桔梗、五味子多糖的分离纯化及活性研究
|
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。
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.
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。
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.
引文
[1]张树政.糖生物学:生命科学中的新前沿[J].生命的化学,1999,19(3):103-107.
[2] FRANZ G. Polysaccharides in pharmacology. Current application and future concepts[J]. Planta Med,1998,55(6):493-497.
[3] TANG W, HEMM I, BERTRAM B. Recent development of antitumor agents fromChinese herbal medicines， Part Ⅱ. High molecular compounds[J]. Panta Med，2003，69(3):193-201.
[4] Wang Xiaoqing, Zhao Yuqing. The research progresss on extraection and puripicationof garlic polysacaride[J]. Modern Chinese Medicine,2009,11(7):4-7(Ch).
[5]李化.超声波技术在中草药成分提取中的应用[J].中草药,2001,24(4):229-230.
[6]郭孝武.一种提取中草药化学成分的方法——超声提取法[J].天然产物研究与开发,1998,11(3):37-40.
[7] Stahmann K P, Monschau N, Sahm H, et al. Structure properties of native and sonicatedcinerean, a β-(1-3)-(1-6)-D-glucan produced by Botrytiscinerea[J]. Carbohydr Res,1995,226(1):115-128.
[8]李岱,张静,孙润广.超声波处理对柴胡多糖提取率、微观形貌特征及生物活性的影响[J].生物加工过程,2009,7（2）:29.
[9]郭赣林,徐深圳,李文浩,等.球等鞭金藻多糖的微波萃取工艺[J].食品科学,2011,32(14):113-117.
[10]范会平,符锋, Giuseppe Mazza,等.微波提取法对樱桃、猕猴桃和枸杞多糖特性的影响[J].农业工程学报,2009,25（10）:355-360.
[11]董彩霞,黄建华,祝勇,等.酶解法提取香菇多糖的探讨[J].光谱实验室,2005,22（5）:947-950.
[12] Xu Bin, Chen Ping, Chen Xin. Study on extraction process of polysaccharideconstituents from Panax japonicus by enzyme method[J]. China journal of Chinesemateria medica,2008,33(13):1549-1551.
[13]沈国良.超临界CO2萃取的研究与应用[J].化工时刊,2001,12:10-13.
[14]宿廷敏,王敏娟,阮时宝.超临界田萃取技术在中药提取中的应用[J].黑龙江医药,2008,21（6）:40-41.
[15]陈明,熊琳媛,袁城.超临界CO2萃取茶多糖的试验研究[J].安徽农业科学,2011,39(1):261-263,269.
[16] Marston A, Slacanin I, Hosttmann K. Centrifugal partition chromatography in theseparation of natural products[J]. Phytochemical Anal,1990,1(1):3-5.
[17] Song Guanglei, Du Qizhen. Isolation of a polysaccharide with anticancer activity fromAuricularia polytricha using high-speed countercurrent chromatography with anaqueous two-phase system[J]. Journal of Chromatography A,2010,1217(38):5930-5934.
[18]蒋志国,杜琪珍,盛利燚.制备型高速逆流色谱分离纯化香菇多糖[J].分析化学,2009,37(3):412-416.
[19]刘春娟,张守勤,张劲松,等.超高压技术提取黄芪多糖的研究[J].中成药,2009,31(9):1359-1363.
[20]高峰,张守勤,刘静波,等.超高压技术提取北虫草多糖的工艺研究[J].食品科学,2009,30(16):41-43.
[21]韩玉珠.高压脉冲电场常温快速提取中国林蛙多糖工艺及机理研究[D].长春:吉林大学生物与农业工程学院,2006.
[22]王莉,陈正行,张兵.不同方法提取米糠多糖工艺的优化研究[J].食品科学,2007，28:112-116.
[23]卢敏,毕艳春.高压脉冲电场提取麸皮多糖影响因素的研究[J].粮食加工,2009,34(1):31-33.
[24] Huang Gang L, Yang Qin, Wang Zhong B. Extraction and Deproteinization ofMannan Oligosaccharides[J]. ZEITSCHRIFT FUR NATURFORSCHUNG SECTIONCA JOURNAL OF BIOSCIENCES,2010,65(5):387-390.
[25] Mu Lu-xia, Zhu Lu, Zhao Ai-hua, et al. Study on extraction and purification ofpolysaccharides from Astragalus membranaceus[J]. Journal of Chinese medicinalmaterials,2009,32(11):1741-1745.
[26] HE Yu-tang, GONG Zhao-jie. Comparative Study of Decolorization of Crude Corn SilkPolysaccharide [J]. Food Science,2009,30(8):50-53.
[27]方积年.多糖的分离纯化及其纯度鉴别与分子量测定[J].药学通报,1984,19(l0):46-52.
[28]廖铭能,苏伟明,于立坚. SephadexG-25用于YML-1脱盐条件优化[J].湛江海洋大学学报,2006,26(01):94-95
[29] LI Juan-mei, NIE Shao-ping, LI Jing-en, et al. Ethanol Fractional Precipitation andCharacterization of Polysaccharides from Herba Moslae[J]. Food Science,2010,31,(19):182-185.
[30]刘庆生,张萍,范志影.离子色谱法检测糖[J].现代科学仪器,2005,1:75-78.
[31]韩美艳.桔梗多糖的提取、分离纯化和结构研究[D].郑州市：郑州大学药学院,2010.
[32]于广利,嵇国利,冯以明,等.刺松藻水溶性多糖的提取分离及理化性质研究[J].中国海洋大学学报,2011,40(11):90-94.
[33] Kambiz Jahanbina, Ahmad Reza Goharib, Sohrab Moinic, et al. Isolation, structuralcharacterization and antioxidant activity of a new water-soluble polysaccharide fromAcanthophyllum bracteatum roots[J]. International Journal of Biological Macro-molecules,2011(49)567-572.
[34] Sheng-quan Huang, Jin-wei Li, Yu-qiang Li, et al. Purification and structuralcharacterization of a new water-soluble neutral polysaccharide GLP-F1-1fromGanoderma lucidum[J]. International Journal of Biological Macromolecules,2011(48):165-169.
[35]刘倩,何法林,江春花,等.松茸多糖两种测定方法的比较研究[J].时珍国医国药,2010,21(5):1058-1059.
[36]贾亮亮,袁丁,何毓敏,等.多糖提取分离及含量测定的研究进展[J].食品研究与开发,2011,32(3):189-192.
