Antifungal Activities of Commercial Rice Wine Extracts of Taiwanese Allium fistulosum

Antifungal activities of the commercial rice wine extracts of Allium fistulosum were analyzed. Antifungal activities were tested against 7 pathogenic fungi by using agar disc diffusion and tube dilution tests. The results show that the commercial rice wine extracts of Allium fistulosum have strong antifungal activity against Aspergillus brasiliensis ATCC 16404, Candida albicans ATCC 10231, Microsporumcanis ATCC 36299, M. gypseum ATCC 24102, Trichophyton mentagrophytes ATCC 9533, T. rubrum ATCC 28188, and T. tonsurans ATCC 28942. The commercial rice wine extracts of different A. fistulosum parts were found to exhibit significant antifungal activities with the minimal inhibitory concentration (MIC) in the range of 0.2 1.0 mg/mL. The antifungal activity of the extracts of different A. fistulosum parts was in the order of AFS (stem) > AFI (plant body) > AFL (leaf) > AFR (root).


Introduction
Pathogenic fungi often cause nosocomial infection and invade the keratinized tissues of humans and animals * Corresponding author.causing several diseases.Opportunistic fungal infections are difficult to treat in immunocompromised patients, such as transplant patients, AIDS patients, cancer patients, and other immunocompromised hosts; moreover, approximately 40% of systemic infections result in serious consequences, such as death [1] [2].Dermatophytes that grow on skin, mucous membranes, hair, nails, feathers, and other body surfaces cause ringworm and related diseases.A variety of pathogenic fungi, such as Aspergillus sp. and Candida albicans, secrete mycotoxins and cause allergic reactions and localized or systemic infection [1]- [3].Only a limited number of antifungal agents (such as polyenes and azoles) are currently available for the treatment of life-threatening fungal infections in modern medicine; however, very few antifungal agents from natural products could effectively suppressed of pathogenic fungi.
Allium fistulosum (Welsh onion), a member of the Allium family, is rich in fiber and can facilitate digestion, prevent constipation and colon diseases, possesses antioxidant and antimicrobial properties, and exert other effects [4]- [8].In addition, allicin, or chemically known as diallyl thiosulfinate, is the bioactive compound derived from the Allium family and can exert antioxidant and antibacterial activities [9] [10].Allicin has been reported to possess good antifungal properties [1] [11] [12]. A. fistulosum plant extract had a MIC of 140 μl/ml against Fusarium oxysporum f. sp.tulipae, a fungus that lives in soil and on plant debris, compared to that of allicin (160 μl/ml) and fluconazole (100 μl/ml) [13].However, the studies of Sohn et al. [14] showed that fistulosides, the dominant compound from A. fistulosum extract, exhibited comparatively lower MIC of 3.1 -6.2 μg/ml against Candida albicans ATCC10231.Taiwanese A. fistulosum extracts obtained using rice wine have been shown to exhibit strong antioxidant and antibacterial activities in our recent study [10].To broaden the inhibitory spectrum the antimicrobial activities against fungal pathogens that commonly caused infection in human were examined, and the MIC and minimum fungicidal concentration (MFC), and allicin content of the A. fistulosum wine extracts was studied in this work.
Rice wine is commonly used in Taiwanese cooking to make food more delicious.We developed a rapid method for obtaining the active constituents of A. fistulosum by using commercial rice wine.Welsh onion was used as a raw material and commercial rice wine Michiu Tou (MT) (34% alcohol) was used for preparing extracts of different plant parts of Welsh onion.The potential use of A. fistulosum extracts as natural antifungals was evaluated by determining their antifungal activities.

Test Materials and Chemicals
A. fistulosum cultivar, Lanyang No.1, grown in Sunshin, Yilan County, Taiwan, was used as the test material.The planting to harvesting duration was approximately 84 -90 d; hence, the A. fistulosum plant (AFI) was planted in April 2013 and harvested in July 2013.The characteristics of the A. fistulosum parts were shown in Table 1.The A. fistulosum root (AFR) length was 3.6 -12.8 cm, A. fistulosum stem (AFS) length was 14.7 -19.2 cm, and A. fistulosum leaf (AFL) length was 38.5 -51.8 cm.These materials were placed in a drying oven at 50˚C.After drying, they were placed in MT wine for 7 d.Whatman filter paper no. 1 was used to filter impurities from the extracts.The filtrate was concentrated under reduced pressure at 50˚C, and the A. fistulosum extracts were dried for 24 h in a vacuum oven.All extracts were evaporated to dryness under nitrogen and used within 24 h for experiments.All solvents and reagents were purchased from Sigma Chemical Co.Potato dextrose agar (PDA), potato dextrose broth (PDB), and Mueller Hinton agar (MHA) media were purchased from Difco Chemical Co., USA.

