【作者】 张文斌；

【导师】 许时婴；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 亚麻籽中含有多种木酚素,其中开环异落叶松树脂酚(SECO)的含量最高,SECO以二糖苷开环异落叶松树脂酚二葡萄糖苷(SDG)的形式存在于亚麻籽中。研究发现亚麻木酚素具有多种生物活性,如抗癌、防治心血管疾病、减缓更年期症状、预防骨质疏松等。开发利用亚麻木酚素对预防这些膳食模式依赖性的疾病和提高人民身体素质具有重要意义。亚麻木酚素的开发利用还可以大大提高亚麻籽的附加值,产生显著的经济效益,促进国内亚麻资源深加工与利用。本文通过对亚麻木酚素的测定、提取、纯化及亚麻籽综合利用方法的系统研究,建立了一条SDG提取和纯化的工艺路线,具有安全、经济、易于工业化的优点,得到了高纯度(95%以上)的木酚素产品。本文从体外细胞培养和化学计算相结合的角度出发,研究了亚麻木酚素的抗乳腺癌功效及其作用机理。通过生物活性实验与理论计算交互验证的方法解释了其量效、构效关系。研究发现,在SDG的测定过程中,将碱水解与有机溶剂提取合并可以更充分地从脱脂亚麻籽壳中提取SDG。研究了木酚素测定中普遍采用的酸水解方法对SDG的影响,纯化的SDG样品经过酸水解主要生成3种产物,低盐酸浓度下产物以SECO及开环异落叶松树脂酚葡糖苷(SG)为主,提高盐酸浓度和作用时间,产物以脱水开环异落叶松树脂酚(ANHSEC)为主,但木酚素总量降低。采用HPLC-PAD-MS可准确测定SDG、SG、SECO与ANHSEC,采用NMR验证了HPLC-PAD-MS的测定结果并鉴别了SDG的构型。以SDG为对照,建立了HPLC定量方法,避免了酸水解导致的损失,路线简便,方法可靠,灵敏度令人满意。本文还采用比色法测定SDG,使其定量更为方便快捷。为富集SDG和综合利用亚麻籽中功能性成分,本文采用湿法脱粘,以粘性脱除率(定义为粘质物脱除率与0.1%粘质物溶液粘度的乘积)为指标,确定了最适脱粘条件:温度70°C,水料比7:1,pH 6.0,时间60 min,脱粘4次。分析了所得粗亚麻籽胶的组成,其多糖与蛋白含量分别为65.4%和8.50%(w/w)。其单糖组成(摩尔比例)为鼠李糖:岩藻糖:阿拉伯糖:木糖:葡萄糖:半乳糖= 5.10:1.00:4.18:8.98:2.13:2.75,木糖含量最高,表明中性多糖含量较高。采用湿法脱壳,在砂轮间距0.40 mm、砂轮转速2800 rpm、水力分级两次条件下,富仁、富壳与混合部分的得率分别为52.6%、22.3%和12.7%,总回收率87.7%,仁中含壳率和壳中含仁率分别为20.9%和11.1%,达到较为满意的程度。分析了亚麻籽脱粘和仁壳分离的影响,发现部分SDG(9.6%)随粉碎的壳层流失,SDG主要分布在富壳部分,其次是混合部分,富仁部分含量很少。采用正交实验设计的方法优化了SDG的搅拌提取条件:提取溶剂为50%乙醇(v/v),温度60°C,液固比20 mL/g,时间3 h。该条件下SDG的得率达1.80 g/100 g脱脂亚麻籽壳粉(DFHF),向提取液中加入0.25 mol/L NaOH时,SDG的得率提高至1.90 g /100 g DFHF。研究了超声辅助提取对亚麻木酚素得率的影响,发现超声功率160 W、时间30 min、温度40°C的条件下,SDG得率可达到2.01 g/100 g DFHF。采用中心组合设计和响应面分析确定微波辅助提取的工艺条件为:乙醇浓度40.9 % (v/v),液固比21.9: 1 (mL/g),超声处理5 min进行预浸,在130 W微波功率下辐照90.5 s。该条件下SDG得率为2.19 g/100 g DFHF,高于常规搅拌提取和超声辅助提取的得率。圆二色性检测表明实验所用微波辐照条件对SDG构型没有影响。MAE大大缩短了提取时间,显著提高了SDG的得率,并节省了能耗。研究了亚麻木酚素粗提物在正己烷、氯仿、乙酸乙酯和水四种溶剂中的分配特性,发现SDG极性较大,主要保留在水相中。以洗脱物紫外吸收特征为评判指标,采用硅胶柱层析法二次洗脱分离出SDG产物。HPLC-PAD-MS检测其纯度为91.85%,硅胶柱层析的回收率为92.4%。采用大孔吸附树脂对SDG粗提物进行了静态吸附和解析。筛选出吸附率和解析率分别为86.4%和95.1%的X-5大孔吸附树脂。采用X-5为分离介质,研究其动态吸附特征及合适的洗脱条件,确定采用乙醇溶液进行梯度洗脱分离SDG,在20%乙醇洗脱组分中检测出SDG,其纯度为65.7%,但整个分离过程回收率偏低,为80.8%。采用Sephadex LH-20凝胶柱分离纯化SDG,确定水作为洗脱溶剂,二次分离得到纯度96.6%的SDG产物,回收率达到97.2%。研究了不同浓度SECO及其在人体内的代谢产物END与ENL对雌激素阳性乳腺癌细胞MCF-7增殖的抑制作用,并同金雀异黄素(GEN)进行对照,发现SECO与GEN在较高浓度(分别为100和40μmol/L)下对MCF-7具有抑制作用,而在低浓度(10~40和10~20μmol/L)下促进MCF-7增殖作用。END和ENL在10~100μmol/L浓度下均表现出对MCF-7增殖的抑制作用。采用流式细胞仪检测了由100μmol/L的SECO、40μmol/L的END、ENL与GEN作用后的MCF-7细胞的周期分布变化,发现G0/G1期细胞比例增加,S期细胞比例降低,G2/M期细胞比例略有增高,说明SECO、END、ENL与GEN可能是通过影响细胞DNA的增殖来实现对MCF-7的抑制作用的。采用放射性配基竞争结合法测定了SECO与α雌激素受体(ERα)的结合活力,发现在10μmol/L浓度下SECO不具有与ERα结合的活力。采用分子对接方法(DOCK)研究了SECO、END、ENL等配基与雌激素受体对接过程中的能量变化(ΔG),结果表明ΔG与配基分子的结合活力间无明显的线性相关性,通过对接研究初步确定了ERα与ERβ的配基结合部位及可能的作用方式。通过比较分子场分析(CoMFA),建立了预测配基分子与ERα、ERβ结合活力的CoMFA模型,结果表明氢键在模型中起重要作用,模型的预测结果与实测结果接近。采用该模型预测了END、ENL及SECO同ERα与ERβ的相对结合活力,结果表明ENL的结合活力远高于SECO,并且三者与ERβ的结合活力均高于与ERα的结合活力。因此,在ERβ分布较多的器官中,SECO发挥功效的途径之一可能是通过代谢产物ENL来实现的。更多还原

【Abstract】 Flaxseed (Linum usitatissimum L) is a rich source for several kinds of lignans. Secoisolariciresinol (SECO,C20H26O6) is the most abundant among these lignans and exists in flaxseed as a diglucoside (SDG). Researches have been carried out and flaxseed lignan (mainly SDG) was found to possess multiple bioactivities including anticancer, preventing cardiovascular disease, releasing postmenopausal symptoms, preventing osteoporosis and antioxidation. Exploitation of flaxseed lignan could make great contribution to prevent these dietary-dependent diseases and improve people’s health. Extraction and purification of flaxseed lignan would add value to flaxseed and make economic benefit. Besides, comprehensive utilization of flaxseed needs deep and systematic research.This paper aims at accurate and fast determination of flaxseed lignan and development of a safe, economic