Indo Asian Journal of Multidisciplinary Research (IAJMR) ISSN:

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1 Available online at Volume 2; Issue - 6; Year 2016; Page: DOI: iajmr Indo Asian Journal of Multidisciplinary Research (IAJMR) ISSN: PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF SELECTED PULSES HUSK EXTRACTS A. Gayathri, M. S. Deepa and M. Fernandus Durai*, Department of Biochemistry, Sacred Heart College (Autonomous), Tirupattur , Tamil Nadu, India. Abstract The present study was aimed to study the phytochemical analysis and antimicrobial activity of different pulses husk extract using methanol (Black Gram, Green Gram, Kidney Beans, Green Peas, Chick Peas and Red Chori). The qualitative analysis for these present phytochemicals was performed by using methanolic extracts of husk. These phytochemicals are used as sources of direct medicinal agents. Phytochemical analysis showed the presence of Alkaloids, Flavonoids, Saponins, Steroids, Tannins, Terpenoids and Phenols in all the extracts varying quantities. Since, the pulses contain high quantities of these new bioactive potential compounds, it is reliable to possess large number of pharmacological values like Antioxidants, Antifungal, Antibacterial, Anti-Inflammatory, Antiulcer, Diuretics activities and are being employed for the treatment of different ailments in the foreign system of medicine. Antibacterial activity of different pulses husk extract against Bacillus cereus.the tested methanolic crude extract of different husk sample shown the antibacterial activity by using the Well diffusion method with different degrees of inhibition was ranged from 8 14 mm. The present research concluded that the Green peas husk possess high antibacterial activity when compared to other pulse. The results showed significant activity of husk as natural antibacterial agent for the control of various bacterial diseases. Key words: Phytochemical screening, Methanol, Pulses and Antibacterial Activity. 1. Introduction Pulses (Family - Fabaceae) are common in diet rich in carbohydrates, proteins and minerals. It is traditionally used in the treatment of Inflammation, Diabetes, Hypertension, Wounds, Pain and Fever. Phytochemicals are the chemicals that present naturally in pulses and plant materials. It is more popular for its countless medicinal uses. Pulses are the almost exclusive source of drugs for majority of the world population. The phytocomponents in Black gram helps in treating aliments like liver diseases, cancer, diabetes, kidney diseases by functioning as antioxidants (Romero Arenas et al., 2013). *Corresponding author: M. Fernandus Durai E. mail: fernandusdurai@shctpt.edu Received: ; Revised: ; Accepted: The bean is the most essential food legume for nearly 300 million people, most of them livein developing countries, due to the crop is also known as "the meat of the poor"`in addition, food is a little expensive for lowincome consumers. The dry common beans (Phaseolus vulgaris L.) are widely consumed throughout the world. The phytochemicals become more popular due to their countless medicinal uses. Phytochemicals play a vital role against number of diseases such as Asthma, Arthritis, Cancer, etc. Unlike pharmaceutical chemicals these phytochemicals do not have any side effects (Shiva Rani et al., 2013). Since, the phytochemicals cure diseases without causing any harm to human beings these can also be considered as man - friendly medicines (Zahid Khorshid Abbas et al.,2015). A natural products from plants called secondary metabolites are the

