【作者】 张君萍；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 药用植物学， 2011， 博士

【摘要】 沙葱为百合科(LiLiaceae)葱属(Allium)植物,学名蒙古韭(Allium Monglicum Regel),别名蒙古葱,是多年生草本植物。沙葱可食用和药用,地上部分入蒙药,能开胃、消食、杀虫,有“菜中灵芝”的美称。沙葱种子中脂肪和总糖含量较高。沙葱种子含油量约为15.07%,其中不饱和脂肪酸含量约90.37%,总糖中以多糖为主,沙葱籽多糖含量约为7.59%。本研究利用超临界CO2萃取技术萃取沙葱籽油,以超临界CO2萃取脱脂后的沙葱籽滤渣为材料,采用超声波法提取沙葱籽多糖(AMRP),并对沙葱籽油和多糖的降血脂生物活性功能进行了研究,结果如下：1.超临界CO2萃取沙葱籽油的工艺优化应用超临界CO2萃取法提取沙葱籽油,在单因素试验的基础上得出萃取压力、萃取温度和萃取时间对沙葱籽油的超临界CO2萃取得率有显著影响。采用响应曲面法(RSM)建立了超临界CO2萃取沙葱籽油萃取率的二次多元回归方程。根据萃取率回归方程对沙葱籽油的超临界萃取工艺参数进行了优选,最优工艺参数为：萃取压力31.50MPa；萃取温度46.00℃；萃取时间101.60min；该条件下最大萃取得率预测值为15.07%,与实测值相符,证实了该方程的预测值与试验值之间具有较好的拟合度。利用优化工艺参数提取沙葱籽油,具有最大的产量值。2.沙葱籽油的脂肪酸成分及理化性质分析沙葱籽油成分采用气相色谱-质谱联用仪(GC-MS)分析,鉴定出9种脂肪酸成分。饱和脂肪酸以棕榈酸(5.73%)为主,占脂肪酸总量的9.63%；不饱和脂肪酸主要为亚油酸(64.50%)和油酸(19.28%),另外还检出单不饱和脂肪酸7-十八碳烯酸(2.84%)、11-二十碳烯酸(2.17%),多不饱和脂肪酸9,15-十八碳二烯酸(1.57%),占脂肪酸总量的90.37%,与维药黑种草子油的主要脂肪酸成分相类似。用常规方法对沙葱籽油的理化性质进行测定,研究结果表明超临界CO2流体萃取法得到的沙葱籽油各项理化指标均良好,产物纯度高,品质好。3.超声波辅助提取沙葱籽多糖的工艺优化以超临界CO2萃取脱脂后沙葱种子滤渣为材料,将RSM用于沙葱籽多糖的超声波提取工艺。对影响沙葱籽多糖提取的主要因素水料比(V/m)、提取温度、提取时间的最佳水平范围及其交互作用进行了研究和探讨,依据回归分析结果,最佳工艺条件是：水料比48.00：1,提取温度73.10℃,提取时间1.60h,超声波功率300W,提取次数2次,沙葱籽多糖得率的理论值为7.59%,与试验值相符。对三氯乙酸(TCA)脱蛋白条件进行了优化,选择TCA终浓度为5%左右,TCA沉淀蛋白的时间为2h或稍长。4.沙葱籽多糖的分离与纯化及结构分析超临界CO2萃取脱脂后的沙葱种子滤渣,通过超声波辅助提取,TCA法除蛋白,乙醇沉淀得AMRP粗品。采用DEAE-52纤维素离子交换柱层析对AMRP粗品进行初步分离,得到AMRP-Ⅰ、AMRP-Ⅱ采用Sephadex G-100凝胶柱层析对AMRP-ⅠAMRP-Ⅱ进一步纯化；AMRP-Ⅰ经Sephadex G-100柱层析只洗脱出单一的峰；AMRP-Ⅱ洗脱曲线为四个明显的洗脱峰,试验共分离得到5个组分。用气相色谱(GC)、紫外-可见光谱(UV-vis)、红外光谱(IR)分析对AMRP-Ⅰ、AMRP-Ⅱ的结构进行初步研究。结果表明：纯化的AMRP为杂多糖,AMRP-Ⅰ由鼠李糖、甘露糖、葡萄糖和半乳糖4种单糖组成,AMRP-Ⅱ由鼠李糖、阿拉伯糖、岩藻糖、甘露糖、葡萄糖和半乳糖6种单糖组成；UV检测显示AMRP-Ⅰ、AMRP-Ⅱ在波长260nm与280nm处未见有核酸和蛋白特征吸收峰,经IR检测证实AMRP-Ⅰ、AMRP-Ⅱ具有多糖类物质的特征吸收峰。5.沙葱籽油、多糖的降血脂功能考察沙葱籽油、多糖对高血脂小鼠降血脂功能研究。采用KM小鼠作为试验动物,将小鼠随机分为5小组：空白对照组(NC)、高脂模型组(HM)、高剂量组(HD)、中剂量组(MD)、低剂量组(LD)。用不同浓度的沙葱籽油、多糖给小鼠灌胃,连续灌胃35天,测定小鼠体重变化、脏器指数和小鼠血清中的总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、动脉硬化指数(AI)、丙二醛(MDA)值；超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性。结果表明：沙葱籽油、多糖连续灌胃35天后,沙葱籽油、多糖对高血脂小鼠有明显的降血脂作用,使小鼠体重和脏器指数下降；各干预组小鼠血清的TC、TG水平比试验前下降显著(p<0.01), LDL-C水平比试验前下降显著(P<0.01或P<0.05),各干预组小鼠的血清HDL-C水平比试验前显著升高(P<0.01或P<0.05),各干预组小鼠的动脉硬化指数(Al)与高脂模型组比较都显著降低(P<0.01)；表明沙葱籽油和多糖均能有效调节小鼠脂质代谢,减少脂质在体内的积累。沙葱籽油、多糖均能显著降低高酯血症小鼠血清MDA(P<0.05)含量,提高血清SOD (P<0.01或P<0.05)、GSH-Px (P<0.01)活性；表明沙葱籽油、多糖均能显著提高机体内源性抗氧化酶活性,降低体内脂质过氧化水平,起到拮抗动脉粥样硬化(Atherosclerosis, AS)的作用。更多还原

【Abstract】 Allium mongolicum Regel, also named Mongolia Leek, belongs to the Allium of Liliaceae and perennial herb. It is a kind of natural wild drug-food herb in China and a kind of Mongolia drug (aerial parts) in Inner Mogolia due to its functions in stimulating appetite and anthelminthic. It is named mythic fugus in vegetable. The contents of fat and total sugar in Allium mongolicum Regel seeds are the highest. Allium mongolicum Regel seeds contain about 15.07%oil, in which approximate 90.37%is unsaturated fatty acids.The polysaccharide is dominated in total sugar, the content of polysaccharide is about 7.59%in Allium mongolicum Regel seeds. In this study, Supercritical CO2 extraction was used to extract oil from Allium Mongolicum Regel seed. Ultrasonic-assisted extraction technology was applied to extract polysaccharides from Allium Mongolicum Regel seeds (AMRP) which were degreased