Asian J. Pharm. Tech. 2013; Vol. 3: Issue 1, Pg 16-19 [AJPTech.]

ISSN- 2231–5705 (Print) www.asianpharmaonline.org ISSN- 2231–5713 (Online)

RESEARCH ARTICLE

Antioxidant Activity of flabellifer (linn.) Fruits.

Pramod H. J., Dr. A.V. Yadav., Raje V.N., Madhuri Mohite*, Ganesh Wadkar. K.L.E. University, Department of Pharmacognosy and Phytochemistry, J.N.M.C Campus, Nehru Nagar, Belgaum, Karnataka-590010, . GIPER, Limb, Satara. *Corresponding Author E-mail: [email protected]

ABSTRACT: The fruit of Borassus flabellifer Linn. belonging to family Areaceae reported to be posses the antioxidant activity. The extracts have been also evaluated for antioxidant activity by using in-vitro methods DPPH (2, 2-Diphenyl-1 picryl hydrazyl) and ABTS (2, 2-azinobis-(3-ethylbenzo-thiazoline-6-sulfonic acid) assay. The phytochemical analysis carried out revealed the presence of saponins, tannins, flavonoids, carbohydrates, amino acids and phenolic compounds in the extracts.

KEY WORDS: Antioxidant, DPPH, ABTS, Borassus flabellifer Linn.

INTRODUCTION: [2] Chemically, oxidation is the removal of electrons and Antioxidants are potent scavengers of free radicals . reduction is the gain of electrons; oxidation is always Antioxidant action includes radical scavenging capacity, coupled with reduction. Many of the reactive oxygen inhibition of lipid peroxidation, metal ion chelating ability species are free radicals. A free radical is an atom or a and reducing capacity. Antioxidants are beneficial molecule with one or more unpaired electrons [1] . Free components that neutralize free radicals before they can radicals oxidatively damage lipids and proteins and attack cells and hence prevent damage to cell proteins, [3] compromise genomic DNA integrity [2] . These oxidants are lipids and carbohydrates . generated during the normal metabolic reactions in the body. The other sources of free radicals and other oxidants Antioxidants are endogenous or exogenous substances are cyclooxygenation, lipooxygenation, lipid peroxidation, which inactivate the free radicals. These substances include neutrophils etc. other oxidants may be involved in the lipid soluble vitamins, ascorbic acid, sulfhydryl containing pathogenesis of diseases such as cancer, diabetes mellitus, compounds and serum proteins. A wide range of anti- cardiovascular and neurological diseases. oxidant has been proposed for use in treatment of human diseases. The current recommendation is to increase the consumption cereals, nuts, fruits and vegetables all of which are good sources of antioxidants [1] .

Received on 11.12.2012 Accepted on 13.01.2013 © Asian Pharma Press All Right Reserved Asian J. Pharm. Tech. 3(1) : Jan.-Mar. 2013; Page 16-19 16 Asian J. Pharm. Tech. 2013; Vol. 3: Issue 1, Pg 16-19 [AJPTech.]

Whole of B. flabellifer Linn . Fruits of B. flabellifer Linn . Pulp of B. flabellifer Linn .

Borassus flabellifer Linn . () is a tall tree (palm) temperature). After the effective extraction, the solvents growing in sandy soil and attaining a height of about 20-30 were distilled off. The extract was then concentrated on meters with a straight trunk [4] . The fruits are large and water bath to avoid the decomposition of natural fibrous, containing usually three nuts like portions each of metabolites. which encloses a [5] . Flowers and fruits during December to August [6]. The plant has been used In-Vitro Antioxidant Activity traditionally as a stimulant, anti-leprotic, diuretic and By DPPH (2, 2-Diphenyl-1 picryl hydrazyl) radical antiphlogistic. The fruits are stomachic, sedative, laxative scavenging activity and ABTS (2, 2-azinobis-(3- and aphrodisiac in nature useful in hyperdipsia, dyspepsia, ethylbenzo-thiazoline-6-sulfonic acid) radical cation flatulence, skin diseases, haemorrhages, fever and general decolourisation assay. debility. The roots and juice of the plant are useful in inflammatory reactions. The ash obtained by burning the 1] Scavenging Activity of DPPH is a good antacid antiperiodic, and is useful in Principle:- heart burn, splenomegaly [7] . The effect of the plant extract of the DPPH was estimated according to the method of Hou et al. with minor Survey of literature revealed that the medicinal plant modification. The principle of the reduction of DPPH free- Borassus flabellifer Linn. have been used as antidiabetic, radical assay is that antioxidants react with the stable DPPH antidote, anti-inflammatory, wound healing, anthelmintic radical and convert it into 1, 1-diphenyl, 2-picryl hydrazine. activity, analgesic and antipyretic. It has been reported that The ability to scavenge the stable DPPH radical is measured the methanolic extract from the male flowers of Borassus by a decrease in the absorbance. flabellifer Linn. was found to inhibit the increase of serum glucose levels in sucrose-loaded rats which may be due to MATERIALS AND METHODS:- presence of spirostane-type steroid saponins. It also has Step I (Preparation of stock solution):- been documented to possess immunosuppressant property. Prepare 0.05Mm solution of DPPH by mixing 9.8 mg of DPPH in 50ml of ethanol and incubate it at normal room MATERIAL AND METHODS:- temperature for 2-3 hrs. The fruits of Borassus flabellifer Linn. were collected from local areas of Pune, Maharashtra and authenticated by P.G. Step II (Preparation of Drug dilution):- Diwakar Joint Director, at Botanical Survey of India (BSI), Stock solution of 1 mg/ml of the drug/plant extract is Govt. of India, Ministry of Environment and Forests, Pune, prepared in distilled water and diluted to get various India. The fruits were then dried in shade at temperatures concentrations (100-1000 µg/ml). The concentration can between 21-30°C for 15 to 30 days, after which these parts thus be further increased depending upon the results. of plant were fine powered (40 size mesh) by hammer. Finally extraction was carried out by the following Step III (Reaction Mixture and Analysis):- procedure. i) Working solution: - Take out 10ml of the stock solution and dissolve it in 40 ml. 0f ethanol. This makes the working Preparation of the extract solution. The extraction was carried out; around 400 gm of powder ii) Reaction Mixture: - Mix 1ml of the working solution was subjected to successive hot continuous extraction with 1ml various extract /drug concentration (100- (soxhlet) with petroleum ether, chloroform, methanol and 1000 µg/ml) prepare in a final volume of 1 ml. Incubate the chloroform-water. (Each time before extracting with next mixture for 30 min. at room temperature. solvent the powdered material was dried at room 17 Asian J. Pharm. Tech. 2013; Vol. 3: Issue 1, Pg 16-19 [AJPTech.]