2 Fernandus Durai /Indo Asian Journal of Multidisciplinary Research (IAJMR), 2(6): end products of primary metabolites such as carbohydrates, amino acid and chlorophyll lipid soon. They are synthesis large variety of chemical substances known as secondary metaboliteswhich includes Alkaloids, Steroids, Flavonoids, Terpenoids, Tannins, Saponins and phenolic compounds etc. Bacillus cereus is a Gram Positive, rod shaped endospore forming bacteria that produces toxins which are responsible for the food poisoning. The Bacillus cereus causes two types of illness Diarrhoea and Emetic toxin which was characterized by nausea and vomiting. 2. Materials and Methods Sample Collection Various pulses samples viz., Black gram, Green gram, Kidney beans, Green peas, Chick peas and Red chori are collected from the agriculture field in Govindapuram, Vellore district, Tamil Nadu, India. De Husking Process Different kinds of Pulses are placed in the separate beaker and water was added into the beaker and it was kept for 10 hrs in the room temperature. Then, the husk was peeled from the soaked legume. After collecting, the husk was dried using the Hot air oven at 55 C. The fully dried sample was powdered using commercial Mixer and Grinder. Extraction of Metabolites Five gram of grained husk was dissolved in 25 ml of methanol and Erlenmeyer flask was kept in a shaker at 30 ºC for 24 hrs. The extracted solvent was filtered using a Whatman filter paper No. 1 and the filtrate poured into a petriplate allowed to air dry at room temperature. The dried extract was collected using 2 ml of methanol and used for further assay. Phytochemical analysis Phytochemical screening was analyzed to detect the presence of bioactive compounds and it was performed by standard procedures. After the addition of specific reagents to the test solution, the tests were detected by visual observation of color change or by precipitate formation. Qualitative phytochemical selection was performed to identify the phytochemical constituents, i.e., Alkaloids, Terpenoids, Saponins, Tannins, Sugars, Phenolic compounds and Flavonoids. Phytochemical Screening Test for Alkaloids About50µl of the husk extract (methanol) was taken and 1 mlof Mayer s reagent was added. Formation of Pale yellow coloured precipitate indicates the presence of alkaloids (Sofowora,1993). Test for Flavonoids About 50 µl of different sample extract was taken and few drops of 10 % Ferric chloride solution was added. Occurrence of green - blue or violet colouration indicates the presence of a phenolic hydroxyl group (Trease and Evans, 2002). Test for Saponins To 50 µl of the huskmethanolic extract, 3 ml of distilled water was added and shaked vigorously in a test tube. The formation of stable foam was taken as an indication of the presence of saponins. Test for Steroids To the 50 µl of husk methanolic extract, 2 ml of acetic acid was added. The solution was cooled well in ice followed by the addition of concentrated sulphuric acid carefully. Colour developed from violet to blue or bluish green indicated the presence of a steroidal ring i.e., a glycone portion of glycoside (Sofowora, 1993). Test for Tannins About 50 µl of different sample was taken and few drops of 1 % Ferric chloride solution was added. Occurrence of a blue - black, green or blue - green precipitate indicates the presence of tannins (Trease and Evans, 2002). Test for Terpenoids To the 50 µl of husk methanolic extract, 2 ml of chloroform and concentrated H 2 SO 4 (3 ml) was added carefully from a layer. Formation of reddish brown colouration shows positive result of the presence of Terpenoids.

3 Fernandus Durai /Indo Asian Journal of Multidisciplinary Research (IAJMR), 2(6): Antimicrobial activity of Methanolic Husk extract against Bacillus cereus The antibacterial activity of selected pulses husks viz., Black gram, Green gram, Kidney beans, Green peas, Chick peas and Red chori was determined by Well diffusion methods (Baueret al., 1966) in accordance with CLSI (2012). About 100 ml of Mueller HintonAgar was poured into a sterile petriplates. The plates were allowed to solidify, 18 hrs grown fresh culture of Bacillus cereus were swabbed using sterile cotton swabs.the test samples (methanolic husk extracts) were poured into the well with various concentrations (i.e., 25, 50, 75 and 100 µg/well). The antibiotic Streptomycin was loaded well and served as control. The plates were incubated at 37 C for 24 hours. The antibacterial activity was determined by measuring the diameter of the zone of inhibition around the well was measured. 3. Results and Discussion Phytochemicals are a group of nonnutrient bioactive compounds naturally found in plant parts such as flowers, leaves, fruits, roots, barks, spices and medicinal plants. Since ancient times, people have been exploring the nature particularly plants in search of new drugs. This has resulted in the use of large number of medicinal plants with curative properties to treat various diseases. The study of plants continues principally for the discovery of various novel pharmaceutically active secondary metabolites which has pharmacologically active substances showing antifungal, antibacterial and anticancer activities. The crude extracts and active pure compounds isolated from plants species used in herbal and traditional remedies. The phytochemical analysis and antimicrobial activity of Vigna radiata is an attempt to unravel the scientific benefits derivable against food pathogens. Microbes are used for these, study were selected on the basis of their implication as agents of food spoilage and food borne diseases. In vitro preliminary screening of the antibacterial activity against food pathogens from methanol extracts of different pulses husk extract was studied by Agar well diffusion method. Plants are known to produce secondary metabolites that complement their structural barrier in their efforts at warding of microbial attack (De Lucca et al., 2005). The results of phytochemical selection of methanolic extract of different pulses huskwas given in the Table 1. Different kinds of pulses were taken in the present research. Amongthese samples, Black gram showed the presence of five phytochemical compounds like Alkaloids, Flavonoids, Steroids, Tannins and Terpenoids. Saponins are completely absent in Black gram. The Kidney beans showed the presence of Flavonoids, Steroids, Tannins and Terpenoids, Alkaloids and Saponins are completely absent in Kidney beans. In Green gram, Alkaloids, Flavonoids, Tannins and Terpenoids are present. The Saponins and Steroids are absent in Green gram. In Green peas and Chick peas, the presence of Steroids and Terpenoids are observed and Saponins, Tannins, Alkaloids and Flavonoids are absent. Then, the Red chori showed the presence of five phyto compounds like Alkaloids, Flavonoids, Steroids, Tannins and Terpenoids. Saponins are completely absent. Phytochemical screening of medicinal plants is very important in identifying new sources of therapeutically and industrially important compounds. It is imperative to initiate an urgent step for screening of plants for secondary metabolites (Somkuwar and Kamble, 2013). In the present study, the antibacterial activityof different pulses husk methanol extracts was determined and the results are showed in Table 2. The antibacterial activity was determined by Well diffusion methods (Bauer et al., 1966). The bacterial species Bacillus cereus were usedin this present study and it showed different degrees of inhibition zone.the result showed the antibacterial activity against test bacteria with zone of inhibition ranged from 8 mm 14 mm. The maximum zone of inhibition was against Gram positive bacteria Bacillus cereus. The final result in this testing report analysed green peas husk is a high antibacterial activity pulse.

