ABSTRACT
Objective: Sweet Tea (ST), derived from the leaves of Lithocarpus polystachyus, is a Chinese folk medicine with wide pharmacological activities. However, the promotive effects of ST water extract on hepatocytes proliferation and its underlying mechanism remains still unknown. In the present study, the beneficial effects of ST water extract on human hepatocytes and its possible mechanism were investigated. Methods: MTT assay was used to detect the safety range of ST; HL7702 cells were divided into four groups: control group, ST low- (50 μg/mL), medium- (200 μg/mL) and high-concentration (800 μg/mL) groups; BrdU ELISA and EDU staining were used to observe DNA content and cell proliferation; Moreover, flow cytometry was applied to analyze the distribution of cell cycle. Furthermore, the expression of cyclin D1, CDK4, HGF/c-Met, Akt, Erk1/2 were detected by Western blot. Results: It was found that ST water extract concentration-dependent promoted human hepatocytes HL7702 cell proliferation within 72 h through accumulating the cells in S phase and G2/M phase. Furthermore, ST water extract up-regulated expression of Cyclin D1 and CDK4 proteins. Moreover, ST water extract not only increased HGF expression and phosphorylation of c-Met level, but also activated the phosphorylation levels of AKT, ERK1/2. Interestingly, both of AKT inhibitor A6730 and ERK1/2 inhibitor U0126 reversed the promotive effects of ST water extract, which further confirmed that activation of AKT and ERK1/2 were involved. Conclusion: The findings reveal that ST water extract promoted HL7702 cells proliferation through the stimulation of cell cycle mediated by activating the AKT- and ERK1/2-related pathway.
ABSTRACT
Objective: To clone the leucoanthocyanidin reductase (LAR) gene of Lithocarpus polystachyus, and analyze the relationship between LAR gene expression level and phloridzin content. Methods: According to the results of L. polystachyus transcriptome sequencing (unigene: DN30711_c0_g1_i1), the full-length cDNA sequence of LAR gene was amplified by PCR and the bioinformatics analysis was carried out. Its expression was detected by quantitative Real-time PCR (qRT-PCR). The phloridzin content of L. polystachyus was measured by UPLC method and the correlation between LAR gene expression and phloridzin content was analyzed by SPSS 18.0 software. Results: The full-length cDNA of the LAR gene was 1 053 bp and contained a complete open reading frame that encoded 350 amino acids. This protein did not exist a transmembrane domain and was localized in the cytoplasm. The LAR protein of L. polystachyus was the number of PCBER_SDR_a family and had a high similarity (95%) to the LAR protein of Quercus suber and their genetic relationship was close. The phloridzin content of L. polystachyus was positively correlated with the expression of LAR gene (P < 0.05). Conclusion: The LAR gene of L. polystachyus was cloned for the first time. It was confirmed that the content of phloridzin was positively correlated with the expression of LAR gene of L. polystachyus, which laid a theoretical and technical basis for revealing the biosynthesis mechanism of phloridzin of L. polystachyus.
ABSTRACT
OBJECTIVE: To examine the antioxidant properties of the total flavonoids from Lithocarpus polystachyus Rehd (TFL) in vitro and in vivo. METHODS: The TFL was obtained by ultrasound-assisted extraction with 70% ethanol and purified with D-101 macroreticular resin. DPPH, ABTS, reducing power and ORAC of TFL were analyzed for antioxidant activity in vitro, with VC and rutin as control. Acute liver injury mice model was induced with carbon tetrachloride (CCl4), the effect of TFL on the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) levels were examined. RESULT: At 1 mg·mL-1 of TFL, the DPPH and ABTS radicals-scavenging activities was 81.25% and 85.54% respectively, while the reducing power was slightly lower than that of VC but higher than that of rutin. In the concentration of 1%, 2% and 3%, the value of ORAC was 47.86, 65.43 and 81.9 μmol TE·g-1 respectively. In mice with CCl4-induced actute liver injury model, TFL at the concentration of 100, 200 and 400 mg·mL-1 significantly decreased the MDA content, and increased the activities of SOD, CAT and GSH. CONCLUSION: The total flavonoids from Lithocarpus polystachyus Rehd. have a strong antioxidant effect.