Study On Proanthocyanidins In Bayberry Leaves

Proanthocyanidins which have a C6·C3·C6 skeleton in their chemical structures blong to a class of natural phenolic compounds found abundantly in the plant kingdom. The most widely distributed proanthocyanidins in plants are of procyanidin type. Proanthocyanidins are known for their notable pharmacological effects, which include anti-oxidative, antimicrobial, and antitumor properties, and confer cardioprotective activities. Bayberry (Myrica rubra Sieb. et Zucc.), cultivated mainly in southern China, is a special fruit in our country. At present, bayberry fruits are the main materials of research and production. Foliar growth of bayberry tree is luxuriant, and leaves remain green throughout the year. Bayberry trees are pruned every year more than once. Generally, the leaves are discarded generally and remain underutilized. Bayberry leaves are rich in phenolics. Extracts of bayberry leaves are reported to have anti-oxidative, antimicrobial, and antiviral properties. Bayberry leaves are natural source with potential economic and medicinal values.In this study, structures of proanthocyanidins extracted from bayberry leaves were characterized. Antioxidant and anticancer activities were also determined and the mechanism was analyzed. Then the safety of bayberry leaf extract was evaluated. Results were summarized as follows:The structural features of bayberry leaf proanthocyanidins of Biqi were investigated by using fractionation on a Sephadex LH-20 column, acid-catalysis in the presence of excess phloroglucinol, HPLC coupled to photodiode array detection or UV detection and electrospray ionization MS (HPLC-DAD-UV/ESI-MS) and NMR analysis. The extractable and unextractable proanthocyanidins (EPAs and UEPAs) were of prodelphinidin type. In fact, epigallocatechin-3-O-gallate (EGCG) and traces of epigallocatechin (EGC) were detected as the extension units, but only EGCG was present in the terminal units. The EPAs were of B-type proanthocyanidins. The mean degree of polymerization (mDP) of bayberry leaf EPAs was 6.5. The UEPAs were highly polymerized prodelphinidins consisting of EGCG and traces of EGC. The EPAs and UEPAs consist mostly of EGCG, which is unusual in the plant kingdom.EPAs and UEPAs of different bayberry leaves were investigated by using NP-HPLC-DAD analysis and acid-catalysis. Composition of prodelphinidins in bayberry leaves with different cultivar and different leaf age was different from each other. In bayberry leaves of Biqi, the contents of EGCG, oligomer, polymer, and UEPAs of immature leaves were higher than those of intermediate and mature leaves. In bayberry leaves of Dongkui, content of UEPAs of immature leaves was highest, and contents of EGCG, oligomer, and polymer of mature leaves were highest. Sum contents of dimer, trimer, and tetramer determined by NP-HPLC-DAD were higher than those of polymers with DP>4 in both Biqi and Dongkui bayberry leaves.Individual prodelphinidin oligomers and other low-molecular-weight phenolic compounds in bayberry leaves were investigated by HPLC-DAD-ESIMS. Four flavan-3-ol monomers, three prodelphinidin dimmers, and two trimers were identified. Only one phenolic acid was identified as gallic acid. Six flavonol glycosides were also identified and myricetin deoxyhexoside (535.4-853.0 mg/100 g) was the major flavonol glycoside. Among the three categories, immature leaves of both cultivars contained the highest level of total phenolics and prodelphinidins.The antioxidant activity of the bayberry leaf extract determined by DPPH·and FRAP assay was comparable with ascorbic acid. Prodelphinidins in the extract had a significant positive correlation with the antioxidant activities of bayberry leaf extract. The correlation coefficients (R) were 0.963 and 0.970 for DPPH·and FRAP assay, respectively. In order to develop a new natural antioxidant, a central composite design was employed when the yield of prodelphinidins was selected as the response value to investigate the extraction. The best extraction condition involving acetone concentration, time, solid-liquid ratio and temperature was obtained for the maximum extraction of prodelphinidins by using response surface methodology (RSM). The extraction yield of prodelphinidins was affected significantly by the extraction variables. The optimal conditions obtained by RSM include 56.93% acetone,31.98 min time,1:44.52 solid-liquid ratio, and 50.00℃temperature. Under the optimal condition, the experimental yield of prodelphinidins was 117.3±5.1 mg/g, which was not different from the predicted value significantly.Immature bayberry leaves of two cultivars, Bigi and Dongkui, were selected to investigate the effect of water temperature and extraction time on contents of total phenolics and proanthocyanidins, composition of proanthocyanidins, and the antioxidant activity of the hot water extract. The results indicated that the contents of total phenolics and proanthocyanidins were higher at higher water temperature and on the longer extraction time, then the contents dropped if the temperature was too high or the extraction time was too long. The contents of total phenolics and proanthocyanidins of Biqi cultivar were lower than that of Dongkui cultivar under the same extraction condition. Generally, higher contents of oligomers can be obtained at higher temperature. However, the content finally dropped when temperature exceeded 75℃. Higher contents of polymers can be obtained at higher temperature and over longer duration.The antioxidant activity of bayberry leaf proanthocyanidins (BLPAs) was evaluated by several antioxidant assays with different antioxidant mechanisms (e.g. DPPH, ABTS, PF, PFAP, Metal chelation). In all tested assays, BLPAs exhibited strong antioxidant activities. The DPPH radical scavenging capacity of BLPAs was weaker than that of gallic acid but stronger than that of grape seed procyanidins and bayberry leaf flavonols. Reducing activity of BLPAs was also stronger than that of grape seed procyanidins and bayberry leaf flavonols. The metal chelating activity of BLPAs was very weak compared to EDTA. Bayberry leaf phenolic (BLP) and BLPAs can inhibit lipid oxidation in oils. The inhibition activity of BLPAs was stronger than BLP and BHT. The mDP of BLPAs had significant effect on their radical scavenging activity while no significant effect on reducing and metal chelating activity.Ovarian (OVCAR-3 and CP-70) and prostate (DU-145 and PC-3) cancer cell lines were selected to estimate anticancer activity of bayberry leaf polyphenols (BLP). The main compounds in BLP were proanthocyanidins. BLP significantly inhibited cell viability in ovarian cancer cell lines while can not inhibit cell viability in prostate cancer cell lines. BLP can inhibit VEGF expression in all cancer cell lines selected. HIF-la and p-AKT levels which were implicated in VEGF expression were also inhibited by BLP. cMyc expression was down regulated by BLP in selected cancer cell lines. Inhabitation of NF-κB expression in cancer cell lines was different from each other. PTEN which play a role in suppressing tumor growth was also be inhibited by BLP. Then it was indicated that the anticancer activity of BLP was a comprehensive action of these factors studied.Through acute toxity test and 30 days feeding study methods, it can be concluded that bayberry leaf extract was safe.In conclusion, bayberry proanthocyanidins were phenolic compounds with unusual structures and strong antioxidant activity. They also had anticancer capacity. Bayberry leaf can be a good source of novel functional food.