37.Studies on the chemical and medicinal value of Vitex negundo Linn.
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This document summarizes a study on the chemical and medicinal value of Vitex negundo Linn leaves. The study found that the leaves contain phytochemicals like glycosides, alkaloids, tannins, and volatile oils. Extracts of the leaves showed antioxidant properties like superoxide scavenging and anti-inflammatory effects in mice models. Glycosides isolated from the leaves also demonstrated antioxidant and anti-inflammatory activities. Gas chromatography-mass spectrometry analysis identified the phytochemical constituents in the volatile oil. The results support the traditional use of V. negundo for its long-term antioxidant, anti-inflammatory, and potential anti-cancer properties.
37.Studies on the chemical and medicinal value of Vitex negundo Linn.
- 1. I.J.A.B.R., VOL. 2(2) 2012: 298-301 ISSN: 2250 - 3579 298 STUDIES ON THE CHEMICAL AND MEDICINAL VALUE OF VITEX NEGUNDO Linn. a Brindha, S., b Thamaraiselvi,T., c Maragathavalli, S., d Annadurai, B. & d Gangwar, S. K. a Department of Bioinformatics, New Prince Arts and Science College, Chennai, b Department of Biochemistry,Government Art College for women,Krishnagiri,TN. c Department of Biochemistry, King Nandi Varman College of Arts and Science, Thellar, d Department of Crop and Horticultural Sciences, Biotechnology Team, College of Dryland Agriculture and Natural Resources, Mekelle University, Mekelle,Ethiopia. ABSTRACT The present study was aimed to study the chemical value and medicinal value of vitex negundo Linn. Herbal medicines are the precursors of many common drugs prescribed in clinical practice modern western industrial countries today. In the present study the selected plant Vitex negundo Linn., belongs to the family Verbenaceae, which is known to possess several medicinal properties. This plant has been extensively investigated by many scientists for its medicinal activities. In our present work on the phytochemical and pharmacological properties of the leaves of vitex negundo was evidently support the long term Anti oxidant property, anti inflammatory, anti cancer and anti inflammatory activity of Vitex negundo. The studies on Vitex negundo leaves were showed good potential to utilize this plant for commercial purposes. KEY WORDS: Vitex negundo, phytochemicals ,secondary metabolites, Thin layer chromatography . INTRODUCTION Plant derived material or preparation with therapeutic or other human health benefits, which contains either raw or processed ingredients from one or more plants. Many of these indigenous plants have shown very significant therapeutic activities like hepatoprotective, anti- inflammatory, antihelmintic etc. (Kokkate, et al., 1988). Principle indigenous systems are homeopathy, herbal medicines (medical herbalism) and aromatherapy. World trade in plant medicines is in billions of dollars. The number of medicinal plants in trade too is astonishing. Germany imports at least 1560 plant species for medicinal purposes (Fransworth, N.K. et al., 1991). The aim of the present study is phytochemical and pharmacological activities of a medicinally well known plant Vitex negundo . Phytochemical studies includes determination of water extractive value of the leaves of Vitex negundo, qualitative analysis of the water extract and thin Layer Chromatographic studies of water extract with isolation and quantization of various phytoconstituents from the leaves of V. negundo. Total Glycoside content, Total Tannin content, Total Alkaloid content, Distillation of volatile oil from the leaves of V. negundo and identification of its various phytoconstituents through GC-MS analysis. Thin Layer Chromatographic studies of the volatile oil. Pharmacological Studies includes Antioxidant studies of the water extract of the leaves were done by using following methods Superoxide scavenging activity, Hydroxyl scavenging activity, Lipid peroxide scavenging activity, Anti-inflammatory activity of the water extract in Balb/c mice. Antioxidant studies of total Glycosides include Superoxide scavenging activity. Hydroxyl scavenging activity Anti-inflammatory activity of total Glycosides in Balb/c mice. Anti-inflammatory activity of the volatile oil in Balb/cmice. MATERIALS AND METHODS A laboratory experiment was carried out to assess the effect of Vitex negundo. Plant The leaves of Vitex negundo Linn., belong to the family Verbenaceae, were collected from Vellore area and the authentification was done at Presidency College Herbarium in Chennai. Animals Mice were supplied by Animal Breeding Station, University of Veterinary and Animal Science, Chennai. Phytochemical evaluation of Vitex negundo Phytochemical investigation of a plant essentially involves a preliminary extraction, separation and isolation of the individual constituents of interest, their purification and characterization. Extraction Extraction is the removal of constituents from the drug. The process involves the separation of medically active portion of plant or animal tissue from the inactive or inert components by the use of selective solvent by standard extraction procedures. The principal methods of extraction are. 1. Maceration 2. Percolation 3. Digestion 4. Infusion 5. Decoction 6. Continuous hot extraction (soxhlet extraction). The continuous hot extraction method was used for the experimental work. This is a classical chemical procedure of obtaining organic constituents from dried plant tissues.
