小刺猴头菌硫酸化多糖的制备与活性研究
|
摘要
小刺猴头[Hericium caput-medusae (Bull.:Fr)Pers.]是一种珍贵的可药用和可食用菌,是长白山自然保护区稀有的菌物资源。小刺猴头菌含有多种营养物质,其中研究较多的是多糖成分,在提高免疫力、抗肿瘤、抗氧化、增强消化功能等方面具有活性。目前,对于小刺猴头菌的研究主要集中在提取方法,结构研究和生物活性上,对小刺猴头菌化学修饰方面的研究甚少。本研究采用两种方法进行硫酸化衍生物的制备,并检测小刺猴头菌多糖及其硫酸化衍生物的生物活性。将推动小刺猴头菌在多糖药物学和生物学方面的发展,为进一步开发小刺猴头菌的药用价值提供理论依据。
采用热水浸提法提取小刺猴头液体深层发酵浸膏多糖,在液料比,提取时间和提取温度单因素试验的基础上,利用SAS 9.2(the SAS System for Windows 9.2)软件设计响应面,以多糖得率作为响应值,优化得最佳提取条件：液料比2.29：1(mL/g),提取时间4.52 h,提取温度79.5℃,多糖得率可达12.30%。经乙醇分步沉淀,根据得率选择75%级分进一步纯化,采用Sevag法去除游离蛋白。透析选择MW 8000～10000的透析袋。选用SepharoseCL-6B凝胶柱层析分级分离多糖。多糖纯度鉴定结果显示分离出的多糖为均一组分多糖,命名为HLSF。采用凝胶柱层析法测得多糖的分子量为4.32×104Da。
采用氯磺酸-吡啶法(Wolfrom method)和浓硫酸法对HLSF进行硫酸化,通过红外光谱分析,具有硫酸酯键的特征吸收,证明硫酸基团已经与多糖结合,将制得的硫酸化衍生物分别命名为HLSFL和HLSFN。通过体外MTT试验和SOD试剂盒分析检测了硫酸化修饰前后多糖样品的活性,体外MTT试验结果显示经硫酸化修饰后的多糖样品对MCF-7细胞的抑制率均比多糖样品活性高,当多糖样品浓度为1200μg/mL时,氯磺酸-吡啶法硫酸化修饰衍生物对MCF-7细胞的抑制率提高了27.00%,浓硫酸法硫酸化修饰衍生物对MCF-7细胞的抑制率提高了21.21%,结果表明经硫酸化修饰后的多糖样品对MCF-7细胞的抑制率明显增强；SOD试剂盒检测硫酸化修饰前后的多糖样品清除超氧阴离子能力,得出经氯磺酸-吡啶法修饰后的多糖样品比未硫酸化的多糖样品活力提高了102.49%,经浓硫酸法修饰后的多糖样品比未硫酸化的多糖样品活力提高了27.53%,结果表明经硫酸化修饰后多糖样品的抗氧化活性均有所增强,而且经氯磺酸-吡啶法制备的硫酸化衍生物活性要比浓硫酸法制备的硫酸化衍生物活性高一些。综上所述,经硫酸化修饰后的多糖样品生物活性更强,氯磺酸-吡啶法比浓硫酸法更适合制备小刺猴头液体深层发酵浸膏多糖硫酸化衍生物。
Hericium caput-medusae is one of the valuable fungi for both medical and edibility, It is scarce fungus natural resources in Mount Changbai natural reserve. Hericium caput-medusae include many kinds of nutritive material, of which polysaccharide component is major studied, its activity has many aspects about increasing immunity、resisting cancer、anti-oxidant、enhancing digestive function and so on. Nowadays, the research about extraction method、structures research、bioactivity aspects, but studying on chemical modification of Hericium caput-medusae on a large scale. This study used two methods to preparation sulfated derivatives, then the biological activities of Hericium caput-medusae and sulfated derivatives were determined. It will impulse the development of Hericium caput-medusae polysaccharide about the aspect of pharmacology and biologic. On development medical value of Hericium caput-medusae to provide accordance with theory.
Warm water extraction was used for hericium caput-medusae liquid submerged fermentation extractum polysaccharides, based on solvent-solid ratio、extracting time、extracting temperature single-factor experiments, The SAS 9.2 software was employed to design response surface, the yield of polysaccharides was used as the responsive values, The optimum extraction conditions by optimize were as follow: solvent-solid ratio 2.29:1 (mL/g), extracting time 4.52 h, extracting temperature 79.5℃, the yield of polysaccharides rate was 12.30%. Through ethyl alcohol fractional precipitation, to choose 75% fraction further purification on the base of yield, removing floating protein by Sevag method. Dialysis select bag filter which is MW 8000-10000. Selection Sepharose CL-6B gel column chromatography was used to fractionation on polysaccharides, collecting the main peak, The result of identification the purity of polysaccharide is uniform polysaccharide, named HLSF. Gel chromatography is used to measure the molecular weight of polysaccharide is 4.32×104Da.
