Griseococcin(1) from Bovistella radicata (Mont.) Pat and Antimicrobial Activity

Bovistella radicata (Mont.) Pat was a natural plant with multiple medicinal values. The puried compound puried from Bovistella radicata (Mont.) Pat by DEAE-cellulose and sephadex LC-20 column which could inhibit main pathogens leading to tinea pedis. Based on the spectral (HPLC, FT-IR, 1D and 2D NMR etc.) studies, puried compounds were identied as the Griseococcin(1) which were naphthoquinone derivatives, the Chemical formula and MW of Griseococcin(1) was determined as C 37 O 10 H 43 N and 661 Da. Minimum inhibitory concentration(MIC) and zone of inhibition(ZOI) of Griseococcin(1) were 31.2, 31.2 µg/ml and 18.06 ± 0.85 mm, 15.01 ± 1.02 mm respectively against main pathogenic fungus Trichophyton rubrum and Trichophyton mentagrophytes. 7.93) to C-3(δ C175.11) and C-9(δ C138.56), from H 2 -13(δH 1.07) to C-8(δ C 135.45) and C-6 (δC 137.21), from H 3 14’ (δH 1.85) to C-2’ (δ C 215.7) and C-4’(OH) (δ C 73.60), from H 3 -15’ (δH 2.11) to C-6’ (δ C 215.70) and C-4’(OH) (δ C 73.60), from H 2 -7’ (δH 1.08) to C-9’ (δC 71.25) and C-13’ (δC 71.18). The 1 H, 1 H three-bond couplings observed in the COSY spectrum from H-8’ (δH 1.94) to H-9’ (δH 3.62) ,from H-10’ (δH 1.29) to H-11’ (δH 3.49), from H-12’ (δH 1.73) to H-13’ (δH 3.51), together with the chemical shifts of the 13 C resonances (C-8’-13’) observed at alternating higher and lower elds, revealed the presence of cyclohexane with alternating hydroxyl and methyl groups. 1 H- 1 H COSY correlations from H 2 -13 (δH1.07, m) to H 2 -14 (δH3.62, m), from H 2 -14 (δH3.62, m) to H 2 -15 (δH 3.49, m) and from H 2 -16 (δH 3.55, m) to H 2 -17(δH 3.51, m) and HMBC correlations from H 2 -13 (δH 1,07, m) to C-15 (δC 166.02), from H 2 -14 (δH3.62, m) to C-16 (δC 166), from H 2 -15(δH3.49, m) to C-17 (δC 166.01) and from


Introduction
Tinea pedis is a chronic fungal infection of the feet (Alteras, et al 1981). Patients that have tinea pedis may be affected by several pathogens, including lamentous fungi named Trichophyton rubrum and Trichophyton mentagrophytes (Koltin and Hitchcock 1997), as well as a yeast named Candida albicans (Erbagci, et al. 2005). T. rubrum is the main pathogenic fungi for tinea pedis, having a prevalence as high as 80% among all tinea-pedis-associated pathogenic microbes (Miyajima et al. 2013). Traditionally, to treat tinea pedis, synthetic fungicides such as uconazole, itraconazole, echinocandins (Daneshmend and Warnock. 1983), and miconazole nitrate, either by oral medication or external use (Patel et al. 2017), 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 (Francis P and Walsh T J. 1992; Stamm et al.1987; Vermes et al. 2000). Similar studies on Itraconazole have demonstrated that it is effective against fungal infections (Denning et al.1997). However, due to side effects or the continuous drug resistance, some oral medications are unsafe for patients (Subissi et al. 2010), and these chemicals also cause potential deleterious effects on the environment due to their residues(Lushchak. 2016; Rajendra et al.2013). In general, plant natural products have been for decades one of the most successful sources of drugs to treat infectious diseases (Genilloud. 2014) and natural products extracted represent a rich resource for screening bioactive compounds (Ribeiro et al. 2018).
Puffballs are widely distributed in many provinces of China, and are various by more than 100 species (Bates et al. 2009 The aim of the present study was to evaluate the antifungal activity of Griseococcin(1) extracted from B. radicata fermentation broth. The antifungal activities were evaluated in terms of their minimum inhibitory concentration (MIC) values and zone of inhibition(ZOI) values (Negi et al. 2003), 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).

Material And Methods
Sample Bovistella radicata (Mont.) Pat collection and strain isolation

Antimicrobial activity
The antimicrobial activity was tested against selected fungi(T.rubrum, T. mentagrophytes, E. occosum, and C. albicans) and bacteria(S. aureus, E. coli and P. aeruginosa from tinea pedis. The examined methods were the minimum inhibitory concentrations (MICs) (Negi et al.2003)and zone of inhibitions (ZOIs).The MIC value of Griseococcin(1) was determined in the 96-well plates by the double micro dilution method(7.8 ~ 250 µg/mL) against pathogens. The zones of inhibition ( ZOI) of Griseococcin(1) (100 µg/ml) was also evaluated (Geetha et al. 2015), Terbina ne and Gentamicin sulfate as the positive control. .

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 In nity 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℃. 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 compound was performed on a Hypersil RP-C18 column (5 µm, 250 × 10.0 mm, Thermo Fisher Scienti c, USA)at a temperature of 25 °C. Injection volume: 20 µL.
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 In vitro antagonistic assay

Discussion
In the present study, selected puffball (Bovistella radicata (Mont.) Pat) showed remarkable antifungal activities. These data are consistent with previous ndings on the minimum inhibitory concentrations (MICs) and zone of inhibition (ZOI) of B. radicata (Ye et al. 2017).
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, hence, the present study about antifungal function of B. radicata is the rst report. The novel antimicrobial activities of B. radicata might be due to different geographic sources of the material used and different strains used (Ali et al. 2003).
In this study, the puri cation extraction Griseococcin(s) from fermentation broth of B. radicata obtained through macroporous resin D-101, celluous DE-52 and sephadex LH-20 column, puri ed fractions were used for biological activity analysis. In vitro assays demonstrated that fraction Griseococcin (1)  The FT-IR spectrum of Griseococcin (1)  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.
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 puri ed. The puri ed compound can restrain main pathogens(T. rubrum and T. mentagrophytes) leading to tinea pedis. The antifungal activity of Griseococcin (1)   The key 1H-1H COSY, HMBC and HSQC correlations of Griseococcin (1) Figure 2 UV spectral and HPLC chromatography of SPAF (A) and puri ed fraction (Griseococcin (1))(B) Figure 3 FT-IR of fractions F2 and F3 Note: line A is F2; line B is F3