Agilent 1260 series system

Dried D. huoshanense powder (0.12 g) was defatted with 80% alcohol at 80°C for 4 h and then extracted with 100 mL of distilled water at 100°C for 1 h, as previously described (Mohan et al., 2020 (link)). The filtered supernatant (1.0 mL) was hydrolyzed with 0.5 mL of 3.0 M HCl at 110°C for 1 h in a sealed glass and neutralized to pH 7.0 with 3.0 M NaOH after being cooled to room temperature. The hydrolyzed products (0.4 mL) were added to a solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) consisting of 0.3 M aqueous NaOH (0.4 mL) and 0.5 M methanol (0.4 mL), and then kept at 70°C for 100 min in an oven. After neutralization with 0.3 M HCl, the PMP derivatives were determined in an Agilent Series 1260 system (Agilent Technologies, Shanghai, China) equipped with a Topsil C18 HPLC column (4.6 mm × 250 mm, 5 μm particle size) and monitored by UV absorbance at 250 nm. Elution was carried out using (A) 0.02 M amine acetate solution and (B) acetonitrile as a mobile phase. The ratio of A to B was 80:20. The flow rate was 1.0 mL/min, the sample injection volume was 10 μL, and the column temperature was 30°C. Glucose (100 μg/ml) and mannose (100 μg/ml) were used as internal standards, and there were three biological replicates for each sample in this experiment were used.

Oleanolic acid, β-amyrin and three saponin monomers (platycoside E, platycodin D3, and platycodin D) standards were purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China). Metabolites were extracted from 0.5 g of P. grandiflorus root treated with MeJA concentrations of 0, 10, 50, 100, or 200 μM by adding 1 ml of methanol, sonicating for 30 min, centrifuging at 12 000 rpm for 10 min at 4°C, and filtering through a 0.22-μm syringe membrane for High-Performance Liquid Chromatography (HPLC) analysis. The HPLC detection was performed on the Agilent Series1260 system (Agilent Technologies Inc., USA), with Techmate C18 column (250 × 4.6 mm, 5 μm). The mobile phase for oleanolic acid and β-amyrin was methanol (A)-water (B) (90:10 v/v), with detection wavelengths of 203 and 215 nm, respectively. The mobile phase for saponin monomers was acetonitrile (A)-water (B) (27:73 v/v), with a detection wavelength of 215 nm. The sample injection volume was 10 μl, the flow rate was 1 ml/min, column temperature was 30°C [51 (link)]. Each sample was performed with three biological replicates.

Deionized water was used throughout the study. Ethanol was analytical grade. ¹H and ¹³C NMR spectra were collected on an Avance III 600 spectrometer (Bruker, Switzerland) with tetramethylsilane (TMS) as an internal standard in pyridine-d₅ (Cambridge Isotope Laboratories, Inc.). An ultra-high-performance liquid chromatograph connected to a triple quadrupole tandem mass spectrometer (UPLC-Xevo TQ MS, Waters, United States) was used to obtain electrospray ionization–mass spectrometry (ESI-MS) data. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), a macroporous resin column (D101, Jiangsu Donghong Chemical Co., Ltd., China), and thin-layer chromatography (TLC) plates (Qingdao Haiyang Chemical Co., Ltd., China) were used to monitor the compound. The TLC spots were visualized under ultraviolet (UV) light at 254 nm and stained by spraying the TLC plates with 5% (v/v) H₂SO₄ in alcohol followed by heating. Semi-preparative high-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 series system (Agilent Technologies, United States) equipped with a diode array detector, and preparative HPLC was conducted with a Hanbon series system (Jiangsu Hanbon Science & Technology Co., Ltd., China) equipped with a UV detector, using two types of C18 columns produced by Agilent (Zorbax SB-C18 column, 5 μm; ϕ 9.4 × 250 mm and ϕ 4.6 × 250 mm).

The organic solvents, including methanol (MeOH), hexane (Hx), ethyl acetate (EtOAc), butanol (BuOH), and methylene chloride (MC) were purchased from Duksan Chemical Co. (Seoul, Korea). The column chromatography used was silica gel 60 (Merck 230–400 mesh, ASTM, Darmstadt, Germany). The preparative TLC was performed using 20 × 20 cm plates coated with 1 mm-thick F254 silica gel (Merck, Darmstadt, Germany). The NMR spectra were recorded on a JEOL ECX-500 spectrometer, operating at 500 MHz for ¹H and 125 MHz for ¹³C NMR spectra (JEOL Ltd., Tokyo, Japan). The determination of high-performance liquid chromatography (HPLC) spectra were recorded on an Agilent 1260 series system (Agilent Inc., Palo Alto, CA, USA) with a photodiode array (PDA) and an evaporative light scattering detector (ELSD).

Column chromatography was performed using silica gel (70–230 mesh; Merck, Darmstadt, Germany). High-performance liquid chromatography was performed using the Agilent 1260 series system (Agilent Technologies, Santa Clara, CA, USA) with a C18 column (Phenomenex Synergi 10 μ Hydro-RP 80A, 10 μm, 4.6 mm × 250 mm, Torrance, CA, USA). Gentianae Scabrae Radix roots were purchased from a commercial herbal medicine market. A voucher specimen, P542, was deposited in the Natural Products Bank at the National Institute for Korean Medicine Development (NIKOM).