- 2. Chemical and medicinal value of Vitex negundo Linn. 299 In this method the powdered material is continuously extracted in a Soxhlet apparatus with a range of solvent beginning from non polar solvents like petroleum ether and ending with the most polar solvent i.e., water so as to ensure a complete extraction. In the soxhlet apparatus the vapour passes through the side tube and reflux returns to the extraction chamber where the solution collects. As this takes place the liquid level will also rise in the return tube, when the liquid reaches the top of the return tube a siphon is set up and content of the extraction chamber are transferred to the flask. Experiment The coarsely powdered, air dried leaves of Vitex negundo was extracted by using the continuous hot extraction method. The solvent used as water. The extract was subsequently used for preliminary phytochemical screening of alkaloid, glycosides, volatile oils, tannins, resins etc METHODS FOR PHYTOCHEMICAL ASSAY a. Total glycosides (Stass-Otto method) Coarsely powdered crude drug (2g) was percolated with dilute ethanol (50%). The tannins were precipitated from the percolate by the addition of a solution of lead acetate until the precipitation was complete. The precipitate was centrifuged. The precipitate of lead tannate was rejected whereas the supernatant was retained. Hydrogen sulphide was bubbled through the supernatant solution to remove the excess of lead as lead sulphide. The solution was filtered to remove the precipitate and then evaporated to dryness to get the residue of total glycosides. (Turner et al.,1975 Amount of total glycoside was estimated and result is in Table no:3. b. Total Tannins 3g of the powder was shaken in 100ml of water for 30min. on a mechanical shaker. The extract was filtered and the residue was washed thoroughly with water. The filtrate was treated with lead acetate to precipitate the tannins as lead tannate. The precipitate was centrifuged, washed with water and suspended in ethanol. Then hydrogen sulphide gas was passed to remove excess of lead. The solution was filtered. The precipitate was discarded and the filtrate was evaporated to dryness on a water bath to a constant weight. Amount of total tannin was estimated and result is given in table no:3 c. Total Alkaloid content Accurately weighed (2 grams) crude drug was extracted with 95% ethanol in a soxhlet extractor till extraction was complete. The extract was evaporated to dryness on a water bath and treated with dilute HCl (50ml). The solution was filtered and the filtrate was made alkaline with sodium carbonate and extracted with 3 portions of chloroform of 25ml each. The chloroform extracts were collected together and evaporated to a constant weight on a water bath. Amount of total alkaloid was estimated and result is in table no:3 Antioxidant assay of glycosides isolated from the leaves of Vitex negundo a. Determination of superoxide scavenging activity of glycoside Superoxide scavenging was determined by nitroblue tetrazolium reduction method. Principle, procedure and calculations of this method were same to that of water extract. One difference was, instead of various concentrations of water extract, here various concentrations of glycosides were taken. Anti-inflammatory activity of glycosides isolated from the leaves Vitex negundo Acute and chronic anti-inflammatory activity of glycoside isolated from the leaves of V. negundo was evaluated by the method of carrageenan and formalin induced paw oedema in mice hind paw. Anti- inflammatory activity of water extract Acute and chronic anti-inflammatory activity was evaluated. The former was done by the method of carrageenan induced paw-oedema in mice and the latter by formalin induced oedema in mice hind paw. a. Carrageenan induced paw oedema in mice Animals were divided in to four groups with four animals in each group. In all group