CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF LIGUSTICUM WALLICHII EXTRACT IN 3T3-L1 AND HEP-G2 CELL LINES

Abstract

Ligusticum wallichii, or widely known as chuangxiong, is one common crude medicinal plant used in traditional Chinese, Japanese and Korean medicines. Several studies reported on its bioactivity were mainly focused on vasodilatation, anti-platelet aggregation, anti-thrombotic, serotonergic activity and anti-proliferative effects. Therefore, the objective of this study was to evaluate the effects and investigate the relevant mechanism of phthalides extract from chuangxiong in human hepatocellular carcinoma (Hep-G2) cell line and 3T3-L1 preadipocytes cell line.

Among those investigated extraction solvents used (water, methanol, ethanol, hexane and ethyl acetate), methanol was selected to produce lyophilized extract for further experiments due to its highest recovery yield. A variety of phthalides constituents were identified in chuangxiong extracts based on their MS data spectra and comparison of UV spectra with the literature. MTT cell viability assay showed that crude phthalides extracts (CPE) did not exhibit cytotoxic activity against Hep-G2 cells at concentrations below 1.0 mg/mL. However, after being purified using XAD4 resin, isolated phthalides extracts (IPE) was found to reduce the cell viability in a dose dependent manner with IC50 value of 271.6 ± 20.7 µg/mL. However, there was no significant difference on LDH assay though it showed a dose dependent increase. The inhibited proliferation mechanism was studied through cell cycle analysis and results revealed that it might associate with the arrest of cell cycle in G0/G1 phase but not apoptosis of cell.

Similar studies were performed on 3T3-L1 preadipocytes and results showed that IPE reduced preadipocytes viability in a dose dependent manner with IC50 value of 476.3 ± 11.6 µg/mL. The inhibition mechanism might closely correlate with an increase of LDH in the medium and a G2/M arrest by DNA cell cycle analysis, but no apoptosis was detected. When IPE was added into the differentiation medium of the adipogenesis study, increased intracellular lipid droplets were observed via morphological images of Oil-Red-O staining. The lipolytic activity of the cells and the glucose consumption assay revealed that IPE stimulated triglyceride release and glucose uptake in 3T3-L1 adipocytes in a concentration dependent manner. These findings suggest that IPE shows some important effects similar to thiazolidinedione, an anti-diabetic therapeutic agent. Therefore, IPE from chuangxiong exhibited bioactive activity against adipocytes and it could be a potential PPAR-¿ ligand, which enhances the adipogenesis process in adipose tissues.