Ursolic acid, a key anticancer compound derived from <i>Prunella vulgaris</i> L., induces apoptosis in HepG2 cells by directly targeting the STAT3 signaling pathway

Prunella vulgaris L., renowned for its anti-cancer properties, has been used for centuries in China due to its rich content of terpenoids, notably ursolic acid (UA), which exhibits potent anti-cancer effects. Despite this, UA has not been recognized as a pivotal component during Prunella vulgaris evaluation, and its specific anticancer mechanism remains elusive. Henceforth, this investigation seeks to explore UA’s role within the 95% ethanol extract of Prunella vulgaris (EEPV) concerning growth inhibition and apoptosis induction in HepG2 cells through a component knockout approach. The results revealed that the half maximal inhibitory concentration (IC50) value of EEPV against HepG2 cells increased from 110.933±5.543 μg/mL to 366.167±22.945 μg/mL after UA knockout, while the IC50 value of UA was only 9.315±0.525 μg/mL. Furthermore, the apoptosis-inducing effect of EEPV significantly decreased following UA knockout but was essentially restored upon UA supplementation. These findings identify UA as the primary active compound in EEPV, driving antiproliferative and proapoptotic responses in HepG2 cells. Network pharmacology coupled with molecular docking implicates signal transducer and activator of transcription 3 (STAT3) in mediating UA-driven programmed cell death within the HepG2 cells. Cellular thermal shift assay revealed that UA could directly interact with STAT3, inhibit STAT3 phosphorylation, reduce nuclear translocation of STAT3, subsequently decrease Bcl-2 expression, and ultimately initiate mitochondrial pathway-induced apoptosis in HepG2 cells; however, no significant impact on STAT3 gene and protein expression was observed. These findings indicate that UA is a pivotal anticancer component within Prunella vulgaris capable of directly binding to STAT3 and interfering with the STAT3/Bcl-2 pathway to induce apoptosis in HepG2 cells. Consequently, UA should be considered a key marker compound for the quality evaluation of Prunella vulgaris.