[37] ZHU Xiao-lin, MA Yong-jian1, FENG Fang, et al. Study on determination ofpolysaccharide of panax quinquefolium by high performance capillary electro-phoresis[J]. Chinese Journal of Biochemical Pharmaceutics,2001,22(6):298-230.
[38] Pelkonen S., Finne J. Polyacrylamide gel electrophoresis of capsular polysaccharides ofbacteria[J]. Methods in Enzymology,1989,179:104-110.
[39]方积年.多糖的分离纯化及其纯度鉴别与分子质量测定[J].药学通报,1984,19:46.
[40]陈轶兰,王友茹,马立人.用高效凝胶渗透色谱法测定多糖的分子量[J].军事医学科学院刊,1988,45（1）:70-73.
[41]魏远安,方积年.高效凝胶渗透色谱法测定多糖纯度及分子量[J].药学学报，1989,24(7):532-536.
[42] Zhang Yu, Gu Ming, Wang Kaiping, et al. Structure, chain conformation and antitumoractivity of a novel polysaccharide from Lentinus edodes[J]. Fitoterapia,2010(81):1163-1170.
[43]吕志华,于广利,赵峡,等.不同标准品对HPGPC法测定多糖相对分子质量的影响[J].中国新药杂志,2002,11(3):220-221.
[44]蒋秋燕,乔旭光.生命科学的新领域——糖生物学[J].山东农业大学学报(自然科学版),2003,34(2):294-298.
[45]刘天龙,许剑琴.多糖现代研究及应用进展[J].中国兽医杂志,2004,40(3):24-26.
[46]金城,张树政.糖生物学与糖工程的兴起与前景[J].生物工程进展,1995,15(3):12-17.
[47] Chen Xiaoming, Nie Wenjian, Yu Guoqing, et al. Antitumor and immunomodulatoryactivity of polysaccharides from Sargassum fusiforme[J]. Food and chemical toxi-cology,2012,50(3-4):695-700.
[48] Kim Jin-Kyung, Cho Myoung Lae, Karnjanapratum Supatra, et al. In vitro and in vivoimmunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera[J]. INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES，2011,49(5):1051-1058.
[49] Xie Gang, Schepetkin Igor A, Quinn Mark T. Immunomodulatory activity of acidicpolysaccharides isolated from Tanacetum vulgare L[J]. INTERNATIONAL IMMU-NOPHARMACOLOGY,2007,7(13):1639-1650.
[50] Masuda Yuki, Ito Koichi, Konishi Morichika, et al. A polysaccharide extracted fromGrifola frondosa enhances the anti-tumor activity of bone marrow-derived dendriticcell-based immunotherapy against murine colon cancer[J]. CANCER IMMUNOLOGYIMMUNOTHERAPY,2010,59(10):1531-1541.
[51] Yoshikawa R, Yanagi H, Hashimoto-Tamaoki T, et al. Gene expression in response toanti-tumour intervention by polysaccharide-K (PSK) in colorectal carcinoma cells[J].ONCOLOGY REPORTS,2004,12(6):1287-1293.
[52]袁从英,车文文,张宁,等.天然植物多糖的研究进展与前景展望[J].中国现代应用药学杂志,2009,12(26):979-982.
[53] Wang Jing, Zhang Quanbin, Zhang Zhongshan, et al. Antioxidant activity of sulfatedpolysaccharide fractions extracted from Laminaria japonica [J]. International Journal ofBiological Macromolecules,2008,42(2):127-132.
[54] Quanbin Zhang, Ning Lia, Gefei Zhou, et al. In vivo antioxidant activity ofpolysaccharide fraction from Porphyra haitanesis (Rhodephyta) in aging mice[J].Pharmacological Research,2003,48(2):151–155.
[55] Miao Ming-san, Gu Li-ya, Fang Xiao-yan, et al. Effect of Phragmites communispolysaccharide on the aged-model mice [J]. China journal of Chinese material,2004,29(7):673-675.
[56]马明华,易杨华,汤海峰.多糖抗病毒作用的研究概况[J].药学服务与研究，2004,4(4):305-308.
[57]岑颖洲,王凌云,马夏军,等.羊栖菜多糖体外抗病毒作用研究[J].中国病理生理杂志,2004,20(5):765-768.
[58]张婷婷,尹鸿萍,盛玉清,等.杜氏藻多糖抗病毒及免疫活性研究[J].药物生物技术,2009,16(4):330-333.
[59] Tatiana Ivanova, Youngsoo Han, Hyeog-Jin Son, et al. Antimutagenic effect ofpolysaccharide ginsan extracted from Panax ginseng[J]. Food and Chemical Toxicology,2006,44(4):517–521.
[60]杨晓杰,付学鹏.蒲公英多糖体外抑瘤和抗突变作用研究[J].时珍国医国药,2009,20(10):2470-2471.
[61]赵先英,刘毅敏,赵华文.多糖抗辐射作用研究进展[J].西南国防医药2007,17(2):253-254.
[62]陈立,董俊兴.多糖抗辐射作用研究进展[J].癌变·畸变·突变,2004，16(6):380-382.
[63] Subramanian M, Chintalwar GJ, Chattopadhyay S.Antioxidant and radioprotectiveproperties of an Ocimum sanctum polysaccharide[J]. REDOX REPORT,2005,10(5):257-264.
[64] Yuan Z, He P, Cui J, et al. Hypoglycemic effect of water-soluble polysaccharide fromAuricularia auricula-judae Quel on genetically diabetic KK-Ay mice[J]. Bioscience,biotechnology, and biochemistry,1998,62(10):1898-1903.
[65] Kiho T, Tsujimura Y, Sakushima M, et al. Polysaccharides in fungi. XXXIII.Hypoglycemic activity of an acidic polysaccharide (AC) from Tremella fuciformis[J].Journal of the Pharmaceutical Society of Japan,1994,114(5):308-315.
[66] Wu Y, Ou-Yang JP, Wu K, et al. Hypoglycemic effect of Astragalus polysaccharideand its effect on PTP1B [J]. Acta Pharmacologica Sinica,2005,26(3):345-352.
[67] Xu Jie, Wang Yuan, Xu De-Sheng, et al. Hypoglycemic effects of MDG-1, apolysaccharide derived from Ophiopogon japonicas, in the ob/ob mouse model of type2diabetes mellitus[J]. International Journal of Biological Macromoleciles,2011,49(4):657-662.
