CHEMICAL CONSTITUENTS FROM AN ENDOPHYTE, Cercosporella sp.

Cercosporella sp., an endophyte, was isolated from Schisandra chinensis Baill., a medicinal plant. Extracts of Cercosporella sp. are nontoxic to human lymphocytic MT-4 cells and exhibited potent anti-HIV activity in an in vitro MTT assay [1]. The selectivity index (SI) value was 5.6. The fungi were cultured in wheat bran culture medium (containing dextrose, monopotassium phosphate, magnesium sulfate) on a rocking bed without light. The cultured fungus was filtered to separate mycelium from fermentation liquor. Mycelia were rinsed to eliminate fermentation liquor before desiccation, and dry mycelia, about 832 g, were extracted twice with 95% ethanol for 1 h by reflux. The solvents were removed under reduced pressure to give a crude extract (120 g). The aqueous suspension of the extract was partitioned successively with light petroleum, EtOAc, and n-BuOH, resulting in light petroleum (56 g), ethyl acetate (13 g), and n-BuOH (12 g) extracts. The ethyl acetate-soluble fraction was concentrated and subjected to 200–300 mesh silica gel column chromatography eluting with a gradient of CHCl3–MeOH (from 0:1 to 1:1 ) to yield thirteen fractions. The subfractions were then purified by successive column chromatography on 300–400 mesh silica gel using light petroleum–EtOAc and CHCl3–MeOH as elution systems, monitored by TLC, which led to the isolation of compounds 1–6. The structures were elucidated by 1H NMR, 13C NMR, HSQC, HMBC, and MS analysis. All these data were in good agreement with the literature data. All compounds were isolated from Cercosporella sp. for the first time. Compounds 1 and 4 exhibited potent anti-HIV activity, and the SI values were respectively 3.22 and 2.53 in the in vitro MTT assay. 5,6,7-Trihydroxyflavone (1) [2], 19 mg; yellow needles; C15H10O5, mp 223–226 C. Mass spectrum (EI, 70 eV), m/z: 270 (M+, 100%), 242 (M – CO), 168, 140, 112, 102, 77, 69. 5,7-Dihydroxy-6-methoxyflavone (2) [3], 28 mg; saffron yellow needles; C16H12O5, mp 231–232 C. PMR spectrum (500 MHz, CD3OD, , ppm, J/Hz): 8.02 (2H, dd, J = 9.0, 2.0, H-2 , H-6 ), 7.57 (3H, m, H-3 , H-4 , H-5 ), 6.76 (1H, s, H-8), 6.28 (1H, s, H-3), 3.93 (3H, s, 6-OCH3). 13C NMR spectrum data (125 MHz, CD3OD, , ppm): 184.0 (C-4), 165.4 (C-7), 159.0 (C-9), 158.3 (C-2), 151.3 (C-5), 133.1 (C-4 ), 132.5 (C-6), 130.3 (C-1 ), 129.3 (C-3 , C-5 ), 127.4 (C-2 , C-6 ), 105.9 (C-10), 105.4 (C-3), 100.4 (C-8), 62.0 (6-OCH3). Mass spectrum (EI, 70 eV), m/z: 284 (M+), 269 (M – 15, 100%), 241, 167, 139, 103, 77, 69. Physcion (3) [4], 38 mg; red needles; C16H12O5, mp 203–205 C. PMR spectrum (600 MHz, CD3OD, , ppm, J/Hz): 12.31 (1H, s, OH), 12.11 (1H, s, OH), 7.63 (1H, dd, J = 1.5, 0.5, H-14), 7.37 (1H, d, J = 2.5, H-10), 7.08 (1H, dd, J = 2.5, 1.0, H-9), 6.69 (1H, d, J = 2.5, H-13), 3.94 (3H, s, H-16), 2.45 (3H, s, H-15). 13C NMR spectrum data (150 MHz, CD3OD, , ppm): 190.8 (C-1), 182.0 (C-2), 162.5 (C-3), 166.6 (C-4), 148.4 (C-5), 165.2 (C-6), 133.2 (C-7), 113.7 (C-8), 124.5 (C-9), 110.3 (C-10), 135.3 (C-11), 108.2 (C-12), 106.8 (C-13), 121.3 (C-14), 22.1 (C-15), 56.1 (C-16). Indole-3-carboxylic Acid (4) [5], 10 mg; flavo-green crystal; C9H7O2N, mp 232–234 C. Mass spectrum (EI, 70 eV), m/z: 161 (M+, 100%), 144 (M – OH), 132 (M – CHO), 116 (M – COOH). 4-Hydroxyphenylacetic Acid (5) [5], 5 mg; colorless needles; C8H8O3, mp 148–150 C. 2-(4 -Hydroxyphenyl)ethanol (6) [5], 60 mg; colorless needles; C8H10O2, mp 89–92 C. Mass spectrum (EI, 70 eV), m/z: 138 (M+), 120 (M – H2O), 107 (M – CH2OH, 100%).