HLSF was sulfated using Wolfrom method and sulfuric acid method, to obtain HLSFL and HLSFN respectively. The result is analyses by infrared spectrum and substitution, it has the special absorption peak about sulfate, to prove the sulphuric acid group has linked with the polysaccharide, the products has named HLSFL and HLSFN respectively. Through the MTT experiment in vitro and SOD kit analysis comparing the activity of polysaccharide sample which is modified or not, the result of MTT experiment in vitro displayed the polysaccharide sample which is sulfated has more activity to the cell inhibition ratio of MCF-7 than the polysaccharide sample. When the polysaccharide concentration is 1200μg/mL, the inhibition ratio of HLSFL is improving to 27.00% to the cell of MCF-7, the inhibition ratio of HLSFN is improving to 21.21% to the cell of MCF-7, The result of sample polysaccharides inhibition ratio of the activity to the cell of MCF-7 by sulfating modification is enhanced obviously. SOD kit analysis comparing the activity of polysaccharide sample to remove superoxide anion which is modified or not. The activity of HLSFL is improved 102.49% than sample polysaccharide, The activity of HLSFN is improved 27.53% than sample polysaccharide, the result indicated the activity of anti-oxidant of sample polysaccharide by sulfating modification is enhanced obviously. Furthermore the activity of the sulfating derivatives by Wolfrom method is more higher than the activity of the sulfating derivatives by sulfuric acid method. As all the result, the bioactivity of sample polysaccharide sulfate is better, Wolfrom method is more fit to prepare polysaccharide sulfate of Hericium caput-medusae liquid submerged fermentation extractum than the sulfuric acid method.
采用热水浸提法提取小刺猴头液体深层发酵浸膏多糖,在液料比,提取时间和提取温度单因素试验的基础上,利用SAS 9.2(the SAS System for Windows 9.2)软件设计响应面,以多糖得率作为响应值,优化得最佳提取条件：液料比2.29：1(mL/g),提取时间4.52 h,提取温度79.5℃,多糖得率可达12.30%。经乙醇分步沉淀,根据得率选择75%级分进一步纯化,采用Sevag法去除游离蛋白。透析选择MW 8000～10000的透析袋。选用SepharoseCL-6B凝胶柱层析分级分离多糖。多糖纯度鉴定结果显示分离出的多糖为均一组分多糖,命名为HLSF。采用凝胶柱层析法测得多糖的分子量为4.32×104Da。
采用氯磺酸-吡啶法(Wolfrom method)和浓硫酸法对HLSF进行硫酸化,通过红外光谱分析,具有硫酸酯键的特征吸收,证明硫酸基团已经与多糖结合,将制得的硫酸化衍生物分别命名为HLSFL和HLSFN。通过体外MTT试验和SOD试剂盒分析检测了硫酸化修饰前后多糖样品的活性,体外MTT试验结果显示经硫酸化修饰后的多糖样品对MCF-7细胞的抑制率均比多糖样品活性高,当多糖样品浓度为1200μg/mL时,氯磺酸-吡啶法硫酸化修饰衍生物对MCF-7细胞的抑制率提高了27.00%,浓硫酸法硫酸化修饰衍生物对MCF-7细胞的抑制率提高了21.21%,结果表明经硫酸化修饰后的多糖样品对MCF-7细胞的抑制率明显增强；SOD试剂盒检测硫酸化修饰前后的多糖样品清除超氧阴离子能力,得出经氯磺酸-吡啶法修饰后的多糖样品比未硫酸化的多糖样品活力提高了102.49%,经浓硫酸法修饰后的多糖样品比未硫酸化的多糖样品活力提高了27.53%,结果表明经硫酸化修饰后多糖样品的抗氧化活性均有所增强,而且经氯磺酸-吡啶法制备的硫酸化衍生物活性要比浓硫酸法制备的硫酸化衍生物活性高一些。综上所述,经硫酸化修饰后的多糖样品生物活性更强,氯磺酸-吡啶法比浓硫酸法更适合制备小刺猴头液体深层发酵浸膏多糖硫酸化衍生物。
Hericium caput-medusae is one of the valuable fungi for both medical and edibility, It is scarce fungus natural resources in Mount Changbai natural reserve. Hericium caput-medusae include many kinds of nutritive material, of which polysaccharide component is major studied, its activity has many aspects about increasing immunity、resisting cancer、anti-oxidant、enhancing digestive function and so on. Nowadays, the research about extraction method、structures research、bioactivity aspects, but studying on chemical modification of Hericium caput-medusae on a large scale. This study used two methods to preparation sulfated derivatives, then the biological activities of Hericium caput-medusae and sulfated derivatives were determined. It will impulse the development of Hericium caput-medusae polysaccharide about the aspect of pharmacology and biologic. On development medical value of Hericium caput-medusae to provide accordance with theory.
Warm water extraction was used for hericium caput-medusae liquid submerged fermentation extractum polysaccharides, based on solvent-solid ratio、extracting time、extracting temperature single-factor experiments, The SAS 9.2 software was employed to design response surface, the yield of polysaccharides was used as the responsive values, The optimum extraction conditions by optimize were as follow: solvent-solid ratio 2.29:1 (mL/g), extracting time 4.52 h, extracting temperature 79.5℃, the yield of polysaccharides rate was 12.30%. Through ethyl alcohol fractional precipitation, to choose 75% fraction further purification on the base of yield, removing floating protein by Sevag method. Dialysis select bag filter which is MW 8000-10000. Selection Sepharose CL-6B gel column chromatography was used to fractionation on polysaccharides, collecting the main peak, The result of identification the purity of polysaccharide is uniform polysaccharide, named HLSF. Gel chromatography is used to measure the molecular weight of polysaccharide is 4.32×104Da.