iii) Result Analysis : - After 20 min. absorbance of reaction Step III (Preparation of Drug dilution):- mixtures was recorded at 517nm. In all the experiment, Stock solution of 1mg/ml of the drug / plant extract is distilled water served as blank and reaction mixtures prepared in distilled water and dilutes to get various without plant extract/ drug dilutions (1ml of working concentrations (100-1000 µg/ml). The concentration can solution) served as control samples. The changes in the thus be further increased depending upon the results. absorbance of the reaction mixtures were measured using a spectrophotometer and the percent scavenging or inhibition Step IV (Reaction Mixture and Analysis):- was calculated according to the formula [8]. i) Reaction Mixture: - Mix 1 ml of the working solution with 1ml of the various Percent Absorbance Absorbance - extract/drug concentrations (100-1000 µg/ml) prepared in a scavenging or = of control of test X 100 final volume of 1ml. inhibition (%) (Absorbance of control) Note: - All procedures Related to DPPH are to be performed in dark. ii) Result Analysis:- Absorbance of reaction mixtures was recorded at 734nm. In 2] ABTS Radical Decolorization Assay:- all the experiment, distilled water served as blank and Principle: - ABTS diammonium salt radical cation reaction mixtures without plant extract/ drug dilutions (1ml decolorization test was performed using spectrophotometric of working solution) served as control samples. The method of pellegrini et al. The principle of the ABTS ((2, 2- changes in the absorbance of the reaction mixtures were azinobis-(3-ethylbenzo-thiazoline-6-sulfonic acid)) measured using a spectrophotometer and the percent diammonium salt cation radical decolourization assay is scavenging or inhibition was calculated according to the that the antioxidant react with ABTS resulting in the formula. decolourization of the ABTS radical in aqueous phase.

RESULTS : MATERIALS AND METHODS:- The preliminary phytochemical screening carried out on Step I (Preparation of stock solution):- aqueous extract of fruits of Borassus flabellifer Linn. Weigh 4 mg. of ABTS and dissolve it in 100ml. of distilled revealed the presence of phytoconstituents such as water, then take 38mg of pot. Persulphate and dissolve it in carbohydrates, amino acids, flavonoids, tannins, saponins, 1ml. distilled water, from this add 88 µl of potassium vitamin C and phenolic compounds. persulphate (K2S208) to 100ml of ABTS solution and keep it for overnight incubation at 4 o c. For assessing In-vitro antioxidant activity DPPH and ABTS

free radical scavenging methods were used on various Step II (Working solution):- concentrations viz 100, 200, 300, 500, 600,700,800,900 and The working ABTS reagent was prepared by diluting the 1000 µg/ml of potent antiulcer extracts from which aqueous stock solution with ethanol to give an absorbance of 0.7 ± extract showed antioxidant activity on concentration 0.05 at 734 nm. dependent manner. The values are shown in Table1.

