羊栖菜多糖的提取和纯化研究
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摘要
羊栖菜多糖(简称SFP)是从马尾藻科植物羊栖菜(Sargassum fusiforme(Harv.)Setchel)中提取得到的，具有抗肿瘤、抗凝血、降血糖、提高肌体免疫功能等生理活性。本文研究用水提法、酸提法、酶法提取羊栖菜多糖，考虑到提取温度、时间、加水量、加酶量等因素的影响，用正交试验优化酶法提取条件。酶法提取的最适温度为50℃，最适pH值为3.0，提取时间为3hr，加酶量为纤维素酶(16000 U／g)8％，果胶酶(20000 U／g)4％。为适应工业化生产需要，确定水提法为煮沸提取时间3hr，提取液pH值为3.0，固液比为1：40。用酶法结合高温浸提多糖提取的工艺条件是：加纤维素酶量为(16000U／g)8％，加果胶酶量为(20000 U／g)4％，pH3.0，酶解时间为3hr，酶解温度为50℃；浸提温度120℃，浸提时间3hr，固液比1：40。采用该工艺的粗多糖提取得率达14.88％，比热水浸提工艺的粗多糖得率提高了117.23％，比资料介绍的四次浸提法提高了75.06％。
本文系统地研究了羊栖菜粗多糖中蛋白质的分离方法，先后选用了Sevag法、酶法结合Sevag法、三氯乙酸法、1万分子截留量膜超滤、阴阳离子交换树脂法。发现用弱碱性阴和弱酸性阳离子交换树脂串联脱除粗多糖中蛋白质，效果理想，蛋白脱去除率可达91.76％，多糖回收率为92.56％，其中(多糖含量／蛋白含量)值高达42.27，经1万截留分子量透析膜透析，最后冷冻干燥后，得到淡黄色的羊栖菜多糖精粉，其中多糖含量达90.27％，蛋白质含量仅为
浙江工业大学硕士学位论文
羊栖菜多糖的提取和纯化研究
3.巧%。该方法还起到部分脱色效果。
将脱蛋白后的多糖精粉用SePhadexG一200葡聚糖凝胶分离成大分子量多
糖部分和小分子量多糖两部分。大分子量多糖部分用SePhacy1S一400共聚糖凝
胶分离，得到一个分子量相当大的组分，经凝胶HPLC纯度鉴定为均一性物
质，命名组分Fl，该组分含量占多糖精粉的28.45%;小分子量多糖部分进行
SePhadexG一1 00葡聚糖凝胶柱层析，结果表明主要有三个组分fl、几、。，
经凝胶HPLC标准分子量对照分析，三个组分分子量分别为8.2万、5.6万和
1.4万;经计算，比例为:37.4%:15.1%:475%。
本实验研究有效地分离了羊栖菜粗多糖中蛋白质，制得羊栖菜多糖精粉，
为羊栖菜在保健食品和药物研究和开发方面提供了良好的基础，同时为羊栖
菜多糖提取和分离的工业化研究和生产提供了理论依据。
Sargassum jusiforme(Harv.) Setchel is one of the plentiful brown seaweeds in east China sea. Its various biological activities (antitumoral, anticoagulant, antiviral, antioxdative, hypoglycemic and so on) have been reported in the literature. To make best use of the precious marine resources, the methods of extraction and purification of polysaccharide (SFP) from Sargassum fusiforme were studied in this paper.