HLSF was sulfated using Wolfrom method and sulfuric acid method, to obtain HLSFL and HLSFN respectively. The result is analyses by infrared spectrum and substitution, it has the special absorption peak about sulfate, to prove the sulphuric acid group has linked with the polysaccharide, the products has named HLSFL and HLSFN respectively. Through the MTT experiment in vitro and SOD kit analysis comparing the activity of polysaccharide sample which is modified or not, the result of MTT experiment in vitro displayed the polysaccharide sample which is sulfated has more activity to the cell inhibition ratio of MCF-7 than the polysaccharide sample. When the polysaccharide concentration is 1200μg/mL, the inhibition ratio of HLSFL is improving to 27.00% to the cell of MCF-7, the inhibition ratio of HLSFN is improving to 21.21% to the cell of MCF-7, The result of sample polysaccharides inhibition ratio of the activity to the cell of MCF-7 by sulfating modification is enhanced obviously. SOD kit analysis comparing the activity of polysaccharide sample to remove superoxide anion which is modified or not. The activity of HLSFL is improved 102.49% than sample polysaccharide, The activity of HLSFN is improved 27.53% than sample polysaccharide, the result indicated the activity of anti-oxidant of sample polysaccharide by sulfating modification is enhanced obviously. Furthermore the activity of the sulfating derivatives by Wolfrom method is more higher than the activity of the sulfating derivatives by sulfuric acid method. As all the result, the bioactivity of sample polysaccharide sulfate is better, Wolfrom method is more fit to prepare polysaccharide sulfate of Hericium caput-medusae liquid submerged fermentation extractum than the sulfuric acid method.
引文
[1]Hawdswoith DL.Ainsworth & Bishby's dictionary of the fungi. [M]. CAB International.1995.
[2]徐锦堂.中国药用真菌学[J].北京医科大学和中国协和医科大学联合出版社,1997.12：448-449.
[3]李丽,李秀玲,李春丰等.黑龙江省大亮子河深林公园8种白腐菌的培养特性研究[J].科技信息,2008.35：807-808.
[4]Alexopoulos.C.J& M.Blackwell & C.W.Mims. Introductory Mycology [M].姚一健,李玉,译.北京：中国农业出版社：2002：518-519.
[5]池玉杰.10种针阔叶树上常见的木材[J].菌物系统,2002,21(1)：116-119.
[6]宋慧.小刺猴头菌化学成分研究及菌种鉴定[D].长春：吉林农业大学,2003：3.
[7]陈宇.小刺猴头菌甾体化合物的化学成分研究[D].长春：吉林农业大学,2008：1.
[8]吴云艳,宋慧.小刺猴头菌子实体多糖的提取工艺[J].湖北农业科学,2007,46(2)：293-294.
[9]罗巅辉,王昭静,梁忠岩.小刺猴头菌子实体多糖的分离、纯化及初步研究[J].福建中医学院学报,2005,15(4)：32-34.
[10]张金亭,黄勇,李金花等.小刺猴头多糖Ⅰ的分离纯化及组成分析[J].中国药学杂志,2005,40(7)：545-546.
[11]苏玲.小刺猴头菌液体深层发酵浸膏糖类物质药理学活性研究[D].长春：吉林农业大学,2008：22.
[12]柳保强.小刺猴头多糖对大鼠慢性萎缩性胃炎的实验研究[D].长春：吉林大学,2006.
[13]Kawagishi H,qima F,Okamoto K.Preparation of Cyathane derivatives with Hericium erinaceus as Stimulators of HGF and antimicrobial agents.Eur Pat EP.613.903
[14]马焕璞,李敬轩,杨风和等.小刺猴头菌丝球粒深层培养及其临床疗效观察[J].微生物学通报,1985,05：198-200.
[15]周东正,王丽兰.小刺猴头药用价值及其提取物的研究[J].中药通报,1983：41-42.
[16]王兰,柏青安,邵坤范.小刺猴头深层培养球提取物的镇静安定作用[J].中药通报,1986,11(4)：54-55.
[17]张恒弼,田望霓,王云剑等.人工小刺猴头菌丝镇静作用的研究[J].药学情报通报,1987：54-55.
[18]何苗,魏敏杰,赵琳等.小刺猴头菌醇提物防治化学诱导的阿尔茨海默病小鼠的作用与机制初探[J].中国医科大学学报,2008,37(2)：184-186.
[19]吴云艳.小刺猴头菌子实体多糖的免疫佐剂效应[J].江西农业大学学报,2008,30(4)：743-745.
[20]焦克芳、陈望忠、曲红.糖类与生命科学研究进展,大学化学,1999,(1)：28-31.
[21]蔡孟深,李中军,Carbohydrate Chemistry[M].北京,化学工业出版社,2006：1.
