthelminthic plant. key words: lamiaceae; leucas aspera; anthelminthic activity; pheretima posthuma; ethanol extract; aqueous extract. introduction parasitic helminthes are worm-like organisms that live and feed off living hosts, receiving nourishment and protection while disrupting their hosts’ nutrient absorption, causing weakness and disease in human and animals inficting heavy production losses. helminths infections are also among the most common infections in human, affecting a large proportion of the world’s population in developing countries and produce a global burden of disease and contribute to the prevalence of malnutrition, anaemia, eosinophilia, and pneumonia which more often physically impair their hosts than kill them. [1] anthelminthics are those agents that expel parasitic worms (helminthes) from the body, by either stunning or killing them. [2] various problems have been evolved with chemotherapeutic control practices such as parasites are developing resistance to several families of chemical anthelminthics, [3] chemical residues and toxicity problems, [4] un-economical and nonavailability of drugs in remote areas. furthermore, it has been recognized recently that anthelmintic substances having considerable toxicity to human beings are present in foods derived from livestock, posing a serious threat to human health. [5] for these various reasons, interest in the screening of medicinal plants for their anthelminthic activity remains of great scientifc signifcance despite extensive use of synthetic chemicals in modern clinical practices all over the world. [6] helminthes infections are commonly found in community and being recognized as cause of much acute as well as chronic illness among the various human beings as well as cattle’s. more than half of the population of the world suffers from various types of infection and majority of cattle’s suffers from worm infections. [7] however, the high cost of modern anthelminthics has limited the effective control of these parasites. in some cases widespread intensive use of sometimes low quality anthelminthics [8] has led to development of resistance and hence a reduction in the usefulness of available anthelminthics. [9] although the use of alternate drugs has also been advocated as a measure to avoid the development of resistant strains ofagarwal, et al.: evaluation of in vitro anthelminthic activity of leucas aspera extracts 78 pharmacognosy journal  august 2011  vol 3  issue 24 helminth parasites, and as a means of reducing the cost of controlling helminthic diseases. [10-13] leucas aspera (willd.) linn. (family: lamiaceae), a herb commonly known as ‘thumbai is distributed throughout india from the himalayas down to ceylon. [14] the plant is used as an insecticide and indicated in traditional medicine for coughs, colds, painful swellings, and chronic skin eruptions. [15] flowers are valued as stimulant, expectorant, aperient, diaphoretic, insecticide and emmenagogue. leaves are considered useful in chronic rheumatism, psoriasis and other chronic skin eruptions. bruised leaves are applied locally in snake bites. [14,16] compounds isolated from the plant include, long-chain aliphatic compounds, a triterpene-leucolactone, sterols- sitosterol, campesterol, stigmasterol and a novel phenolic compound. [17-20] however, anthelminthic activity of leucas aspera whole plant extract is not scientifcally and reported. to justify the traditional claims of leucas aspera, wemade an effcient attempt to assess the anthelminthic activity of leucas aspera. materials and methods drugs and chemicals the standard drug piperazine citrate (sd fine chemicals ltd., mumbai). ethanol was purchased from hong, yang chemical corporation, china. plant resource leucas aspera plant material was collected from agricultural felds of tinsukia, assam, india. the plant was authenticated by prof. v. krishna, kuvempu university. fresh plant material was washed thoroughly in tap water to remove traces of soil and other contaminants. it is then shade dried. further, the whole plant was chopped fnely and was shade dried, powdered