Indian Journal of Natural Products and Resources Vol. 2(4), December 2011, pp.464-471

Pharmacognostical and physicochemical standardization of ethnopharmacologically important seeds of Lepidium sativum Linn. and tinctoria R. Br.

P Bigoniya*, C S Singh and A Shukla Radharaman College of Pharmacy, Radharaman Group of Institutes, Fatehpur Dobra, Bhadbada Road, Ratibad, Bhopal-462 002, Madhya Pradesh, .

Received 19 October 2010; Accepted 28 July 2011

Quality control standardizations of the various medicinal used in traditional medicine is becoming more important today in view of the commercialization of formulations based on these plants. Lepidium sativum Linn. seeds are used as tonic, carminative in chronic liver enlargement and spleen diseases. The bruised seeds mixed with lime juice are applied to the relief the local inflammation and rheumatic pain. Wrightia tinctoria R. Br. seeds are astringent, acrid, thermogenic, carminative, digestive, stomachic, antidysenteric, constipating, depurative, anthelmintic, aphrodisiac, febrifuge and diuretic. WHO recommends various physicochemical and phytochemical evaluation parameters for quality control of medicinal plants. In view of their medicinal importance and taxonomic confusion, morphology and microscopy, physico-chemical parameters, fluorescence analysis, preliminary phytochemical screening and quantitative estimation were performed to establish the salient diagnostic characters. The morphological, microscopical and physicochemical standards developed in this study will provide referential information for identification of these crude drugs and standardization.

Keywords: Lepidium sativum, Garden cress, Wrightia tinctoria, Pharmacognosy, Physicochemical, Fluorescence, Phytochemical. IPC code; Int. cl. (2011.01)—A61K 36/00

Introduction every medicine in the market because the scope After decades of serious obsession with the modern for variation in different batches of medicine is medicinal system, people have started looking at the enormous. Due to natural heterogeneity, plant ancient healing systems like Ayurveda, Siddha and material may vary in its phytochemical contents and Unani to treat the various types of ailments. This is therefore in its therapeutic effect according to because of the adverse effects associated with different places of collection, with different times in a synthetic drugs. Herbal traditional medicines have year for collection, with collection at the same time gained considerable momentum worldwide during the and places but in different years and with different past decade and play a paramount role in health care environmental factors surrounding the cultivation programs especially in developing countries. Ancient of a particular medicinal plant. Adding to this Indian literature incorporates a remarkably broad variability it is the fact that in herbal medicine several definition of medicinal plants and considers all plant plants may be used together in the same preparation. parts to be potential sources of medicinal substances1. These factors substantiate basic need of standardized However, a key obstacle, which has hindered the quality control tests for herbal preparations to ensure acceptance of the alternative medicines in the quality of the product. There is an internationally developed countries, is the lack of documentation and increasing demand for documentation of research non-compliance of GMP guidelines basically due to work carried out on traditional medicines2. With this poor standardization status. It is very important that a backdrop, it becomes extremely important to make an system of standardization must be established for effort towards standardization of the plant material used as traditional medicine for proper marketing —————— authorization and approval. Morphological *Correspondent author, E-mail: [email protected] Phone: +91-0755-2477941, +91-0755-2896237 authentication is not sufficient to ensure quantitative Fax No: +91-0755-2896664 consistency of bioactive or marker compounds BIGONIYA et al.: PHARMACOGNOSTIC STANDARDIZATION OF L. SATIVUM AND W. TINCTORIA SEEDS 465

