Efectivida Angunas Plantas Hinues 4982

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

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

www.worldjournalofscience.com

SCREENING OF VARIOUS PLANT EXTRACTS FOR ANTIFUNGAL

ACTIVITY AGAINST CANDIDA SPECIES

Amit Kumar1, Vikrant Bhatii3, Ajay Kumar4, Sandip Patil2, Vikesh Bhatia2 andAnil

Kumar2

1Assistant 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: [email protected]

AbstractCandida 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 fromHamirpur, 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.

Introduction

Medicinal plants are part of humanmedicine since the dawn of civilization. These

plants are making backbone of traditional medicinalsystems in India (Nayak, 2011). The Use of plantextracts to treat microbial infections is also reportedin our ancient Ayurvedic compendium CharakSamhita and Sushrat Samhita (Chatterjee, 1994).Atkinson et al., 1946, Dhar et al., 1968, and otherseveral researchers have carried out screening ofplant extract for antimicrobial activities (Atkinson,1946, Dhar, 1968).Due to increased prevalence of

drug resistant microorganisms there is great need tosearch for new effective drugs having natural orsynthetic origin (Pai, 1994). Plant extracts and theirproducts are clinically safer than antibiotics

(Kelmanson, 2000, Srinivasan, 2001). In the presentstudy five different medicinal plants were screenedfor their antifungal activity against Candida species.The isolates used in the study were collected fromNational Culture Collection of Pathogenic FungiPGIMER, Chandigarh. Five different medicinalplants were collected from different parts ofHimachal Pradesh and extracts were prepared andtested for their antifungal activity against Candida.


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Significant results were obtained during the study.This study will help us to design new naturaltherapeutic strategies against Candida associatedinfections

Material and methods

IsolatesNine Isolates used in study were collected

from National Culture Collection of PathogenicFungi (N.C.C.P.F) Post Graduate Institute ofMedical Education and Research (PGIMER)Chandigarh, India. Strains were maintained onSabourauds Dextrose Agar (Himedia Mumbai)slants and preserved in 10 % glycerol at -20oC. Subculturing was done regularly to maintain the freshcultures for the experiments

Plant materialFive different plants selected for antifungal

activity were Vitex negundo (leaf),Adathoda vasica(leaf), Azadirachta indica (leaf), Mentha piperita(leaf), Curcuma longa (rhizome). Fresh leaves ofplants collected randomly from District solan,Himachal Pradesh (India) except the Azadirachtaindicawhich was collected from Hamirpur, HimachalPradesh (India). The plants were identified andauthenticated at Department of forestry, Dr. Y.S.Parmar University, Solan, Himachal Pradesh(India).

Preparation of Plant ExtractsFresh plant leaves were washed under

running tap water and ethanol (30-40%). Leaves weresoaked for 10 minutes in sterilized distilled water andshade dried. They were ground to fine powder by usingpestle and mortar. The powder was stored in air tightbottles (Mohan M.C.H. 2009). Plants aqueous extractwas prepared by mixing 15.0 gm of dry powder of plantleaves with 100 ml of sterile distilled water in a roundbottom flask for 4-5days with occasional shaking.Extract was then filtered through a muslin cloth forcoarse residue and finally filtered through WhatmannNo.1 filter paper and stored in an airtight container at4oC for further use. Ethanolic extract was prepared by

mixing 15.0 gm of dry powder of plant leaves with 100ml of 95% ethanol and kept at room temperature for 5days in a round bottom flask with occasional shaking.After five days period, the extract was filtered througha muslin cloth and finally filtration was done throughWhitmans No.1 filter paper and stored in an airtightbottle at 4oC for further use(Hassawi, 2006).Air-driedand powdered plant material (10 gm of each) in 100 mlmethanol were taken to prepare methanolic extractand kept on a rotary shaker for 24 hours then filtered

and centrifuged at 5000 rpm for 15 minutes. Thesupernatant was collected and stored in bottle at 4o Cfor further use (Parekh, 2008).

Preparation of inoculumInoculum was prepared by taking 5-8

colonies of fungal strain from the fresh cultures andsuspended in 5 ml saline water in the test tube andvortexed (Shihabudeen, 2010).