Three methods for extraction of SFP are discussed. The conditions of extracting SFP by boiling water, acid water and cellulase were investigated, result shows the optimum extraction temperature of enzymatic treatment is 50℃ optimum time is 3hr, optimum pH value is 3.0, add cellulase (16000U/g ) 8%, pectinase (20000U/g) 4%. The optimum extraction temperature of boiling water treatment is 102℃, optimum time is 3hr, weight of material: weight of water=l :40. Enzyme and high temperature extraction was combined to improve the low extraction rate of acid extraction and neutral boiling water extraction, and the yield could attain 14.88%. The best experiment factors are received Cellulase (16000U/g ) 8%, pectinase (20000U/g) 4%, pH3.0, weight of material : weight of water=1:40. The time of enzymolysis 3hr> the time of extraction 3hr the temperature of extraction 120℃. Compared with other extraction methods,
enzymatic treatment made yield of polysaccharide increasing 117.23% and up to 75.06%, which was higher than the method of four time extractions mentioned in literatures
Several methods includeing, Sevag method, enzyme decomposition with Sevag method, trichloroacetic acid(TCA) sedimentation, ultrafiltration , anion and cation exchange resin chromatography were studied to remove the protein in the SFP. Based on experimental results and general consideration, it was suitable to adopt using anion and cation exchange resin in series chromatography once. After that, 91.76% protein of crude polysaccharides could be removed, the recovery rate of polysaccharide was 92.56%, and the ratio value of polysaccharides content to protein content was up to 42.27.Then after dialysis and freeze dryness, light yellow powder was produced. In this powder, polysaccharide and protein content was 90.27% and 3.15%.
Higher molecular and lower molecular weight sections were gained by using Sephadex G-200 gel chromatograph. Then a polysaccharide fraction containing 1.5% protein was gained by Sephacryl S-400 chromatographing to the higher molecular. The purity of this fraction was proved by HPGPC and its molecular was more than 2×106 dalton and was named F1. The portion of F1 was 28.45% of fine polysaccharides. And the low molecular weight was chromatographed by Sephadex G-100 gel filtration, and three fraction f1, f2, f3 were gained , the molecular weight of them was 8.2×104(37.4%), 5.6×104 (15.1%), 1.4× 104(47.5%), the weight ratio was 38.2%, 16.1%, 45.7% separately.
In this paper, purified powder of SFP was obtained by isolated protein in rude SFP effectively. The research offers some scientific basis for producing healthy foods and medicines. It provides theoretical foundation for industrialization of extraction and isolation polysaccharides from Sagasssum fusiforme.
本文系统地研究了羊栖菜粗多糖中蛋白质的分离方法，先后选用了Sevag法、酶法结合Sevag法、三氯乙酸法、1万分子截留量膜超滤、阴阳离子交换树脂法。发现用弱碱性阴和弱酸性阳离子交换树脂串联脱除粗多糖中蛋白质，效果理想，蛋白脱去除率可达91.76％，多糖回收率为92.56％，其中(多糖含量／蛋白含量)值高达42.27，经1万截留分子量透析膜透析，最后冷冻干燥后，得到淡黄色的羊栖菜多糖精粉，其中多糖含量达90.27％，蛋白质含量仅为
浙江工业大学硕士学位论文
羊栖菜多糖的提取和纯化研究
3.巧%。该方法还起到部分脱色效果。
将脱蛋白后的多糖精粉用SePhadexG一200葡聚糖凝胶分离成大分子量多
糖部分和小分子量多糖两部分。大分子量多糖部分用SePhacy1S一400共聚糖凝
胶分离，得到一个分子量相当大的组分，经凝胶HPLC纯度鉴定为均一性物
质，命名组分Fl，该组分含量占多糖精粉的28.45%;小分子量多糖部分进行
SePhadexG一1 00葡聚糖凝胶柱层析，结果表明主要有三个组分fl、几、。，
经凝胶HPLC标准分子量对照分析，三个组分分子量分别为8.2万、5.6万和
1.4万;经计算，比例为:37.4%:15.1%:475%。
本实验研究有效地分离了羊栖菜粗多糖中蛋白质，制得羊栖菜多糖精粉，
为羊栖菜在保健食品和药物研究和开发方面提供了良好的基础，同时为羊栖
菜多糖提取和分离的工业化研究和生产提供了理论依据。
Sargassum jusiforme(Harv.) Setchel is one of the plentiful brown seaweeds in east China sea. Its various biological activities (antitumoral, anticoagulant, antiviral, antioxdative, hypoglycemic and so on) have been reported in the literature. To make best use of the precious marine resources, the methods of extraction and purification of polysaccharide (SFP) from Sargassum fusiforme were studied in this paper.
