ANVESHANA PHARMACOGNOSTIC AND PHYTOCHEMICAL ANALYSIS OF GOKSHURA (TRIBULUS TERRESTRIS LINN) W.S.R. TO CARDIOPROTECTIVE ACTIVITY

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1 ANVESHANA Research Article AYURVEDA MEDICAL JOURNAL ISSN: PHARMACOGNOSTIC AND PHYTOCHEMICAL ANALYSIS OF GOKSHURA (TRIBULUS TERRESTRIS LINN) W.S.R. TO CARDIOPROTECTIVE ACTIVITY Shaialja J. R. Reader and HOD, Dept. of Agada Tantra, N. K. Jabshetty Ayurvedic Medical College PG Centre, Bidar, Karnataka, India Corresponding Author: ABSTRACT In the current day clinical practice CVD are the commonest cause for mortality, accounting up to 16.7 million (29%) of people around the globe. By 2020 AD, CVD will become the leading cause for both death and disability. Among multitude of pharmacological activities Hrudya karma is one of the important karma envisaged by the Acharyas; hence Hrudya dashemani and certain other Hrudya dravyas to protect heart and to prevent CVD are mentioned. Gokshura (Tribulus terestris linn) is one among them and it is said to have hrudya karma by nigantukaras and both are mentioned in the context of hrudroga chikithsa in many formulations. Several phyto constituents present in Gokshura will help protect heart in different ways. Hence in the present study a detailed analysis of Gokshura both pharmacognostically and phytochemically has been made. Key words: Phytochemical, Pharmacognostical, Cardio vascular disease. INTRODUCTION In the current day clinical practice CVD are the commonest cause for mortality, accounting up to 16.7 million (29%) of people around the globe. By 2020 AD, CVD will become the leading cause for both death and disability 1.a,b. Gokshura botanically identified as Tribulus terrestris Linn belongs to Zygophyllaceae family. It is found widely distributed in warmer regions of the world. It is a common weed of road sides, chiefly in hot, dry or sandy localities. Specially in India, it is found common in dry and hotter parts of the country viz, Rajasthan, Andhra Pradesh, Gujarat 2,3,4 etc. It is a small, prostrate, hirsute, and annual or biennials herb grows up to 90cm in length. Leaves are abruptly simple, pinnate, opposite. Leaflets: 3-6 or more pairs, almost sessile or with short petioles, 0.2 to 0.5inch in length, rounded or oblique at the base, mucronate at the apex. Flowers: Bright yellow, solitary, pseudo axillary or leaf opposed. Fruits: Fruit is a Schizocarp, stalked, light or greenish yellow five ribbed or angled 2,5,6,7. The plant containing major chemical constituents like Diosgenin (a steroidal saponin), Glycosides, Oleic acid, calcium 2,8 etc. The drug being madhura rasa, madhura vipaka, sheeta veerya acts as thridosha shamaka, rasayana, balya, bhrimhana, vrushya, mootrala 9,10. etc. Even though different parts

2 of the plant are used medicinally fruit is one of the important useful part which exhibits several therapeutic effects like diuretic activity (Gujral et al), anti-cancer activity (Behdir and Khan et al), anti-angina activity (Bowen, W. Tongen, M. Tongker) 2,4,5,11 etc. The present study is aimed to carryout pharmacognostical and phytochemical analysis of Gokshura w.s.r to its cardio protective activity. MATERIALS AND METHODS 1. Plant material collection: The authenticated dried fruits of tribulus terrestris linn were collected from market. Foriegn matter is separated and cleaned. OBSERVATIONS AND DISCUSSION 2. Analysis: The external fruit morphology was observed and studied for the parameters-color, odour, texture and taste. Microscopical study was carried out after soaking the drug in water for 12 hrs. Chloral hydrate, Iodine, Phloroglucinol solution, Ferric chloride solution and Sudan III solutions were used for staining the different sections separately and observed under the microscope. The dried fruit powder was used for determination of ash values, extractive values and Ph according to standard procedures 12. Alcoholic extract was prepared by soxhalation technique and subjected to phytochemical analysis, TLC and HPTLC. Table 1: Orgenoleptic observation of Gokshura 1. Shabdha Easily separating cocci 2. Rupa Light Yellowish 3. Rasa Slight bitter and sweetish 4. Gandha Characteristic odour 5. Identifying features Schizocarp, light yellow, five ribbed, stalked, covered with pubescent hairs. Table 2: UV analysis of the drug Gokshura Sl. Gokshura Sample with reagent No. Visible rays UV 365 m UV 254 m 1 Powder as such Yellowish Brown Yellowish Brown Dark yellowish brown 2 Powder with water Yellowish Brown Yellowish Brown Blackish brown 3 Powder with Dil. Hcl Yellowish Brown Yellowish Brown Blackish brown 4 Powder with ethanol Light Brown with yellow tinge Light Brownish yellow Brownish yellow Macroscopic Characters: Fruits are, schizocarp dried, ripe, stalked, light yellow, five ribbed or angled more or less spherical in structure and covered with short stiff or pubescent hairs, prominent short stiff spines, pointed down wards. Tips of spines almost meet in pairs, whole together forming pentagonal frame work around the fruit. Ripe fruit separates in to five segments of each cocci and each appears as single fruit. Each coccus semi lumen or Plano convex in structure, armed with a pair of spines, containing four or more seeds. Fig 1: Fruit and flowers of Gokshura AAMJ / Vol. 1 / Issue 5 / Sep Oct

