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Food and Chemical Toxicology 47 (2009) 26062612

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Food and Chemical Toxicology


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Antioxidant and antidermatophytic activities of essential oil and extracts of Magnolia liliora Desr.
Vivek K. Bajpai, Jung In Yoon, Sun Chul Kang *
Department of Biotechnology, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t
This study was carried out to assess the antioxidant and antidermatophytic activities of the essential oil and extracts of Magnolia liliora Desr. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC50 values = 10.11 and 16.17 lg/ml, respectively) as compared to butylatedhydreoxyanisole (BHA), (IC50 value = 18.27 lg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (96.13 mg/g of dry wt) as compared to the other extracts. Further, the oil (1000 lg/disc) and extracts (1500 lg/disc) revealed 42.3663.12% and 19.0754.14% antidermatophytic effect, respectively along with their respective MIC values ranging from 62.5 to 500 and 250 to 2000 lg/ml against the members of Trichophyton and Microsporum spp. Also the oil had strong detrimental effect on spore germination of tested fungal pathogens as well as concentration and time dependent kinetic inhibition of Microsporum canis KCTC 6348. The results of this study justify a potential role of M. liliora to serve as a natural antioxidant and antidermatophytic agent. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 23 June 2009 Accepted 21 July 2009

Keywords: Magnolia liliora Antidermatophytic activity Antioxidant activity Essential oil Phenolic contents

1. Introduction Oxidation is essential to many living organisms for the production of energy to fuel biological processes. However, oxygen-centered free radicals and other reactive oxygen species (ROS), which are continuously, produced in vivo, result in cell death and tissue damage. The role of oxygen radicals has been implicated in several diseases, including cancer, diabetes and cardiovascular diseases, ageing, etc. (Halliwell and Gutteridge, 1999). Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress (Ozsoy et al., 2008). There is an increasing interest in natural antioxidants, e.g., polyphenols, present in medicinal and dietary plants, which might help prevent oxidative damage (Silva et al., 2005). Human body has multiple mechanisms especially enzymatic and non-enzymatic antioxidant systems to protect the cellular molecules against reactive oxygen species (ROS) induced damage (Anderson, 1999). However the innate defense may not be enough for severe or continued oxidative stress. Hence, certain amounts of exogenous antioxidants are constantly required to maintain an adequate level of antioxidants in order to balance the ROS in human body. There is widespread agreement that some synthetic

* Corresponding author. Address: Department of Biotechnology, College of Engineering, Daegu University, Kyoungsan, Kyoungbook 712-714, Republic of Korea. Tel.: +82 53 850 6553; fax: +82 53 850 6559. E-mail address: sckang@daegu.ac.kr (S.C. Kang). 0278-6915/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.025

antioxidants such as butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) need to be replaced with natural antioxidants due to their potential health risks and toxicity (Safer and Al-Nughamish, 1999). Hence, compounds especially from natural sources capable of protecting against ROS mediated damage may have potential applications as antioxidants in prevention and/or curing of diseases. The presence of phenolic compounds (phenolic acids, polyphenols and avonoids) in plants, herbs and spices, is gaining increasing attention because of their various functions, such as antioxidant activity and health benets (Sacchetti et al., 2005). Consumption of natural aromatic plant extracts rich in phenolics is expected to prevent the risk of many free radical-mediated diseases because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of foods (Young and Woodside, 2001). The incidence of dermatophytic infections has increased considerably during the past several decades (Jessup et al., 2000). Dermatophytes are responsible for serious human pathogenic disorders in various parts of the world and, although control measures are available, they are of limited effectiveness. Conventional antifungal agents such as chlorhexidine and imidazole derivatives have limited uses in the pregnant and the young due to their common side effects such as hepatotoxicity, nausea, diarrhea and impotency (Curtis, 1998). As it becomes necessary to identify and develop novel antimicrobial agents, a number of plant essential oils and extracts have