[68] Zhang HN, Lin ZB. Hypoglycemic effect of Ganoderma lucidum polysaccharides [J].Acta Pharmacologica Sinica,2004,25(2):191-195.
[69]宋超,宋淑亮,刘学红.天然降血糖多糖的研究进展[J].山东医药,2011,51(41):113-114.
[70]韩春然,马永强,唐娟.黑木耳多糖的提取及降血糖作用[J].食品与生物技术学报,2006,25(5):111-114.
[71]韦璐,秦小明,林华娟,等.金花茶多糖的降血脂功能研究[J].食品科技,2008,7:247-249.
[72]中华人民共和国药典[M].北京:人民卫生出版社,2010:259-260
[73] Masahito OKA, Nagayo OTA, Yoshiki MINO, et al. Studies on the ConformationalAspects of Inulin Oligomers [J]. Pharmaceutical Society ofJapan,1992,40(5):1203-1207.
[74] Y D Yoon, J S Kang, S B Han, et al. Activation ofmitogenactivated protein kinasesand AP-l by polysaccharide isolated from the radix of platycodon grandiflorum in RAW264.7cells [J]. International Immunopharmacology,2004,4(12):1477-1487.
[75] Jae Ho Choi, Yong Pil Hwang, Hyun Sun Lee, et al. Inhibitory effect of PlatycodiRadix on ovalbumin-induced airway inflammation in a murine model of asthma [J].Food and Chemical Toxicology,2009,47:1272-1279.
[76] Sang B. Han, Se H. Park, Ki H. Lee, et al. Polysaccharide isolated fron the radix ofPlatycodon grandiflorum selectively activates B cells and macrophages but not Tcells[J]. International Immunopharmacology,2001,1:1969-1978.
[77] Kyung Jin Lee, Ji Young Kim, Jae Ho Choi, et al. Inhibition of tumor invasion andmetastasis by aqueous extract of the radix of Platycodon grandiflorum[J]. Food andChemical Toxicology,2006,44:1890-1896.
[78] Chul Yung Choi, Ji Young Kim, Young Sup Kim, et al. Aqueous extract isolated fromPlatycodon grandiflorum elicits the release of nitric oxide and tumor necrosis factor-afrom murine macrophages[J]. International Immunopharmacology,2001,1:1141-1151.
[79]杨放,袁军,付平.五味子的研究概况[J].华西药学杂志,2003,18(6):438-440.
[80]张占英,许书琦.北五味子多糖对小鼠抗疲劳和耐缺氧的作用[J].东北林业大学学报,2011,12(39):98-99,103.
[81] Gao Xiaoxv, Meng Xianjun, Li Jihai, et al. Isolation, Characterization and Hypo-glycemic Activity of an Acid Polysaccharide Isolated from Schisandra chinensis(Turcz.) Baill[J]. LETTERS IN ORGANIC CHEMISTRY,2009,6(5):428-433.
[82] Miao Mingsan, Gao Jianlian, Zhang Guangwei, et al. Effect of Schisandra chinensispolysaccharide on intracerebral acetylcholinesterase and monoamine neurotransmittersin a D-galactose-induced aging brain mouse model [J]. NEURAL REGENERATIONRESEARCH,2009,4(9):687-693.
[83] Chen Yong, Tang Jinbao, Wang Xiaoke, et al. An immunostimulatory polysaccharide(SCP-IIa) from the fruit of Schisandra chinensis (Turcz.) Baill[J]. International journalof biological macromolecules,2012,50(3):844-848.
[84]尹艳,高文宏,于淑娟.多糖提取技术的研究进展[J].食品工业科技,2007,2(20):248-250.
[85] FukudaK, UematsuT, HamadaA, et.al.ThePolysaecharidefromUmPteromyces jaPonicus[Jl. Chem·Phann.Bull(TOkyo),1975,23(9):1955-1959.
[86]徐大伦,欧昌荣,黄晓春,等.浒苔多糖脱蛋白方法的研究[J].水产科学,2005,24(5):26-27.
[87]韩舜愈,宋雪梅,祝霞,等.除去燕麦β-葡聚糖粗提液中蛋白质方法的比较研究[J].食品科学,2006,27,(12):300-303.
[88] Bradford MM．A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding[J]．Anal Biochem,1976,72（2）:248-254．
[89] Jae Ho Choi, Yong Pil Hwang, Hyun Sun Lee, et al. Inhibitory effect of Platycodi Radixon ovalbumin-induced airway inflammation in a murine model of asthma [J]. Food andChemical Toxicology,2009,47:1272-1279.
[90] Gerald J, Cleich MD, Rochester Ninn. Mechanism of eosinophil associated infla-mmatlon [J]. J Allergy Clin lirimuno1,2000,105(4):651-62
[91]刘艳玲.嗜酸性粒细胞机制研究进展与临床应用[J].国外医学·儿科学分册,2002,29(2):57.
[92]向希雄.畅肺防喘方对哮喘大鼠气道重构和炎症介质的影响的试验研究[D].武汉：湖北中医学院,2009.
[93] Jose PJ, Griffiths-Johnson DA, Collins PD, et al. Eotaxin1:a Potent eosinophi1chemoattraetant cytokine detected in a guinea Pig model of allergic airwaysinflammation. J Exp Med,1994,179:881-887.
[94]赵振东,刘镜愉,等.邻苯二甲酸醉哮喘大鼠模型制备及其肺组织中细胞间粘附分子-lmRNA的表达[J].北京医科大学学报,1998,30(6):521-524.
[95]李慧,等.肺表面活性物质对哮喘大鼠粘附分子的影响[J].实用医杂志,2003,(4):288-289.
[96]杨锡强.细胞因子在支气管哮喘发病机理中的作用[J].中华儿科杂志,1993,31(6):373
[97]王晓岩,迟宝荣,陈漉.喘息康对支气管哮喘大鼠外周血浆IFN-γ, IL-5, Eos和Eotaxin1的影响[J].第四军医大学学报,2008,29(8):682-684.
[98]王艳妮,常小红. Th1/Th2功能失衡与支气管哮喘[J].医学信息,2011,24(6):390.
[99] Romagniani S. Regulation and deregulation of human IgE synthesis[J]. Immunol Tooday,1990,(11):316.
[100] Gabriele G, Gerald J, Dale T, et al. Roles of interleuckin-13and interferon-γin lunginflammation[J]. Chest,2002,121(2):88-95.
