Research Article Ravi Kant Upadhyay et al. / Journal of Pharmacy Research 2011,4(4),1153-1156 Available online through ISSN: 0974-6943 http://jprsolutions.info Antifungal activity of 16 plant essential oils against S.cerevisiae, Rhizopus stolonifer and Aspergillus flavus Ravi Kant Upadhyay*,Pratibha Dwivedi and Shoeb Ahmad Department of Zoology, D D U Gorakhpur University, Gorakhpur, 273009. India Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011 ABSTRACT In the present investigation antifungal activity of 16 different essential oils have been tested in vitro against S.cerevisiae, Rhizopus stolonifer and Aspergillus flavus.For screening antifungal susceptibility both MIC, MFC values and inhibition zone diameters were determined in Yeast culture. Spore counting and mycelial growth were measured in Rhizopus stolonifer and Aspergillus flavus by applying E-test. The MIC and MFC values obtained in presence of essential oils were in a range of 0.012-0.050µg/ml in S.cerevisiae.Besides this the maximum inhibition zone diameter was obtained 32 mm in Ashoka oil in S.cerevisiae. It was found significantly much higher than that of antifungal drug Griseofulvin. According to agar disc diffusion method, more potent essential oils screened are Sesame, Saunf, Sandal wood and Maulsri, which have shown very high susceptibility against fungal species. Besides this,there was obsereved lesser mycelial growth and no. of spores in presence of each essential oil in comparison of Griseofulvin . These essential oils have significantly altered the level of different biomolecules found in Yeast (S.cerevisiae ). There was obsereved a significant reduction in DNA level i.e. 48.93-74.58% in presence of each essential oil in Yeast culture. The maximum reduction was obtained in Dhania oil i.e. 74.58% at 32µl concentration and minimum reduction i.e.48.93% was obtained in presence of Neem oil at the same concentration. In RNA level the maximum reduction was found to be 91.78% in Ajwaine oil and minimum reduction i.e. 83.68% in presence of Saunf essential oil. The maximum reduction in Protein lelvel i.e. 90.18% in presence of Dhania essential oil at 32µl concentration.

Key words: Essential oil, MIC, MFC, S.cerevisiae, Rhizopus stolonifer Aspergillus flavus INTRODUCTION For controlling growth of infectious agents most of the synthetic drugs have failed to control concentration of each and every essential oil. For this purpose two-fold dilution of the essential infection due to rising drug resistance in microbes. Therefore, to fight against pathogenic oils was done in Luria Browth. Fungal growth inhibition was determined in presence of diseases researchers have isolated active ingredients from plant species which work as effective various serial concentrations of each essential oil in a range of 32µl/ml to 0.0078µl/ml by antifungal agents and explored their antimicrobial activities for the treatment of infectious using serial micro dilution method. The essential oil was added to fresh suspension of each pathogenic diseases (Gurgel, 2005). However, these plant derived antimicrobial agents are bacterial strain after following the serial dilution up to 10-10. Each oil was assayed in triplicate. used as alternative medicine. Essential oils are plant constituents which possess very high The MIC value was considered as the lowest concentration of the essential oil at which no antibiotic potential against micro-organisms (Darokar et al., 1998). Essential oils are aromatic turbidity in the culture media found visible after 24 hrs of incubation at 37oC temperature. oily liquids obtained from various plant parts such as flowers, buds, seeds, leaves twig, fruits, peel and roots. Essential oils are secondary metabolites having chemical structure C10H16 and DISC DIFUSSION TEST possess enormous power to kill microbial pathogens. Few essential oils possesses anti- Disc diffusion tests recommended by NCCLS were performed for determination of antifungal diabetic, anti inflammatory and strong antifungal activity (Mimica-Dukic et al., 2004). These activity of essential oils. For testing antifungal activity of various essential oils Muller-Hinton oils possess few important constituents like triterpines, tetraterpenes, hemipterans and agar was used. It was supplemented with 0.2% glucose and 0.5-µg/ml methylene blue dyes. sesqueterpenes. Both terpenes and terpenoids were found active against bacteria, viruses and The pH of the medium was kept between 7.2 and 7.4. The turbidity of the medium was protozoa. Among tri-terpenoids beutilinic acid is one of the strong antimicrobial agents, which standardized to 0.5 MacFarland units using a densitometer and plates were incubated at 37oC inhibit the growth of so many microorganisms. Besides this essential oils possess anti-cancer for 24 hrs. For comparsion strong antifungal agent Griseofulvin was used. The zone inhibition (Sylvestre et al, 2006), anti-tumoral (Sousa et al., 2004), and anti-bacterial activity (Burt, diameter was compared with NCCLS reference antimicrobial drugs. By regression analysis 2004). Besides this, essential oils are also used in food preservation (Faid et al., 1995) and tentative zone size was established for essential oil susceptibility (19mm or <= susceptible, show growth inhibitory and systemic activity against pathogens (Buttner et al., 1996). 