ISSN Vol.02,Issue.19, December-2013, Pages:

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1 ISSN Vol.02,Issue.19, December-2013, Pages: Studies on Antifungal Properties of Some Plant Extracts (Garlic, Fenugreek, Ginger) Against of Clinical Isolate Candida Species ASHWAK JASIM 1, YASHAB KUMAR 2, JANE C.BENJAMIN 3, HARISON MASIH 4 1 Department of Pathological Analysis, Medical and Health Technical College, Foundation of Technical Education, Ministry of Higher Education and Scientific Research, Baghdad, IRAQ. 2 Department of Microbiology and Fermentation Technology, Jacob school bioengineering, Sam Higginbottom Institute of Agriculture, Technology and Sciences Allahabad, U.P-INDIA. Abstract: The antifungal properties of different plant extracts were studied to screen their ability to inhibit the growth of opportunistic pathogenic fungi of Candida species (C. albicans, C. glabrata, C. parapsilosis). The isolates were subjected to antifungal susceptibility towards Amphotericin B, Clotrimazole, Fluconazole, Itraconazole, Ketoconazole, and Nystatin. All the isolates were found to be susceptible to Ketoconazol. Further the isolates were tested for susceptibility against different plant extracts i.e. aqueous (Aq), ethanolic (E) extracts of Garlic, Fenugreek, and Ginger using agar well-diffusion method. The aqueous extracts of garlic were more effective than Ethanolic extract. MIC value determination showed mg/ml for Candida spp. Fresh aqueous garlic extract has the low MIC values as compared with Ethanolic extract. MFC value for aqueous garlic extracts were found mg/ml, gm/ml and mg/ml for C.albicans, C.glabrata, and C.parapsilosis respectively. In this present study all three plants have been found to exhibit a greater antifungal activity against human pathogenic fungi (Candida spp). Therefore this study has justified the traditional use of these plants in managing fungal diseases. Keywords: Antifungal Activity, Antifungal Susceptibility, Aqueous, Ethanol, Garlic, Fenugreek, Ginger, MIC, MFC. I. INTRODUCTION The pathogenic fungi in the genus Candida are capable of causing a variety of superficial and deep-seated mycoses. Candida species are now recognized as major agent of hospital-acquired infection (Douglas 2003). Candida albicans is the organism most often associated with serious fungal infection and it is showing increased resistance to traditional antifungal agents (Hawser and Douglas 1995). Recently, Candida species, such as C. parapsilosis and C. glabrata also show dramatic increases in fungal infections and antifungal resistance (Nguyen et al., 1996; Barchiesi et al., 2005). The spread of multidrugresistant strains of Candida and the reduced number of drugs available makes it necessary to search for new sources of antifungal and compounds that inhibit these resistance mechanisms. This has led to a search for therapeutic alternatives, particularly among medicinal plants and compounds isolated from them used for their empirically antifungal properties. The practice Complementary and alternative medicine is now on the increase in developing countries in response to World Health Organization directives culminating in several preclinical and clinical studies that have provided the scientific basis for the efficacy of many plants used in folk to treat infections (Akko et al., 2003). The aim of this study was to determine the antifungal susceptibility pattern of the test organisms and evaluate and compare the antimicrobial activity of different extracts of fresh garlic, fenugreek, and ginger also determine the MIC (Minimum Inhibitory Concentration) and MFC (Minimal fungicidal concentrations) of the extracts. II. MATERIALS AND METHODS A. Sample collection Garlic (Allium sativum), ginger (Zingiber officinale) and fenugreek (trigonella foenuum- graecum) used in the present study were purchased from the local market, Allahabad, U.P., India. B. Fungal strains 3 different characterized drug resistant fungal strains including Candida albicans, Candida glabrata, and Candida parapsilosis were obtained from by the Microbial Culture Collection Bank, Department of Microbiology and Fermentation Technology, Sam Higginbottom Institute of Agriculture Technology and Sinces, Allahabad. The strains were maintained on sabuoroid dextrose agar slants the culture was kept under refrigerated conditions and was sub cultured every 15 days. C. Preparation of extracts Aqueous, Ethanolic garlic extract was prepared according to Tsao et al., (2001). Similarly, Fenugreek and 2013 SEMAR GROUPS TECHNICAL SOCIETY. All rights reserved.