[101]谢宝秋.气道重建与支气哮喘[J].国外医学,1999,3:124-125.
[102]李树钧,戚好文,新宇.地塞米松对气道重建影响的研究[J].中华结核和呼吸杂志,2001,24(3):152-154.
[103]王文建,杨莉.地塞米松对支气管哮喘模型大鼠气道重建的影响[J].郑州大学学报(医学版).2007,42(1):130-132.
[104]侯方丽.黑米花色昔对CCl4诱导小鼠肝损伤的保护作用及其抗氧化机制[D].武汉:华中农业大学食品科学技术学院,2009.
[105]王玉卓.灰树花多糖对四氮化碳肝损伤的保护作用及其机制的研究[D].济南:山东大学医学院细胞生物学研究所,2010.
[106] Wu J, Norton PA. Animal models of liver fibrosis［J］. Scand J Gastroenterol,1996,31(12):1137-1143.
[107] PLAA G L. Chorinated methanol and liver injury: highlights of the past50years［J］.Annu Rev Pharmacol Toxicol,2000,4042-4065．
[108] Taniguchi M, Takenchi T, Nakatsuka R, et al. Molecular process in acute liver injuryand regeneration induced by carbon tetrachloride[J]. Life Sci.2004,75(13):1539-1549.
[109] Boll M, Weber LW, Becker E, et al. Mechanism of carbon tetrachloride-inducedhepatotoxicity[J]. Z Naturforeh,2001,56(7-8):649-659.
[110] Brent JA, Rumaek BH. Role of free radiea1s in toxic hepatic injury[J].ClinicalToxicology,1993,31:173-196.
[111] Ozer J, Ratner M, Shaw M, et al. The current state of serum biomarkers ofhepatotoxieity[J].Toxicology.2008,245(3):194-205.
[112]吴希美,周汉良,卞如濂.一氧化氮与肝细胞氧化应激[J].国外医学(生理、病理科学与临床分册),1997,17(3):279.
[113] Rocke DC, Chung JJ. Regulation of inducible nitric oxide synthase and nitric oxidehepatic injury and fibrogenesis[J]. Am J Physiol,1997;273(1Pt1):124-30
[114]孙设宗,唐微,张红梅,等.山药多糖对小鼠CCL4肝损伤的保护作用[J].郧阳医学院学报.2008,27(6):502-504.
[115]范荣车,任涛,刘成柏,等.不同溶剂提取五味子多糖体外生物活性研究[J].中成药,2008,30(3):432-434.
[116] Mavundza EJ, Tshikalange TE, Lall N, et al. Antioxidant activity and cytotoxicityeffect of flavonoids isolated from athrixia phylicoides[J]. Med. Plants Res，2010，4（23）:2584-2587.
[117] Lin SC, Chang CMJ, Deng TS. Enzymatic Hot Pressurized Fluids Extraction ofPolyphenolics from Pinus taiwanensis and Pinus morrisonicola[J]. Taiwan. Inst. Chem.Engrs.2001,40:136-142.
[118] Hakkim FL, Arivazhagan G, Boopathy R. Antioxidant property of selected Ocimumspecies and their secondary metabolite content[J]. Journal Of Medicinal PlantsResearch,2008,2(9):250-257.
[119] Wang J, Zhang QB, Zhang ZS, Li ZE. Antioxidant activity of sulfated polysaccharidefractions extracted from Laminaria japonicaInt[J]. Biol. Macromol,2008,42:127-132.
[120]李春阳,许时婴,王璋．DPPH法测定葡萄籽原花青素清除自由基的能力［J］．食品与生物技术学报,2006,25(2):102-106．
[121]沈明花,崔海丹,王欣彤.榛蘑多糖的抗氧化作用研究[J].食品科技,2010,35(5):77-78
[122] CetellenN., BemierJ.L., Catteac J.P., et al. Antioxidant ProPerties of hydroxyl-flavones[J]. Free Radical.Bio.Med.,1998,20(l):35-43.
[123] SunT., HoC.T.. A ntioxidantac tivities of buekwheat extracts[J]. Food Chem.,200590:743-749.
[124] Brand-Williams W, Cuvelier M E, Berset C. Use of a freee radical method to evaluateantioxidant activity［J］.Lebensm Wiss Technol,1995,28(1):25-30．
[125] C.A.Riee-Evans, N.J.Miller, P.G. Bolwell, et al.Therelative Antioxidants activities ofPlant-derived polyphenolic fiavonoids[J].Free Radical Researeh.1995,22:375-383.
[126] K.X.Zhu, H.M.Zhou, H.F.Qian. Antioxidant and free radical-scavenging activities ofwheat Germ protein hydrolysates (WGPH)prepared with alealase[J]. Process Bioche-mistry,2006,41:1296-1302.
[127] Yuan, J.F., Zhang, Z.Q., Fan, Z.C., et al. Antioxidant effeets and cytotoxicity of threepurified polysaccharides from Ligusticum chuanxiong Hort[J]. Carbohyd.Polym.2008,74(4),822-827.
[128]邓源,袁伯俊,张淑英.解酒灵对乙醇致鼠肝急性损伤的保护作用及机制的探讨[J].中国新药杂志,2000;9(6):388-391.
[129] Yuan, J.F., Zhang, Z.Q., Fan, Z.C., et al. Antioxidant effeets and cytotoxicity of threepurified polysaccharides from Ligusticum chuanxiong Hort[J]. Carbohyd.Polym.2008,74(4),822-827.
[130] Constantin M, Bromont C, FickatR, et al. Studies on the activity of bepridil as ascavenger of free radicals[J]. Biochem Pharmacol.199040(7):1615-1622.
[131] Rekka E, et al. Effect of hydroxyethyl rutosides and related compounds on lipidperoxidation and free radical scavenging activity. Some structural aspects. The Journalof pharmacy and pharmacology.1991,43（7）:486-491.
[132]张作法.桑枝活性成分分离纯化及其药理学作用的研究[D].杭州市;浙江大学动物科学学院,2008.
[133] Volpi N, Tarugi P. Influence of chondroitin sulfate charge density, sulfate groupposition, and molecular mass on Cu2+-mediated oxidation of human low-densitylipoprotein: effect of normal human plasma-derived chondroitin sulfate[J].Journal ofBiochemistry,1999,125(2):297-299.
[134] Sun YX, Kennedy JF. Antioxidant activities of different polysaccharide conjugates(CRPs) isolated from the fruiting bodies of Chroogomphis rutilus (Schaeff.: Fr.) O. K.Miller. Carbohydrate Polymers,2010,82(2):510-514.
