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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2. McGahon, AJ. et al. (1995) Methods Cell Biol. 46:153–185.
3. Tamura, H. et al. (2003) Int J Mol Med. 11:369–374.
4. Lee, YI. et al. (2004)J Biol Chem. 279:15460–15471.
5. Mosmann, T. et al. (1983)J. Immunol. Methods 65:55–63.
6. Masanet, J. et al. (1988) Toxicol. in Vitro 2:275–282.
