2.1 Structure elucidation
Eight polyketides were isolated from rice fermentation products of Neohelicosporium griseum GZCC 23–0142. Their structures are shown in Fig. 2.
Vertixanthone (1) was obtained as a yellow crystal; ESI-MS at m/z 293.0 [M + Na]+, 563.1 [2M + Na]+, 269.0 [M-H]-, HR-ESI-MS at m/z 293.04229 [M + Na]+, 563.09495 [2M + Na]+, 269.04675 [M-H]- (calcd for C15H10O5, 270.05266). 1H NMR and 13C NMR data are shown in Figures S1 ~ 2. The data above closely parallels 1-hydroxy-10-methoxy-dibenz[b,e]oxepin-6,11-dione (a) [8] and vertixanthone [9], making it challenging to establish whether its structure is compound a or vertixanthone (Fig. 3). Consequently, we performed additional tests on the compound’s HSQC and HMBC spectra (Figures S1 ~ 2). The molecular formula, 1D-NMR, and HSQC correlation (Figure S1 ~ S3) indicated signals for an exchangeable proton, six aromatic protons, a methoxyl, and eight non-protonated carbons, five of which are oxygenated. The key HMBC correlation from H-3 (δH 7.95 ppm) to C-1 (δC 133.2 ppm) revealed that C-1 (δC 133.2 ppm) is not an oxygenated carbon (Figs. 2 ~ 4), thus establishing the structure of compound 1 as vertixanthone rather than compound a.
Diaportheone A (2) was obtained as a light-yellow crystal; [a]25 D-58.9 (c 0.1, CHCl3). ESI-MS at m/z 241.0 [M + Na]+, HR-ESI-MS at m/z 241.04749 [M + Na]+, 217.05118[M-H]−(calcd for C12H10O4, 218.05771). 1D-NMR data are shown in Figures S6 ~ 7. These data are consistent with those reported in reference [10–11], so the structure of compound 2 was identified as Diaportheone A.
1,3,6,8-tetrahydroxyanthraquinone (3) was obtained as a red crystal; ESI-MS at m/z 271.0 [M-H]-, HR-ESI-MS at m/z 271.02578 [M-H]- (calcd for C14H8O6, 272.02578). 1D-NMR data are shown in Figures S9 ~ 10. These data are consistent with the reference [12–13], so compound 3 was identified as 1,3,6,8-tetrahydroxyanthraquinone.
Lecanoric acid (4) was obtained as a white amorphous powder; ESI-MS at m/z 317.3 [M-H]-, HR-ESI-MS at m/z 317.06809 [M-H]- (calcd for C16H14O7, 318.06809). 1D-NMR data are shown in Figures S12 ~ 13. These data are consistent with the reference [14], so compound 4 was identified as lecanoric acid.
Decarboxycitrinone (5) was obtained as a white solid. ESI-MS at m/z 221.1 [M + H]+, 463.1 [2M + Na]+, HR-ESI-MS at m/z 221.08128 [M + H]+, 243.06303 [M + Na]+, 219.06724[M-H]-, (calcd for C12H12O4, 220.07426). 1D-NMR data are shown in Figures S15 ~ 16. These data are consistent with the reference [15–16], so compound 5 was identified as decarboxycitrinone.
6,8-Dihydroxy-4-hydroxymethyl-3,5-dimethyl-isochromen-1-one (6) was obtained as a colourless oil. ESI-MS at m/z 235.1 [M-H]-, HR-ESI-MS at m/z 235.06223 [M-H]- (calcd for C12H12O5, 236.06223). 1D-NMR data are shown in Figures S18 ~ 19. These data are consistent with the reference [16–17], so compound 6 was identified as 6,8-dihydroxy-4-hydroxymethyl-3,5-dimethyl-isochromen-1-one.
Decarboxyhydroxycitrinone (7) was obtained as colourless needles. ESI-MS at m/z 235.1 [M-H]-, HR-ESI-MS at m/z 237.07927 [M + H]+, 259.05806 [M + Na]+, 235.06211 [M-H]- (calcd for C12H12O5, 236.07069). 1D-NMR data are shown in Figures S21 ~ 22. These data are consistent with the reference [18], so compound 7 was identified as decarboxyhydroxycitrinone [18].
Ergosterin (8) was obtained as a white needle crystal; 1D-NMR data are shown in Figures S24 ~ 25. The above data are consistent with the reference [6, 19], so the structure of compound 8 was identified as ergosterin.
2.4 Biological Activity
Compounds 1–8 were tested for their antimicrobial activity against Candida albicans, Gram-positive bacteria Staphylococcus aureus, and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. They were also tested for cytotoxicity against four mammalian cell lines: human ovarian cancer cell A2780, human non-small cell lung cancer cell A549, human liver cancer cell HePG2, and human cervical cancer cell HELA. Compounds 1–4 exhibited moderate activity against Pseudomonas aeruginosa, with an MIC of 62.5 µg mL-1 and an MBC of 250 µg mL-1. Compound 3 showed weak activity against Staphylococcus aureus, displaying an MIC of 125.0 µg mL-1 and an MBC of 500.0 µg mL-1 (Table 1). In-vitro cytotoxic testing revealed that compounds 1, 3, and 4 possessed cytotoxic activity against the HELA cell line, with corresponding IC50 values of 30.8, 13.7, and 14.1 µM. Compounds 1, 3, and 4 also showed considerable cytotoxicity against the A549 cell line, with IC50 values of 24.7, 7.4, and 10.3 µM, respectively (Table 2).
Table 1
Minimum inhibitory concentrations (MIC, µg mL-1) and Minimum Bactericidal concentrations (MBC, µg mL-1) of 1–4 against bacterial test organisms.
Compound
|
P. aeruginosa
|
S. aureus
|
E. coli
|
C. albicans
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
1
|
62.5
|
250.0
|
-
|
-
|
-
|
-
|
-
|
-
|
2
|
62.5
|
250.0
|
-
|
-
|
-
|
-
|
-
|
-
|
3
|
62.5
|
250.0
|
125.0
|
500.0
|
-
|
-
|
-
|
-
|
4
|
62.5
|
250.0
|
-
|
-
|
-
|
-
|
-
|
-
|
5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
6
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
7
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
8
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Gentamicin
|
0.2
|
0.78
|
0.05
|
0.1
|
-
|
-
|
-
|
-
|
(–) = no activity investigated |
Table 2
Cytotoxicity of 1–8 against mammalian cell lines [half maximal inhibitory concentration (IC50): µM].
Compound
|
HELA
|
A549
|
A2780
|
HePG2
|
1
|
30.8
|
24.7
|
-
|
-
|
2
|
NT
|
NT
|
NT
|
NT
|
3
|
13.7
|
7.4
|
-
|
-
|
4
|
14.1
|
10.3
|
-
|
-
|
5
|
NT
|
NT
|
NT
|
NT
|
6
|
NT
|
NT
|
NT
|
NT
|
7
|
NT
|
NT
|
NT
|
NT
|
8
|
NT
|
NT
|
-
|
NT
|
Adriamycin
|
3.2
|
7.1
|
1.1
|
4.3
|
(–) = no activity investigated, NT = not tested |