Three methods for extraction of SFP are discussed. The conditions of extracting SFP by boiling water, acid water and cellulase were investigated, result shows the optimum extraction temperature of enzymatic treatment is 50℃ optimum time is 3hr, optimum pH value is 3.0, add cellulase (16000U/g ) 8%, pectinase (20000U/g) 4%. The optimum extraction temperature of boiling water treatment is 102℃, optimum time is 3hr, weight of material: weight of water=l :40. Enzyme and high temperature extraction was combined to improve the low extraction rate of acid extraction and neutral boiling water extraction, and the yield could attain 14.88%. The best experiment factors are received Cellulase (16000U/g ) 8%, pectinase (20000U/g) 4%, pH3.0, weight of material : weight of water=1:40. The time of enzymolysis 3hr> the time of extraction 3hr the temperature of extraction 120℃. Compared with other extraction methods,
enzymatic treatment made yield of polysaccharide increasing 117.23% and up to 75.06%, which was higher than the method of four time extractions mentioned in literatures
Several methods includeing, Sevag method, enzyme decomposition with Sevag method, trichloroacetic acid(TCA) sedimentation, ultrafiltration , anion and cation exchange resin chromatography were studied to remove the protein in the SFP. Based on experimental results and general consideration, it was suitable to adopt using anion and cation exchange resin in series chromatography once. After that, 91.76% protein of crude polysaccharides could be removed, the recovery rate of polysaccharide was 92.56%, and the ratio value of polysaccharides content to protein content was up to 42.27.Then after dialysis and freeze dryness, light yellow powder was produced. In this powder, polysaccharide and protein content was 90.27% and 3.15%.
Higher molecular and lower molecular weight sections were gained by using Sephadex G-200 gel chromatograph. Then a polysaccharide fraction containing 1.5% protein was gained by Sephacryl S-400 chromatographing to the higher molecular. The purity of this fraction was proved by HPGPC and its molecular was more than 2×106 dalton and was named F1. The portion of F1 was 28.45% of fine polysaccharides. And the low molecular weight was chromatographed by Sephadex G-100 gel filtration, and three fraction f1, f2, f3 were gained , the molecular weight of them was 8.2×104(37.4%), 5.6×104 (15.1%), 1.4× 104(47.5%), the weight ratio was 38.2%, 16.1%, 45.7% separately.
