Screening of Phyto-Chemical Constituents, Trace Metal Concentrations and Antimicrobial Efficiency of Rauvolfia Tetraphylla Vs
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IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504 INTERNATIONAL JOURNAL OF PHARMACEUTICAL, CHEMICAL AND BIOLOGICAL SCIENCES Available online at www.ijpcbs.com Research Article SCREENING OF PHYTO-CHEMICAL CONSTITUENTS, TRACE METAL CONCENTRATIONS AND ANTIMICROBIAL EFFICIENCY OF RAUVOLFIA TETRAPHYLLA VS. Nandhini and G. Vijistella Bai* Post graduate and Research Department of Botany, Kunthavai Naacchiyar Govt. Arts College for Women (Autonomous), Thanjavur, - 613 007, Tamil Nadu, India. ABSTRACT Phytochemical constituents and trace metals concentrations, and antimicrobial activity of ethanolic extract of Rauvolfia tetraphylla leaves were investigated. The phytochemical screening of the crude extract revealed the presence of steroids, reducing sugars, sugars, alkaloids, flavonoids, saponins, tannins and amino acids. The leave powder was subjected to analyze the trace metals using atomic absorption spectroscopy (AAS). The average concentrations of Cd, Cr, Cu, Fe, Ni, Pb and Zn were BDL, 0.03, 0.2, 0.5, BDL, 0.06 and 0.5 mg kg-1, respectively. And also the crude ethanol extract of Rauvolfia tetraphylla were analyzed for in-vitro antimicrobial activity against certain pathogens and the zone of inhibition were compared with control drug Methicillin and Itraconazole. The Salmonella typhimurium is the most sensitive strains (17mm) to test solution while smallest inhibition were recorded in Micrococcus luteus and Cryptococcus sp. (11 mm). Keywords: Rauwolfia tetraphylla, Antimicrobial activity, Phytochemistry, Trace metals. INTRODUCTION shrubs in the Apocynaceae family. Indian medicinal plants are well-known Approximately 85 species in the genus can universally for its main role in the primary mainly be found in tropical regions. Rauvolfia health care systems. Most of the rural people tetraphylla and Rauvolfia serpentina is the South still rely largely on traditional system to cure African quinine plant species. Rauvolfia their ailments. Medicinal plant in their tetraphylla contains a number of bioactive surrounding environments are collected and phytochemicals and is mainly known for its used largely. As they provide low cost or no cost phytochemical reserpine, which was widely for practicing knowledge on such medicinal used as an antihypertensive drug. But very least systems have been in the process of research has been published on the leaves of transmittance from one generation to the other. plant Rauvolfia tetraphylla. It has increased the efficacy and reliability of the Many plants throughout the world, including medicinal uses as it has been in continuous some with documented medicinal properties testing among the population over the years. In contain chemicals that are toxic to recent years, secondary plant metabolites or microorganisms (Hudson, 1987). The phytochemicals, previously with unknown acceptance of traditional medicine as an pharmacological activities, have been alternative form of health care and the extensively investigated as a source of medicinal development of microbial resistance to the agents (Krishnaraju et al. 2005). There were available antibiotics has led researchers to many reports on the antimicrobial activity of investigate the antimicrobial activity of plant extracts against human pathogenic medicinal plants. The use of plant extracts and bacteria (White et al. 2002; Mishra et al. 2009; phytochemical, with established antimicrobial Haris Kumar et al. 2010). properties, could be of great significance in Rauvolfia is a genus of evergreen trees and preventive and therapeutic approaches. The 47 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504 most important antimicrobial compounds of heated. Formation of green or purple color plants are alkaloids, flavonoids, tannins and indicates the presence of sugars. phenolic compounds. Although synthetic and semi-synthetic antimicrobial drugs abound in Alkaloids: A 3 ml of test solution was taken various markets today, there is a need for with 2N HCl. Aqueous layer formed was continuous search for new ones to cope with the decanted and then added with one or a few increased evolution of multiple antimicrobial drops of Mayer’s reagent. Formation of white resistant strains of organisms (Hart et al. 1998). precipitate or turbidity indicates the presence of The increasing prevalence of multi-drug alkaloids. resistant strains of bacteria and the recent appearance of strains with reduced Phenols: A 3 ml of test solution in alcohol was susceptibility to antibiotics raised the specter of added with one drop of neutral ferric chloride “untreatable” bacterial infections and adds (5%) solution. Formation of intense blue color urgency to the search for new