【作者】 张梦娟；

【导师】 徐怀德；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2007， 硕士

【摘要】 天麻（Gastrodia elata Blume）属兰科非自养型植物的干燥块茎,是我国传统的名贵中药,主产于陕西、云南、贵州及四川等省。中医临床主要用于治疗头痛眩晕、惊风抽搐、肢体麻木、面肌痉挛、高血压、冠心病等疾病。目前,国内天麻方面的研究仅限于天麻素,而关于天麻中多糖的相关信息未见任何系统的报道。故本研究旨在对天麻多糖的提取分离和性质活性方面做初步的摸索,为今后天麻多糖的利用和开发奠定基础。本试验以天麻作为原料,系统的研究了天麻多糖水提取和超声波提取的技术条件,对天麻多糖进行了分离纯化研究,并对其抗氧化和抑菌的活性进行了评价,对纯化后所得两种多糖的理化性质和单糖组成进行了探讨,研究的主要结果如下:1.建立了以蒽酮-硫酸法测定天麻多糖含量的测定方法。2.采用传统水提取和超声波技术提取天麻多糖,在单因素试验的基础上,采用响应面分析法对天麻多糖提取工艺进行优化。传统水提取的最优工艺参数为:提取温度为58℃,提取时间36min,料水比1:37,天麻多糖提取率为9.25%。超声波水提取天麻多糖的最优工艺参数为:提取温度为66℃,提取时间34min,料水比1:45,天麻多糖提取率为32.78%。超声波浸提时,粗多糖提取率均比传统热水浸提时高。3.天麻多糖的分离纯化。经试验确定了以下纯化路线:进行乙醇分级醇析,分别收集乙醇浓度为0-40%和40%⁸0%之间沉淀出的多糖,得到粗多糖G-40和G-80;再经木瓜蛋白酶-Sevag法脱蛋白,透析法除去小分子杂质及盐类等步骤,逐级提高样品的纯度,得到多糖GeB40和GeB80;最后经DEAE-52纤维素柱层析,选取一定最大洗脱峰范围进行多糖的收集,得到多糖GeB40-1和GeB80-4。紫外光谱结果表明该多糖组分不含蛋白质等杂质。4.多糖GeB40-1的理化性质:白色粉末状;可溶于水;不溶于无水乙醇、乙醚、丙酮、氯仿、正丁醇等有机溶剂;不含有淀粉类物质、还原糖类物质和蛋白质核酸类物质;红外光谱分析表明分子结构中存在α-型糖苷键;分子量大于8000D;比旋光度为[α]²⁰ =+150.5°;熔点为270.3-271.1℃。采用离子色谱法分析单糖组成结果为:该多糖由L-鼠李糖、D-半乳糖、D-葡萄糖、D-木糖和D-甘露糖五种单糖组成,五种单糖摩尔数之比L-鼠李糖:D-半乳糖:D-葡萄糖:D-木糖:D-甘露糖=29:1:42:11:2。5.多糖GeB80-4的理化性质:浅黄色粉末状;可溶于水;不溶于无水乙醇、乙醚、丙酮、氯仿、正丁醇等有机溶剂;不含有淀粉类物质、还原糖类物质和蛋白质核酸类物质;红外光谱分析表明分子结构中存在α型糖苷键;分子量大于8000D;比旋光度为[α]20 = -94.7°;熔点为278.5-279.9℃。采用离子色谱法分析其单糖组成结果为:该多糖由L-鼠李糖、D-半乳糖和D-葡萄糖三种单糖组成,三种单糖摩尔数之比L-鼠李糖:D-半乳糖:D-葡萄糖=2.5:1:25。6.抗氧化活性试验研究了天麻多糖GeB40和GeB80对O₂^-·、·OH、DPPH·的清除能力以及其还原能力。结果表明GeB40和GeB80都具有O₂^-·、·OH、DPPH·的清除能力和较强的还原能力,且清除率随浓度的增加都有所增强,GeB40对O₂^-·的清除能力强于GeB80;GeB80对·OH和DPPH·的清除能力以及还原能力都强于GeB40。7.抑菌试验表明天麻多糖GeB40、GeB80和天麻乙醇提取物对细菌、真菌均有一定的抑菌效果。GeB40对大肠杆菌有明显的抑制作用,对根霉有一定的抑制作用;GeB80的抑菌作用强于GeB40,对青霉、根霉等霉菌和金黄色葡萄球菌有较强抑菌效果,对肠炎沙门氏菌和枯草芽孢杆菌也有一定抑制作用;天麻乙醇提取物的抑菌活性强于天麻多糖GeB40和GeB80,对金黄色葡萄球菌、大肠杆菌和青霉的抑制作用明显,对根霉和肠炎沙门氏菌也有一定抑制作用。8.采用ACE抑制率的体外检测法测定天麻多糖的降血压活性。结果表明,天麻多糖GeB40和GeB80都具有很好的降血压功效,GeB80降血压活性高于GeB40,在浓度为10 mg·mL^-1时两者的抑制率都达到50%以上。本文的创新点:1.首次利用超声波提取、木瓜蛋白酶-Sevag法脱蛋白、透析以及DEAE-52柱层析纯化等先进工艺,获得天麻多糖。2.用离子色谱法测定天麻多糖的单糖组成,采用ACE抑制率的体外检测法研究其降血压活性均具有一定创新性。更多还原