In this paper, purified powder of SFP was obtained by isolated protein in rude SFP effectively. The research offers some scientific basis for producing healthy foods and medicines. It provides theoretical foundation for industrialization of extraction and isolation polysaccharides from Sagasssum fusiforme.
引文
[1] 谭周进，谢达平．多糖的研究进展，食品科技，2003(3)：10～12
[2] 王克夷．灰姑娘的马车已经来了，生命的化学，2001，21(3)：257～259
[3] Pamlela S, Chi H w. Toward automated synthesis of Oligosaccharides and Glycoprotein, Science, 3.21.2001,291(5512):2351～2356
[4] Anne D, Howard R M. Glycoprotein structure determination by mass spectrometry. Science, 3.21,2001,291 (5512):2351～2356
[5] Carolyn R B, Laura L K. Chemical Glycobiology. Science, 3.21, 2001,29(5512):2357～2363
[6] Pauline M R, Time E, Peter C, et al. Glycosylation and immune system. Science, 3.21,2001, 291(5512):2370～2375
[7] Marsin J R, Topalova H. Radioprotection by polysaccharide. Pharmacol. Ther. 1987, 39: 255～266
[8] Siddhanta Ak.Marine algal polysaccharide-functions and utilization. Trend Carbohydr Chem[C]. Carbohydr Conf 9 th 1993, India: Surya International Publications,1995:125～131
[9] Brown MR, Jeffrey SW, Volkman JK, et al. Nutritional Properties of microalgae for maxiculture. Aquaculture, 1997, 151(1-4): 315
[10] 李兆杰，薛长湖．岩藻聚糖硫酸酯降血脂及抗氧化作用的研究，营养学报，1999，21(3)：280．
[11] 李福川，唐志红，崔博文，等．三种海带多糖的降糖作用，中国海洋药物，2000，5：12．
[12] Brockbank KGM. Algae-derived polysaccharide as cryoprotective agent and its use in cryopreservation of cellular mattey[P]. U.S. US5071, 741,1991-10-10
[13] Okai Yasuji,Ishizaka Shigeaki, Higashi-Okai Kiyoka,et al.Detection of immunomochulating activities in an extract of Japanese edible seaweed, Laminaria japocuca(Makonbu). J Sci Food Agric, 1996, 72(4):455
[14] 王文涛．海藻多糖对小鼠淋巴细胞增殖反应及白细胞介素2产生的影响．中药药理学通讯，1992，(3)；47．
[15] Nazarova IV, Sheochenko NM. Immunomodulatory properties of polysaccharides from red algae: influence on the complement system. Biol Morya, 1998,24(1):47
[16] 童为人，团雪梅．补体活化在动脉粥样硬化形成中的意义．国外医学，免疫学分册，1999，22(1)：47．
[17] 王常青，杨桂兰．两种海藻多糖对大鼠脂质代谢和血小板功能的比较．中华预防医学杂志，1997，31(6)：342．
[18] 陈毓强，吴炳南，四种多糖化合物的抗癌作用及其对免疫功能的影响．癌症，1997，16(3)：198．
[19] Lee YS, Kim DS,Piyu BH, et al. Artitmuor and immunomodulating effects of seaweeds toward sarcoma-180 cells. Hau'guk Yong Yang Siklyong Hakhoechi, 1992,21(5):544
[20] 宋剑秋，徐誉泰．海带硫酸多糖对小鼠腹腔巨噬细胞的免疫调节作用．中国免疫学杂志，2000，16(2)；70．
[21] 滕霞，丛建波，田晓华，等 海藻硫酸多糖抗氧化及抗肿瘤作用的实验研究．营养学报，1998，20(1)：48．
[22] 郑刚，郝军，贾钢锐，等 褐藻胶对大鼠实验性肝纤维化的防治．中国海洋药物，1997，16(1)：30．
[23] 陈家童，张斌，白玉华，等．红藻多糖抗AIDS病毒作用的体外实验研究．南开大学学报(自然科学版)，1998，31(4)：21．
[24] Arad S, Huliheil M, Tal J. Antiviral agents[P]. Israel. PCT Int Appl WO 97/006 89,1997-01-09
[25] 齐慧玲，魏绍云，王继伦．Sevag法去除白及多糖中蛋白的研究，天津化工，2000(3)：20～21
