Extraction,Purification And Antioxidant Activity Of Triterpenic Acids From Blackened Jujube(Zizyphus Jujuba Mill.)

Blackened jujube,is a new type of deep processing products of red jujube,without adding any additives through the control of temperature and humidity,formed by the jujube after a period of high temperature ripening,its color is black brown,soft texture,sweet and sour taste.Compared with the original jujube,its taste,nutritional value and function have new changes.Triterpenes was one of the main bioactive components in jujube fruit,which has the effects of anti-oxidation,lowering blood lipid,anti-fatigue,anti-cancer and anti-inflammatory.At present,the research on triterpenoid acids in jujube at home and abroad is limited to natural jujube fruit,but the research on triterpenoid acids in jujube black and their biological activities has not been reported,which restricts their further application research.In this work,the efficient extraction and purification techniques of triterpenoids from blackened jujube were studied and the composition of the purified product was analyzed,and then,the antioxidant capacity of triterpenic acids were determined by in vitro chemical assay and in vitro cell assay using HUVECs cell as model,to expand dateplum persimmon triterpenoid acid application in food,cosmetics and related products.The main findings and conclusions were as follows:1.Changes of color and main triterpenoid acids in blackening process of jujube.The changes of color and contents of three triterpenoid acids in blackening period from 0 h to 108h were studied.The results showed that the color of jujube changed from red to black during the blackening period of 0～108 h,and the change of color was mainly concentrated in the 48h before blackening.The content of triterpenoid acid in jujube was increased by vanillin-perchloric acid colorimetry and high performance liquid chromatography during the blackening period from 0 to 108 h.Betulinic acid increased from 151.61μg/g to 159.52μg/g,and reached the highest value of 208.74μg/g at 72 h.The content of oleanolic acid increased from 161.42μg/g at 0 h to 208.84μg/g at 108 h,which was 1.3 times higher than that of jujube.Ursolic acid increased from 15.70μg/g at 0 h to 26.35μg/g at 108 h,and the content of ursolic acid increased by about 2 times.2.Optimization of extraction method and conditions of triterpenic acids from blackened jujube.Ultrasonic-assisted extraction method was used to extract triterpenic acids from blackened jujube.Based on the single factor experiment,the process conditions were optimized by Box-Behnken response surface methodology.The results showed that the optimal extraction conditions were as follows:50%ethanol concentration,solid-liquid ratio of 1∶23（g/mL）,ultrasonic time of 30 min and ultrasonic power of 300 W.Under these conditions,the content of triterpenoid acid was（1.313±0.01）mg/g.3.Isolation and purification of triterpenic acids from blackened jujube.The adsorption and desorption properties of five resins（AB-8,D-101,S-8,X-5 and HPD-100）for triterpenoid acids of blackened jujube were compared,and the optimal resin was selected.The effects of loading solution and eluent conditions and elution conditions on purification efficiency were studied,and the purification parameters were optimized.The results showed that D-101 was the best resin,and its purification parameters were as follows:loading concentration 25.5μg/mL,loading volume 130 mL,loading flow rate 2.0 mL/min,eluent dosage 80 mL,eluent ethanol concentration 95%,eluent flow rate 1.0 mL/min.Under these conditions,the recovery rate of triterpenoid acid was（78.58±0.67）%,and the purity of triterpenoid acid after purification was increased by 2.49 times.4.Antioxidant properties of chemical reagent method.Take Vc as the control,the antioxidant activities of triterpenoid acid before and after purification were compared.The results showed that within the experimental concentration range,The IC₅₀values of DPPH·were 0.571,0.053 mg/mL,ABTS⁺·0.186,0.059 mg/mL,and·OH were 0.900,0.850 mg/mL,respectively.The IC₅₀values of O^2-·were 0.745,0.594 mg/mL,respectively.The total reducing power showed a dose-effect relationship with the sample concentration.5.Antioxidant properties of cellular method.HUVECs cell model was used to investigate whether the purified triterpenoid acid could inhibit H₂O₂-induced HUVECs cell damage.MTT test,cell morphology observation,DCFH-DA test,Rh123,Hoechst 33258 test were used for detection.The results showed that the PTA could protect cell morphology,increase cell survival rate,decrease intracellular ROS,increase mitochondrial membrane potential,and reduce cell apoptosis,and there was a dose-response relationship.These results can provide a basis for the study of the structure-activity relationship of melanized jujube triterpenes,and demonstrate their potential as a new antioxidant in functional food and pharmaceutical industries.