Investigations of cytotoxicity of Lippia dulcis Trev. on the human hepatoma cell line HepG2

Lippia dulcis Trev. is an intensely sweet plant native to tropical America and contains the sesquiterpenoid sweetener (+)-hernandulcin in the essential oil [1].

The effects of the essential oil (I) and hernandulcin (II) were investigated in the human hepatocellular carcinoma cell line HepG2. Cells were incubated with I at 50,150 and 250µg/ml and with II at 25, 68 and 150µg/ml for 4–48h. Morphologic evaluation of apoptotic cells was performed by fluorescent staining (acridine orange/ethidium bromide) [2]. The formation of DNA ladder in HepG2 cells treated with I and II was monitored by electrophoresis on 1.5% agarose gels [3], staurosporine was used as positive control [4]. The cytotoxicity was determined with MTT assay and LDH assay (lactate dehydrogenase leakage), which is a measure of membrane integrity after incubating the cells with different concentrations of I (50–250µg/ml) and II (6.25–150µg/ml) for 4h [5,6].

Apoptotic morphology was observed in HepG2 cells treated with I and II from a concentration of 150 and 68µg/ml respectively. DNA fragmentation analysis demonstrated that the apoptosis of cells appeared already after 4h of treatment and that the action of I and II is both dose- and time course-dependent. I and II did not increase LDH leakage, a significant (P<0.05) increase in LDH leakage was found only after incubating the cells with the highest concentration (250µg/ml) of I. Whereas MTT results showed that the proliferation of HepG2 cells was significantly inhibited by I at 150, 200 and 250µg/ml and II at 50,100 and 150µg/ml. We suggest that I and II show at higher concentration cytotoxicity and induce mainly apoptosis in HepG2 cell lines.

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