[2] FRANZ G. Polysaccharides in pharmacology. Current application and future concepts[J]. Planta Med,1998,55(6):493-497.
[3] TANG W, HEMM I, BERTRAM B. Recent development of antitumor agents fromChinese herbal medicines， Part Ⅱ. High molecular compounds[J]. Panta Med，2003，69(3):193-201.
[4] Wang Xiaoqing, Zhao Yuqing. The research progresss on extraection and puripicationof garlic polysacaride[J]. Modern Chinese Medicine,2009,11(7):4-7(Ch).
[5]李化.超声波技术在中草药成分提取中的应用[J].中草药,2001,24(4):229-230.
[6]郭孝武.一种提取中草药化学成分的方法——超声提取法[J].天然产物研究与开发,1998,11(3):37-40.
[7] Stahmann K P, Monschau N, Sahm H, et al. Structure properties of native and sonicatedcinerean, a β-(1-3)-(1-6)-D-glucan produced by Botrytiscinerea[J]. Carbohydr Res,1995,226(1):115-128.
[8]李岱,张静,孙润广.超声波处理对柴胡多糖提取率、微观形貌特征及生物活性的影响[J].生物加工过程,2009,7（2）:29.
[9]郭赣林,徐深圳,李文浩,等.球等鞭金藻多糖的微波萃取工艺[J].食品科学,2011,32(14):113-117.
[10]范会平,符锋, Giuseppe Mazza,等.微波提取法对樱桃、猕猴桃和枸杞多糖特性的影响[J].农业工程学报,2009,25（10）:355-360.
[11]董彩霞,黄建华,祝勇,等.酶解法提取香菇多糖的探讨[J].光谱实验室,2005,22（5）:947-950.
[12] Xu Bin, Chen Ping, Chen Xin. Study on extraction process of polysaccharideconstituents from Panax japonicus by enzyme method[J]. China journal of Chinesemateria medica,2008,33(13):1549-1551.
[13]沈国良.超临界CO2萃取的研究与应用[J].化工时刊,2001,12:10-13.
[14]宿廷敏,王敏娟,阮时宝.超临界田萃取技术在中药提取中的应用[J].黑龙江医药,2008,21（6）:40-41.
[15]陈明,熊琳媛,袁城.超临界CO2萃取茶多糖的试验研究[J].安徽农业科学,2011,39(1):261-263,269.
[16] Marston A, Slacanin I, Hosttmann K. Centrifugal partition chromatography in theseparation of natural products[J]. Phytochemical Anal,1990,1(1):3-5.
[17] Song Guanglei, Du Qizhen. Isolation of a polysaccharide with anticancer activity fromAuricularia polytricha using high-speed countercurrent chromatography with anaqueous two-phase system[J]. Journal of Chromatography A,2010,1217(38):5930-5934.
[18]蒋志国,杜琪珍,盛利燚.制备型高速逆流色谱分离纯化香菇多糖[J].分析化学,2009,37(3):412-416.
[19]刘春娟,张守勤,张劲松,等.超高压技术提取黄芪多糖的研究[J].中成药,2009,31(9):1359-1363.
[20]高峰,张守勤,刘静波,等.超高压技术提取北虫草多糖的工艺研究[J].食品科学,2009,30(16):41-43.
[21]韩玉珠.高压脉冲电场常温快速提取中国林蛙多糖工艺及机理研究[D].长春:吉林大学生物与农业工程学院,2006.
[22]王莉,陈正行,张兵.不同方法提取米糠多糖工艺的优化研究[J].食品科学,2007，28:112-116.
[23]卢敏,毕艳春.高压脉冲电场提取麸皮多糖影响因素的研究[J].粮食加工,2009,34(1):31-33.
[24] Huang Gang L, Yang Qin, Wang Zhong B. Extraction and Deproteinization ofMannan Oligosaccharides[J]. ZEITSCHRIFT FUR NATURFORSCHUNG SECTIONCA JOURNAL OF BIOSCIENCES,2010,65(5):387-390.
[25] Mu Lu-xia, Zhu Lu, Zhao Ai-hua, et al. Study on extraction and purification ofpolysaccharides from Astragalus membranaceus[J]. Journal of Chinese medicinalmaterials,2009,32(11):1741-1745.
[26] HE Yu-tang, GONG Zhao-jie. Comparative Study of Decolorization of Crude Corn SilkPolysaccharide [J]. Food Science,2009,30(8):50-53.
[27]方积年.多糖的分离纯化及其纯度鉴别与分子量测定[J].药学通报,1984,19(l0):46-52.
[28]廖铭能,苏伟明,于立坚. SephadexG-25用于YML-1脱盐条件优化[J].湛江海洋大学学报,2006,26(01):94-95
[29] LI Juan-mei, NIE Shao-ping, LI Jing-en, et al. Ethanol Fractional Precipitation andCharacterization of Polysaccharides from Herba Moslae[J]. Food Science,2010,31,(19):182-185.
[30]刘庆生,张萍,范志影.离子色谱法检测糖[J].现代科学仪器,2005,1:75-78.
[31]韩美艳.桔梗多糖的提取、分离纯化和结构研究[D].郑州市：郑州大学药学院,2010.
[32]于广利,嵇国利,冯以明,等.刺松藻水溶性多糖的提取分离及理化性质研究[J].中国海洋大学学报,2011,40(11):90-94.
[33] Kambiz Jahanbina, Ahmad Reza Goharib, Sohrab Moinic, et al. Isolation, structuralcharacterization and antioxidant activity of a new water-soluble polysaccharide fromAcanthophyllum bracteatum roots[J]. International Journal of Biological Macro-molecules,2011(49)567-572.
[34] Sheng-quan Huang, Jin-wei Li, Yu-qiang Li, et al. Purification and structuralcharacterization of a new water-soluble neutral polysaccharide GLP-F1-1fromGanoderma lucidum[J]. International Journal of Biological Macromolecules,2011(48):165-169.
[35]刘倩,何法林,江春花,等.松茸多糖两种测定方法的比较研究[J].时珍国医国药,2010,21(5):1058-1059.
[36]贾亮亮,袁丁,何毓敏,等.多糖提取分离及含量测定的研究进展[J].食品研究与开发,2011,32(3):189-192.