and easy-to-scale-up process to extract and isolate SDG (with purity higher than 95%). Besides, an integrate process for utilization of flaxseed has also been taken into consideration. As to the inhibition effect on breast cancer and its mechanism, in vitro cell culture experiment and chemical calculation are combined to cross validation and to explore its dose-effect and structure-activity relationship.For the determination of SDG, we found combination of basic hydrolysis and solvent extraction would get high SDG yield and the result is more accurate. Acid hydrolysis of purified SDG would cause three main hydrolysates, namely secoisolariciresinol glucoside (SG), SECO and anhydrosecoisolariciresinol (ANHSEC). With lower HCl concentration, SECO and SG were produced mainly and ANHSEC became the main product when acid concentration and heating duration was improved. HPLC-PAD-MS was adopted to determine SDG, SG, SECO and ANHSEC, and NMR was used to identify configuration of SDG. The quantitative method using SDG as standard avoided artifacts caused by acid hydrolysis was considered as simple, reliable and sensible. A colorimetric method was also developed to determine SDG quickly and easily.To integrate recover bioactive components in flaxseed, a wet process was proposed to get rid of the mucilage. Yield and static viscosity of the extract were determined and produced as a comprehensive index, demucilage ratio, to evaluate different levels of each factor. Demucilage conditions were optimized as following: temperature of 70°C, liquid to solid ratio of 7:1, pH 6.0, soaking duration 1 h and soaking times four. Major components of crude flaxseed gum, i.e., polysaccharide and protein were determined as 65.4% and 8.50% respectively. Monosaccharide composition was determined as rhamnose: fucose: arabinose: xylose: glucose: galactose = 5.10: 1.00: 4.18: 8.98: 2.13: 2.75 in mole ratio. High content of xylose means that crude gum contains neutral polysaccharide of high content. Hull and kernel were separated with a wet process. Flaxseed were breaking with a grinding wheel under the conditions that wheel distance 0.40 mm and wheel speed 2800 rpm. Broken flaxseed was fractionated by hydrocyclone for two times. Kernel-rich fraction, hull-rich fraction and mixture fraction were recovered and the yields of them were 52.6%, 22.3% and 12.7% respectively. Kernel content of hull-rich fraction and hull content of kernel-rich fraction were 20.9% and 11.1% respectively. Effect of Demucilage and dehull on SDG was investigated, and it was found little amount of SDG (9.6%) leaked with fine hull, most of the SDG was enriched in hull-rich fraction, mixture fraction has higher content of SDG than kernel-rich fraction.Conditions for stirring extraction of SDG from defatted flaxseed hull flour (DFHF) were optimized using orthogonal experimental design. Under the optimized conditions, i.e., 50% (v/v) aqueous ethanol as solvent, temperature of 60°C, liquid to solid ratio 20 mL/g and stirring duration of 3 h, 1.80 g SDG was obtained from 100 g DFHF. When adding NaOH to solvent with final concentration of 0.25 mol/L, SDG yield was improved to 1.90 g /100 g DFHF. Ultrasound-assisted extraction (UAE) of SDG was investigated and under the conditions that ultrasonic power as 160 W, time duration as 30 min, temperature as 40°C, SDG yield of 2.01 g/100 g DFHF. Central composite design and response surface analysis were carried out to optimize a microwave-assisted extraction (MAE) process for SDG. With ethanol concentration of 40.9 % (v/v), liquid to solid ratio of 21.9: 1 (mL/g), presoaking with ultrasound