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been investigated extensively to achieve higher levels of human safety standards (Baker et al., 1989; Willocks et al., 1991). The screening of such natural products should offer potential resources since their use is widespread with much of the research population relying on them. Magnolia liliora Desr (Magnoliaceae) is a 34 m deciduous shrub propagated and distributed in many parts of East Asia and North America. It has been reported that M. liliora has the benecial effects on several ailments such as analgesic, anodyne, carminative, febrifuge, sedative, and tonic (Duke and Ayensu, 1985). Previously, we reported the chemical composition and antibacterial properties of the essential oil and various organic extracts of M. liliora against foodborne and spoilage bacteria (Bajpai et al., 2008). In the present investigation, we assessed the antioxidant activity of the essential oil and various extracts of M. liliora as well their antidermatophytic potential against a panel of skin infectious fungal pathogens.
2. Materials and methods 2.1. Chemicals and reagents Gallic acid, L-ascorbic acid (AA), FolinCiocalteaus phenol reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and butylated hydroxyanisole (BHA) were obtained from SigmaAldrich (St. Louis, MO). All other chemicals and solvents were of highest commercial grade. 2.2. Fungal pathogens The human skin infectious fungal pathogens used in this study were Trichophyton rubrum KCTC 6345, Trichophyton rubrum KCTC 6375, Trichophyton rubrum KCTC 6352, Trichophyton mentagrophytes KCTC 6085, Trichophyton mentagrophytes KCTC 6077, Trichophyton mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349, which were obtained from Korean Agricultural Culture Collection (KACC), Suwon, Republic of Korea. All the strains were maintained on SBA (Sabourauds agar, Difco) at 4 C. 2.3. Preparation of the spore suspension and test sample The fungi were grown on sabourauds agar (SBA) plates in dark at 28 2 C for 79 days, after which time spores were harvested from sporulating colonies and suspended in sterile distilled water containing 0.1% (v/v) Tween 20. The concentrations of spores in suspension were determined using a hematocytometer and adjusted to 1.0 108 spores/ml. To prepare the stock solutions, essential oil was dissolved in 5% dichloromethane, whereas extracts were dissolved in their respective 5% solvents (n-hexane, chloroform, ethyl acetate, and methanol), which were further diluted to prepare test samples, where the nal concentration of the solvent was 0.5% (v/v). 2.4. Antioxidant activity assay

2.6. Antidermatophytic activity assay The atnidermatophytic activity of essential oil and the extracts (n-hexane, chloroform, ethyl acetate and methanol) was assessed by disc diffusion method using sabourauds agar (SBA) in 9 cm petri dishes (Duru et al., 2003). Sterile Whatman paper discs of 6 mm diameter were pierced in the agar, equidistant, and near the border, where the essential oil (1000 lg/disc) and the extracts of n-hexane, chloroform, ethyl acetate and methanol (1500 lg/disc) were used separately. Controls were prepared in the same solvent employed to dissolve the samples. An agar plug of fungal inoculum (6 mm in diameter) was removed from a 10-day old previous cultures of all the fungal pathogens tested and placed upside down in the center of the petri dishes. The plates were incubated at 28 2 C for 79 days, until the growth in the control plates reaches the edges of the plates. The plates were used in triplicate for each treatment. The relative growth inhibition of treatment compared to control was calculated by percentage, using the following formula: Inhibition (%) = {1 radial growth of treatment (mm)/radial growth of control (mm)} 100. 2.7. Antidermatophytic susceptibility assay The susceptibility of dermatophytes was determined by minimum inhibitory concentration determination method (Murray et al., 1995). The minimum inhibitory concentrations (MICs) of essential oil and the extracts (n-hexane, chloroform, ethyl acetate and methanol) were determined by twofold serial dilution against T. rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, M. canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349. Four micro-liter samples from essential oil and extracts were dissolved in the same solvent employed to isolate and extract the essential oil and organic extracts, respectively. These solutions were serially diluted with their respective 5% solvent and were added to sabourauds broth (SDB) at nal concentrations of 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 lg/ml, respectively. A 10 ll spore suspension (1.0 108 spores/ml) of each test pathogens was inoculated in the test tubes in SDB medium and incubated at 28 2 C for 27 days. The control tubes containing SDB medium were inoculated only with fungal spore suspension. The minimum concentrations at which no visible growth was observed were dened as the MICs, which were expressed in lg/ml. 2.8. Spore germination assay For spore germination assay of T. rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, M. canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349 (Leelasuphakul et al., 2008), test samples of oil (4 ll) were dissolved in 5% dichloromethane to obtain 31.25, 62.5, 125, 250, 500, 1000, 2000 and 4000 lg/ml concentrations of the oil, where the nal concentration of the solvent was 0.5%. The samples were inoculated with spore suspension of each fungal pathogen containing 1.0 108 spores/ml. From this, aliquots of 10 ll spore suspension from each were placed on separate glass slides in triplicate. Slides containing the spores were incubated in a moisture chamber at 28 C for 4 h. Each slide was then xed in lactophenol-cotton blue and observed under the microscope for spore germination. The spores generated germ tubes were enumerated and percentage of spore germination was calculated. The control (0.5%) dichloromethane was tested separately for spore germination of different fungi. 2.9. Growth kinetics assay

