摘要
萝卜硫素(sulforaphane,1-异硫氰酸基-4-甲磺酰基丁烷,简称SF)是一种异硫氰酸酯类化合物,由西兰花中的萝卜硫苷经黑芥子酶酶解得到。研究表明,SF不仅在肿瘤发生的多个阶段发挥抑癌作用,而且还是潜在的肿瘤治疗药物,在医药和保健食品等方面越来越引起人们的重视,SF的制备和活性研究可以为研制具有预防癌症的功能性食品或药品提供理论基础和实验依据。
本论文系统地研究了SF的制备工艺、稳定性和诱导细胞凋亡活性。以西兰花种子为原料,通过对酶解-提取工艺进行优化,提高了萝卜硫素产量；筛选了SP850树脂为吸附树脂,并对其吸附性能进行了研究；制备了SF的羟丙基-β-环糊精包合物(SF/HP-β-CD),研究了SF及其包合物的稳定性；对SF诱导B16和U251细胞凋亡及其机制等也进行了研究。主要内容如下：
以西兰花种子为原料,通过Plackett-Burman设计、最陡爬坡和中心组合实验对SF的酶解、提取工艺进行了优化。根据Plackett-Burman设计,从11个变量中筛选出影响最大的3种因素为提取料液比、水解时间和水解料液比。在此基础上,用最陡爬坡路径逼近最大产SF区域,最后通过中心组合实验及响应面分析得出优化工艺为：水解时间13min,水解料液比(g：m1)为1：2.9,萃取料液比(g：m1)为1：17.5。在此条件下,SF的得率最高,达14.8mg/g。
通过吸附平衡实验、吸附热力学及吸附动力学方法,研究了水相中大孔吸附树脂对SF的吸附性能。根据SF在大孔吸附树脂上的吸附率、解吸率等,筛选了大孔吸附树脂SP850为吸附树脂。吸附平衡实验表明,SP850吸附SF的最大平衡吸附容量在研究范围内随温度升高而减少,且吸附等温线更加遵循Freundlich方程,可初步推测SF在SP850活性位点上的吸附为多分子层吸附。计算了SF吸附热力学函数,(?)G<0,(?)S<0,(?)H<0,表明SP850吸附SF是一个放热过程,吸附能自发进行,随着温度升高,AG不断增加,表明在研究温度范围内降低温度有利于SF吸附。吸附动力学实验表明,吸附过程可以用拟二阶模型拟合。颗粒内扩散模型分析表明,SP850吸附SF过程受内扩散控制,在考察浓度范围内,内扩散速率常数随SF初始浓度增加而增大。SF在SP850上的吸附为快吸附,20mmin达到吸附平衡,液膜扩散是吸附初期的主要速率控制步骤。
制备了SF/HP-B-CD包合物,研究了SF及其包合物的热降解动力学规律。采用HP-B-CD包合SF,通过红外光谱、SF/HP-β-CD核磁分析和示差扫描量热法(DSC)等方法对包合物进行表征。从红外光谱官能团强度变化、核磁氢谱的H原子位移变化和DSC分析表明HP-B-CD和SF发生了包合作用。研究了SF和SF/HP-B-CD在不同的pH(2.2、3.0、4.0、5.0和6.0)和不同加热温度(60、75、82和90℃)下的降解动力学,结果表明SF在低pH值和低温下,稳定性更高,包合作用提高了SF的稳定性,SF及其包合物的降解符合一级降解动力学方程。通过Arrhenius方程求出SF及其包合物在不同pH值和温度下,活化能为70.7-94.5kJ/mol,包合物的活化能比SF单体的要高。提出了一种描述SF及其包合物在不同pH值和温度下的降解模型,验证试验表明,该模型可以很好的预测SF在不同条件下的降解过程。提出了一种简化的指数方程,可以很好的代替Arrhenius方程。
探讨SF诱导B16和U251细胞凋亡及其可能的机制。MTT实验表明SF能抑制B16和U251细胞增殖,抑制B16细胞和U251细胞的IC50分别为6.1+1.0μmol/L和16.4±1.4μmol/L。细胞凋亡实验表明SF能诱导B16和U251细胞凋亡,且呈一定的剂量依赖关系。SF诱导U251细胞凋亡所需的浓度要大于B16细胞。通过荧光定量PCR检测了B16和U251细胞中p21WAF1/CIP1和CyclinD1表达情况,并通过Western blot检测了Bax和Cyclin D1的表达情况及组蛋白H3和H4的乙酰化程度变化情况。结果表明SF能引起B16细胞中p21WAF1/CIP1和Bax基因表达上调,CyclinD1表达下调,B16细胞中组蛋白H3和H4乙酰化程度提高。而SF作用U251细胞后,p21WAF1/CIP1表达上调,Cyclin D1表达下调,Bax表达没有明显影响,组蛋白H3和H4的乙酰化程度没有变化。根据上述结果,推测了SF诱导B16细胞凋亡的可能机制。
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.
引文
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