infection-fighting indicates the presence of phenols. strategies. In contrast to synthetic drugs, antimicrobial of plant origin usually are not Flavonoids: A 3 ml of test solution in alcohol associated with many side effects and have an was added with a bit of magnesium and one (or) enormous anti-infective potential in numerous two drops of concentrated HCl and heated. infectious diseases. In present study aims to Formation of red or orange color indicates the analyze the phytochemical constituents, trace presence of flavonoids. metals and antimicrobial activity of the Rauvolfia tetraphylla. Saponins: A 3 ml of test solution was added with water and shacked. Formation of foamy MATERIALS AND METHODS lather indicates the presence of Saponins. Plant Material Tannins: A 3 ml of test solution was added with The plant materials were collected from water and lead acetate. Formation of white Thanjavur District of Tamil Nadu in India during precipitate indicates the presence of tannins. the period of January to February 2013. The shade dried plant powders (100 g) were Anthroquinones: A 3 ml of test solution was successively extracted with ethanol by soxhlet added with magnesium acetate. Formation of apparatus and is used as test sample. pink color indicates the presence of anthroquinones. Phytochemical screening Qualitative analysis Amino Acids: A 3 ml of test solution was added The solvent extracts were subjected to routine with 1% ninhydrin in alcohol. Formation of blue qualitative chemical analysis to identify the or violet color indicates the presence of amino nature of phytochemical constituents present in acids. sample (Koperuncholan et al. 2011). Catechins: A 3 ml of test solution in alcohol was Steroids: Three ml of test solution and added with Ehrlich reagent and a few drops of minimum quantity of chloroform was added concentrated HCl. Formation of pink color with 3-4 drops of acetic anhydride and one drop indicate the presence of catechins. of concentrated H2SO4. Purple color thus formed changes into blue or green color indicating the Quantitative analysis of phytoconstituents presence of steroids. The chlorophyll pigments in the leaves were estimated following the method of Arnon Triterpenoids: A 3 ml of test solution was (1949). After pre-cleaning, weighted fresh leaf added with a piece of tin and 2 drops of thionyl material was homogenized and extracted thrice chloride. Formation of violet or purple colour in chilled 80% acetone (v/v). The volume of the indicates the presence of triterpenoids. acetone extract was made up to a known one and the optical density was read at 645nm and Reducing Sugars: A 3 ml of test solution was 663nm wavelengths on a spectrophotometer. added with a 2 ml of Fehling’s reagent and 2 ml The concentration of the chlorophyll pigments of water. Formation of reddish orange color was calculated and is expressed in mg/g fresh indicates the presence of reducing sugar. weight. Amino acids were estimated by Ninhydrin method which is calorimetrically Sugars: A 3 ml of the test solution was added measured at 570nm (Hwang, 1975). Proteins with very small quantity of anthrone reagent were estimated by Bradford method and the and a few drops of concentrated H2SO4 and absorbance was measured at 595 nm against 48 IJPCBS 2014, 4(1), 47-52 Vijistella Bai et al. ISSN: 2249-9504 blank/ sample (Bradford, 1976). Carbohydrates were performed in triplicate and the average were estimated by anthrone method which can values are presented. Methicillin – 10mcg (for be measured by using colorimetrically at 620nm bacteria) and Itraconazole – 10mcg (for fungus) (or) by using a red filter (Morris 1948). All the was used as positive control. All the media, trials were performed thrice and the mean standard discs and sterile disc were purchased values were presented. from Hi-Media (Mumbai, India). Trace metal analysis RESULTS AND DISCUSSION The Rauvolfia tetraphylla plant sample was Phytochemical constituents of secondary collected from the Thanjavur district, Tamil metabolites Nadu. The leaves were carefully removed and The present study revealed that the medicinal washed with sterile distilled water. The cleaned plant such as Rauvolfia tetraphylla contains leaves were dried in shadow area and were bioactive compounds. The phytochemical grinned with mortar and pestle. After drying, 1 g constituents were screened by qualitative and of plant samples was treated with aqua-regia quantitatively methods and the results are mixture in Teflon bomb and was incubated at presented in Table 1 and 2. In Tannin analysis, 140 °C for 2-3 days. After incubation, the the brownish green colour formation indicates reaction mixture was filtered with Whatman the presence of Tannin. Similarly, based on the No.1 filter paper. Then the extraction was test presence or absence of colour change indicate for trace metals (Fe,