[37] ZHU Xiao-lin, MA Yong-jian1, FENG Fang, et al. Study on determination ofpolysaccharide of panax quinquefolium by high performance capillary electro-phoresis[J]. Chinese Journal of Biochemical Pharmaceutics,2001,22(6):298-230.
[38] Pelkonen S., Finne J. Polyacrylamide gel electrophoresis of capsular polysaccharides ofbacteria[J]. Methods in Enzymology,1989,179:104-110.
[39]方积年.多糖的分离纯化及其纯度鉴别与分子质量测定[J].药学通报,1984,19:46.
[40]陈轶兰,王友茹,马立人.用高效凝胶渗透色谱法测定多糖的分子量[J].军事医学科学院刊,1988,45（1）:70-73.
[41]魏远安,方积年.高效凝胶渗透色谱法测定多糖纯度及分子量[J].药学学报，1989,24(7):532-536.
[42] Zhang Yu, Gu Ming, Wang Kaiping, et al. Structure, chain conformation and antitumoractivity of a novel polysaccharide from Lentinus edodes[J]. Fitoterapia,2010(81):1163-1170.
[43]吕志华,于广利,赵峡,等.不同标准品对HPGPC法测定多糖相对分子质量的影响[J].中国新药杂志,2002,11(3):220-221.
[44]蒋秋燕,乔旭光.生命科学的新领域——糖生物学[J].山东农业大学学报(自然科学版),2003,34(2):294-298.
[45]刘天龙,许剑琴.多糖现代研究及应用进展[J].中国兽医杂志,2004,40(3):24-26.
[46]金城,张树政.糖生物学与糖工程的兴起与前景[J].生物工程进展,1995,15(3):12-17.
[47] Chen Xiaoming, Nie Wenjian, Yu Guoqing, et al. Antitumor and immunomodulatoryactivity of polysaccharides from Sargassum fusiforme[J]. Food and chemical toxi-cology,2012,50(3-4):695-700.
[48] Kim Jin-Kyung, Cho Myoung Lae, Karnjanapratum Supatra, et al. In vitro and in vivoimmunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera[J]. INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES，2011,49(5):1051-1058.
[49] Xie Gang, Schepetkin Igor A, Quinn Mark T. Immunomodulatory activity of acidicpolysaccharides isolated from Tanacetum vulgare L[J]. INTERNATIONAL IMMU-NOPHARMACOLOGY,2007,7(13):1639-1650.
[50] Masuda Yuki, Ito Koichi, Konishi Morichika, et al. A polysaccharide extracted fromGrifola frondosa enhances the anti-tumor activity of bone marrow-derived dendriticcell-based immunotherapy against murine colon cancer[J]. CANCER IMMUNOLOGYIMMUNOTHERAPY,2010,59(10):1531-1541.
[51] Yoshikawa R, Yanagi H, Hashimoto-Tamaoki T, et al. Gene expression in response toanti-tumour intervention by polysaccharide-K (PSK) in colorectal carcinoma cells[J].ONCOLOGY REPORTS,2004,12(6):1287-1293.
[52]袁从英,车文文,张宁,等.天然植物多糖的研究进展与前景展望[J].中国现代应用药学杂志,2009,12(26):979-982.
[53] Wang Jing, Zhang Quanbin, Zhang Zhongshan, et al. Antioxidant activity of sulfatedpolysaccharide fractions extracted from Laminaria japonica [J]. International Journal ofBiological Macromolecules,2008,42(2):127-132.
[54] Quanbin Zhang, Ning Lia, Gefei Zhou, et al. In vivo antioxidant activity ofpolysaccharide fraction from Porphyra haitanesis (Rhodephyta) in aging mice[J].Pharmacological Research,2003,48(2):151–155.
[55] Miao Ming-san, Gu Li-ya, Fang Xiao-yan, et al. Effect of Phragmites communispolysaccharide on the aged-model mice [J]. China journal of Chinese material,2004,29(7):673-675.
[56]马明华,易杨华,汤海峰.多糖抗病毒作用的研究概况[J].药学服务与研究，2004,4(4):305-308.
[57]岑颖洲,王凌云,马夏军,等.羊栖菜多糖体外抗病毒作用研究[J].中国病理生理杂志,2004,20(5):765-768.
[58]张婷婷,尹鸿萍,盛玉清,等.杜氏藻多糖抗病毒及免疫活性研究[J].药物生物技术,2009,16(4):330-333.
[59] Tatiana Ivanova, Youngsoo Han, Hyeog-Jin Son, et al. Antimutagenic effect ofpolysaccharide ginsan extracted from Panax ginseng[J]. Food and Chemical Toxicology,2006,44(4):517–521.
[60]杨晓杰,付学鹏.蒲公英多糖体外抑瘤和抗突变作用研究[J].时珍国医国药,2009,20(10):2470-2471.
[61]赵先英,刘毅敏,赵华文.多糖抗辐射作用研究进展[J].西南国防医药2007,17(2):253-254.
[62]陈立,董俊兴.多糖抗辐射作用研究进展[J].癌变·畸变·突变,2004，16(6):380-382.
[63] Subramanian M, Chintalwar GJ, Chattopadhyay S.Antioxidant and radioprotectiveproperties of an Ocimum sanctum polysaccharide[J]. REDOX REPORT,2005,10(5):257-264.
[64] Yuan Z, He P, Cui J, et al. Hypoglycemic effect of water-soluble polysaccharide fromAuricularia auricula-judae Quel on genetically diabetic KK-Ay mice[J]. Bioscience,biotechnology, and biochemistry,1998,62(10):1898-1903.
[65] Kiho T, Tsujimura Y, Sakushima M, et al. Polysaccharides in fungi. XXXIII.Hypoglycemic activity of an acidic polysaccharide (AC) from Tremella fuciformis[J].Journal of the Pharmaceutical Society of Japan,1994,114(5):308-315.
[66] Wu Y, Ou-Yang JP, Wu K, et al. Hypoglycemic effect of Astragalus polysaccharideand its effect on PTP1B [J]. Acta Pharmacologica Sinica,2005,26(3):345-352.
[67] Xu Jie, Wang Yuan, Xu De-Sheng, et al. Hypoglycemic effects of MDG-1, apolysaccharide derived from Ophiopogon japonicas, in the ob/ob mouse model of type2diabetes mellitus[J]. International Journal of Biological Macromoleciles,2011,49(4):657-662.
[68] Zhang HN, Lin ZB. Hypoglycemic effect of Ganoderma lucidum polysaccharides [J].Acta Pharmacologica Sinica,2004,25(2):191-195.