4 Fernandus Durai /Indo Asian Journal of Multidisciplinary Research (IAJMR), 2(6): Table 1: Phytochemical screening of different pulses husk extract Phytochemical Different Pulses Husk Extract Using Methanol Test s Black Gram Kidney Beans Green Gram Green Peas Chick Peas Red Chori Alkaloids Flavonoids Saponins Steroids Tannins Terpenoids = Present; - = Absent Table - 2: Antibacterial activity of different pulses husk extract against Bacillus cereus Different husk Extract Control (mm) Zone of inhibition (mm) 25 µg 50 µg 75 µg 100 µg Black Gram Kidney Beans Green Gram Green Peas Chick Peas Red Chori References 1) De Lucca, A. J.,T. E. Cleveland and D. E. Wedge Derived antifungal proteins and peptides. Canadian Journal of Microbiology, 51 (12): ) Dipali Somkuwar, O and Vilas A. Kamble Phytochemical screening of ethanolic extracts of stem, leaves, flower and seed kernel of Mangifera Indica L. International Journal of Pharma and Biosciences, 4 (2): ) Jaya Prakash, A., G. Yamini Sudhalakshmi, S. Fouzia Banu and S. Gopalakrishnan Phytochemical screening and antibacterial activity of Vigna radiate against bacterial pathogens involved in food spoilage and food borne diseases. Journal of Academia and Industrial Research,1 (6): ) Jukanti, A. K., P. M. Gaur, C. L. L. Gowda and R. N. Chibbar International CropsResearch Institute for the Semi-Arid Troipcs. British Journal of Nutrition, 108: S11 - S26. 5) Rama Swamy Nanna, Mahitha Banala, Archana Pamulaparthi and Archana Kurra Evaluation of phyto chemicals and fluorescent analysis of seed and leaf extracts of Cajanus cajan. International Journal of Pharmacology Science, 22 (1): ) Romero Arenas, O., M. A. Damian Huato and J. A. Rivera Tapia The Nutritional value of Beans (Phaseolus vulgaris) and its importance for Feeding of Rural communities in Puebla - Mexico. International Research Journal of Biological Sciences,2 (8): ) Rohit Kumar Bargah Preliminary test and phytochemical screening of crude ethanolic and aqueous extract of Moringa pterygosperma. Journal of Pharmacognosy and Phytochemistry, 4 (1): ) Sahira Banu, K and L. Cathrine General techniques involved in phytochemical analysis. International Journal of Advanced Research in Chemical Science, 2:

5 Fernandus Durai /Indo Asian Journal of Multidisciplinary Research (IAJMR), 2(6): ) Shiva Rani, S. K., Neeti Saxena and P. Udaysree Antimicrobial activity of Black pepper (Piper nigrum L). Global Journal of Pharmacology, 7 (1): ) Zahid Khorshid Abbas, A., A. Shalini Saggu, I. Mohamed and B. C. Sakeran Phytochemical, antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves. Saudi Journal of Biological Sciences, 22: