B. Muthukumar et al. / Drug Invention Today 2014,6(2),120-122

Research Article Available online through ISSN: 0975-7619 http://jprsolutions.info

In vitro Antibacterial Activities of caledulacea Less () Leaf Extract on Pathogenic Bacterial Strains

B. Muthukumar1, M. N. Abubacker ,2 and K. Gunasekaran3 1,3 PG & Research Department of Botany, National College, Tiruchirappalli 2PG & Research Department of Biotechnology, National College, Tiruchirappalli.

Received on: 08-05-2014; Revised on: 10-07- 2014; Accepted on:23-08-2014

ABSTRACT Antibacterial activity of leaf extracts of Wedelia caledulacea Less (Asteraceae) was evaluated against two gram positive (Bacillus subtilis and Staphylococcus aureus) and two gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic bacterial strains. The crude leaf extracts cold aqueous, hot aqueous and ethanolic extracts were tested by disc diffusion method. Though all the extracts were found effective, the ethanolic extract showed maximum antibacterial activities against the test bacterial strains followed by hot aqueous extract and cold aqueous extract.

KEYWORDS: Wedelia caledulacea, Antibacterial activity, Disc diffusion method

INTRODUCTION Medicinal being the effective source of both traditional and countries use traditional medicines derived from medicinal plants. modern medicines are genuinely useful for primary health care over Therefore such plants should be investigated thoroughly to deter- the years. World Health Organisation (WHO) advocated traditional mine their structural and functional properties as well as the effi- medicines as safe remedies for ailments of both microbial and non- ciency of various parts.10 microbial origins.1 Some antibiotics have become almost obsolete because of drug resistance2 and consequently new drugs must be Wedelia caledulacea Less (Asteraceae), a herb, leaves are used for sought for. Herbal treatment is one possible way to treat disease dyeing grey hair and as hair tonic, roots yield black dye, an attempt caused by multidrug resistant bacteria.3 was made to evaluate the potential of W. caledulacea leaf cold aqueous, hot aqueous and ethanol extracts for potential antibacterial The genetic ability of pathogenic bacteria to develop resistance against activity against two gram positive Bacillus subtilis and Staphylo- commonly used antibiotics is a major medical problem and challenge coccus aureus and two gram negative Escherichia coli and worldwide, posing a big threat to human society.4-6 This has necessi- Pseudomonas aeruginosa. tated a search for novel antibacterial substances from various natural sources from flowering plants. A wide variety of plant secondary MATERIALS AND METHODS metabolites have been identified as active principles for the treatment The plant material used in this study is the leaves of W. caledulacea of various ailments.7,8 A number of studies have been conducted for collected from the herbal garden of Department of Botany, National the selection of the crude plant extracts in a therapeutic treatment of College, Tiruchirappalli, Tamil Nadu, India. The leaves were initially bacterial infections.9 About 80 per cent population of the developed rinsed with distilled water and dried on paper towel in laboratory under shade and used for the present study. *Corresponding author. Dr. M. N. Abubacker Associate Professor & Head Preparation and Preservation of Plant Extract PG & Research Department of Biotechnology, National College, Tiruchirappalli - 620001, Cold aqueous extract: 50 g of the sample W. caledulacea leaves was Tamil Nadu, India.

Drug Invention Today Vol.6.Issue.2.July -December 2014 120-122 B. Muthukumar et al. / Drug Invention Today 2014,6(2),120-122 soaked in 200 ml cold water in a conical flask stoppered with paper that all the bacterial strains were sensitive to all six antibiotics tested cork and left undisturbed for 24 h. Then filtered off using sterile in this study (Table-1). All the values given are the mean of the three Whatman No. 1 filter paper into a sterile conical flask and subjected sets of observations. to water-bath evaporation, when the aqueous solvent was evapo- Table-1. Antibiogram - Diameter of the inhibitory zone (mm) rated at its boiling temperature of 100 C. The standard extract ob- Antibiotics Bacterial Strains tained was then stored in a refrigerator at 4 C for further use.11 (g/ml) Gram-positive Gram-negative B. subtilis S. aureus E. coli P. aeruginosa