12mm or >= resistant, 13mm-18mm = dose dependent). Moreover, eugenol is a well characterized alcoholic component occur in clove oil was found highly toxic to fungi (Duke et al., 1985) and bacteria (Thomsori et al., 1978). Cinnamon bark DETERMINATION OF MINIMUM FUNGICIDAL CONCENTRATION (MFC) oil is used against respiratory tract myocoses (Singh et al., 1995). Essential oils obtained from For determination of MFC values growth inhibitory assays were performed. For this purpose the leaves of so many plants have shown inhibitory activity against bacterial, fungal and viral inoculum size was adjusted to prepare a final colony number as 108 colony forming units infections (Karaman et al., 2001). (CFU/ml) in sterile agar plates. All incubations of tests and control cultures were performed at 37 oC for 24 hrs. For comparison both negative and positive controls were set and fungal Essential oils from many plants are known to possess, antifungal activity (Kalemba and colony number was counted in all test and control discs. The least concentration at which no Kunica, 2003), These plant origin natural products show lesser side effects in patients and are visible growth was obtained in agar plates was considered as MFC value. For evaluation of proved highly lethal to drug resistant pathogenic strains (Barkers and Rogers, 2006; Klepser, growth inhibition two parallel concentrations were set up for each and every essential oil. 2006, Aperis, et al., 2006, Mondello et al., 2006). In spite of good efforts made on antifungal Fungicidal growth obtained in presence and absence of various quantities of essential oils was activity of essential oils in the past antifungal activity of so many essential oils is still considered for growth inhibition. After 24 hrs fungal culture was harvested to determine DNA, unknown. Due to limited information available in this area in the present investigation RNA and Protein level at various time periods. antifungal potential of various plant essential oils has been evaluated against S.cerevisiae, Rhizopus stolonifer and Aspergillus flavus and MIC and MFC values, spore counting and DETERMINATION OF LENGTH OF HYPHAE (MYCELIA) AND NUMBER OF mycelial growth were determined in presence of each essential oil separately. Besides this effect SPORES BY APPLYING SLIDE CULTURE METHOD of essential oils were also investigated on certain biomolecules such as DNA,RNA and For determination of mycelial growth slide culture of molds was done on Sabouraud’s agar Proteins.. medium. Media contained 1% peptone, 4% glucose and 2% agar. The pH of medium was MATERIALS AND METHODS adjusted to 5.6 to inhibit the bacterial growth. For slide culture a sheet of filter paper was put in the bottom of Petri dish. On filter paper a U shaped sterile slide was kept over this rod A ANTIFUNGAL SUSCEPTIBILITY TESTING cut piece of 5mm square block of the Sabouraud’s agar were inoculated with spores of mold In the present investigation, antifungal activity of various essential oils was evaluated against colony. Inoculated block of agar was put in middle of the slide. It was covered by a cover glass certain medically important fungi such as Saccharomyces cerevisiae by agar disc diffusion and keep inside the Petri dish up to 48 hrs. After 48 hrs of incubation length of hyphae was method (Yoshida et al., 1987). For evaluation of antifungal activity of various fungal strains observed under microscope after staining with lacto phenol spores and cotton blue. Growth serial dilution method was used. inhibitory activity was determined in fungal cells at various increasing concentrations of each essential oil. For observation of effect on mycelial growth 2µl of each and every essential oil was coated on agar cubes separately and growth was recorded at 24 hrs. DETERMINATION OF MIC VALUES DETERMINATION OF BIOCHEMICAL INHIBITION BROTH DILUTION METHOD Biochemical inhibition will be measured in terms of macromolecules i.e. DNA, RNA and Broth dilution susceptibility tests were performed to determine the minimum inhibitory protein respectively. Biochemical estimation of proteins will be done by Lowry et al., while DNA and RNA by Diphenylamine and Orcinol method (Schneider, 1957) respectively. Each assay will be performed after termination of experiment at 24 hrs and for the same 10 ml of the growth medium will be centrifuged. Pellet will be dissolved in 5% TCA and centrifuged again *Corresponding author. at 6000 rpm for 30 minutes. 200 ml of dissolved pellet will be used for estimation of DNA and Ravi Kant Upadhyay RNA. For determination DNA and RNA contents absorbance will be taken at 590 and 660 nm Department of Zoology, respectively. D D U Gorakhpur University, STATISTICAL ANALYSIS OF THE DATA All statistical calculations were expressed as mean ± SE of three replicates. Student’s‘t’- test Gorakhpur, India 273009 was applied to locate significant difference in oil activity (Sokal and Rohlf, 1973).

