【作者】 龚志华；

【导师】 施兆鹏；

【作者基本信息】 湖南农业大学 ， 茶学， 2010， 博士

【摘要】 茯苓是一种资源丰富、多糖含量高、成分较为单一的典型多糖菌类植物,其多糖具有抗病毒、抗肿瘤、抗炎症、增强免疫和防石利尿等多种生理功能。目前国内外对茯苓的研究与开发主要集中于传统中药制剂开发和茯苓糖类物质的化学组成、分子修饰、生物活性以及提取制备技术等研究方面。研究表明,茯苓主体多糖成分β-茯苓聚糖是一类含有少量β-(1→6)葡聚糖支链的β-(1→3)-D-葡聚糖,分子结构结合紧密,水溶性较差,生物活性较低。采用新型多糖降解技术,将茯苓多糖转化为分子量较小且生物利用率较高的活性多糖是茯苓及其多糖高效开发利用的关键。本研究集成辐照降解、现代光谱、柱色谱分离纯化以及用于功能与安全性评价的高通量筛选、细胞培养、动物实验等先进技术,对茯苓辐照降解产物、茯苓水溶性多糖高效制备、辐照降解产物抗氧化与调节糖脂代谢的生物活性及安全性等内容进行了系统研究,获得了以下结果：1、首次采用60Co辐照降解茯苓多糖,并运用扫描电镜、水溶性糖分析、紫外光谱扫描、红外光谱扫描、凝胶渗透色谱、DEAE-52纤维素柱层析等先进技术方法,’系统研究了辐照处理对茯苓多糖降解的微观效应。结果表明,茯苓分别经600 kGy、800 kGy、1000 kGy、1200 kGy剂量辐照后,水溶性总糖、水溶性多糖含量显著增加,并呈剂量效应,其中1000kGy处理分别增加18倍、19倍以上,降解产物主要为含有羰基基团的分子量较小的酸性多糖,其数均相对分子量在10128-11881之间、重均相对分子质量在23995-27917之间,且基本组成与茯苓原料多糖的基本组成类似。2、系统比较了热水提取、超声波辅助提取、微波辅助提取三种物理方法以及β-葡聚糖酶、植物复合酶SPE-002两种酶法辅助提取茯苓水溶性多糖的效果。结果显示,酶法辅助提取是较好的方法,且采用p-葡聚糖酶较好,其最优参数为酶用量6.5%、提取时间60 min、提取温度50℃、提取体系pH值5.0,茯苓水溶性多糖得率达13.31%。微波辅助提取是三种物理方法中的最优方法,其最优参数为中低火(231 w)、时间60min、固液比1：25,茯苓水溶性多糖得率为3.66%。3、首次应用p-葡聚糖酶辅助降解辐照后茯苓多糖,结果表明,低剂量辐照-酶解具有一定协同作用,各因素影响大小依次为辐照剂量>酶促降解温度>酶促降解时间>酶促降解体系pH值>酶用量,其中辐照剂量因素影响显著。采用辐照剂量1000 kGy,在最优酶解参数条件下,茯苓水溶性多糖得率为43.57%,茯苓1000 kGy辐照后直接提取的水溶性多糖得率为45.15%。由此可以推断p-葡聚糖酶对1000 kGy与1200 kGy辐照剂量处理产生的水溶性茯苓多糖进行了“过度降解”,产生一些分子量更小的物质。为提高水溶性茯苓多糖得率,采用1000 kGy剂量辐照处理即可,而不需采用辐照-酶解协同降解。4、不同辐照剂量处理茯苓水提取物的体外抗氧化功能结果表明,茯苓辐照后,还原能力增强,并对超氧阴离子自由基、羟基自由基、DPPH自由基均具有显著的清除作用,且呈一定剂量效应,以1000 kGy辐照剂量处理的茯苓水提取物的综合抗氧化能力最强。5、首次运用高通量筛选和细胞试验技术研究了辐照降解茯苓多糖产物调节糖脂代谢的功能。结果显示,1000 kGy辐照剂量处理的茯苓水提取物对LXR核受体模型、PPARy核受体模型、FXR激活受体模型、PPARa激活核受体模型均有一定的调节糖脂代谢功能,其中对前三个核受体模型的活性明显,而其80%乙醇醇沉物仅对LXR核受体模型、PPARy核受体模型有一定的生物活性,说明辐照处理可以增强茯苓调节糖脂代谢功能。利用1 mL/L 20%脂肪乳剂处理48 h能够成功复制细胞模型,可应用于非酒精性脂肪肝药物筛选,经过1000 kGy辐照处理的茯苓能够显著降低非酒精性脂肪肝细胞内的TG及ALT水平,并明显优于其它处理。茯苓辐照后,降血糖功能增强,并主要体现在抑制AGEs形成、抑制醛糖还原酶活性和促进葡萄糖消耗与摄取等几方面,其中1000 kGY辐照处理的水溶性多糖可通过促进葡萄糖消耗与摄取而直接降低血糖,也可通过抑制蛋白质非酶糖基化产物的形成来防止高糖诱导的并发症发生。6、高通量筛选、细胞毒性试验和动物安全性评价结果证实,经辐照处理的茯苓水提取物与水溶性多糖提取物安全无毒。更多还原

