PHARMACOGNOSTICAL & PHARMACEUTICAL STUDY Aims & Objectives: To carry out the pharmacognostical & physico-chemical analysis of Flax seed & Flax seed oil including TLC. Material & Method Flax seeds were purchased from Lallubhai Vrajlal Gandhi Ltd., Ahmedabad in the month of December 2010. Pharmacognostical evaluation of flax seed including organoleptic, transverse sections, powder microscopy and histo-chemical tests were carried out. Transverse sections were made by free hand sections, powder microscopy flax seed was carried out with and without staining 1. Photomicrographs were taken using Corlzeiss Binocular Microscope attached with camera. Oil was obtained (25%) by grinding & expression pressing method 2. Thin Layer Chromatography: Thin Layer Chromatographic study of the Unsapofiable matter of Flax seed oil was carried out on Silica gel G plate using Hexane: Diethyle ether: Gl.Acetic acid 5:4.5:0.5 (V/V) as solvent system. The developed plate, after drying was sprayed with LB reagent (Libberman buchard) spray reagent followed by heating at 110 C for 10 minutes. From the chromatogram the number of spots and their s were calculated. Sample preparation: Unsaponified matter 3 of Flax seed oil was diluted with diethyl ether and used for spotting. Mobile phase: Hexane: Diethyle ether: Gl.Acetic acid 5:4.5:0.5 (V/V) Stationary phase: Silica Gel G (Merck) Spray Reagent: LB reagent (Libberman buchard) Visualization: a) At Short UV - 254 nm & Long UV - 366 nm 155
Results Organoleptic characters: Organoleptic characters like mucilaginous sweetish in taste, whitish yellow in colour and bland odour. Transverse Section: Transverse section of seed showed testa consists of isodiametric cells with mucilaginous outer walls, collenchymatous cells of middle layer of seed coat cylindrical, single layered, yellowish brown, longitudinally elongated, thick, and lignified and with pitted walls, single layer of flattened polygonal pigment cells with reddish-brown contents, aleurone grains in the cotyledons, abundant globule of fixed oil. (figure 3,4,5) Powder Microscopy: Microscopical characteristics of powdered drug observed were yellow coloured fibre, pitted walls, lignified sclerides, rounded collenchymatous cells in hypodermis and polygonal epidermis cells filled with mucilage, pigment layer, square cells with orange brown mass, aleurone grains & fatty oil globules etc. (figure 6-14) Histochemical evaluation: Transverse sections of flax seed were subjected to various Histochemical tests 4 (table.1). Table 1 Histo-Chemical Tests Sr. Reagent Observation Characteristics Result No. 1 Phloroglucinol+Conc. No Calcium oxalate -- HCL effervescence 2 Phloroglucinol+Conc. Pink Lignified sclerides ++ HCL 3 Sudan Red III Red Oil globules in the cells of endosperm and cotyledon ++ Physico chemical Parameters: The sample of flax seed was first subjected to Fixed oil content 5 parameter and the result showed that it contains 42.05% fixed oil which confirms the genuinity of the drug as per API standards. After that the oil was expressed from the same sample by grinding & expression pressing method and Physicochemical parameters of the flax seed oil subjected to various parameters i.e. Refractive index, loss on drying, acid, specific gravity etc. (table 2) 156
Table 2 - Physico chemical parameters of flax seed oil No. Test Sample Flax seed oil 1 Loss on drying 6 0.19% w/w 2 Refractive Index at 40 0 C 7 1.4835 3 Specific Gravity 40/40 0 C 8 0.9215 4 Acid 9 0.33 5 Iodine 10 170.90 6 Saponification 11 192.77 Thin Layer Chromatography: Thin Layer Chromatographic study of the Unsapofiable matter of Flax seed oil was carried out on Silica gel G plate using Hexane: Diethyle ether: Gl.Acetic acid 5:4.5:0.5 (V/V) as solvent system. The details of the chromatogram i.e. s have been presented in table 4 (figure 15). Table 4 - TLC Profile of Flax seed oil on Silica Gel GF 254 Sample Flax seed Oil Solvent System Hexane: Diethyle ether: Gl.Acetic acid 5:4.5:0.5( V/V) Short UV 254nm Long UV 366nm After spray Libberman Buchard reagent 1 0.27 1 0.34 1 0.04 2 0.41 2 0.53 2 0.12 3 0.5 3 0.68 3 0.28 4 0.7 4 1.33 4 0.44 5 1.03 5 0.62 6 1.32 6 0.89 7 1.47 157
