（锡金）微孔草[Microula sikkimensis（Clarke）Hemsl ]种子油成分、活性及应用研究

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	（锡金）微孔草[Microula sikkimensis（Clarke）Hemsl ]种子油成分、活性及应用研究
	曹越
学位类型	博士
导师	索有瑞
	2010-06
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	生态学
关键词	锡金微孔草超临界co2萃取高效液相色谱-质谱脂质体紧密连接
摘要	1、采用超临界CO2萃取技术，提取锡金微孔草种子油，研究萃取过程中萃取的工艺参数，如萃取温度、压力、时间及原料粒度等对种子油萃取得率的影响，确定了超临界CO2萃取微孔草种子油的最佳工艺参数为：萃取温度40℃；萃取压力20MPa；萃取时间为120 min。萃取率约为27%-28%。 2、利用气相色谱(GC)分析方法对超临界CO2萃取的锡金微孔草种子油进行分析。结果发现锡金微孔草种子油中含有大量的不饱和脂肪酸。不饱和脂肪酸包括油酸、亚油酸和亚麻酸，含量均值高达89.4%。对提取的微孔草种子油理化性质和稳定性进行了研究。 3、利用荧光衍生试剂1,2-苯并-3,4-二氢咔唑-9-乙基对甲苯磺酸酯（BDETS）作为柱前衍生化试剂, 在Eclipse XDB-C8色谱柱上, 采用梯度洗脱对微孔草种子油中游离脂肪酸(FFA)衍生物进行基线分离。利用柱后在线的串联质谱以大气压化学电离源(APCI)正离子模式实现了各组分的质谱定性。荧光检测的激发和发射波长分别为ex =260nm，em = 380nm。脂肪酸的线性回归系数0.9994～0.9999, 检测限为14.32～26.82fmol。建立的方法具有良好的重现性。结果表明锡金微孔草种子油中含有大量的游离不饱和脂肪酸；并且超临界CO2提取工艺对其中的脂肪酸的提取率有着重要的影响。 4、通过KM小鼠和Wistar大鼠半数致死量(LD50)、最大耐受量等安全性毒理学指标的实验检测，动物实验表明锡金微孔草种子油每日口服剂量可达36.60g/kg，相当于临床成年人日服用剂量的732倍，说明锡金微孔草种子油属无毒性保健食品，服用安全。并且对卫生学指标进行了检测与评价，为微孔草种子油的开发提供了科学依据和实验资料。微孔草种子油可调节小鼠的血脂水平，降低血脂作用显著。锡金微孔草种子油各剂量组均能提高小白鼠血清中HDL-C的含量，并且存在明显量效关系。锡金微孔草种子油高、中剂量对HDL/TC比值有明显的升高作用，对LDL/HDL比值有明显的降低作用。 5、利用薄膜蒸发—超声工艺制备微孔草种子油脂质体。配方为: 2.6g卵磷脂，0.4g胆固醇，1g微孔草种子油，溶解在30mL氯仿中，制备温度为35℃；减压蒸发1.5小时，滴加pH为6.5的双蒸水15mL；4℃条件下，制得水性混悬液；超声处理(50KHz×2min/次×3次)；使用0.45μm和0.22μm的滤膜过滤。此条件下产物包封率可达75%以上，平均粒径为240～420nm。温度、粒径、制备工艺和储存条件都是影响微孔草种子油脂质体稳定性的重要因素。国内外都进行了相关制备技术的研究，制备工艺和产品已形成一定的局面。 6、脂质体是一种定向药物载体，具有生物膜的特性和功能。肠道紧密连接（Tight junction，TJ）是维持肠黏膜上皮细胞间屏障的重要结构基础。靶向性是脂质体作为药物载体最突出的特征。细胞间紧密连接蛋白-1（Zonula Occludens-1）、封闭蛋白或遮光蛋白（Occludin）、上皮-钙粘附蛋白（E-Cadherin），是肠道结构的重要成分蛋白。在啮齿枸橼酸杆菌感染条件下，发现微孔草种子油脂质体对这三种蛋白的表达和形态分布存在效应关系。推测微孔草种子油脂质体较强的被吸收能力，使高浓度脂类营养物迅速进入肠道细胞和组织，加速和增强了肠道本身的生理机能，提高了肠道应变的缓冲力。
其他摘要	1. Seed oil of Microula sikkimensis (Clarke) Hemsl was obtained by supercritical carbon oxide. The effects of various parameters, such as pressure, temperature, particle sizes and extraction time, on the supercritical carbon dioxide extraction efficiency of Microula sikkimensis seed oil were studied. The results showed that under pressure of 20 MPa, temperature 40℃, the dynamic extraction time of 120min. The yield of oil was 27%-28%. 2. Using gas chromatography (GC) analysis of the supercritical CO2 extraction sikkimensis seed oil for analysis. The results showed Microula sikkimensis seed oil contains large amounts of unsaturated fatty acids. Unsaturated fatty acids include oleic acid, linoleic acid and linolenic acid, Mean levels as high as 89.4%. The extracted Microula sikkimensis seed oil and stability of physical and chemical properties were studied. 3. Separation of free fatty acids in Microula sikkimensis seed oil extracted by supercritical carbon oxide was accomplished with a pre-column derivation method using 1,2-benzo-3,4- dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as labeling regent followed by high-performance liquid chromatography with fluorescence detection. The target compounds were identified by mass spectrometry with atmospheric pressure chemical ionization (APCI in positive-ion detection mode). Separtion of derivatized fatty acids have been optimized by a reversed-phase Eclipse XDB-C8 column in conjunction with a water-acetonitrile gradient elution (ex=260nm，em=380nm). Excellent linear responses were observed with correlation coefficients 0.9994～0.9999. Detection limits are 