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1153-1156 Ravi Kant Upadhyay et al. / Journal of Pharmacy Research 2011,4(4),1153-1156 RESULTS average 6 times inhibition was obtained in tests in comparison to control (Table 3 and 4). EFFECT OF ESSENTIAL OIL ON FUNGAL GROWTH There was observed a constant inhibition in mycelial growth up to 120 hrs. Antifungal activity of all 16 different essential oils were also determined in yeast culture i.e. Saccharomyces cerevisiae. In case of Saccharomyces cerevisiae growth inhibition was deter- Table 3. Effect of essential oils on mycelial growth of Aspergillus flavus in mm mined both in Luria Broth and Luria Agar in presence of each essential oil and one broad spectrum antifungal drug i.e. Griseofulvin. Essential oil 24 hrs 48 hrs 72 hrs 96 hrs 120 hrs

Asoke oil 2-4 2-4 2-4 2-8 2-8 DETERMINATION OF MIC AND MFC VALUES Saunf oil 2-4 2-6 2-6 2-6 2-6 The MIC and MFC values of 16 plants essential oils were determined against Saccharomyces Elachi oil 2-6 2-6 2-8 2-8 2-8 cerevesiae .These are presented in Table 1. All different essential oils have shown very low Sesame oil 2-4 2-4 2-4 2-6 2-6 MIC and MFC value. The maximum MIC values obtained in presence of Saunf, Dhania, Sandal wood oil 2-4 2-4 2-4 2-4 2-4 Orange, Clove and Maulsri essential oil i.e. 0.05µl/ml and minimum MIC values obtained Dhania oil 2-4 2-8 2-8 2-8 2-8 Olive oil 2-4 2-4 2-4 2-4 2-4 in presence of Ashoka, Sesame, and Sandal wood oil i.e. 0.01µl/ml against Saccharomyces Orange oil 2-4 2-6 2-6 2-6 2-6 cerevisiae. It is lower than the antifungal drug i.e. 41.22 µg/ml concentration (Table 1). Ajwaine oil 2-6 2-8 2-8 2-8 2-8 Clove oil 2-4 2-4 2-4 2-6 2-6 The maximum MFC values obtained in presence of Ashoke, Elachi, Sesame, Dhania, Soyabean Bavchi oil 2-6 2-6 2-6 2-62 2-6 and Jaifal essential oil against Saccharomyces cerivisiaeis i.e. 0.25µl/ml and minimum MFC Maulsri oil 2-6 2-6 2-6 2-6 2-6 Almond oil 2-6 2-8 2-8 2-8 2-8 value was obtained in presence of Saunf ,Orange and Maulsri against Saccharomyces cerivisiae Neem oil 2-5 2-8 2-8 2-8 2-8 i.e. 0.10 µl/ml,which are lower than the anti fungal drug Griseofulvin i.e. 55.5µg/ml Jaifal oil 2-4 2-4 2-4 2-4 2-4 concentration (Table 1). Soyabean oil 2-5 2-6 2-6 2-6 2-6 Control 12-24 12-34 14-44 14-48 14-57 Table 1. MIC and MFC values in presence of various essential oils in Yeast culture (Saccharo- myces cervisiae) Similarly mycelial growth was also noted in Aspergillus flavus, Again almist 80% inhibition in mycelial size was obtained in tests in comparision to control. Essential oil MIC* MFC* Table 4. Effect of essential oils on mycelial growth of Rhizopus stolonifer in mm Asoke oil 0.012 0.250 Saunf oil 0.050 0.100 Elachi oil 0.050 0.250 Essential oil 24 hrs 48 hrs 72 hrs 96 hrs 120 hrs Sesame oil 0.012 0.250 Sandal wood oil 0.012 0.250 Asoke oil 2-4 2-6 2-6 