Puerarin

To analyze CQA contents of plant tissues, 20 mg dry weight of sunflower sprout tissue and 20 mg fresh weight of N. benthamiana leaves were extracted with 1 ml of 80% (v/v) methanol containing an internal standard, 0.05 g L⁻¹ puerarin. The reactions were mixed vigorously at 15°C for 15 min by shaking at 1,500 rpm and then centrifuged at 12,000 × g for 15 min. Supernatant was collected and filtered through 0.2 μm nylon syringe filters.
A Shimadzu UFLC system equipped with an SPD-M20A photodiode array detector (Shimadzu, Japan) and Kinetex® C18 (250 mm × 4.6 mm, 5 μm; Phenomenex®, USA) was used to analyze 10 μl of the extract from sunflower sprouts and N. benthamiana leaves. Chromatographic separation was performed using 0.1% (v/v) TFA in water (solvent A) and 0.1% (v/v) TFA in acetonitrile (solvent B) as the mobile phase. The following elution gradient was used: 5% B for 5 min, 5–15% B for 10 min, a 25-min hold, 15–100% B for 4 min, a 2-min hold, 100–5% B for 4 min, and a 5-min hold. The flow rate was set at 1.5 ml min⁻¹, and the column oven temperature was maintained at 40°C. UV spectra were acquired in the range of 190–800 nm, and chromatograms were obtained at 320 nm. Peaks corresponding with the retention time and UV spectrum of a commercial standard were identified as CQAs. Amounts of each CQA were calculated according to the calibration curve in the range of 0.5–0.007825 mg ml⁻¹. Puerarin was used as an internal standard (Sigma-Aldrich, USA). All CQA standards used in this study were purchased from Carbosynth, England.
Additionally, to confirm identities of the CQAs, the components were analyzed using an Agilent UHPLC system (Agilent Technologies, USA) using Kinetex® C18 (250 mm × 4.6 mm, 5 μm; Phenomenex®, USA). The following elution gradient was used: 0–5% B for 5 min, 5–15% B for 30 min, a 65-min hold, 15–100% B for 5 min, a 5-min hold, 100–5% B for 5 min, and a 10-min hold. The flow rate was set at 0.5 ml min⁻¹, and the column oven temperature was maintained at 40°C. For MS/MS analysis, QTRAP® 4,500 MS/MS System (AB Sciex™, USA) in multiple reaction monitoring (MRM) and negative ionization mode (ESI-) was used. Operating conditions for MS analysis were as follows: heat block temperature of 500°C, curtain nitrogen gas 30 psi, nebulizer and auxiliary gases of 50 psi, collision nitrogen gas at medium position, ionization voltage of −4,500 V, and entrance potential (EP) of −10. For the tested compounds, the following transition under optimal instrumental conditions of collision energy (CE) of −35 eV, declustering potential (DP) of −50 V, and collision cell exit potential (CXP) of −12 V.

Using the chemical‐protein binding method, the pivotal targets were screened out and identified for puerarin‐based molecular docking analysis. After searching for specific proteins through the PDB database, 5R84 protein was selected to dock with the puerarin compound. The ChemBio3D Draw in Chem Bio Office 2010 software was used to conduct the three‐dimensional structure of puerarin before docking the molecular structures using AutoDock Vina software. The rationality of the docking parameter setting was assessed according to the root‐mean‐square deviation (RMSD) of the ligand molecule. The RMSD ≤ 4 Å was the threshold for the conformation of the ligand molecule.²⁶, ²⁷

Using the R language packages, such as ‘ClusterProfiler’, ‘ReactomePA’, ‘org.Hs.eg.Db’ and ‘GOplot’ in the R language (3.6.1), the enrichment analysis and visualization of the biological process and KEGG pathways of the intersection targets were performed and obtained accordingly. Moreover, the information on gene annotation resulted from ‘org.Hs.eg.Db’, p‐value cut‐off = 0.05, q‐value cut‐off = 0.05 for enrichment prior to plotting the corresponding bubble chart, histogram and Circos circle chart.²⁴, ²⁵

The obtained product, puerarin palmitate, was purified by liquid–liquid extraction. The immobilized enzyme in the reaction mixture that reacted under optimal conditions was isolated through filtration, and the reaction solution was dried using a vacuum desiccator. The powder obtained by drying was dissolved in 35 mL of an acetonitrile/n-heptane (2:5, v/v) solution at 60 °C. The unreacted acyl donor in the n-heptane phase was then removed by separating the acetonitrile phase. The separated acetonitrile phase was then dried in a vacuum desiccator. To remove unreacted puerarin, the obtained powder was dissolved in 40 mL of an ethyl acetate/water (3:5, v/v) solution at 60 °C. Puerarin palmitate was finally obtained by separating and drying the ethyl acetate phase in which the puerarin ester had been dissolved. All the processes were repeated three times to improve the purity of the product. Afterwards, it was purified using thin-layer chromatography (TLC) and column chromatography on silica to obtain a higher-purity product. A mixture of ethyl acetate:chloroform:methanol (5:4:1, v/v/v) was used as the eluent.