Available online at www.jpsscientificpublications.com Life Science Archives (LSA) ISSN: 2454-1354 Volume 1; Issue - 3; Year 2015; Page: 161-165 Research Article In vitro STUDIES ON INHIBITORY EFFECT OF Tragia involucrata AGAINST METHICILLIN RESISTANT Staphylococcus aureus Abstract E. Gowthami * and G. Sudha supriya, PG and Research Department of Microbiology, Sri Akilandeswari Women s College, Wandiwash, Tiruvannamalai District, Tamil Nadu, India. Ayurveda is a medical system primarily practiced in India that has been known for nearly 5000 years. It includes diet and herbal remedies, while emphasizing the body, mind and spirit in disease prevention and treatment. India is one of the 12 mega biodiversity centers having over 45,000 plant species. wadays, higher plants are playing a prominent role in the treatment of innumerable diseases including cancer, lymphosarcoms, AIDS, senile dementia and auto-immune diseases. Classically, higher plants are occupying a key position in the production of new therapeutic agents. Thus, the plant drugs are able to occupy an important niche in modern medicine. MRSA has now become established outside the hospital environment and is appearing in community populations without identifiable risk factor. Methicillin Resistant Staphylococcus aureus (MRSA) strains that cause such infections are called community acquired MRSA. This present study aims to determine the antibacterial properties of Tragia involucrata extract against MRSA. The leaves of Tragia involucrata has been tested against MRSA. Its ethanolic extract shown higher activity in comparison with other fractions of Ethyle acetate and Hexane. This is an encouraging result in regards to the ability of MRSA to be resistant to most antibiotics. Article History Received : 28.04.2015 Revised : 06.05.2015 Accepted : 08.05.2015 1. Introduction Ayurveda is a medical system primarily practiced in India that has been known for nearly 5000 years. It includes diet and herbal remedies, while emphasizing the body, mind and spirit in disease prevention and treatment (Morgan, 2002). India is one of the 12 mega biodiversity centers having over 45,000 plant species (Anonymous, 1998). wadays, higher plants are playing a *Corresponding author: E. Gowthami, PG and Research Department of Microbiology, Sri Akilandeswari Women s College, Wandiwash, Tamil Nadu, India. Key words: Ayurveda, Tragia Involucrata, Antibacterial properties and Methicillin Resistant Staphylococcus aureus (MRSA). prominent role in the treatment of innumerable diseases including cancer, lymphosarcoms, AIDS, senile dementia and auto-immune diseases. Classically higher plants are occupying a key position in the production of new therapeutic agents. Thus, the plant drugs are able to occupy an important niche in modern medicine. The Staphylococcus aureus is a Gram positive, spherical shaped bacteria (0.5-1.5 µm in diameter), which occur singly, in pairs or tetrad. They are short chained with three to four cells forming irregular clusters (Brock et al., 2001; Adejuwon et al., 2010). The Staphylococcus genus includes at least forty species. Most are harmless and reside normally on the skin and mucous
E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161-165, 2015 162 membranes of humans and other organisms (Harris et al., 2002). It may also be found in many parts of our environment, including dust, water air and faeces and on clothing or utensils. Staphylococcus aureus is considered to be a poor competitor in complex microbial populations and is frequently inhibited or overgrown by other faster growing microorganisms (spoilage organisms) in foods. Therefore, foods that present the greatest risk of Staphylococcal food poisoning are those where the normal microflora has previously been destroyed (e.g. cooked products) or inhibited (e.g. foods that contain a high concentration of salt) but that have subsequently been contaminated by aureus (Bremer et al., 2004). MRSA is known to be one of the most prevalent nosocomial pathogens throughout the world to be capable of causing a wide range of hospital-linked infection. Some strains of MRSA have been designated epidemic strains; these are associated with a higher prevalence and have been shown to spread within hospitals, between hospitals, and between countries (Humphrevs et al., 1990, Aires De souse et al., 1998). MRSA has now become established outside the hospital environment and is appearing in community populations without identifiable risk factor. MRSA strains that cause such infections are called community acquired MRSA (Hreold et al., 1998). Thus, this present study was planned to determine if Tragia involucrate extract has antibacterial properties against MRSA. 2. Materials and Methods 2.1. Plant collection Fresh leaves of Tragia involucrata were collected from the Podhigai hills, Western Ghats, Tamil Nadu, India. 