Antifungal assayAntifungal assay was performed by using

Disc diffusion method. Sabourauds dextrose agarplates with yeast extract and without yeast extract(Himedia) were used for aqueous and methanolicextracts respectively (Mohan et al., 2009, Parekh etal., 2008). Ethanolic extracts were tested on Nutrientagar plates (Himedia) (Hassawi, 2006). 0.2 ml ofinoculum was inoculated and spread uniformly oneach plate. Inoculum was allowed to dry for 5minutes. Different concentrations of plant extracts (3,

5, 10 l) were loaded on sterile individual discs(Himedia). The loaded discs were placed on thesurface of medium and the extract was allowed todiffuse at least for 5 minutes.Theplates were kept forincubation at 28oC for 24-48 hours. Fucanazole(Cipla, Ltd.) (5l) was used as positive control andnegative control containing respective solvents wereused for every assay. Plates were observed after 24-48 hours incubation for appearance of zones ofinhibition around the discs. Antifungal activity wasevaluated by measuring diameter of zones ofinhibition (in millimeters) of fungal growth

(Shihabudeen, 2010, Nayak, 2011). All theexperiments were carried out in triplicate.

Results

Selected plant extracts were checked forthe antimicrobial activity in nine different Candidaspecies. Aqueous and Ethanolic extract of each plantshowed effective antimicrobial activity againstdifferent species of Candida while no antimicrobialactivity was there in methanolic extract except inCurcuma longa against C. albicans B-1622/09.

Aqueous and Ethanolic extract of Adathoda vasicashowed effective antimicrobial activity (total 8 cases)followed by Vitex negundo and Curcuma longa (total6 cases). Ethanolic extract of Adathoda vasicashowed antimicrobial activity in 5 Candida sp.followed by aqueous extract of Curcuma longa in 4Candida sp. Very less antimicrobial activity wasshown by various extracts of Mentha piperita (Table-1).


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Table 1. Diameter of zone of Inhibition (in mm) showed by various plant extracts (volume

used 5l)

Discussion

Candida is a yeast and part of the normalmicroflora of human body. Candida is known asopportunistic fungal pathogens as it causes infectionwhen person become immunocompromised,immunonosuppressed or diabetic. In immunecompromized individuals Candida causes diseaseslike oral thrush, intestinal candidiasis, vaginal thrushand onchomycosis (Chamaine, 2005, K J Kwon-Chung, John E. Bennett, 1992, Chander, 1996).

These diseases are highly infectious and have a highmortality rate all over the world. Because of theemergence of the resistant strains against commonlyused antifungal agents the researchers are nowpaying attention to develop natural antifungal agents.Our study involves the in vitro evaluation of threedifferent solvent extracts of five different medicinalplants against nine strains ofCandida.

Vitex

Negundo

Adathoda

Vasica

Azadirachta

indica

Mentha

Piperita

Curcuma

longa

FucanazoleTest Strain

Used

AqueousExtract

EthanolicExtract

MethanolicExtract

AqueousExtract

EthanolicExtract

MethanolicExtract

AqueousExtract

EthanolicExtract

MethanolicExtract

AqueousExtract

EthanolicExtract

MethanolicExtract

AqueousExtract

EthanolicExtract

MethanolicExtract

C. albicans

B-1622/09

6 3 2.5 4 5 1 3.5 2 1 3 2 1.5 2.5 1 3 6

C. albicansB-1599/09

5 3 0.5 7 3 2 2 2 3 4 1 2 2.5 3.5 5 5

C. tropicalis

B-1389/093 3 1 2 2 1 6 1 3 2.5 2 1 2 1.5 2 4

C. tropicalisB-1410/09

2 3 2 1 5 2.5 2 3.5 1.5 3 1 2.5 5 3 2.5 4

C.guilliermondiiB-1343/09

3 2 1 6 3 1 4 4 3.5 4 5 - 5 3 2 5

C.guilliermondii

B-1418/09

4 2.5 1.5 3 5 2.5 8 3 0.5 3.5 1.5 1 6 5 2 4

C. glabrataB-1366/09

2.5 6 3 3.5 5 0.5 4.5 4 2 6 2 0.3 4 4 2.5 4

C. glabrata

B-1303/09

2 5 1 2 6 0.5 3.5 1 3 2 2 2 4 - 2.5 3

C.parapsilosis

B-1597/09

3 4 1.5 5 8 2.5 1 3 1.5 3 4 2 2.5 2 1 4


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Fig 1.Azadirachta indica aqueousextract