The antioxidant activity of essential oil and the extracts (n-hexane, chloroform, ethyl acetate and methanol) of M. liliora was measured on the basis of the scavenging activities of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (Archana et al., 2005). IC50 values (concentration of sample required to scavenge 50% of free radicals) were calculated from the regression equation, prepared from the concentration of the essential oil and organic extracts, and percentage inhibition of free radical formation/ percentage inhibition DPPH was assayed. Synthetic antioxidant reagents, butylatedhydroxyanisole (BHA) and L-ascorbic acid were used as positive controls and all tests were carried out in triplicate. 2.5. Determination of total phenolic contents The amount of total phenolics was determined with the FolinCiocalteu reagent (Lister and Wilson, 2001). First a standard curve was plotted using gallic acid as a standard. Different concentrations of samples were prepared in 80% of methanol. Hundred microliter of sample was dissolved in 500 ll (1/10 dilution) of the FolinCiocalteu reagent and 1000 ll of distilled water. The solutions were mixed and incubated at room temperature for 1 min. After 1 min, 1500 ll of 20% sodium carbonate solution was added. The nal mixture was shaken and then incubated for 2 h in the dark at room temperature. The absorbance of samples was measured at 760 nm and the results were expressed in mg of gallic acid/g (GAE) of dry weight of samples.

M. canis KCTC 6348, which appeared to be more resistant fungus as compared to the T. mentagrophytes KCTC 6077 and M. canis KCTC 6591 to the essential oil in spore germination study, was chosen as test fungus for kinetic study and evaluation of antidermatophytic activity of essential oil (Rana et al., 1997). Ten microliter spore suspension (1.0 108 spores/ml) of this fungal pathogen was inoculated to different concentrations of essential oil (31.25, 62.5, 125 and 250 lg/ml) in a test tube and a homogenous suspension was made by inverting the test tubes 34 times. After the specic intervals of 30, 60, 90, 120, 150 and 180 min, the reaction mixture was ltered through Whatman No. 1 lter paper and the retained spores were washed two or three times with sterile distilled water. The lter was then removed and spores were washed off into 10 ml of sterile distilled water. From this 100 ll of spore suspension was taken onto the glass slide and incubated at 28 2 C for 24 h. The spores generated germ tubes were enumerated and percentage of spore germination was calculated. Control sets were prepared in 0.5% dichloromethane with sterile distilled water. All experiments were conducted in triplicate. 2.10. Statistical analysis All data were expressed as means standard errors of triplicate measurements. Correlation between antioxidant activity and total phenolic content was carried out using the correlation and regression in the Excel program. Differences were considered signicant at p < 0.05.

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3. Results 3.1. Antioxidant activity The DPPH free radical scavenging activity of essential oil and the extracts (n-hexane, chloroform, ethyl acetate and methanol) of M. liliora has been shown in Fig. 1. The IC50 values of essential oil and the extracts were compared with the standards ascorbic acid (AA = 6.55 lg/ml) and butylated hydroxyanisole (BHA = 18.27 lg/ml). A lower IC50 value indicates a greater antioxidant activity. The IC50 values of essential oil and various extracts were recorded in the range of 10.11178.10 lg/ml. The free radical scavenging activities of essential oil and ethyl acetate extract (IC50 = 10.11 and 16.17 lg/ml) were found superior to all other extracts. However, low polar extracts exhibited low DPPH scavenging activity. 3.2. Total phenolic contents The amount of total phenolic contents of the extracts (n-hexane, chloroform, ethyl acetate and methanol) of M. liliora was tested, and occurred in the range of 13.4196.13 mg GAE/g of dry sample (Fig. 2). The total phenolic contents of various extracts of methanol, ethyl acetate, chloroform and hexane were noted to be 49.27, 96.13, 39.32 and 13.41 mg GAE/g of dry sample, respectively. The results showed that the total phenolic contents in ethyl acetate extract (96.13 mg GAE/g of dry sample) were the highest as compared to the other extracts. 3.3. Antidermatophytic activity The oil of M. liliora exhibited a moderate to high antidermatophytic activity against the tested fungal pathogens. As shown in Table 1, essential oil (1000 lg/disc) showed potent inhibitory ef-