[26] 王卫国，赵永亮，韩山宝．香菇多糖分离纯化技术研究，中国食用菌，2002，21(2)：30～32
[27] 王仲孚等．钝顶螺旋藻多糖SPPC-1的分离纯化、性质及活性的研究，中国海洋药物，2001，81 (3)：171～20
[28] 刘成梅等．百合多糖脱蛋白方法的研究，食品科学，2002，23(1)：89～90
[29] 于宙，钟洁，吴克．金针菇多糖的分离和分析，安徽农业科学，1998，386～388
[30] 孙向军，姚晓敏，闵锐．酶法提取螺旋藻粗多糖的研究，上海农学院学报，1999，17(2)：115～118
[31] 傅明辉，佘纲哲，彭达聪．喇叭藻(T．ornate．J．Ag)多糖的分离及纯化，2000，17(4)：204～25
[32] 陈怡．天然多糖的研究概况，世界科学技术，2000，(6)：52～57
[33] 鞠海，张建民，魏锋等．天然多糖的分离、纯化和结构鉴定，国外医药-植物分册，2000，15(3)：107～113
[34] 周鹏，谢明勇，傅博强．多糖的结构研究，南昌大学学报(理科版)，2001，25(2)：197～204
[35] Chioritti A, Aiao ML,Kraft GT, et al. Cell wall polysaccharides from Australian red algae of the fanuly Solieriaceae (Gigartinales, Rhodophyta):Highly methylated earrageenans from the genus Rhabdonia. Bot Man, 1996,39(1):47
[36] Brosch-Salonmon S, Hoftberger M, Holzing A, et al. Ultrastructural localization of polysaccharides and N-acetyl-D-galactosamine in the secretory pathway of green algae(pesmiddiaceae). J Exp Bot, 1998,49(319): 145
[37] Nardella A, Chaubet F, Boisson-Vidal C, et al. Anticoagulant low molecular wt. focans produced by radical process and ion exchange chromatography of high molecular weight fucans extracted from the brown seaweed Ascophyllum nodosum. Carbohydr Res, 1996,289,201
[38] 李时珍，本草纲目．北京：人民卫生出版社影印版，1957：1377
[39] 阴健主编．中药现代研究与临床应用．第三卷．北京：中医古籍出版社，1997：237
[40] Harlod C B, Michael J W. Introduction to the Algae 2. New Jersey: Prentice Hall Inc Englewood Cliffs, 1985: 20
[41] 李八方，功能食品与保健食品，青岛：青岛海洋大学出版社，1997：412～450
[42] 范饶，韩丽君，周天成等．海洋与湖沼，1995．26(2)；199
[43] 钱浩，胡巧玲，羊栖菜的成分分析研究，中国海洋药物，1998(3)：33～34
[44] 季字彬，张海滨，刘中海．羊栖菜多糖对荷瘤小鼠红细胞免疫功能的影响．中国海洋药物，1995(2)：10
[45] 季字彬等．羊栖菜多糖对门P388小鼠红细胞免疫促进作用的机制研究，中国海洋药物，1998(2)：14
[46] 蒋谷人等，我国马尾藻热水提取物抗肿瘤实验研究．海洋药物，1986(2)：17
[47] 张全斌．铁祖洪．褐藻多糖硫酸酯化学研究的进展，中国海洋药物，1996(4)：38
[48] 刘明哲等．海藻提取物对实验性高脂大鼠模型的药效学研究．中国现代应用药学杂志，1998(10)：9
[49] 刘明哲等，海藻Ⅱ号对心血管疾病防治的研究．海洋生物活性物质研究与开发，(李光友等编)2000：71
[50] 李八方等．羊栖菜水提取物及其食品对肌体生长发育的影响．中国海洋药物，1999(4)：35
[51] 西出英一，内田直行，日本产褐藻类中含有多糖．Nippon Suisan Gakkaishi 1987，53(6)：1083～1088
[52] 陈绍媛，莫为民，潘远江，陈耀祖，海洋药物研究(Ⅲ)——羊栖菜多糖，兰州大学学报，1998，34(4)：23～25
[53] Kyung-Im Kim, Hae-Duk Seo, Hyun-Sun Lee, et al. Studying in anticogulant polysaccharide extracted
from Hizikia fusiform, Journal of the Korean Society of food science and nutrition, 1998, 27(6):1204～1210
[54] 黄伟坤．食品检验与分析，北京：轻工业出版社，1989: 3～34
[55] A Fouad Abdel- Fattan et al. Studies of the purification and some properties of Sargassum, a sulfated heteropolysaccharide from Sargassum linifolium. Carbohydro. Res., 1974, 33:1
[56] 王惟香．辽宁师范大学学报(自然科学版)，1998，21(4)：315～317
[57] 张维杰．复合多糖生化研究技术，上海科学技术出版社，1987，7
[58] 食品分析，轻工业出版社，1983：93；216～219
