Griseococcin (1) from Bovistella radicata (Mont.) Pat and antifungal activity

Background To evaluate the antimicrobial and microbicidel activity of B. radicata fermentation broth, the broth was purified by DEAE-cellulose and sephadex LC-20 column. The compounds were submitted to spectral analyses (HPLC, FT-IR, 1D and 2D NMR etc.). Results The purified compounds were identified as the Griseococcin(s) which were naphthoquinone derivatives, the Chemical formula and MW of Griseococcin (1) was determined as C37O10H43N and 661 Da. only Griseococcin (1) has good antimicrobial activity among the Griseococcin(s). The zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of Griseococcin (1) were used to investigate the antimicrobial activity. Antifungal activity of Griseococcin (1) was significant, especially for main pathogenic fungus Trichophyton rubrum and Trichophyton mentagrophytes, MFC/MIC of Griseococcin (1) was 1, while MFC/MIC of postive control was greater than 4, the fungicidal effect of Griseococcin (1) was better than that of positive control. Conclusions In this paper, the secondary metabolite compound Griseococcin (1) from B. radicata was purified. The purified compound can restrain main pathogens (T. rubrum and T. mentagrophytes) leading to tinea pedis. The antifungal activity of Griseococcin (1) was similar to that of the positive control and the fungicidal effect of Griseococcin (1) was better than that of positive control, it might be suitable for pharmaceutical industries.


Background
Tinea pedis is a chronic fungal infection of the feet [1]. Patients that have tinea pedis may be affected by several pathogens, including filamentous 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 fluconazole, 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 flucytosine and AMB (amphotericin B) were moderately effective in fighting 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]. In general, plant natural products have been for decades one of the most successful sources of drugs to treat infectious diseases [14] and natural products extracted represent a rich resource for screening bioactive compounds [15].
Puffballs are widely distributed in many provinces of China, and are various by more than 100 species [16].
The aim of the present study was to evaluate the antimicrobial activity of Griseococcin (1) extracted from B. radicata fermentation broth. The antimicrobial and microbicidel activities were evaluated in terms of their minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) or minimum bactericidal concentration (MBC) and zone of inhibition (ZOI) values [22], the physico-chemical characterization (HPLC, UV, FT-IR) of Griseococcin (1) and the chemical constituents responsible for this activity were also studied (1D and 2D NMR).

Results
Fermentation, extraction and purification of active compound from B. radicata 20% NaCl elution fraction from fermentation broth of B. radicata was named as SPAF by DEAE-cellulose column. The strongest antimicrobial activity fraction from SPAF was Griseococcin (1) by Sephadex LH-20 column. The UV max of all the fraction was 215 nm, the HPLC chromatograms of SPAF and Griseococcin (1) were shown in Fig. 1(a~b). The chromatogram of B showed a single and symmetrical peak for Griseococcin (1) (Fig.  1b).
This connectivity was also secured by the observation of the HSQC correlations from H 3 -14′ to C-3′ and from H 3 -15′ to C-6′. Therefore, the complete structure of naphthoquinone was determined as shown in Fig. 2c.

Physico-chemical characterization of Griseococcin (1)
Griseococcin (1) was white powder and its solubility was 0.063 g/ml in water. It could be slight soluble in methanol and DMSO, but insoluble in n-hexane, dichloromethane, chloroform, ethyl acetate and acetone.
Due to side effects and the continuous drug resistance, commercial reagents might be unsafe for patients [11], Therefore, the development of fungicidal therapies is crucial, above results (MIC, MFC or MBC and MFC/ MIC or MBC/MIC) add more value to Griseococcin (1).

Discussion
In the present study, Griseococcin (1) purified from selected puffball (Bovistella radicata (Mont.) Pat) had remarkable antifungal activities. These data are consistent with previous findings on the minimum inhibitory concentrations (MICs) and zone of inhibition (ZOI) of B. radicata fermentation [20].
According to the Chinese Pharmacopeia, the puffball can restrain S. aureus and P. aeruginosa. The antifungal function of puffball has not been reported previously, hence, the present study is interesting and original. The novel application of B. radicata might be due to different geographic sources of the material used and different strains used [25]. In this study, the purification extraction Griseococcin(s) from fermentation broth of B. radicata obtained through celluous DE-52 and sephadex LH-20 column. In Vitro study on antifungal effects of Griseococcin (1) on fungi showed that the most sensitive fungi strains were the main pathogenic fungi (T. rubrum and T. mentagrophytes) causing tinea pedis, ZOIs were 18.06 ± 0.85 and 15.01 ± 1.02 mm, MICs were 31.2 ± 2.7 and 31.2 ± 1.8 μg/ ml, MFCs were 31.2 ± 3.1 μg/ml and 31.2 ± 2.1 μg/ml, MFC/MICs were 1 and 1 against T. rubrum and T. mentagrophytes. ZOI values of positive control (Terbinafine) were 20.67 ± 1.58 mm 28.33 ± 2.15 mm, MICs were 15.6 ± 1.6 and 7.8 ± 1.2 μg/ml, MFCs were 93.6 ± 2.1 and 39.0 ± 2.2 μg/ml, MFC/MICs were 6 and 5 respectively. The antifungal effect of Griseococcin (1) was similar with that of positive control, the fungicidal effect of Griseococcin (1) was better than that of positive control. The most sensitive bacterial species for Griseococcin (1) was S. aureus and E. coli, MICs and MBCs were 62.5 ± 1.5, 125 ± 2.3 and 125 ± 3.1, 250 ± 2.1 μg/ml respectively, P. aeruginosa and B. subtilis were more resistant. MFC/ MICs and MBC/MICs of Griseococcin (1) were less than positive control which meant that antibacterial activity Griseococcin (1) was better than that of the commercial drugs. This study is important for the development of new drugs with low toxicity, overcoming drug resistance and recurrence.  The FT-IR spectrum of Griseococcin (1)  Previously, many authors reported the various biological activity of fermentation broth from puffball like anticancer activity [26,27], antioxidant activity [28], antifatigue effect [29], etc. In the present study, the antifungal activity of B. radicata was another important biological function. The biological activities of organic compounds are related to their molecular weight, functional groups, the length of chain, the composition of group and the number of branches, hydrophilic and hydrophobic group. It means that the structure-activity relationship should be disclosed.

