Pharmacognosy Pharmacognostic Standardization, Physico- and Phytochemical Evaluation of Amaranthus Spinosus Linn. Root Jhade D, Ahirwar D, Jain R, Sharma NK, Gupta S School of Pharmacy, Chouksey Engineering College, Bilaspur, Chhattisgarh, India Address for correspondence: Mr. Sandeep Gupta; E-mail: [email protected] ABSTRACT Amaranthus spinosus Linn. (Amaranthaceae) is found throughout India. This tree species has been of interest to researchers because it is a medicinal plant employed in the Indian traditional system of medicine. Pharmacognostic standardization; physico-and phytochemical evaluation of the roots of Amaranthus spinosus was carried out, to determine its macro-and microscopical characters, and also some of its quantitative standards. Microscopical studies were done by using the trinocular microscope. Total ash, water-soluble ash, acid-insoluble ash, sulfated ash values, and alcohol-and water-soluble extractive values were determined for physico-chemical evaluations. A preliminary phytochemical screening was also done to detect different phytoconstituents. Microscopically, the root showed cork, cortex, stellar region, and calcium oxalate crystals. Powder microscopy showed anamalous secondary growth in between the xylem vessels and Calcium Oxalate crystals in the cortex region. Total ash was approximately three times more than acid insoluble and water soluble ash. The ethanol soluble extractive was approximately the same as the water soluble extractive. Thin Layer Chromatography (TLC) of the Petroleum-ether extract using Benzene : Ethyl acetate (6 : 1), showed six spots. In the chloroform extract, using Benzene : Ethyl acetate (4 : 1) nine spots were seen, and in the ethanol extract, using Chloroform: Methanol (93 : 7), only four spots were observed, using Iodine vapor as a viewing medium. Phytochemically, the root exhibited terpenes, alkaloids, glycosides, and sugars. These findings might be useful to supplement information with regard to its identification parameters, which are assumed significant in the way of acceptability of herbal drugs, in the present scenario, which lacks regulatory laws to control the quality of herbal drugs. Key words: Amaranthus spinosus Linn., pharmacognostic standardization, physicochemical evaluations INTRODUCTION tropical countries. The roots are used as a laxative, as emollient poultice,[1] as antimalarial,[2] antioxidant,[3] anti- Amaranthus spinosus Linn. (Amaranthaceae)[1] is an inflammatory, antimicrobial, and antidiuretic agents, and annual herb found throughout India and also in many also in hepatic disorders.[4-6] The water extract of the plant showed significant immunostimulating activity.[7] Access this article online Quick Response Code: Pharmacognostic studies have not been reported for the Website: roots of this plant. Therefore, the main aim of the present www.jyoungpharm.in study is to study the macro, microscopic, and some other pharmacognostic characters and physicochemical standards of DOI: 10.4103/0975-1483.83770 the roots of A. spinosus Linn., which could be used to prepare a monograph for the proper identification of the plant. Journal of Young Pharmacists Vol 3 / No 3 221 Jhade, et al.: Pharmacognostical studies on Amaranthus spinosus Linn. root MATERIALS AND METHODS Photomicrograph Microscopic descriptions of the selected tissues Plant material were supplemented with micrographs. Photographs of different magnifications were taken with Nikon The plant specimens for the study were collected from Lab Photo 2 (Two) Microscopic unit. For normal the bank of the Arpa river, Bilaspur, (Chhattisgarh, observations, a bright field was used. For the study of India) 22006’35.83”N and 82008’06.23”E, and were crystal, starch grains, and lignified cells, polarized light positively identified and authenticated by the Botanist was employed. As these structures have a birefringent Dr. Shiddhamallaya N, Regional Research Institute (Ay.), property under polarized light they appear bright against Central council for research in Ayurveda and Siddha, a dark background.[11] Ashoka Pillar, Jayanagar, Bangalore. The voucher specimen No. was (RRCBI / mus.5-27), Reference No. (RRI / Physicochemical evaluations BNG / SMP / Drug Authentication / 2008–‘09 / 959), Physicochemical parameters of A. spinosus root powder Dated 28 / 02 / 2009. Care was taken to select healthy were determined[12] and reported as total ash, water- fully grown plants with normal organs. The samples of the soluble ash, acid-insoluble ash, and sulfated ash values. different organs were cut suitably, removed from the plant, Alcohol and water-soluble extractive values were thoroughly washed with water to remove the adherent determined to find out the amount of water and alcohol- impurities, and dried in sunlight. soluble components. The moisture content and pH was also determined. Macroscopical characterization Preliminary phytochemical screening Macroscopical studies of the root were carried out using The coarse root powder of A. spinosus (25 g) was the naked eye, and the shape, color, taste, and odor of roots subjected to soxhlet for successive solvent extraction. were determined and reported. The extract was concentrated and subjected to various chemical tests to detect the presence of different Microscopical characterization phytoconstituents.[13,14] Sectioning Selected samples of the dried root were stored in a solution RESULTS containing formalin (5 ml), acetic acid (5 ml), and 70% Macroscopical study v/ v ethyl alcohol (FAA) (90 ml). After 24 hours of fixing, the specimens were dehydrated with a graded series of The root was long, easily breakable by hand, about 10 to tertiary-Butyl alcohol as per the method.[8] Infiltration 12 cm in length, and 0.1 to 0.3 mm in breadth. The surface of the specimens was carried out by gradual addition was brown in color, but inside it was cream in color. The of paraffin wax (50 – 60°C m.p.) until the tertiary-Butyl fractures were slightly fibrous [Figure 1]. It had a slightly alcohol solution attained supersaturation. The specimens sweetish taste and agreeable odor. were casted into paraffin blocks. The paraffin-embedded specimens were sectioned with the help of a Senior Rotary Microscopical study Microtome, RMT-30 (Radical Instruments, India). The thickness of the sections was kept between 10 and 12 μm. The transvers section of the root was circular in The dewaxing of the sections was carried out as per the outline and showed the outer cork, cortex, and stellar procedure described by Johanson.[9] The section was regions [Figures 2 and 3]. Abundant clustered crystals stained with phloroglucinol-hydrochloric acid (1 : 1) and of Calcium oxalate were present in the cortex region mounted in glycerin. A separate section was prepared and [Figure 4]. Centrally, the stellar region was present with stained with iodine solution for the identification of starch well-developed xylem and phloem [Figures 2 and 5]. The grains. Powder [Sieve mesh 60 (Sixty)] of the dried roots medullary rays were multiseriate and well-developed. The was used for the observation of powder microscopical cork was six-to-eight layered and the cortex was narrow, characters. The powdered drug was separately treated five-to-seven layered. with phloroglucinol-hydrochloric acid (1:1) solution, acetic acid, and iodine solution to determine the presence of One outstanding anatomical peculiarity is the anomalous Sclerenchymatous interfasicular tissue, parenchymatous growth, which is thick in the axis, and takes place by the tissue, and xylem vessels.[10] development of a succession of collateral vascular bundles 222 Journal of Young Pharmacists Vol 3 / No 3 Jhade, et al.: Pharmacognostical studies on Amaranthus spinosus Linn. root Figure 1: External morphology of the A. spinosus root Figure 2: Microscopical view of the T. S. of the A. spinosus root at ×10 × ×40. [C: Cork, CT: Cortex, X: Xylem, P: Phloem, VB: Vascular bundle, SRC: Sclerenchyma] Figure 3: Microscopical view of the cortex and cork, enlarged at Figure 4: Microscopical view of the cortex with calcium oxalate crystals ×10 × ×40 at ×10 × ×40. [COC: Calcium Oxalate Crystals, CT: Cortex] Figure 5: Microscopical view of the Phloem enlarged at ×10 × ×40. Figure 6: Microscopical view of the vascular bundle, enlarged at [SRC: Sclerenchyma, P: Phloem] ×10 × ×40 from rings or arcs of secondary meristamatic tissue in the consists of parenchyma in some species or the