mechanically. the powdered plant material was transported in a vacuum sealed container to bangalore for further experimental studies. this plant material was subjected to cold extraction using ethanol as the solvent system for about 96 h, after every 24 h fresh ethanol was added and ethanol containing the extract was separated, followed by double distilled water (with 5% ethanol, to avoid microorganism contamination) for 96 h. both the extracts were fltered and concentrated in vacuum under reduced pressure and allowed for complete evaporation of the solvent on water bath and fnally vacuum dried. the yield of ethanol crude and aqueous extract for 1 kg of powdered plant material was 36 g and 45 g respectively. test organism indian adult earthworms (pheretima posthuma) collected from the indo-american hybrid seeds, bangalore. the earthworms weremaintained under normal vermicomposting medium with adequate supply of nourishment and water, for about two weeks. before the initiation of experiment the earthworms were washed with normal saline. adult earthworms of approximately 4 cm in length and 0.2 ‐ 0.3 cm in width were used for the experiment. this organism was selectedmodel for anthelminthic activity due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings. [21,22] extract preparation for experiment the porously powdered plant material was used for extract preparation. after extraction, the crude extract was stored in dessicator until further use. ethanol extract and standard drug piperazine citrate were dissolved in 0.5% dmso in normal saline (v/v). whereas, the crude aqueous extract was directly dissolved in normal saline and used for evaluation for anthelminthic activity. antihelminthic activity the anthelminthic activity of whole plant extracts of leucas aspera was evaluated as per the method reported by dash et al. [23] twelve groups of animals with three earthworms in each groups, each earthworm were separate released into 20 ml of desired formulation in normal saline, group 1 earthworm were released in 20 ml normal saline in a clean petri plate. group ii, iii, iv, v, vi earthworms were released in 20 ml normal saline containing 50, 100, 150, 200 and 250 mg/ml of ethanol extract respectively. similarly, group vii, viii, ix, x, xi earthworms were released in 20 ml normal saline containing 50, 100, 150, 200 and 250 mg/ml of aqueous extract respectively. group xii earthworms were released in 20 ml normal saline containing standard drug piperazine citrate (50 mg/ml). earthworms were observed; the time taken for paralysis and the time taken for death was monitored and documented in minutes. paralysis time was analyzed based on the behavior of the earthworm with no revival body state in normal saline medium. death was concluded based on total lose of motility with faded body color. [24] the result of anthelminthic activity is depicted in table 1. statistical analysis the data of anthelminthic evaluations were expressed as mean s.e.m of three earthworms in each group. the statistical analysis was carried out using one way anova followed by tukey’s t-test. the difference in values at p 0.01 was considered as statistically signifcant. the analysis of variance (anova) was performed using ezanova (version 0.98) software to determine the mean and standard error of paralysis and death time of the earthworms. results and discussion leucas aspera is a well known medicinal plant and is widely used in folk medicine/ ayurvedic system of medicine. inpharmacognosy journal  august 2011  vol 3  issue 24 79 agarwal, et al.: evaluation of in vitro anthelminthic activity of leucas aspera extracts references 1. chartier c, soubirac f, pors i, silvestre a, hubert j, couquet c, cabaret j. prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern france. j. helminthol. 2001; 75:325-330. 2. chaturvedi m, dwivedi s, dwivedi a, barpete pk, sachan r. formulation and evaluation of polyherbal anthelmintic preparation, ethnobot. leaflets. 2009; 13:329-331. 3. chartier c, soubirac f, pors i, silvestre a, hubert j, couquet c, cabaret j. prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern france. j. helminthol. 