摘要
壳聚糖是一种具有降脂作用的阳离子多糖,在国内外受到广泛的关注。但是壳聚糖由于原料以及制备方法等不同,产品性质差别大,导致其生理活性也迥然有异;而且虽然市场上已有壳聚糖降脂产品热销,但其降脂作用机理目前还不是很清楚。因此,本论文在制备具有显著降脂效果的壳聚糖的基础上,从体外降脂作用和动物体内分布与排泄、影响脂质代谢的分子机制、降低脂质过氧化等方面,对壳聚糖的降脂机理进行了系统深入的研究。本研究对壳聚糖在功能性食品的开发以及天然降血脂药物的开发具有理论学术意义和实际应用价值。
采用碱熔法制备了一系列不同脱乙酰度(DD)、相对分子质量和粒径的壳聚糖,在体外模拟人体胃肠消化环境中,对其与脂肪、胆固醇以及胆盐的结合能力进行了研究,并采用动物实验比较了不同脱乙酰度和不同相对分子质量壳聚糖的降脂效果,结果显示,体外实验中,壳聚糖结合油的能力较为显著,且结合能力随着脱乙酰度或相对分子质量的增大而增强;不同壳聚糖结合胆盐量的范围为1.75～7.75 mg/g,当样品脱乙酰度相同时,结合胆盐能力随着相对分子质量的增加而增强;壳聚糖的粒径对胆固醇结合能力影响较大,片状壳聚糖显示了最差的结合能力;动物实验中,对于DD73%、81%和90%的三种壳聚糖,脱乙酰度较高的壳聚糖能更有效地抑制体重增长、显著地降低血清总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C),升高高密度脂蛋白(HDL-C)水平,对血清甘油三酯(TG)水平影响较小;相对分子质量对减肥效果影响较明显,高相对分子质量比低相对分子质量的壳聚糖更显著降低了大鼠的饲料利用率和体重增长量以及血清TG水平。
对壳聚糖在动物体内的分布与排泄进行了研究。建立了异硫氰酸荧光素标记壳聚糖(FITC-CIS)在小鼠血浆和组织样品中的检测方法,并给小鼠口服FITC-CIS后在不同时间观察壳聚糖在组织和血液中的分布、消化道中含量变化以及尿液、粪便中的排泄,结果表明,检测方法适用于FITC-CIS在动物体内的药动学研究;FITC-CIS主要分布在小鼠的肝、肾和肌肉中,在给药1 h后,肝中浓度最高,随后的时间点中,均为肾中含量最高,肝脏是脂肪代谢的重要场所,壳聚糖快速进入肝脏影响了脂类的代谢,发挥了降脂作用;在胃中,壳聚糖浓度随时间逐渐降低,且发现了壳聚糖对脂肪液滴的吸附,在小肠和大肠中,浓度最高的时间点分别为1 h和2 h;粪便和尿液中,壳聚糖浓度最高的时间点分别与大肠和肾脏中壳聚糖浓度相对应,粪便中在FITC-CIS口服后2.5～5 h之间排泄最多,其中的壳聚糖以原药形式存在,而尿液中壳聚糖在6～12 h之间排泄最多,且发现部分壳聚糖被降解。这些结果直接证明了壳聚糖能在消化道中结合脂肪从而不经消化直接随粪便排出,且有一部分壳聚糖能发生降解,对体内脂质代谢过程进行调节。
用降脂效果最佳的壳聚糖饲喂高脂血症大鼠和正常大鼠,比较其对高脂血症的预防和治疗效果,并以降血脂药物考来烯胺作为阳性对照,结果表明,与高脂对照相比,壳聚糖组大鼠体脂含量显著下降,肝脏指数显著下降,脂肪肝症状显著减轻,血清中TG、TC和LDL-C浓度显著降低,HDL-C浓度显著上升,肝脂含量(TC和TG)也显著降低,说明壳聚糖具有显著的降脂效果;大鼠摄入含有壳聚糖的高脂饲料后,粪便中排出的脂肪和胆固醇的量均显著高于高脂对照组的大鼠,且粪脂中脂肪酸成分与饲料中脂肪酸成分相同,表明壳聚糖能减少机体对食物中脂肪的吸收;肝脏肝脂酶和脂蛋白脂酶活性显著增强表明壳聚糖能调节脂质代谢酶活性;壳聚糖能预防长期摄入高脂食物所引起的高脂血症,也能减轻已患高脂血症的大鼠的血脂水平以及肝脏脂肪累积;等量的考来烯胺比壳聚糖能更有效地降低血清胆固醇水平,并且完全抑制了脂肪肝的形成,但是也能显著升高血清TG水平,且副作用明显。
从分子水平研究了壳聚糖对大鼠肝脏中脂质代谢相关蛋白的调节,结果表明,壳聚糖能显著上调高脂饲料引起的低密度脂蛋白受体(LDL-R)mRNA下降以及过氧化物酶体增殖物激活受体α(PPARα)的表达,轻微上调卵磷脂胆固醇酰基转移酶(LCAT)和7α羟化酶(CYP7A1)的mRNA水平,下调β-羟基β-甲基戊二酸单酰CoA还原酶(HMG CoA还原酶)的mRNA水平,表明壳聚糖能在分子水平上调节胆固醇和脂质代谢的动态平衡,发挥降脂功效。
从体外和体内研究了壳聚糖抗脂质氧化的作用及机理,结果表明,添加量为0.02%的壳聚糖对猪油和粗榨菜油均有抗氧化作用,但不如等量的常规抗氧化剂维生素C(Vc)效果明显,而当添加量增大后,壳聚糖与Vc在实验后期抗氧化能力接近,表明壳聚糖具有体外抗氧化能力,但效果不是很明显,可能与壳聚糖的性质有关;壳聚糖能显著降低高脂饮食引起的血清游离脂肪酸(FFA)浓度上升,降低脂质过氧化物代谢产物丙二醛(MDA)的含量,提高机体主要的抗氧化酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)的活性,表明壳聚糖在体内通过调节抗氧化酶活性降低脂质过氧化程度,从而促进降脂效果。
本论文的研究明确了具有显著降脂作用的壳聚糖性质,建立了一种新型的荧光标记壳聚糖在血浆和组织样品中的检测方法,从壳聚糖对脂质代谢基因表达的影响结合体内分布排泄和抗脂质过氧化以及体外结合脂质能力等途径进一步阐明了壳聚糖的降脂机理。
Chitosan, a cation polysaccharide, has exhibited marked hypolipidemic effect, which attracts much attention in the world. However, the physicochemical properties of chitosans vary with the reaction conditions and materials, and then affect the physiological effects. Moreover, the hypolipidemic mechanism is still unclear although it has been provided as a weight-loss product. In this study, a series of chitosans with different properties were prepared and chosen in vitro and in vivo, then one with the best hypolipidemic effect was used to study its mechanism, which will be of great significance for the utilization and pharmacological investigation of chitosan.