acute inflammation was induced by sub plantar injection of 0.02ml of freshly prepared 1% suspension of carrageenan in normal saline in right hind paw of mice. One group was kept as the control, the second group kept as the standard reference and they have administered orally with 10mg/kg diclofenac. The third group received 100mg/kg and fourth group 250mg/kg of water extract orally 1 hour before to the sub plantar injection of carrageenan. The paw thickness was measured initially before carrageenan injection and during six consecutive hours (1 hour interval) after carrageenan challenge by using vernier calipers. Oedema was calculated as the difference between the two measurements. Reduction of swelling was determined by comparing the changes in hind paw volume in drug treated, standard and control mice. b. Formalin induced chronic paw oedema in mice The animals were divided in to four groups with four animals in each group. In all groups, chronic inflammation was induced by sub- plantar injection of 0.02ml of 2% formalin in the right hind paw of mice. One group was kept as control while the second group referred as standard, which is treated with 10mg/kg diclofenac orally one hour prior to the sub plantar injection. The third group received 100mg/kg and fourth group received 250mg/kg of water extract orally one hour before to the formalin injection. The administration of the diclofenac and extract was continued for six consecutive days. Degree of inflammation was measured using vernier calipers before and 6 days after formalin challenge. Thin Layer Chromatography (TLC) TLC of the volatile oil was performed using silica gel poured on a glass plate as stationary phase and Benzene: Ethylacetate (95:5) as a mobile phase and Rf value was calculated. Gas chromatograph/mass spectrometer (gc/ms) analysis Gas chromatograph is the ideal technique for the separation of thermally stable and volatile compounds. The separated compounds are converted to ions in the mass spectrometer and are separated according to mass to charge ratio. The mass spectrum is plotted as detector output against mass. RESULTS
- 3. I.J.A.B.R., VOL. 2(2) 2012: 298-301 ISSN: 2250 - 3579 300 Chronic Anti-Inflammatory Activity of Water Extract of V.negundo in Formalin Induced Inflammation 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Before Initial 1st day 2nd day 3rd day 4th day 5th day 6th day Time interval Pawthickness Control Standard 100mg/kg 250mg/kg The results obtained by the evalution of leaves extract and the percentage of phytochemicals given in the table 1 and 2. The preliminary phytochemical screening indicated the presence of glycosides, alkaloids and tannins are showed in the table 2. There are various kinds of complex secondary metabolites such as glycosides, alkaloids, flavonoids, volatile oils are having pharmaceutical significance. (Ramawat, et al., 1999). Increased antioxidant intake may help to reduce oxidant stress and to minimize the development of asthmatic symptoms. (Miric, M. et al., 1991) A series of acylation reactions were performed on this compound to increase its cytotoxic potency. One of these derivatives viz 5-3’–dihexanoyloxy- 3,6,7,4– tetramethoxy flavone has comparative cytotoxic potency to vitexicarpin this indicate the presence of anticancer property of vitex negundo (Diaz, F. et al., 2003) Table 3 fig. 1 shows the oral administration of the water extract close to 100mg/kg and 250mg/kg was found to inhibit the carrageenan and formalin induced paw oedema this was indicated by the presence of anti-inflammatory activity of Vitex negundo (Chawla, A. S. et al., 1992) & Green Wald, R.A., 1991). Water extract of V. negundo was found to scavenge the superoxides generated by photo reduction of riboflavin. The concentration of water extract of V.negundo needed for 50% inhibition (EC50) of superoxide radicals were found to be 99g/ml this