Lc 20a liquid chromatograph

The extraction and analysis of flavonoid compounds were carried out as described by Li et al. [29 (link)]. Briefly, the frozen tissue powder (0.5 g) was ground in 1.5 mL phenolic compound, extracting a solution containing 70% methanol and 2% formic acid at 0–4 °C. After centrifugation at 10,000 g for 20 min, the supernatant was passed through a 0.22-μm syringe filter prior to analysis.
The Inertsil ODS-3 column (5.0 μm particle size, 4.6 mm × 250 mm, GL Sciences Inc., Tokyo, Japan) was used for the separation, preceded by an Inertsil ODS-3 Guard Column. A 10-μL sample of the filtered supernatant was injected into an LC-20A Liquid Chromatograph with a diode array detector (Shimadzu Corporation, Tokyo, Japan). Solvent A consisted of 10% formic acid (11.36% 88% formic acid) in water and solvent B consisted of 10% formic acid and 1.36% water (11.36% 88% formic acid) in acetonitrile. The gradients used were 95% A (0 min), 85% A (25 min), 78% A (42 min), 64% A (60 min), and 95% A (65 min) with a post-run time of 10 min. The flow rate was 1.0 mL min⁻¹, and the column temperature was set at 35 °C. Flavonoid compounds were detected at 365 nm for flavonols and 520 nm for anthocyanins.

We took a mixture of Danshen and Chuanxiong (10 kg each), added it with 160 L water, and boiled it for 2 h. Afterwards, the extracted solution was poured out, and the remaining residue was extracted twice, in which 120 L water was added each time and boiled for 1.5 h. The three extracts were merged to concentrate to 15 L at 60°C in a single-effect concentrator, and the solution after concentration was transferred to a rotary evaporator to concentrate to approximately 9 L at 60°C. About 35 L of 95% ethanol solution was added to the concentrated liquor, and was allowed to stand overnight. The supernatant was taken and concentrated to about 5 L at 60°C using a rotary evaporator. The concentrate was placed into a vacuum drying oven, dried thoroughly at 60°C, and powdered homogeneously with a powder machine to obtain solid powder of about 1.5 kg (raw dose of 12.85 g/g). Subsequently, 1.0 g of the powder was accurately weighed, brought to 20 mL with 50% methanol added, and sonicated for 10 min. It was then filtered through a 0.45-μm microporous filter column, from which an appropriate amount was injected into HPLC-MS instrument for analysis (Shimadzu LC-20A liquid chromatograph, Shimadzu, Japan; API 3200 LCMS/MS Mass Spectrometry System, American AB SCIEX, USA).