[69]宋超,宋淑亮,刘学红.天然降血糖多糖的研究进展[J].山东医药,2011,51(41):113-114.
[70]韩春然,马永强,唐娟.黑木耳多糖的提取及降血糖作用[J].食品与生物技术学报,2006,25(5):111-114.
[71]韦璐,秦小明,林华娟,等.金花茶多糖的降血脂功能研究[J].食品科技,2008,7:247-249.
[72]中华人民共和国药典[M].北京:人民卫生出版社,2010:259-260
[73] Masahito OKA, Nagayo OTA, Yoshiki MINO, et al. Studies on the ConformationalAspects of Inulin Oligomers [J]. Pharmaceutical Society ofJapan,1992,40(5):1203-1207.
[74] Y D Yoon, J S Kang, S B Han, et al. Activation ofmitogenactivated protein kinasesand AP-l by polysaccharide isolated from the radix of platycodon grandiflorum in RAW264.7cells [J]. International Immunopharmacology,2004,4(12):1477-1487.
[75] Jae Ho Choi, Yong Pil Hwang, Hyun Sun Lee, et al. Inhibitory effect of PlatycodiRadix on ovalbumin-induced airway inflammation in a murine model of asthma [J].Food and Chemical Toxicology,2009,47:1272-1279.
[76] Sang B. Han, Se H. Park, Ki H. Lee, et al. Polysaccharide isolated fron the radix ofPlatycodon grandiflorum selectively activates B cells and macrophages but not Tcells[J]. International Immunopharmacology,2001,1:1969-1978.
[77] Kyung Jin Lee, Ji Young Kim, Jae Ho Choi, et al. Inhibition of tumor invasion andmetastasis by aqueous extract of the radix of Platycodon grandiflorum[J]. Food andChemical Toxicology,2006,44:1890-1896.
[78] Chul Yung Choi, Ji Young Kim, Young Sup Kim, et al. Aqueous extract isolated fromPlatycodon grandiflorum elicits the release of nitric oxide and tumor necrosis factor-afrom murine macrophages[J]. International Immunopharmacology,2001,1:1141-1151.
[79]杨放,袁军,付平.五味子的研究概况[J].华西药学杂志,2003,18(6):438-440.
[80]张占英,许书琦.北五味子多糖对小鼠抗疲劳和耐缺氧的作用[J].东北林业大学学报,2011,12(39):98-99,103.
[81] Gao Xiaoxv, Meng Xianjun, Li Jihai, et al. Isolation, Characterization and Hypo-glycemic Activity of an Acid Polysaccharide Isolated from Schisandra chinensis(Turcz.) Baill[J]. LETTERS IN ORGANIC CHEMISTRY,2009,6(5):428-433.
[82] Miao Mingsan, Gao Jianlian, Zhang Guangwei, et al. Effect of Schisandra chinensispolysaccharide on intracerebral acetylcholinesterase and monoamine neurotransmittersin a D-galactose-induced aging brain mouse model [J]. NEURAL REGENERATIONRESEARCH,2009,4(9):687-693.
[83] Chen Yong, Tang Jinbao, Wang Xiaoke, et al. An immunostimulatory polysaccharide(SCP-IIa) from the fruit of Schisandra chinensis (Turcz.) Baill[J]. International journalof biological macromolecules,2012,50(3):844-848.
[84]尹艳,高文宏,于淑娟.多糖提取技术的研究进展[J].食品工业科技,2007,2(20):248-250.
[85] FukudaK, UematsuT, HamadaA, et.al.ThePolysaecharidefromUmPteromyces jaPonicus[Jl. Chem·Phann.Bull(TOkyo),1975,23(9):1955-1959.
[86]徐大伦,欧昌荣,黄晓春,等.浒苔多糖脱蛋白方法的研究[J].水产科学,2005,24(5):26-27.
[87]韩舜愈,宋雪梅,祝霞,等.除去燕麦β-葡聚糖粗提液中蛋白质方法的比较研究[J].食品科学,2006,27,(12):300-303.
[88] Bradford MM．A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding[J]．Anal Biochem,1976,72（2）:248-254．
[89] Jae Ho Choi, Yong Pil Hwang, Hyun Sun Lee, et al. Inhibitory effect of Platycodi Radixon ovalbumin-induced airway inflammation in a murine model of asthma [J]. Food andChemical Toxicology,2009,47:1272-1279.
[90] Gerald J, Cleich MD, Rochester Ninn. Mechanism of eosinophil associated infla-mmatlon [J]. J Allergy Clin lirimuno1,2000,105(4):651-62
[91]刘艳玲.嗜酸性粒细胞机制研究进展与临床应用[J].国外医学·儿科学分册,2002,29(2):57.
[92]向希雄.畅肺防喘方对哮喘大鼠气道重构和炎症介质的影响的试验研究[D].武汉：湖北中医学院,2009.
[93] Jose PJ, Griffiths-Johnson DA, Collins PD, et al. Eotaxin1:a Potent eosinophi1chemoattraetant cytokine detected in a guinea Pig model of allergic airwaysinflammation. J Exp Med,1994,179:881-887.
[94]赵振东,刘镜愉,等.邻苯二甲酸醉哮喘大鼠模型制备及其肺组织中细胞间粘附分子-lmRNA的表达[J].北京医科大学学报,1998,30(6):521-524.
[95]李慧,等.肺表面活性物质对哮喘大鼠粘附分子的影响[J].实用医杂志,2003,(4):288-289.
[96]杨锡强.细胞因子在支气管哮喘发病机理中的作用[J].中华儿科杂志,1993,31(6):373
[97]王晓岩,迟宝荣,陈漉.喘息康对支气管哮喘大鼠外周血浆IFN-γ, IL-5, Eos和Eotaxin1的影响[J].第四军医大学学报,2008,29(8):682-684.
[98]王艳妮,常小红. Th1/Th2功能失衡与支气管哮喘[J].医学信息,2011,24(6):390.
[99] Romagniani S. Regulation and deregulation of human IgE synthesis[J]. Immunol Tooday,1990,(11):316.
[100] Gabriele G, Gerald J, Dale T, et al. Roles of interleuckin-13and interferon-γin lunginflammation[J]. Chest,2002,121(2):88-95.
[101]谢宝秋.气道重建与支气哮喘[J].国外医学,1999,3:124-125.
