4-Hydroxybenzoic Acid and ß-Sitosterol from Cucurbita ficifolia Act as Insulin Secretagogues, Peroxisome Proliferator-Activated Receptor-Gamma Agonists, and Liver Glycogen Storage Promoters: In Vivo, In Vitro, and In Silico Studies.

Insulin secretion and GLUT4 expression are two critical events in glucose regulation. The receptors G-protein-coupled receptor 40 (GPR40) and peroxisome proliferator-activated receptor-gamma (PPARγ) modulate these processes, and they represent potential therapeutic targets for new antidiabetic agent's design. Cucurbita ficifolia fruit is used in traditional medicine for diabetes control. Previous studies demonstrated several effects: a hypoglycemic effect mediated by an insulin secretagogue action, antihyperglycemic effect, and promoting liver glycogen storage. Anti-inflammatory and antioxidant effects were also reported. Moreover, some of its phytochemicals have been described, including d-chiro-inositol. However, to understand these effects integrally, other active principles should be investigated. The aim was to perform a chemical fractionation guided by bioassay to isolate and identify other compounds from C. ficifolia fruit that explain its hypoglycemic action as insulin secretagogue, its antihyperglycemic effect by PPARγ activation, and on liver glycogen storage. Three different preparations of C. ficifolia were tested in vivo. Ethyl acetate fraction derived from aqueous extract showed antihyperglycemic effect in an oral glucose tolerance test and was further fractioned. The insulin secretagogue action was tested in RINm5F cells. For the PPARγ activation, C2C12 myocytes were treated with the fractions, and GLUT4 mRNA expression was measured. Chemical fractionation resulted in the isolation and identification of ß-sitosterol and 4-hydroxybenzoic acid (4-HBA), which increased insulin secretion, GLUT4, PPARγ, and adiponectin mRNA expression, in addition to an increase in glycogen storage. 4-HBA exhibited an antihyperglycemic effect, while ß-sitosterol showed hypoglycemic effect, confirming the wide antidiabetic related results we found in our in vitro models. An in silico study revealed that 4-HBA and ß-sitosterol have potential as dual agonists on PPARγ and GPR40 receptors. Both compounds should be considered in the development of new antidiabetic drug development.