2001; 75:325-330. 4. muhammad g, abdul j, khan mz, saqib m. 2004. use of neostigmine in massive ivermectin toxicity in cats. vet. hum.toxicol. 2004; 46:28-9. 5. paras malik, usha gavani, paarakh pm. anthelmintic activity of annona squamosa linn leaves. pharmacology online. 2009; 2:601-604. 6. von bingen h. naturkunde—reproduction of a medieval book. 2 nd edn. mueller-wiss, buchgesellschaft: salzburg; 1974. 7. chaturvedi m, dwivedi s, dwivedi a, barpete pk, sachan r. formulation and evaluation of polyherbal anthelmintic preparation, ethnobot. leaflet. 2009; 13:329-331. 8. monteiro am, wanyangu sw, kariuki dp, brain r, jackson f, mckellar qr. pharmaceutical quality of anthelmintics sold in kenya. vet. rec. 1997; 142:396-398. 9. waller pj. anthelmintic resistance. vet. parasitol. 1997; 72:391-405. 10. kelly jd, hall ca. resistance of animal helminths to anthelmintics. adv. pharm.ther. 1979; 16:89-128. 11. okon ed, ogunsusi ra, fabiyi jp. survey and feasibility studies on fascioliasis and parasitic gastroenteritis of ruminants in nigeria. federal livestock department of nigeria report, lagos; 1980; 1-55. 12. taylor ma, hunt kr. anthelmintic drug resistance in the uk. vet. rec. 1989; 125:143-147. 13. coles gc,roush rt.slowing the spread of anthelmintic resistant nematodes of sheep and goats in the united kingdom.vet. rec. 1992; 139:505-510. 14. rai v, agarwal m, agnihotri ak, khatoon s, rawat ak, mehrotra s. pharmacognostical evaluation of leucas aspera. nat. prod. sci. 2005; 11:109-114. 15. chopra rn, nayar sl, chopra ic. glossary of indian medicinal plants. new delhi: niscair, csir; 2002. 16. shirazi am. studies on leucas aspera. indian j pharm. 1947; 9:116-117. 17. misra tn, singh rs, pandey hs, singh s. long chain compounds from leucas aspera . phytochemist. 1992; 31:1809-1810. 18. misra tn, singh rs, prasad c, singh s.two aliphatic ketols from leucas aspera . phytochemist. 1993; 32:199-201. the present study solvents namely ethanol and water were used sequentially for crude extraction of leucas aspera whole plant. to justify the ethnomedical claims of leucas aspera we made an effcient attempt in evaluating the anthelminthic property of leucas aspera. earthworm belonging to control group showed paralysis time as 64.33min and death time as 200min. aqueous extract at the concentration of 50mg/ml showed the time of paralysis and death at 117 and 168 min respectively. for concentration of 100 mg/ml, the paralysis and the death time was found to be 100.67 and 154 min respectively. at the concentration of 150, 200 and 250 mg/ml, time taken to paralysis was 62, 56 and 50.33 min respectively and death time 123.33, 115 and 96.67min respectively. among the various concentrations tested, aqueous extract at 250 mg/ml showed effcient anthelminthic activity (table 1). on the other hand ethanol extract at the concentration of 50 mg/ml showed the time of paralysis and death at 20 and 26.33 min respectively. for concentrations at 100, 150, 200 and 250 mg/ml paralysis was shown at 16, 10.67, 6.33 and 4.33 min respectively and death occurred at 23, 14, 11 and 9.33min respectively. among all the concentrations ethanol extract tested, concentration at 250 mg/ml gave signifcant results. standard drug at 50 mg/ml showed paralysis at 31.33 min and death time was 40.67 min (table 1). this investigation revealed that ethanol extract of leucas aspera showed signifcant anthelminthic activity against pheretima posthuma when compared aqueous extract. ethanol extract also proved to be effcient than the standard drug. this investigation supported the ethnomedical claims of leucas aspera as anthelminthic plant. acknowledgement the authors are grateful to department of biotechnology, the oxford college of science, bangalore, for providing the facilities to carry out the entire experiment. table 1: in vitro anthelminthic activity of ethanol and aqueous extracts of leucas aspera against pheretima posthuma test samples concentration (mg/ml) time taken for paralysis (min) time taken for death (min) control (normal saline) 64.33 0.88 200.00 2.60 ethanol extract of vinca