A rapid method and mechanism to identify the active compounds in Malus micromalus Makino fruit with spectrum-effect relationship, components knock-out and molecular docking technology.

Fingerprints of 20 batches of Malus micromalus Makino fruit were established by HPLC coupled with hierarchical cluster analysis (HCA) and principal component analysis (PCA) to estimate the common peaks on the basis of traditional similarity evaluation methods. Chromatographic peaks were identified as p-coumaric acid (P2), ferulic acid glycoside (P6), 4-O-ß-Glucopyranosyl-cis-coumaric acid (P8), phloretin-2'-xyloglucoside (P10), phloridzin (P11) and quercetin-3-O-α-rhamnoside (P12) by UPLC-MS/MS method. The results of tyrosinase kinetics experiments showed that: P2 and the concentration of P11 was greater than 0.50 mmol/L mainly had a competitive inhibitory effect on tyrosinase, and the concentration of phlorizin was less than at 0.25 mmol/L, it has a mixed inhibitory effect. P8 was mainly a non-competitive activation type in the concentration range, while P12 was a mixed activation type. The results of tyrosinase molecular docking showed that: P2, P8, P11, P12 was located in the active center of the hydrophobic pocket of the enzyme. They bound to tyrosinase residues by hydrogen bonds and interacted with many hydrophobic residues around them to maintain the structure of the complex. This research provides a rapid method to determine the active compounds in edible plants with the technology of spectrum-effect relationship, component knock-out and molecular docking.

Anti-Hepatoma Compound Determination by the Method of Spectrum Effect Relationship, Component Knock-Out, and UPLC-MS2 in Scheflera heptaphylla (L.)Frodin Harms and Its Mechanism.

Scheflera heptaphylla (L.)Frodin, a kind of Traditional Chinese Medicine, is commonly used in anti-inflammatory, analgesic, anti-viral, anti-tumor, and hemostasis. This study aimed to determine the anti-hepatoma components and its mechanism from the leaves of S. heptaphylla. The spectrum-effect relationships were analyzed by the method of Partial least squares, indicating that P1, P2, and P10 were positively correlated to inhibitory activity of Huh7 cells. Whereas others were negatively correlated. The technologies of component knock-out and UPLC-MS2 were used to determine compounds as 3,4-Dicaffeoylquinic acid (P6), 3,5-Dicaffeoylquinic acid (P7), 3α-Hydroxy-lup-20(29)-ene-23,28-dioic acid (P10, named Compound A). The results forecasted that Compound A had the best correlation with inhibitory activity. The effects of Compound A on the activities of human hepatoma cells (Huh7, SMMC-7721, HepG 2) and normal hepatocytes (L0-2, Chang liver) were evaluated. Cell apoptosis was observed with inverted microscope and flow cytometer. In addition, the proteins, related to apoptosis, were detected by Western blot. The results showed that Compound A (400 nM) could significantly inhibit the activity of three hepatoma cells (P < 0.001) with slight toxicity to normal hepatocytes, and the IC50 values were 285.3 and 315.1 nM, respectively, which were consistent with the prediction of spectrum-effect relationships. After treatment with Compound A, the number of hepatoma cells decreased significantly. And the apoptosis rate of Huh7 cells increased significantly (P < 0.001) in Compound A (200, 400 nM) groups, SMMC-7721 and HepG 2 were directly necrotic. Compound A groups could significantly improve the level of intracellular reactive oxygen species (ROS) (P < 0.05, P < 0.001) in Huh7 with no effect on normal hepatocytes. The content of apoptotic protein (Bax and Bim) in mitochondria was significantly increased in Compound A groups (P < 0.001). On the contrary, the content of anti-apoptotic protein (Bcl-xL and Mcl-1) decreased significantly (P < 0.001). These results demonstrated that Compound A was the main anti-hepatoma active component in the S. heptaphylla leaves. It achieved the effect of promoting apoptosis of Huh7 cells by regulating the levels of ROS and Bcl-2 family protein in mitochondrial apoptosis pathway.

Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with HPLC and Artificial Neural Network Analysis for Ganoderma lucidum.

Ganoderma lucidum is widely used in traditional Chinese medicine (TCM). Ganoderic acid A and D are the main bioactive components with anticancer effects in G. lucidum. To obtain the maximum content of two compounds from G. lucidum, a novel extraction method, an ionic liquid-based ultrasonic-assisted method (ILUAE) was established. Ionic liquids (ILs) of different types and parameters, including the concentration of ILs, ultrasonic power, ultrasonic time, rotational speed, solid-liquid ratio, were optimized by the orthogonal experiment and variance analysis. Under these optimal conditions, the total extraction yield of the two compounds in G. lucidum was 3.31 mg/g, which is 36.21% higher than that of the traditional solvent extraction method. Subsequently, an artificial neural network (ANN) was developed to model the performance of the total extraction yield. The Levenberg-Marquardt back propagation algorithm with the sigmoid transfer function (logsig) at the hidden layer and a linear transfer function (purelin) at the output layer were used. Results showed that single hidden layer with 9 neurons presented the best values for the mean squared error (MSE) and the correlation coefficient (R), with respectively corresponding values of 0.09622 and 0.93332.

Ionic Liquid-Based Ultrasonic-Assisted Extraction to Analyze Seven Compounds in Psoralea Fructus Coupled with HPLC.

Psoralea Fructus is widely used in traditional Chinese medicine (TCM), and the content of psoralen, isopsoralen, neobavaisoflavone, bavachin, psoralidin, isobavachalcone, and bavachinin A is the main quality control index of Psoralea Fructus because of its clinical effects. Thus, a fast and environmentally-benign extraction method of seven compounds in Psoralea Fructus is necessary. In this work, an ionic liquid-based ultrasonic-assisted method (ILUAE) for the extraction of seven compounds from Psoralea Fructus was proposed. Several ILs of different types and parameters, including the concentration of ILs, concentration of ethanol (EtOH), solid-liquid ratio, particle size, ultrasonic time, centrifugal speed, and ultrasonic power, were optimized by the Placket-Burman (PB) design and Box-Behnken response surface analysis. Under this optimal condition, the total extraction yield of the seven compounds in Psoralea Fructus was 18.90 mg/g, and significantly greater than the conventional 75% EtOH solvent extraction.