3 Fig 2: Fruit as a whole and separated cocci Microscopy of fruit Gokshura (microscopy of single cocci) PORTION ENLARGED MICROSCOPY TRI PAR TRI MES MES EPI T.S. OF THE FRUIT (GROUND PLAN)SINGLE COCCI PORTION ENLARGED PORTION ENLARGED SHOWING ENDOCARP PORTION ENLARGED SHOWING OIL GLOUBLES AND COTYLEDON PORTION STC END OG PAR Fig 3: T.S of the fruit showed outer pericarp, which is differentiated in to epicarp, mesocarp and endocarp. Outer surface of the epicarp that is epidermis is covered by simple, nonglandular, elongated simple tuffts of trichomes. The mesocarp region is parenchymatous, many layered, cells are thin walled, closely arranged.the endocarp is 3 4 layered composed of Table 3: Physical Constants of the Drug sclerenchymatous cells. Seed is small outer region consists of single layered epidermis and cotyledon, and is made up of thin walled, compactly arranged paranchymatous cells and these cells show abundant oil globules. Sl. No. Test Permissible limit Obtained percentage for Gokshura 1 Total ash Not more than 15% 12.24% 2 Acid insoluble ash Not more than 2% 1.92% 3 Water soluble extractive Not less than 10% 12.69% 4 Alcohol soluble extractive 6% 10.56% 5 Foreign matter Not more than 2% 1.37%w They were in the permissible limits % and 12.24% ash value in Gokshura indicates presence of inorganic constituents like calcium, Iron, chlorides, and sulphates. AAMJ / Vol. 1 / Issue 5 / Sep Oct

4 Phyto chemical Table 4: Phyto-chemical analysis Tests Procedure Observation Results Alkaloids Dragendroffs test The extract was dissolved in dilute sulphuric acid, and to this acidic solution, Dragendroffs reagent was added. Reddish-brown precipitate indicates presence of alkaloids Absent Glycosides Saponins Tannin Modified Borntragers test Foam Test Froath test Ferric chloride test To the extract 5ml of 5% ferric chloride solution and 5ml of dilute HCL were added and boiled on water bath. After cooling, benzene or any organic solvent was added and shaken well. The banzen layer was separated and equal volume of dilute ammonia was added. To the filtrate, Few ml of water was added and shaken well. To the filtrate, few drops of sodium bicarbonate solution were added. The mixture was shaken vigorously and left for few minutes. To the alcoholic extract dissolved in water +few drops of 5% FeCl 3 solution. Pinkish red colour of Ammonical layer indicates the presence of glycosides. Froth is produced. Honeycomb like froth was produced. Blue, black Brownish or green precipitate indicates presence. Absent Steroids Flavonoids Carbo hydrates Proteins Salkowski reaction Ferric chloride test a. Benedicts test b. Fehlings test Biuret s test 2ml chloroform solution+1ml Con. H 2SO 4 added from side of test tube 2ml of FeCl 3+2 drops of Alc.Ext. 0.5ml Alc.Ext+ 5 ml of benedicts solution, boil for 5min 2ml of Alc. Ext+ 1 ml of fehlings sol. (Mixture of sol. A and B), boil for few min. To the filtrate, Biuret s reagent was added. Red colour indicates the presence. Blackish red indicates the presence. Reddish brown ppt indicates presence Red ppt. Indicates the presence. Violet colour indicates the presence Table 5: Chemical analysis for inorganic constituents Tests Procedure Observation Results Iron To the filtrate, few drops of 2%potassium Ferro cyanide solution were added. Dark blue coloration was observed. Sulphate White crystalline barium sulphate To the filtrate, few drops of 5% was formed which is insoluble in barium chloride were added. hydrochloric acid. Chlorides To the filtrate prepared in dilute nitric acid, few drops of 10% silver nitrate solution were added. White ppt of silver chloride was formed which is soluble in dilute ammonia solution. Carbonates To the filtrate, mercuric chloride was added. Brownish red precipitate was formed. Absent Calcium To 10ml filtrate, 1 drop of dilute ammonium hydroxide and saturated ammonium oxalate solution were added. White precipitate of calcium oxalate was formed; it is soluble in hydrochloric acid. AAMJ / Vol. 1 / Issue 5 / Sep Oct

