Isocoumarins and Dihydroisocoumarins From the Endophytic Fungus Biscogniauxia capnodes Isolated From the Fruits of Averrhoa carambola

Abstract

An endophytic fungus Biscogniauxia capnodes was isolated from a popular edible fruit Averrhoa carambola. The fungus was fermented in potato dextrose broth for 3 weeks, and then the culture broth and mycelium were extracted with ethyl acetate. Chromatographic separation of this extract furnished 2 isocoumarins, reticulol (1) and 6-O-methyl-reticulol (2), and 2 dihydroisocoumarins, 5-methylmellein (3) and 7-hydroxy-5-methylmellein (4). Compound 1 showed moderate antioxidant activity against 2,2′-diphenyl-1-picrylhydrazyl radicals (IC₅₀ value, 58 μg/mL). This is the first report of the isolation of B. capnodes as an endophyte, as well as the compounds 1 to 4 from B. capnodes.

Endophytes, microorganisms that reside in the tissues of living plants, have been attracting a growing interest as potential sources of novel natural products for exploitation in medicine, agriculture, and industry. In a continuation of our search for bioactive compounds from endophytic fungi from Sri Lankan flora, ¹ we have investigated the secondary metabolites produced by an endophytic fungus, Biscogniauxia capnodes (Family: Xylariaceae), isolated from the fruits of Averrhoa carambola L. (Oxalidaceae), commonly called star fruit. A variety of carotenoid-derived compounds have been reported from star fruit. ² We previously reported the chemistry and phytotoxic activity of the constituents of A. carambola fruits. ³

An endophytic fungus isolated from A. carambola fruits was identified as B. capnodes by morphological studies and sequence analysis of internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA). This is the first report of the isolation of B. capnodes from A. carambola fruits. Biscogniauxia capnodes was cultured in potato dextrose broth (PDB). The ethyl acetate (EtOAc) extract of the broth and mycelium showed antifungal activity against Cladosporium cladosporioides, antioxidant activity against 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radicals (42.5% inhibition at 25 ppm), high brine shrimp lethality (96.7% at 125 ppm), and phytotoxicity in the lettuce (Lactuca sativa) seed germination assay (shoot inhibition 45.3% and root inhibition 55.3% at 200 ppm). Chromatographic separation of the EtOAc extract furnished two isocoumarins, reticulol (1) ⁴ and 6-O-methylreticulol (2), ⁵ and 2 dihydroisocoumarins, 5-methylmellein (3) ⁶ and 7-hydroxy-5-methylmellein (4) (Figure 1). ⁷ The structures of compounds 1 to 4 were elucidated by the analysis of nuclear magnetic resonance (NMR) spectroscopic data and by comparison with reported data, as well as mass spectrometry (MS) data (Table S1). Among these secondary metabolites, compound 3 was most abundant. Compounds 3 and 4 were found to have (3R)-absolute configuration on the basis of their negative optical rotations (3: [α]_D ²⁵ −98.9 (c, 1.87, CHCl₃) (lit. [α]_D −90 (CHCl₃) ⁸ and 4: [α]_D ²⁵ −88.9 (c, 0.18, CHCl₃) (lit. [α]_D −57.4 (CHCl₃). ⁷ It seems likely that the fungi biosynthesize preferentially (3R)-enantiomers of 3 and 4, since the (3S)-antipodes have not been reported from natural sources thus far. Compounds 1 to 4 were subjected to the above-mentioned bioassays, but no significant activities were observed except for reticulol (1), which exhibited a moderate DPPH radical scavenging activity with an IC₅₀ value of 58 µg/mL (IC₅₀ of a positive control butylated hydroxyanisole was 5.5 µg/mL).

Figure 1

Structures of compounds 1 to 4.

Compounds 1 to 4, all possessing an isocoumarin skeleton, are rather common fungal metabolites, except for compound 4. Reticulol (1) has been reported as an inhibitor of cyclic adenosine 3′,5′-monophosphate phosphodiesterase. ⁹ Further, reticulol was suggested to inactivate Topo I, which is involved in tumor metastasis, and exhibit excellent cytotoxicity against B16F10 melanoma when combined with adriamycin. ¹⁰ Recently, reticulol has been reported to decrease degranulation and histamine release significantly. ¹¹ 6-O-Methylreticulol (2) was previously isolated from Streptomyces mobaraensis ¹² and the liverwort Wettsteinia schusterana. ¹³ α-Glucosidase inhibitory activity of 2 has been reported recently. ¹⁴ 5-Methylmellein (3), isolated from the endophytic fungus Biscogniauxia mediterranea that was separated from the medicinal cactus Opuntia humifusa was shown to have antifungal activity against the plant pathogen Phomopsis obscurans. ⁶ 7-Hydroxy-5-methylmellein (4) was previously reported from the marine-derived fungi Acremonium sp. and Nodulisporium sp. ⁷ This paper represents the second isolation of this rare dihydroisocoumarin.

Experimental

General

¹H and ¹³C NMR were recorded on a JEOL JMN-AL300 (300 MHz for ¹H and 75 MHz for ¹³C) spectrometer (JEOL, Tokyo, Japan) in CDCl₃ solution. Fast atom bombardment mass spectra were obtained on a JEOL JMS-700 spectrometer (JEOL, Tokyo, Japan), while electrospray ionization mass spectra were recorded on Hitachi Chromaster 5610 MS detector (Hitachi, Tokyo, Japan).

Isolation and Identification of the Endophytic Fungus

Healthy, matured, and ripened fruits of A. carambola were collected from a home garden in Kandy in 2014. The fruits were rinsed gently in running tap water and triple sterilized with 2% sodium hypochlorite (NaOCl) and 75% ethanol and finally washed with sterile distilled water. A few segments of the inner fleshy part of the fruit were placed on potato dextrose agar medium and allowed to stand at room temperature for 2 to 3 days in the dark. The emerging fungus was subcultured serially to obtain a pure culture of the fungus, which was identified as B. capnodes by sequence analysis of ITS1 and ITS4 regions of the rDNA gene. Search using Basic Local Alignment Search Tool indicated that the sequence of the ITS region matched 95% that of B. capnodes (Gene Bank Acc. No. EF026131.1). Fungal identification was carried out by GeneTech Institute, Colombo. Photographic evidence of the fruits of A. carambola and B. capnodes strain (TAC-2014) is deposited at the National Institute of Fundamental Studies.

Fermentation of Fungus, Extraction, Isolation of Compounds, and Bioassays

Biscogniauxia capnodes was cultured by inoculating flasks containing 400 mL of PDB medium. The flasks were allowed to stand at room temperature for 10 days, then shaken every other day on a laboratory shaker. After 21 days, the culture media were filtered and the filtrate extracted with EtOAc (×3). The residual mycelium was extracted with EtOAc using a sonicator. Based on the thin-layer chromatography (TLC) analysis both EtOAc extracts were combined. The combined extract was screened for antifungal activity against C. cladosporioides by a TLC bioautography method, ¹⁵ DPPH radical scavenging activity with a spectrophotometric method, ¹⁶ brine shrimp (Artemia salina) lethality, ¹⁷ phytotoxicity using lettuce seed germination assay, ¹⁸ and α-amylase inhibitory activity using a spectrophotometric method. ¹⁹ Chromatographic separation of the EtOAc extract (1.46 g) over silica gel, Sephadex LH-20, and preparative TLC furnished reticulol (1, 5 mg), 6-O-methylreticulol (2, 11 mg), 5-methylmellein (3, 36 mg) and 7-hydroxy-5-methylmellein (4, 4 mg).