【Abstract】 Poria cocos (Schw.) Wolf is a typical polysaccharides fungus plant, which is of resource-rich, high-polysaccharide,and simplex-components. and pachman with anti-virus, anti-tumor, anti-inflammatory, enhancing immunity, anti-stones and diuresis and other physiological functions. At present, the domestic and international research and development of Poria cocos, are primarily focused on these aspects, which include the development of traditional Chinese medicine preparations and the research of chemical composition, molecular modification and biological activity of Poria carbohydrates, and the technology of extraction and preparation.Studies have shown that Poria cocos main polysaccharideβ-pachyman are a kind ofβ-(1→3)-D-glucan with a little (1→6)-β-glucan branched chain, which is a tight molecular structure, poorly water-soluble,low biological activity. The key way to exploit and utilize Poria cocos and pachymose efficiently is to adopt a new degradation technology,which make pachyman convert to be smaller molecular weight with higher activity and bioavailability of polysaccharides.Integrated with radiation degradation, modern spectroscopy, column chromatography separation and purification, evaluation of function and safety such as high-throughput screening, cell culture, animal experiments and other advanced technologies, this paper make a deep and systemic study on radiation degradation products of Poria cocos, efficient preparation of Poria water-soluble polysaccharide, the biological activity of antioxidation and regulating glucose and lipid metabolism and safety of products in Poria cocos by radiation degradation. The following results were obtained.1, first time,60Co irradiation degradation was used on pachman, at the same time, scanning electron microscopy, analysis water-soluble sugar, UV spectrum scanning, IR scanning, gel permeation chromatography, DEAE-52 cellulose column chromatography and other methods of advanced technology were used. It were studied systemically on the micro effects of polysaccharides.The results show that the content of soluble total sugar and water-soluble polysaccharide in Poria cocos increased significantly after treated with 600kGy, 800 kGy,1000 kGy,1200 kGy dose of irradiation, and shown a dose effect.Of which after 1000 kGy treatment the content of soluble total sugar and water-soluble polysaccharide increased more than 18 times,19 times respectively. And the main degradation products were acid polysaccharides which contained carbonyl group and smaller molecular weight.The number average molecular weight were between 10128 and 11881, and weight average molecular weight were between 23995 and 27917, and their basic compositions are similar to polysaccharide of Poria raw material.2, It made a Systematic comparison of efficient preparation of water-soluble polysaccharide in Poria cocos, including the hot water extraction, ultrasonic extraction, microwave-assisted extraction for the three physical method and P-glucanase, plant enzyme SPE-002 two kinds of enzyme assisted extraction.The results showed that enzyme assisted extraction is a better way, and better use of P-glucanase, the optimal parameters for the enzyme dosage of 6.5%, extraction time 60 min, extraction temperature 50℃, extraction system, pH 5.0, Poria water-soluble polysaccharide reached 13.31%. Microwave-assisted extraction is the best of three methods of physical methods, the optimal parameters for the low fire (231 W), time 60 min, solid to liquid ratio 1:25, Poria water-soluble polysaccharide was 3.66%.3, first time,β-glucanase was used to assisted degrade pachman after irradiation. The results showed that low doses of radiation-enzyme has a synergistic effect, the order of the factors affecting were the radiation dose> enzymatic degradation temperature> enzymatic degradation time> the pH value enzymatic degradation of system> amount of enzyme, among them the radiation dose was a significant effect factor.With radiation dose 1000 kGy, the optimal conditions for zymohydrolysis parameters, poria water-soluble polysaccharide was 43.57%. while it reached 45.15%, if extracted directly after irradiation dose 1000 kGy. It Can be inferred that after the 1000 kGy or 1200 kGy radiation dose treatment on the water-soluble polysaccharides,β-glucuronidase made a "excessive degradation"on them,and resulting in some smaller molecular weight substances. In order to improve the yield of water-soluble pachman,1000 kGy dose of irradiation is ok, without using radiation-enzymatic collaborative degradation.4, In vitro antioxidant function of Poria water extract with different radiation dosage were studied, the results showed that, Poria irradiated, reducing power increased, and superoxide anion radical, hydroxyl radical, DPPH radical scavenging were significant effects and showed a dose effect. The comprehensive antioxidant capacity of 1000 kGy radiation dose is best.5, For the first time, high-throughput screening and cell experiment technique were adopted to research the function in regulating glucolipid metabolism of irradiation degradation products of pachman. The results showed that,1000 kGy irradiation dose treatment on the water extract of Poria cocos have a particular glucolipid metabolism regulation biological activity on the bioactivities of the LXR nuclear receptor model, PPARγ nuclear receptor model, FXR activated receptor model, PPARαactivated nuclear receptor model, especially in the first three activities of nuclear receptor model, while its 80% ethanol sediments of water-soluble carbohydrates only exert some bioactivity on LXR nuclear receptor model, PPARy nuclear receptor model. It is indicated that irradiation can enhance the function of regulating glucose and lipid metabolism of pachman. Using 1 ml/L 20% fat emulsion processing cells 48 h, it can successfully clone the cell-model applied to screen non-alcoholic fatty liver drug. after 1000 kGy irradiated, Poria can significantly reduce the levels of TG and ALT of non-alcoholic fatty liver cell, and it is significantly better than other treatments. Poria cocos after irradiation, the function of hpyerglycemic is enhancement, and the main reflections were on suppressing the AGEs formation, inhibiting aldose reductase activity and the promotion or consumption of glucose uptake and other aspects. of which,1000 kGy irradiation water-soluble polysaccharide can not only promote the uptake of glucose consumption and lower blood sugar directly, but also inhibite the protein non-enzymatic glycation products formation to prevent high glucose-induced complications.6.High-throughput screening, cell toxicity and animal safety evaluation test results confirmed that, after irradiation, water extract and water-soluble polysaccharide extracts of Poria is safe and non-toxic.更多还原