Ginsenosides

Starch was measured via an enzyme hydrolysis method. Starch was hydrolyzed into dual sugars by amylase, hydrolyzed into monosaccharides by hydrochloric acid, and finally determined by reducing sugar, which is converted to starch (Rose et al., 1991 (link)).
The contents of pyruvate in the sample were determined according to the methods of Lin et al. (1995 ). Protein was removed from the samples by TCA precipitation, and in the resulting sample, pyruvate reacted with 2,4-nitrophenylhydrazine. The product turned red in the presence of an alkali solution, and the intensity of the color change was measured by a spectrophotometer. A standard curve for calibration was obtained using sodium pyruvate as a reagent with a gradient of concentrations of pyruvic acid. Absorbance values were obtained to generate a standard curve to calculate the pyruvate concentration.
For glutathione (GSH), roots were ground in liquid nitrogen and homogenized in 1 mL 5% (w/v) m-phosphoric acid containing 1 mM diethylene triamine pentaacetic acid (DTPA) and 6.7% (w/v) sulfosalicylic acid. Root extracts were centrifuged at 12,000 × g for 15 min at 4°C. GSH contents were determined according to the methods of Kortt and Liu (1973 (link)) and Ellman (1959 (link)) with some modifications.
The ascorbic acid (AsA) content was determined according to Egea et al. (2007 (link)) with slight modifications. Ginseng roots were ground in an ice bath with 10 mL 5% metaphosphoric acid stored at 4°C, and then the final mix was homogenized by vortex. The final solution was maintained on the ice bath, in darkness, for 30 min and then centrifuged at 20,000 × g for 25 min at 4°C. Ascorbate was spectrophotometrically detected by measuring absorbance at 254 nm with a UV detector. For quantification of the compound, a calibration curve in the range of 10–100 mg kg⁻¹ was prepared from standard ascorbic acid. Results were expressed as mg 100 g⁻¹ FW.
Root extracts were centrifuged at 12,000 × g for 15 min at 4°C. The extraction and determination of ginsenosides was performed following the method of Yu et al. (2002 (link)).

Amylase (AMY, EC 3.1.1.2) activity was detected by the 3,5-dinitrosalicylic acid colorimetric method (Hao et al., 2007 ). Malate dehydrogenase (MDH, EC 1.1.1.37) activity was examined as described by Husted and Schjoerring (1995 (link)), with some modifications. Ten microliter samples were added to a 3 ml reaction mixture containing 0.17 mM oxalacetic acid and 0.094 mM β-NADH disodium salt in 0.1 M Tris buffer, pH 7.5. The reaction was measured by the decrease in absorbance at 340 nm for 180s in a spectrophotometer (Hitachi U-2001 Japan), the same reaction system only with sample buffer added in was used as a blank. Superoxide dismutase (SOD, EC 1.14.1.1) activity was measured according to the method of Zhang and Kirkham (1996 (link)), and Xu and Huang (2004 (link)). One unit of SOD activity is defined as the amount of SOD required to cause 50% inhibition of nitroblue tetrazolium (NBT) reduction at 560 nm min-1. Catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC.1.11.1.7) activity were determined based on the method of Chance and Maehly (1955 (link)) as described in detail for creeping bentgrass in Xu and Huang (2004 (link)). Enzyme activities were based on the absorbance change of the reaction solution per minute at a given wavelength for each enzyme: CAT at 240 nm and POD at 470 nm.
The activities of farnesyl diphosphate synthase (FDPS, EC. 2.5.1.10), cycloartenol synthase (CAS, EC. 5.4.99.8), squalene epoxidases (SE, EC:1.14.13.132), and squalene synthase (SS, EC. 2.5.1.21) involved in ginsenosides biosynthesis, were quantified by an indirect competitive enzyme-linked immunosorbent assay (ELISA). The optical density (OD) values of each sample were read by a BioTek ELx800 microplate reader at 450 nm. The primary concentration of each test sample was calculated from the linear regression equation based on the OD values of the standards.

The experiment was divided into HepG2 (–) and HepG2 (+) groups. DMEM media containing Re, CK, Rh2 (S), and Rg3 (S) ginsenosides were added into the upper culture chambers. Ginsenosides were converted into metabolites by the hepatic cells in the top layer and then diffused into the lower cell chambers where the target cells were stimulated by the drugs and their metabolites. The concentrations of ginsenosides are shown in Table 1. After 24 h of ginsenoside stimulation, the cell toxicity in the bottom chambers was analyzed using Calcein/PI cell viability assay kit. Dead cells were stained red, whereas the viable cells were stained green. Quantitative data on cell vitality were tested using the CCK-8 assay. A 50 μL cell culture medium with 10% CCK-8 reagent was added to the bottom chambers to analyze A549, MCF-7, and HL7702 cells. The solutions were collected into a 96-well plate, and their absorbances were measured using a microplate reader at 450 nm.
Cell apoptosis was assayed using the Annexin V-FITC apoptosis detection kit. Briefly, HepG2 (–) and HepG2 (+) groups were treated with DMEM media containing 0.5% DMSO and ginsenosides, respectively. The concentrations of ginsenosides are shown in Table 2. In total, 5 × 10⁵ cells were collected and washed twice using PBS. Cells were centrifuged at 1,000 g for 5 min at 4°C. The cells were then resuspended in 195 μL of binding buffer and incubated with 1 μL of Annexin V-FITC and 5 μL of PI staining solution for 15–20 min at room temperature. Cell apoptosis was measured using flow cytometry.

Polycarbonate porous membrane (Whatman, UK), Polydimethylsiloxane (PDMS, Dow Corning, USA), SU-8 3035 negative photoresist (MicroChem, USA), Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA), fetal bovine serum (FBS, Gibco, USA), trypsin and EDTA (Gibco, USA), rat tail type-I collagen (BD, USA), Calcein/PI cell viability assay kit (Beyotime, China), cell counting kit-8 (CCK-8, APExBIO, USA), Annexin V-FITC apoptosis detection kit (Beyotime, China), Capecitabine (CAP, purity≥98%, Sigma, USA), Re, Rg3(S), Rh2(S) and CK ginsenosides (purity≥98%) were purchased from Sichuan China. All chemical reagents were mass spectrometry reagent grade.

Data were expressed as mean ± SEM of each independent replication. For comparison of three or more replications, data were analyzed by t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. In compositional analysis of ginsenosides of P. ginseng extracts the two-way ANOVA were performed. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).