Studies On Sulforaphane:Preparation,Degradation Kinetics And Its Activities On Inducing Apoptosis

Sulforaphane (1-isothiocyanate-(4R)-(methylsulfinyl) butane, SF) can be obtained through hydrolysis.of glucoraphanin by endogenous myrosinase. Studies show that SF not only restrains tumor during different stages of carcinogenesis, but also is a potential drug in the oncotherapy, thus SF has been of increasing interest for nutraceutical and pharmaceutical industries. Researches on preparation and activity of SF can provide theoretical and experimental basis for development of functional food for cancer prevention or drug for cancer treatment.In this paper, the production of SF and its stability and activities on inducing apoptosis were studied. Sulforaphane yield were optimized from broccoli seed using response surface methodology; Macroporous resin SP850was selected for SF adsorption, and SF adsorption performance of macroporous resin SP85O were studied; The inclusion complex of SF with hydroxypropyl-β-cyclodextrin (SF/HP-β-CD) were prepared, and thermal degradation of sulforaphane and its inclusion complex were studied; And then the effects of SF on apoptosis and its mechanism in B16mouse melanoma cells and U251human glioma cells were also studied. The main contents of this paper include:The production of SF from broccoli seed was optimized using response surface methodology. Three major factors (hydrolysis time, water volume and ethyl acetate volume) were screened out through Plackett-Burman (PB) factorial design. The methods of steepest ascent combined with central composite design (CCD) were employed for optimization of the SF production process. The optimal extraction conditions for SF production were a hydrolysis time of13min, a hydrolysis weight/volume ratio of1:2.9(g:ml) and an extraction weight/volume ratio of1:17.5(g:ml). The maximum SF yield was14.8mg/g.The adsorption equilibrium, thermodynamic and kinetics of adsorbing SF in aqueous phase by macroporous resin were studied. On the basis of the adsorption and desorption tests of SF, macroporous resin SP850resin was selected for adsorption kinetics and isotherms. Batch equilibrium experiments demonstrate that, in the examined temperature range, the equilibrium adsorption capacity of the SP850decreases with the increase of adsorption temperature. Based on the analysis of the adsorbing isotherms, the adsorption data of adsorbing SF on SP850fit the Freundlich equation well. We deduced that the adsorption of SF on macroporous resin SP850was multi-molecular layers adsorption. The thermodynamic parameters fitting reveal that△G<0,△S<0,△H<0, which indicates that the SF adsorption is exothermic, along with the increase of temperature came an increase in△G, indicated that low temperature will favor the adsorption process. Adsorption kinetics studies show that the adsorption process can be fitted with a pseudo second-order model. The intra-particle diffusion model indicated that the process of SF adsorption on SP850macroporous was controlled by the diffusion procedure, and the intra-particle diffusion rate constant Increases with the increase of initial SF concentration. Increase the temperature could accelerate the adsorption rate, but the adsorption capacity will be lowered. Film diffusion is the main controlling step in the initial adsorption.The pure SF and inclusion complex of SF with hydroxypropyl-β-cyclodextrin (SF/HP-B-CD) were prepared, and thermal degradation of SF and its inclusion complex were studied. FTIR,’H NMR and differential scanning calorimetry (DSC) analysis were performed to prove the formation of the inclusion complex SF/HP-B-CD inclusion complex. Then the depletion rates of SF and SF/HP-B-CD were investigated at temperatures of60,75,82and90℃and pH values of2.2,3.0,4.0,5.0and6.0. The results showed that SF and SF/HP-β-CD were more stable at lower pH values and temperatures, and SF/HP-B-CD was more stable than SF. The SF and SF/HP-B-CD degradation was observed to follow first order kinetics, and the temperature-dependent rate constants for SF and SF/HP-B-CD inclusion complex in aqueous solution were well described by the Arrhenius equation with corresponding activation energies of70.7to94.5kJ/mol, depending on the pH values. Then two models to describe the retention ratio of SF and SF/HP-B-CD at different pH values, depletion time and temperatures were proposed, a good agreement between the actual and the calculated retention values of SF was obtained. Moreover, the rate constant vs temperature relationships, which yield linear Arrhenius plots, could be described by a simpler exponential equation.The effects of SF on apoptosis and its mechanism in B16mouse melanoma cells and U251human glioma cells were studied. MTT assay showed that the proliferation of B16and U251cells were significantly inhibited by SF. The IC50of B16and U251after SF treatment for48h were6.1±1.0μmol/L and16.4±1.4μmol/L, respectively. Annexin V-PI double staining assay demonstrated that SF induced apoptosis in B16and U251cells, and dose-dependent manner could be found. The mRNA expression of p21WAF1/CIP1and Cydin D1in B16and U251after SF treatment for48h were measured with Quantitative real-time PCR, and the protein expression of Bax, Cyclin D1and acetylation degree of H3and H4changes were detected by Western blot. The results show that in B16cells, p21WAF1/CIP1and Bax was upregulated, Cyclin Dl was downregulated, and an increase in the degree of histone H3and H4acetylation were found after SF treatment for48h. In U251cells, however, though p21WAF1/CIP1upregulated, Cyclin D1downregulated, acetylation of histone H3and H4were not found after SF treatment for48h. The possible mechanisms of B16apoptosis were deduced.