World Journal of Science and Technology 2011, 1(10): 43-47 ISSN: 2231 2587 www.worldjournalofscience.

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SCREENING OF VARIOUS PLANT EXTRACTS FOR ANTIFUNGAL ACTIVITY AGAINST CANDIDA SPECIES
Amit Kumar1, Vikrant Bhatii3, Ajay Kumar4, Sandip Patil2, Vikesh Bhatia2 and Anil Kumar2 Professor, Faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, 173212, India. 2Faculty of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, 173212, India. 3Post Graduate student, Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, 173212, India. 4Head, Department of Microbiology, Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, 173212, India. Corresponding author e-mail: amit.thakur2035@gmail.com Abstract
Candida is a part of normal micro flora of human body and is commonly isolated from blood stream infection. Since very early in human history, people have relied on medicinal plants to cure them of various ills. The Use of plant extracts to treat microbial infections are reported in our ancient Ayurvedic Medicinal system. Taking into account, five different plants; Vitex negundo (leaf), Adathoda vasica (leaf), Azadirachta indica (leaf), Mentha piperita (leaf) and Curcuma longa (rhizome) were selected for antifungal activity against Candida. Fresh leaves of plants were collected randomly from Solan, Himachal Pradesh, India except Azadirachta indict which was collected from Hamirpur, Himachal Pradesh (India). Nine clinical Candida isolates were collected from National Culture Collection of Pathogenic fungi (N.C.C.P.F) P.G.I.M.E.R Chandigarh India. Various extracts of the selected plants were tested for their antifungal activity against Candida species. A significant antifungal activity was shown by all five plant extracts. This study will help us to design new natural therapeutic strategies against Candida associated infections. Keywords: Candida, Plant extracts, antifungal agent.
1Assistant

Introduction
Medicinal plants are part of human medicine since the dawn of civilization. These plants are making backbone of traditional medicinal systems in India (Nayak, 2011). The Use of plant extracts to treat microbial infections is also reported in our ancient Ayurvedic compendium Charak Samhita and Sushrat Samhita (Chatterjee, 1994). Atkinson et al., 1946, Dhar et al., 1968, and other several researchers have carried out screening of plant extract for antimicrobial activities (Atkinson, 1946, Dhar, 1968). Due to increased prevalence of

drug resistant microorganisms there is great need to search for new effective drugs having natural or synthetic origin (Pai, 1994). Plant extracts and their products are clinically safer than antibiotics (Kelmanson, 2000, Srinivasan, 2001). In the present study five different medicinal plants were screened for their antifungal activity against Candida species. The isolates used in the study were collected from National Culture Collection of Pathogenic Fungi PGIMER, Chandigarh. Five different medicinal plants were collected from different parts of Himachal Pradesh and extracts were prepared and tested for their antifungal activity against Candida.

World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 43-47

Significant results were obtained during the study. This study will help us to design new natural therapeutic strategies against Candida associated infections

and centrifuged at 5000 rpm for 15 minutes. The supernatant was collected and stored in bottle at 4o C for further use (Parekh, 2008). Preparation of inoculum Inoculum was prepared by taking 5-8 colonies of fungal strain from the fresh cultures and suspended in 5 ml saline water in the test tube and vortexed (Shihabudeen, 2010). Antifungal assay Antifungal assay was performed by using Disc diffusion method. Sabourauds dextrose agar plates with yeast extract and without yeast extract (Himedia) were used for aqueous and methanolic extracts respectively (Mohan et al., 2009, Parekh et al., 2008). Ethanolic extracts were tested on Nutrient agar plates (Himedia) (Hassawi, 2006). 0.2 ml of inoculum was inoculated and spread uniformly on each plate. Inoculum was allowed to dry for 5 minutes. Different concentrations of plant extracts (3, 5, 10 l) were loaded on sterile individual discs (Himedia). The loaded discs were placed on the surface of medium and the extract was allowed to diffuse at least for 5 minutes. The plates were kept for incubation at 28oC for 24-48 hours. Fucanazole (Cipla, Ltd.) (5l) was used as positive control and negative control containing respective solvents were used for every assay. Plates were observed after 2448 hours incubation for appearance of zones of inhibition around the discs. Antifungal activity was evaluated by measuring diameter of zones of inhibition (in millimeters) of fungal growth (Shihabudeen, 2010, Nayak, 2011). All the experiments were carried out in triplicate.

