Network Pharmacology and Zebrafish Model Elucidate the Hypoglycemic Mechanism of Major Compounds in Cyclocarya paliurus

Diabetes Mellitus is a complex metabolic disorder, primarily characterized by persistent high blood sugar levels, and it is becoming increasingly prevalent with numerous associated complications. The leaves of Cyclocarya paliurus (Batal.) Iljinskja, traditionally prepared as a tea beverage in China, is frequently used in folk medicine for managing metabolic syndromes, particularly diabetes and hyperlipidemia. However, the main active components responsible for its hypoglycemic effect and their underlying mechanisms remain unclear. The current study aimed to clarify the main chemical components of the aqueous extract of C. paliurus leaves and to explore their mechanisms of action. The primary constituents from the aqueous extract of C. paliurus leaves were isolated and identified using macroporous adsorption resin, preparative liquid chromatography, and nuclear magnetic resonance technology. The contents of these identified compounds in the leaves were quantified using HPLC. An integrated approach combining network pharmacology and molecular docking was initially used to predict the potential molecular targets and associated signaling pathways responsible for the hypoglycemic activity of the compounds, with subsequent experimental validation performed in a hyperglycemic zebrafish model. From the aqueous extract, a total of seven compounds were obtained and subsequently identified as Chlorogenic Acid (CA), Quercetin-3-O-β-D-glucuronide (Q3GA), Astragalin, 3,4-Dicaffeoylquinic Acid (3,4-DCA), Afzelin, Quercetin, and Kaempferol. Their contents in C. paliurus leaves, as determined by HPLC, were 24.88 mg/g, 30.87 mg/g, 1.21 mg/g, 1.19 mg/g, 5.24 mg/g, 2.43 mg/g, and 1.34 mg/g, respectively. Network pharmacology analysis identified AKT1, TNF, and IL1B as key targets for the hypoglycemic effects of both the aqueous extract and the seven individual compounds. These findings were further supported by RT-PCR experiments in a zebrafish model, which showed that blood glucose regulation occurs through the downregulation of TNF and IL1B and the upregulation of AKT1 protein. The aqueous extract is rich in Chlorogenic Acid, Quercetin, and their derivatives, all of which display significant hypoglycemic activity.