treatment for 5 min, microwave power of 130 W and irradiation time of 90.5 s, SDG yield of 2.19 g/100 g DFHF was obtained. MAE was superior to stirring extraction and UAE in SDG yield and time-saving. Circular dichroism of SDG with MAE was determined and it was found MAE didn’t change its configuration.Partition of crude SDG extract was carried out with 4 kind of solvent, i.e., n-hexane, chloroform, acetic ether and water, SDG was found mostly in water fraction. By Judging UV spectra, SDG was purified on a silica gel column for 2 times. Purity of the eluate was determined by HPLC-PAD-MS as 91.85%, and 92.4% SDG was recovered. Static adsorption and desorption capacity pf several kinds of macroporous adsorption resin (MAR) were determined and X-5 was found to possess a high adsorption and desorption rate as 86.4% and 95.1% respectively. X-5 was used to isolate SDG and dynamic elution characteristics were investigated. Gradient concentration of aqueous ethanol was adopted to separate SDG from the crude extract. SDG was eluted with 20% (v/v) aqueous ethanol and purity was determined with HPLC-PAD-MS as 65.7%. 80.8% SDG was recovered after MAR separation. Sephadex LH-20 column chromatography was used to purify SDG. Elution conditions were investigated and water was found to give the best resolution. After separation on Sephadex LH-20 column for two times, 97.2% SDG was recovered and eluate purity was determined as 96.6%. Effect of different concentration of SECO and its metabolites, enterodiol (END) and enterolactone (ENL) on proliferation of an estrogen receptor (ER) positive cancer cell, MCF-7 was studied. Genistein (GEN) was used as the counter. We found that SECO and GEN could inhibit proliferation of MCF-7 at high concentration (100 and 40μmol/L respectively). However, proliferation of MCF-7 was promoted when 10~40μmol/L SECO or 10~20μmol/L GEN was added to culture media. Two kinds of mammalian lignans, END and ENL showed dose-dependent inhibitory effect on proliferation of MCF-7 in concentration range of 10~100μmol/L. Flow cytometry method was adopted to analyze the changes of MCF-7 cell cycle after treatment with 100μmol/L SECO, 40μmol/L END, 40μmol/L ENL and 40μmol/L GEN. Results showed cell ratio of both G0/G1 and G2/M stages increased while that of S stage decreased. Therefore, SECO, END, ENL and GEN may exert its inhibitory effect by control proliferation of DNA proliferation.The method by competitive binding of irradiative ligand was used to determine relative binding affinity (RBA) of SECO to ERα. It was found that at concentration of 10μmol/L, SECO could not bind to ERα. Docking study of different ligands including SECO, END, ENL with two types of ER, i.e., ERαand ERβwas carried out. Energy change (ΔG) of the system during docking was compared and we found that there was no obvious linear relationship betweenΔG and log (RBA). However, ligand binding domain (LBD) was determined and possible interactions between ligands and ER were analyzed. RBA of ligands to ERαand ERβwere predicted by comparative molecular field analysis (CoMFA). It was found that hydrogen bond plays an important role in the interaction between ligands and both ER. Predictive values of optimized CoMFA model was close to observed values. RBA of END, ENL and SECO to ERαand ERβwere predicted by the CoMFA models, we found that RBA of ENL was much higher than that of SECO for both estrogen receptors. And all three ligands showed higher RBA to ERβthan that to ERα. Therefore, we suppose that in the tissues where ERβwas distributed, SECO may exert anticancer bioactivity by its metabolite ENL.更多还原