14.32～26.82fmol. The facile BDETS derivatization coupled with mass spectrometry allowed the development of a highly sensitive method for the analysis of free fatty acids obtained in seed oil by supercritical carbon oxide extraction. The established method is of high efficiency for seed oil extraction and high sensitivity for the determination of fatty acids in seed oil. And the SFE-CO2 extracted conditions had obviously effect on the contents of fatty acid in the seed oil. 4. KM mice and Wistar rats by the median lethal dose (LD50), the maximum tolerated dose toxicology and other safety parameters of the test. Animal experiments show that Microula sikkimensis seed oil daily oral doses of up to 36.60g/kg, Clinical use of adult equivalent to daily oral doses of 732 times, Description Microula sikkimensis seed oil is non-toxic health food, take safety. And on hygiene indicators detection and evaluation, was Microula sikkimensis seed oil development provides scientific basis and experimental information. Microula sikkimensis seed oil adjustable mice lipid levels, Reduce Lipid significantly. Microula sikkimensis seed oil could increase the dose mice serum HDL-C levels, and there are clear dose-effect relationship. Microula sikkimensis seed oil high dose and moderate dose of HDL/TC ratio was significantly increased role. On the LDL / HDL ratio were significantly lower effect 5. Using thin film evaporation-ultrasound technology, producing Microula sikkimensis seed oil Liposomes. Formula: 2.6g lecithin, 0.4g cholesterol, 1g Microula sikkimensis seed oil, Dissolved in 30ml chloroform, 35 vacuum evaporation of 1.5 ℃hours, Dropping ddH2O 15mL(pH 6.5), 4 condition, the water suspension; Ultrasonic ℃treatment (50KHz×2min/times×3times), Using the membrane filter(45μm and 0.22μm). Under these conditions, the product of more than 75% encapsulation efficiency, the average particle size of 240～420nm. Temperature, particle size, preparation and storage conditions all affect the Microula sikkimensis seed oil Liposomes Stability of important factors. Home and abroad for the preparation of technology-related research, production process and products have obtained good results. 6. Liposome is targeted drug delivery, with the features and functionality biofilm. Intestinal tight junctions (TJ) is the maintenance of intestinal epithelial cell barrier of the important structural basis. Targeting as a drug carrier is liposomes the most prominent features. Zonula Occludens-1, Occludin and E-Cadherin is an important structural component of intestinal protein. Citrobacter infections in rodents under，Microula sikkimensis seed oil Liposomes for these three species distribution of protein expression and effect relationship exists. Speculated that Microula sikkimensis seed oil Liposomes absorption capacity to be strong. High concentrations of lipid nutrients to rapidly enter the intestinal cells and tissues, High concentrations of lipid nutrients to rapidly enter the intestinal cells and tissues, Increased intestinal metabolism of the buffer force.
学科领域	植物学
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3389
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	曹越. （锡金）微孔草[Microula sikkimensis（Clarke）Hemsl ]种子油成分、活性及应用研究[D]. 北京. 中国科学院研究生院,2010.

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