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1.
RESEARCH POSTER PRESENTATION
DESIGN © 2012 www.PosterPresentations.com The control and eradication of wound infection is a challenging aspect in the management of wounds. Despite the use of synthetic antimicrobial agents, drug resistance and toxicity hinder the activity of these antimicrobial agents and thereby increase the chances of infection and delay wound healing processes. Additionally, the microbial enzymes secreted by these pathogens degrade the extracellular matrix at wound site. The present study investigates the influence of Triphala on antimicrobial activity and enzyme inhibition activity which will be used in wound healing studies. The methanolic extract of triphala was prepared and its antimicrobial activity was tested against Pseudomonas aeruginosa. The activity of triphala extract against metalloprotease were studied by Zymography. Enzymatic activities were detected as clear bands of gelatin lysis against a dark background. The 18 ± 2mm clear zone in disc diffusion assay and minimal inhibitory concentration (MIC) of 7.8125mg/ml against pseudomonas control strains clearly showed the antibacterial activity of Triphala. Zymography analysis exhibited greater reduction in d metalloprotease activity at ≥1500μg/ml. By virtue of the inhibitory effect of Triphala on Pseudomonas aeruginosa strains and their enzymes such as proteases, it could be, potentially used as a new therapeutic agent for pseudomonas infected dermal wounds. The results hence highlighted the beneficial effects of the topical application of Triphala in the acceleration of wound healing and its effect on controlling wound infection at wound site. ABSTRACT INTRODUCTION RESULTS Protease Inhibition By Methanolic Extract of Triphala DISCUSSION Inhibitory activity of triphala against P. aeruginosa and provides some scientific rationale for its use as antimicrobial agents on infected dermal wounds. As an important observation in our study, we have found both methicillin resistant and sensitive S. aureus were also inhibited at the same concentration of triphala extract. Although the triphala has rich in polyphenols and ascorbic acid. The poly phenols might be the responsible for antimicrobial action due the presence of EGCG (epigallocatechin gallate) as one of the condensed tannins. The literature proved that Epigallocatechin gallate has the specific mechanism for antimicrobial activity. The epigallocatechin gallate more amount binds to S. aureus than that of gram negative bacterium E.coli and sensitivity of EGC treated S. aureus to high ionic strength was more than towards and low osmotic pressure. The epigallocatechin gallate binds to the peptidoglycan layer in the cell wall of S.aureus. Peptidoglycan is a cross-linked complex formed from various polysaccharides and peptides. Thirty to fifty layers of peptidoglycan form part of the cell wall of S.aureus offering osmotic protection, assisting in cell division and also act as a primer for further biosynthesis of peptidoglycan. In the case of pseudomonas as gram negative bacterium, The thin layer of peptidoglycan is available for binding with active constituents in triphala. However, The mechanism of antimicrobial action remains obscure. The reduction in the activity of pseudomonas metalloprotease by the triphala extract gives additional support to its potential use as a therapeutic agent for wound treatment. ACKNOWLEDGEMENT Bharathi Ravi would like to express her gratitude to her organization Lifecell International private limited for presenting this work in this international conference. As the development of bacterial resistance to antibiotics and the controversies regarding the use of topical antiseptics persists, thousands of phytochemicals were sucessfully isolated from plants that inhibit all types of microorganisms yet safe and broadly effective without inducing microbial resistance. According to WHO, nearly 20,000 medicinal plants are present in 91 countries. New compounds inhibiting microorganisms such as benzoin and emetine have been isolated from plants . The antimicrobial compounds from plants may inhibit bacterial growth by different mechanisms than those presently used antimicrobial agents and may have a significant clinical value in treatment of resistant microbial strains . To date, widespread opinion among wound care practitioners is that aerobic or facultative pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and beta-hemolytic streptococci are the primary causes of delayed healing and infection in both acute and chronic wounds . Triphala is a traditional Ayurvedic herbal formulation that consists of the dried fruits of three medicinal plants, Terminalia chebula, Terminalia bellirica, and phyllanthus emblica. Triphala and/or its constituent plants have been reported to possess numerous biological and pharmacological activities such as antioxidant, antibacterial, antifungal, antiviral, antimalarial, anti-mutagenic, anticancer, radio protective, anti-allergic, cardiotonic, hypocholesterolaemic, capillary strengthening and hepatoprotective. The antimicrobial potential of Triphala is tested against various wound pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and beta-haemolytic streptococci. But the antimicrobial activity of Triphala against Pseudomonas aeruginosa is focused in this work and presented in this conference. *Centre for Biotechnology, Anna University, Chennai-600025, India. # Lifecell International Private Ltd, Chennai, India. Email:bharathi.p.ravi@gmail.com S.Kirubanandan*, Bharathi Ravi# and S.Renganathan* Enzyme Inhibition and Antimicrobial Activity of Triphala against Pseudomonas aeruginosa EXPERIMENTAL METHODS Microorganisms Triphala extract Antibiotic S. aureus P. aeruginosa 18 mm 20 mm 34 mm (methicillin) 30 mm (ciprofloxacin) Fig 1- Enzyme Inhibition by Triphala Extract Microorganisms Minimum concentration S. aureus P. aeruginosa Streptococcus pyogenes 7.8 mg 7.8 mg 31.25mg Table 1 - Disc Diffusion Assay by Triphala Extract Fig 2- Disc Diffusion Assay by Triphala Extract Table 2 – Minimum Inhibitory Triphala Extract Preparation of alcohol extract of Triphala 100 g of powder was extracted in 500 mL of methanol by stirring overnight and was centrifuged at room temperature. The supernatant was collected and evaporated to dryness under reduced pressure in a rotary evaporator. The yield of this methanolic extract was 12.5%. The concentrated extract was aliquoted in amber-colored bottles and kept in desiccators for further use. The dried extract was dissolved in 10% Dimethyl Sulfoxide (DMSO) and used to assay the antibacterial activity. Determination of antibacterial activity The minimal inhibitory concentration (MIC) of the extract was determined by the broth tube dilution method and The antibacterial sensitivity test was performed by disc diffusion method. Sterile blank discs (6 mm diameter) were impregnated with minimum inhibition concentrations of triphala extract against standard strains. Standard methicillin disc and discs treated with DMSO were used as control. Inhibition zone diameters around each of the disc were measured and recorded at the end of the incubation time. Enzyme inhibition assay The overnight microbial cultures were transferred in to sterile 50 mL conical tubes and centrifuged at 5000 rpm for 5 minutes. To the culture supernatant, solid ammonium sulfate was added slowly with stirring to achieve 80% saturation. The resulting precipitate was collected by centrifugation and dissolved in 0.02 M phosphate buffer, pH 6.8. From this, 1mL was transferred in to sterile vials and incubated with different concentration of triphala extract (100µg-2000µg/mL) for overnight at 37oC. 1mL of 10% DMSO was used as control. The activity of triphala extract against metalloprotease was studied by Zymography. Enzymatic activities were detected as clear bands of casein/gelatin lysis against dark background..
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