2 ASHWAK JASIM, YASHAB KUMAR, JANE C.BENJAMIN, HARISON MASIH ginger Extracts: Aqueous and ethanolic were prepared by the method of Saxena and Methela, (1996). The mixture was filtered through Whatman s No.1 paper to get the crude extracts. All the extracts were stored at 4 0 C. D. Susceptibility testing Antifungal susceptibility test was performed to detect the resistance and susceptibility of the test isolates against various antifungal it was done by Kirby-Bauer disc diffusion method. (Bauer et al., 1966) Melted and cooled saboaroids dextrose agar media was poured in sterile petriplates and swabbed with 100 µl of overnight culture of the test organism. Under aseptic conditions, anti fugues discs namely Amphotricin B,Clotrimazole, Fluconazole, Itraconazoel, Ketoconazole, Nystatin were placed on the surface of the inoculated plates with the help of sterile forceps following 24 h incubation at 37 ± C aerobically in upright position. Zone around the discs was measured in millimeter (mm) and results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) standards (Wayne, 2003). E. Antifungal Activity of extracts by Agar Well diffusion Test: The antifungal activity of different extracts against Candida species was evaluated by using agar well diffusion method (Perez, and Anesini., 1993). Sabouraud Dextrose agar (SDA) plates was inoculated with 100 µl of overnight culture of the test organism and spread with the help of sterile swabs. Wells of 8mm was punctured with the help of a sterilized cork borer into the pre-solidified Sabouraud Dextrose agar plates containing the test organism.. Using the micropipette, 100 µl of each extracts (aqueous and ethanol) was poured into the different wells of the inoculated plates. Standard antifungal (Fluconazole, 1 mg/ml) will use as positive control and fungal plates was incubated at 37 o.cfor 72 h. The diameters of zone of inhibition was measured. F. Determination of Minimum Inhibitory Concentration (MIC) and Minimum fungicidal concentration (MFC) The minimum inhibitory concentration (MIC) of different garlic, fenugreek and ginger extracts was determined by micro dilution method using serially diluted (2 folds) plant extract according to the National Committee for Clinical Laboratory Standards (NCCLS) (National Committee for Clinical Laboratory Standards,2000). Equal volume of each extract and nutrient broth was mixed in a test tube. Specifically 0.1ml of standardized inoculums ( cfu/ml) was added in each tube. The tube was incubated aerobically at 37ºC for 24_48HRS. Two control tubes were maintained for each test batch, these included antifungal control (tube containing extract and growth media without inoculums) and organism control (tube containing the growth medium, saline and the inoculums). The lowest concentration (highest dilution) of the extract that produced no visible fungal growth (no turbidity) when compared with the control tube was regarded as MIC. However, the MFC was determined by sub-culturing the test dilution on to SDA medium and incubated further for 72 h, (Rubio et al., 2005). the highest dilution that yielded no signal fungal colony on the solid medium was taken as MFC. III. STATISTICAL ANALYSIS The data recorded during the course of investigation were analyzed by Analysis of variance technique (Gomez and Gomez, 1976). The significant and nonsignificant treatment effects were judged with the help of F (variance ratio) table. IV. RESULTS AND DISCUSSION In the present study, using the disc-diffusion method, 3 isolates of Candida ie Candida albicans (MCCB0290), Candida glabrata (MCCB0286), Candida parapsilosis (MCCB0289) were tested for antifungal susceptibility pattern. All the 3 isolate of Candida species were found to be resistant against Amphotricin B and sensitive to against Ketoconazol Clotimozole was found to be effective, inhibiting the growth of C.glabrata and C.parapsilosis but C.albicans showed resistance against it C. parapsilosis was found sensitive against Fluconazol but C.albicans and C.glabrata were found to be resistant Itraconazole and Nystatin were found to be effective antifungal of against C.albicans but C.glabrata and C.parapsilosis showed resistance towards them. (Table1). Similar pattern of susceptibility has been reported by several workers for Candida species (Narin, 2003, Ritcher et al.2005;shimokawa et el.2005;rex et al.2001;barchiesi et al.1998; Kahan et al.2006; ) Prolonged and extensive use of antibiotics in the medical centers and Hospitals leads Candida species to build resistance towards them. Fig1: Antifungal Susceptibility Profile of C.albicans MCCB0290. Abbreviations: Fu-Fluconazole; Ap-Amphotericin B; Kt- Ketoconazole; Co-Clotrimazole; It-Itraconazole; Ns- Nystatin