responsible for the therapeutic effects. Advances in undertaken to generate standardized data on various chemical and instrumental techniques have made it pharmacognostical, phyto and physico-chemical easier to estimate phytochemical parameters of crude characteristics of the plant materials. The outcome of the drugs. The process of standardization can be achieved present study will be helpful in identification, by stepwise pharmacognostic studies3. authentication and quality control of the plant materials. Keeping in view the above mentioned problems, an attempt has been made to standardize the Materials and Methods ethnopharmacologically useful seeds of Lepidium Plant material sativum Linn. (Garden Cress) and Wrightia tinctoria The studies were executed in the fresh specimens R. Br., commonly available and widely used in central of seeds of Chansur and Indrajau, collected India, on the basis of pharmacognostical and other from their natural habitat, surrounding Bhopal, M. P., physio-chemical characteristics. India during Oct-Nov of the year 2009-2010. L. sativum Linn. (Family-Brassicaceae) is The seed of L. sativum was authenticated by commonly known as Chansur. This is a small, taxonomist Dr. H. B. Singh of Herbarium Department, herbaceous, glabrous annual, 15-45 cm high plant NISCAIR, New Delhi (voucher specimen no. cultivated as salad supplement throughout India. The NISCAIR/RHMD/Consult/2009-10/1232/36) and seeds are reddish in colour, oblong, somewhat angular seed of W. tinctoria was authenticated by Dr. T. and slightly curved on one side with rugous surface4. Hussain of Herbarium and Angiosperm Taxonomy Chansur is traditionally reported to have aperient, Department, NBRI, Lucknow and a specimen voucher alternative, tonic, aphrodisiac and carminative no. 97314 was assigned. Voucher specimens of the property. A cold infusion is used to relieve of seeds have been retained in the department for hiccough5. The seeds are used in treatment of chronic reference purpose. The collected seeds were washed, liver enlargement and spleen diseases6. As a shade dried and pulverized with mechanical carminative, they are given as an adjunct to pulverizer for size reduction. The size reduced seed purgatives. The bruised seeds mixed with lime juice powder was then passed through mesh 40-60 and used are applied to the relief the local inflammation and for determination of physicochemical parameters and rheumatic pains5. The are mildly stimulant, preparation of different solvent extracts. The fresh diuretic and serviceable in scorbutic diseases7. seed samples were used for macroscopic and Wrightia tinctoria R. Br. (Family-), microscopic studies. commonly known as Indrajau is a small deciduous tree distributed in Central India, Burma and Timor. Macroscopic and microscopic analysis This plant grows in abundance in dry, hilly and rocky The external morphology of seed such as nature, areas of Tamil Nadu, Andhra Pradesh, Madhya color, odour and taste were noted and other structural Pradesh and Rajasthan. The seeds resemble the seeds peculiarities like size, shape and texture were of Jau (Barley), this is the reason it is known as observed by using simple microscope. In microscopy, Indrajau8. They are useful in vitiated conditions of the desired part of fresh seeds were cut into pieces of pitta and kapha, dyspepsia, bilious affections, 2-5 mm without compression and immediately flatulence, colic stomach pain, diarrhoea, leprosy, transferred into FAA solution (95% ethyl alcohol: psoriasis, haemorrhoids, helminthiasis, fever, burning glacial acetic acid: formalin: water in 50:5:10:35) for sensation and dropsy9. The plant is very useful as one day to kill and fix the tissues. The pieces were stomachic, in the treatment of abdominal pain, skin embedded with paraffin wax. The paraffin embedded diseases, as antidiarrhoeal and antihaemorrhagic10. specimens were sectioned with the help of rotary Bark and seeds are astringent, acrid, thermogenic, microtome having thickness of 10-12 μm. Dewaxing carminative, digestive, stomachic, antidysenteric, of the sections was performed by customary constipating, depurative, anthelmintic, aphrodisiac, procedure12. The sections were stained with hemalum febrifuge and diuretic11. and safranin. A drop of HCl and phloroglucinol were Correct identification and quality assurance of the used to detect lignified cell in the cut sections13. The starting materials is an essential prerequisite to ensure microphotographs were captured using trinocular reproducible quality, which will provide safety and microscope (Jyoti Scientific, Gwalior) with digital efficacy of herbal medicine. This study was Olympus camera. 466 INDIAN J NAT PROD RESOUR, DECEMBER 2011