【Abstract】 Gastrodia elata Blume is a saprophyte widespread in Chinese province Shanxi, Yunnan, Guizhou, Sichuan, etc. This drug is considered to have very beneficial properties. In clinical practice, this plant is used to treat vertigo, blackout, headache, hemiplegia, neuralgia, paralysis, lumbago, hypertension, coronary heart disease and it also has sedative actions. At present, the research of Gastrodia elata Blume is merely limit to Gastrodin. There is no systemic report on the research of Gastrodia elata Blume Polysaccharide. The objective of this paper is to lay a foundation for utilization and exploitation of Gastrodia elata Bl.ume polysaccharide.In order to study the polysaccharide of Gastrodia elata Blume, Gastrodia elata Blume was taken as material. The research contains extraction, ultrasonic extraction and purification, chemical and physical properties and composition of monosaccharide of Gastrodia elata Blume polysaccharide, Gastrodia elata Blume polysaccharide of antioxidation and anti-bacterial activity. The major results were shown as follows:1. The quantifying of Gastrodia elata Blume polysaccharide was used by the anthrone-sulfuric acid method, and determined the polysaccharide’s content.2. Gastrodia elata Blume polysaccharide was obtained by using hot water extraction and ultrasonic wave extraction. On the basis of one-factor tests, the method of response surface analysis was carried out to determine the optimal condition of the extraction. The using hot water extraction optimal condition were: extraction 36min at 58℃with 1:37 ratio of Gastrodia elata Blume to water. Under the optimized condition, the extraction rate of Gastrodia elata Blume Polysaccharide was 9.25%.The ultrasonic wave extraction optimal condition were:ultrasonic extraction temperature 66℃, time 34min and the ratio of Gastrodia elata Blume to water was 1:45. Under the optimized condition, the extraction rate of Gastrodia elata Blume Polysaccharide was 32.78%.The yield and content of polysaccharide by using ultrasonic wave extraction were higher than using hot water. The method of ultrasonic wave was optimum to extract polysaccharide. 3. The purification of Gastrodia elata Blume polysaccharide. The successive procedures were as follows: first,G-40 was precipitated with 0%~40%(v/v) ethanol,G-80 was precipitated with 40%~80%(v/v) ethanol,secondly, G-40 and G-80 were deproteinized by enzyme combined with Sevag method and dialysised with distilled water, thirdly, GeB40-1 and GeB80-4 was further purified by DEAE-52 cellulose column chromatography and collected according to the peak of the elution curve of water. The UV spectrum’s result indicated this polysaccharide component does not contain protein and other impurities.4. The physical and chemical properties of Polysaccharide GeB40-1:white powder; diss- olves in water;doesn’t dissolve in some organic solvent such as ethanol,aether and acetone; doesn’t contain Starch,reducing sugar,protein and nucleic acid;the data of IR revealed that GeB40-1 is chiefly linked byα-glycosidic bonds;the average molecular weight is more than 8000 Dalton;the specific rotation is +150.5°; the melting point is 270.3-271.1℃. Ion Chroma- tography analyze monosaccharide composition of polysaccharide:GeB40-1 is composed of L-rhamnose,D-galactose,D-glucose,D-xylose and D-mannose with the molar ratio of 29:1:42: 11:2.5. The physical and chemical properties of Polysaccharide GeB80-4: yellow powder; dis- solves in water;doesn’t dissolve in some organic solvent such as ethanol,aether and acetone; doesn’t contain Starch,reducing sugar,protein and nucleic acid;the data of IR revealed that GeB80-4 is chiefly linked byα-glycosidic bonds;the average molecular weight is more than 8000 Dalton;the specific rotation is -94.7°; the melting point is 278.5-279.9℃. Ion Chromato- graphy analyze monosaccharide composition of polysaccharide:GeB80-4 is composed of L-rhamnose,D-galactose and D-glucose with the molar ratio of 2.5:1:25.6. The antioxidative activity of Gastrodia elata Blume polysaccharide:GeB40 and GeB80 had the ability of scavenging oxygen free radicals,hydroxyl free radical,DPPH·radical and reducing ability. Oxygen free radicals scavenging ability of GeB40 was higher than GeB80; Hydroxyl free radical,DPPH·radical scavenging ability and reducing ability of GeB80 were higher than GeB40.7. The antibacterial activity of GeB40,GeB80 and ethanol extracts from Gastrodia elata Blume were shown as follows: GeB40,GeB80 and ethanol extracts from Gastrodia elata Blume had Bacterial Inhibition and Fungal Inhibition. GeB40 had strong inhibitory effect on Escherichia coli and Rhizopussp. GeB80 had strong inhibitory effect on Rhizopussp., Penicilliumsp. and Staphylococcus. aureus. GeB80 had strong inhibitory effect on Rhizopussp.,Penicilliumsp. and Staphylococcus. aureus. Ethanol extracts had strong inhibitory effect on Staphylococcus. aureus,Escherichia coli and Penicilliumsp. GeB80 had stronger inhibitory effect than GeB40,and Ethanol extracts was stronger than GeB80 and GeB40. 8. The detecting method in vitro of ACE inhibition ratio was adopted on the study of Gastrodia elata Blume polysaccharide’s antihypertensive activity. The test result indicate: GeB40 and GeB80 had good antihypertensive activity, GeB80 stronger than GeB40. When the Concentration of GeB40 and GeB80 was 10 mg·mL-1, ACE inhibition ratio of their were over 50 %.The innovation points were as follows:1. The Gastrodia elata Blume polysaccharide optimum parameter was obtained by using hot water extraction and ultrasonic wave extraction,separation and purification including deproteinization, dialysised and column chromatographic.2. The studies of Gastrodia elata Blume polysaccharide involved monosaccharide composition and antihypertensive activity.更多还原