[22]Haruki Yamada, Noride shinizu Characterization and Immunologicalactivitids of a novel acidic polysaccharide [J].Chem Pharm Bull,1990,38(10):2771.
[23]Naoko ohara, Katsutoshi Takada.Characterization of two novel polysaccharides having Immunological activities from the root of Panaxginseng[J].Biol Pharm Bull,1993, 16(11):1087
[24]Mates J M, Sanchez-Jimenez F M.Role of reactive oxygen species in Apoptosis; implication for cancer therapy[J].Int J Biochem Cell Biol,2000,32(2):157-170
[25]Locigno R, Castronovo.Reduced glutathione system:role in cancer development, prevetion and reatment [J].Int J Oncol,2001,19(2):221-236.
[26]BravardA, Ageron-Blane Agnes, AlvarezS, et al.Correlation between antioxidant status, tumorigenicity and radiosensitivity in sister rat cell lines[J].Carcinogenesis,2002,23(5): 705-711.
[27]肖建辉,将依辉,梁宗琦等.食药用真菌多糖研究进展[J].生命的化学,2002,22(2)：148.
[28]陈传红,金卫根,李荣同等.真菌多糖药理作用研究进展[J].时珍国医国药,2006,17(6)：933-934.
[29]高坡.小刺猴头菌化学成分研究及菌种鉴定[D].长春：吉林农业大学,2003：3.
[30]赵玉红,郑磊,毛凤彪等,利用Box-Behnken设计优化鹿茸糖胺聚糖的提取工艺[J].东北林业大学学报,2010,38(5)：93-96.
[31]陈艳,程胜,王晓丽等,正交多项式回归设计优化微波提取海藻糖工艺[J].食品科学,2010,31(22)：119-123.
[32]褚立军,但飞君,鄢文芳等,超声波提取白根独活多糖工艺研究[J].安徽农业科学,2010,38(12)：6216-6217.
[33]秦蕾.沙棘叶多糖的提取、功能及其分子修饰的研究[D].哈尔滨：东北林业大学, 2010：20-25.
[34]陶跃中.茯苓多糖深层发酵、提取的参数控制及羧甲基化改性研究[D].开封市：河南大学,2010：38.
[35]谭敏,邱细敏,陆艳艳等.白术多糖脱蛋白脱色工艺[J].Chinese Jou mal of New Drugs.2010,19(22):2100-2102.
[36]孔祥辉,张介驰,戴肖东等.黑木耳多糖的提取与纯化[J].生物技术.2005,15(1)：81-82.
[37]陈昕,尹鸿萍.等电点除蛋白的pH值对螺旋藻酸性多糖硫酸基团含量的影响[J]药物生物技术.2004,11(6)：381-384.
[38]扈瑞平,张兴夫,杜玲等.沙葱多糖Sevag法除蛋白工艺的研究[J].内蒙古大学学报,2009,40(6)：658-662.
[39]刘娜女,张静,李岱等.利用傅立叶红外光谱和原子力显微镜研究超声提取对鸡腿菇多糖结果的影响[J].生物加工过程.2010,8(1)：56-60.
[40]林德祥,邱巨香,陈叶明.正交试验法优选云芝多糖醇沉工艺的研究[J].南京中医药大学学报.2005,21(5)：328-330.
[41]段穗芳,郑宗坤.超滤提纯香菇子实体中的香菇多糖[J].深圳大学学报.1999,16(4)：66-69.
[42]顾亚萍,钱和.茶叶多糖纯度鉴定及结构分析的研究进展[J].食品研究与开发.2008,29(7)：166-171.
[43]孙勇民.酸浆宿萼水溶性多糖的分离纯化及理化性质分析[J].食品研究与开发.2007,28(7)：118-120.
[44]韩果萍.马齿苋多糖的分离纯化及抗氧化活性研究[D].西安：陕西大学,2003：Ⅰ.
[45]钟维庚,阮美娟,殷利娟等.苦瓜渣酶解制备多肽的研究[J].食品工业科技.2011,32(1)：240-242.
[46]栗衍华,谭成玉,王秀武等.硫酸化多糖的制备及其生物活性的研究进展[J].精细与专用化学品.2006,14(16)：6-9.
[47]姚秋萍硫酸多糖结构和性质的探讨：[D].长春：吉林大学,2006.
[48]程斌.海带硫酸多糖的提取纯化降解及活性研究：[D].曲阜市：曲阜师范大学,2009.
[49]谌素华,王维民.褐藻岩藻聚糖硫酸醋调血脂活性研究进展.食品学会年会论文集
[50]李晓坤.黄芪硫酸酯多糖的合成方法研究：[D].长春：东北师范大学,2007.
[51]王鲁.人参皂苷硫酸化修饰及其免疫活性的研究：[D].长春：吉林大学,2007.
[52]梁中焕.硫酸化修饰对白术多糖理化性质及生物活性影响的研究：[D].长春：东北师范大学,2008.
[53]张安强,马新等.硫酸酯化猴头菌多糖的结构与构象分析[J].食品与发酵工业,2009,35(3)
[54]初丽伟.人参硫酸酯多糖的合成方法及其有机氯多残留的分析方法研究：[D].长春：东北师范大学,2008.