Material and methods

Isolates Nine Isolates used in study were collected from National Culture Collection of Pathogenic Fungi (N.C.C.P.F) Post Graduate Institute of Medical Education and Research (PGIMER) Chandigarh, India. Strains were maintained on Sabourauds Dextrose Agar (Himedia Mumbai) slants and preserved in 10 % glycerol at -20oC. Sub culturing was done regularly to maintain the fresh cultures for the experiments Plant material Five different plants selected for antifungal activity were Vitex negundo (leaf), Adathoda vasica (leaf), Azadirachta indica (leaf), Mentha piperita (leaf), Curcuma longa (rhizome). Fresh leaves of plants collected randomly from District solan, Himachal Pradesh (India) except the Azadirachta indica which was collected from Hamirpur, Himachal Pradesh (India). The plants were identified and authenticated at Department of forestry, Dr. Y.S. Parmar University, Solan, Himachal Pradesh (India). Preparation of Plant Extracts Fresh plant leaves were washed under running tap water and ethanol (30-40%). Leaves were soaked for 10 minutes in sterilized distilled water and shade dried. They were ground to fine powder by using pestle and mortar. The powder was stored in air tight bottles (Mohan M.C.H. 2009). Plants aqueous extract was prepared by mixing 15.0 gm of dry powder of plant leaves with 100 ml of sterile distilled water in a round bottom flask for 4-5days with occasional shaking. Extract was then filtered through a muslin cloth for coarse residue and finally filtered through Whatmann No.1 filter paper and stored in an airtight container at 4oC for further use. Ethanolic extract was prepared by mixing 15.0 gm of dry powder of plant leaves with 100 ml of 95% ethanol and kept at room temperature for 5 days in a round bottom flask with occasional shaking. After five days period, the extract was filtered through a muslin cloth and finally filtration was done through Whitmans No.1 filter paper and stored in an airtight bottle at 4oC for further use (Hassawi, 2006). Air-dried and powdered plant material (10 gm of each) in 100 ml methanol were taken to prepare methanolic extract and kept on a rotary shaker for 24 hours then filtered

Results
Selected plant extracts were checked for the antimicrobial activity in nine different Candida species. Aqueous and Ethanolic extract of each plant showed effective antimicrobial activity against different species of Candida while no antimicrobial activity was there in methanolic extract except in Curcuma longa against C. albicans B-1622/09. Aqueous and Ethanolic extract of Adathoda vasica showed effective antimicrobial activity (total 8 cases) followed by Vitex negundo and Curcuma longa (total 6 cases). Ethanolic extract of Adathoda vasica showed antimicrobial activity in 5 Candida sp. followed by aqueous extract of Curcuma longa in 4 Candida sp. Very less antimicrobial activity was shown by various extracts of Mentha piperita (Table1).

World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 43-47

Table 1. Diameter of zone of Inhibition (in mm) showed by various plant extracts (volume used 5l) Test Strain Used Vitex Negundo Adathoda Vasica Azadirachta indica Mentha Piperita Curcuma longa Fucanazole

Methanolic Extract

Methanolic Extract

Methanolic Extract

Methanolic Extract

C. albicans B-1622/09 C. albicans B-1599/09 C. tropicalis B-1389/09 C. tropicalis B-1410/09

6 5 3 2

3 3 3 3 2

2.5 4 0.5 7 1 2 1 2 1 6

5 3 2 5 3 5

1 2 1

3.5 2 2 6 2 1

1 3 3

3 4

2 1

1.5 2.5 1 2 1

Methanolic Extract

Ethanolic Extract

Ethanolic Extract

Ethanolic Extract

Ethanolic Extract

Ethanolic Extract

Aqueous Extract

Aqueous Extract

Aqueous Extract

Aqueous Extract

Aqueous Extract

6 5 4

2.5 3.5 5 2 1.5 2 3 3 5 4 -

2.5 2 1 5

2.5 2 1 4

3.5 1.5 3 4 3 3.5 4

2.5 5 5 6

2.5 4 2 2 5 4

C.guilliermondii 3 B-1343/09 C.guilliermondii 4 B-1418/09 C. glabrata B-1366/09 C. glabrata B-1303/09 C.parapsilosis B-1597/09

