Bactericidal activity of Flavonoids isolated from Muntingia calabura

The investigation was carried out for the isolation and characterization of the compounds from heart wood of root and root bark of Muntingia calabura. We have isolated six compounds; three from each extract were identified as flavonoids. The bactericidal activity of these compounds found significant against tested bacterial strains (gram-positive and gram-negative). Among the tested compounds, 8-methoxy, 3ʹ,5ʹ7ʹ-trihydroxyflavone and 3,5,7-trihydroxyflavone (Galangin) showed paramount activity against Methicillin-resistant Staphylococcus aureus (MRSA). The results were compared with known standards gentamycin sulphate and cefixime. Key-words: Bactericidal, Broth dilution method, Flavonoids, MRSA, Muntingia calabura INTRODUCTION Flavonoids are polyphenolic secondary metabolites, ubiquitously found in nature. Over 4,000 flavonoids have been identified from different sources. The potential therapeutic applications of these metabolites have been considerable interest in recent years . Antibacterial resistance a “ticking time bomb” of public heath, serious threatening issue, whenever a simple infection turns to fatal and if tomorrow it extends its current course could become even worst. Majority of the plant metabolites in drug discovery has come from the diverse structures of the medicinal plants. These are often perceived as immense drug-likeness and more biological friendliness and making them good candidates in drug development . M. calabura is native to Southern Mexico and Central America, distributed all over the tropical regions of the world and especially, In India. M. calabura crude extracts for the treatment of various human disorders requires a proper scientific evaluation and documentary reports by How to cite this article Gorripati S, Rajashekar K, Dasu D, Jupaka A, Thupurani MK. Bactericidal activity of Flavonoids isolated from Muntingia calabura. Int. J. Life Sci. Scienti. Res., 2018; 4(3): 1827-1833 Access this article online www.ijlssr.com Sridhar ; Zakaria et al. [11] of active principle responsible. Several researchers round the globe have been isolated and identified the compounds of this plant as flavonoids . Till the date, only few of these compounds have been evaluated for its therapeutic properties and still there are several compounds stand remained for scientific evidence based utilization . Thus, the compounds isolated in current study have been further determined for their attributed biological activity. With regard microbial resistance and plant derived drugs, the current investigation has been documented about bactericidal activity of flavonoids isolated from heart wood root and root bark of M. calabura. MATERIALS AND METHODS Plant MaterialHeart wood of root and root bark of M. calabura was collected from College premises of Chaitanya Degree and Postgraduate College (Autonomous), Hanamkonnda, Warangal District, Telangana, India. The authenticity of the plant was carried out by Prof. V.S. Raju, Taxonomist, Plant systems laboratory, Department of Botany, Kakatiya University, Warangal, India (Voucher number: Dep/B/KU/WGL/MC014/2013). The plant material was chopped into smaller fragments, dried under shade and grinded in homogenizer to coarse powder. Research Article Int. J. Life Sci. Scienti. Res. eISSN: 2455-1716 Gorripati et al., 2018 DOI:10.21276/ijlssr.2018.4.3.14 Copyright © 2015 2018| IJLSSR by Society for Scientific Research under a CC BY-NC 4.0 International License Volume 04 | Issue 03 | Page 1828 Chemicals usedAll the chemicals required for media preparation and bactericidal assay were purchased from Hi-media chemical laboratories, Mumbai, India and for analytical grade. Extraction and separation of compoundsThe plant material was finely powdered (500 g) and extracted with chloroform in a soxhlet apparatus. The extract was concentrated under reduced pressure. The resultant gummy product was further used for separation of compounds by column chromatography. Bacterial Strains and their Growth“Gram-positive” strains MRSA NCTC 13616, Bacillus subtilis ATCC 6633, Bacillus cereus, ATCC 14579 and “Gram-negative” strains Klebsiella pneumoniae ATCC 43816, Escherichia coli ATCC 8739, Proteus vulgaris ATCC 13315 were procured from American type culture collection, USA. MRSA was purchased from culture collections, UK. All bacterial strain stored at -80C were streaked on Luria-Bertani (LB) agar plates (Hi-media Laboratories, Mumbai, India) and incubated at 37C for 20 to 24 h. A few isolated colonies were harvested from each plate and suspended in 5 ml of LB broth contained in a 15 ml of sterile plastic tube. The tube was capped tightly and incubated with gentle shaking (140 rpm) at 37C for 20 h. Preparation of bacteria for bactericidal assayBroth culture (1 ml) of test organisms was added separately to a 1.9 ml eppendorf tube, bacterial sedimentation was achieved by centrifugation at 12,000 rpm for 30 sec. The pellet was re-suspended using 1 ml of sterile PBS by gentle aspiration in and out of a transfer pipette. The optical density (OD) of the pellet was determined at 620 nm in spectrophotometer. The OD at 620 of the sample was adjusted approximately to 0.8 to 0.9 by the addition of PBS. Ten microliters of the diluted sample was subjected for serial dilution with PBS so that these dilutions would produce approximately 1,500 to 2,000 bacteria per 50ml sample. The ODs of the samples results in 60 to 200 CFU/mL. Preparation of compound stocks and their dilutions10,000 mg of each isolated compound was dissolved in one liter of PBS. Further, 1 mL of this solution was diluted in 9 mL of PBS to generate 1000 mg/L stock. This stock was used for serial dilutions to produce the concentrations ranging from 0.1–200 mg/mL. Cefixime and Gentamycin sulphate are used as positive control (10 μg/L). Solubility was achieved by adding few drops of saturated NaHCO3. The dilution of the compounds was achieved by dissolving 1 mg of compound in 1L of WFI (water for Injection). 1 mL of this dilution was dissolved in 9 mL of WFI to produce 10 μg/L concentrations and used in the study. Bactericidal assayThe assay was conducted to assess the bactericidal activity of the isolated compounds through microtiter plate described previously. The assay reaction mixture consisted of PBS (50 mM sodium phosphate, 150 mM NaCl [pH 7.0]) test compounds at various concentrations and the bacterial strains were prepared in sterile 96-well microtiter plates (Nunc, Inc). The wells were filled with 100 μl diluted test compounds in PBS and 50 μl of the diluted bacterial strains and incubated with gentle shaking (140 rpm) at 37C for various incubation periods [0 (baseline), 2, 4, 8, 12, and 24 h) (time-kill studies)] 24 h. Subsequently, positive and negative controls, was prepared and screened. Following incubation, a 20 μl aliquot from each well was spotted at the top of a square plate containing Nutrient agar medium. The plate was labeled and tapped gently to facilitate the movement of the liquid. There were approximately 200 cells in the spotted (20 μl) sample. Plates were placed uncovered in biohood until the sample liquid dried (ca. 10 min) and incubated overnight at 37C. CFU of test organisms were visible after 18 to 24 h and was counted. The experiments were performed in duplicate, and CFU for each streak were enumerated with a colony counter. The control value to determine the percentage of bacteria killed per well. The percentage of the bacteria killed was plotted graphically, and the percentage of the test compound resulting decrease in the number of CFU at each dilution of test compounds was compared with the average of positive number of CFU (BA50) was determined.