3 Studies on Antifungal Properties of Some Plant Extracts (Garlic, Fenugreek, Ginger) Against of Clinical Isolate Candida Species TABLE 1: ANTIFUNGAL SUSCEPTIBILITY PROFILE OF THE CANDIDA SPECIES. A. Antimicrobial Activity of Plant Extracts As shown in figure 2 the 3 Candida species strain were then tested for their antifungal susceptibility towards different plant extracts namely aqueous and Ethanolic from fresh Garlic cloves, Ginger and Fenugreek seeds, using Agar well-diffusion method. Each Candida species strains showed varied results when subjected to the extracts. The results were the average of the three observations. The inhibition zone diameter (mm) of aqueous and ethanolic extracts on different fungal strains varies, depending on Candida species and type of extract C.albicans MCCB (0290), C.glabrata MCCB0286, and C.parapsilosis MCCB (0289),used in research study were inhibited by three extracts tested. Among all the tested spices, shows that garlic aqueous extract exhibited highest antifungal activity against all clinical strains. Garlic aqueous extract was more effective as compared to ethanol extract in inhibiting the fungal growth. The most susceptible isolate towards aqueous was found to be C.parapsilosis MCCB0289 with an inhibition zone of 48 mm for the three consecutive observations and 36 mm against Candida albicans MCCB0290 and 35mm against Candida glabrata MCCB Fig3: Fenugreek extracts on C.albicans TABLE-2: FIG-1 ANTIFUNGAL ACTIVITY OF DIFFERENT EXTRACTS OF GARLIC, FENUGREEK AND GINGER AGAINST CLINICAL STRAINS OF CANDIDA SPECIES BY DISK DIFFUSION METHOD BY (MM) Fig2: Antifungal activity of the plant(s) extracts Aq: Aqueous extract; E: Ethanol extract Includes diameter of well (8mm). Due to extracts: F (cal ),= 5.612> F (tab) at 5%=3.33 Significant; CD= Due to isolates: F (cal ),=0.096 < F (tab) at 5%=4.10 -non Significant; CD= The ethanolic Garlic extract was least effective exhibited inhibition zones of 26 mm against C. glabrata MCCB0286 and maximum zone of 30 mm against C. parapsilosis MCCB0289 where as it remained ineffective against C.albicans MCCB0290. Fenugreek ethanol extracts are more effective against the clinical strains showed maximum susceptibility against C. parapsilosis at inhibition zone 35mm and 30 mm against both C. albicans, C.glabrata while C.albicans maximum albicans, C.glabrata while C.albicans maximum susceptibility to fenugreek aqueous extract it give inhibition zone for Aq 12mm and

4 ASHWAK JASIM, YASHAB KUMAR, JANE C.BENJAMIN, HARISON MASIH non activity against C.glabrata, C.parapsilosis. Ginger ethanolic effective on C.glabrata with maximum inhibition zone 30mm and inhibition zone 26mm on C.albicans. While showed non antifungal activity against C. parapsilosis. However the aqueous ginger extract was found ineffective against all the tested fungi. On comparing the data with respect to the treatments, the differences were found to be statistically significant (P<0.05) for the extracts treatment while with respect to the isolates and extracts, the differences were found to be statistically non significant (P>0.05). Fig4: Ginger extracts on C.glabrata Fig5: Garlic extracts on C.parapsilosis The present study thus stated that all the plants are effective against fungal infections caused by Candida species. The data collected and evaluated represents that aqueous and ethanol Garlic, ginger and fenugreek extract in the: (table-2,) The results of antimicrobial effect of ginger and garlic in this study are accordance with most the reports published regarding ginger