Fig.6 The MS/MS fragmentation pathway of p-Coumaric acid (a),
4-O-β-Glucopyranosyl-cis-coumaric acid (c).
On this basis, the enzymatic reaction kinetics is used to explore the influence of knock-out components on the tyrosinase catalyzed reaction and the kinetic model analysis, and with the help of molecular docking The technology predicts the binding mode of active ingredients and tyrosinase, and clarifies the active ingredients that inhibit/activate tyrosinase in the fruit of Begonia xifuensis and their mechanism of action.
Fig. 7 Lineweaver-Burk plots of tyrosinase after the addition of p-coumaric acid (a), phloridzin (b ),quercetin-3-O-α-rhamnoside (c) and 4-O-β-glucopyranosyl-cis-coumaric acid (d).
Fig. 8 Molecular docking interaction of tyrosinase with p-coumaric acid(a), phloridzin(b),quercetin-3-O-α-rhamnoside(c) and 4-O-β-glucopyranosyl-cis-coumaric acid(d)
Note:(a, b, c, d) Plane projection diagram of the interaction between compound and tyrosinase.
Through the study of spectrum-effect relationship, it is found that the fruit of Begonia xifuensis has the best inhibitory effect on tyrosinase activity at a concentration of 0.5 g/mL ; PLS-DA analysis showed that the inhibitory effects of P2, P3, P5 and P6 on tyrosinase activity in the characteristic chromatogram of Xifu crabapple fruit were positively correlated (R>0.1); the inhibitory effects of P8 and P9 on tyrosinase activity showed a positive correlation. Negative correlation (R<-0.1). P2, P6, P8, P10, P11, and 12 were identified by UPLC-MS/MS as p-coumarin acid, ferulic acid-acyl glucoside, coumarin acid-4-O-glucoside, and phallokin-2, respectively '-Xylose glucoside, phlorizin and quercetin-3-O-rhamnoside. The results of tyrosinase kinetics experiments showed that coumaric acid (P2) and phlorizin (P11) (P11>0.50 mmol/L) have a competitive inhibitory effect on tyrosinase, and the concentration of phlorizin is less than 0.25 When mmol/L, it has a mixed inhibitory effect. Coumaric acid-4-O-glucoside (P8) and quercetin-3-O-rhamnoside (P12) are mainly expressed in the concentration range of 0-9 mmol/L and 0.3-11 mmol/L, respectively Non-competitive activation type, while quercetin-3-O-rhamnoside (P12) is a mixed activation type. The molecular docking results of tyrosinase showed:p-coumaric acid (P2), coumaric acid-4-O-glucoside (P8), phlorizin (P11), quercetin-3-O-rhamnoside ( P12) is located in the active center of the hydrophobic pocket of the enzyme, which binds to tyrosinase residues by hydrogen bonds, and has a hydrophobic interaction with numerous surrounding hydrophobic residues to maintain the structure of the complex together.
Using spectrum-effect relationship, ingredient knockout and high-resolution mass spectrometry identification technology to quickly identify active ingredients in complex traditional Chinese medicine systems, expound the overall view of traditional Chinese medicine, and explore the fields of active ingredients/ingredient groups , The team has formed a mature research strategy. The research results have been published in Food and Chemical Toxicology (https://doi.org/10.1016/j.fct.2019.110754);
Food Science and Human Wellness (https://doi.org/10.1016/j.fshw.2021.02.019);
Food and Chemical Toxicology (https://doi.org/10.1016/j.fct.2019.110754);
International Journal of Molecular Sciences (https://doi.org/10.3390/ijms19113439);
Frontiers in pharmacology (https://doi.org/10.3389/fphar.2020.01342) and other magazines. And with this technological innovation and integration, it won the second prize of Henan Science and Technology Progress Award in 2020.