Isolation, Purification, Structure And Antioxidant Capacity In Vitro Of Polyphenols From Rabbiteye Blueberry(Vaccinium Ashei)

Polyphenols from blueberry leaves have many biological functions such as antioxidation, anti-virus, anti-cancer, anti-diabetes, anti-hypertension, and reducing blood lipid. In China, there are few researches and utilizations on polyphenols from blueberry leaves. The majority of blueberry leaves were disposed as garbage, which result in the waste of natural resources. The study of the extraction and purification of blueberry polyophenols and the analysis of its composition are of great significance to the development of natural medicine and functional food making from blueberry leaves. In order to establish definite theoretical foundation for the synthetic utilization of blueberry leaves, the extraction, purification, isolation and structure identification of leaves of rabbiteye blueberry(Vaccinium ashei) were investigated in the present study.1. The extraction conditions of polyphenols from blueberry leaves were optimized by response surface method. The optimal extraction conditions were as follows:ethanol concentration was62.05%, extraction temperature was67.54℃, solid-liquid ratio was1:23.65, and extraction time was2.06h. Under the optimized condition, the extraction yield was89.02%. Besides, a quadratic equation model for polyphenols extraction was set up. The model played an important role in predicting the extraction rate of polyphenols from blueberry leaves.2. The static adsorption and desorption of eight types of macroporous resins towards polyphenols from blueberry leaves were investigated. The results showed that HPD400was the most suitable to purify blueberry leaves polyphenols. When140mL of samples (3mg/mL, pH2) flowed through the HPD400resin at a speed of1mL/min, the adsorption reached dynamic equilibrium. The adsorbed polyphenols could be completely eluted with 80mL of40%ethanol at a speed of1mL/min, with a desorption rate of90.50%. After the purification by HPD400resin, the purity of polyphenols increased from38.75%to69.38%.3. High performance liquid chromatography-Diode array detector-Elaectrospray Ionization-tandem Mass spectrometry (HPLC-DAD-ESI-MS tandom) was used to analyze the components of polyphenols from blueberry leaves. The results showed that polyphenols from blueberry leaves include Oligomeric Proanthocyanidin (B-type procyanidin dimer, A-type procyanidin dimer, A-type procyanidin trimer), chlorogenic acid and its derivatives (Neochlorogenic acid, Cryptochlorogenic acid, Di-O-caffeoylquinic acid), quercetion and its derivatives (rutin, quercetin-3-O-hexoside, quercetin-3-O-gluconide, Quercetin-3-O-pentoside, quercetin-3-O-(4"-HMG)-rhamnoside), kampferol and its derivatives (Kampferol-3-O-rutinoside, Kampferol-3-O-hexoside, Kampferol-3-O-gluconide, Kampferol-(HMG) rhamnoside), and two types of O-p-coumaroylquinic acid.4. After purification by macroporous resins, blueberry polyohenols was subjected to separation using silica gel column and Sephadex LH20. Three pure compounds were obtained and identified as kampferol, quercetion, and quercetin-3-O-galactoside (hyperoside) by ESI-MS,1H HMR, and13C HMR.5. The ABTS+radical scavenging capacity, DPPH-radical scavenging activity,-OH scavenging activity, O2-·scavenging capacity, nitrite scavenging activity, reducing power, FRAP value, and ORAC value of polyphenols from rabbiteye blueberry were investigated. Results showed that polyphenols from blueberry leaves had strong antioxidant capacity. After purification by macroporous resins, the antioxidant capacity was greatly improved. In addition, the antioxidant capacities increased in the order:quercetion> kampferol> hyperoside, and all the three compounds showed higher antioxidant capacities than the positive control rutin. Thus polyphenols from blueberry leaves could be used to develop natural antioxidants due to its superb antioxidant capacity.