【作者】 盛瑜；

【导师】 关铭；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 本课题通过单因素试验和四因素三水平[L9(34)]的正交试验，确定了桔梗粗多糖和五味子粗多糖的最佳提取方法，并在此基础上，用sevag法除蛋白杂质，透析袋除小分子杂质，利用柱层析法进一步分离纯化多糖，最终得到分子量均一的桔梗多糖PRPS-a，PRPS-b和PRPS-c以及五味子多糖ASPS-a-1，ASPS-b-2和ASPS-b-3。本课题从体内和体外两个方面研究和探讨了桔梗多糖的抗哮喘活性。在体内实验中，以卵蛋白诱导的哮喘大鼠为模型，应用不同浓度的桔梗多糖进行干预，测定哮喘大鼠体内的炎性细胞、细胞因子和炎症介质含量的变化，进而观察桔梗多糖对哮喘大鼠的治疗作用。体外实验中，将桔梗多糖PRPS-a，PRPS-b，PRPS-c作用于ET-1致敏的人气道平滑肌细胞（HASMCs）增殖模型，观察它们对其增殖的作用。本课题系统考察了五味子多糖的抗肝损伤活性及抗氧化活性。选取肝指数，胸腺指数，脾指数，血清ALT，AST，ALP，Alb，T-AOC等为观察指标，探究了五味子多糖对CCl4所致小鼠急性肝损伤的影响；并对肝组织中MDA，SOD，NO及肝脏组织病理学变化进行了相关研究。在抗氧化实验中，较为系统探究了五味子多糖ASPS-a-1，ASPS-b-2和ASPS-b-3对DPPH、羟自由基、超氧阴离子的清除作用及金属螯合能力。实验结果表明，桔梗多糖可显著抑制哮喘大鼠Eotaxin1和ICAM-1的表达，减少血液及气道内EOS数量及百分比，显著降低哮喘大鼠血清和BALF中IL-4含量，升高IFN-γ含量，并能够有效的抑制HASMCs的增殖。由此提示，桔梗多糖抗哮喘机制可能与其通过调节哮喘Thl/Th2失衡，进而影响细胞因子的分泌而抑制HASMCs的增殖，使之具有较强的抗哮喘活性有关。五味子多糖能够减轻肝组织增重，增加Alb含量和提高T-AOC、SOD活性，降低肝组织中MDA的含量，降低NO的活性，减轻肝细胞肿胀、脂肪变、炎细胞浸润的程度，并具有抗氧化活性和金属螯合能力，这提示五味子多糖能够通过清除自由基、抑制其生成来保护肝脏，从而减少CCl4对肝脏造成的损伤。本课题以我国传统中药桔梗和五味子的多糖为研究对象，在确定其提取分离和纯化工艺的基础上，较系统的研究了它们的理化性质及相关药效活性。实验结果表明，桔梗多糖具有抗哮喘活性；五味子多糖具有抗肝损伤活性。依据国家科技部“重大新药创制”的理念，本研究结果，力求为建立中药药效物质基础及物质资源库提供桔梗和五味子多糖的较系统基础研究资料，为桔梗多糖成为防治哮喘有效药物，五味子多糖成为保肝药物及抗氧化制剂提供理论依据。更多还原

【Abstract】 Platycodonis Radix and Schisandrae Chinensis Fructus are famous medicinal plants inChina. Platycodonis Radix has been used in traditional oriental medicine as an expectorantand antitussive and used to treat coughs, colds, upper respiratory infections, sore throats,tonsillitis, and chest congestion. According to reports, the the polysaccharides of Platycodongrandiflora has immunomodulatory and anti-tumor effects. Schisandrae Chinensis Fructushas the function of replenishing and promoting production of body fluid, tonifying thekidney and relieving mental strain. It is reported that the polysaccharides of Schisandra hasanti-fatigue, hypoxia, lowering blood sugar and immunomodulatory effects. So far thestudies on Platycodonis Radix and Schisandrae Chinensis Fructus have been focused onsaponin and lignans. So we need to systematic study on the polysaccharides of them.Thesubject studied on extraction, separation, purification and biological activities ofpolysaccharides from Platycodon Grandiflorum and Schisandra Chinensis.This subject is divided into six parts. The first part is to set up and optimize the ways ofextracted crude polysaccharides from Platycodonis Radix and Schisandrae ChinensisFructus.The second part is the purification of them. This part also studies on the properties ofthem. The third part is that polysaccharides from Platycodonis Radix (PRPS) acts on the ratasthma model. The forth part is that observe the effect of PRPS on the HASMCsproliferation model which is sensitized by endothelin-1(ET-1). In the fifth part,polysaccharides from Schisandrae Chinensis Fructus (ASPS) acts on CCl4acute hepaticinjury mice of model. The last part is to abserve antioxidation activity of them.The first part of the experiment used the hot reflux method to extract the the crudepolysaccharides from Platycodonis Radix, and used room-temperature immersion method ofalkaline to extract crude polysaccharide from Schisandrae Chinensis Fructus. In order tofurther optimize the extraction process of the polysaccharides, the experimental designedtwo orthogonal experiments of L9(34). The results showed that the yield of crudepolysaccharides from Platycodonis Radix was12.66%at the conditions of85%ethanolrecipitation,1:10solid-liquid ratio and2h for3times for extraction.The yield of crudepolysaccharides from Schisandrae Chinensis Fructus was1.18%at the conditions of90% ethanol recipitation,1:6solid-liquid ratio,2h for extraction and the concentration of NaOHwas2M. In the two extraction process, the energy consumption was low, the conditions weremild. They were efficient extraction methods. Polysaccharides of Platycodonis Radix andSchisandrae Chinensis Fructus have a variety of pharmacological activities. The optimization of theextraction process laid the foundation for the further development and application of polysaccharides.And it provided a rich source of polysaccharides to separate and purify.In the second part, the crude polysaccharides were purified by sevag deprotained. Theseparation and purification of the polysaccharides were used the means of columnchromatography. In the end, we gained five polysaccharides called PRPS-a, PRPS-b,PRPS-c, ASPS-a-1, ASPS-b-2and ASPS-b-3. They were isolated from Platycodonis Radixand