fect on the growth of T. rubrum KCTC 6345 (53.39%), T. rubrum KCTC 6375 (52.15%), T. rubrum KCTC 6352 (56.12%), T. mentagrophytes KCTC 6077 (62.63%), T. mentagrophytes KCTC 6316 (63.12%), M. canis KCTC 6591 (59.33%) and M. canis KCTC 6348 (62.19%). T. mentagrophytes KCTC 6085 and M. canis KCTC 6349 displayed less susceptibility to essential oil and exerted 44.43% and 42.36% of antidermatophytic effect as fungal mycelial growth inhibition percentage, respectively. T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316 and M. canis KCTC 6348 were found to be the most inhibited fungal pathogens by the essential oil. Besides, methanol extract (1500 lg/disc) also exhibited strong antidermatophytic effect as fungal mycelial growth inhibition (36.1251.17%) against all the fungal pathogens tested (Table 2). Also ethyl acetate and chloroform extracts exerted signicant antidermatophytic effect against all the pathogens tested with their respective fungal mycelial growth inhibition percentage of 38.2754.143% and 32.0147.23%. However, low to moderate antidermatophytic effect of hexane extract was observed against the tested fungal pathogens with fungal mycelial growth inhibition percentage of 19.0730.12% (Table 2). 3.4. Minimum inhibitory concentrations (MICs) According to the results given in the Table 1, the minimum inhibitory concentrations (MICs) dened as the lowest concentrations of the essential oil of M. liliora that resulted in complete growth inhibition of T. rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, M. canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349 were found to be 125, 125, 125, 500, 62.5, 125, 62.5, 62.5 and 500 lg/ml, respectively. T. mentagrophytes KCTC 6077, M. canis KCTC 6591, M. canis KCTC 6348, T. mentagrophytes KCTC 6316, T. rubrum KCTC 6345, T. rubrum KCTC 6375 and T. rubrum KCTC 6352 were found to be

Fig. 1. DPPH radical scavenging activity of essential oil and various organic extracts of Magnolia liliora Desr. EO: essential oil, HXE: hexane extract, CHE: chloroform extract, ETE: ethyl acetate extract, MNE: methanol extract, AA: ascorbic acid (control), BHA: butylated hydroxyanisole (control). Data are represented as the mean S.D. of triplicate experiments.

Fig. 2. The amount of total phenolic content (mg/g dry weight) in various organic extracts of Magnolia liliora Desr.

V.K. Bajpai et al. / Food and Chemical Toxicology 47 (2009) 26062612 Table 1 Antidermatophytic activity of essential oil of Magnolia liliora against skin infectious fungal pathogens. Fungal pathogen Essential oila Radial growth (mm) Trichophyton Trichophyton Trichophyton Trichophyton Trichophyton Trichophyton Microsporum Microsporum Microsporum rubrum KCTC 6345 rubrum KCTC 6375 rubrum KCTC 6352 mentagrophytes KCTC 6085 mentagrophytes KCTC 6077 mentagrophytes KCTC 6316 canis KCTC 6591 canis KCTC 6348 canis KCTC 6349 21.5 1.51 22.13 0.34 20.12 1.33 25.49 0.57 17.60 1.20 17.12 0.43 18.61 0.59 17.52 0.47 26.47 0.57
b

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MICd Radial growth inhibition (%) 53.39 1.23a 52.15 1.15a 56.12 1.37ab 44.43 1.11c 62.63 1.51ab 63.12 1.13ab 59.33 1.35ab 62.19 1.17ab 42.36 1.15c
c

125 125 125 500 62.5 125 62.5 62.5 500

Values are given as mean S.D. of triplicate experiment and considered to be signicantly different at p < 0.05. a Essential oil (tested volume 1000 lg/disc). b Radial growth of fungal pathogens. c Radial growth inhibition percentage. d Minimum inhibitory concentration (values in lg/ml).

