Int J Pharma Res Health Sci. 2018; 6 (2): 2497-01 DOI:10.21276/ijprhs.2018. 2018. 02.28 Shanmugavadivu M et al. CODEN (USA)-IJPRUR, e-ISSN: 2348-6465 International Journal of Pharma Research and Health Sciences Available online at www.pharmahealthsciences.net Original Article Preliminary Phytochemical Screening and Anti- bacterial Activity of Grape Seeds Methanolic Extract Vidhya Jaganathan, Shanmugavadivu Muthusamy*, Sandhya Ganesh Department of Biotechnology, Dr. N.G.P. Arts and Science College, Kalapatti road, Coimbatore-641048, Tamil Nadu, India. ARTICLE INFO ABSTRACT Received: 06 Apr 2018 Objective: The aim of the study is to screen the phytochemicals present in the methanolic extract of grape seed and to determine its antibacterial activity against both gram-positive Accepted: 20 Apr 2018 and gram-negative bacteria. Methodology: The present study involves extraction, preliminary photochemical analysis and antibacterial activity of grape seeds belonging to the family Vitaceae for its medicinal value. A qualitative photochemical analysis of methanol extract of grape seeds was performed to identify the presence of alkaloids, carbohydrates, cardiac glycosides, saponins, phenols, flavonoids, proteins, amino acids, tannins, anthraquinones and steroids. Disc diffusion method was conducted to determine the inhibitory effect of grape seed methanolic extract against four bacterial species such as gram positive, Staphylococcus aureus; Streptococcus pyogenes and gram negative, Pseudomonas aeruginosa; Klebsiella pneumoniae. Results: Preliminary phytochemical screening of methanolic extract of grape seed showed the presence of alkaloids, carbohydrate, tannins, flavonoids, proteins, terpenoids, anthraquinones and phenols. Whereas, cardiac glycoside, saponins, amino acids and steroids were absent. Maximum zone of inhibition was observed against Streptococcus pyogene(18mm), followed by Klebsiella pneumoniae (17mm) and ______ Pseudomonas aeruginosa (16mm). It was also observed that grape seed methanolic extract showed only 14mm zone of inhibition against Staphylococcus aureus. Conclusion: As grape seeds found to have tannins, phenols and flavonoids, it can be a potent bioactive agent. The methanolic extract of grape seed was proven to be effective in inhibiting both Gram-positive and Gram-negative bacteria hence it was suggested that it is used in treating the infections as an antibiotics. Key words: Grape seed, Vitis vinifera, methanolic extract, phytochemicals, antibacterial activity. 1. INTRODUCTION Grape (Vitis vinifera), the member of Vitaceae family, is one of the widely cultivated and most important fruit crops in the world 1. This plant is native to most of Europe and northern and central Asia, but has widely naturalized elsewhere in the Corresponding author * world. Grape cultivation is believed to be originated in Shanmugavadivu M. Armenia near the Caspian Sea in Russia, from where it Assistant professor, spreads westward to Europe and eastward to Iran and Department of Biotechnology, Dr. N.G.P. Arts and Science College, Kalapatti road, Coimbatore-641048, Afghanistan. It was introduced in India in 1300 AD by Tamil Nadu, India. Moghul invaders from Iran and Afghanistan2. Grapes can be E mail: [email protected] 2497 IIIIIIIII© International Journal of Pharma Research and Health Sciences. All rights reserved Int J Pharma Res Health Sci. 2018; 6 (2): 2497-01 categorized into grapes with edible seeds, seedless, wine Preparation of methanolic extract: 5g of grape seed grapes, table grapes, and raisin grapes. It is also one of the powder was dissolved in 100ml of methanol and kept in most commonly consumed fruits in the world both as fresh orbital shaker overnight at 120rpm. Then the mixture was fruit and processed fruit. Grape therapy, also known as filtered using whatmann No.1 filter paper and the filtrate was ampelo therapy is a form of naturopathic medicine or evaporated at room temperature to get dried powder. The alternative medicine that involves heavy consumption of yield of dried extract was calculated using the formula, grapes, including seeds, and parts of the vine, including Yield (%) = (W1 X 100)/W2 leave4.About 80% of the grape production is used in juice- Where, W1 was the weight of extract after evaporation and making, and also in this process seeds remained as by- W2 was the dry weight of the sample. product which was formulated for an animal feed. Grape Preliminary phytochemical Screening: seed is a well-known dietary supplement and contains Phytochemical examinations were carried out for the vitamins, minerals, and polyphenols. They are also rich in methanolic extract as per the standard methods11,12,13. sugars, flavonoids, anthocyanins and proanthocyanins, Detection of Carbohydrates: organic