Schisandrae Chinensis Fructus for the first time. Their molecular weights were2567Da,439746Da,2839Da,103300Da,61600Da,34700Da tested by HPSEC. The uronic acidcontent of ASPS-a-1, ASPS-b-2and ASPS-b-3were7.8%,39.4%and43.5%. There was nouronic acid in PRPS-a, PRPS-b, PRPS-c. The monosaccharide composition of them wasanalyzed by GC. PRPS-a was composed of fructose, glucose and galactose with a molarpercentage ratio of41:35:24. PRPS-b was composed of arabinose, fructose and glucose witha molar percentage ratio of35:43:22. PRPS-c was composed of arabinose, fructose andgalactose with a molar percentage ratio of28:39:33. The monosaccharide composition ofASPS-a-1, ASPS-b-2and ASPS-b-3were Ara:Xyl:Glu:GalA=22:15:51:6,Ara:Xyl:Glu:GalA=14:18:33:35,Ara:Xyl:Glu:GalA=16:9:32:41.In the third and forth parts, anti-asthma activity of PRPS was tested in vivo and in vitro.In vitro experiment, the rat asthmatic model was established by sensitizing and challengingSD rats with aerosolized ovalbumin (OVA) and the intervention of PRPS was performed.The changes of the levels of IL-4, IFN-γ, EOS in broncho alveolar lavage fluid (BALF) andserum were observed. The expression level of Eotaxin1and ICAM-1were detected. Theresults showed that PRPS can significantly inhibit the expression of Eotaxin1, ICAM-1inasthmatic rats and reduced the number of EOS in blood and airway. In addition, PRPS cansignificantly reduce the level of IL-4and increase the level of IFN-γ in blood and serum inrat asthmatic model. This proved that PRPS can regulate the disorder in the balance betweenTh1and Th2cytokines which makes it has anti-asthma activity. In vivo experiment, the cellsproliferating model of human ASMCs were established by ET-1and the intervention ofPRPS-a, PRPS-b and PRPS-c was performed. MTT method was used to detect inhibition toHASMCs. The experimental results showed that PRPS-b and PRPS-c could inhibit HASMCs proliferation obviously. The inhibitory activity of PRPS-a wasn’t evident. Theactivity difference depends on the monosaccharide composition of PRPS-a, PRPS-b andPRPS-c. The polysaccharide has powerful inhibitory activity which containing arabinose andfructose. This part ultimately proved that PRPS has anti-asthma activity which consistentwith the medicinal efficacy of relieving cough and asthma of Platycodonis Radix. So thepolysaccharides are the active site of Platycodonis Radix.In the last two parts, antihepatitis and antioxidant activity of alkaline S. chinensispolysaccharides (ASPS) were studied. The model of acute liver injury was induced by CCl4and the intervention of ASPS was performed. The activities of T-AOC, NO and SOD, thelevel of Alb and MDA, as well as the pathological changes of the liver were determined. Theresults showed that different doses of ASPS can reduce the weight of liver tissue, the level ofMDA and the activities of NO. It can also increase the level of Alb and the activities ofT-AOC, SOD. The pathological sections of liver showed that the swelling of liver cells, fattychange and inflammatory cells infiltration were alleviated by ASPS. These results suggestedthat ASPS can role in the resistance to the liver injury. The free radical scavenging and metalchelate activity of ASPS-a-1, ASPS-b-2and ASPS-b-3were detected. The results provedthat they have strong antioxidant activity. In addition, the antioxidant activity of them relatedto the content of furfural acid. These two chapters determined that ASPS can protect the liverby inhibiting free radicals generation and radical scavenging from injury caused by CCl4.In conclusion, this project took polysaccharides of Platycodonis Radix and SchisandraeChinensis Fructus as the research object. It determined their extraction and purificationprocess, studied on their physicochemical properties and pharmacological activity. Itconfirmed the PRPS, PRPS-a, PRPS-b and PRPS-c have antiasthmatic activity. It alsoproved that ASPS, ASPS-a-1, ASPS-b-2and ASPS-b-3have antihepatitis activity andantioxidant activity.These results provide the data of polysaccharides from PlatycodonisRadix and Schisandrae Chinensis Fructus to establish the material basis of traditionalChinese medicine efficacy and material resources library. The object provides a theoreticalbasis for the polysaccharides becoming. effective drugs against the disease, the Schisandrapolysaccharide as a hepatoprotective drug and antioxidant provides a theoretical basis.更多还原