Isolation,Purification And Biological Activity Evaluation Of Glucosinolates And Their Enzymolysis Product Of Maca And Differentiation Of Maca From Different Origins By PCA

Glucosinolate is a kind of secondary metabolite rich in Lepidium meyenii(Maca),and it is also a kind of compound widely existing in cruciferous plants.Glucosinolate and its enzymolysis products have a variety of biological activities,such as anti-cancer,anti-inflammatory and antibacterial.It is of great significance to improve enzymatic hydrolysis efficiency by controlling enzymatic hydrolysis conditions.Macamide,a kind of compound only existing in Maca,is the same as glucosinolate to be used as an important index to evaluate the quality of Maca.It is of great significance to identify Maca from different origin and evaluate the quality of Maca according to the content of macamide and macaene.In this thesis,the extraction process of glucosinolate was optimized according to the yield of glucosinolate by palladium chloride method and UV-Vis spectrophotometry.Glucosinolate in Maca was enriched and purified by macroporous resin and acidic alumina.The biological activities of the purified products and their enzymolysis products were evaluated.Finally,based on the content of macamide and macaene,PCA method was established to identify the origin of Maca and evaluate the quality of Maca.(1)In this chapter,based on the principle of color reaction between palladium chloride and glucosinolate,the effects of three single factors including methanol mass concentration,solid-liquid ratio and ultrasonic time on the extraction yield of glucosinolate from Maca were studied by UV-vis spectrophotometry.Then,orthogonal test was designed to optimize the extraction conditions of glucosinolate.The optimal extract condition of glucosinolate is that using 70%methanol as solution,Maca was extracted with the ratio of liquid to Maca under ultrasonic concussion for30 min.The yield of glucosinolate is 1.51%when Maca was extracted at the optimal condition.Palladium chloride coloration method was verified by methodology.The results show that the palladium chloride coloration method used to detect glucosinolate has good stability,high precision,good repeatability and recovery.Therefore,this method is reliable for the determination of total glucosinolates in Maca.(2)Static adsorption-desorption method was applied to assess to the properties adsorption-desorption of D201,D261 and D301 resins.D301 resin was considered to be the most suitable to purification of glucosinolate in Maca among them.Hence,D301 resin and acidic alumina were used to purify glucosinolate in fresh Maca and dried Maca.The purified product was characterized by UPLC-MS,FT-IR and UV-Vis,respectively.The results show that purification of glucosinolate with D301resin and acidic alumina can remove a large number of impurities and enrich glucosinolate effectively.After purification,the sample mainly contains three glucosinolates including GTL,GLH and GSB.The contents of GTL,GLH and GSB in fresh Maca are 375.61±9.67?g/m L,57.61±2.48?g/m L and 94.04±1.72?g/m L,respectively,while those in dried Maca are 299.29±2.66?g/m L,43.36±1.31?g/m L and 64.01±1.60?g/m L,respectively.The 525.62±1.729.37?g/m L content of total glucosinolate in fresh Maca is more than 406.66±5.57?g/m L content of total glucosinolate in dried Maca.(3)Antioxidant abilities of GLC_S,MCE and BITC were evaluated by DP PH·scavenging ability,ABTS·scavenging ability and iron reducing ability.Anti-inflammatory ability of GLCs and BITC were evaluated by NO inhibition rate test.MTS method(3-(4-5-dimethylthiazol-2-yl)-5(3-carboxymeth-oxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium)was used to evaluate anticancer activities of GLCs and BITC against five tumor cells including leukemia HL-60,lung ca ncer A549,liver cancer SMMC-7721,breast cancer MCF-7721 and colon cancer SW480.The results show that MCE has good antioxidant activity,and GLCs has lower antioxidant activity than that of MCE.These three antioxidati on assays of BITC were carried out at the same condition,but there are no p henomena when the concentration of BITC is less than 10 mg/m L.GLCs had no anti-inflammatory activity and inhibitory activity on five tumor cells.Howe ver,BITC has strong anti-inflammatory ability and good inhibitory activity on five tumor cells.IC₅₀of BITC on leukemia HL-60,lung cancer A549,liver ca ncer SMMC-7721,breast cancer MCF-7 and colon cancer SW480 are 0.886±0.065?g/m L,1.021±0.032?g/m L,0.941±0.023?g/m L,1.880±0.088?g/m L and1.200±0.057?g/m L,respectively.Its IC50 was significantly lower than that of cisplatin(DDP)and higher than that of paclitaxel(Taxol).(4)UPLC-MS was used for qualitative and quantitative analysis of macamide and macaene separated by silica gel column chromatography.It was found that Maca from four origins contained five kinds of macamide and two kinds of macaene and there were significant differences in contents of macamide and macaene in Maca from different origins.Based on contents of macamide and macaene,Maca from different origins can be effectively identified by PCA method.This method provides a reference for rapid identification of Maca from different origin and evaluation of Maca quality.