An endophytic Microbispora sp. GMKU363 was isolated from a root of Thai medicinal plant ‘Lin Ngu Hao’ (Clinacanthus siamensis Bremek.) according to the reported protocol.6 The strain was identified as a member of the genus Microbispora on the basis of 99.9% 16S ribosomal RNA gene sequence identity (1387 nucleotides; GenBank accession number GU459171) with the Microbispora mesophila JCM 3151T type strain (accession number AF002266).

Strain GMKU363 was cultured on Bn-2 slant agar medium consisting of soluble starch 0.5%, glucose 0.5%, meat extract (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) 0.1%, yeast extract (Difco Laboratories, Detroit, MI, USA) 0.1%, NZ-case (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 0.2%, NaCl 0.2%, CaCO3 0.1%, agar 1.5% and was inoculated into 500-ml K-1 flasks each containing 100 ml of the V-22 seed medium (pH 7.0) consisting of soluble starch 1%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, triptone (Difco Laboratories) 0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.05% and CaCO3 0.3%. The cultures were cultivated on a rotary shaker (200 r.p.m.) at 30 °C for 4 days. The seed culture (3 ml) was transferred into 500-ml K-1 flasks each containing 100 ml of the A-11M production medium (pH 7.0) consisting of soluble starch 2.5%, glucose 0.2%, yeast extract 0.5%, polypeptone (Wako Pure Chemical Industries, Ltd.) 0.5%, NZ-amine 0.5%, CaCO3 0.3% and Diaion HP-20 (Mitsubishi Chemical Co.) 1%. The cultures inoculated in flasks were cultured on a rotary shaker (200 r.p.m.) at 30 °C for 6 days, and the whole culture broth was extracted with 100 ml of 1-butanol on each flask by shaking for an additional hour. The organic layer was evaporated to give 3.0 g of crude extract from 1.5 l of culture. The crude extract (3.0 g) was subjected to silica gel column chromatography with a step gradient of CHCl3-MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1 and 0:1 v/v). The concentration of the fraction eluted with a 2:1 mixture of CHCl3-MeOH provided 0.23 g of dark viscous oil, which was further purified by preparative HPLC (Cosmosil AR-II, San Diego, CA, USA, 250 × 10 mm2) using 30% MeCN in distilled water at 4 ml min−1 to give linfuranone A (2.0 mg, tR=12.1 min).

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