2.2. Preparation of Tragia involucrata plant extract The leaves were carefully washed with tap water, rinsed with distilled water, and air-dried for 1 hour. Then leaves are separated and dried in room temperature for one week. Then, they were ground into powder and stored in room temperature. 2.3. Direct extraction Direct extraction with hexane, ethyl acetate and methanol was performed following the method of Eloff (1998). In this method, finely ground plant material was extracted with chloroform, ethyl acetate and methanol in the ratio of 1:10 in conical flask in shaking condition for overnight. The extract was filtered through the Whatman. 1 filter paper in a separate container. The process was repeated 3 times using the same plant material and fresh solvent. The solvent was removed by placing the extracts in distillation unit in the respective temperature. The extracted residues were weighed and re-dissolved in different solvents to yield 10 mg/ml solutions ready for further analysis. 2.4. Antibacterial activity Antibacterial activity was performed by Well diffusion assay (Eloff, 1998) using 4 different concentrations (250 µg/ml, 500 µg/ml, 750 µg/ml and 1000 µg/ml) of the plant extract, and one negative control (DMSO). 2.5. Determination of Minimum Inhibitory Concentration (MIC) (Broth dilution assay) (Cos et al., 2006) Broth Dilution assays is the standard method used to compare the minimum inhibition efficiency of the antimicrobial agents. 2.6. Detection of Phytochemical compounds 2.6.1. Detection of alkaloids Detection of alkaloids was performed using Mayer s test 2.6.2. Detection of flavonoids Three methods were used to test for the detection of flavonoids. 0.5 g of extract was dissolved in 5 ml of Distilled water and filtered. Dilute ammonia (5 ml) was added to 1 ml of the extract filtrate. Concentrated sulphuric acid (1 ml) was added. Yellow colorations that disappear on standing indicate the presence of flavonoids. 2.6.3. Detection of tannins About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and
E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161-165, 2015 163 observed for brownish green or a blue-black coloration. 2.6.4. Detection of saponins test. Detection of saponins was done by Foam 2.6.5. Detection of proteins test. Detection of proteins was done by Millon s 2.6.6. Detection of glycosides Detection of glycosides was done by Borntrager s test. 2.6.7. Detection of reducing sugars Detection of reducing sugars was done by Fehling s test. 2.6.8. Detection of phenolic compounds Detection of phenolic compounds was done by Ferric chloride test. 2.7. Quantitative phytochemical analysis of T. involucrata 2.7.1. Estimation of Total phenol content Total phenol content in various solvent extracts of leaves was determined by Folin- Ciocalteu s reagent method (Mc Donald et al., 2001). 2.7.2. Estimation of Total flavonoid content Estimation of Total flavonoid content was determined using the Dowd method (Meda et al., 2005). 2.7.3. Thin layer chromatography Thin layer chromatography is used to separate the compound present in the crude extract. 3. Results and Discussion 3.1. Extraction of plant material The leaves of Tragia involucrata were collected and as described earlier, extracts were obtained. 3.2. Antibacterial activity - Well diffusion assay The results obtained from well diffusion assay suggest that the methanol extract of T. involucrata was efficient in inhibiting MRSA strains with a maximum zone of inhibition (ZOI) of 22 mm at a concentration of 1000 µg/ml against MRSA 1968. The ethyl acetate and hexane extracts possessed less inhibitory action, comparatively. Furthermore, the Minimum Inhibitory Concentration (MIC) of methanol extract of T. involucrata was studied to be 250 µg/ml. Hence, the methanol extract was used for further IC 50 determination (Table 1). Concentration (µg) Table 1: Effect of T. involucrata on MRSA strains Zone of inhibition (mm) Hexane Ethyl acetate Methanol 1 2 3 4 1 2 3 4 1 2 3 4 1. 250 - - - - 9 - - - 15 15 17 16 2. 500 - - 9-10 - 10-17 17 18 17 3. 750 9-10 - 11-11 9 19 18 20 19 4. 1000 10-11 - 13-12 11 23 20 21 21 5. Std 25 21 23 19 21 21 22 19 25 23 25 25