[55]任丽靖.党参多糖及其硫酸化党参多糖的结构及抗氧化活性的初步研究：[D].西安市：陕西师范大学,2008.
[56]徐述明.猪苓多糖硫酸酯化及其研究：[D].武汉：武汉理工大学2006.
[57]李公斌,王振宇.黑木耳多糖硫酸酯化制备工艺及红外光谱分析[J].东北林业大学学报2008,36(12).
[58]孔倩,周婷等.南瓜水溶性多糖的制备及硫酸酯化初步研究[J].食品科学2009,30(16).
[59]孙永旭等.Sulfated modification of the water-soluble polysaccharides from Polyporus albicans mycelia and its potential biological activities 2009 International Journal of Biological Macromolecules 14-17.
[60]杨伟,鲁梅芳,金玉妍.灰树花菌丝体多糖的硫酸酯化及其降血脂作用[J].天津科技大学学报2009,24(4).
[61]张体祥,刘捷等.硫酸化红薯叶多糖的制备工艺及其抗氧化活性研究[J].食品科技2009,34(8).
[62]Ueno Y,etal.Anantitum or activity of the Sclertia of Grifora umbel late(Fr)PILAT [J].Carbohydr Res,1982,101:106-107.
[63]田庚元,李寿桐,宋麦立等.牛膝多糖硫酸酯的合成及其抗病毒活性.药学学报,1995,30(2)：107-111
[64]张丽萍,汉丽萍,王月秋等.硫酸化高山红景天多糖(RSASL)的制备及鉴定.分子科学学报,1999,15(4)：205-209
[65]杨淑娟,胡元亮,郭利伟等.正交试验优选党参多糖的硫酸化工艺研究[J].中草药.2010,41(7)：1091-1094.
[66]曹倩倩,朱蓓薇,杨静峰等.扇贝糖原酶法提取及其硫酸化修饰条件[J].大连工业大学学 报.2010：1-4.
[67]张帆,王德云,赵晓娜.响应面法探讨氯磺酸-吡啶法修饰条件对硫酸化当归多糖取代度的影响[J].天然产物研究与开发.2010,22：830-835.
[68]申进文,冯雅岚,余海尤等.硫酸化分级香菇多糖抗氧化活性研究[J].菌物学报.2010,29(3)：449-453.
[69]张丽萍,王月秋,姜世成等.离子色谱法测定高山红景天多糖硫酸酷中硫酸根含量的研究[J].分子科学学报,2000,16(3)：161—164.
[70]王莉,张远,朱丽丹等.[J].食品工业科技,2010,31(10)：243-246.
[71]赵鹏.款冬花多糖提取工艺研究及结构鉴定[D].西安市：西北大学,2010
[72]游庆红,尹秀莲.响应面法优化桑黄多糖提取工艺研究[J].中国酿造,2010,218(5)：67-69.
[73]蔚荣海,崔喜艳,于少华主编.基础生化实验与技术[M].吉林大学出版社,2000,9：42-43.
[74]罗巅辉,王昭晶,梁忠岩.小刺猴头子实体多糖的分离、纯化及初步研究[J].福建中医学院学报,2005,15(4)：32-34.
[75]张惟杰.糖复合物生化研究技术[M].杭州：浙江大学出版社,1999：79-80.
[76]柳洪芳,王新宇,吕金超.榆耳多糖的分离纯化、结构鉴定及抗肿瘤活性研究[J].中国生化药物杂志,2010,31(5)：293-296.
[77]霍贤,红花水溶性多糖CTP的结构研究[D].长春市,东北师范大学,2005：7.
[78]周明军,褐藻糖胶提取纯化、结构分析及抗氧化研究[D].杭州市,浙江大学,2006：41.
[79]郝桂堂,夏枯草多糖的分离分析[D].无锡市,江南大学,2007：14.
[80]周勇,桑黄水溶性多糖的分离纯化及结构的初步研究[D].长春市,吉林农业大学,2007：33.
[81]张雅君,党参水溶性多糖CPPS3的结构研究[D].长春市,东北师范大学,2005：8.
[82]耿慧,碱提猴头发酵菌丝体多糖的分离纯化及结构研究[D].长春市,东北师范大学,2003：9.
[83]屈静,低分子量黄芪多糖的分离纯化和结构分析[D].长春市,东北师范大学,2010：9.
[84]李天宝,碱提肠浒苔水溶性多糖的结构及活性研究[D].长春市,东北师范大学,2007：6.
[85]刘丹,紫茉莉根碱提水溶性多糖MJL的结构研究[D].长春市,东北师范大学,2010：7
[86]颜邦干,香菇多糖的分离纯化及其分子结构改性的研究[D].杭州市,浙江大学,2006：43.
[87]丁剑,猴头菌属不同菌种发酵多糖的分离纯化与结构研究[D].长春：吉林农业大学,2008：29.
[88]Ueno Y,Okamoto Y,Yamauchi R etal.An antitumor activity of the alkali-soluble polysaccharide and its derivatives obtained from the Sclertia of Grifora umbellate (Fr) PILAT.Carbohydr Res,1982,101:160-167.
[89]柳洪芳.榆耳多糖的分离纯化、结构鉴定及抗肿瘤活性的研究[D].长春：吉林农业大学.2009：21.