Hot aqueous extract: 50 g of the sample W. caledulacea leaves was Amikacin (30) 30 24 10 28 soaked into 200 ml of hot water which was then boiled for 30 min11 and Ciprofloxacin (5) 20 20 12 30 kept in a conical flask for 24 h undisturbed. The other steps were the Doxycycline (30) 28 10 20 26 Gatifloxacin (10) 24 22 26 22 same as followed in case of cold aqueous extract. Kanamycin (30) 20 12 10 20 Norfloxacin (10) 10 18 20 18 Ethanol extract: 50 g of shade dried W. caledulacea leaf material was Antibiogram assay of different extract of W. caledulacea ground in 200 ml of ethanol. The mixture was kept for 24 h in tightly The antibacterial screening of the plant extract against B. subtilis, E. sealed sterile bottle at room temperature, protected from sunlight and coli, P. aeruginosa and S. aureus revealed that ethanol extract was mixed with a sterile glass rod. This mixture was filtered through more effective than those of hot aqueous and cold aqueous extracts Whatman No. 1 filter paper and the residue adjusted to the required against all the bacterial strains tested for this work. concentration (50 ml of ethanol for the residue of 50 g of powdered plant material) with the extraction fluid for further extraction and it The ethanol extract of W. caledulacea leaves showed maximum anti- was repeated thrice and a clear colourless supernatant extraction bacterial activity against P. aeruginosa followed by E. coli, B. subtilis liquid was finally obtained. The extract liquid was subjected to rotary and S. aureus. The cold aqueous extract of leaves showed highest evaporation in order to remove ethanol. The semisolid extract ob- efficacy against P. aeruginosa followed by B. subtilis, E. coli and S. tained was stored in an airtight container at 4 C in refrigerator for further use. aureus, whereas in the case of hot aqueous extract, the maximum sensitivity was shown by E. coli followed by P. aeruginosa, B. All the dried extracts were exposed to UV light (200-400 nm) for 24 h subtilis and S. aureus (Table-2). All the values given are the mean of and checked frequently for sterility by streaking on nutrient agar three sets of data. plates (Chessbrough, 2000).12 Table-2 . Antibacterial assay of aqueous and ethanol leaf extracts of W. caledulacea Antibacterial Assay Antibiotics (g/ml) Diameter of inhibition zones (mm) Gram-positive Gram-negative B. subtilis S. aureus E. coli P. aeruginosa Disc Diffusion Method: Antibiogram was conducted by disc diffu- sion method13,14 using standard antibiotic disc as positive control Cold aqueous extract 1 2 1 0 1 2 1 4 (Hi-media). The surface of the media were inoculated with bacteria Hot aqueous extract 1 3 1 2 1 5 1 4 Ethanol extract 16 14 18 20 from a pure nutrient broth culture maintained in the Microbiology Sterile distilled water (Control) 0 0 0 0 Laboratory, Department of Biotechnology, National College, Ethanol (Control) 0 0 0 0 Tiruchirappalli. The test extract was loaded in the plain bio-discs (Hi- media) and were placed on the bacteria inoculated agar. After 24 h of The ethanol extracts of the leaves of W. caledulacea showed greater incubation at a specific temperature (32  1 C) for B. subtilis and 37 antibacterial activity than the corresponding water extracts. This find-  1 C for E. coli, P. aeruginosa and S. aureus, the culture plates were ing is interesting because in the traditional method of treating a bac- examined and the diameters of the inhibition zones were measured in terial infection, decoction of the plant parts or boiling the plant in mm. water is employed, whereas according to present study preparing an extract with an organic solvent was shown to provide an antibacterial RESULTS AND DISCUSSION activity in accordance with the results obtained by Nair et al. (2005).15 Antibiogram of some common antibiotics against test microorgan- These observations may be attributed to two reasons: firstly, the isms nature of biological active components whose activity can be en- Antibiogram of the gram-positive and gram-negative bacteria revealed hanced in the presence of ethanol, secondly the stronger extraction