rosea  50 20.00 0.58** 26.33 0.88** 100 16.00 0.58** 23.00 0.58** 150 10.00 0.33** 14.00 0.58** 200 6.33 0.33** 11.00 0.58** 250 4.33 0.88** 9.33 0.33** aqueous extract of vinca rosea  50 117.00 1.15** 168.33 4.06** 100 100.67 1.45** 154.00 8.14** 150 62.00 1.53 ns 123.33 2.60** 200 56.00 0.58** 115.00 2.65** 250 50.33 0.88** 96.67 2.33** piperazine citrate  50 31.33 1.86** 40.67 0.88** values are the mean s.e.m. of three earthworms. symbols represent statistical signifcance. * p 0.05, ** p 0.01, ns: not signifcant as compared to compared to control group.agarwal, et al.: evaluation of in vitro anthelminthic activity of leucas aspera extracts 80 pharmacognosy journal  august 2011  vol 3  issue 24 22. vigar z. atlas of medical parasitology. in: 2 nd ed. p.g. publishing house, singapore: 1984; 216 ‐ 217. 23. dash gk, suresh p, kar dm, ganpaty s, panda sb. evaluation of evolvulus alsinoids linn for anthelmintic and antimicrobial activities. j. nat. rem. 2002; 2:182-185. 24. tambe vd, nirmal sa, jadhav rs, ghogare pb and bhalke rd. anthelmintic activity of wedelia trilobata leaves. indian j. nat. prod. 2006; 22:27-29. 19. misra tn, singh rs, pandey hs, singh s. a novel phenolic compound from leucas aspera. indian j. chem. 1995; 34:1108-1110. 20. pradan bp, chakraborty dk, subba gc. a triterpenoid lactone from leucas aspera . phytochemistry 1990; 29:1693-1965. 21. thorn gw, adams rd, braunwald e, isselbacher kj and petersdrof rg. harrison’s principles of internal medicine. in: mcgraw hill co., new york: 1977; 1088 ‐ 1089.pharmacognosy journal  august 2011  vol 3  issue 24 81 o r i g i n a l a r t i c l e p h c o g j . *address for correspondence: devang j. pandya. ‘prakruti’, maruti nagar-2, airport road, rajkot-360001, gujarat, india. e-mail id: pandyadevang82@yahoo.com doi: 10.5530/pj.2011.24.16 2008. herbarium of the collected sample was prepared and deposited in department of pharmacognosy, rk college of pharmacy (no. rkcp/cog/01/2008). authentication was done by dr. n. r. sheth, head of department of pharmaceutical sciences, saurashtra university. preparation of extracts successive extraction of 1 kg powder of the entire plant was carried out using four solvents in the decreasing order of their polarity index: petroleum ether, chloroform, methanol and distilled water. complete extraction of the powder with each solvent was carried out in round-bottom fask at a temperature 50c. the yield of the dried extracts was found to be 10.1%w/w, 8.5%w/w, 7.5%w/w and 14.1%w/w respectively. their concentrations were adjusted in the solvents according to their dose. for investigation of each activity, the experimental animals were divided into six groups, with six animals in each group: normal control, standard (agar-agar), petroleum ether extract, chloroform extract, methanol extract, aqueous extract. pharmacological study the pharmacological study was approved by the institutional animal ethics committee (rkcp/cog/rp/10/06) and carried out according to cpcsea guidelines. all animals weremaintained under environmentally controlled conditions of 24 1 c and 12 h-light and 12 h-dark cycle. the animals were acclimatized to laboratory conditions for 1 month before starting the pre-clinical trials. all studies were performed under standard conditions of temperature, light, humidity and noise. evaluation of laxative activity of oxystelma esculentum pandya dj 1 *, anand is 2 1 r. k. college of pharmacy, rajkot, gujarat, india. 2 shri sarvajanik pharmacy college, mehasana, gujarat, india. a b s t r a c t oxystelma esculentum is a perennial twiner growing near water-logged areas in the indian subcontinent. it is used traditionally as a laxative. the present work deals with the investigation of laxative potential of various extracts of o. esculentum. the plant was successively extracted with solvents of varying polarities, which served as the test extracts. laxative effect was checked in wistar rats using different models. the petroleum ether extract was found to possess the most effective laxative activity, thereby supporting the traditional claim of the plant as a laxative. phytochemical