was indicate the presence of antioxidant property of Vitex negundo. Degradation of deoxyribose mediated by hydroxyl radical generated by the Fe3+ /ascorbate/EDTA/H2O2 system was also found to be inhibited by the V. negundo extract. The concentration of water extract needed for 50% inhibition (EC50) was found to be 80. Water extract of was found to inhibit lipid peroxides generated by the induction of Fe2+ /ascorbate/Fe3+/ADP/ascorbate in rat liver homogenates. The concentration of the water extract needed for the 50% inhibition (EC50) was found to be 95g/ml TABLE 1. Extractive Value As per Hot Soxhlet Extraction Solvent Percentage Water 26% Preliminary Phytochemical Screening The results obtained in the qualitative chemical examination of the extract was showed in Table No: 2 Plant Constituents Water extract Test/Reagents Colour Result 1. Alkaloids -Dragendorff’s reagent Yellow +ve 2. Tannins- Ferric Chloride Solution Greenish black +ve 3. Sterols- Concentrated Sulphuric acid Red ring +ve 4. Glycosides -Libermann’s – Burchard reagent Pink Colour +ve 5. Flavonoids- Ethanolic extract + Magnesium ribbon + Concentrated HCl Orange Colour +ve 6. Resins- concentrated Nitric acid No colour change -ve 7. Carbohydrates- Molisch’s reagent Pinkish ring +ve 8. Test for polysaccharides- Anthrone test Green ring +ve 9. Protein- Pottasium Iodide + Iodine solution Yellow colour +ve FIGURE-1
- 4. Chemical and medicinal value of Vitex negundo Linn. 301 TABLE 2. Chronic anti-inflammatory activity of Glycosides Groups Paw thickness before formalin nection (cm) Paw thickness after 6days (cm) Increase in paw thickness(cm) % of inhibition Group-I Control 0.21 0.01 0.445 0.006 0.225 0.008 - Group-II Standard - Diclofenac(10mg/kg) 0.238* 0.017 0.285*** 0.013 0.047 0.005 79% Group-III Glycosides (250mg/kg) 0.224* 0.012 0.243*** 0.005 0.019 0.002 91% TABLE: 3 Amount of total glycoside, Tannins and Total Alkaloids was estimated Constituent Percentage w/w Total glycosides Total Tannins Total Alkaloids 25% 15% 10% DISCUSSION Medicinal plants contribute nearly 25% of the prescribed drug in the world market. In recent years screening of such plants for biological activities has resulted in the development of therapeutics used in the treatment of cancer, AIDS and others. A large number of medicinal plants are exploited from natural flora for the commercial production of the drugs. In recent study revealed that the phytochemical and pharmacological activities of a medicinally well known plant Vitex negundo , the results of present phytochemical and pharmacological observations may evidently support the long term Anti oxidant property, anti inflammatory, anti cancer and anti inflammatory activity of Vitex negundo. The studies on Vitex negundo leaves showed good potential to utilize this plant for commercial purposes. REFERENCES Chawla, A. S., Sharma, A. K., Handa, S. S. Dhar, K. L. (1992) Tyrosinase inhibitory lignans from the methanol extract of the roots of Vitex negundo Linn. and their structure-activity relationship.J. Nat prod. 55(2): 163-167. Diaz, F., Chavez, D., Lee, D., Tan, G.T., Kardono, L. B., Riswan, S., Fair Child, C.R., Fransworth, N. R., Cordell, G.A., Pezzuto, J. M., Kinghor, A.D. (2003) Cytotoxic flavone analogues of vitex carpin, from V. negundo. J. Nat products. 66 (6): 865-7. Fransworth, N.K., Soejarto, D.D. and Bingel, A. S. (1991) Medicinal plants in therapy. Bulletin of world health organization. p.80. Greek Wald, R..A. (1991) Animal model for evaluation of arthritic drugs. Meth. Find Clin Pharmacol. p.75. Kokate, C. K., Purohit, A. P., Gokhale, S. B. (1988) Pharmacognosy. Vol. (6):12-28. Miric, M. and Haxhiu, M. A., Effect of vitamins on Exercise – Induced Broncho constriction. Can. J. Anaesth . 43(2):94-97. Ramawat, K.G., Merillon, J.M. (1999) Biotechnology– Secondary metabolites. Science Publishers, Enfield, NH, USA, 1999 p. 39-69, 193, 305.