2-6 2-6 Dhania oil 0.050 0.100 Saunf oil 1-2 2-4 2-4 2-5 2-5 Olive oil 0.012 0.250 Elachi oil 2-4 2-4 2-6 2-6 2-6 Orange oil 0.050 0.100 Sesame oil 2-4 2-4 2-4 2-6 2-6 Ajwaine oil 0.012 0.250 Sandal wood oil 2-4 2-5 2-5 2-5 2-5 Clove oil 0.050 0.250 Dhania oil 1-3 2-5 2-5 2-5 2-6 Bavchi oil 0.012 0.250 Olive oil 1-3 1-3 1-3 2-5 2-5 Maulsri oil 0.050 0.100 Orange oil 1-2 1-2 2-4 2-4 2-4 Almond oil 0.050 0.250 Ajwaine oil 1-2 1-2 2-4 2-4 2-4 Neem oil 0.012 0.250 Clove oil 1-2 2-5 2-5 2-5 2-5 Jaifal oil 0.012 0.250 Bavchi oil 1-2 2-5 2-6 2-6 2-6 Soyabean oil 0.050 0.250 Maulsri oil 1-2 2-6 2-6 2-6 2-6 Almond oil 2-4 2-4 2-5 2-5 2-5 Graceofulvin Neem oil 2-4 2-5 2-5 2-5 2-5 MIC-Minimum Inhibitory Concentration Jaifal oil 2-4 2-4 2-6 2-6 2-6 MFC-Minimum Fungicidal Concentration Soyabean oil 1-3 1-3 2-4 2-5 2-5 Control 12-25 13-33 14-48 14-51 14-55 DETERMINATION OF INHIBITION ZONE DIAMETER *For each treatment 2µl of each essential oil was coated on agar cube of (1 mm3 size),*Mycelial Antifungal activity was evaluated by agar disc diffusion method. For testing antifungal activity growth was measured in mm each oil was coated separately on filter paper discs ( Whatmann no.1) of 6mm in size and kept o in the center of agar coated plate. After 24 hrs of incubation at 37 C size of inhibition zone EFECT ON BIOMOLECULAR PARAMETERS diameter was measured . The inhibition zone diameter obtained at 2,4,8,16 and 32µl concen- Effect of essential oils was also measured in yeast (Saccharomyces cerevisiae) cultures after tration i.e. 6mm, 9mm, 18mm, 29mm and 32mm in presence of Ashoke oil against treating them with variable dose of essential oils separately.Effects were meausered in form of Saccharomyces cerivisiae .The inhibition zone diameter was found to be increased with alteration occurred in DNA, RNA and Protein levels. In all the cases level of biomolecules increasing concentration of essential oils. However, maximum inhibition zone diameter was obtained was much lower than the standard antifungal drug Griseofulvine (Figure 1A, B, C). obtained in presence of Ashoke oil, i.e. 32mm at 32µl concentration against Saccharomyces cerevisiae and minimum inhibition zone diameter was obtained in presence of Olive oil i.e. 110 20mm .While in presence of other essential oils on an average 20-32mm inhibition zone 100 diameter was obtained which is much higher than the that of standard antifungal drug Griseofulvin at 320 µg concentration (Table 2). Griseofulvin showed the inhibition zone 90 diameter i.e. 6 mm, 9 mm, 13 mm,14 mm,and 16 mm at 20, 40, 80, 16 and 320 µg 80 concentration (Table 2). 