E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161-165, 2015 164 3.3. Broth dilution assay The data obtained from broth dilution assay depicts that the methanol extract of T. involucrata inhibited MRSA with IC 50 value of 500 µg/ml. Also, it was studied that the percentage inhibition varied between 35 and 73% for concentration range of 100 1000 µg/ml (Table 2). Table 2: IC 50 of T. involucrata on MRSA U1968 Concentration (µg) Inhibition (%) 1 100 35.826 2 200 37.381 3 300 38.527 4 400 43.219 5 500 49.692 6 600 55.346 7 700 62.93 8 800 66.84 9 900 69.18 10 1000 73.34 3.4. Phytochemical screening of crude extracts of T. involucrata The preliminary phytochemical screening of methanol extract T. involucrata showed the presence of alkaloids, terpenoids, glycosides, flavonoids, tannins, saponin, reducing sugar whereas the proteins were completely absent (Table 3 and Table 4). Table 3: Qualitative phytochemical screening of methanol extract of T. involucrata Phytochemicals Results 1 Alkaloids ++ 2 Terpenoids ++ 3 Tannins ++ 4 Saponins + 5 Flavonoids +++ 6 Proteins - 7 Glycosides ++ 8 Reducing Sugar + 9 Phenols +++ Table 4: Quantitative estimation of phytochemicals Phytochemical Composition (mg/ml) 1 Total Phenols 25.271 GAE/g 2 Total Flavonoids 97.32 QE/g 3.4. Thin Layer Chromatography (TLC) The thin layer chromatography was carried out using the methanol extract of Tragia involucrata was loaded on per-coated (silica). The TLC plate which has ethyl acetate: hexane solvent (1.8:0.2) as a solvent system. Spots were observed under UV light and in iodine chamber. The result revealed that the ratio 1.8:0.2 was significant in separating compounds where presence of 6 major compounds at the R f values of 0.45, 0.34, 0.32, 0.31, 0.24 and 0.09. Methicillin-resistant aureus (MRSA) has become increasingly widespread as a major cause of both nosocomial and community infections. The recent increase in the methicillinresistant and multiple resistant strains at large hospitals have started. Hence, an accurate and rapid detection of methicillin resistance in Staphylococci is therefore important, not only for choosing the appropriate antibiotic therapy for the individual patient, but also for the control of the endemicity of the MRSA. This situation has placed limits on our options to treat infections by this organism. Glycopeptide derivatives, such as vancomycin and teicoplanin, are now considered to be agents of last resort for the treatment of MRSA infections (Sucilathangam et al., 2012). However, there are increasing numbers of reports indicating the emergence of vancomycin-resistant aureus (VRSA) strains exhibiting two different resistance mechanisms. A similar study showed inhibition zones ranging from 11 17 mm by the ethanolic extract of Quercus infectoria demonstrated the largest inhibition zone. Similar result was observed in a study conducted by Chusri and Voravuthikunchai (2007). The phytochemical profile of T. involucrata is well corroborating with previous
E. Gowthami / Life Science Archives (LSA), Volume 1, Issue 3, Page 161-165, 2015 165 literature and hence it can be derived that this plant has bioactive compounds like Terpenoides, Glycosides, Carbohydrate, Alkaloids, Phenolic compound, Flavanoids, Saponins, and Tannins form the characteristic nature for its medicinal property (Satish et al., 2013). 4. Conclusion In conclusion, the results of the present study reveal that the medicinal plant Tragia involucrata could be considered an effective source of Methicillin-resistant Staphylococcus aureus (MRSA) therapeutics, however further mechanistic studies are required to study the bioactive components present in the medicinal plant Tragia involucrata. 5. References 1) Adejuwon AO, Ajayi AA, Akintunde A and Olutiola PO, 2010. Antibiotics resistance and susceptibility pattern of a strain of Staphylococus aureus associated with acne. International Journal of Medicine and Medical Sciences. 2(9): 277-280. 2) Asima Chatterjee, Satish Chandraprakash, 1997. The treatise of Indian medicinal plants. New Delhi: Publications and Information Directorate; 3(2): 120 170. 3) Harborne, J.B., 1984. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis, 2 nd edition, Chapman and Hall, London. 4) Heidanadia Zulkefli, Jamaludin Mohamad & Nurhayati Zainal Abidin,2013. Antioxidant Activity of Methanol Extract of Tinospora crispa and Tabernaemontana corymbosa. Sains Malaysiana 42(6)(2013): 697 706. 5) McDonald,, Prenzler, P.D., Autolovich, M., Robards K. (2001). Phenolic content and antioxidant activity of olive extracts. Food Chem. 73:73-84. 6) Morgan, K., 2002. Medicine of the Gods: Basic Principles of Ayurvedic Medicine [http://www.compulink.co.uk/mandrake/a yurveda.htm]. 1.Srikanth K, T Murugesan et al., 2002. Effect of Trichodesma indicum extract on cough reflex induced by sulphur dioxide in mice. Phytomedicine, Volume 9, Issue 1, Pages 75-77. 2.Vedavathy., 2003. Scope and importance of traditional medicine. Indian J Traditional Knowledge 2(20), 236-241.