OBJECTIVE  To analyze the serum chemical composition of rats after intragastric administration of water extract of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis based on liquid chromatography-mass spectrometry (LC-MS) technology, predict the effect of salt processing on the treatment of type 2 diabetes in Anemarrhenae Rhizoma-Pheellodendri Chinensis combined with network pharmacology, and preliminarily verify it through in vitro experiments.

  METHODS  Rats were continuously intragastrically administered with crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair water extract twice, with an interval of 1 h. After 60 min of the last administration, the blood was taken from the abdominal aorta, and the protein was precipitated by methanol. After dissolution, the chromatographic column was Shim-pack GIST C₁₈ (4.6 mm×150 mm, 5 μm); the mobile phase A was 0.1% formic acid water, and the mobile phase B was 0.1% formic acid-acetonitrile; gradient elution, positive and negative ion full scan mode, mass scan range 100-1 500 m/z. Combined with the secondary spectrum of the database and literature, the blood components of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair were analyzed and identified. The disease targets of type 2 diabetes were retrieved, and the protein interaction network analysis, GO and KEGG pathway enrichment analysis were performed on the intersection targets of blood components and diseases. The "blood components-targets" network diagram was constructed, and the selected core components and core targets were verified by molecular docking using AutoDock software. In the verification experiment, HepG2 cells were used as the experimental object, and the insulin resistance model was induced by high insulin and high glucose. CCK8 method was used to test the effect of Rhizoma Anemarrhenae-Phellodendri Chinensis on cell proliferation before and after salt processing. Western blot was used to detect the expression of PI3K-AKT signaling pathway-related proteins.

  RESULTS  15 prototype components and 1 mangiferin metabolic component were identified in the serum of rats. 17 prototype components and 1 mangiferin metabolite were identified in the rat serum of the water extract of Anemarrhenae Rhizoma-Phellodendri Chinensis after salt processing. The contents of mangiferin, berberine and 3-isobutylglutaric acid in the blood components after salt-processing were higher than those in the raw products. According to the results of KEGG and GO, the treatment of type 2 diabetes may be related to the transcriptional regulation of RNA polymerase, inflammatory response, AGE-RAGE, PI3K-AKT pathway and insulin resistance. Cell experiments showed that the ratio of p-PI3K/PI3K, p-AKT/AKT and GLUT4 protein expression could be up-regulated before and after salt processing, and the effect of salt processing group was better than that of the crude group.

  CONCLUSION  This experiment preliminarily revealed the components of Anemarrhenae Rhizoma-Phellodendri Chinensis drug pair entering the blood before and after salt exposure, and suggested that ferulic acid, berberine, berberrubine, mangiferin, mTOR, SIRT1, EGFR and PPARA may be the main components and targets of Rhizoma Anemarrhenae-Phellodendri Chinensis after salt processing to enhance the therapeutic effect of type 2 diabetes. The mechanism may be to enhance the role of PI3K-AKT and other related signaling pathways, providing an important reference for the pharmacodynamic material basis and clinical application of Anemarrhenae Rhizoma-Phellodendri Chinensis before and after salt processing.