Screening of Antifungal Substances from Bovistella Radicata (Mont.) Pat and Their Antifungal Effect


 Background To analyse the antifungal active compounds in B. radicata alcohol extract, the alcohol extract was purified by column chromatography (macroporous resinD-101) and the active compounds was named as SPAF-1(the spore powder active fraction). Results Alcohol extracts and SPAF-1 were submitted to GC-MS analysis, there were two characteristic peaks(peak1 and peak2) in Gas chromatogram. By comparing in NIST, the compound were 2-propyl-1-pentanol corresponding to peak1 and decanal, n-decanol and 2E-decanol corresponding to peak2. The main constituents were decanal(24.3%) n-decanol(27.9%), 2E-decenol (21.2%) and 2-Propyl-1-pentanol (13.6%). Their MIC values were 62.5 µg/ml, 31.2 µg/ml, 31.2 µg/ml and 250 µg/ml against T. rubrum respectively. Furthermore, transmission electron microscope (TEM) analysis showed altered surface morphology in the majority of T. rubrum cells after treatment with SPAF-1. Conclusions In this paper, we successfully separate SPAF-1 from alcohol extract of B. radicata. The antifungal effect of SPAF-1 is similar to positive control, the main component were decanal, n-decanol, 2E-decenol and 2-Propyl-1-pentanol, the anti-tinea pedis effect of them was obvious.


Background
Tinea pedis is a chronic fungal infection of the feet [1] . Patients that have tinea pedis may be affected by several pathogens, including lamentous fungi named Trichophyton rubrum and Trichophyton mentagrophytes [2] , as well as a yeast named Candida albicans [3] . T. rubrum is the main pathogenic fungi for tinea pedis, having a prevalence as high as 80% among all tinea-pedis-associated pathogenic microbes [4] . Traditionally, to treat tinea pedis, synthetic fungicides such as uconazole, itraconazole, echinocandins [5] , and miconazole nitrate, either by oral medication or external use [6] , have been used to treat this disease. Vermes et al. (2000) found that ucytosine and AMB (amphotericin B) were moderately effective in ghting against invasive fungal infections [7][8][9] . Similar studies on Itraconazole have demonstrated that it is effective against fungal infections [10] . However, due to side effects or the continuous drug resistance, some oral medications are unsafe for patients [11], and these chemicals also cause potential deleterious effects on the environment due to their residues [12][13] , which has prompted researchers to develop newer and safer antifungal agents. Generally speaking, natural products extracted from plants represent a rich resource for screening bioactive compounds [14] .
The aim of the present study was to evaluate the antifungal activity of the spore powder active fraction (SPAF) extracted from Bovistella radicata (Mont.) Pat and its fractions against different species of tinea pedis pathogens, including T. rubrum and T. mentagrophytes. The differences of antifungal activities and GC ngerprints between spore and sporophore powder were determined. The antifungal activities were evaluated in terms of their minimum inhibitory concentration (MIC) values and zone of inhibition(ZOI) values [21] , and the chemical constituents responsible for this activity were identi ed.

Results
In Vitro Antifungal Activity Assay The puffballs (B. radicata, C. gigantean, C. lilacina, L. fenzlii Reich, and L. pyriforme) exhibited different antifungal activity against T. rubrum, T. mentagrophytes, E. occosum, and C. albicans. The results were shown in Table 1. Alcohol extract of B. radicata spore powder showed good activity against the tested microorganisms (MIC was 62.5 and 125 µg/ml), while alcohol extract of B. radicata sopophore powder and other puffballs (C. gigantean, C. lilacina, L. fenzlii Reich, and L. pyriforme) showed weak activity against the fungus (MIC was about 250 and 500 µg/ml).  Note T spore powder groups treated with alcohol, U sporophore powder groups treated with alcohol, Terbina ne is positive control.
As shown in Table 1, antifungal effect of B. radicata sporophore powder and other puffball alcohol extract performed poor antifungal activity. The zone of inhibition (ZOI) was observed in PDA medium inoculated with T. rubrum, after co-cultivation with different alcohol extracts from B. radicata and L.fenzlii Reich, The results were showed in Fig. 1.   Figure1. zone of inhibition(ZOI) of spore and sporophore powder from B.radicata From Fig. 1, the inhibitory effects on the growth of T. rubrum suggested that antifungal compound maybe was from B. radicata spore powder.