70 60 Table 2. Inhibition zone diameters obtained in presence of different essential oils in agar disc diffusion method in Yeast culture (Saccharomyces cervisiae) ( mm) 50 40 Essential oil 2µl 4µl 8µl 16µl 32µl 30 Asoke oil 6 9 18 29 32 Saunf oil 6 13 16 20 27 20 Per cent DNA evel Elachi oil 10 18 22 25 30 10 Sesame oil 9 14 18 25 31 Sandal wood 8 18 18 24 31 0 Dhania oil 8 13 18 21 27 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Gf Olive oil 8 11 16 18 20 A Orange oil 7 13 16 17 23 Essential oils and Antibiotics 110 Ajwaine oil 8 15 17 21 25 Clove oil 8 12 16 22 29 100 Bavchi oil 8 13 16 19 24 90 Maulsri oil 7 9 15 21 27 Almond oil 7 11 15 17 23 80 Neem oil 6 8 14 17 21 70 Jaifal oil 9 14 18 23 30 Soyabean oil 6 7 11 15 29 60 Control Nill Nill Nill Nill Nill 50 Graceofulvin 6 9 13 14 16 40 *The strength of activity is presented as resistant (> 7mm), Intermediate ( > 12mm ), and Suscep- 30 tible ( > 18 mm).*In negative control no antifungal used, *In positive control antifungal are used 20 for compariso Per cent RNA level 10 EFFECT ON MYCELIAL GROWTH 0 For observation of effect on mycelial growth 2µl of each and every essential oil was coated on C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Gf agar cubes separately and growth was recorded at 24 hrs up to 12 hrs. Almost all 16 different essential oils have significantly inhibited the mycelial growth in E-test. However, on an B Essential oils and Antibiotics Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1153-1156 Ravi Kant Upadhyay et al. / Journal of Pharmacy Research 2011,4(4),1153-1156 110 High antifungal susceptibility is also proved by higher inhibition zone diameters obtained in 100 presence of each essential oil in comparison to antifungal drug. Maximum inhibition zone diameter was obtained i.e. 54 mm in presence of Ashoke oil against Saccharomyces cerevisiae 90 at 32µl concentration. It was much higher than that of standard antifungal drug Griseofulvin 80 i.e. 33 mm at 320 µg concentration (Table 2). 70 All essential oils have significantly (p<0.05) altered the level of different molecules found in 60 Saccharomyces cerevisiae. There was observed a significant decrease in DNA level in presence 50 of each essential oil in yeast cells (Figure 1A).Essential oils have shown higher reduction in 40 DNA level in comparison of Griseofulvin.The maximum reduction was obtained in Dhania 30 oil i.e.74.58% at 32µl concentration and minimum reduction i.e. 48.93% in presence of Neem oil at the same concentration. Similarly antifungal activity was also reported in ethane extract 20 of root Leuzea carthamoides (Asteraceae) against C. albicans, A.fumigatus (Chobot et al., 10 Per cent Protein level 2003) and Centaurea hermanni (Asteraceae) ( Sur- Altiner et al., 1997). Similarly, Clove and 0 Cinnamon oil have shown antifungal activity (Duke et al, 1985) against fungi. However, C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Gf Griseofulvin showed 33.01% reduction in DNA level at 320µg concentration, which was C Essential oils and Antibiotics found to be lower than the essential oils (Figure 1A).

