The flow rate was set at 0.4 mL/min and wavelength was 203 nm. Seventeen saponins (Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2S, Rg2R, Rg3S, Rg3R, Rh1, Rh2S, Rh2R, C-K, F1, F2; Sigma–Aldrich, St Louis, MO, USA) were used as standards for this purpose. Rg1, Rf, and Rc were the main contents, the retention time of Rg1 was 16.12 min, that of Rf was 19.18 min, and that of Rc was 19.53 min. The concentration

of Rg1 was 3.73%, that of Rf was 3.57%, and that of Rc was 1.87% (Fig. 1). Progress of analytical measurements in the study is shown in Table 1.The participants were asked to visit every CHIR-99021 mouse 2nd wk. Blood pressure, body weight, waist circumference, and body composition were measured at every visit, and blood analysis and stool analysis were checked on the 1st visit day (wk 0) and last day (wk 8). Blood pressure and heart rate were measured using an automatic digital sphygmomanometer. Wearing a hospital gown, body weight and height were measured to the nearest 0.1 kg and 0.5 cm, respectively. Waist circumference was measured

three times according to the World Health Organization method [21] by the same observer. Body composition was measured at every visit using the bioelectrical impedance analysis method (InBody 3.0; Biospace, Seoul, Korea). This device measures impedance through eight tactile electrodes placed on palms, thumbs, heels, and soles. Each participant stood upright, stepping onto the foot electrodes and loosely gripping the pipe-shaped hand electrodes with arms held vertically. Lean body mass, body mass index, PLX4032 and percent fat were measured and recorded. Blood tests including fasting glucose, high-density lipoprotein-cholesterol, triglyceride, and total cholesterol were performed prior to the start of the experiment and 8 wk later. At baseline, participants with high fasting blood glucose (>140 mg/dL) or possible liver problems (aspartate aminotransferase or alanine

aminotransferase >100 IU/L) were excluded. The participants were asked to bring their stool samples on the 1st visit day (wk 0) and last day (wk 8) in the stool-sampling container. The fresh human stools were collected and immediately stored Phenylethanolamine N-methyltransferase at –70°C. Genomic DNA were extracted from fecal samples of participants using a Fast DNA SPIN extraction kit (MP Biomedicals, Santa Ana, CA, USA), and fragments of the 16S rRNA gene (V1–V3) were amplified from the extracted DNA. The amplifications were performed according to previous reports using a barcoded fusion primer [22] and [23] using a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA). The amplified products were visualized on 2% agarose gel electrophoresis using the Gel Doc system (Bio-Rad). Amplicons were purified using the QIA quick PCR purification kit (Qiagen, Valencia, CA, USA) and quantified using the PicoGreen dsDNA Assay kit (Invitrogen, Carlsbad, CA, USA).