Preparation of extracts washed with dilute ammonium hydroxide and filtered. Coarse powders (25 g) of each seed were defatted The total alkaloid residue was dried and weighed20. individually with sufficient quantity of petroleum ether (40-60°C) with the aid of Soxhlet apparatus for Flavonoid estimation Aluminium chloride colorimetric technique was 24 h. The defatted seed cakes (5 g each) were 21 then extracted separately with 100 ml each of ethyl used for flavonoids estimation . Each extract (0.5 ml acetate, chloroform methanol, ethanol and water for of 1:10 g/ml) in methanol was separately mixed with 48 h by maceration and then filtered to obtain 1.5 ml of methanol, 0.1 ml of 10% aluminum respective extracts. The petroleum ether fraction chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml obtained after defatting was recovered as petroleum of distilled water. It was left at room temperature for ether extract after filtration. The extracts in different 30 min after which the absorbance of the reaction solvent were collected separately and volume reduced mixture was measured at 415 nm with a double beam under low pressure. 25 ml of the each extract UV/Visible spectrophotometer, (SHIMADZU, model was used to determine the percentage extractive no. 1700 series, Japan). The calibration curve was plotted by preparing the quercetin solutions at values of seeds in different solvents. The remaining 22 extract was stored in air tight glass container at 4-8°C concentrations 12.5 to 100 g/ ml in methanol . for fluorescence analysis. Saponin estimation

Physicochemical studies Crude sample (20 g) was put into a conical flask The percentage of foreign matter, loss on drying, and 100 ml of 20% aqueous ethanol added. The total ash and acid insoluble ash were determined samples were heated over a hot water bath for 4 h according to the method described in Indian with continuous stirring at about 55°C. The mixture Pharmacopoeia and the WHO guidelines on quality was filtered and the residue re-extracted with another control methods for medicinal plants materials14, 15. 200 ml 20% aqueous ethanol. The combined extracts The dried seed powders were subjected to were reduced to 40 ml over water bath at about 90°C. fluorescence analysis, as it is and also after treating The concentrate was transferred into a 250 ml separating funnel and 20 ml of diethyl ether was separately with 1 N of HCl, HNO3, H2SO4, NaOH, KOH, alcoholic NaOH, alcoholic KOH and ammonia added and shaken vigorously. The aqueous layer was against normal and ultra-violet light (254 nm). Colour recovered while the ether layer was discarded. The reaction of petroleum ether, ethyl acetate, chloroform, purification process was repeated three times, and methanol and water extract was also observed in then 60 ml of n-butanol was added. The combined n- normal light and UV light (254 nm)16, 17. butanol extracts was washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was Preliminary phytochemical screening heated in a water bath. After evaporation the samples Preliminary phytochemical screening of the seed were dried in the oven to a constant weight and the extracts in different solvents has been performed to saponin content was calculated as percentage23. detect the phytoconstituents like; alkaloid, amino acid, carbohydrate, glycoside, inulin, mucilage, Estimation of total phenols tannin, starch, saponin, steroid, triterpenoid and The total phenols of both the extracts were flavonoid18, 19. measured at 765 nm by Folin Ciocalteu reagent. The dilute methanolic extract (0.5 ml of 1:10 g/ml) or Quantitative estimation of phytoconstituents gallic acid (standard phenolic compound) was mixed

Alkaloid estimation with Folin Ciocalteu reagent (5 ml, 1:10 diluted with Seed powder was weighed (5 g) into a 250 ml distilled water) and aqueous sodium carbonate (4 ml, flask; fitted with stopper and 200 ml of 10% acetic 1 M). The mixture was allowed to stand for 15 min acid in ethanol was added and allowed to stand for and the total phenols were determined by 4 h. This was filtered and concentrated on a water spectrophotometer at 765 nm. The standard curve bath to one-quarter of its original volume. was prepared using 0, 50, 100, 150, 200, 250 mg/ ml Concentrated ammonium hydroxide solution was solutions of gallic acid in methanol: water (50:50). added drop wise to the extract until the precipitation Total phenol values were expressed in terms of gallic was complete. The whole solution was allowed to acid equivalent (mg/g of dry mass), which is a settle down to collect the precipitate, which was then common reference compound22. BIGONIYA et al.: PHARMACOGNOSTIC STANDARDIZATION OF L. SATIVUM AND W. TINCTORIA SEEDS 467