Chitosans with different degree of deacetylation (DD), molecular weight (Mw) and particle size were prepared by alkali fusion, then their binding capacities with fat, cholesterol and bile salts were studied in the conditions simulated human digestive tract and their hypocholesterolemic effects were also tested in rats. The results showed that the fat-binding capacities of chitosans strengthened with the increment of DD or Mw; the range of bile salt-binding capacities was 1.75~7.75 mg/g and the capacities strengthened with the Mw; particle size was the major factor influencing the cholesterol-binding capacity of chitosan and flake form showed the worst binding capacity; among 73%, 81% and 90% deacetylated chitosans, the last one could more effectively suppress body weight gain, and significantly reduce serum TC, LDL-C and elevate HDL-C level; molecular weight of chitosan had greater influence on the weight-loss effect compared with DD, and chitosan with higher Mw more significantly reduced feed efficiency and body weight gain, and plasma TG level.
The distribution of chitosan in the tissues and plasma was studied. The determination method of FITC-CIS in the plasma and tissues was established, and the concentration in the digestive tract and the excretion in the feces and urine at the different time were observed following oral-administration of FITC-CIS in mice. The results showed that FITC-CIS was distributed mainly in the liver, kidney and muscle, the concentration in the liver was highest after1 h and that in the kidney was highest at the other time; in the stomach, the concentration of chitosan decreased with the time past by and fat in the diet was adsorbed by chitosan; the highest concentration was at 1 h and 2 h respectively in the small and large intestine; time with highest concentration in the feces or urine was corresponding with that in the large intestine or kidney, respectively; in the feces, the excretion of chitosan was most at 2.5~5 h and chitosan appears in the original form; in the urine, chitosan was excreted most at 6~12 h and degradated partially. The results proved directly that chitosan could bind fat in the diet in the digestive tract and then excrete with feces, and a portion of chitosan would be degradated in the body and regulate lipid metabolism.
Chitosan with the best hypolipidemic effect was fed to rats accompanied with high-fat diet at the beginning and after the hyperlipemia to test the prevention and improvement effect. The results showed that compared with rats fed high-fat control diets, chitosan exhibited marked hypolipidemic effect, the body fat and liver index reduced significantly, and fatty liver symptom relieved; serum TG, TC and LDL-C concentrations reduced and HDL-C elevated significantly; liver TC and TG concentrations also lowered significantly; rats receiving chitosan had more excretion of fecal fat and cholesterol than those fed high-fat control diet, and the fatty acid composition in the fecal fat was the same as that in the diet, which indicated chitosan could reduce the absorption of fat in the diet; liver HL and LPL activities increased significantly, which indicated chitosan could regulate the lipase activity; chitosan could not only prevent the hyperlipemia induced by long term-administered high-fat diet but also relieve serum lipid level and liver fat accumulation in hyperlipemia rats; compared with cholestyramine which is a hypocholesterolemic medicine, chitosan with the same addition had worse effect, but it was safe and had no side effects.
The molecular regulation of chitosan on several proteins correlated with lipid metabolism in the liver of rat was studied. The results showed that chitosan could significantly up-regulate LDL-R and PPARαmRNA level, moderately up-regulate LCAT and CYP7A1 mRNA level and down-regulate HMG CoA reducase mRNA level, which indicated chitosan could regulate the dynamic balance of cholesterol metabolism in the molecular level and then exert the hypolipidemic effect.
The anti-oxidation activity of chitosan was studied in vitro and in vivo. The results showed that chitosan at an addition of 0.02% had anti-oxidation effect on the lard and crude rape oil, but the activity was worse than ascorbic acid, and when the addition increased, chitosan and ascorbic acid had similar activity; chitosan could significantly reduce serum FFA and MDA concentrations and elevate SOD, CAT and GSH-PX activities which were major anti-oxidation enzymes in the body, which indicated that chitosan regulated the anti-oxidation enzyme activities and then reduced the lipid peroxidization.
In this study, chitosan with best hypolipidemic activity was chosen; the determination method of a novel fluorescence labeled chitosan in the plasma and tissues was established; the hypolipidemic mechanism of chitosan was elucidated on the mRNA expression of proteins correlated with lipid metabolism, the distribution and excretion of chitosan in mice, the reduction of lipid peroxidization and fat-binding activity in vitro.
引文
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