Ophiopogon japonicus strains from different cultivation regions exhibit markedly different properties on cytotoxicity, pregnane X receptor activation and cytochrome P450 3A4 induction

Abstract

Maidong, known as Ophiopogon japonicus, is one of the two basic ingredients of Shenmai injection, which is a widely used herbal preparation in traditional Chinese medicine (TCM) for the treatment of atherosclerotic coronary heart disease and viral myocarditis. Previously, the ethanol extract of Maidong activated the pregnane X receptor (PXR) signaling pathway and induced the cytochrome P450 3A4 (CYP3A4) reporter gene and raised the concern of herb-drug interactions (HDIs) when Maidong was used in combination with prescribed drugs metabolized by CYP3A4. Therefore, the present study further investigated and compared the differences of the ethanol and aqueous extracts (ee- and ae-, respectively) of two Maidong strains, known as Zhe Maidong (ZM) and Chuan Maidong (CM). Cytotoxicity, PXR activation and CYP3A4 induction by the 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-diphenytetrazoliumromide assay, reporter gene assay and reverse transcription-quantitative polymerase chain reaction analysis were examined. The observations showed that ee-ZM demonstrated a significantly higher cytotoxicity, a relatively weaker PXR activation capability and a markedly stronger CYP3A4-inducing capacity than ee-CM. Compared to ae-CM, ae-ZM exhibited only a slight or no difference on cytotoxicity and CYP3A4 induction, while a significant lower level of PXR activation was apparent. Collectively, Maidong from different producing areas possess different properties upon cytotoxicity and the drug-metabolizing enzyme inducing effect, and attention should be paid to the selection of Maidong strains from different planting regions into TCM preparations for reducing potential adverse reactions and HDIs.

Keywords: Ophiopogon japonicus; cytochrome P450 3A4; herb-drug interactions; pregnane X receptor; traditional Chinese medicine.

Figure 1.

Verification of mRNA expression of the pregnane X receptor (PXR) and cytochrome P450 3A4 (CYP3A4) in human hepatocellular liver carcinoma (HepG2) and LS-174T cells. The relative mRNA level of PXR and CYP3A4 in HepG2 and LS-174T cells was detected by reverse transcription-quantitative polymerase chain reaction. GAPDH was used as internal control.

Figure 2.

Cytotoxicity of CM and ZM extracts. The effect of CM and ZM extracts on cell viability was determined by the 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-diphenytetrazoliumromide assay. When not indicated by fold lines, * and ** represent significant differences from the vehicle (dimethyl sulfoxide) treated group; *P<0.05, **P<0.001. CM, Chuan Maidong; ZM, Zhe Maidong; ee, ethanol extract; ae, aqueous extract.

Figure 3.

Activation of human pregnane X receptor (PXR) by CM and ZM extracts. The activation effects on human PXR by extracts of CM and ZM were evaluated by the reporter gene assay. Rifampicin (RIF; 10 µM) was used as a positive control. Following treatment, firefly luciferase activity was determined and normalized against Renilla luciferase activity. *P<0.05, **P<0.001. CM, Chuan Maidong; ZM, Zhe Maidong; ee, ethanol extract; ae, aqueous extract.

Figure 4.

Induction of cytochrome P450 3A4 (CYP3A4) mRNA by CM and ZM extracts. The induction of CYP3A4 mRNA by extracts of CM and ZM was investigated and compared by RT-qPCR analysis. Dimethyl sulfoxide (DMSO; 0.1%) and 10 µM rifampicin (RIF) were used as the blank and positive control, respectively. When not indicated by fold lines, * and ** represent significant differences from the vehicle (DMSO) treated group; *P<0.05, **P<0.001. CM, Chuan Maidong; ZM, Zhe Maidong; ee, ethanol extract; ae, aqueous extract.