Determination of swelling index W. tinctoria (Indrajau) A fine seed material (1 g) was placed into a 25 ml Seeds are 1.3-2 cm long, 1-2 mm wide, pointed at glass-stoppard measuring cylinder. The internal apex, linear, glabrous, light yellowish-grey in colour. diameter of the cylinder was 16 mm, the length of the Seeds are crowned with a deciduous coma or a tuft of graduated portion was approximately 125 mm, white silky hairs of more than 3.8 cm long at the base by marked in 0.2 ml divisions from 0 to 25 ml in an which they can disseminate in air. The seed resemble the upwards direction. 25 ml of water was added into the seed of jau, thus it is known as Indrajau (Plate 2). cylinder containing material and mixture was shaken thoroughly at interval of every 10 min for 1 h. Sample Microscopic characters of seeds was allowed to stand for 3 h at room temperature. Detailed microscopic examination of a drug helps Volume was measured in ml occupied by the plant to identify the organized cellular structure of drugs material, including any sticky mucilage. The mean material by their known histological characters. Use value was calculated, related to 1 g of plant material15. of various reagents or stains help to distinguish cellular structures depending on their chemical nature. Results and Discussion Histological studies were made from microtome sections of fresh crude drugs to characterize testa, Morphological evaluation of seeds endosperm, embryo, vascular bundles, trichomes, The organoleptic studies indicated the importance seed oil, pericarp, sclerenchyma, epidermis, of characteristics such as typical tongue sensitizing cotyledons, aleurone grains and lipid globules. The aromatic taste, aromatic odour, etc. which are useful transverse sections of seed showed the following diagnostic characters. Morphological characters of characteristics: both the species are given: Chansur L. sativum (Chansur) The transverse section of seed showed presence of Seeds are small, oval-shaped, pointed and testa, tegmen, alleurone layer, endosperm and embryo triangular at one end, smooth, about 3-4 mm long, (Plates 3.1 & 3.2). Testa was thick, 1-2 layered and 1-2 mm wide, reddish brown in colour. A furrow appeared yellowish brown whereas; tegmen layer was present on both surfaces extending up to two thirds attached to inner side of testa layer and appeared as downward, a slight wing like extension present on single layer. Endosperm was composed of thick walled both the edges of seed. Soaked in water seed coat polygonal cells. Embryo appeared as innermost swells and gets covered with transparent, colorless, structure surrounded by endosperm cells. The cells of mucilage with mucilaginous taste (Plate 1). embryo were small in size and polygonal in shape.

Plate 1—Lepidium sativum plant and seed 468 INDIAN J NAT PROD RESOUR, DECEMBER 2011

Plate 2—Wrightia tinctoria plant and ssed

Plate 3.1— Transverse section of Chansur seed Plate 3.2—Representative diagram of Chanssur seed Indrajau collection of plant material from non polluted area24. In normal seed, the outer cover testa was present Loss on drying turned out to be 6.44% in chansur and and the inner layer tegmen is attached to inner side of 3.42% in indrajav which is not too high, hence could testa layer. Transverse section showed distinct discourage bacterial, fungal or yeast growth25. endosperm layer from embryo provided with aleurone As ash value is useful in determining authenticity layer. Small embryo occurs in groves, consisting and purity of drugs and also these values are shield shaped cotyledon known as scutellum important quantitative standards26. Content of total (Plates 4.1 & 4.2). ashes in all powder were found to be relatively lower Physicochemical evaluation i.e. 5.40% in Chansur and 5.02% in Indrajav which The physico-chemical parameters help in judging may be due to low content of carbonates, phosphates, the purity and quality of the drug. The powder drugs silicates and silica. This is also in accordance with were evaluated for its physico-chemical parameters low content of acid insoluble ash in Chansur (0.34%) like foreign matter, loss on drying, total ash, acid and Indrajau (1.06%). The total ash is particularly insoluble ash and different extractive values. Foreign important in the evaluation of purity of drugs, the matters were found to be 1.25% in Chansur and presence of or absence of foreign inorganic matter 1.36% in Indrajav. This may be due to first hand such as metallic salts or silica. The results suggest that BIGONIYA et al.: PHARMACOGNOSTIC STANDARDIZATION OF L. SATIVUM AND W. TINCTORIA SEEDS 469