[59] 张鹤林，张吉德，吴萍萍，等．羊栖菜抗肉毒素中毒、抗肿瘤有效成分的研究．海洋药物，1988，4：18～20
[60] 阮积惠，羊栖菜的药用功能的研究现状，中国野生植物资源，2002，20(6)：8～10
[61] 张展，刘建国，刘吉东．羊栖菜的研究述评，海洋水产研究，2003，23(3)：67～74
[2] 王克夷．灰姑娘的马车已经来了，生命的化学，2001，21(3)：257～259
[3] Pamlela S, Chi H w. Toward automated synthesis of Oligosaccharides and Glycoprotein, Science, 3.21.2001,291(5512):2351～2356
[4] Anne D, Howard R M. Glycoprotein structure determination by mass spectrometry. Science, 3.21,2001,291 (5512):2351～2356
[5] Carolyn R B, Laura L K. Chemical Glycobiology. Science, 3.21, 2001,29(5512):2357～2363
[6] Pauline M R, Time E, Peter C, et al. Glycosylation and immune system. Science, 3.21,2001, 291(5512):2370～2375
[7] Marsin J R, Topalova H. Radioprotection by polysaccharide. Pharmacol. Ther. 1987, 39: 255～266
[8] Siddhanta Ak.Marine algal polysaccharide-functions and utilization. Trend Carbohydr Chem[C]. Carbohydr Conf 9 th 1993, India: Surya International Publications,1995:125～131
[9] Brown MR, Jeffrey SW, Volkman JK, et al. Nutritional Properties of microalgae for maxiculture. Aquaculture, 1997, 151(1-4): 315
[10] 李兆杰，薛长湖．岩藻聚糖硫酸酯降血脂及抗氧化作用的研究，营养学报，1999，21(3)：280．
[11] 李福川，唐志红，崔博文，等．三种海带多糖的降糖作用，中国海洋药物，2000，5：12．
[12] Brockbank KGM. Algae-derived polysaccharide as cryoprotective agent and its use in cryopreservation of cellular mattey[P]. U.S. US5071, 741,1991-10-10
[13] Okai Yasuji,Ishizaka Shigeaki, Higashi-Okai Kiyoka,et al.Detection of immunomochulating activities in an extract of Japanese edible seaweed, Laminaria japocuca(Makonbu). J Sci Food Agric, 1996, 72(4):455
[14] 王文涛．海藻多糖对小鼠淋巴细胞增殖反应及白细胞介素2产生的影响．中药药理学通讯，1992，(3)；47．
[15] Nazarova IV, Sheochenko NM. Immunomodulatory properties of polysaccharides from red algae: influence on the complement system. Biol Morya, 1998,24(1):47
[16] 童为人，团雪梅．补体活化在动脉粥样硬化形成中的意义．国外医学，免疫学分册，1999，22(1)：47．
[17] 王常青，杨桂兰．两种海藻多糖对大鼠脂质代谢和血小板功能的比较．中华预防医学杂志，1997，31(6)：342．
[18] 陈毓强，吴炳南，四种多糖化合物的抗癌作用及其对免疫功能的影响．癌症，1997，16(3)：198．
[19] Lee YS, Kim DS,Piyu BH, et al. Artitmuor and immunomodulating effects of seaweeds toward sarcoma-180 cells. Hau'guk Yong Yang Siklyong Hakhoechi, 1992,21(5):544
[20] 宋剑秋，徐誉泰．海带硫酸多糖对小鼠腹腔巨噬细胞的免疫调节作用．中国免疫学杂志，2000，16(2)；70．
[21] 滕霞，丛建波，田晓华，等 海藻硫酸多糖抗氧化及抗肿瘤作用的实验研究．营养学报，1998，20(1)：48．
[22] 郑刚，郝军，贾钢锐，等 褐藻胶对大鼠实验性肝纤维化的防治．中国海洋药物，1997，16(1)：30．
[23] 陈家童，张斌，白玉华，等．红藻多糖抗AIDS病毒作用的体外实验研究．南开大学学报(自然科学版)，1998，31(4)：21．
[24] Arad S, Huliheil M, Tal J. Antiviral agents[P]. Israel. PCT Int Appl WO 97/006 89,1997-01-09
[25] 齐慧玲，魏绍云，王继伦．Sevag法去除白及多糖中蛋白的研究，天津化工，2000(3)：20～21
[26] 王卫国，赵永亮，韩山宝．香菇多糖分离纯化技术研究，中国食用菌，2002，21(2)：30～32
[27] 王仲孚等．钝顶螺旋藻多糖SPPC-1的分离纯化、性质及活性的研究，中国海洋药物，2001，81 (3)：171～20