acids, tannin, mineral salts and vitamins 3. In recent Fehling’s Test: years, grape seed extract has become increasingly popular on 1ml of crude extract was hydrolysed with dil. HCl, the market as a nutritional supplement especially in the neutralized with alkali and heated with Fehling’s A and B Australia, Korea, Japan and the United States because of solutions. Formation of red precipitate indicates the presence their rich phenolic compounds and their beneficial effects on of reducing sugars. human health5.The grape seed also shown to exhibit Benedict's test: bioactivities such as antioxidant, anti-inflammatory, anti- 1ml of crude extract was mixed with few drops of Benedict’s bacterial, anti-cancer, antiviral, cardio protective, reagent (alkaline solution containing cupric citrate complex) hepatoprotective, neuroprotective, antiaging and anti- and boiled in water bath, observed for the formation of diabetic. The oil extracted from grape seeds is also used in reddish brown precipitate to show a positive result for the cosmetic, culinary, pharmaceutical and medical purposes 6,7. presence of carbohydrate. Seeds, skin, and stem from grape exhibit different phenolic Detection of Proteins (Xanthoprotein Test): profiles. Seeds have the highest total phenolic content with 1ml of crude extract was treated with few drops of conc. flavanol as the most abundant compound8. As grape seeds Nitric acid. Formation of yellow colour indicates the are a natural source of flavanols, they can be added during presence of proteins. wine fermentation to improve the antioxidant activity of Detection of Amino acids (Ninhydrin Test): wines. Catechin is usually the most important individual 1ml of crude extract when boiled with 0.2% solution of flavonol that present in both grape skin and seeds. The Ninhydrin, would result in the formation of purple colour consumption of grape derived dietary flavonoids in the form suggesting the presence of free amino acids. of grape extracts and grape seeds powder has been shown to Detection of Alkaloids (Wagner’s Test): effectively suppress oxidative stress and prevent oxidative 1ml of crude extract was dissolved in dilute HCl. and damage 9. filtered. Filtrates were treated with Wagner’s reagent (iodine Rich phenolic compounds present in grape seeds have the in potassium iodide). Formation of brownish/ reddish ability to damage microbial cells by exerting an influence on precipitate indicates the presence of alkaloids. the selective permeability of the plasma membrane, which Detection of Cardiac glycosides (Keller-Killani test): results in the leakage of vital intracellular substances. Grape 1ml of crude extract was treated with 2ml of glacial acetic seed extract has exhibited as a promising source for the acid containing one drop of ferric chloride solution. This was manufacture of new generation of antibacterial agents for underlayed with 1 ml of concentrated sulphuric acid. A dental use without exerting an influence on the biological brown ring on the interface indicates a deoxysugar equilibrium in the oral cavity 10. characteristic of cardenolides. A violet ring may appear The aim of this present study is to screen the phytochemicals below the brown ring, while in the acetic acid layer, a present in the methanolic extract of grape seed and to greenish ring may form just gradually throughout thin layer. determine its antibacterial activity against both gram- Detection of Saponins (Froth Test): positive and gram-negative bacteria. 1ml of crude extract was diluted with distilled water to 2ml and this was shaken vigorously for 15 minutes. Formation of 2. MATERIALS AND METHODS 1 cm layer of foam indicates the presence of saponins. Collection of plant material: The Grape (Vitis vinifera) was Detection of Flavonoids (Lead Acetate test): procured from local fruit market. Seeds were separated from 1ml of crude extract was treated with few drops of lead grape fruits and dried in shadow. The dried seeds were acetate solution. Formation of yellow colour precipitate ground to powder. indicates the presence of flavonoids. 2498 IIIIIIIII© International Journal of Pharma Research and Health Sciences. All rights reserved Int J Pharma Res Health Sci. 2018; 6 (2): 2497-01 Test for Tannins (Ferric chloride test): anthraquinones and phenols.Whereas, cardiac glycoside, 1ml of crude extract when mixed with ferric chloride saponins, amino acids and steroids were absent (Table-1). solution would give blackish red colour which indicates the presence of tannins. Table 1: Phytochemical screening of grape seed methanolic extract Phytochemical tests Grape seed (Methanol Test for Terpenoids (Salkowshi test): extract) 1ml of crude extract was dissolved in 2 ml of chloroform Alkaloids + and evaporated to dryness. 2 ml of concentrated