Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris
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International Journal of Research in Pharmacy and Biosciences Volume 7, Issue 1, 2020, PP 1-9 ISSN 2394-5885 (Print) and ISSN 2394-5893 (Online) Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris Joushan Ara*, A T M Yusuf and Md. Shahidul Islam Department of Pharmacy, University of Science and Technology Chittagong (USTC), Chattogram, Bangladesh *Corresponding Author: Joushan Ara, Lecturer, Department of Pharmacy, University of Science & Technology Chittagong (USTC), Bangladesh. ABSTRACT Though some plants found in nature are poisonous but there are some plants which serve us as source of energy and effective drugs. Considering the medical importance and source of origin, the plant Alstonia scholaris has been subjected for fractionation with different solvents. The different fractions of ethanolic extract of Alstonia scholaris were evaluated for the antibacterial, antifungal activities and cytotoxic activity as well as biological activity. Phytochemical properties of leaves of Alstonia scholaris were also examined. The different solvent fractions showed the presence of tannins, glycosides, steroids and alkaloids. The different fractions of ethanolic extract of Alstonia scholaris were evaluated for the total phenolic content, total flavonoid content, the antibacterial, antifungal activities and cytotoxic activity. The different fractions of ethanolic extract of Alstonia scholaris were also undertaken to investigate for antibacterial activity using well diffusion method on Gram positive (Staphylococcus aureus) as well as Gram negative bacteria (Eacherichia coli). Crude ethanol fraction exhibited highest activity against S. aureus and E. coli, showed the inhibition zone with diameters 14mm and 13 mm respectively. Whereas ethyl acetate, chloroform and Dia-ion resin adsorbed fraction exhibited lesser activity, while petroleum ether fraction showed no inhibition. For screening biological activities, cytotoxicity test was also performed with various extractives of the Alstonia scholaris. From the result it was observed that ethyl acetate fraction had the highest cytotoxicity activity against Artemiasalina (brine shrimp) with LD50 values 944.730, 2819.016, 83.994, 19.012 and 12.213 ppm after 6, 12, 18, 24 and 30 hours’ treatment respectively. Keywords: Phytochemistry, Cytotoxicity test, Alstonia scholaris Chloroform. INTRODUCTION the food materials so essential for sustenance of the life of animals but also certain other In the beginning of the nineteenth century, the substances, such as alkaloids, vitamins, mankind stepped into the industrial life, partly glycosides, toxalbumins, essential oils, resins, neglecting the dependence on plant kingdom. As bitter principles etc. which are necessary for a result, the ecological balance of the plant and growth, maintenance and protection of life. the animal life is disturbed and the world is Many of these are essential for metabolic being threatened by the industrial pollution and activities (3), many are medicines to human and hazards (1). This is why the modern world is animal life. Many of these are harmful to animal interestingly tending to go back to the pre- life, at least under certain conditions. Plants industrialized days, when the mankind used to containing medicinal properties are commonly depend on the plant kingdom for their food, known as medicinal plants (4). The plants shelter, medicine and other essential commodities. containing these principles are capable of acting This is perhaps, the only way to protect the deleteriously, are popularly known as poisonous ecological balance. plants. A poisonous plant is one which, as a The raw materials of the plant kingdom as whole or as a part thereof, under all or certain mentioned above are directly or indirectly conditions, and in a manner and in amount likely produced by the plants, but are very seldom to be taken or by brought into contact with an used by themselves and serve human beings in organism, 'will exert harmful effects or cause many ways (2). These are called the secondary death either immediately or by reason of metabolites or the natural products. By the cumulative action of the toxic property, due to metabolic activity of plants produces not only the presence of known or unknown chemical International Journal of Research in Pharmacy and Biosciences V7 ● I1 ● 2020 1 Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris substances in it (5). Although some of these different ethnic communities in India have used plants are once poisons, medicines and food or different species of Alstonia in the treatment of fodder. The genus Alstonia belongs to the various human ailments (7). The common family Apocynaceae. It includes totally 43 Bengali name of the plant Alstonia scholaris are species of which two species namely, A. Satiani, Chattin, Chatium. Alstonia scholaris is scholaris (L.) R. Br. and A. venenata R. Br. are widely distributed in dried forest of India, represented in South India (6). These two Western Himalayas, Western Ghats and in the species can be identified with their habits, shape Southern region (8). It grows more or less in all and texture of the leaves, fruit size and papilla districts of Bangladesh. It grows Malaysia, of the seeds. When these plants are used in Australia and Solomon Islands as an ornamental. It herbal formulations, their botanical identity is distributed in Pakistan, Thailand, Philippines, needs to be established beyond any ambiguity. Indonesia (9) and tropical regions of Africa and The genus Alstonia finds a prominent place in Asia Habitat found to be in deciduous and different Indian systems of medicine. The evergreen forests, also in plains (10). Figure1. Alstonia scholaris plant FLOW CHART OF PLANT EXTRACTION Figure2. Flow chart for the preparation of different fraction 2 International Journal of Research in Pharmacy and Biosciences V7 ● I1 ● 2020 Phytochemical and the Antibacterial, Antifungal and Cytotoxic Activities of Different Fractional Extracts of Alstonia Scholaris METHODS AND MATERIALS yellow or red precipitate was the indication for the presence of tannins. In this study, all the reagents and chemicals were used were purchased from THOMAS BAKER Test for Steroids (MUMBAI, INDIA), BDH (ENGLAND), Libermann-Burchard’s Test FLUKA (SWITZERLAND) and E. MERCK (GERMANY). Rectified spirit and absolute A small amount of different fractionates of the alcohol were available from Carew and company, plant materials was dissolved in 1 mL of Darsana, Chuadanga. The Solvents used mainly in chloroform, 2 mL of acetic anhydride and 1 mL of concentrated sulphuric acid were added to the this work are benzene, acetone, tetrahydrofuran solution. Formation of a greenish color which (THF), ethyl acetate, chloroform, n-hexane, terns blue on standing was the indication for the petroleum ether, methanol, absolute alcohol, presence of steroids. toluene etc. The solvents were dried and distilled when necessary. Test for Alkaloids The Alstonia scholaris plant leaves were Color Test collected from the cultivated neighboring areas About 0.5g of the extract was stirred with 5 mL of BCSIR, Rajshahi. The collected leaves were of 1% hydrochloric acid on a steam bath and cleaned with water. was filtered. 1 mL of the filtrate was treated The whole extraction process is shown in the with a few drops of Dragendorff‟s (Bismuth following flow chart. potassium iodide solution) reagent. Formation of orange-red precipitate was the indication for The Alstonia scholaris leaf extracts of the presence of alkaloids. Petroleum ether, Ethyl acetate, Chloroform, Methanol and Ethanol solvents were used for the Biological Studies are Performed in Two Ways phytochemical analysis for the identification of Anti-bacterial& Anti-fungal screening (in various classes of chemical compounds using vitro) crude methanol extract and different the standard protocol. fractions. SCREENING PROCEDURE Cytotoxicity assay study Test for Saponins DETERMINATION OF ANTI-BACTERIAL & ANTI-FUNGAL ACTIVITY OF THE TEST Frothing Test AGENT BY MEASURING THE ZONE OF About 0.1g of different fractionates of the plant INHIBITION materials was taken vigorously with water. The discs were placed in such a way that they Production of a persistent frothing (which were not closer than 15 mm to the edge of the remains stable on heating) was the indication for plate and for enough apart to prevent the presence of saponins. overlapping the zones of inhibition. Test for Glycosides After 24 hours incubation, the antibacterial General Test activities of the antibiotic extracts were determined by measuring the zone of inhibition A small amount of different fractionates of the in term of mm by a transparent scale. Inhibitory plant materials was taken in a test tube and was zone obtained by samples were compared to that dissolved in 1 mL of water. A few drops of of the standard disc. aqueous sodium hydroxide solution were then added to the test tube. Development of yellow DETERMINATION OF MINIMUM INHIBITORY color was the indication for the presence of CONCENTRATIONS OF THE ANTIBACTERIAL glycosides. AGENTS Test for Tannins Minimum inhibitory concentration (MIC) may be defined as the lowest concentration of an Lead Acetate Test antimicrobial drug to inhibit the growth of the About 5 mL of aqueous solution of different organism. The data derived from the test can be fractionates of the plant material was taken in a corrected with