other, pericycle. The bundles are embedded in the parenchymatous lignified or unlignified parenchyma. Frequently the ground ground tissue. One conjunctive tissue, between the bundles, tissue is wholly lignified in some growth zones [Figure 6]. Journal of Young Pharmacists Vol 3 / No 3 223 Jhade, et al.: Pharmacognostical studies on Amaranthus spinosus Linn. root Powder microscopy Table 1: Physicochemical analysis of the root of  Amaranthus spinosus Linn. The microscopic study of the powder revealed the Physicochemical parameters Value presence of an anomalous secondary growth, with Total ash 6.60% w/w Acid insoluble ash 2.09% w/w sclerenchymatous interfasicular tissue, with concentric Water soluble ash 2.44% w/w zones of parenchymatous tissue. There was a presence of Water soluble extract 5.03% w/w abundant clustered crystals of calcium oxalate in the cortex Ethyl alcohol soluble extract 6.60% w/w region. There was a presence of anamalous secondary Moisture content 2.07% growth with concentric conjunctive tissue in between the pH 6.9 xylem vessels. *w/w: weight/weight Table 2: Phytochemical analysis of Amaranthus  Physicochemical parameters spinosus Linn. root Test for Petroleum – Chloroform Ethyl alcohol A. spinosus root powder showed the presence of total ash – constituent ether extract extract extract 6.60% w/w, acid-insoluble ash – 2.09% w/w, water-soluble Alkaloid Negative Negative Positive ash – 2.44% w/w, water-soluble extractive – 5.03%w/w, Steroid Negative Negative Negative alcohol-soluble extractive – 6.60% w/ w, moisture content Terpene Positive Positive Positive Flavanoid Negative Negative Negative – 2.07%, and pH – 6.9 [Table 1]. Glycoside Negative Negative Positive Sugars Negative Negative Positive Preliminary phytochemical studies Saponin Negative Negative Negative Tannin Negative Negative Negative Phytochemical analysis showed the presence of terpene Colour and Yellow oily Green gum Dark green in all three extracts. The alcohol extract also showed consistancy solid gum *Positive: present, Negative: absent a positive report for alkaloids, glycosides, and sugars [Table 2]. The TLC of Petroleum – ether (60 – 80°C) Table 3: Thin layer chromatography pattern of various  extract of the drug on Silica gel 60 F pre-coated sheets extracts of Amaranthus spinosus Linn. root 254 using Benzene : Ethyl acetate (6 : 1) showed six spots in Extract Adsorbent Solvent system Viewing R Values f. Iodine vapor. In the chloroform extract, using Benzene : medium Petroleum– Silica gel 60 F Benzene : Ethyl Iodine 0.02,0.14,0.24 Ethyl acetate (4 :1), nine spots were seen, and in ethanol 254 ether pre-coated sheets acetate (6 : 1) vapor extract, using the Chloroform : Methanol (93 : 7) solvent 60 – 80°C 0.36,0.40,0.62 system, only four spots were observed using the same Chloroform Silica gel 60 F Benzene : Ethyl Iodine 0.11,0.18,0.24 254 pre-coated sheets acetate (4 : 1) vapor 0.33,0.42,0.48 viewing medium [Table 3]. 0.54,0.67,0.85 Ethanol Silica gel 60 F Chloroform : Iodine 0.20,0.29,0.37, 254 pre-coated sheets Methanol (93 : 7) vapor 0.46 DISCUSSION *R: Retention factors f The macroscopic study of the root indicated that its Total ash was approximately three times more than acid color, odor, and taste may be an important characteristic insoluble and water soluble ash. The ethanol-soluble feature for identifying the plant. The anatomy of the extractive was approximately the same as the water-soluble root was studied by taking a transverse section. The extractive. transverse section of the root was circular in outline and showed an outer cork, cortex, and stellar regions. Phytochemically, the root was found to contain alkaloids, It also showed the presence of abundant clustered glycosides, terpenes, and sugars. The TLC of the crystals of Calcium oxalate in the cortex region and petroleum–ether extract using Benzene : Ethyl acetate well-developed xylem and phloem in the central stellar (6 : 1), which showed six spots. In the chloroform extract, region. The medullary rays were multiseriate and well- using Benzene : Ethyl acetate (4 : 1), nine spots were seen, developed. and in the ethanol extract, using Chloroform : Methanol (93 : 7), only four spots were observed using Iodine