by supercritical CO2 extraction, and to investigate the effects of the reduced blood ester of the Allium Mongolicum Regel seed oil and polysaccharides on the high ester mice. The contents as follows:1. Optimization for Supercritical CO2 Extraction of Allium Mongolicum Regel Seed OilThe results showed that extraction pressure, temperature, and time have significant influences on the yield of Allium Mongolicum Regel seed oil based on the results of the single-factor design. Response surface methodology (RSM) was used for establishing the model of a second order quadratic equation for the extraction yield of Allium Mongolicum Regel seed oil by supercritical CO2 extraction. The optimal parameters for the supercritical CO2 extraction determined by the regressive equation were pressure of 31.50MPa, temperature of 46.00℃, time of 101.60min,Under the optimized conditions, the predicted value of seeds oil extraction from Allium Mongolicum Regel was 15.07%, which was in consistent with the experimental values, it showed that the regressive equation fited the extraction process perfectly. It had maximal extraction yield of Allium Mongolicum Regel seed oil with optimized technique parameters.2. The Analysis of Fatty Acid Compositions and Physicochemical Properties of Allium Mongolicum Regel Seed OilThe fatty acid composition of Allium mongolicum Regel seed oil extracted by supercritical CO2 was determined by gas chromatography-mass spectrometry (GC-MS). It showed that the seed oil of Allium mongolicum Regel contained 9 fatty acids, and the fatty acids were composed of 90.37%unsaturated fatty acids and 9.63%saturated fatty acids. Furthermore, linoleic acid (64.50%), oleic acid (19.28%) and palmitic acid (5.73%) were the main fatty acids in the Allium Mongolicum Regel seed oil. In addition, monounsaturated fatty acids 7-octadecenoic acid (2.84%) and 11-eicosenoic acid (2.17%), Polyunsaturated fatty acid 9,15-Octadecadienoic acid (1.57%) were found in seed oil of Allium mongolicum Regel. The main fatty acid compositions of Allium mongolicum Regel seed oil were similar with that of oil from Nigella glandulifera Freyn seed which was Uighur’s traditional medicine. Measured the physical and chemical properties of Allium Mongolicum Regel seed oil with the conventional methods, the results showed that the oil extracted by supercritical CO2 was high purity and good quality.3. Optimization for Ultrasonic-assisted Extraction Technology of Allium Mongolicum Regel seed PolysaccharidesThe Allium Mongolicum Regel seed after degreasing by supercritical CO2 extraction was used as the main raw material, Response surface methodology was applied to optimize the Ultrasonic-assisted extraction conditions of polysaccharides from Allium Mongolicum Regel seeds. The effects of the main variables namely:the solvent/solid ratio, extraction temperature and extraction time as well as the mutual interaction between variables were studied. The predicted value of polysaccharides was up to 7.59%under the conditions as follows:solvent/solid ratio 48.00:1, extraction temperature 73.10℃, extraction time 1.60h, which was in consistent with the experimental values. The conditions of deproteinization by