Figure 1. . Comparison of DNA, RNA and protein levels in Saccharomyces cerevisiae treated The maximum reduction in RNA i.e. 91.78% in presence of Ajwaine oil and minimum with minimum concentration (2µl) of various essential oils i.e. (1)Ashoka (2)Saunf (3)Elachi reduction i.e.78.50% in presence of Ashoka essential oil at 32 µl concentration (Figure 1B). (4)Sesame (5)Sandalwood (6) Dhania (7) Olive (8)Orange (9)Ajwaine (10) Clove (11) Bavchi In antifungal drug 79.43% reduction obtained at 320 µg concentration. (Figure ). (12)Maulsri (13)Almond (14)Neem (15)Jaifal (16) Soyabean essential oil and antifungal drug Griseofulvin (Gf-20µg) (A) DNA level (B) RNA level (C) Protein level The maximum reduction in protein level was obtained 90.81% in presence of Olive oil in Saccharomyces cerevisiae at 32 µl concentration. While, Griseofulvin showed 74.67% reduc- EFFECT ON DNA LEVEL tion in protein level in Saccharomyces cerevisiae at 320 µg concentration (Table 5; Figure Significant decrease was observed in DNA level in presence of each essential oil in fungal 1C). Comparison of DNA, RNA and Protein levels in Saccharomyces cerevisiae have shown cultures (Table 3). Ashoka, Saunf, Elachi and Sesame essential oils have shown 58.99%, in Figure 1A, 2B and 3C. 48.93%, 54.92% and 58.99% reduction at 32µl concentration in Saccharomyces cerevisiae. The maximum reduction was obtained in Dhania oil i.e.74.58% at 32µl concentration and Besides this, antifungal susceptibility was determined in Aspergillus flavus and Rhizopus minimum reduction i.e. 48.93% in presence of Neem oil at the same concentration (Table 3). stolonifer. For which both mycelial growth and spore counting was performed. Again their Reduction in DNA level obtained in presence of antifungal drug i.e. Griseofulvin is given in was obtained significantly lesser mycelial growth in Aspergillus flavus and Rhizopus stolonifer (Table 3) i.e. 17.98%, 22.95%, 26.01%, 27.92% and 33,01% at 20 µg, 40 µg, 80 µg, 160 in presence of each essential oil in comparison to control and Griseofulvin (Table 4 and 5). µg and 320 µg concentration (Figure 1A). All these inhibitory effects suggest susceptibility of essential oils to the fungal cells. However, EFFECT ON RNA LEVEL susceptibility may be to distruction of microbial cells in cultivation and inhibition of meta- The reduction obtained in presence of Ashoka, Saunf, Elachi and Sesame essential oil i.e. bolic activity (Rusenova and Paranov, 2009). However, few growth inhibitors like azole 78.50%, 83.68%, 89.17% and 87.99% at 32µl concentration in Saccharomyces cerevisiae. antifungal drugs inhibit the cell growth by distruction of sterol biosynthesis (Kelly et al, Similarly Maulsri, Almond, Neem and Jaifal essential oils have shown 85.91%, 86.01%, 1995). Essential oils cause conformational changes in the bacterial membrane structure and 85.79% and 87.86% reduction at same concentration. The maximum reduction was obtained result in fludification of cells (Griffin et al, 1999). Essential oils inhibit the function of free i.e. 91.78% in presence of Ajwaine oil while minimum reduction i.e.78.50% in presence of radicals and peroxidation of membrane lipids and stimulate the activity of anti-oxidant Ashoka essential oil at 32 µl concentration. In presence of antifungal drug 79.43% reduction enzymes (Gutierrez et al, 2003). However, the main antimicrobial mechanism appears to be was obtained at 320 µg concentration. (Figure 1B). related to its capacity to interact with the sulpha hydryl group (-SH) of other active compounds (Reuter et al., 1996). In fact garlic oil alone more powerful than its main components and it EFFECT ON PROTEIN LEVEL also shows synergy among the different compounds (Reuter et al., 1996; Gara et al., 2000; Protein level was found to be decreased i.e. 85.70%, 84.68%, 86.36% and 89.09% in presence Ross et al., 2001).Besides this, normally few growth inhibitors like azole antifungal drugs of Ashoka, Elachi, Sesame and Sandal wood essential oil at 32 µl concentration in Saccharo- inhibit the cell growth by disruption of sterol biosynthesis (Kelly et al, 1995). Both hydrocarbans myces cerevisiae seperetly.The maximum reduction in protein level was obtained i.e. 90.81% and phenolic compounds such as the Thymol and Carvacrol posses very strong antimicrobial in presence of Olive essential oil at 32 µl concentration and while minimum reduction i.e. activity in which the hydroxyl group allows the entry of water molecules through membrane 80.02 % in presence of Orange peel essential. In presence of antifungal drug Griseofulvin that result in to the death of microbes (Griffin