antimicrobial activity with garlic by Panzaru et al. (2009) Observed Garlic the Results indicated that only the aqueous extract of Garlic cloves showed inhibition zone against 9 bacterial isolates ranging from 6-30 mm however only Candida parapsilosis was inhibited by Garlic extract (30 mm).and Belguith et al. (2010), observed Garlic and ginger fresh aqueous and ethanol Garlic extracts against antifungal activity on Candida albicans and the zone inhibition for aqueous at (14.3mm), ethanolic 11.6mm) and for ginger inhibition zone at (12.3mm), ethanolic (15mm). Figure 3, 4 and 5 shows the Fenugreek, Ginger and Garlic extracts on C.albicans, C.glabrata and C.parapsilosis respectively. B.Determination of Minimum Inhibitory Concentration (MIC) and Minimum fungicidal concentration (MFC) The MIC values different garlic, fenugreek, and ginger. The results showed that MIC of different extract started from the aqueous garlic extracts were serially diluted to get a decreasing concentration for garlic of 0.1 gm/ml to gm/ml of the nutrient broth against clinical strains ranged from the Aqueous garlic extracts when seeded with 100 µl/ ml of the test broth culture; exhibited MIC at gm/ml for C.parapsilosis, C.albicans, gm/ml C.glabrata and same result against C.albicans gm/ml at 24hrs, 48hrs respectively. Ethanolic Garlic extract exhibited the minimum inhibitory effect at gm/ml for C.glabrata, and same result against C.parapsilosis gm/ml Ethanolic and non effective against C.albicans. Indicated that all tested strains were susceptible to garlic aqueous, Ethanolic extract but most effective was garlic aqueous extract. from aqueous Fenugreek extract determination by the dilutions of extract from 0.2gm/ml to gm/ml had lower exhibited the minimum inhibitory effect at 0.1 mg/ml of the broth on Candida albicans while Ethanol of Fenugreek extract had lower exhibited the minimum inhibitory effect at 0.025gm/ml on C.albicans and same MIC value against C.glabrata C.parapsilosis at 0.025gm/ml, to 24hrs and 48hrs respectively. In case of different ginger extracts, the lowest MIC value for C.albicans (0.05gm/ml) and same MIC value against C.glabrata was observed with ginger ethanol while for C.parapsilosis non activity,however aqueous ginger non activity to all clinical strains. (Table-3, 4) Similar findings were observed in the study conducted by Iwalokun et al. (2004) investigated the antimicrobial properties of aqueous garlic extract against 10 Candida spp were studied. Antifungal activity of AGE by macrobroth dilution method was characterized by -minimum inhibitory concentration (MIC) ranges of MIC values at 24 and 48 hours, respectively. Minimum fungicidal concentrations were found to be 14.9 and 15.5mg/mL, respectively, at these

5 Studies on Antifungal Properties of Some Plant Extracts (Garlic, Fenugreek, Ginger) Against of Clinical Isolate Candida Species incubation periods. A minimum Inhibitory Concentration (gm/ml) activity of aqueous, ethanolic Garlic, fenugreek and ginger extracts are shown in table 3 and 4. TABLE 3: MIC OF AQUEOUS GARLIC, FENUGREEK EXTRACTS ON DIFFERENT CANDIDA SPECIES (IN GM/ML). Minimum fungicidal concentration (MFC) was determined as the highest dilution (lowest concentration) at which no growth occurred on the plates. The least concentration at which no visible growth was observed in the SDA plates was considered as MFC in the (Table.6, 7). TABLE 6: MFC OF AQUEOUS FENUGREEK EXTRACTS AGAINST CANDIDA ALBICANS (IN 100 ΜL) TABLE 4: MIC OF ETHANOLIC PLANT EXTRACTS ON DIFFERENT CANDIDA SPECIES (IN GM/ML). TABLE 7: MFC OF ETHANOLIC GARLIC EXTRACTS AGAINST CANDIDA SPECIES TABLE 5: MFC OF AQUEOUS GARLIC EXTRACT AGAINST CANDIDA SPECIES (IN 100 ΜL) TABLE 8: MFC OF ETHANOLIC FENUGREEK EXTRACTS AGAINST CANDIDA SPECIES For determination of Minimum fungicidal concentration (MFC), the tubes that were incubated for final MIC minimum inhibitory concentration.100μl aliquot from the tubes showing no growth were sub cultured on SDA plates and inoculated SDA plates incubated at 37 0 C for 72 hrs and observed for the development of the colonies to determine if the inhibition was reversible or permanent.