5 Thin Layer Chromatography (TLC) Materials required: Plant extracts, Precoated T.L.C plates, capillary tubes, T.L.C chambers, hot air oven, solvents, spray reagents Procedure 11, 12, 13 : The T.L.C. chamber was thoroughly cleaned and dried before use. Suitable solvent system was poured into the chamber and the lid was closed. The chamber was kept undisturbed for about an hour for saturation. The T.L.C. plates were kept in hot air oven for 15 minutes at a temperature not exceeding 100 o c to make to devoid of moisture. The individual extracts were dissolved in respective solvents. The plates were spotted with the help of capillary tubes, 1cm away from the base. A distance of 1cm was maintained between each spot. The spotted plate was gently immersed in the TLC chamber containing the saturated solvent system in such a way that the solvent had linear contact with the plate. The solvent was allowed to rise up to the required distance (¾ Th of the plate). Plate was taken out and the solvent front (distance travelled by the solvent) was immediately marked with a pencil line. The plates were sprayed suitable detecting agents and dried in hot air oven for few minutes and the spots were observed. The Rf values of the spots were calculated by using the formula, Rf = Distance travelled by the spot Distance travelled by the solvent front Extract used Phyto constituents Mobile phase Solvent front Rf values Glycosides Benzene, Ethanol (7:3) 4.7cms 0.9 Alcoholic extract Benzen, Ethylacetate Flavonoids (4:1) 5.3cms 0.6 Flavonoids extract High Performance Thin Layer Chromatography (HPTLC): It is a major advancement of TLC principle requiring shorter time and better resolution. The basic difference between conventional TLC & HPTLC is only in particle and pore size of the sorbets. Procedure: TLC plate- Pre coated silica gel on aluminium plates (E.Merck- silica Gel 60 F 254) was used. Glycosides extract The extracts were dissolved in respective solvents and filtered thoroughly to get clear solution. The plates were spotted using TLC applicator (Linomat), placed in the TLC chamber containing suitable solvent systems and allowed to elute. When the mobile phase reached 3/4 th of the plate, the plate was taken out, dried AAMJ / Vol. 1 / Issue 5 / Sep Oct

6 and observed under UV chamber at 254NM and 366NM wavelength. The Rf values of the spots were calculated. The plates were then sprayed with suitable reagents to detect the spots. HPTLC findings of the drug Gokshura DRUG CONSTITUENT SOLVENT Rf VALUE Gokshura Diosgenin Toluene :ethyl acetate(8:2) 0.29 DISCUSSION Ayurveda gives due importance to second chathushpada i.e. dravya which is the radicle of ayurvedic pharmacology. Among multiple pharmacological activities, hrudya karma is one of the prime activities. Generally hrudyam is hrudayaya hitam. Dalhana considered the concept of hrudya in three aspects like which is pleasant to mind, which is good for heart and congenial for heart. Whereas Vagbhata considers hrudya karma as the drugs which are having hrudrogahara karma and agreeable for heart. The drugs attributed with hrudya karma are amla rasa dravyas but Gokshura (Tribulus terrestris) is also having hrudya karma according to nighantukaras, Gokshura is mentioned as hrudrogahara in Bhavaprakasha and Dhanvantari nighantu. Cardio tonic drugs generally increase myocardial contractibility and output in a hypo dynamic heart without corresponding increase in oxygen consumption. In toto cardio protective drug may act in different ways like anticoagulant, vasodilator, cardiac stimulant etc. The drug Gokshura showed physico chemical standards according to API and within normal limits. The UV analysis Spots on the plate shows presence of diosgenin carried out confirms the genuinity of Gokshura. Phyto chemical analysis carried out for Gokshura showed the presence of steroids, flavonoids, carbohydrates, saponins, glycosides and proteins. The TLC study showed the presence of glycosides and flavonoids in the alcoholic extract of Gokshura. The HPTLC study carried out showed the presence of diosogenin (steroidal saponin) in Gokshura. Flavonoids basically contain free hydroxyl groups and exert a good physiological effect on capillaries and they have coronary dilatory effect on heart. They have pronounced to have anti-oxidant or cytoprotective and free radical scavenging activity. Glycosides, especially present in Gokshura can probably improve the function of heart through atrio ventricular conduction and vagal tone. Saponins present in Gokshura are steroidal saponins, probably they have immunomodulatory activity which is needed as vyadhihara rasayana. And they have coronary dilating effect and improve coronary circulation. Iron is having anti-oxidant activity and may help in maintain the normal structure of heart specially left ventricle. AAMJ / Vol. 1 / Issue 5 / Sep Oct