screening of this extract revealed the presence of important classes of compounds like cardenolides, favonoids, phenolics, sterols and triterpenoids. this bioactivity-guided phytochemical screening can guide further therapeutic investigations and isolation of therapeutically important compounds from oxystelma esculentum. key words: laxative, oxystelma esculentum, oxystelma secamone, periploca esculenta introduction oxystelma esculentum r. br. syn. oxystelma secamone linn., periploca esculenta roxb., periploca secamone linn., sarcostemma secamone bennet, sarcostemma esculentum linn., asclepias rosea r. br., is a perennial twiner found throughout the plains of the indian sub-continent near water-logged areas. [1] the plant is used as laxative, laxative, antiseptic, depurative, anthelmintic, antiulcer, aphrodisiac, hepatoprotective and useful in leucoderma and bronchitis. decoction of plant is used in ulcer, sore-throat and itches. milky juice is used as galactogogue, antiperiodic, antiulcer and as a vulnerary. leaves are used as antiperiodic. its root is prescribed in jaundice. fruit is bitter, tonic, expectorant, anthelmintic. fruit juice is used in muscle pain, gonorrhoea, cough and leucoderma, and given to children as astringent. [2,3] the present work deals with not only investigating the laxative activity of the plant, but also fnding themost potent extract and performing its phytochemical screening, so as to guide further fractionation of therapeutically potent constituents from this plant. materials and methods collection and authentication oxystelma esculentum in fowering & fruiting stage was collected from barda hills near porbandar, gujarat, india, in octoberpandya and anand: evaluation of laxative activity of oxystelma esculentum 82 pharmacognosy journal  august 2011  vol 3  issue 24 discussion the present study shows that the petroleum ether extract of oxystelma esculentum has themost potent, statistically signifcant and dose-dependent laxative activity amongst all extracts, comparable with agar-agar (standard) at the dose of wistar rats of either sex weighing 200-220 g were kept in individual cages during one week. any rat producing wet feces was rejected. the rats were fasted for 12 h before dosing but were given water ad libitum. three animals per group were placed in one metabolic cage (each cage is provided with a wire mesh at the bottom and a funnel to collect the urine; stainless-steel sieves are placed in the funnel to retain feces). normal control group received normal saline (25 ml/kg). standard control group received 300 mg/kg agar-agar (pharma pvt. ltd.) orally. two groups of three animals were used for each dose of the test extract. three animals of the test extract groups received orally a dose of 200 mg/kg and the remaining three animals from each of these groups received dose of 400 mg/kg body weight. [4] after administration of the test extracts, the feces were weighed upto 8h and 16h (table 1, graph 1). the same animals were used after a washing period of 4 months for observing the laxative effects of the various extracts in constipated rats. the same procedure was repeated, but loperamide (pharma pvt. ltd., 5 mg/kg) was used to induce constipation after 1h of administration of each extract. feces were weighed upto 8h and 16h (table 2, graph 2). the results were expressed as the mean (g) of total feces. [5] results were calculated as mean standard deviation (sd). statistical analysis of control and test data was performed by one-way anova followed by dunnett’s test (sigma-stat software). a probability value of p 0.01 was considered statistically signifcant. phytochemical screening petroleum ether extract was found to have the most potent and statistically signifcant laxative activity. this extract was subjected to phytochemical screening involving established methods for detecting various classes of phytoconsituents (table 3). [6-11] results graph 1: comparison of laxative potential of various extracts graph 2: comparison of laxative potential of various extracts in loperamide-induced constipation model table 1: laxative