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不同分子量和构型(α、β)壳聚糖减肥降脂功能研究
Jin Qiu
学位类型硕士
导师李鹏程
2017-05-09
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词壳聚糖 减肥 降脂 分子量 3t3-l1前脂肪细胞
其他摘要    肥胖是由于卡路里的摄入与消耗不平衡造成的,在过去的几十年里,全球肥胖人群数量呈爆发式增长,使肥胖成为全球性健康隐患。然而现阶段市面上的减肥药品虽数量种类繁多,却仍存在治疗效果不佳、副作用大、易反弹等问题,使减肥药市场上既存在挑战又存在机遇。壳聚糖是一种碱性阳离子多糖,具有资源产量丰富、细胞亲和性、生物相容性好、安全无毒等独特优势,在国内外减肥降脂研究领域受到广泛的关注。平均分子量(Mw)和分子构型是评价壳聚糖的重要指标,影响壳聚糖的物理性质、化学性质、生理活性等,然而现阶段对壳聚糖减肥降脂活性的研究大多没有明确给出所使用壳聚糖的平均分子量及构型,也没有对不同平均分子量、不同分子构型壳聚糖的减肥降脂活性进行对比。
    本论文从体外油脂胆固醇吸附活性、胰脂肪酶活性抑制活性,3T3-L1前脂肪细胞分化抑制活性,对饮食诱导肥胖SD大鼠减肥降脂活性三个方面进行了壳聚糖减肥降脂功能研究。主要研究结果如下:
(1)通过体外模拟胃肠道消化环境,研究不同平均分子量α-、β-壳聚糖对油脂、胆固醇的吸附活性。结果表明平均分子量为~1、~3、~5、~7、~9 kDa的α-、β-壳寡糖均具有一定的体外油脂、胆固醇结合能力。总体而言,α-壳寡糖的体外油脂、胆固醇结合能力高于β-壳寡糖。1 g样品α-壳寡糖平均可以吸附2-8 g油脂或50-65 mg胆固醇。并且,壳聚糖的油脂、胆固醇吸附能力与其平均分子量、分子构型有关,平均分子量为~3000 Da的α-壳寡糖的油脂、胆固醇吸附能力最强,分别为7.08 g.g-1和63.48 mg.g-1,当平均分子量增大或减小,其吸附能力下降。
(2)通过对硝基苯酚法测定不同分子量α-壳聚糖、α-壳寡糖及β-壳寡糖对胰脂肪酶活性抑制能力。结果表明,不同浓度的五种分子量α-壳聚糖(Mw=~1800k、~1500k、~1200k、~900k、~600k、~300kDa)、α-壳寡糖(Mw=~1k,~3k,~5k,~7k,~9kDa)和β-壳寡糖(Mw=~1k,~3k,~5k,~7k,~9kDa)均具有一定的胰脂肪酶抑制活性。虽然16种样品的胰脂肪酶活性抑制率均小于对照组奥利司他(5 mg/mL,抑制率93.39%),但奥利司他属于合成药物,已被证实存在副作用,壳聚糖属于天然产物,健康安全,有很好的应用前景。
(3)通过体外诱导3T3-L1前脂肪细胞分化,模拟前脂肪细胞分化增殖过程,以不同平均分子量的α-、β-壳聚糖为样品,研究其对3T3-L1前脂肪细胞分化的抑制作用。实验结果表明:六种低分子量的α-壳寡糖(A1-6)、B1组的β-壳寡糖的细胞毒性较小,B2-5组的β-壳寡糖、~1800 kDa的α-壳聚糖、2340 kDa的β-壳聚糖细胞毒性较大。以细胞毒性较小、3T3-L1前脂肪细胞分化过程中的脂质生成抑制效果最好的A1组α-壳寡糖进行进一步实验,结果表明:细胞水平上来说,浓度为12.5,25,50 μg/mL的A1均能显著抑制3T3-L1前脂肪细胞成脂分化及分化后的脂质堆积过程,降低前脂肪细胞的分化率,减小成熟脂肪细胞中的TAG浓度;分子水平上来说,浓度为25,50 μg/mL的A1能够显著抑制C/EBPα的表达,抑制脂肪细胞分化及脂质堆积,达到减肥目的。
(4)通过饮食诱导肥胖的SD大鼠减肥降脂实验模型,以α-壳聚糖(A组)、α-壳寡糖(B组)及配方产品(海洋一号HY-1,C组)为样品,以高(1000 mg/kg)、中(500 mg/kg)、低(250 mg/kg)三种剂量进行灌胃,以奥利司他作为阳性对照,研究α-壳聚糖的减肥降脂功能。实验结果表明:A中剂量组和C中剂量组能减少肾脏周围脂肪。三种样品会使肥胖大鼠睾丸附近脂肪增多。A高剂量组和C低剂量组能升高血糖,A中剂量组、B高剂量组、B中剂量组均能降低血糖。B组和C组能降低肥胖大鼠甘油三酯的含量。三组样品均能降低肥胖大鼠总胆固醇含量,升高LDL-C含量。A高剂量组、A中剂量组、B中剂量组、B低剂量组、C低剂量组和C中剂量组能降低HDL-C含量。肝脏、心脏、肠系膜脂肪、皮下脂肪切片观察显示,给药组能抑制脂肪细胞的生长,并一定程度上减少组织损伤。
    本研究明确了不同构型、不同平均分子量的壳聚糖的减肥降脂功能,并从细胞、基因水平对机理进行初步探究,为开发壳聚糖减肥功能食品及天然减肥药物提供理论依据。
;     Obesity is caused by an imbalance between caloric intake and expenditure. In recent decades, it has become a worldwide burden to public health. However, though there are already so many kind of anti-obesity drugs, most of them are characterized with side effects, non-significant effect and rebound. Therefore, the study and application of weight-losing products are full of twists, as well as challenges. Chitosan, as the second most abundant polysaccharide in nature, is confirmed with anti-obesity activity, which attracts much attention. Average molecular weight and molecular conformation are two important indexs for the evaluation of chitosan, which affect the physical properties, chemical properties and physiological activity of chitosan. However, now most studies about the anti-obesity activity of chitosan haven’t specified the average molecular weight and molecular conformation of the chitosan uesd in the study, and haven’t compared the anti-obesity activity and lipid-lowering activity of chitosan with different average molecular weight and molecular conformation.