vapor The powder microscopical examination showed anamalous as a viewing medium. secondary growth in between the xylem vessels and the presence of abundant clustered crystals of calcium oxalate The constant physical evaluation of drugs is an important in the cortex region. parameter in detecting adulteration or improper handling 224 Journal of Young Pharmacists Vol 3 / No 3 Jhade, et al.: Pharmacognostical studies on Amaranthus spinosus Linn. root of drugs. The total ash is particularly important in the ACKNOWLEDGMENT evaluation, for the purity of the drugs, that is, to identify I express my sincere thanks to Dr. Dheeraj Ahirwar, M.Pharm., the presence or absence of foreign inorganic matter such Ph.D., Principal, School of Pharmacy, Chouksey Engineering as metallic salts and/or silica. The moisture content of the College, Bilaspur (C.G.), who took an interest in looking into drug is not too high, thus it can discourage bacterial, fungi our research needs, thus providing us with the best available or yeast growth, as the general requirement for moisture resources. content in crude drug is not more than 14% w/w . [7] The ash values, extractive values, and moisture content of REFERENCES the roots were determined. The results are depicted in [Table 2]. Pharmacognostic standardization including 1. Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd ed. Vol 3. Dehradun, India: International book distributors; 1999. p. 2057-8. the physicochemical evaluation in Tables 1 and 2 is 2. Hilou A, Nacoulma OG, Guiguemde TR. In vivo antimalarial activities meant for identification, authentication, and detection of of extracts from Amaranthus spinosus L. and Boerhaavia erecta L. in mice. adulteration, as also for the compilation of quality control J Ethnopharmacol 2006;16:236-40. 3. Amin I, Norazaidah Y, Hainida KI. Antioxidant activity and phenolic content standards for crude drugs.[15] As the plant, Amaranthus of raw and blanched Amaranthus species. Food Chem 2006;94:47-52. spinosus Linn. is useful in traditional medicine for the 4. Olajide O, Ogunleye B, Erinle T. Antiinflammatory properties of Amaranthus spinosus leaf extract. Pharm Biol 2004;42:521-5. treatment of some ailments, it is important to standardize 5. Stintzing FC, Kammerer D, Schieber A, Adama H, Nacoulma OG, Carle it for use as a drug. R. Betacyanins and phenolic compounds from Amaranthus spinosus and Boerhaavia erecta L. Z Naturforsch C 2004;59:1-8. 6. Van Dunen MB. Antimicrobial activity of Boerhaavia diffusa L. (Nyctagynaceae). The pharmacognostic constants for the roots of this Pharmacopée et Médecine Traditionnelle Africaines 1985;3:23-5. plant, the diagnostic microscopic features, and the 7. African Pharmacopoeia. General Methods for Analysis. 1st ed. Vol 2. (OAU/ numerical standards reported in this study can be useful STRC) Lagos; 1986. p. 123. 8. Sass JE. Elements of Botanical Microtechnique. New York: Mc Graw Hill; for the compilation of a suitable monograph for its proper 1940. identification. 9. Johanson DA. Plant Microtechniques. New York: Mc Graw Hill; 1940. 10. Kokate CK. Practical Pharmacognosy. Delhi: Vallabh Prakashan; 1997. 11. Esau K. Plant Anatomy. New York: John Wiley and Sons; 1964. CONCLUSION 12. The Ayurvedic Pharmacopoeia of India. 1999;1:191-2. 13. Khandelwal KR. Practical book of pharmacognosy. Pune, India: Nirali prakashan; 2005. The present study on the pharmacognostic standardization 14. Harborne JB. Phytochemical Methods. London: Chapman and Hall; 1998. and the physico-and phytochemical evaluation of the 15. Kokate CK, Purohit AP. A text book of pharmacognosy. Pune, India: Nirali Amaranthus spinosus root might be useful to supplement Prakashan; 2006. information with regard to its identification parameters, How to cite this article: Jhade D, Ahirwar D, Jain R, Sharma NK, Gupta S. which are assumed significant for the acceptability of herbal Pharmacognostic standardization, physico- and phytochemical evaluation of drugs in the present scenario, which lacks regulatory laws Amaranthus spinosus Linn. root. J Young Pharmacists 2011;3:221-5. to control the quality of herbal drugs. Source of Support: Nil, Conflict of Interest: None declared. Journal of Young Pharmacists Vol 3 / No 3 225