2.5 1.5 3 3 1

2.5 8

0.5 3.5 1.5 1 2 3 6 2 2 2 4

2.5 6 2 3 5 4

3.5 5 2 6 8

0.5 4.5 4 0.5 3.5 1 2.5 1 3

0.3 4 2 2 4

2.5 4 2.5 3 1 4

1.5 5

1.5 3

2.5 2

Discussion

Candida is a yeast and part of the normal microflora of human body. Candida is known as opportunistic fungal pathogens as it causes infection when person become immunocompromised, immunonosuppressed or diabetic. In immune compromized individuals Candida causes diseases like oral thrush, intestinal candidiasis, vaginal thrush and onchomycosis (Chamaine, 2005, K J KwonChung, John E. Bennett, 1992, Chander, 1996). World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 43-47

These diseases are highly infectious and have a high mortality rate all over the world. Because of the emergence of the resistant strains against commonly used antifungal agents the researchers are now paying attention to develop natural antifungal agents. Our study involves the in vitro evaluation of three different solvent extracts of five different medicinal plants against nine strains of Candida.

of Azadirachta indica. Indian J Pharmacol., 37, 386-389. 3. Chander, J., (1996). A Text book of Medical Mycology .1st edn. New Delhi Interprint. p-213220. 4. Chatterjee, A. and Pakrashi, S., (1994). The Treatise on Indian Medicinal Plants, p-76. 5. Dhar, M.L., Dhar, M.M, Dhawan, B.N, Mehrotra, B.N. and Ray, C., (1968). Screening of Indian plants for biologicak activity.Part-1. Indian J. Exp. Biol. 6; 232-47. 6. Hassawi, D. and Kharma, A., (2006). Antimicrobial Activity of Some Medicinal Plants against Candida albicans. Journal of Biological Sciences.6 (1):109-114. 7. Kelmanson, J.E. and Staden, J.Z., (2000). Medicinal plants with antimicrobial activity. Journal of Ethnopharmacol., 69, 241-246. 8. Kwon-Chung, K.J. and Bennett, J.E., (1992). Medical mycology. Second edition Lea & Febiger, p-280-326. 9. Mohan M.C.H., Rao, S.M. and Kumari, P., (2009). Antimicrobial Activity of Selected Indian Medicinal Plants. Jr. of Microbiol. Biotech. Env. Sc, 11 (2), 355-360. 10. Nayak, A., Nayak, R.N, Soumya, B., Bhat K. and Kudalkar, M., (2011). Evaluation of antibacterial and anticandidal efficacy of aqueous and alcoholic extracts of Neem (Azadirachia India) and in vitro study. IJPAP.1; 230-235. 11. Pai, M.R., Acharya, L.D. and Varun N., (2004). Evaluation of antiplaque activity of Azadiachta Indica leaf extract get a 6 week clinical study. Journal of Enthnopharmacology, 90, 99-103. 12. Parekh, J. and Chanda, S., (2008). In vitro antifungal activity of methanol extracts of some Indian medicinal against pathogenic yeasts ant molds. Afr. J. of Biotech., 7(23), 4349-4353. 13. Shihabudeen, M.S., Hans Priscilla, D. and Thirumurugan, K. (2010). Antimicrobial activity and phytochemical analysis of selected Indian folk medicinal plants. International Journal of Pharma Sciences and Research, 10, 430-434.

Fig 1. Azadirachta indica aqueous extract showing zone of inhibition against Candida guilliermondii B-1418/09
Different plant extracts showed different zone of inhibition against the opportunistic Candida species. The diameter of zone of inhibition for all the extracts were in the range of 0.5 to 10 mm. Highest zone of inhibition was noted in the aqueous extract of concentration of 5l for Azadirachta indica (8 mm) against Candida guilliermondii B-1418/09 followed by Candida tropicalis B-1389/09 (6mm) and Candida glabrata B-1366/09 (4.5mm) as compared with standard Fucanazole (5l). This showed that aqueous extract of Azadirachta indica was effective against antibiotic resistant Candida (Table-1). In the current study various extracts obtained from Adathoda vasica exhibited good antifungal activity against all the Candida strains. The methanolic extracts of Adathoda vasica were resistant at 5l concentration for 8 Candidal strains. The plants without antifungal activity were due to absence of secondary metabolites (Sinha, 2002). Adathoda vasica, Curcuma longa and Vitex negundo showed strong antifungal activity.

Conclusion
The results noticed in the study showed that the extracts obtained from the selected medicinal plants collected from different areas of Himachal Pradesh had anti-Candidal activity and can be used in preparation of novel natural therapeutic drugs against Candida associated infections.

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World Journal of Science and Technology | www.worldjournalofscience.com | 2011 | 1(10): 43-47