Fermentation, extraction and purification of active compound from B. radicata
Fermentation broth of B. radicata was filtered and centrifuging at 6000 rpm, on 3rd day, the fermentation of B. radicata showed the antimicrobial activity against main pathogenic fungi of tinea pedis, Trichophyton rubrum and Trichophyton mentagrophytes. The fermentation was purified firstly using DEAE-cellulose column and eluted by different concentration NaCl(10%-30%) and get different fractions, 20% NaCl elution fraction showed best antifungal activity against pathogens. Furthermore, 20% NaCl elution fraction was purified by sephadex LC-20 column, different fractions(Griseococcin(s)) were obtain and antifungal activity of Griseococcin (1) was strongest. The UVmax of all these fractions were 215nm, the HPLC chromatograms of SPAF and Griseococcin (1) were shown in figure1(A~B). The chromatogram of B showed a single and symmetrical peak for Griseococcin (1)(fig1.B)
1D and 2D NMR of Griseococcin (1)
Griseococcin (1) was isolated as a white amorphous solid powder with the molecular formula of C37H43NO10 derived from the high-resolution electrospray ionization mass spectrum (HR-ESI-MS). The complete assignments for all protons and carbons was shown in Table 1. The 13C NMR spectra of Griseococcin (1) displayed signals of thirty seven carbons, including five carbonyl carbons (δC215.7–175.1), five aromatic/olefinic methine carbons (δC 128.86, δC215.7–175.1), seven non-protonated aromatic/olefinic carbons (δC 161.06-109.99), four methyl carbons (δ C20.27)), and four olefin carbons (δC 166.01). The 1H NMR spectrum of 1in D2O exhibited signals of four methyls at δ H 2.14 (3H, s, H-14’), δ H 2.12 (3H, s, H-15’), δ H 1.06 (3H, s, H-16’)and 1.07 (3H, s, H-17’), five aromatic protons δH 7.80 (1H, s, H-1), δH 7.93(1H, s, H-5), δH 7.72 (1H, s, H-6), δH 7.81 (1H, s, H-8) and 7.66 (1H, s, H-12)], four hydroxyl groups at δ H 8.37 (1H, br s, 4’-OH), δ H 7.81 (1H, br s, 9’OH) and δ H 7.80 (1H, br s, 11’-OH) and 9.63 (1H, br s, 13’-OH).
The structure of Griseococcin (1) was deduced by comprehensive analysis of 1H-1HCOSY, HMBC, and HSQC spectra (Fig.2A). In Griseococcin (1), the naphthoquinone substructure could be identified by the observation of HMBC correlations from H-8(δH 7.80) to C-6 (δ C 137.21), C-4 (δ C 138.60) and C-13 (δ C 30.18), from H-1(δH 7.81) to C-3 (δ C 175.11), C-12(δ C 166.07) and C-1’(δ C 28.40), from H-5(δH 7.93) to C-3(δ C175.11) and C-9(δ C138.56), from H2-13(δH 1.07) to C-8(δ C 135.45) and C-6 (δC 137.21), from H3-14’ (δH 1.85) to C-2’ (δ C 215.7) and C-4’(OH) (δ C 73.60), from H3-15’ (δH 2.11) to C-6’ (δ C 215.70) and C-4’(OH) (δ C 73.60), from H2-7’ (δH 1.08) to C-9’ (δC 71.25) and C-13’ (δC 71.18). The 1H, 1H 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 13C resonances (C-8’-13’) observed at alternating higher and lower fields, revealed the presence of cyclohexane with alternating hydroxyl and methyl groups.1H-1H COSY correlations from H2-13 (δH1.07, m) to H2-14 (δH3.62, m), from H2-14 (δH3.62, m) to H2 -15 (δH 3.49, m) and from H2-16 (δH 3.55, m) to H2 -17(δH 3.51, m) and HMBC correlations from H2-13 (δH 1,07, m) to C-15 (δC 166.02), from H2-14 (δH3.62, m) to C-16 (δC 166), from H2-15(δH3.49, m) to C-17 (δC 166.01) and from H2-16(δH3.55, m) to C-18 (δC 23.15) identified coupled olefins. The key HMBC correlations from H2-1’ (δH1.94, m) to C-3’ (δC 23.4), from H -3’ (δH2.14, m) to C-5’ (δC 29.05), from H3-14’ (δH1.85, m) to C-2’ (δC 215.7)and C-4’-OH (δC73.6), from H3-15’ (δH2.11, m) to C-6’ (δC 215.7) and C-4’-OH identified two meta position carbonyl group and one ortho position hydroxyl group(Fig
2B).
This connectivity was also secured by the observation of the HSQC correlations from H3-14’ to C-3’ and from H3-15’ 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 it’s solubility was 0.063g/ml in water. It could be slight soluble in methanol and DMSO, but insoluble in n-hexane, dichloromethane, chloroform, ethyl acetate and acetone.
The FT-IR spectrum of Griseococcin (1) showed (Fig. 3) a intense and broad characteristic absorption peaks at 3417.2 cm−1 represented the stretching vibration of O–H. The weak absorption peaks at 2356 and 2925.5 cm−1 were resulted from the stretching vibration of C–H. The absorption bands at 1637.4 and 1618.1 cm−1 are due to the vibration of C=C and C=O in the ester group. The absorptions peaks at 1456.1,1414 and 624 cm−1 were attributed to the presence of an internal C–H deformation. The strong absorption peak at 866 was resulted from aromatics. In conclusion, the typical absorption peak indicated that Griseococcin (1)was naphthoquinone with group O–H,C-H,C=C,C=O and so on [23].
In vitro antagonistic assay
Griseococcin (1) was assessed for antifungal activity against selected T.rubrum, T. mentagrophytes, E. floccosum, C. albicans and antibacterial activity against selected Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa. The results were shown in table 2, it displayed strong antifungal activity against T.rubrum, T. mentagrophytes with ZOI values of 18.06±0.85, 15.01±1.02mm and MIC values of 31.2, 31.2 mg/ml, as compared to the positive control Terbinafine with ZOI and MIC values of 20.67 ± 1.58, 28.33 ± 2.15mm and 15.6, 7.8 μg/mL, respectively. But while antibacterial activity was weak.