Research On The Separation And Purification Of Flavonoids From Ampelopsis Grossedentata By Fermentation

Ampelopsis grossedentata as raw material,the method of hot water extraction was used to extract flavonoids.And combine fermentation and ultrafiltration with Sephadex LH-20 and recrystallization to separate and purify flavonoids.The structure of flavone monomer was identified by color reaction,ultraviolet spectroscopy,infra-red spectrum and HPLC methods.Extracting dihydromyricetinfrom Ampelopsis grossedentataby water extraction method.The orthogonaltestand single factor experimentwere used to optimize the best extraction condition.The ratio of liquid and material is 20:1,extraction time is 90 min, extraction temperature is 90℃,holding time is 1d.The extraction yield could reach 12.9% in this condition.Wine yeast SY was chosed for farther fermentation from four kinds of yeasts by comparation of flavonoids contents in experimental group and control group.Optimize the fermentative medium by single factor experiment.The medium of wine yeast SY was 1%glucose,2%NH4Cl,0.1% Ca Cl2,0.1% MgSO4 and 0.05% KH2PO4.Then confirm the effects of fermentation time,fermentation temperature and inoculation amount three factors on contents of reducing sugar by single factor experiment.And the effect order was fermentation time,fermentation temperature and inoculation amount.The interation between fermentation time and inoculation amount was significant.Optimize the fermentation factors by RSM.And the optimization was fermentation time 23 h, fermentation temperature 29.5℃,inoculation amount 5%.The contents of reducing sugar was 0.25mg/mL in this condition.Compare with the contents of water extraction decreased by 77.3%.The purity of flavonoids was 45.6%.The methods ultrafiltration, Sephadex LH-20 and recrystallization was used to farther separation and purification.Optimize the condition by single factor experiment.The ultrafiltration pressure was 0.2MPa and ultrafiltration temperature was 40℃. The purity of flavonoids was 25% than before.88.5mg flavone monomer was obtaind by the combination of Sephadex LH-20 method,ultraviolet spectrophotometry and HPLC.The purity was high as 98.23%.Recrystal the crystal rime in two different ways.Purify with acetone and recrystallization with water is better.The purity of dihydromyricetin is high to 98%, recovery rate was 59.2%.Finally identify the structure of flavone monomer by color reaction,ultraviolet spectroscopy,infra-red spectrum and HPLC methods.And the results showed this compound was dihydromyricetin.The combination of fermentation method and traditional extraction and purification methods provided the theoretical basis for the development of Ampelopsis grossedentata.