4.2. Saponin Extraction and HPLC analysis

Standards of saponins (i.e., Rg1, Re, Rd, Rb1, Rb2, Rc, and R1) were purchased from Shanghai Tauto Biotech Company (Shanghai, China). These standards have more than 98.0% purity, and their numbers are 16,042,724; 160,907; 160,924; 160,930; 160,606,121; 16,081,931; and 160,923, respectively. The standard stock solutions were dissolved with methanol (Analytical Grade, Fisher, Hampton, NH, USA).Saponins were extracted through ultrasonic extraction in accordance with previously described methods [15]. In brief, the samples were crushed, and 0.1 g of each sample was soaked in 1.0 mL of methanol solution. After more than 10 s of vortexing and 10 min of sonication, the mixture was frozen at −20 °C for 1 h and then centrifuged at 10,000 rpm for 10 min. The upper layer was collected, filtered through a 0.45 µm organic micropore filter, and transferred for HPLC analysis.

The samples were analyzed with an Agilent HPLC 1260 series system (Agilent, Santa Clara, CA, USA) equipped with a VWD detector, a quaternary pump, a column compartment, and an autosampler. A C₁₈ reversed phase Eclipse XDB column (5 μm, 4.6 mm × 250 mm, Agilent, Santa Clara, CA, USA) was used for the separation. The gradient was composed of acetonitrile (A) and water (B), and the linear gradient was set as follows in accordance with the Pharmacopoeia of the People’s Republic of China: 0–12 min for 19% A and 12–60 min for 19% A to 36% A. The following parameters were also used: sample injection volume of 10 µL, wavelength of 203 nm, column temperature of 25 °C, and flow rate of 1.0 mL·min⁻¹.