褐藻新品种厚叶海带岩藻聚糖硫酸酯的结构分析及其对脂质代谢的影响

The studies will be carried out on the structure characteristics and lipometabolic effect for the fucoidan purified from the new brown seaweed of Kjellmaniella crassifolia Miyabe, which contains about 4-5 times of fucoidan compared with that of Laminaria japonica. MALDI-TOFMS and mass-spectrometry ESIMS/MS will be applied for elucidating the complex structural patterns of the monomer composition and their substitution of glycosylation, sulphation and/or O-acetylation based on the monosaccharide composition of the fucoidan analyzed by Gas chromatography and the characteristics of the fucoidan strcuture illustrated by infrared spectrometer and nuclear magnetic resonance. In addition, the structure characteristics of fucoidan from Kjellmaniella crassifolia Miyabe produced in North of Japan will be compared. The changes of peroxisome proliferators activatied receptor PPAR-α , and acyl-CoA oxidase-1 (ACO-1) and carnitine palmitoyltransferase-1 (CPT-1) in lipolytic process modulated by PPAR-α will be detected dynamically by real time transcription and western blotting methods. The major transcription factor involved in the activation of lipogenic genes, mRNA level of SREBP-1c, and that of its two representative target genes, SCD-1 and FAS will be determined at meantime. PPAR-γ,which plays a pivotal role in fat cell differentiation and lipid storage and the key enzyme in lipoprotein and adipocyte metabolism will also be also dynamically analyzed by the method of RT-PCR for detecting the fucoidan acting as dual PPAR agonists. The accomplishment of this project will establish the structure characteristics of fucoidan from Kjellmaniella crassifolia cultured in China and demonstrating its hypolipidemic metabolic mechanism. They will provide scientific base for high efficient utilization of this newly cultured brown seaweed.

项目对褐藻优势新品种厚叶海带（Kjellmaniella crassifolia Miyabe）褐藻多糖硫酸酯(fucoidan)进行结构分析及其对脂质代谢的影响。在气相色谱分析其单糖组成、红外光谱、核磁共振分析其结构的基础上，采用MALDI-TOFMS 和ESIMS/MS分析单糖间及其硫酸根连接结构，比较其结构特征；利用实时-PCR与蛋白印迹法动态检测高脂大鼠肝脏中过氧化酶体增殖物激活受体PPAR-αmRNA的表达及由其调节的肝脏特异性肉碱棕榈酰转移酶(CPT-1)和脂酰辅酶A氧化酶(ACO-1)的活性，动态检测调节元件结合蛋白SREBP-1c变化及其调控的脂肪酸合成酶（FAS）、硬脂酰辅酶A去饱和酶1（SCD-1）的变化；动态检测PPAR-γ及脂蛋白代谢关键酶脂蛋白脂肪酶LPL变化。建立厚叶海带fucoidan的结构理论数据，阐明其对脂肪代谢影响。为厚叶海带培育高效利用奠定基础。

优质褐藻笼目海带（又名厚叶海带，Kjellmaniella crassifolia Miyabe）因富含褐藻聚糖硫酸酯而备受关注。项目组连续五年引入培育栽培了笼目海带，优化工艺技术高效提取分离了笼目海带褐藻聚糖硫酸酯(fucoidan)；并对其组织结构进行了分析，包括单糖组成、红外分析、核磁分析；建立笼目海带fucoidan的结构理论数据，研究结果表明笼目海带褐藻聚糖硫酸酯的单糖组成中以岩藻糖为主，还含有7%以上的葡萄糖醛酸； 红外光谱和核磁共振光谱分析表明其具有典型的褐藻聚糖硫酸酯特征。利用粗糖和分离纯化的笼目海带褐藻聚糖硫酸酯进行了降血脂功效研究，结果证明笼目海带褐藻聚糖硫酸酯的降血脂效果显著，经过4周灌胃大鼠的三酸甘油酯水平显著降低，经过12周灌胃大鼠的三酸甘油酯水平、胆固醇、低密度脂蛋白胆固醇含量显著降低；且高密度脂蛋白胆固醇显著升高。利用实时-PCR与蛋白印迹法动态检测高脂大鼠肝脏中过氧化酶体增殖物激活受体PPAR-αmRNA的表达、动态检测调节元件结合蛋白SREBP-1c变化及其调控的脂肪酸合成酶（FAS）、硬脂酰辅酶A去饱和酶1（SCD-1）的变化；以及动态检测PPAR-γ及脂蛋白代谢关键酶脂蛋白脂肪酶LPL变化，结果证明了笼目海带褐藻聚糖硫酸酯各剂量组在整个实验过程中，可有效地调节肝脏中与胆固醇代谢有关的HMG-CoA、LCAT mRNA的表达，抑制胆固醇的合成与逆转运，从而减少肝脏TC含量；随着灌胃褐藻聚糖硫酸酯时间的延长至12周时，笼目海带的各剂量组都显著地抑制了SREBP-lc mRNA的表达，增加了SCD1mRNA的表达，降低内源性脂肪的生成，实现对脂质代谢的积极调控。同时对β-氧化过程中的基因PPARα、PPARγ、LPL mRNA表达也起到一定的作用，提高机体的抗氧化能力，促进机体对脂肪的排出。研究完成了项目计划内容要求，形成SCI收录文6篇，其中一区发表文章1篇；为笼目海带（厚叶海带）培育高效利用奠定基础。