7 Calcium strengthens the cardiac muscles and enhances their inotropic and chronotropic effects. Madhura rasa of gokshura which is balya, tarpana and rasayana property of Gokshura may be attribute to show its better cardio protective activity. In cardiac problems there can be sodium retention which cause edema and congestion requires diuretics. Gokshura being mutrala and rasayana can be reduce the symptoms by avoiding excess fluid without the depletion of Rasadhathu. CONCLUSION In the current study entiteled Pharmacognostic and phytochemical analysis of Gokshura (Tribulus terrestris Linn) WSR to cardio protective activity, the drug Gokshura was studied in detail for its pharmacognostical aspect and phytochemically specially with reference to its cardioprotective activity. It is found that Gokshura containing different phytochemicals like flavonoids, glycosides, steroidal saponins etc. A small attempt has been made to explain how Gokshura with its rasa panchaka and phytochemicals can help in cardiovascular diseases. Further study is required to evaluate Gokshura both experimentally and clinically in cardicac aliments. REFERENCES 1. International cardiovascular disease statistics. a) WHO cardiovascular disease prevention and control, b) Atlas of heart disease and stroke WHO, September Shastry J.L.N. Dravyaguna Vijnana Vol II Foreward by Prof. K. C. Chunekhar, 2 nd Edition, Varanasi, Chaukhamba Orientalia. 3. Shivaranjana V. V. and Indira Balachandran, Ayurvedic drugs and their plant sources, Mohan Primlani for oxford and IBH publishing co. pvt. Ltd, New Delhi. 4. Arya Vaidya Sala, P. S. Varier s Vaidyaratnam, Indian Medicinal Plants- A Compendium of 500 Species-I, Reprint, Kottakal, Orient Longman Limited. 5. Kirthikar and Basu, Illustrated Indian Medicinal Plants, Vol -5, 2 nd ed, Sri Sat Guru Publications, Allahabad 6. A Dictionary of Indian Raw Materials and Industrial Products-Raw materials. The Wealth of India. Vol X Reprint New Delhi. 7. Sharma P. C, Yelne M. B, T. J. Dennis, Data Base On Medicinal Plants used In Ayurveda Vol 3, New Delhi, 8. Mukund Sabnis V. D. Chemistry and Pharmacology of Ayurvedic medicinal plants, 1st ed 2006, Chaukambha amarabharathi prakashan, Varanasi 9. Bhavamishra, Bhavaprakasa Nighantu, commentory and edited by Dr. Chunekar K. C, Dr. Pandey G. S, and Varanasi: Chaukambha Sanskrit Sansthan 10. Narahari Pandit, Raja Nighantu, edited with Dravyaguna Prakashika by Dr. Indradio Tripathi, Introduction by Acharya Vishwanath Dwivedhi, 2 nd edition Varanasi 11. a. MAPA-Medicinal and aromatic plant abstracts. b Ayurvedic Pharmacopoeia of India, Vol- I, Ministry of Health and Family Welfare, Govt of India, 1 st Edition, Dept of Indian Science of Medicine and Homeopathy, New Delhi. 13. Kokate C. K, Purohit. A. P, Gokhale S. B, Pharmacognosy, 14 th ed, Nirali Prakashana How to cite this article: Shailaja J. R: Pharmacognostic and Phytochemical Analysis of Gokshura w.s.r. to Cardioprotective Activity. AAMJ 2015; 1: Source of Support: Nil. Conflict of Interest: None declared AAMJ / Vol. 1 / Issue 5 / Sep Oct