    In this study, a series of α-、β-chitosan with different average molecular weight (Mw) were prepared. Its fat-binding capacity, cholesterol-binding capacity, inhibitory effects to pancreatic lipase activity, inhibitory effects on cell proliferation and adipogenesis in 3T3-L1 cells and anti-obesity effects in high-fat diet-induced obese rats were studied. Rresearch results are as follows:
(1) A biopharmaceutical model of the digestive tract was used to determine the fat- and cholesterol-binding capacity of α- and β-chitosan. α- and β-chitosan with different Mw (~1、~3、~5、~7、~9 kDa) were prepared by oxidative degradation assisted with microwave irradiation. The results showed that all of the α- and β-chitosan samples exhibited fat- and cholesterol-binding capacities. Within the testing range, 1 g of α-WSC sample could absorb 2–8 g of peanut oil or 50–65 mg of cholesterol, which are both significantly higher than the ability of cellulose to do the same. Meanwhile, both Mw and molecular conformation affect the binding capacity of chitosan, as α-chitosan of ~3 kDa shows the highest fat- and cholesterol-binding capacities (7.08 g.g-1 and 63.48 mg.g-1, respectively), and the binding ability of α-chitosan declines as its Mw increases or decreases from 3000 Da.
(2) The pancreatic lipase assays were based on p-nitrophenyl derivatives. All the samples (α-chitosan of ~1、~3、~5、~7、~9 kDa and ~1800、~1500、~1200、~900、~600、~300 kDa and β-chitosan of ~1、~3、~5、~7、~9 kDa) were proven to be able to inhibit pancreatic lipase activity to some extent. The inhibition ratio of all the samples are smaller than orlistat. While orlistat is proved to have some side effects, chitosan, as marine-derived active compounds, has better application prospects.
(3) The effects of α- and β-chitosan with different Mw on cell proliferation and adipogenesis in 3T3-L1 cells, intracellular lipid accumulation, and the expression of C/EBPα were studied. The results presented that on the cellular level, chitosan could significantly inhibite 3T3-L1 preadipocytes into adipogenic differentiation and lipid accumulation process, reduce the concentration of TAG in mature adipocyte; on the gene level, chitosan could inhibit the expression of C/EBPα.
(4) The anti-obesity effect and lipid-lowering activity of α-chitosan with different Mw were studied using high-fat diet-induced obese rats model. The result indicated that all three samples (α-chitisan, α-chitosan oligosaccharide and a composite product, HY-1) could reduce the weight of obese rats with obvious appetite suppressant, reduce CHO level in the serum and improve LDL-C level in the serum. In general, the anti-obesity effect of the composite product was the best which even better than orlistat.
    This study affirmed the anti-obesity activity and lipid-lowering activity of chitosan, researched the relationship between its anti-obesity effect and its Mw and its molecular conformation, and explored its mechanism of reducing weight from the level of cells and gene. This study is of great significance for the utilization and pharmacological investigation of chitosan’s anti-obesity effect and lipid-lowering activity.
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136614
专题实验海洋生物学重点实验室
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GB/T 7714
Jin Qiu. 不同分子量和构型(α、β)壳聚糖减肥降脂功能研究[D]. 北京. 中国科学院大学,2017.
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