Plate 4.1—Ttansverse section of Indrajau seed Plate 4.2—Representative diagram of Indrajau seed

Table 1—Fluorescence analysis of Chansur and Indrajav seed powder Seed Observation Powder + Solvents Under Dry Powder + Powder + Powder + Powder + Powder + Powder + Powder + Powder + Powder + Powder Water HCl HNO3 H2SO4 NaOH KOH Alc. NaOH Alc. Ammonia KOH Chansur Normal light Brownish Khakhi Saddle Golden Pale Dark Sienna Yellow Yellow Peru yellow brown rod golden rod red U.V. light Brownish Light Light Golden Light Blackish Dark Lawn Lawn Yellowish yellow green green rod green brown brown green green green Indrajau Normal light Khakhi Khakhi Dark Dark Dark Dark Gold Golden Golden Golden brown golden rod brown golden rod rod rod rod U.V. light Earthy Green Dark Olive Dark Olive Lime Lime Lawn Light white brown green brown drab green green green green

Chansur and Indrajau seeds have high water soluble Table 2—Fluorescence analysis of Chansur and Indrajav seed with extractive value (mucilaginous and 39.84%) in different solvents comparison to the alcohol (15.36 and 29.92%), Seed Extract Normal Light UV. Light petroleum ether (1.288 and 1.820%), chloroform Chansur Petroleum ether Light yellow Light green (1.656 and 1.956%), ethyl acetate (1.288 and 1.940%) Ethyl acetate Pale golden rod Pale green and methanol soluble (6.540 and 5.436%) extractive Chloroform Khakhi Spring green values, respectively. It indicates the possibility of Methanol Gold Greenish yellow considerable amount of polar compounds and Indrajav Petroleum ether Peru Olive drab presence of large quantity of water soluble Ethyl acetate Saddle brown Brownish black constituents such as sugar, glycosides, phenolics and Chloroform Saddle brown Brownish black tannins in the seeds27. Methanol Sienna Brownish black

Fluorescence analysis of drug powder and extracts that are fluorescent28. Some constituents show The result of fluorescence studies of seed powder fluorescence in the visible range in day light. The using different reagents are given in Table 1 and ultra violet light produces fluorescence in many that of the extracts is compiled in Table 2. natural products (e.g. alkaloids like berberine), which Fluorescence is an important phenomenon exhibited is not visible in day light. If the substances themselves by various chemical constituents present in plant are not fluorescent, they may often be converted into material. Many phytochemical fluorescence are seen fluorescent derivatives after reacting with different when suitably illuminated. The fluorescence colour reagents hence some crude drugs are often assessed is specific for each compound. A nonfluorescent qualitatively in this way and it is an important compound may fluorescent if mixed with impurities parameter of pharmacognostical evaluation29. The 470 INDIAN J NAT PROD RESOUR, DECEMBER 2011