activity of various extracts in rats groups fecal output (g) at 8h fecal output (g) at 16h normal control 0.5 0.1 1.1 0.1 standard (agar-agar) 1.1 0.1 2.5 0.1 pet ether ext 200 mg/kg 0.8 0.1 1.9 0.1 pet ether ext 400 mg/kg   1 0.1 2.4 0.1 chloroform ext 200 mg/kg 0.6 0.1 1.3 0.2 chloroform ext 400 mg/kg 0.7 0.2 1.4 0.2 methanol ext 200 mg/kg 0.6 0.2 1.1 0.2 methanol ext 400 mg/kg 0.8 0.1 1.4 0.2 aqueous ext 200 mg/kg 0.7 0.1 1.5 0.1 aqueous ext 400 mg/kg 0.8 0.1 1.7 0.2 values are expressed as mean sd number of animals (n) 6 table 2: laxative activity of various extracts in loperamide-induced constipation model groups fecal output (g) at 8h fecal output (g) at 16h normal control 0.2 0.05 0.5 0.05 standard (agar-agar) 0.8 0.05 1.4 0.05 pet ether ext 200 mg/kg 0.5 0.05 0.9 0.05 pet ether ext 400 mg/kg 0.7 0.05 1.3 0.05 chloroform ext 200 mg/kg 0.2 0.1 0.5 0.10 chloroform ext 400 mg/kg 0.3 0.1 0.5 0.15 methanol ext 200 mg/kg 0.3 0.08 0.6 0.07 methanol ext 400 mg/kg 0.4 0.1 0.7 0.15 aqueous ext 200 mg/kg 0.4 0.06 0.8 0.10 aqueous ext 400 mg/kg 0.5 0.08 1.1 0.10 values are expressed as mean sd number of animals (n) 6pharmacognosy journal  august 2011  vol 3  issue 24 83 pandya and anand: evaluation of laxative activity of oxystelma esculentum be responsible for the laxative effect. this bioactivity-guided phytochemical screening can serve as a gauge for further study of therapeutic effects and isolation of therapeutically important compounds from oxystelma esculentum. references 1. guha dn, bakshi bk. flora of murshidabad district, west bengal, india. jodhpur: scientific publishers, 1984. 2. kirtikar kr, basu bd. indian medicinal plants, vol. iii. new delhi: m/s periodical experts, 1975. 3. watt g. dictionary of economic products of india, vol. v. new delhi: periodical experts, 1891. 4. durairaj a, mazumder uk, gupta m, ray sk. effects of methanolic extract of oxystelma esculentum on diuresis and urinary electrolytes excretion in rats. iranian j pharmacol ther. 2007; 6:207-11. 5. ogunti eo, elujoba aa. laxative activity of cassia alata. fitoterapia 1993; 64:437-9. 6. feigl f. spot tests in organic analysis, 4 th ed. london: elsevier, 1956. 7. fishcher r. praktikum der pharmakognosic, 3 rd ed. berlin: springer verlag, 1952. 8. geissman a. modern methods of plant analysis, vol. iii. berlin: springer verlag, 1955. 9. harborne jb. phytochemical methods, 2 nd ed. london: chapman & hall ltd., 1973. 10. list ph, horhammer l. hager hand buch der pharmazeutischem praxis, band 1. berlin: springer verlag, 1967. 11. robinson t. the organic constituents of higher plants, their chemistry and interrelationships. minneapolis: burgers publishing company, 1964. 12. hughes s, higgs nb,turnberg la. loperamide has antisecretory activity in the human jejunum in vivo. gut. 1984; 25:931-5. 13. lawrence rs, carol asa, stephen gm, john sf. mechanism of the antidiarrheal effect of loperamide. gastroenterology. 1984; 86:1475-80. 400mg/kg. agar-agar exerts its laxative action by accumulation of water in the intestinal loop, increasing the bulk of the stools and stimulating the gastrointestinal motility. also, loperamide abolishes diarrhea by acting on intestinal motility and consequently reducing the water and stools entering the colon. [12,13] the laxative activity of petroleum ether extract is comparable to agar-agar, indicating a mechanism of action similar to it, thereby overcoming loperamide-induced constipation. this proves the traditional claims of this plant as a potent laxative drug. phytochemical screening of petroleumether extract revealed the presence of cardenolides, favonoids, phenolics, sterols and triterpenoids, which may table 3: phytochemical screening of petroleum ether extract phytoconstituent test result alkaloids dragendorff ’s test wagner’s test hager’s test mayer’s test –ve –ve –ve –ve flavonoids shinoda test fluorescence test ve ve phenolics ferric chloride test folin ciocalteu test ve ve sterols and triterpenoids libermann burchardt test salkowski test ve ve carotenoids antimony trichloride test –ve cardenolides kedde’s test baljet’s test legal’s test ve ve ve