showing zone of inhibition against

Candida guilliermondii B-1418/09

Different plant extracts showed differentzone of inhibition against the opportunistic Candidaspecies. The diameter of zone of inhibition for all theextracts were in the range of 0.5 to 10 mm. Highest

zone of inhibition was noted in the aqueous extract ofconcentration of 5l forAzadirachta indica (8 mm)against Candida guilliermondiiB-1418/09 followed byCandida tropicalis B-1389/09 (6mm) and Candidaglabrata B-1366/09 (4.5mm) as compared withstandard Fucanazole (5l). This showed thataqueous extract ofAzadirachta indica was effectiveagainst antibiotic resistant Candida (Table-1). In thecurrent study various extracts obtained from

Adathoda vasica exhibited good antifungal activityagainst all the Candida strains. The methanolicextracts ofAdathoda vasica were resistant at 5l

concentration for 8 Candidal strains. The plantswithout antifungal activity were due to absence ofsecondary metabolites (Sinha, 2002). Adathodavasica, Curcuma longa and Vitex negundo showedstrong antifungal activity.

Conclusion

The results noticed in the study showed thatthe extracts obtained from the selected medicinalplants collected from different areas of HimachalPradesh had anti-Candidalactivity and can be used in

preparation of novel natural therapeutic drugs againstCandida associated infections.

References

1. Atkinson, N. and Ramsturd, K.M., (1946).Australian J. Exp. Biol. Med. Sci.24;49.

2. Chamaine A., Loyd, C.L, Menon, T. andUmamasheshweri., (2005). Anticandidal activity

of Azadirachta indica. Indian J Pharmacol., 37,386-389.

3. Chander, J., (1996). A Text book of MedicalMycology .1st edn. New Delhi Interprint. p-213-220.

4. Chatterjee, A. and Pakrashi, S., (1994). TheTreatise 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 Indianplants for biologicak activity.Part-1. Indian J. Exp.Biol. 6; 232-47.

6. Hassawi, D. and Kharma, A., (2006).Antimicrobial Activity of Some Medicinal Plantsagainst Candida albicans. Journal of BiologicalSciences.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 IndianMedicinal 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 ofantibacterial and anticandidal efficacy of aqueousand alcoholic extracts of Neem (AzadirachiaIndia) and in vitro study. IJPAP.1; 230-235.

11. Pai, M.R., Acharya, L.D. and Varun N., (2004).Evaluation of antiplaque activity of AzadiachtaIndica leaf extract get a 6 week clinical study.Journal of Enthnopharmacology, 90, 99-103.

12. Parekh, J. and Chanda, S., (2008). In vitroantifungal activity of methanol extracts of some

Indian medicinal against pathogenic yeasts antmolds. Afr. J. of Biotech., 7(23), 4349-4353.

13. Shihabudeen, M.S., Hans Priscilla, D. andThirumurugan, K. (2010). Antimicrobial activityand phytochemical analysis of selected Indianfolk medicinal plants. International Journal ofPharma Sciences and Research, 10, 430-434.

14. Sinha, K., Mishra, N.P., Singh, J. and Khanya,S.P.S. (2002). Tinospora Cordifolia (Gudushi) a


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reservoir plant for therapeutic applications. Areview. Indian journal of traditional Knowledge.3(3); 257-270.

15. Srinivasan, D., Thirumurugan, N., Hansi, T.and Persumalasamy, P.L., (2001). Antimicrobialactivity of certain Indian medicinal plants used infolkloric medicine. J. Ethnopharmacol.74:217-220.

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Efectivida Angunas Plantas Hinues 4982