Isolation,Identification And Activity Evaluation Of Ethanol Extract From Browned Lotus Roots

Browning is a common biochemical phenomenon and an important indicator of quality control of fruit and vegetables.Enzymatic browning is a reaction process in which phenolic enzymes catalyze phenols to form colored polymers under aerobic conditions.Previous studies have found that substrates for enzymatic browning are diverse in fruit and vegetables,which may be related to different enzymes,but whether browning products affect human nutrition or not is still uncertain.In order to clarify the changes of nutrient composition after lotus root browned,isolation、identification and activity evaluation were carried out for the browned products of lotus root,and then the antioxidant capacity,the effect on the proliferation of tumor cells and immune cell function were evaluated.The results are as follows:(1)Isolation and purification of lotus root browned product.Lotus root slices were packaged in trays to obtain browned samples and stored at 20℃ for 5 days.The browned sample is pulverized and the ethanol solution is extracted and assisted by ultrasonic to obtain crude extract 1(CE1).Similarly,crude extract 0(CEO)is prepared by fresh lotus root;the composition of CEO and CE1 was analyzed by high performance liquid chromatography(HPLC).It was confirmed that the browned products were mainly distributed in the retention time of 50～65 min;Then,the CE1 was separated and purified by macroporous resin chromatography;The components of T1-4 were eluted with 30%,50%,70%and 100%ethanol respectively.It was found that the browned products were mainly concentrated in the component T1.Finally,the silica gel column chromatography was used to purify the component T1,and the different components F1-5 were eluted by different concentrations of ethanol.Finally,the browned substances in each component were mainly distributed around the retention time of 60 min.(2)Structural identification of lotus roots browned products.The material composition and structural characteristics of the browned product were analyzed by UV,FTIR,HPLC and UPLC-Q-TOF-MS.The results show that CE1 contains a large number of unsaturated bonds because its UV absorption wavelength is 210-250 nm,which is a typical conjugated diene absorption region;compared with CEO,the FTIR spectrum of the CE1 has been red-shifted,and the characteristic absorption peak of the alcohol fingerprint region is also changed significantly;What’s more,the absorption peak of CE1 is more;this indicates that the browned product has a complicated structure,many polyhydroxy compounds and been unstable.Chemical constituents of CE1 mainly include sugar,phenols,triterpenes,etc.In particular,coumarins and catechins are associated with browned,which are identified by UPLC-MS method.Compared with CE1,there were significant differences in CEO components,total sugar and organic acid content were lower than browned ethanol extract CE1,and there were differences in the types of betulinic acid,whose were hydroxylation reactions.(3)Activity evaluation of lotus roots browned products.The antioxidant activity of the lotus root browned product,the cancer cell proliferation inhibition ability and the macrophage immunostimulatory effect were evaluated,with the vitro model adopted.Compared with CEO,CE1’s 1,1 diphenyl-2-trinitrophenylhydrazine(DPPH)free radical scavenging capacity increased by 35%,but the total antioxidant capacity(FRAP)of those had no significant difference;Similarly,CE1 has a certain growth inhibition effect on SGC7901 gastric cancer cells(38.055%),but has no significant effect on HepG2 tumor cell proliferation;The ethanol extract of lotus root has significant immunostimulatory activity,but the browned product may have no stimulating effect on macrophage function,combined with TNF-a expression and NO production.In summary,the composition changes of lotus root browned mainly involve phenols,sugar and polymer components,and the effects on different tumor cells may be related to components.However,the browned product may affect the nutritional health function of lotus root,and even enhance the anti-oxidation and inhibit the growth of gastric cancer cells to some extent This study initially explored the composition changes and associated nutritional effects in the process of lotus root browned,which could offer new ideas for the quality control of lotus root products.