Abstract
BACKGROUND: Acanthopanax trifoliatus (L.) Voss or phak-paem, a traditional Thai plant in the Araliaceae family, has been reported to promote adaptogenic effects for a long time. The leaves have been used as a tonic to improve general weakness and to treat tuberculosis, lung hemorrhages, bruises, ulcers and contusions. The young leaves and the shoots are also eaten as vegetables. METHODS: A. trifoliatus leaf extract was prepared, and its physical and chemical properties were qualitatively controlled. Three formulations, tablets, drinks and bubbles, were developed from A. trifoliatus standardized leaf extract. The in vitro antioxidant effects of the A. trifoliatus leaf extracts and formulations were evaluated using a DPPH scavenging assay. The hepatoprotective effects of A. trifoliatus leaf extract and formulations were evaluated in the HepG2 cell line using a hepatoprotective assay, the detection of intracellular reactive oxygen species (ROS) levels and the quantification of apoptotic cells and necrotic cells. The mechanism of action for hepatoprotective effects was determined by detecting antioxidant and apoptosis markers using quantitative real-time polymerase chain reaction (qPCR). RESULTS: Standardized extracts of A. trifoliatus leaves were prepared from quality-controlled raw material. The physical properties of the extract were controlled, including loss on drying, total ash, and acid-insoluble ash. It had specific chromatographic fingerprint with total phenolic and total flavonoid contents of 13.44 ± 0.22 g of chlorogenic acid equivalent in 100 g of extract (g% CAE) and 6.14 ± 0.45 g of rutin equivalent in 100 g of extract (g% RE), respectively. According to high-performance liquid chromatography (HPLC) analysis, the extract contained mono-caffeoylquinic acid 1.33 ± 0.00 g% CAE, di-caffeoylquinic acid, 1.82 ± 0.00 g of a cynarin equivalent in 100 g of extract (g% CYE) and flavonoid 0.48 ± 0.00 g% RE. Tablets, drinks and bubbles were developed from A. trifoliatus standardized leaf extract. A. trifoliatus leaf extract, tablets and drinks showed strong DPPH scavenging effects, with EC(50) values of 38.88 ± 1.19, 62.99 ± 1.80 and 81.19 ± 1.18 µg/mL, respectively. Treatment with the A. trifoliatus extract at a concentration of 12.5-100 μg/mL reduced hepatocyte toxicity by 30-40% in a dose-dependent manner, as determined by a tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity assay. The three formulations of A. trifoliatus extracts also showed hepatoprotective effects of approximately 5-25% at concentrations ranging from 12.5-100 μg/mL. The mechanisms of action for hepatoprotective effects were found to include a reduction in intracellular ROS production; the prevention of t-BHP-induced apoptosis; an increase in the expression of antioxidant genes such as catalase, superoxide dismutase-1 (SOD-1) and NADPH quinone dehydrogenase-1 (NQO-1); a reduction in the expression of apoptotic genes, including the proapoptotic genes AIFM-1 and BAK-1 and the apoptotic genes CASP-8 and CASP-3; and the upregulation of the antiapoptotic gene BCL-2. CONCLUSION: Standardized A. trifoliatus leaf extract and its formulations, tablets, drinks and bubbles were prepared and quality controlled. They promoted in vitro antioxidant activities with hepatoprotective effects in HepG2 cell line.