HPLC determination of oleanolic acid content in Hedyotis diffusa Willd. and its anti-HepG-2 cell activity

Objective To establish the method for quantitative determination of oleanolic acid in Hedyotis diffusa Willd., and study its inhibitory effect on human hepatoma HepG-2 cells. Methods HPLC with a column of Angilent ZORBAX Eclips Plus C18 (4.6 × 250 mm, 5 um) is used; column temperature: 20°C. Detection wavelength: 210nm. Anti-hepatoma activity of Hedyotis diffusa Willd. is analyzed by observing changes in cell morphology under inverted microscope and by MTT assay. Results Oleanolic acid shows a good linearity within an 11.06-154.84 ug range; its recovery is 98.51%, with a RSD of 1.18% (n = 6). Under inverted microscope, cells in the control group are grown adherently, with intact membranes. Growth density of cells in the Hedyotis diffusa Willd. test groups becomes gradually low with increasing drug concentration. Cell surface is wrinkled. In the high concentration group, most cells are disrupted, cell morphology is not intact, and number of adherent cells is reduced. MTT assay results show that the viability of HepG2 cells decreases with increasing concentration of Hedyotis diffusa Willd.; inhibition rate reaches 52.67% in the high dose group after treating HepG2 cells for 48 h. HepG2 cell inhibition rate exhibits rather obvious dose-response relationship. Conclusion HPLC method is accurate and reliable for the determination of oleanolic acid content in Hedyotis diffusa Willd.; Hedyotis diffusa Willd. can effectively inhibit the proliferation of human hepatoma HepG-2 cells.