et al., 1999; Davidson and Naidu, 2000 ; 74.67% reduction was obtained at 320µg concentration (Figure 1C). Dorman and Deans, 2000 ; Cox et al., 2001) but few essential oil components such as terpenes, terpenoids and phenyl propanoids show action against bacteria and disrupt the cell DISCUSSION membrane by lipophilic action (Griffin et al, 1999 ; Sangwan et al, 2001 ; Davidson and Naidu Essential oils are highly useful natural products which are easily available and possess wide 2000 ; Dorman and Deans 2005). Similarly tannins show broad spectrum antimicrobial spectrum antimicrobial potential against number of infectious agents. These are considered as activity and inhibit the growth of microbes by enzyme inhibition, oxidative phosphorylation blends of secondary metabolites which are obtained from the plant volatile fraction by steam reduction and iron deprivation (Parekh and Chanda, 2007) while thymols show antimicrobial distillation (Gershenzon and Croteau, 1991). Essential oils obtained from the leaves of so activity due to their phenolic character, which causes membrane-disruption (Helander et al, many plant parts have shown inhibitory activity against bacterial, fungal and viral infection. 1998). In general phenols interacts with protein through hydrogen bridges and ionic or During life time plants achieve antifungal resistance and use it as their own defense from fungal hydrophobic interactions (Prescott, 2004), whereas non-phenolic compounds interact through pathogens (Wojtaszek, 1997). In the present investigation various concentrations of the carbonyl group like Cinnamaldehyde (Ouattara et al., 1997). Other aldehyde compounds may essential oils exhibited significant antifungal effects in vitro against S. cerevisiae, Rhizopus also interact with nucleic acids and proteins, resulting in their inactivation by forming cross stolonifer and Aspergillus flavus. In suspension culture all the essential oils have shown bridges or by alkylation (Prescott et al., 2004). Besides this essential oils have shown anti significantly higher growth inhibition than the antifungal drug Griseofulvin. On an average convalescent (Viana et al, 2000), antiseptic activity (Burt, 2004) acute and sub-chronic toxicity essential oils have shown 25-70% growth inhibition while it was 21-33% in case of (Orafidiya et al, 2004). Griseofulvin(Table 1). Maximum growth inhibition was obtained in presence of Olive oil i.e. 70%. In the present investigation all 16 essential oils exhibited significant antifungal activity against Rhizopous stolonifer, A. niger and Saccharomyces cerevisiae in vitro. Essential oils have Besides this, all different essential oils have shown very low MIC and MFC values against shown very low MIC and MFC values. Growth reduction, which was found to be dose Saccharomyces cerivisiae than antifungal drug Griseofulvin (Table 2) which suggests that dependent. It was increased with increasing concentration of each essential oil. It proves the essential oils possess some active components which might have very high susceptibility to susceptibility of essential oils against fungal stain which may be due to presence of important fungal cells. MIC vales obtained were very low i.e. 0.012-0.050 µg/ml (Table. 1). Again it antifungal constituents in the essential oils.These growth inhibitory effects may be useful for was found to be lesser than the antifungal drugs i.e. 0.100-0.250 µg/ml. Very low MIC values subsiding the fungal infection. were obtained in presence of Saunf, Dhania, Orange, Clove, Maulsri, Ashoka, Sandalwood and Sesame were found i.e. 0.75-1.83 µl/ml against Saccharomyces cerevesiae (Table 1). Simi- ACKNOLEDGEMENT larly the hexane extract of Sphaeranthus indicus showed MIC as 2.5 and 0.625 mg/ml against Authors are highly thankful to University Grants Commission, New Delhi, for providing A. niger and Botrytis cinerea (Duraipandiyan et al, 2009). Similarly the antifungal activity financial assistance. against Trichophyton spp, Epidermophyton floccosum and Microsporum cooki were reported in thiophene compound isolated from Tagetes patula (Asteracee) (Romagnoli et al., 1998). REFERENCES Similarly, essential oils isolated from the leaves of Eucalyptus species have shown antifungal 1. Abdelrahim SI, Almagboul AZ, Omer ME, Elegami A. “Antimicrobial activity of Psidium guajava activity (Assarch et al., 2007). Similarly guava leaf extract inhibited the growth of Streptococ- L.”, Fitoterapia, 73, 2002, 713-715. cus. aureus in disk diffusion method (Adelrahim et al, 2002). Similar antimicrobial activity 2. 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