Table 3—Preliminary phytochemical sceening of Chansur and Indrajau seeds with different solvents Seed Solvent Phytoconstituents Inulin Starch Starch Tannin Steroid Saponin Alkaloid Mucilage Mucilage Glycoside Flavonoid Amino acid Amino acid Triterpenoid Carbohydrate Chansur Petroleum ether - + - - + + - - - - + - Chloroform - - + + - + - + + + - - Ethyl acetate - + - - - + - - - - + - Methanol + + - - + - + - - - + + Water - + + + + - - - - + + Indrajau Petroleum ether - + - - + + + - + - + - Chloroform - + + + + + + + - + - - Ethyl acetate - + - - - + + - - - + - Methanol + + - - + - - - - + - + Water + + + - + + - - + - + + “+” = Presence of constituent “-’’ = Absence of constituents results of fluorescence analysis of seed powders Swelling index showed their characteristic fluorescent colour. Swelling index of Chansur was observed 15 ml possibly due to presence of mucilage in seed. Preliminary phytochemical test for extracts W. tinctoria is commonly used as adulterant Preliminary phytochemical investigation was of an important antidysentric drug Holarrhena undertaken for the identification of different type of antidysenterica (Linn.) Wall., another Apocynaceae chemical constituents present in the seeds. Results of plant. Therapeutic properties of both the plants are preliminary phytochemical screening are compiled in quite similar. The differentiation of species are mostly Table 3. Screening of chloroform extract indicated the presence of carbohydrate, glycoside, mucilage, done by morphological basis like size of , colour starch and steroid in both the seed extracts. of flower and root, taste of root and bark etc. Seeds of H. antidysenterica have hair at apex whereas Methanolic extract of seeds showed the presence of 30 alkaloid, inulin, flavonoid and amino acid. Tannin W. tinctoria has tuft of hair at the base of seed . was found only in methanolic extract of Chansur. Whereas, aqueous extracts of both the seeds showed Conclusion the presence of carbohydrate, inulin and mucilage. This study establishes not only pharmacognostic Triterpenoid and flavonoid was present in aqueous and phytochemical characterizations of seed but extract of both the seeds. Amino acid, mucilage and also microscopic and fluorescence characters of both triterpine were present in ethyl acetate extract of the seeds. These characteristics can be used further both the seed. Petroleum ether extract of both the as identification and authentication parameters of the seeds showed presence of amino acid, inulin, seeds. Both the seed are found to be rich in flavonoid mucilage and triterpene, whereas Indrajau also and saponins having wide spectrum of bioactivity. contains tannin and saponin. The seed studied here can be seen as a potential source of useful therapeutics. Further studies are Quatitative estimation of phytoconstituents going on these seeds in order to isolate, identify, Quantitative estimation indicate that Chansur seed characterize and elucidate the structure of bioactive have higher percentage yield of flavonoid (2.50%) compounds along with their pharmacological and saponin (2.96%), whereas the percentage yields activity. of alkaloid and total phenol recorded were minimal

(0.29 and 0.94, respectively). The percentage yield of References flavonoid and saponin in indrajau was found to be 4.4 1 Darshan S and Ved DK, A balanced perspective for and 1.96% whereas yield of alkaloid and total phenol management of Indian medicinal plants, Indian For, 2003, were 0.17 and 0.78% , respectively. 129, 275-288. BIGONIYA et al.: PHARMACOGNOSTIC STANDARDIZATION OF L. SATIVUM AND W. TINCTORIA SEEDS 471