[90]徐静,傅颖媛,黄彬红等.金边瑞香浸膏体内外抗肿瘤作用[J].中国新药杂志.2008,17(23)：2019-1026.
[91]LIU Gao-Qiang, WANG Xiao-Ling.Structure-activity relationship and anticancer mechanisms of Ganoderma lucldum polysaccharides[J].菌物学报,2006,25(3):430-438.
[92]Wasser, S.P.and Weis, A.L.Medicinal properties of substances occurring in higher Basidiomycete mushrooms:current perspectives(review).International Journal MedicinalMushrooms,1999,1：31-62.
[93]毛逸嵘,灰树花多糖及其硫酸化衍生物的结构鉴定和生物活性研究[D].上海,华东师范大学,2009：29.
[94]张珏,灵芝多糖的硫酸化修饰及其衍生物抗肿瘤活性的初步研究[D].长春市,江南大学,2005.
[95]陈益,半夏多糖的结构与抗肿瘤活性研究[D].西安,陕西师范大学,2007,41.
[2]徐锦堂.中国药用真菌学[J].北京医科大学和中国协和医科大学联合出版社,1997.12：448-449.
[3]李丽,李秀玲,李春丰等.黑龙江省大亮子河深林公园8种白腐菌的培养特性研究[J].科技信息,2008.35：807-808.
[4]Alexopoulos.C.J& M.Blackwell & C.W.Mims. Introductory Mycology [M].姚一健,李玉,译.北京：中国农业出版社：2002：518-519.
[5]池玉杰.10种针阔叶树上常见的木材[J].菌物系统,2002,21(1)：116-119.
[6]宋慧.小刺猴头菌化学成分研究及菌种鉴定[D].长春：吉林农业大学,2003：3.
[7]陈宇.小刺猴头菌甾体化合物的化学成分研究[D].长春：吉林农业大学,2008：1.
[8]吴云艳,宋慧.小刺猴头菌子实体多糖的提取工艺[J].湖北农业科学,2007,46(2)：293-294.
[9]罗巅辉,王昭静,梁忠岩.小刺猴头菌子实体多糖的分离、纯化及初步研究[J].福建中医学院学报,2005,15(4)：32-34.
[10]张金亭,黄勇,李金花等.小刺猴头多糖Ⅰ的分离纯化及组成分析[J].中国药学杂志,2005,40(7)：545-546.
[11]苏玲.小刺猴头菌液体深层发酵浸膏糖类物质药理学活性研究[D].长春：吉林农业大学,2008：22.
[12]柳保强.小刺猴头多糖对大鼠慢性萎缩性胃炎的实验研究[D].长春：吉林大学,2006.
[13]Kawagishi H,qima F,Okamoto K.Preparation of Cyathane derivatives with Hericium erinaceus as Stimulators of HGF and antimicrobial agents.Eur Pat EP.613.903
[14]马焕璞,李敬轩,杨风和等.小刺猴头菌丝球粒深层培养及其临床疗效观察[J].微生物学通报,1985,05：198-200.
[15]周东正,王丽兰.小刺猴头药用价值及其提取物的研究[J].中药通报,1983：41-42.
[16]王兰,柏青安,邵坤范.小刺猴头深层培养球提取物的镇静安定作用[J].中药通报,1986,11(4)：54-55.
[17]张恒弼,田望霓,王云剑等.人工小刺猴头菌丝镇静作用的研究[J].药学情报通报,1987：54-55.
[18]何苗,魏敏杰,赵琳等.小刺猴头菌醇提物防治化学诱导的阿尔茨海默病小鼠的作用与机制初探[J].中国医科大学学报,2008,37(2)：184-186.
[19]吴云艳.小刺猴头菌子实体多糖的免疫佐剂效应[J].江西农业大学学报,2008,30(4)：743-745.
[20]焦克芳、陈望忠、曲红.糖类与生命科学研究进展,大学化学,1999,(1)：28-31.
[21]蔡孟深,李中军,Carbohydrate Chemistry[M].北京,化学工业出版社,2006：1.
[22]Haruki Yamada, Noride shinizu Characterization and Immunologicalactivitids of a novel acidic polysaccharide [J].Chem Pharm Bull,1990,38(10):2771.
[23]Naoko ohara, Katsutoshi Takada.Characterization of two novel polysaccharides having Immunological activities from the root of Panaxginseng[J].Biol Pharm Bull,1993, 16(11):1087
[24]Mates J M, Sanchez-Jimenez F M.Role of reactive oxygen species in Apoptosis; implication for cancer therapy[J].Int J Biochem Cell Biol,2000,32(2):157-170
[25]Locigno R, Castronovo.Reduced glutathione system:role in cancer development, prevetion and reatment [J].Int J Oncol,2001,19(2):221-236.
[26]BravardA, Ageron-Blane Agnes, AlvarezS, et al.Correlation between antioxidant status, tumorigenicity and radiosensitivity in sister rat cell lines[J].Carcinogenesis,2002,23(5): 705-711.
[27]肖建辉,将依辉,梁宗琦等.食药用真菌多糖研究进展[J].生命的化学,2002,22(2)：148.
