Structural Elucidation And Effect On Melanogenesis And Apoptosis Of Proanthocyanidins Extracted From Chinese Bayberry Leaves

Chinese bayberry (Myrica rubra Sieb. et Zucc., Myricaceae) is a subtropical evergreen fruit tree widely grown in Southern China. Recent research has mainly focused on the function of myricetin isolated from M. rubra Sieb. et Zucc. leaves. Researches on proanthocyanidins of Chinese bayberry leaves are limited. Proanthocyanidins (PAs) are the second most abundant plant secondary metabolites after lignin. Previous works of our lab indicated that Chinese bayberry leaves are rich in proanthocyanidins, most of which are prodelphinidins. The separation of PAs is complicated because of the enormous variety of similar isomeric oligomers, making it difficult to investigate the proanthocyanidins extracted from Chinese bayberry leaves.In this study, proanthocyanidins extracted from bayberry leaves in different degree of polymerization were isolated. Their function of melanogenesis inhibition and mechanism of apoptosis induction were also investigated. In order to make better use of Chinese bayberry leaves proanthocyanidin extract, the effect of environmental conditions and chemicals on their stability were evaluated. The main contents and results were summarized as follows:Part Ⅰ. Chinese bayberry leaves were extracted with aqueous acetone (80:20 acetone/water, v/v) and then purified by Sephadex LH-20 column to produce crude proanthocyanidin extract of Chinese bayberry leaves (PCBLs). NMR data suggest that PCBLs are mostly composed of (epi)gallocatechin gallate units. Matrix-assisted laser desorption time-of-flight MS data indicate 95 possible prodelphinidin structures, ranging from dimers to tridecamers. Preparative normal-phase HPLC and further analysis by reverse-phase HPLC together with electrospray ionization MS enabled detection of 20 compounds, including seven newly identified compounds in Chinese bayberry leaves. This is the first known report of detection of tetrameric PAs and A-type PAs in Chinese bayberry leaves. A trimer consisting of two (E)GC units and one (E)GCG unit was also first identified.The fractions collected from preparative normal-phase HPLC were combined and divided as oligomeric proanthocyanidins (OPAs) and polymeric proanthocyanidins (PPAs). Fractions containing dimers and trimers were considered as OPAs, which made up almost 23.9% of the PCBLs, whereas fractions containing tetramers and higher polymerizations of PAs were considered as PPAs and composed almost 47.8% of the PCBLs. Then, the effect of PCBLs, OPAs and PPAs on melanogenesis and apoptosis.Part Ⅱ. PCBLs, OPAs and PPAs showed potent effect on the inhibition of mushroom tyrosinase activity, tyrosinase activity in B16 cells and melanin content in B16 cells. Although, the effect of PPAs on the inhibition of tyrosinase activity in B16 cells is not as good as OPAs, the inhibition effect of PPAs on mushroom tyrosinase activity and melanin content in B16 cells are better than OPAs. Also, PCBLs proved to be a good antioxidant and could prevent prawns melanogenesis.Part Ⅲ. In the present work, we demonstrated that PCBLs, OPAs and PPAs induced apoptosis in a concentration-dependent manner in OVCAR-3 cells. This finding was confirmed by Hoechst 33342 DNA staining and Annexin V+/PI- staining. According to total apoptotic cells percentage, PPAs were more effective than OPAs in inducing apoptosis. Based on increased apoptosis in treated cells, our next aim was to examine the involvement of caspases that play a major role in the execution of apoptotic events. Accordingly, we investigated the levels of active Caspase by Caspase Glo in cell lysates. The result of this study showed that PCBLs, OPAs and PPAs have a very strong and concentration-dependent effect on the induction of active Caspase 3/7,8,9 levels. Treatment of cells with 20 or 40 μ/mL concentrations of PPAs showed the most prominent increase in the activation of Caspase 3/7,8,9. The effect of the overall extract from Chinese bayberry leaves (PCBLs) showed no effect on p53 and AKT expression. However, the purified proanthocyanidins (OPAs and PPAs) showed potent effects on these two important targets. Although the bioavailability such as metabolites and distribution of proanthocyanidin especially polymers higher than tetramers remained unknown, our results showed that PPAs (tetramers and higher polymerizations proanthocyanidins) showed higher cytotoxic and apoptotic activities than OPAs (dimers and trimers), in general.Part Ⅳ. Natural light, D65 light and UV light all weakened the stability of proanthocyanidins to some extent. Temperature between 30℃ to 80℃ had no significant effect on the stability in a short time. Proanthocyanidins was stable when the pH value was in the range of 4.3 to 8.0. In addition, proanthocyanidins was sensitive to Cu2+, Fe2+, Mg2+ and Pb2+. As the humectant, glycerol stabilized the proanthocyanidin better than sodium hyaluronate. Methyl 4-hydroxybenzoate had almost no effect on proanthocyanidins. VC significantly improved the stability of proanthocyanidins as a good antioxidant.In conclusion, proanthocyanidins extracted from Chinese bayberry leaves can effectively inhibit melanogenesis and induce cell apoptosis with their main structure unit being EGCG. They have good stability and are eligible to be added into food and cosmetic products. Therefore, this is a natural product source with broad development prospect.