2 Dahanukar S A, Kulkarni R A and Rege N N, Pharmacology 17 Kokoshi C J, Kokoshi R J and Sharma F T, Fluorescence of of medicinal plants and natural products, Indian J powdered vegetable drugs under ultraviolet radiation, J Amer Pharmacol, 2000, 32, 81-118. Pharm Assoc, 1958, 47, 715-717. 3 Ozarkar K R, Studies on anti-inflammatory effects of two 18 Harbone J B, Phytochemical Methods- A Guide to Modern herbs Cissus quadrangularis Linn. and Valeriana wallichii Techniques of Plant Analysis, Chapman and Hall Ltd, DC. using mouse model, Ph. D Thesis, University of London, 1998, 42, 129, 189, 203. Mumbai, Mumbai, 2005. 19 Kokate CK, Practical Pharmacognosy, Nirali Prakashan, 4 The Ayurvedic Pharmacopoeia of India, Ministry of Health Pune, India, 4th Edn, 2000, p. 107–111. and Family Welfare, Controller of Publications, New Delhi, 20 Harbone J B, Phytochemical Methods, Chapman and Hall India, 2000, p. 37-38. Ltd, London, 1973, p. 49-188. 5 The Wealth of India-A Dictionary of Indian Raw Material 21 Chang C, Yang M, Wen H and Chem J, Estimation of and Industrial Products, National Institute of Science total flavonoid content in propolis by two complementary Communication and Information Resources, CSIR, New colorimetric methods, J Food Drug Anal, 2002, 10, Delhi, 1998, Vol. 6, p. 71-72. 178-182. 6 Khory R, Materia Medica of India and Their Therapeutics, 22 Mujeeb M, Siddique N A, Najmi A K and Akram M, Komal Prakashan, New Delhi, 1999, p. 63-64. Evaluation of antioxidant activity, quantitative estimation of 7 Kirtikar K R and Basu B D, Indian Medicinal Plants, Bishen phenols and flavonoids in different parts of Aegle marmelos, Singh Mahendra Pal Singh, Dehra Dun, 2004, Vol. 1, p. 174. Afr J Plant Sci, 2010, 4(1), 1-5. 8 Nadkarni K M, Indian Materia Medica, Popular Prakashan, 23 Obdoni B O and Ochuko P O, Phytochemical studies and Bombay, 1976, Vol. 1, p. 1296. comparative efficacy of the crude extracts of some 9 Joshi M C, Patel M B and Mehta P J, Some folk medicines of homeostatic plants in Edo and Delta states of Nigeria, Global Dangs, Gujarat State, Bull Med Ethnobot Res, 1980, 1, 8-24. J Pure Appl Sci, 2001, 8, 203-208. 10 Singh V P, Sharma S K and Kare V S, Medicinal plants from 24 Khandelwal K R, Practical Pharmacognosy, Techniques and Ujjain District Madhya Pradesh, Indian Drugs, 1980, 17(2), Experiments, Nirali Prakashan, Pune, 13th Edn, 2005. 7-12. 25 African Pharmacopoeia, General Methods for Analysis, 11 Shah G L and Gopal G V, Ethnomedical notes from the tribal OAU /STRC Scientific Publications, Lagos, 1st Edn, 1986, inhabitants of the north Gujarat (India), J Ecotoxicol Bot, Vol. 2, p. 123. 1988, 6, 193-221. 26 Kokate C K, Purohit A P and Gokhale S B, Pharmacognosy, 12 Sass J E, Elements of Botanical Microtechnique, Mc Graw Nirali Prakashan, Pune, 34th Edn, 2006. Hill Book Company, New York, 1940, p. 523. 27 Nawagish M, Ansari S H, Ahmad A, Preliminary 13 Easu K, Anatomy of Seed Plants, John Wiley and Sons pharmacognostical standardization of Lawsonia inermis Publication, New York, 2nd Edn, 2002, p. 45-464. Linn. seeds, Res J Bot, 2007, 2(3), 161-164. 14 Indian Pharmacopoeia, Government of India, Ministry of 28 Pimenta A M, Montenegro M C, Araujo A N and Martinez J Health, Controller of Publications, New Delhi, India, 3rd Edn, C, Application of sequential injections analysis to 1985, Vol. 2, p. 74. pharmaceutical analysis. J Pharm Biomed Anul, 2006, 40, 15 Quality Control Methods for Medicinal Plant Material, By 16-34. WHO Geneva, A.I.T.B.S. Publishers and Distributors, New 29 Ansari S H, Essentials of Pharmacognosy, Birla Publications Delhi, 2002, p. 8-24, 51 Pvt. Ltd., New Delhi, 1st Edn, 2006. 16 Chase CR and Pratt RS, Fluorescence of powdered vegetable 30 Chopra R N, Nayar L and Chopra I C, Glossary of Indian drugs with particular reference to development of a system of Medicinal Plants, Council of Scientific and Industrial identification, J Amer Pharmacol Assoc, 1949, 38, 32. Research, New Delhi, India. 1956, p. 198.