SPAF-1 preparation from B. radicata alcohol extract by macroporous resin D-101
We focused on B. radicata spore powder alcohol extract for further extraction and puri cation. This portion was separated by column chromatography using macroporous resin D-101 with 90% ethanol as eluent. The antifungal effectiveness of alcohol extract from B. radicata, eluent fractions with distilled water(EF) and 90% ethanol (SPAF-1) were assessed against T. rubrum (the main pathogenic fungi of tinea pedis). The antifungal assay con rmed that the alcohol extract and SPAF-1 exhibited high antifungal effectiveness, while EF exhibited weak antifungal activity (MIC = 250 mg/l). MIC of alcohol extract was 62.5 mg/l. While SPAF-1( 90% ethanol eluent fraction) exhibited the most complete activity with a 100% inhibition rate at a concentration of 31.2 mg/l. HPLC analysis of alcohol extract and SPAF-1 from B. radicata As stated above, alcohol extract and SPAF-1 from B. radicata have obvious antifungal activity, while the eluent fractions(EF) has weak antifungal activity. It meaned that the active ingredient(SPAF-1) could be puri ed by macroporous resin D-101. Alcohol extract and SPAF-1 were analysed by HPLC. The characteristic peak1(2.42 min) and peak2(16.04 min) were shown in Fig. 2B (SPAF-1), the results also demonstrated macroporous resin D-101 was a good selectivity for puri cation of SPAF-1. The antifungal substances in B. radicata were also analyzed by GC-MS, among the GC-MS chromatograms of alcohol extract of spore powder, sporophore powder and SPAF-1. More than 50 compounds were identi ed from spore and sporophore powder of B. radicata by GC-MS. Fitting analysis results of spore and sporophore GC chromatograms showed that spore chromatograms presented characteristic peak P1(8.791 min) and P2(17.825 min) (Fig. 3), the characteristic peak time was consistent with SPAF-1. The test results were submitted to the National Institute of Standards and Technology (NIST) library, we analyzed the mass chromatograms of SPAF-1, the results were showed in Table 2. Peak1 revealed a peak of m/z 116 (M + H), by comparing in NIST, the substance were 2-propyl-1-pentanol. Peak2 revealed peaks of m/z 158,145,130,141 (M + H), by comparing in NIST, the substances may include decanal, n-decanol and 2E-decanol, The results are showed in Table 2.

FT-IR analysis of SPAF-1
The FT-IR spectral results of alcohol extract, SPAF-1 and EF are shown in Fig. 4, the differences range among them are from 1100 cm − 1 to 1800 cm − 1 and 720 cm − 1 to 900 cm − 1 . The broad stretch of frequency from 3500 -3200cm − 1 was assigned to the hydroxyl group. There are no stretching vibrations at 1600,1580, 1500 and 1450 cm − 1 which is considered to no aromatics in alcohol extract, EF and SPAF-1. In addition, the band obtained at ~ 1433 cm − 1 (SPAF-1) and 1652 cm − 1 (alcohol) is assigned to the stretching vibrations of unsaturated -C = O and the stretch from 1300 cm − 1 to 1000 cm − 1 was attributed to -C-O. The -C = O and -C-O stretching vibrations could be due to aldehyde group and hydroxyl group. The absorbance signals at 2930, 2932, and from 2300 to 2500 cm − 1 could be explained by a long fat chain existing in the compounds. Decanal, n-decanol, 2E-decanol and 2-Propyl-1-pentanol as components of SPAF-1 were veri ed by FT-IR. To investigate the changes in shape and ultrastructure of T.rubrum cells, cells were examined after cocultivation with SPAF-1 by transmission electron microcopy. As shown in Fig. 5A. The membrane of the non-inoculated control cells was intact with uniformly distributed cytoplasm and electron density inside the cells. In contrast, more than 30% of T. rubrum cells showed concentrated cytoplasm and altered cell morphology after co-cultivation with SPAF-1 (Fig. 5B).As indicated by the red arrow 1, the cell membrane is badly shrunk out of wall. As indicated by the red arrow 2, the cell wall is obviously thinner and the edge is blurred comparing with the control. which may increase membrane permeability and cause leakage of intracellular substances.

In Vitro Antagonistic Activity of Puffball
The examined methods were the minimum inhibitory concentrations (MICs) (Negi et al.2003)and zone of inhibitions (ZOIs). MIC value was determined in the 96-well plates by the double micro dilution method(7.8 ~ 250 µg/mL) against pathogens. ZOI (100 µg/ml) was also evaluated [33] , Terbina ne and Gentamicin sulfate as the positive control.

Alcohol extraction by Soxhlet system
Puffball samples include spore and sporophore powder. The Soxhlet system includes extraction bottle, extraction tube and condenser. When extracting, the samples were wrapped in a degreased lter paper bag and put into the extraction tube. Alcohol was added into the extraction bottle, the extraction bottle was heated, alcohol was gasi ed, risen, condensed, dripped into the extraction tube, the bioactive components were extracted into the extraction bottle. so that the cycle reciprocates untile the extraction is complete.