Antifungal Activity of Taiwanese A. fistulosum
Fungal strains used were Aspergillus brasiliensis ATCC 16404, Candida albicans ATCC 10231, Microsporum canis ATCC 36299, M. gypseum ATCC 24102, Trichophyton mentagrophytes ATCC 9533, T. rubrum ATCC 28188, and T. tonsurans ATCC 28942.These strains were purchased from the Bioresource Collection and Research Center of the Food Industry Research Institute in Hsinchu City, Taiwan.A tube dilution test [15] and an agar disc diffusion test [16] [17] were performed to study the antifungal activity of A. fistulosum extracts against the aforementioned seven fungal strains.Fifty microliters of the A. fistulosum extract at 1 mg/ml was applied to an ethanol-sterilized paper disc (8 mm in diameter) and placed onto the PDA agar plates.After incubation at 30˚C for 24 h, the inhibition zone around the disc was measured [16] [17].Nystatin at a concentration of 50 μg/ml was used as the control in the antifungal assay.Inhibition zones of the extract-coated discs and the control-coated discs were compared.In addition, the minimal inhibitory concentration (MIC) of the samples was determined using the broth dilution method [3] by employing serially diluted A. fistulosum extracts.Subsequently, fungal cultures were prepared in the PDB and incubated at 30˚C for 24 h.The media containing various A. fistulosum extracts were diluted with distilled water to obtain concentrations in the range of 2 to 0.05 mg/ml.The mixture was incubated at 30˚C for 24 h to determine the minimal concentration at which fungal cell growth was fully inhibited.The MIC and minimum fungicidal concentration (MFC), the lowest concentration of A. fistulosum extracts required to inhibit microbial growth and kill them were determined.MFC was defined as the concentration of antifungal agents at which the number of colony forming units was zero [5].

HPLC Assay of Allicin
HPLC analysis of allicin was performed using the Agilent 1100 HPLC UV-VIS (DAD) detector (Heisenburg), Finnigan LCQ-DECA (CURIE) spectrometer, and the Phenomenex Luna C18(2) HPLC assay column (dimensions: 150 mm × 4.6 mm; particle size: 5 μm).The mobile phase was acetonitrile and distilled water in a 30:70 ratio, and the flow rate was 1.0 mL/min.Samples were analyzed using UV detection at 195 nm.The injection volume was 1 mL, and the column temperature was maintained at 25˚C.All samples were filtered through a 0.45 μm filter before HPLC analysis.The eluate was detected using a UV detector at 25˚C.A standard solution containing authentic allicin was used for calibrating the retention time and standard curve.

Statistical Analysis
Data from triplicate experiments were subjected to analysis of variance for a completely random design by using SAS.The data are presented as the mean ± standard deviation of triplicate determinations.Means were compared using the Scheffe's test, and differences were considered significant when p < 0.05.

Plant Material Characteristics
The moisture content of AFI, AFR, AFS, and AFL was 91.6%, 85.5%, 93.2%, and 93.6% (Table 1), respectively.The moisture content of these test A. fistulosum was similar to that of other Taiwanese Welsh onions, which is up to 92% approximately.The results show that the extraction yields of AFI, AFS, and AFL was 42.3%, 43.6%, 42.4%, respectively, which are higher than those of AFR (15.5%).Furthermore, the moisture content and extraction yields of these A. fistulosum parts were also similar to that obtained in our previous study [9] [10].