Table 2 Antidermatophytic activity of (1500 lg/disc) of various organic extracts of Magnolia liliora against skin infectious fungal pathogens. Fungal pathogen Radial growth inhibition HXEa mm Trichophyton Trichophyton Trichophyton Trichophyton Trichophyton Trichophyton Microsporum Microsporum Microsporum rubrum KCTC 6345 rubrum KCTC 6375 rubrum KCTC 6352 mentagrophytes KCTC 6085 mentagrophytes KCTC 6077 mentagrophytes KCTC 6316 canis KCTC 6591 canis KCTC 6348 canis KCTC 6349
e

CHEb %
f

ETEc % 46.21 2.3a 47.19 2.3ab 32.01 2.7c 33.33 3.1c 39.14 3.1c 43.13 2.9bc 41.19 2.1c 47.15 2.3ab 47.23 2.7ab mm 24.11 1.5 21.20 1.3 26.56 1.2 28.12 1.7 27.31 1.7 22.52 1.8 22.56 1.3 21.55 1.7 22.49 1.9 % 47.15 2.2a 54.14 1.9a 41.22 2.3c 38.27 2.2c 39.23 2.4c 51.25 2.5ab 51.66 2.6ab 53.12 2.1ab 51.32 2.4ab

MNEd mm 22.95 1.5 26.71 1.3 27.61 1.3 29.12 1.6 26.11 1.8 23.51 1.3 24.02 1.3 23.04 1.4 22.85 1.5 % 49.13 2.8a 42.12 2.3c 39.16 2.5c 36.12 2.2c 42.23 2.5c 51.17 2.5ab 47.26 2.lab 49.01 2.2b 48.21 2.6bc

mm 24.21 1.3 24.52 1.2 31.01 1.5 30.52 1.4 27.56 1.8 26.12 1.7 26.92 1.5 24.12 1.4 24.22 1.6

33.14 1.1 32.25 1.2 35.52 1.7 33.23 1.3 33.59 1.6 33.26 1.2 36.56 1.3 33.82 1.4 32.11 1.5

27.12 1.7a 29.32 1.3ab 21.13 2.7c 27.13 2.7ac 26.23 2.6ac 27.16 2.8ac 19.07 2.3c 22.13 1.8c 30.12 2.8ab

Values are given as mean S.D. of triplicate experiment and considered to be signicantly different at p < 0.05. a HXE, hexane extract. b CHE, chloroform extract. c ETE, ethyl acetate extract. d MNE, methanol extract. e Radial growth of fungal pathogens. f Percentage of radial growth inhibition.

Table 3 Determination of minimum inhibitory concentration (MIC) of various organic extracts of M. liliora against skin infectious fungal pathogens. Fungal pathogen MICa HXEb Trichophyton Trichophyton Trichophyton Trichophyton Trichophyton Trichophyton Microsporum Microsporum Microsporum rubrum KCTC 6345 rubrum KCTC 6375 rubrum KCTC 6352 mentagrophytes KCTC 6085 mentagrophytes KCTC 6077 mentagrophytes KCTC 6316 canis KCTC 6591 canis KCTC 6348 canis KCTC 6349 1000 1000 2000 1000 1000 2000 1000 1000 2000 CHEc 500 500 1000 1000 500 1000 1000 500 1000 ETEd 500 250 500 500 250 500 250 250 500 MNEe 500 500 1000 500 250 250 500 500 1000

gens, with their respective MIC values ranging from 250 to 1000, 250 to 500 and 500 to 2000 lg/ml (Table 3). As control, the solvent did not effect the growth of the sample strains at the concentration used in this study. However, low to moderate antidermatphytic effect (10002000 lg/ml) of hexane extract was observed against the tested fungal pathogens as a minimum inhibitory concentration. 3.5. Spore germination The results obtained for essential oil from the spore germination assay of each of the test fungi are shown in Fig. 3. Dichloromethane (0.5%, v/v) as a control did not inhibit the spore germination of any of the fungal pathogens tested. The essential oil signicantly inhibited the fungal spore germination at the varied concentrations. A 100% inhibition of fungal spore germination was observed in T. mentagrophytes KCTC 6077, M. canis KCTC 6591 and M. canis KCTC 6348 at 250, 250 and 500 lg/ml concentrations of essential oil, respectively. Essential oil also exhibited a potent inhibitory effect on the spore germination of T. rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6316, T. mentagrophytes KCTC 6085 and M. canis KCTC 6349 in the range of 6090% at concentrations ranging from 125 to 1000 lg/ml.

Values are given as mean S.D. of three experiments. a Minimum inhibitory concentration (lg/ml). b HXE, hexane extract. c CHE, chloroform extract. d ETE, ethyl acetate extract. e MNE, methanol extract.

the most susceptible fungal pathogens to the essential oil of M. liliora. Also the methanol, ethyl acetate and chloroform extracts displayed potential effect of antidermatophytic activity as minimum inhibitory concentrations against the tested fungal patho-

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Fig. 3. Effect of different concentrations (lg/ml) of the essential oil of Magnolia liliora Desr. on spore germination of tested fungi.