HPTLC DENSITOGRAM (Flax seed oil) 158
Table 5 Physico-chemical parameters of Ashwagandharishta No. Test Ashwagandharishta Standard range prescribed in API 1 Specific Gravity 40/40 0 C 12 1.089 1.05-1.20 2 Solid content 13 38.39 Not less than 18.5% w/v 3 Ph (by paper) 14 3.5 3.50-4.50 4 Alcohol content 15 7.20% 5-10% v/v The sample of Ashwagandharishta was subjected to various physico-chemical parameters and all the parameters were found within the standard range prescribed by Ayurvedic Pharmacopoeia of India 16. Table 6 - TLC Profile of Ashwagandharishta on Silica Gel GF 254 Sample Ashwagandharishta Solvent System Hexane: Diethyle ether: Gl.Acetic acid 5:4.5:0.5( V/V) Short UV 254nm Long UV 366nm After spray Libberman Buchard reagent 1 0.01 1 0.01 1 0.21 2 0.06 2 0.06 2 0.35 3 0.17 3 0.17 3 0.46 4 0.25 4 0.25 4 0.53 5 0.38 5 0.38 5 0.64 6 0.52 6 0.52 159
HPTLC DENSITOGRAM (Ashwagandharishta) 160
Reference 1 Trease GE, Evans WC. Pharmacognosy. 12 th ed. (Eastbourne, U.K : Bailliere Tindall); 1983. Pg. 95-99, 512-547. 2 Dr. Krishnachandra Chunekar, Bhavaprakash Nighantu, Chaukhambha Bharati Academy, Varanasi), 2006, pg. 654. 3 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 202. 4 KR Khandelwal, Practical Pharmacognosy Techniques and Experiments, (Nirali Prakashana, Pune), 2006, pg. 76-78. 5 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, (Government Of India, Ministry Of Health And Family Welfare, Department Of Ayurveda, Yoga & Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 141. 6 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 141. 7 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 190. 8 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 190. 9 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, (Government Of India, Ministry Of Health And Family Welfare, Department Of Ayurveda, Yoga & Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 201. 10 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, (Government Of India, Ministry Of Health And Family Welfare, Department Of Ayurveda, Yoga & Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 200. 11 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, (Government Of India, Ministry Of Health And Family Welfare, Department Of Ayurveda, Yoga & Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 199. 12 Anonymous, The Ayurvedic Pharmacopoeia Of India, Part - II (Formulations), Volume I, First Edition, Naturopathy, Unani, Siddha And Homoeopathy, New Delhi), 2007, pg. 190. 161
13 The Ayurvedic Pharmacopoeia of India, Part II, Vol. II, First edition (Formulations), Appendix -3, 3.8, Govt. of India, Ministry of Health and Family Welfare, Dept. of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homeopathy (AYUSH), New Delhi, 2008, pg. 221. 14 The Ayurvedic Pharmacopoeia of India, Part II, Vol. II, First edition (Formulations), Appendix -3, 3.2, Govt. of India, Ministry of Health and Family Welfare, Dept. of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homeopathy (AYUSH), New Delhi, 2008, pg. 212. 15 The Ayurvedic Pharmacopoeia of India, Part II, Vol. II, First edition (Formulations), Appendix -3, 3.17, Govt. of India, Ministry of Health and Family Welfare, Dept. of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homeopathy (AYUSH), New Delhi, 2008, pg. 225. 16 The Ayurvedic Pharmacopoeia of India, Part II, Vol. II, First edition (Formulations), Govt. of India, Ministry of Health and Family Welfare, Dept. of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homeopathy (AYUSH), New Delhi, 2008, pg. 14-15. 162