Antifungal Activity of Taiwanese A. fistulosum
Growth inhibition caused by compounds in A. fistulosum extracts was apparent as a clear zone around the paper disk where no fungi could be recovered.A larger zone of inhibition around the control-disc indicates that the fungi are more sensitive to Nystatin.As expected the blank disc (34% (v/v) ethanol) shows no clear zone at all.AFS extracts had the highest activity against T. rubrum and T. tonsurans, with an inhibition zone diameter of 24.0 ± 1.1 mm and 20.3 ± 1.3 mm, respectively, for an MIC of 0.2 mg/mL and MFC of 0.4 mg/mL for both (Table 2).For A. brasiliensis, the inhibition zone diameter, MIC, and MFC of the AFI extracts were 13.3 ± 0.7 mm, 0.4 mg/mL, and 0.8 mg/mL, respectively.The MIC range for A. brasiliensis was 0.4 -0.8 mg/mL, whereas the MFC range was 0.8 -1.0 mg/mL.AFS extracts had the weakest antifungal activity against C. albicans and M. canis, with an inhibition zone diameter of 12.0 ± 0.3 mm and 12.3 ± 0.6 mm, respectively, for a MIC of 0.8 mg/mL and MFC of 1.0 mg/mL.The MIC was 0.4 mg/mL whereas the MFC was 0.8 mg/mL for both AFI and AFS extracts against M. gypseum and T. mentagrophytes.In general, the antifungal activities of Taiwanese A. fistulosum extracts against the seven pathogenic fungi were in the order of AFS > AFI > AFL > AFR.
Yamada and Azuma [3] used agar dilution and broth dilution methods for in vitro evaluation of antifungal activity of allicin against Candida, Trichophyton, and Microsporum species and found that the MIC ranged from 1.57 to 6.25 μg/ml.Sohn et al. [14] reported antifungal activity of the prominent compound, fistulosides from A. fistulosum, with the MIC ranged from 3.1 to 6.2 μg/ml and the MFC ranged from 3.1 to 6.2 μg/ml.Khodavandi et al. [12] used allicin to demonstrate its intrinsic antifungal activity, and the MIC of allicin against six Candida species ranged from 0.05 to 25 μg/ml.Aala et al. [11] evaluated the in vitro efficacy of pure allicin alone against six dermatophyte isolates, and the MIC ranged from 0.098 to 25.0 μg/ml.Kim et al. [1] studied the antifungal activity of allicin alone and its synergistic effects with the antifungal agents.They proposed that allicin had antifungal activity but an extremely high MIC against pathogenic fungi and could reduce the MIC of amphotericin B while retaining its efficacy.Commercial rice wine extracts of different A. fistulosum parts in our study exhibited different antifungal activities against A. brasiliensis, C. albicans, M. canis, M. gypseum, T. mentagrophytes, T. rubrum, and T. tonsurans, probably because the amount of allicin differed in each extract.In general, the antifungal activities of Taiwanese A. fistulosum extracts against the seven pathogenic fungi were in the order of AFS > AFI > AFL > AFR.Most chemical antifungal drugs were prone to cause side effects.Our study demonstrated that the commercial rice wine extracts of Allium fistulosum had the antifungal activities.We can use to enhance the extraction and purification technology to the development of the natural antifungal agent in future.Therefore, HPLC analysis was performed to determine the allicin content of the extracts and its inhibitory activity.minimal inhibitory concentration (mg/mL); MFC, minimum fungicidal concentration (mg/mL) was defined as the concentration of the antifungal agent at which the number of colony forming units was zero.* Nystatin was used as control.The concentration was 50 μg/mL.** Blank was 34% ethanol of the commercial MT wine.*** In. zone represents the inhibition zone diameter (mm) of extracts.Each test was performed in triplicate, and data are presented as the mean ± standard deviation (SD).Data with different superscript lowercase letters in the individual column are significantly different at p < 0.05, according to the Scheffe's test.**** Not detected.

Correlation of Antifungal Activity and Allicin Content in Wine Extracts of Different A. fistulosum Parts
For the commercial MT rice wine extracts from different A. fistulosum parts, the allicin content ranged from 89.6 to 95.9 μg/mL.The allicin content was the highest in the AFS extract (95.9 ± 2.5 μg/mL).The allicin content of the AFI and AFL extracts were 93.5 ± 2.1 μg/mL and 94.5 ± 2.7 μg/mL, respectively.The allicin content was the lowest in the AFR extract (89.6 ± 2.5 μg/mL).As shown in Figure 1, the correlation between the inhibition zone diameter and allicin content in different A. fistulosum part extracts obtained using MT wines was determined.The r 2 values between the inhibition zone diameter and allicin content for A. brasiliensis, C. albicans, M. canis, M. gypseum, T. mentagrophytes, T. rubrum, and T. tonsurans were 0.55, 0.67, 0.71, 0.61, 0.86, 0.64, and 0.68, respectively.The calibration results are more or less acceptable and show a positive correlation when these r 2 values are equal and bigger than 0.6.Samuel et al. [18] evaluated the antifungal activity of Allium sativum bulb extract against T. rubrum, and a positive correlation was observed between the inhibitory zone diameter and the allicin content.In previous studies [17] [19], allicin extracts were obtained from different plants of the genus Allium, including garlic and A. fistulosum, by using hot water and alcohol.In these Allium plant extracts, the 20% -30% alcoholic extracts had the highest allicin content.Our study had found similar results and indicated that different A. fistulosum parts extracted using commercial MT wine have different allicin contents.The antifungal activity depends on the allicin content of the A. fistulosum part extracts.The r 2 values between the inhibition zone diameter and allicin content for these test strains have indicated a positive correlation.

