Isolation And Purification Of Glucoerucin, Hydrolysis Product Erucin And Study Of Their Antioxidant Activity

Glucosinolates (GLs) are the sulfur-containing secondary metabolitesfound in plants during their growth. Epidemiological data show thatisothiocyanates (ITCs) are considered as the natural products which havewonderful effects on the prevention and treatment to cancer. It is found thatisothiocyanates can not only inhibit the formation of a variety of cancer cells,but also can increase the levels of glutathione in the human body. They are thestrongest inducer of phaseⅡenzymes in nature. In this paper, we studied theseparation and purification conditions of glucoerucin and erucin from rocketseeds; the process of using glucoerucin to prepare sulforaphane; alsocompared and researched the antioxidant activity of glucoerucin (GER),glucoraphanin (GRA), glucoraphenin (GRE), erucin (ERN), sulforaphane(SFN), GRE-ITC by scavenging DPPH radical, OH free radical and thereducing power.The separation and purification conditions of glucoerucin and erucin byresin and preparative chromatographic were researched. Through the screening of resin, we found that D261resin was in favor of selection ofglucoerucin and the eluent was100mM/L KCl solution. The experimentalconditions of purification glucoerucin by preparative chromatographic were asfollows: the mobile phase was2%methanol solution, the flow rate was8ml/min and the injection was100mg. When we used macroporous resinSP850to purify erucin, the best adsorption rate was8ml/min. After washedthe impurities with deionized water,80%ethanol was used to elute erucin, andthe flow rate was10ml/min. Moreover, preparative chromatography was usedto further purify erucin, and the conditions were as follows: mobile phase was25%methanol solution, the detection wavelength was254nm, and the flowrate was8ml/min.Glucoerucin was used to prepare sulforaphane with myrosinase. Firstly,glucoerucin was oxidated into glucoraphanin (GRA) by hydrogen peroxide,and then GRA could be hydrolyzed to sulforaphane by the myrosinase. At thesame concentration, the volume ratio of glucoerucin and hydrogen peroxidewas1:7, and the time of oxidation reaction was3h at room temperature.Myrosinase were extracted from broccoli seeds. At the same time, oxidatedGRA was added to the hydrolyzed liquid of broccoli seed, and the content ofsulforaphane was significantly increased.Scavenging activity on DPPH radicals is time and concentrationdependent. The inhibition of DPPH radical was decreased as the followingorder: GER>GRE>ERN>GRE-ITC>GRA>SFN. The second-order rate constant (k₂) was9.04M^-1s^-1and EC₅₀was2.5mM/L for the scavengingreaction with DPPH radical. Experiments demonstrate that purified GLs andITCs did have the ability of scavenging OH radical. The a-tocopherol was asthe contrast, reducing power of GLs and ITCs was compared and exhibited thefollowing order: a-tocopherol> GER> ERN> GRE> GRE-ITC> GRA>SFN. The purified GER and ERN consistently exhibited higher antioxidantactivity based on the tests performed.