Conclusions
Future work concentrating on determining the antifungal mechanisms of Griseococcin (1) will be performed, which will be helpful in laying a foundation for overcoming the drug resistance that pathogens quickly develop against tinea pedis.
In this paper, the antifungal secondary metabolite compound Griseococcin (1) from B. radicata were studied. The compound from Bovistella radicata (Mont.) Pat was purified. Molecular weight and molecular formula of the purified compound (Griseococcin (1)) were 661 Da and C 37 H 43 NO 10 respectively, it can restrain main pathogens (T. rubrum and T. mentagrophytes) leading to tinea pedis. The antifungal activity of Griseococcin (1) was similar to that of the positive control.

Fermentation, extraction and purification of Bovistella radicata (Mont.) Pat
The mature B.radicata should be dried at 40°C for at least 2 days, and taken out when its weight is no longer changed, the sporophore and spore powder were ground together and filtered through a 100 mesh sieve. The mixed powder of B.radicata was inoculated into 100 mL of potato dextrose broth (PDB) in 250 ml flask. The flask was kept in rotary shaker at 25°C with 115 rpm for 72 h. The pH and moisture content of PDB was also determined according Maguireboyle (2014) and Mcauliffe (2016) [30,31]. For every 12 h, the fermentation was taken to perform antimicrobial activity against main pathogens T. rubrum and T. mentagrophytes by zone of inhibition (ZOI) method. Then the fermentation were centrifuged at 7000 rpm for 20 min and filtered over Whatman No.4 paper to get the final clear supernatant and preserve at 4°C. 50 ml clear supernatant was purified firstly using 100 ml DEAEcellulose column and eluted by different concentration NaCl (10-30%) to get different fractions. 20% NaCl elution fraction showed best antifungal activity against pathogens and was named as SPAF. Furthermore, SPAF (20% NaCl elution fraction) was purified by sephadex LH-20 column. Different purified fractions (named Griseococcin(s)) were obtained from SPAF, only Griseococcin(1) (500 μg/ml) has antifungal activity and it's biochemical characteristics and spectral (HPLC, FT-IR, 1D and 2D NMR etc.) studies were assessed.

Antimicrobial activity
The examined methods were the minimum inhibitory concentrations (MICs) [22], minimum bactericidal concentration (MBCs) or minimum fungicidal concentration (MFCs) [32,33] and zone of inhibitions (ZOIs) [34].  (1) (100 μg/ml) was also evaluated. The prepared Griseococcin (1) was filled into the wells. After incubating for 24 h at 37°C, the measurements were done basically from the edge of the zone to the edge of the well [34].

General experimental procedures
The UV max absorption spectrum of SPAF was analyzed at full-wave spectra (200-900 nm) by UV/vis 2802 spectrophotometer. The FT-IR spectrum of Griseococcin(s) were recorded on a Thermo Nicolet Spectrum FT-IR in a range of 4000-400 cm − 1 with KBr pellets. HR-ESI-MS data were obtained on an Agilent 1260 Infinity LC coupled to a 6230 TOF. 20 mg of the dried sample was dissolved in 0.55 mL of deuteroxide (99.99% D) in a NMR tube. 1D and 2D NMR spectra were acquired on an AVANCE-600 NMR spectrometer (Bruker Inc., Rheinstetten, Germany) at 50°C. The chemical shifts were given in δ (ppm) and referenced to the solvent signal (D 2 O-d 6 , δ H 2.50, δ C 39.5). Column chromatography (CC) was conducted on DEAE-cellulose and Sephadex LH-20. The fractions Griseococcin(s) were also monitored by HPLC (Agilent 1260 chromatography system, USA) which was equipped with a diode array detector (DAD). The DAD detector was set at 215 nm to acquire chromatograms. The separation of the