Table No.1 Effect of Aqueous and Methanolic extracts on DPPH and ABTS antioxidant method Concentration of DPPH (%) inhibition DPPH (%) inhibition ABTS (%) inhibition ABTS (%) inhibition Extracts µg/ml Aqueous Extract Methanolic Extract Aqueous Extract Methanolic Extract 100 40.27±0.34 37.23±0.54 28.71±0.34 31.23±0.54 200 43.46±1.3 39.42±0.23 36.91±0.8 34.32±0.98 300 51.97±0.56 42.49±0.65 47.65±0.56 38.42±0.54 400 54.27±0.67 48.61±1.12 554.88±0.46 41.68±0.87 500 59.82±0.54 50.67±0.56 57.87±0.63 47.63±0.78 600 63.18±0.12 54.14±0.21 59.17±0.18 53.31±0.34 700 67.27±0.45 56.38±0.98 61.71±0.45 52.42±0.65 800 72.83±0.89 61.55±0.54 64.65±0.87 61.52±0.23 900 75.93±0.23 68.31±0.18 71.23±0.43 61.54±0.65 1000 81.34±1.067 71.25±0.4 74.12±0.43 63.49±0.87

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DISCUSSION : REFERENCE: 1. Satoskar RS, Bhandarkar SD, Rege NN, Pharmacology and The free radical chain reaction is widely accepted as a th common mechanism of lipid peroxidation. Radical Pharmacotherapeutics. Pouplar Prakashan, 2007.12 ed.1072. 2. Srinivasan RM, Chandraseka J N, Nanjan MS, Suresh B, Free scavengers may directly react with and quench peroxide radical Scavenging activity of Spomoea obscura (L.) Ker-gawl. radicals to terminate the peroxidation chain reactions and Journal of Natural Remedies.2007; 7(2): 184-88. improve the quality and stability of food products. Assays 3. Beris H, Anti-oxidant effects a bases of drug selection .Drugs based upon the use of DPPH• and ABTS•+ radicals are 1991; 42:569-605. among the most popular spectrophotometric methods for 4. Kapoor LD, Hand book of Ayurvedic medicinal first ed. Washington: CRC Press; 2005, 82. determination of the antioxidant capacity of foods, 5. Van Rheedede’s Hortus malabarus, English ed. Vol. I 23-24. beverages and vegetable extracts. Both chromogens and 6. Arya Vaidya sala. Indian Medicinal plants ; Orient [9] radical compounds can directly react with antioxidants . Longaman Ltd 1996; 293. 7. Virupaksha JH and Shivkumar H. Anti-ulcer activity of Fengreek Herbal drugs containing antiradical constituents are gaining in Aspirin plus pylorus ligation model. Indian Journal of Natural product. 23(3):3-7. importance in prevention and treatment of diseases and free 8. Shirwaikar A, Ashwatha Ram, Mohapatra P, Antioxidant and radical scavengers like phenolics are known for their Antiulcer activity of aqueous extract of a polyherbal therapeutic activity. formulation, Indian Journal of Exp Bio 2006 ; 44 :474-80. 9. Tuba A. k. I˙lhami Gulcin, Antioxidant and radical scavenging Antiradical activity of Borassus flabellifer Linn. extract was properties of Curcumin. Chemico-Biological Interactions. 2008; tested by its ability to bleach the stable DPPH radical. This 174: 27–37. 10. Ghoghari AM, Bagul MS, Anandjiwala S, Chauhan MG, Rajani assay is being used widely as a preliminary test which M; Free radical scavenging activity of Aspidium cicutarium provides information on the reactivity of test compound rhizome. Journal of Natural Remedies.2006; 6(2):131-34. with a stable free radical since odd electron of DPPH gives 11. Audipudi AV. and Bhaskar V.S. Chakicherla. Antioxidative and strong absorption band at 517 nm and when it is quenched Antimicrobial Activity of Methanol and Chloroform Extracts of by the extract, there is a decrease in absorbance [10] . Gmelina Arborea Roxb. International Journal of Biotechnology and Biochemistry.2010; 6 (1): 139-144. 12. Kannabiran K, Mohankumar T, Gunaseker V. Evaluation of CONCLUSION: Antimicrobial Activity of Saponin Isolated from Solanum The antioxidant activity could be attributed due to the Xanthocarpum and Centella asiatica . International Journal of presence of high content of crude flavanoids, saponins and Natural and Engineering Sciences.2009; 3(1):22-25. [11] 13. Ghedini PC &. Almeida CE. Butanolic Extract of Aster phenolic compounds . From literature survey phenolic squamatus aerial Parts is the active fraction responsible to the compounds and saponins has been reported to possess antiulcer and gastric acid antisecretary effects. Latin American [12, 13] antioxidant activity . Journal of Pharmacy.2007; 26(6):889-92. Hence, to put into a nutshell, more significant antioxidant activity of aqueous extract may be due to the presence of phenolic compounds, flavonoids or saponins.

ACKNOWLEDGEMENTS: The authors are grateful to the Principal, Department of Pharmacognosy and Phytochemistry, K.L.E. College of Pharmacy Belgaum and GIPER, Limb Satara for their encouragement and providing special permission to use the research facilities to undertake this programme. 19