Fig. 4. Kinetics of inhibition of Microsporum canis KCTC 6348 spores by the essential oil of Magnolia liliora Desr.

3.6. Growth kinetics assay The antidermatophytic kinetics of the essential oil against M. canis KCTC 6348 is shown in Fig. 4. Exposure of M. canis KCTC 6348 spores to different concentrations of the essential oil for a period of 0180 min caused varying degree of inhibition of spore germination. An increase in fungicidal activity was observed with increase in exposure time and concentration. The essential oil at 31.25 and 62.5 lg/ml showed antifungal activity but not rapid killing and about 4050% inhibition was observed at exposure time of 120 min. However, there was a marked increase in the killing rate at 125 and 250 lg/ml after 30 and 60 min of exposure, and 100% inhibition of spore germination was observed on 150 and 180 min exposure, respectively.

4. Discussion The DPPH radical is a free radical, which has been widely used as a tool to estimate free radical scavenging activity of antioxidants. Antioxidants, on interaction with DPPH, either transfer electrons or hydrogen atoms to DPPH, thus neutralizing the free radical character (Archana et al., 2005). In some cases, in the present study, essential oil and ethyl acetate extract showed higher antioxidant activities as compared to the standard BHA and ascorbic acid. This is due to most bioactive compounds such as polyphenols including tannins and avonoid existed in highpolar extracts (Karmanoli, 2002). Polyphenols are one of the major plant compounds with antioxidant activity. The antioxidant activity of phenolic compounds is reported to be mainly due to their redox properties (Galato et al., 2001), which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. Organic extracts may be more benecial than isolated constitu-

ents, because other compounds present in the extracts can change the properties of bioactive individual component (Borchers et al., 2004). Besides, phenolics were also found to be one of the most plentiful classes of constituents in ethyl acetate extract of M. liliora. This is due to the presence of high bioactive compounds in ethyl acetate extract as compared to other non-polar extracts. The key role of phenolic compounds to scavenge free radicals has been emphasized in several reports (Rauha et al., 2000; Archana et al., 2005). The estimated lifetime risk of acquiring a dermatophyte infection is between 10% and 20%. Besides, methods for reducing side effects of treatments include use of natural antifungal agents. Essential oils and extracts have long been known and used throughout the world for treatment of many conditions, including skin conditions, and there is at least some evidence that natural antimicrobials such as essential oil and extracts may tend to have less deleterious side effects than corresponding synthetic drugs (Tavares et al., 2008). In general, plant-derived essential oils and extracts are considered as non-phytotoxic compounds and potentially effective against several microorganisms including many fungal and bacterial pathogens (Pandey et al., 1982; Chung et al., 2007). Since ancient times, interests have been given on the development of safer antifungal agents to control severe fungal diseases by the essential oils and extracts (Chung et al., 2007; Prasad et al., 2004). In brief, the hydrodistillated oil of M. liliora was consisted of oxygenated mono- and sesqueterpenes, and mono- and sesqueterpene hydrocarbons (Bajpai et al., 2008). In recent years, several researchers have reported the mono- and sesquiterpenes, and mono- and sesquiterpene hydrocarbons as the major components of essential oils, which have enormous potential to strongly inhibit microbial pathogens (Gudzic et al., 2002; Cakir et al., 2004). The active antimicrobial compounds of essential oils are terpenes, which are phenolic in nature, it would seem reasonable that their