[102]李树钧,戚好文,新宇.地塞米松对气道重建影响的研究[J].中华结核和呼吸杂志,2001,24(3):152-154.
[103]王文建,杨莉.地塞米松对支气管哮喘模型大鼠气道重建的影响[J].郑州大学学报(医学版).2007,42(1):130-132.
[104]侯方丽.黑米花色昔对CCl4诱导小鼠肝损伤的保护作用及其抗氧化机制[D].武汉:华中农业大学食品科学技术学院,2009.
[105]王玉卓.灰树花多糖对四氮化碳肝损伤的保护作用及其机制的研究[D].济南:山东大学医学院细胞生物学研究所,2010.
[106] Wu J, Norton PA. Animal models of liver fibrosis［J］. Scand J Gastroenterol,1996,31(12):1137-1143.
[107] PLAA G L. Chorinated methanol and liver injury: highlights of the past50years［J］.Annu Rev Pharmacol Toxicol,2000,4042-4065．
[108] Taniguchi M, Takenchi T, Nakatsuka R, et al. Molecular process in acute liver injuryand regeneration induced by carbon tetrachloride[J]. Life Sci.2004,75(13):1539-1549.
[109] Boll M, Weber LW, Becker E, et al. Mechanism of carbon tetrachloride-inducedhepatotoxicity[J]. Z Naturforeh,2001,56(7-8):649-659.
[110] Brent JA, Rumaek BH. Role of free radiea1s in toxic hepatic injury[J].ClinicalToxicology,1993,31:173-196.
[111] Ozer J, Ratner M, Shaw M, et al. The current state of serum biomarkers ofhepatotoxieity[J].Toxicology.2008,245(3):194-205.
[112]吴希美,周汉良,卞如濂.一氧化氮与肝细胞氧化应激[J].国外医学(生理、病理科学与临床分册),1997,17(3):279.
[113] Rocke DC, Chung JJ. Regulation of inducible nitric oxide synthase and nitric oxidehepatic injury and fibrogenesis[J]. Am J Physiol,1997;273(1Pt1):124-30
[114]孙设宗,唐微,张红梅,等.山药多糖对小鼠CCL4肝损伤的保护作用[J].郧阳医学院学报.2008,27(6):502-504.
[115]范荣车,任涛,刘成柏,等.不同溶剂提取五味子多糖体外生物活性研究[J].中成药,2008,30(3):432-434.
[116] Mavundza EJ, Tshikalange TE, Lall N, et al. Antioxidant activity and cytotoxicityeffect of flavonoids isolated from athrixia phylicoides[J]. Med. Plants Res，2010，4（23）:2584-2587.
[117] Lin SC, Chang CMJ, Deng TS. Enzymatic Hot Pressurized Fluids Extraction ofPolyphenolics from Pinus taiwanensis and Pinus morrisonicola[J]. Taiwan. Inst. Chem.Engrs.2001,40:136-142.
[118] Hakkim FL, Arivazhagan G, Boopathy R. Antioxidant property of selected Ocimumspecies and their secondary metabolite content[J]. Journal Of Medicinal PlantsResearch,2008,2(9):250-257.
[119] Wang J, Zhang QB, Zhang ZS, Li ZE. Antioxidant activity of sulfated polysaccharidefractions extracted from Laminaria japonicaInt[J]. Biol. Macromol,2008,42:127-132.
[120]李春阳,许时婴,王璋．DPPH法测定葡萄籽原花青素清除自由基的能力［J］．食品与生物技术学报,2006,25(2):102-106．
[121]沈明花,崔海丹,王欣彤.榛蘑多糖的抗氧化作用研究[J].食品科技,2010,35(5):77-78
[122] CetellenN., BemierJ.L., Catteac J.P., et al. Antioxidant ProPerties of hydroxyl-flavones[J]. Free Radical.Bio.Med.,1998,20(l):35-43.
[123] SunT., HoC.T.. A ntioxidantac tivities of buekwheat extracts[J]. Food Chem.,200590:743-749.
[124] Brand-Williams W, Cuvelier M E, Berset C. Use of a freee radical method to evaluateantioxidant activity［J］.Lebensm Wiss Technol,1995,28(1):25-30．
[125] C.A.Riee-Evans, N.J.Miller, P.G. Bolwell, et al.Therelative Antioxidants activities ofPlant-derived polyphenolic fiavonoids[J].Free Radical Researeh.1995,22:375-383.
[126] K.X.Zhu, H.M.Zhou, H.F.Qian. Antioxidant and free radical-scavenging activities ofwheat Germ protein hydrolysates (WGPH)prepared with alealase[J]. Process Bioche-mistry,2006,41:1296-1302.
[127] Yuan, J.F., Zhang, Z.Q., Fan, Z.C., et al. Antioxidant effeets and cytotoxicity of threepurified polysaccharides from Ligusticum chuanxiong Hort[J]. Carbohyd.Polym.2008,74(4),822-827.
[128]邓源,袁伯俊,张淑英.解酒灵对乙醇致鼠肝急性损伤的保护作用及机制的探讨[J].中国新药杂志,2000;9(6):388-391.
[129] Yuan, J.F., Zhang, Z.Q., Fan, Z.C., et al. Antioxidant effeets and cytotoxicity of threepurified polysaccharides from Ligusticum chuanxiong Hort[J]. Carbohyd.Polym.2008,74(4),822-827.
[130] Constantin M, Bromont C, FickatR, et al. Studies on the activity of bepridil as ascavenger of free radicals[J]. Biochem Pharmacol.199040(7):1615-1622.
[131] Rekka E, et al. Effect of hydroxyethyl rutosides and related compounds on lipidperoxidation and free radical scavenging activity. Some structural aspects. The Journalof pharmacy and pharmacology.1991,43（7）:486-491.
[132]张作法.桑枝活性成分分离纯化及其药理学作用的研究[D].杭州市;浙江大学动物科学学院,2008.
[133] Volpi N, Tarugi P. Influence of chondroitin sulfate charge density, sulfate groupposition, and molecular mass on Cu2+-mediated oxidation of human low-densitylipoprotein: effect of normal human plasma-derived chondroitin sulfate[J].Journal ofBiochemistry,1999,125(2):297-299.
[134] Sun YX, Kennedy JF. Antioxidant activities of different polysaccharide conjugates(CRPs) isolated from the fruiting bodies of Chroogomphis rutilus (Schaeff.: Fr.) O. K.Miller. Carbohydrate Polymers,2010,82(2):510-514.