Drug Invention Today Vol.6.Issue.2.July -December 2014 120-122 B. Muthukumar et al. / Drug Invention Today 2014,6(2),120-122 capacity of ethanol could have produced greater number of active tions for a post-antimicrobial era. Science, 257: 1050-1055. constituents responsible for antibacterial activity.9 5. Nen, H. C. 1992. The crisis in antibiotic resistance. Science, 257: 1064-1073. 6. Yurdakok, K., Sahin, N., Ozmert, E. and Berkman, E. 1997. The antimicrobial activities can be enhanced if the active compo- Shigella gasteroenteritis: Clinical and epidemiological as- nents are purified and adequate dosage determined for proper admin- pects and antibiotic susceptibility. Acta. Prediator. J. 39: istration. This may go a long way in preventing the administration of 681-683. inappropriate concentration, a common practice among many tradi- 7. Taylor, J. L. S., Rabe, T., McGaw, L. J., Jager, A. K., Van tional medical practitioners. Staden, J. 2001. Towards the scientific validation of tradi- tional medicinal plants. Plant Growth Regul. 34: 23-37. 8. Ncube, N. S., Afolaya, A. J. and Okoh, A. T. 2008. Assess- CONCLUSION ment techniques of antimicrobial properties of natural com- The results of this study highlighting the significance of antibacterial pounds of plant origin: Current methods and future trends. components from ethanolic leaf extract of W. caledulacea will find a Afr. J. Biotechnol. 7: 1797-1806. place in the formulation of herbal medicine to control bacterial medi- 9. Bhattacharjee, I., Chatterjee, S. K., Chatterjee, S. and Chandra, ated infections. G. 2006. Antibacterial potential of Wedelia caledulacea sol- vent extracts against some pathogenic bacteria. Mem Insti- tutions. Oswaldo Cruze Rio de Janerio, 6: 645-648. ACKNOWLEDGEMENTS 10. Ellof, J. N. 1998. Which extractant should be used for the Authors wish to thank DST-FIST, Government of India, New Delhi for screening and isolation of antimicrobial components from providing the infrastructure facilities to the Department of Botany, plants. J. Ethanopharmacol. 60: 1-6. National College, Tiruchirappalli, Tamil Nadu. Author also expresses 11. Akueshi, C. O., Kadin, C. O., Kkneshi, E. U., Agina, S. E. and thanks to Sri. K. Ragunathan, Secretary and Dr. K. Anbarasu, Princi- Ngurnkwem, B. 2002. Antimicrobial potentials of Hyptis pal, National College, Tiruchirappalli for their encouragement. sauvedens Poit (Lamiaceae). Nigeria J. Bot. 15: 37-41. 12. Chessbrough, M. 2000. Medical Laboratory Manual for Tropical Countries. Linaere House, Jordan Hill, Oxford, pp. REFERENCES 260. 1. WHO 1978. The promotion and development of traditional 13. Baucr, A. W., Kirby, W. M., Sheris, J. C. and Turck, M. 1966. medicine. Technical Report Series, pp. 622. Antibiotic susceptibility testing by a standardized single 2. Ekpendu, T. O., Akshomeju, A. A. and Okogun, J. I. 1994. disc method. Am. J. Clin. Pathol. 45: 149-158. Antiinflamatory antimicrobial activity, Lett. Appl. Microbiol., 14. NCCLS (1993). Performance standards for antimicrobial disc 30: 379-384. susceptibility tests. Approved standard, NCCLS Publica- 3. Olukoya, D. K., Idika, N., Odugbemi, T. 1993. Antibacterial tions M2-A5 , PA, US. activity of some medicinal plants from Nigeria. J. 15. Nair, R., Kalariya, T., Sumitra, C. 2005. Antibacterial activity Ethnopharmocol., 39: 69-72. of some selected Indian medicinal flora. Turk. J. Biol. 29: 41- 4. Cohen, M. L. 1992. Epidemiology of drug resistance implica- 47.

Source of support: DST-FIST, Government of India, New Delhi , Conflict of interest: None

Drug Invention Today Vol.6.Issue.2.July -December 2014 120-122