6 ASHWAK JASIM, YASHAB KUMAR, JANE C.BENJAMIN, HARISON MASIH TABLE 9: MFC OF ETHANOLIC FENUGREEK EXTRACTS AGAINST CANDIDA SPECIES garlic and Chinese leek oils. Journal of Medical Microbiology 50: [7] Saxena, J. and Methela, C.S., (1996). Antifungal activity of new compound from Nepata IeucophyIIa and Nepata Clarkii.. Journal of Applied and Environmental. Microbial. 62 (2): [8] Onyeagba, R.A., Ugbogu, O.C., Okeke, C.U. and Iroakasi, O., (2004). Studies on the antimicrobial effects of garlic, ginger and lime. African Journal of Biotechnology 3(10): [9] Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turk, M. (1966). Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology 45: V. CONCLUSION Aqueous (Aq.) Garlic extracts and Ethanolic (E) Fenugreek extracts being highly potential for inhibiting on opportunistic pathogenic growth of Candida sp. indicates the fact that both these extracts contains potent antifungal agents which can be used for the treatments against community and hospital-acquired infections and their management. The extent to both extracts showed maximum antifungal activity against all the Candida species isolates at such low concentrations and high frequency of inhibiting the growth constantly for each of the strains. VI. REFERENCES [1] Douglas, L.J., (2003). Candida biofilms and their role in infection.trends of Microbiology.2: [10] Wayne, P.A., (2003).National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement M100-S13. NCCLS, USA. [11] Perez, C and Anesini, C. (1994). In vitro antibacterial activity of Argentine folk medicinal plants against Salmonella typhi. Journal of Ethno pharmacology44(1): [12] Gomez, K.A., & Gomez, A.A., (1976). Statistical Procedures for agricultural Research with Special Emphasis on rice. Philippines. International Rice Research 2_15.html. [2] Hawser, S.P., and Douglas, L.J., (1995). Resistance Candida albicans biofilms to antifungal agents in-vitro. Antimicrobiology Agents chemotherapy. 39: [3] Nguyen, M.H. (1996). The changing face of Candidemia: Emergence of Non- Candida albicans and antifungal resistance. American Journal of Medical. 100: [4] Barchiesi, F. (1999) in- vitro activity of five antifungal agents against uncommon clinical isolates of Candida spp. Journal of Antimicrobial Chemotherapy. 43: [5] Ako- Nai, A.K., Ikem, I.C., Aziba, A., Ajayi, A.A and Opined, O.A. (2003) Bacteriological examination of chronic osteomyelitis cases in IIE-Ife, South Western Nigeria. African Journal Clinical Express Microbial. 4(2):4151. [6] Tsao, S. and Yin, M.,(2001). In-vitro antimicrobial activity of four diallyl sulphides occurring naturally in