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antifungal mode of action might be related to that of other compounds. Most of the studies on the mechanism of phenolic compounds have focused on their effects on cellular membranes. Actually, phenolic compounds not only attack cell walls and cell membranes, thereby affecting the permeability and release of intracellular constituents but they also interfere with membrane function. Thus, active phenolic compounds might have several invasive targets which could lead to the inhibition of human infectious fungal pathogens. In our study, the essential oil of M. liliora showed potential antidermatophytic effect against the tested skin fungal pathogens. This research work also describes the complex effect of essential oil on fungal spore germination and exhibited a wide range of antidermatophytic activity. During the kinetic study of M. canis KCTC 6348, it appeared that exposure time of the essential oil had a little effect on the fungicidal activity at lower concentration but at the concentration of 125 and 250 lg/ml, the fungicidal action was very rapid and showed 100% spore germination inhibition of M. canis KCTC 6348. These activities could be attributed to the presence of major components of the oil such as methylcyclopropane, phenyethyl alcohol, levoxine, 2-b-pinene, caryophyllene oxide, b-caryophyllene, a-bourbonene, farnesene, a-terpineol and ahumulene, and these ndings are in agreement with the previous research literature reported by others (Tavares et al., 2008; Chung et al., 2007). Also, the results of the antifungal screening showed that the extracts of methanol, ethyl acetate and chloroform have potential antidermatophytic activity against some of the skin fungal pathogens. This might be attributed to the presence of several bioactive compounds in various extracts of M. liliora as evident by the ndings of others (Urzua et al., 1995; Perry and Foster, 1994). Previous research articles on the analysis and antifungal properties of the essential oils of some species have shown that they had a varying degree of antidermatophytic effects against some of the skin fungal pathogens due to their different chemical compositions (Tavares et al., 2008; Chung et al., 2007). Besides, millions of people throughout the world are affected by supercial fungal infections, which are the most common skin diseases. These infections, which occur in both healthy and immunocompromised persons, are caused mainly by dermatophytes. In this study, it was observed that both oil and extracts of M. liliora have great potential to strongly inhibit the members of the Trichophyton, and Microsporum spp. causing certain supercial fungal infections of the skin known as tinea infections. Certain essential oils, plant extracts and phytochemicals act in many ways on various types of disease complex, and may potentially contribute in the eld of human mycology as the supplement to control human pathogenic fungi in future as the fast and reliable alternatives. The results of this study suggested that M. liliora can be used as a leading factor in a wide range of activities against many dermatophytes, where these pathogens have developed resistance against the specic fungicides (griseofulvin, imidazoles, clotrimazole, and benzimidazoles, biosynthesis inhibitors) (Ghannoum and Rice, 1999). The development of natural antifungal agents would also help to decrease the negative impact of synthetic agents such as residues, resistance, and environmental pollution. In this respect, natural antimicrobials may be effective, selective, biodegradable, and less toxic to the environment.

extracts of M. liliora to evaluate proper action mechanism of the observed activities. Conict of interest statement The authors declare that there are no conicts of interest. Acknowledgement This research was supported by the Daegu University Research Grant, 2008. References
Anderson, D., 1999. Antioxidant defences against reactive oxygen species causing genetic and other damage. Mut. Res. 350, 103108. Archana, B., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chem. 90, 727733. Bajpai, V.K., Rahman, A., Dung, N.T., Huh, M.K., Kang, S.C., 2008. In vitro inhibition of food spoilage and foodborne pathogenic bacteria by essential oil and leaf extracts of Magnolia liliora Desr. J. Food Sci. 73, 314320. Baker, J.H., Goodpasture, H.C., Kuhns, H.R., Rinaldi, M.G., 1989. Fungemia caused by an amphotericin B-resistant isolate of Sporothrix schenckii. Arch. Pathol. Lab. Med. 113, 12791281. Borchers, A.T., Keen, C.L., Gerstiwin, M.E., 2004. Mushrooms, tumors, and immunity: an update. Exp. Biol. Med. 229, 393406. Cakir, A., Kordali, S., Zengin, H., Izumi, S., Hirata, T., 2004. Composition and antifungal activity of essential oils isolated from Hypericum hyssopifolium and Hypericum heterophyllum. Flavour Frag. J. 19 (1), 6268. Chung, P.H., Lee, C.W., Chou, J.Y., Murugan, M., Shieh, B.J., Chen, H.M., 2007. Antifungal activity of crude extracts and essential oil of Moringa olifera Lam. Bioresour. Technol. 98, 232236. Curtis, C., 1998. Use and abuse of topical dermatological therapy in dogs and cats. Part 1. Shampoo Ther. Pract. 20, 244251. Duke, J.A., Ayensu, E.S., 1985. Reference Publications, Medicinal Plants of China Inc., ISBN: 0-917256-20-4. Duru, M.E., Cakir, A., Kordali, S., Zengin, H., Harmandar, M., Izumi, S., Hirata, T., 2003. Chemical composition and antifungal properties of essential oils of three Pistacia species. Fitoterapia 74, 170176. Galato, D., Ckless, K., Susin, M.F., Giacomelli, C., Ribeiro Do Vallle, R.M., Spinelli, A., 2001. Antioxidant capacity of phenolic and related compounds: correlation among electrochemical, visible spectroscopy methods and structureantioxidant activity. Redox Rep. 6, 243250. Ghannoum, M.A., Rice, L.B., 1999. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol. Rev. 12 (4), 501517. Gudzic, B., Djokovic, D., Vajs, V., Palic, R., Stojanovic, G., 2002. Composition and antimicrobial activity of the essential oil of Hypericum maculatum Crantz. Flavour Frag. J. 17 (5), 392394. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, Oxford. Jessup, C.J., Warner, J., Isham, N., Hasan, I., Ghannoum, M.A., 2000. Antifungal susceptibility testing of dermatophytes: establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates. J. Clin. Microbiol. 38 (1), 341344. Karmanoli, K., 2002. Secondary metabolites as allelochemicals in plant defence against microorganisms of the phyllosphere. In: Reigosa, M., Pedrol, N. (Eds.), Allelopathy from Molecules to Ecosystems. Science Publishers Inc., NH, USA, pp. 277288. Leelasuphakul, W., Hemmanee, P., Chuenchitt, S., 2008. Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicillium digitatum Sacc.) of citrus fruit. Postharvest Biol. Technol. 48 (1), 113121. Lister, E., Wilson, P., 2001. Measurement of Total Phenolics and ABTS Assay for Antioxidant Activity (Personal Communication). Crop Research Institute, Lincoln, New Zealand. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolke, R.H., 1995. Manual of Clinical Microbiology, sixth ed. ASM, Washington. Ozsoy, N., Can, A., Yanardag, R., Akev, N., 2008. Antioxidant activity of Smilax excelsa leaf extracts. Food Chem. 110, 571583. Pandey, D.K., Tripathi, N.N., Tripathi, R.D., Dixit, S.N., 1982. Fungitoxic and phytotoxic properties of the essential oil Caesulia axillaris Roxb. Angewa. Bot. 56, 259267. Perry, N.B., Foster, L.M., 1994. Antiviral and antifungal avonoids, plus triterpene from Hebe cupressoides. Planta Med. 60, 491492. Prasad, N.R., Anandi, C., Balasubramanian, S., Pugalendi, K.V., 2004. Antidermatophytic activity of extracts from Psoralea corylifolia (Fabaceae) correlate with the presence of a avonoid compound. J. Ethnopharmacol. 91, 2124. Rana, B.K., Singh, U.P., Taneja, V., 1997. Antifungal activity and kinetics of inhibition by essential oil isolated from leaves of Aegle marmelos. J. Ethanopharmacol. 57, 2934.

