5-Prenyltryptophol, a new inhibitor of bone morphogenetic protein-induced alkaline phosphatase expression in myoblasts, produced by Streptomyces colinus subsp. albescens HEK608

The producing microorganism S. colinus subsp. albescens HEK608, classified on the basis of the partial sequence of 16S rRNA genes (data not shown), was isolated from soil collected in Hegurajima, Wajima-shi, Ishikawa Prefecture, Japan. A loopful of spores of strain HEK608 was inoculated into 100 ml seed medium consisting of 1.0% starch, 0.5% glucose, 0.3% NZ-Case (Sigma-Aldrich, St Louis, MO, USA), 0.2% yeast extract (Oriental Yeast, Itabashi-ku, Tokyo, Japan), 0.5% Bacto-tryptone (Becton Dickinson and Company, Franklin Lakes, NJ, USA), 0.1% K₂HPO₄, 0.05% MgSO₄ 7H₂O, 0.3% CaCO₃ (adjusted to pH 7.0 before sterilization) in a 500-ml Erlenmeyer flask. The inoculated Erlenmeyer flask was incubated in a rotary shaker (210 r.p.m.) at 30 °C for 3 days to obtain the seed culture. For the production of 5-PT, the culture was initiated by transferring 3 ml seed culture into 25 × 500 ml Erlenmeyer flasks containing production medium (0.5% glucose, 2.0% glycerol, 2.0% soluble starch, 1.5% pharmamedia (Southern Cotton Oil, Richmond, TX, USA), 0.3% yeast extract, 1% Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), non-adjusted pH) and fermentation was carried out in a rotary shaker (210 r.p.m.) at 30 °C for 7 days.

The culture broth (2.5 l) was centrifuged at 8000 r.p.m. for 15 min to obtain the mycelium. The mycelium was treated with acetone (2.5 l) for 1 h, and acetone extracts were filtered to remove the mycelium. After concentration of the extracts to remove acetone, the aqueous solution was adjusted to pH 9.0 and extracted with EtOAc (2.5 l). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give brown materials (1.24 g). The materials were dissolved in a small amount of MeOH and purified by octa decyl silyl (ODS) column chromatography (i.d. 3.5 × 16 cm, Chromatorex ODS, 100–200 mesh; Fuji Silysia Chemical, Aichi-ken, Japan). The active materials were eluted stepwise with CH₃CN–H₂O (0, 20, 40, 60, 80 and 100%, 160 ml each). 5-PT was recovered from the 80% CH₃CN–H₂O fraction, which was concentrated under reduced pressure to give brown oily materials (48.8 mg). The materials containing 5-PT were dissolved in MeOH and finally purified by HPLC under the following conditions: column, CAPCELL PAK C18 (i.d. 20 × 250 mm; Shiseido, Tokyo, Japan); mobile phase, a linear gradient from 40 to 70% CH₃CN for 45 min; flow rate, 8 ml min⁻¹; detection, UV 210 nm. 5-PT was eluted as a peak with a retention time of 24 min. The fraction of the peak was collected and concentrated to dryness to give pure 5-PT (1.2 mg) as a yellow powder.

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