Studies On The Anti-tumor Effect Of Active Components From Bayberry Bark In Vitro

Myrica(Myrica rubra Sieb. et Zucc) is a genus of small trees and shrubs in the family Myricaceae. Common names include Bayberry, Bay-rum tree, Candleberry, Sweet Gale, and Wax-myrtle. It is one of main fruits in our country which is cold resistance and likes wet. The bark of bayberry contains various compounds such as polyphenols, flavonoids, diarylheptanoid, tannin, triterpene et al., which possessed variety of pharmacological activities. The anticancer activity of the bark of bayberry extract was determinded in this paper. The components which possessed biological activity in bark of bayberry were revealed. It was established that they had a synergistic effect and the best ratio of substances which possessed biological activity was determinded. Further more, their synergistic effect mechanism was revealed. The main conclusions are as follows:(1) The bark of bayberry was extrated by n-Butanol. Then two extracts butanol extraction phase (BuE) and water extraction phase (WaE)) were obtained. The concentration (W/%) of polyphenols and flavonoids in WaE were (36.01±0.8148)%and (33.15±0.7374)%. In BuE they were (61.01±0.1165)%and (31.20±0.7987)%. Through DPPH radical scavenging assays it was found that the concentrations of IC50of WaE and BuE were164.8μg/mL and114.8μg/mL. By high performance liquid chromatography analysis, it was identifid that the main components in BuE were myricetin, quercetin and myricitrin. The concentrations (W/%) in BuE respectively were (3.75±0.58)%,(29.28±1.27)%and (6.06±0.355)%, and1:5.35:1.7in mole ratio.(2) Through HepG2cell proliferation inhibition study, it was proved that BuE had strong inhibitory effect on HepG2cell proliferation. Among myricetin, myricitrin and myricetin combined with myricitrin the effect of myricitrin was the weakest. In24h, the IC50of cell proliferation inhibition of myricetin, myricitrin and myricetin combined with myricitrin respectively were841.03μmol/L,1578.13μmol/L and652.79μmol/L. In48h, the IC50of three samples respectivly were321.67μmol/L,908.20μmol/L and286.92μmol/L; the IC50of BuE was116.54μg/mL. In72h, the inhibitory effect of myricetin combined with myricitrin was better than the single use of myricetin, but there was not obvious synergistic effect between them. The IC50of BuE was80.05μg/mL. By Annexin V-FITC/PI double staining method it was found that the induction of apoptosis in HepG2cell line by the three samples was in a dose-dependent manner. In high concentrations, the induction of apoptosis rate of myricetin, myricitrin and myricetin combined with myricitrin respectively were38.04%,13.77%and38.67%. The induction of early apoptosis induced by myricetin combined with myricitrin was stronger but the induction of late apoptosis induced by myricetin used alone was stronger.(3) Through YTS-1cell proliferation inhibition study, it was observed that the IC50of cell proliferation inhibition of myricetin, myricitrin and myricetin combined with myricitrin respectively were240.10μmol/L,272.05μmol/L and378.82μmol/L in48h, in addition the IC50were198.95μmol/L,209.15μmol/L and251.06μmol/L in72h. The effect of YTS-1cell proliferation inhibition was in a time-and dose-dependent manner. There was not significant synergistic effect between myricetin and myricitrin. Through PI staining assays it was showed that the G2/M phase of YTS-1cell cycle was significantly blocked after the treatments of the three samples in high concentrations (P<0.01). In addition, in cell apoptosis experiments it was demonstrated that the induction of apoptosis in YTS-1cell line by the three samples in low concentrations was not strong, and in the highest concentration the induction of apoptosis was significant (P<0.01). The induction of apoptosis rates of myricetin, myricitrin and myricetin combined with myricitrin respectively were31.70%,25.50%and25.19%. The induction of early apoptosis induced by myricetin was stronger meanwhile the induction of late apoptosis induced by myricetin combined with myricitrin was stronger.(4) Through B16cell proliferation inhibition study, it was showed that the IC50of cell proliferation inhibition of myricetin. myricitrin and arbutin respectively were131.55μmol/L,138.44μmol/L and154.03μmol/L in48h and90.40μmol/L,88.6μmol/L and101.55μmol/L in72h. In the tyrosinase activity inhibitory effect experiments it was proved that the inhibition rate on tyrosinase activity by myricitrin was (74.52±3.248)%. Through the detection of melanin contents it was showed that myricitrin could significantly inhibit the melanin synthetic action. The effects of myricitrin and arbutin were so proximate that myricitrin could be used as a potential excellent whitening agent.(5) Through PC-3cell proliferation inhibition study, it was revealed that the IC50of cell proliferation inhibition of myricetin, myricitrin and myricetin combined with myricitrin respectively were94.48μmol/L,267.1μmol/L and418.25μmol/L in48h. The IC50of cell proliferation inhibition of BuE and WaE were95.21μg/mL and118.3μg/mL in48h. In the research of synergistic effect between myricetin and myricitrin, it was revealed that the cell proliferation inhibition of myricetin, myricitrin (75μmol/L) and myricetin combined with myricitrin (each concentration was37.5μmol/L) respectively were40.53%,13.34%and72.28%. The cell proliferation inhibitory effect was in a time-and dose-dependent manner and there was significant synergistic effect between myricetin and myricitrin. Through the acridine orange staining analysis, it was observed that by the different concentrations of drugs treatments PC-3cells showed apoptosis-like changes, with a time-and dose-dependent. By Annexin V-FITC/PI double staining method it was found that the effect inducing PC-3apoptosis by the three samples was in a dose-dependent manner and among them myricetin was the strongest inducer. Otherwise, the induction of late apoptosis induced by myricetin was stronger but the induction of early apoptosis induced by myricitrin was stronger. There was significant synergistic effect between myricetin and myricitrin.(6) Through the study of synergistic effect between myricetin and myricitrin in PC-3cell line, it was reaveled that the cell proliferation inhibitory effect by the treatments of myricetin and myricitrin was in a time-and dose-dependent manner. The effect of myricetin combined with myricitrin was the strongest and the best mole ratio was1:5. Through PI staining assays it was observed that the G0/G1phase of PC-3cell cycle was significantly blocked after the treatments of the three samples (P<0.01) and among them the effect of myricetin combined with myricitrin was the strongest when the best mole ratio was1:5. Further more, in Annexin V-FITC/PI staining analysis the induction of apoptosis of both compounds were not obvious in a low concentration. But when the mole ratio were increased to1:5and1:7the induction of apoptosis became significant. When the mole ratio was1:7the late apoptosis effect was stronger and the mechanism of synergistic effect between myricetin and myricitrin was shifted. Through TUNEL analysis for late cell apoptosis, it was established that between myricetin and myricitrin there was a significant synergistic effect and when the molar ratio was1:5or1:7the late induction of apoptotic was strong.In conclusion, myricetin and myricitrin possesed potential anti-tumor activities in different tumor cells with a selective manner. In addition, antitumor effect of BuE mixture was significant. Further more, there was significant synergic effect between myricetin and myricitrin. The best ratio was1:5which was coincided to the naturally existence in BuE. It was proved that in BuE the main anti-cancer bioactive substances were myricetin and myricitrin. The synergistic effect between them may be the most effective reason for why a natural product mixture is most effective in most cases.