5. Conclusion The results of present study conclude that M. liliora derived essential oil and extracts could be used as a source of natural antioxidants as well as potential antifungal agents to control human infectious fungal pathogens. Further studies are required to isolate and characterize the bioactive molecules present in various organic

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V.K. Bajpai et al. / Food and Chemical Toxicology 47 (2009) 26062612 Tavares, A.C., Goncalves, M.J., Cavaleira, C., Cruz, M.T., Lopes, M.C., Canhoto, J., Salgueiro, L.R., 2008. Essential oil of Daucus carota subsp. Halophilus: composition, antifungal activity and cytotoxicity. J. Ethnopharmacol. 119, 129134. Urzua, A., Torres, R., Bueno, C., Mendoza, L., 1995. Flavonoids and diterpenoids in the trichome resinous exudate from Psudeognaphalium cheivanthifolium, P. Heterotrichium and P. vira vira. Biochem. Syst. Ecol. 23, 459. Willocks, L., Leen, C.L.S., Brettle, R.P., Urquart, D., Russel, T.B., Milne, J.R., 1991. Fluconazole resistance in AIDS patients. J. Antimicrob. Chemother. 28, 937939. Young, I.S., Woodside, J.V., 2001. Antioxidants in health and disease. J. Clin. Pathol. 54 (3), 176186.

Rauha, J.P., Remes, S., Heinonen, M., Hopia, A., Kahkonen, M., Kujala, T., Pihlaja, K., Vuorela, H., Vuorela, P., 2000. Antimicrobial effects of Finnish plant extracts containing avonoids and other phenolic compounds. Int. J. Food Microbiol. 56, 312. Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., 2005. Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621632. Safer, A.M., Al-Nughamish, A.J., 1999. Hepatotoxicity induced by the anti-oxidant food additive butylated hydroxytoluene (BTH) in rats: an electron microscopical study. Histol. Histopathol. 14, 391406. Silva, B.A., Ferreres, F., Malva, J.O., Dias, A.C.P., 2005. Phytochemical and antioxidant characterization of Hypericum perforatum alcoholic extracts. Food Chem. 90, 157167.