The chemical constituents of alcohol extraction by GC-MS
The alcohol extracts were recorded on a GC-MS(Gas Chromatography-Mass Spectrometer) system. One microliter of alcohol extract of spore and sporophore powder was injected into a DB-5MS capillary column coated with 5% diphenyl cross-linked 95% dimethylpolysiloxane (30 m × 250 µm inner diameter, 0.25 µm lm thickness; J&W Scienti c, Folsom, CA, USA). The alcohol extract were injected in the splitless mode. Helium was used as the carrier gas. The front inlet purge ow was 1 mL/min, and the gas ow rate through the column was 20 mL/min. The initial temperature was kept at 40 ℃, held for 3.0 min, then raised to 150 ℃ at a rate of 10 ℃/min. The temperature was kept for 10 min at 150 ℃. The mass spectrometer was operated in electronionization (EI) mode at 70 eV. The injection, transfer line, and ion source temperatures were 150, 250, and 230 ℃, respectively. The mass spectrometry data were acquired in full-scan mode with an m/z range of 10-500 u at a rate of 20 u spectra per second after a solvent delay of 210 s. Peaks were identi ed by comparing with the mass spectra data from the National Institute of Standards and Technology (NIST) spectral library.
HPLC analysis of alcohol extract and SPAF-1 from Bovistella radicata (Mont.) Pat Alcohol extract from B. radicata was also analyzed via high-performance liquid chromatography (HPLC) coupled with an ultraviolet (UV) detector (Agilent, 1260 In nity II Prime, USA). The aim was to identify the most active compounds in the fractions of the puffballs. A C18 reversed-phase column (Hypersil Gold 25 mm × 2.1 mm,1.8 µm, Thermo Scienti c, Massachusetts, USA) was used with the following solvent system: A = acetonitrile, B = 0.15% ammonium acetate-water. The gradient elution was 7% A in 5 min, 7-10% A in 3 min, 10% A in 2 min, 10-15% A in 5 min, 15% A in 3 min, 5-15% A in 2 min. The LC system was operated at a ow rate of 1.0 mL/min for 20 min. The injection volume was 5 µL, and the detection was at 220 nm.

FT-IR analysis of SPAF-1
The FT-IR(Fourier transform infrared spectroscopy) spectra of samples were recorded using a NICOLET 5700 Fourier Transform Infrared Spectrometer (provided by Nicolet Instrument Co., U.S.A) using potassium bromide (KBr) pellets. The pellets were designed by blending the sample and KBr at a ratio of 1:100 and were ground into particles smaller than 0.1 mm. The FT-IR measurement scanned the range from 400 to 4000 cm − 1 . A He-Ne laser source operating at 0.5 W was utilized for sample excitation.

Transmission Electron Microscopy
Overnight cultures of T. rubrum. were on PDA. The wells (5.0 mm in diameter) were cut from PDA medium, The tested compounds (SPAF-1) were added to the wells, the wells added normal saline were used as controls, then incubated at 28℃ for 48 hours. Following the incubation period samples were pick up from PDA and xed overnight in 2.5% glutaraldehydein PBS (phosphate-buffered saline), washed three times in PBS and post xed overnight in 1% osmium tetroxide. Following ethanol dehydration, probes were embedded in Epon resin (Sigma-Aldrich), cut on a ultramicrotome Leica UC7 and contrasted in uranyl acetate and lead citrate. Transmission electron microscopy studies were performed using a Philips CM100 electron microscope [34] .

Discussion
In the present study, B. radicata from Jiangxi province, China, shows remarkable antifungal activities. These data are consistent with previous ndings on the minimum inhibitory concentrations (MICs) of B.
According to the Chinese Pharmacopeia, the main anti-microorganism activity of the puffball is against S. aureus and P.aeruginosa. The antifungal function of puffballs has not been reported previously. The novel antimicrobial activities of B. radicata might be due to different geographic sources of the material used and different strains used [22] .
In vitro assays demonstrated that alcohol extract and SPAF-1 from B. radicata were able to suppress the mycelial growth of T. rubrum and T. mentagrophytes. The MICs of SPAF-1 from alcohol extract are 31.2 µg/ml and 31.2 µg/ml against T. rubrum and T. mentagrophytes, respectively. Terbina ne was used as a positive control in this study with MIC values 62.5 and 31.2 µg/ml. The antifungal effect of SPAF-1 was similar with that of positive control.

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All these aliphatic alcohols and aldehydes obtained from B. radicata alcohol extracts increase the uidity of the membrane and are known to inhibit the key enzyme of saturated fatty acids [31] . The aliphatic alcohols also have one hydroxyl group. The hydroxyl group mainly determines the hydrophilicity of the molecule, resulting in much easier interaction with its target in a living organism [32] .
Future work concentrating on determining the antifungal mechanisms of aliphatic alcohols and aldehydes will be performed, which will be helpful in laying a foundation for overcoming the drug resistance that pathogens quickly develop against antibiotic drugs.