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10 which had been cultured in ordinary M9 medium ,it 25°C on a reciprocal shaker (170 strokes /min) in the dark for 5 days. The cells were cultured under the same condition for further 14 days. The paraffin layer was diluted with 120 ml n-hexane, and the medium was extracted with another 120 ml n-hexane. The combined organic phases were extracted with 100 ml 0.5 N NaOH After washing with 2 × 100 ml n-hexane and incubation at room temperature for 10 min (hydrolysis), the aqueous phase was acidified with 5N HCl to pH 3 and extracted with 2 × 120 ml n-hexane. After evaporation of the solvent, the residue (shikonin) was dissolved in methanol and further purified by HPLC using a RP-18 column (250 × 4 mm, 5 μ) and a gradient of methanol in 1% aqueous HCOOH as solvent system. 5.5 mg labelled shikonin were obtained and subjected to NMR analysis.
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