Relationship between Allicin Content and Antifungal Activity
To determine the inhibitory effect of allicin towards the test fungi, commercial pure allicin (99%) were prepared at concentrations of 10 −5 mg/mL to 10 −1 mg/mL, and the correlation between the inhibition zone diameter (mm) and allicin content (mg/mL) was determined (Figure 2).The r 2 values of the correlation between the inhibition zone diameter and allicin content for A. brasiliensis, C. albicans, M. canis, M. gypseum, T. mentagrophytes, T. rubrum, and T. tonsurans were 0.94, 0.60, 0.82, 0.81, 0.93, 0.93, and 0.95, respectively, thus indicating a strong positive correlation.The results suggested that allicin exhibited good fungicidal activity against all the test fungi except C. albicans.
Table 3 showed the results of antifungal activity of allicin determined using the MIC and MFC in the broth   dilution method.The commercial allicin had the highest antifungal activity against A. brasiliensis and T. mentagrophytes, with the MIC of 0.1 μg/mL and MFC of 1.0 μg/mL.The MIC and MFC for M. gypseum, T. rubrum, and T. tonsurans were 1.0 μg/mL and 10.0 μg/mL, respectively.The MIC for M. canis was 10.0 μg/mL, whereas the MFC was 50.0 μg/mL.Among the test fungi, allicin had the weakest inhibitory activity towards C. albicans, which the MIC and MFC were 10.0 μg/mL and 100.0 μg/mL, respectively.Yamada and Azuma [3] studied the antifungal activity of allicin by using the agar dilution methods in Sabouraud glucose medium, in which the MIC of allicin against Candida, Cryptococcus, Ttichophyton, Epidermophyton, and Microsporum ranged from 3.13 to 25.0 μg/ml.Khodavandi et al. [12] investigated the antifungal activity of allicin against Candida species, and the MIC of allicin alone against six Candida species within the range of 0.05 -25 μg/ml.Aala et al. [11] evaluated the in vitro efficacy of pure allicin alone against six dermatophytes, and the MIC of allicin ranged from 0.098 to 25.0 μg/ml.Figure 2 showed the r 2 values of the correlation between the inhibition zone diameter and allicin content for these test strains have a strong positive correlation.The results suggested that allicin exhibited good fungicidal activity against all the test fungi except C. albicans.The commercial allicin had the highest antifungal activity against A. brasiliensis and T. mentagrophytes, and had the weakest inhibitory activity towards C. albicans.The present study used extracts of different A. fistulosum parts and obtained results consistent with those of the aforementioned studies.

Conclusion
The commercial MT wine extracts of Taiwanese A. fistulosum exhibited antifungal activities, and the MIC and MFC of allicin were within the range of 0.2 -1.0 and 0.4 -2.0 mg/mL, respectively.This study also evaluated the in vitro efficacy of pure allicin used alone against seven dermatophytes, and the MIC and MFC of the allicin content were 0.1 -10 and 1 -100 μg/mL, respectively.A strong positive correlation was observed between the

Figure 1 .
Figure 1.Correlation between antifungal inhibition zone and allicin content of extracts of different A. fistulosum parts.

Figure 2 .
Figure 2. Correlation between antifungal inhibition zone and allicin content.

Table 1 .
The characteristics of different A. fistulosum parts.

Table 2 .
Antifungal activities of extracts of different A. fistulosum parts extracts Obtained using commercial MT wine.

Table 3 .
Antifungal activities of allicin content according to MIC and MFC.