[28]陈传红,金卫根,李荣同等.真菌多糖药理作用研究进展[J].时珍国医国药,2006,17(6)：933-934.
[29]高坡.小刺猴头菌化学成分研究及菌种鉴定[D].长春：吉林农业大学,2003：3.
[30]赵玉红,郑磊,毛凤彪等,利用Box-Behnken设计优化鹿茸糖胺聚糖的提取工艺[J].东北林业大学学报,2010,38(5)：93-96.
[31]陈艳,程胜,王晓丽等,正交多项式回归设计优化微波提取海藻糖工艺[J].食品科学,2010,31(22)：119-123.
[32]褚立军,但飞君,鄢文芳等,超声波提取白根独活多糖工艺研究[J].安徽农业科学,2010,38(12)：6216-6217.
[33]秦蕾.沙棘叶多糖的提取、功能及其分子修饰的研究[D].哈尔滨：东北林业大学, 2010：20-25.
[34]陶跃中.茯苓多糖深层发酵、提取的参数控制及羧甲基化改性研究[D].开封市：河南大学,2010：38.
[35]谭敏,邱细敏,陆艳艳等.白术多糖脱蛋白脱色工艺[J].Chinese Jou mal of New Drugs.2010,19(22):2100-2102.
[36]孔祥辉,张介驰,戴肖东等.黑木耳多糖的提取与纯化[J].生物技术.2005,15(1)：81-82.
[37]陈昕,尹鸿萍.等电点除蛋白的pH值对螺旋藻酸性多糖硫酸基团含量的影响[J]药物生物技术.2004,11(6)：381-384.
[38]扈瑞平,张兴夫,杜玲等.沙葱多糖Sevag法除蛋白工艺的研究[J].内蒙古大学学报,2009,40(6)：658-662.
[39]刘娜女,张静,李岱等.利用傅立叶红外光谱和原子力显微镜研究超声提取对鸡腿菇多糖结果的影响[J].生物加工过程.2010,8(1)：56-60.
[40]林德祥,邱巨香,陈叶明.正交试验法优选云芝多糖醇沉工艺的研究[J].南京中医药大学学报.2005,21(5)：328-330.
[41]段穗芳,郑宗坤.超滤提纯香菇子实体中的香菇多糖[J].深圳大学学报.1999,16(4)：66-69.
[42]顾亚萍,钱和.茶叶多糖纯度鉴定及结构分析的研究进展[J].食品研究与开发.2008,29(7)：166-171.
[43]孙勇民.酸浆宿萼水溶性多糖的分离纯化及理化性质分析[J].食品研究与开发.2007,28(7)：118-120.
[44]韩果萍.马齿苋多糖的分离纯化及抗氧化活性研究[D].西安：陕西大学,2003：Ⅰ.
[45]钟维庚,阮美娟,殷利娟等.苦瓜渣酶解制备多肽的研究[J].食品工业科技.2011,32(1)：240-242.
[46]栗衍华,谭成玉,王秀武等.硫酸化多糖的制备及其生物活性的研究进展[J].精细与专用化学品.2006,14(16)：6-9.
[47]姚秋萍硫酸多糖结构和性质的探讨：[D].长春：吉林大学,2006.
[48]程斌.海带硫酸多糖的提取纯化降解及活性研究：[D].曲阜市：曲阜师范大学,2009.
[49]谌素华,王维民.褐藻岩藻聚糖硫酸醋调血脂活性研究进展.食品学会年会论文集
[50]李晓坤.黄芪硫酸酯多糖的合成方法研究：[D].长春：东北师范大学,2007.
[51]王鲁.人参皂苷硫酸化修饰及其免疫活性的研究：[D].长春：吉林大学,2007.
[52]梁中焕.硫酸化修饰对白术多糖理化性质及生物活性影响的研究：[D].长春：东北师范大学,2008.
[53]张安强,马新等.硫酸酯化猴头菌多糖的结构与构象分析[J].食品与发酵工业,2009,35(3)
[54]初丽伟.人参硫酸酯多糖的合成方法及其有机氯多残留的分析方法研究：[D].长春：东北师范大学,2008.
[55]任丽靖.党参多糖及其硫酸化党参多糖的结构及抗氧化活性的初步研究：[D].西安市：陕西师范大学,2008.
[56]徐述明.猪苓多糖硫酸酯化及其研究：[D].武汉：武汉理工大学2006.
[57]李公斌,王振宇.黑木耳多糖硫酸酯化制备工艺及红外光谱分析[J].东北林业大学学报2008,36(12).
[58]孔倩,周婷等.南瓜水溶性多糖的制备及硫酸酯化初步研究[J].食品科学2009,30(16).
[59]孙永旭等.Sulfated modification of the water-soluble polysaccharides from Polyporus albicans mycelia and its potential biological activities 2009 International Journal of Biological Macromolecules 14-17.
[60]杨伟,鲁梅芳,金玉妍.灰树花菌丝体多糖的硫酸酯化及其降血脂作用[J].天津科技大学学报2009,24(4).
[61]张体祥,刘捷等.硫酸化红薯叶多糖的制备工艺及其抗氧化活性研究[J].食品科技2009,34(8).
