Paeoniflorin

Acetonitrile (100%), formic acid (99.9%), and distilled water were purchased from J.T. Baker (HPLC grade, USA). Reference compounds of albiflorin and paeoniflorin were purchased from Wako (Japan). z-Ligustilide was purchased from ChromaDex (USA). Nodakenin and decursin were divided from Ministry of Food and Drug Safety (Korea).

The reference standards, gallic acid, benzoic acid, and coumarin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, glycyrrhizin, and 6-gingerol were purchased from Wako (Osaka, Japan). Benzoylpaeoniflorin and liquiritin apioside were purchased from Biopurify Phytochemicals (Chengdu, China) and Shanghai Sunny Biotech (Shanghai, China). A standard stock solution of these components were dissolved in methanol at concentrations of 1.0 mg/mL. For HPLC analysis of the GJT extract, 100 mg of lyophilized GJT extract was dissolved in 20 mL of distilled water and then the solution was diluted to 10-fold for quantitative analysis of paeoniflorin. Solutions were filtered through a SmartPor GHP 0.2 μm syringe filter (Woong Ki Science Co., Seoul, Korea) before application to the column.

The GBH extract was analyzed using a Shimadzu Prominence LC–20A system (Kyoto, Japan), which consisted of a solvent delivery unit, an online degasser, a column oven, a sample autoinjector, and a photodiode array (PDA) detector. The data were acquired and processed using LCsolution software (Version 1.24, SP1, Kyoto, Japan). Amygdalin (PubChem CID: 656516, purity 99.0%), gallic acid (PubChem CID: 370, purity 99.0%), and coumarin (PubChem CID: 323, purity 99.0%) were purchased from Merck KGaA (Darmstadt, Germany). Albiflorin (PubChem CID: 51346141, purity 99.8%), paeoniflorin (PubChem CID: 442534, purity 98.8%), cinnamic acid (PubChem CID: 444539, purity 99.0%), and paeonol (PubChem CID: 11092, purity 99.9%) were obtained from Wako (Osaka, Japan). The eight marker components were separated on a Waters SunFire C₁₈ column (250 × 4.6 mm, 5 μm, Milford, MA, USA) and maintained at 40°C. The mobile phases consisted of water (A) and acetonitrile (J. T. Baker, Phillipsburg, NJ, USA) (B), both containing 1.0% (v/v) acetic acid (Merck KGaA, Darmstadt, Germany). The gradient elution of the mobile phase was as follows: 10–60% B for 0–30 min, 60–100% B for 30–40 min, 100% B for 40–45 min, and 100–10% B for 45–50 min. The flow-rate and injection volume were 1.0 ml/min and 10 μl, respectively.

Eight reference standards (albiflorin, cinnamaldehyde, cinnamic acid, daidzin, glycyrrhizin, liquiritin, paeoniflorin, and puerarin) were purchased from Wako (Osaka, Japan) and the purities of these reference standards were greater than 98.0%, as evaluated via high-performance liquid chromatography (HPLC). The HPLC-grade reagents, methanol, acetonitrile, and water were obtained from J.T. Baker (Phillipsburg, NJ, USA). Analytical-grade glacial acetic acid was procured from Junsei (Tokyo, Japan).