trichloroacetic acid (TCA) were optimized. TCA final concentration was about 5%, and precipitation time was 2h or longer.4. Speration, Purification and Structural Analysis of Allium Mongolicum Regel Seed PolysaccharidesThe crude AMRP was obtained through Ultrasonic-assisted extraction, ethanol precipitation, and protein removed with TCA method from Allium Mongolicum Regel seeds which were degreased by supercritical CO2 extraction. The DEAE-Cellulose column was used to separate AMRP, and two AMRP components were obtained and respectively named AMRP-Ⅰand AMRP-Ⅱ. The Sephadex G-100 column was used to furter separate AMRP-Ⅰand AMRP-Ⅱ, One component was obtained from AMRP-Ⅰand four components were obtained from AMRP-Ⅱ. AMRP-Ⅰand AMRP-Ⅱwere analyzed structurally by gas chromatography (GC), infrared absorption spectrum (IR) and ultraviolet-visible spectroscopy (UV-vis). The results showed that the two polysaccharides were highly heterogeneous, AMRP-Ⅰwas mainly composed of rhamnose, mannose, galactose and inositol, AMRP-Ⅱwas mainly composed of rhamnose, arabinose, fucose, mannose, glucose and inositol. No characteristic absorption of nucleic acid or protein in AMRP-Ⅰand AMRP-Ⅱwas observed in their UV-vis spectrums. AMRP-Ⅰand AMRP-Ⅱexhibited characteristic IR absorption of polysaccharides.5. Studies on Reducing Blood Lipids Function of the Allium Mongolicum Regel Seed Oil and PolysaccharideTo investigate the effects of reducing blood lipids of the Allium Mongolicum Regel seed oil and polysaccharides on the high blood lipids mice. KM mice were divided into five groups namely normal control group (NC), hyperlipidemia model group (HM), high-dose group (HD), middle-dose group (MD) and low-dose group (LD). The body weight and viscera coefficient of mice were tested. The concentration of serum triglyceride (TG), cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), arteriosclerosis indes (AI), malondildehyde (MDA) and the activity of superoxidedismutase (SOD), glutaathione perioxidase (GSH-PX) were analysed after 35 days. The Allium Mongolicum Regel seed oil and polysaccharides showed significantly the reducing blood lipids, and the body weight and viscera coefficient of mice were lessened. The contents of TG and TC were significantly lower than that before the experiment (P<0.01), and the level of the LDL-C was also lower (P<0.01 or P<0.05). The level of the HDL-C in the two interference groups was all evidently higher than that before the experiment (P<0.01 or P<0.05). AI of mice in the two interference groups was all obviously lower than the hyperlipidemia model group (P<0.01). It can be concluded that the Allium Mongolicum Regel seed oil and polysaccharides regulate the lipids metabolism in mice effectively, reduce the accumulation of lipids in body. The Allium Mongolicum Regel seed oil and polysaccharided could reduce the content of MDA of serum (p<0.05), increase the activity of SOD (P<0.01 or P<0.05) and GSH-PX (P<0.01). It can be concluded that the Allium Mongolicum Regel seed oil and polysaccharides could promote the activities of endogenous anti-oxidation enzymes and reduce the level of peroxidation in body, which attribute to the effect of preventing AS.更多还原