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The preparative scale biotransformation: The preparative scale biotransformation of 1 by P. cactorum ATCC 32134 were carried out by a two-stage procedure in potato dextrose medium (PDA). Cultures were incubated with shaking at 180 rpm at 28 °C on rotary shakers. one milliliter of inoculum derived from 24 h old stage I culture was used to initiate stage II cultures, which were incubated for 24 h before receiving 5 mg of ruscogenin in 1 ml of acetone, a total of 120 mg of 1 was distributed evenly among 24 culture flasks, and incubations were conducted as before. The cultures were extracted with EtOAc at 120 h after addition of substrate. The extract was subjected to silica gel column chromatography eluted with chloroform-methanol (10:1, v/v) to afford the product (compound 4, 30 mg).
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2 in a 96-well plate at 37 °C for 15 min. Then, 50 μL of factor Xa chromogenic substrate (0.5 mM) containing 100 mM EDTA (pH 8.4) was added, and the absorbance was measured at 405 nm. Purified reconstituted human TF was used to generate a standard curve.
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