Ginseng extract was prepared according to the method described by Bae et al [12]. Briefly, the dried root of Panax ginseng Meyer (1 kg) produced at Kumsan (Chungnam, Korea) was extracted selleck screening library with 70% ethanol twice, concentrated, and freeze-dried (yield, 18%). The extracted powder contained 8.9% ginsenoside Rb1 and 1.4% ginsenoside Rd. The ethanol extract was suspended in water and successively extracted with hexane and butanol. The butanol fraction was

separated by silica gel column chromatography to yield ginsenosides Rb1 (purity > 92%, 52 mg) and Rd (purity > 94%, 8 mg). NUTRIOSE, a mixture of glucose polymers with a fairly narrow molecular weight range (number-average molecular weight, LY294002 research buy 200–4000 Da; weight-average molecular weight, 4000–6000; degree of polymerization, 12–25), was kindly donated from Roquette (Lestrem, France). Rat fecal specimens (n = 5, approximately 0.2 g) were collected in plastic cups and suspended in 1.8 mL cold saline [19]. The fecal bacterial suspension was centrifuged at 500 × g for 5 min, and the resultant supernatant was sonicated and centrifuged at 10,000 × g for 30 min. The resultant supernatant was used as a crude enzyme solution. To investigate the effect of diet on the metabolic activation of ginsenoside Rb1 to ginsenoside Rd by intestinal microbiota cultured in Gifu anaerobic

broth (GAM broth), the fresh stool specimen was suspended in GAM broth and centrifuged at 500 × g. The resultant supernatant was inoculated in dextrose (1%) or NUTRIOSE (1%) containing GAM broth (glucose-free broth) and cultured for 24 h. The cultured media was collected by centrifugation

(10,000 × g, 20 min). The precipitate was used as the crude enzyme for assaying the metabolism of ginsenoside Rb1 to Rd. The generated Rd was assayed by high performance liquid chromatography (HPLC). For assaying the contribution Y-27632 of fecal activity in the metabolism of ginsenoside Rb1 to ginsenoside Rd, a reaction mixture (2 mL) containing 0.2 mL of the fecal culture prepared from freshly collected rat feces (n = 5) and 0.2 mL of 0.1mM ginsenoside Rb1was incubated at 37°C for 1 h, which was followed by the addition of 2 mL of MeOH to stop the reaction. The reaction mixture was centrifuged at 3000 × g for 10 min, and the levels of ginsenoside Rb1 and its metabolite ginsenoside Rd in the resultant supernatant were analyzed by HPLC. The HPLC system was as follows: a Hewlett Packard series 1050 module, a UV detector (Ramsey, MN, USA) set at 203 nm, a Hypersil ODS column (4.6 × 150 mm i.d., 5.0 μm; Agilent, Santa Clara, CA, USA), linear-gradient mixture of 30% water and 70% acetonitrile for 15 min as elution solvent, flow rate of 1.0 mL/min, and injection volume of 20 μL. Male Sprague–Dawley rats (210–240 g) were supplied by the Orient Experimental Animal Breeding Center (Gyunggi-do, Korea).