[62]Ueno Y,etal.Anantitum or activity of the Sclertia of Grifora umbel late(Fr)PILAT [J].Carbohydr Res,1982,101:106-107.
[63]田庚元,李寿桐,宋麦立等.牛膝多糖硫酸酯的合成及其抗病毒活性.药学学报,1995,30(2)：107-111
[64]张丽萍,汉丽萍,王月秋等.硫酸化高山红景天多糖(RSASL)的制备及鉴定.分子科学学报,1999,15(4)：205-209
[65]杨淑娟,胡元亮,郭利伟等.正交试验优选党参多糖的硫酸化工艺研究[J].中草药.2010,41(7)：1091-1094.
[66]曹倩倩,朱蓓薇,杨静峰等.扇贝糖原酶法提取及其硫酸化修饰条件[J].大连工业大学学 报.2010：1-4.
[67]张帆,王德云,赵晓娜.响应面法探讨氯磺酸-吡啶法修饰条件对硫酸化当归多糖取代度的影响[J].天然产物研究与开发.2010,22：830-835.
[68]申进文,冯雅岚,余海尤等.硫酸化分级香菇多糖抗氧化活性研究[J].菌物学报.2010,29(3)：449-453.
[69]张丽萍,王月秋,姜世成等.离子色谱法测定高山红景天多糖硫酸酷中硫酸根含量的研究[J].分子科学学报,2000,16(3)：161—164.
[70]王莉,张远,朱丽丹等.[J].食品工业科技,2010,31(10)：243-246.
[71]赵鹏.款冬花多糖提取工艺研究及结构鉴定[D].西安市：西北大学,2010
[72]游庆红,尹秀莲.响应面法优化桑黄多糖提取工艺研究[J].中国酿造,2010,218(5)：67-69.
[73]蔚荣海,崔喜艳,于少华主编.基础生化实验与技术[M].吉林大学出版社,2000,9：42-43.
[74]罗巅辉,王昭晶,梁忠岩.小刺猴头子实体多糖的分离、纯化及初步研究[J].福建中医学院学报,2005,15(4)：32-34.
[75]张惟杰.糖复合物生化研究技术[M].杭州：浙江大学出版社,1999：79-80.
[76]柳洪芳,王新宇,吕金超.榆耳多糖的分离纯化、结构鉴定及抗肿瘤活性研究[J].中国生化药物杂志,2010,31(5)：293-296.
[77]霍贤,红花水溶性多糖CTP的结构研究[D].长春市,东北师范大学,2005：7.
[78]周明军,褐藻糖胶提取纯化、结构分析及抗氧化研究[D].杭州市,浙江大学,2006：41.
[79]郝桂堂,夏枯草多糖的分离分析[D].无锡市,江南大学,2007：14.
[80]周勇,桑黄水溶性多糖的分离纯化及结构的初步研究[D].长春市,吉林农业大学,2007：33.
[81]张雅君,党参水溶性多糖CPPS3的结构研究[D].长春市,东北师范大学,2005：8.
[82]耿慧,碱提猴头发酵菌丝体多糖的分离纯化及结构研究[D].长春市,东北师范大学,2003：9.
[83]屈静,低分子量黄芪多糖的分离纯化和结构分析[D].长春市,东北师范大学,2010：9.
[84]李天宝,碱提肠浒苔水溶性多糖的结构及活性研究[D].长春市,东北师范大学,2007：6.
[85]刘丹,紫茉莉根碱提水溶性多糖MJL的结构研究[D].长春市,东北师范大学,2010：7
[86]颜邦干,香菇多糖的分离纯化及其分子结构改性的研究[D].杭州市,浙江大学,2006：43.
[87]丁剑,猴头菌属不同菌种发酵多糖的分离纯化与结构研究[D].长春：吉林农业大学,2008：29.
[88]Ueno Y,Okamoto Y,Yamauchi R etal.An antitumor activity of the alkali-soluble polysaccharide and its derivatives obtained from the Sclertia of Grifora umbellate (Fr) PILAT.Carbohydr Res,1982,101:160-167.
[89]柳洪芳.榆耳多糖的分离纯化、结构鉴定及抗肿瘤活性的研究[D].长春：吉林农业大学.2009：21.
[90]徐静,傅颖媛,黄彬红等.金边瑞香浸膏体内外抗肿瘤作用[J].中国新药杂志.2008,17(23)：2019-1026.
[91]LIU Gao-Qiang, WANG Xiao-Ling.Structure-activity relationship and anticancer mechanisms of Ganoderma lucldum polysaccharides[J].菌物学报,2006,25(3):430-438.
[92]Wasser, S.P.and Weis, A.L.Medicinal properties of substances occurring in higher Basidiomycete mushrooms:current perspectives(review).International Journal MedicinalMushrooms,1999,1：31-62.
[93]毛逸嵘,灰树花多糖及其硫酸化衍生物的结构鉴定和生物活性研究[D].上海,华东师范大学,2009：29.
[94]张珏,灵芝多糖的硫酸化修饰及其衍生物抗肿瘤活性的初步研究[D].长春市,江南大学,2005.
[95]陈益,半夏多糖的结构与抗肿瘤活性研究[D].西安,陕西师范大学,2007,41.