[28] 刘成梅等．百合多糖脱蛋白方法的研究，食品科学，2002，23(1)：89～90
[29] 于宙，钟洁，吴克．金针菇多糖的分离和分析，安徽农业科学，1998，386～388
[30] 孙向军，姚晓敏，闵锐．酶法提取螺旋藻粗多糖的研究，上海农学院学报，1999，17(2)：115～118
[31] 傅明辉，佘纲哲，彭达聪．喇叭藻(T．ornate．J．Ag)多糖的分离及纯化，2000，17(4)：204～25
[32] 陈怡．天然多糖的研究概况，世界科学技术，2000，(6)：52～57
[33] 鞠海，张建民，魏锋等．天然多糖的分离、纯化和结构鉴定，国外医药-植物分册，2000，15(3)：107～113
[34] 周鹏，谢明勇，傅博强．多糖的结构研究，南昌大学学报(理科版)，2001，25(2)：197～204
[35] Chioritti A, Aiao ML,Kraft GT, et al. Cell wall polysaccharides from Australian red algae of the fanuly Solieriaceae (Gigartinales, Rhodophyta):Highly methylated earrageenans from the genus Rhabdonia. Bot Man, 1996,39(1):47
[36] Brosch-Salonmon S, Hoftberger M, Holzing A, et al. Ultrastructural localization of polysaccharides and N-acetyl-D-galactosamine in the secretory pathway of green algae(pesmiddiaceae). J Exp Bot, 1998,49(319): 145
[37] Nardella A, Chaubet F, Boisson-Vidal C, et al. Anticoagulant low molecular wt. focans produced by radical process and ion exchange chromatography of high molecular weight fucans extracted from the brown seaweed Ascophyllum nodosum. Carbohydr Res, 1996,289,201
[38] 李时珍，本草纲目．北京：人民卫生出版社影印版，1957：1377
[39] 阴健主编．中药现代研究与临床应用．第三卷．北京：中医古籍出版社，1997：237
[40] Harlod C B, Michael J W. Introduction to the Algae 2. New Jersey: Prentice Hall Inc Englewood Cliffs, 1985: 20
[41] 李八方，功能食品与保健食品，青岛：青岛海洋大学出版社，1997：412～450
[42] 范饶，韩丽君，周天成等．海洋与湖沼，1995．26(2)；199
[43] 钱浩，胡巧玲，羊栖菜的成分分析研究，中国海洋药物，1998(3)：33～34
[44] 季字彬，张海滨，刘中海．羊栖菜多糖对荷瘤小鼠红细胞免疫功能的影响．中国海洋药物，1995(2)：10
[45] 季字彬等．羊栖菜多糖对门P388小鼠红细胞免疫促进作用的机制研究，中国海洋药物，1998(2)：14
[46] 蒋谷人等，我国马尾藻热水提取物抗肿瘤实验研究．海洋药物，1986(2)：17
[47] 张全斌．铁祖洪．褐藻多糖硫酸酯化学研究的进展，中国海洋药物，1996(4)：38
[48] 刘明哲等．海藻提取物对实验性高脂大鼠模型的药效学研究．中国现代应用药学杂志，1998(10)：9
[49] 刘明哲等，海藻Ⅱ号对心血管疾病防治的研究．海洋生物活性物质研究与开发，(李光友等编)2000：71
[50] 李八方等．羊栖菜水提取物及其食品对肌体生长发育的影响．中国海洋药物，1999(4)：35
[51] 西出英一，内田直行，日本产褐藻类中含有多糖．Nippon Suisan Gakkaishi 1987，53(6)：1083～1088
[52] 陈绍媛，莫为民，潘远江，陈耀祖，海洋药物研究(Ⅲ)——羊栖菜多糖，兰州大学学报，1998，34(4)：23～25
[53] Kyung-Im Kim, Hae-Duk Seo, Hyun-Sun Lee, et al. Studying in anticogulant polysaccharide extracted
from Hizikia fusiform, Journal of the Korean Society of food science and nutrition, 1998, 27(6):1204～1210
[54] 黄伟坤．食品检验与分析，北京：轻工业出版社，1989: 3～34
[55] A Fouad Abdel- Fattan et al. Studies of the purification and some properties of Sargassum, a sulfated heteropolysaccharide from Sargassum linifolium. Carbohydro. Res., 1974, 33:1
[56] 王惟香．辽宁师范大学学报(自然科学版)，1998，21(4)：315～317
[57] 张维杰．复合多糖生化研究技术，上海科学技术出版社，1987，7
[58] 食品分析，轻工业出版社，1983：93；216～219
[59] 张鹤林，张吉德，吴萍萍，等．羊栖菜抗肉毒素中毒、抗肿瘤有效成分的研究．海洋药物，1988，4：18～20
[60] 阮积惠，羊栖菜的药用功能的研究现状，中国野生植物资源，2002，20(6)：8～10
[61] 张展，刘建国，刘吉东．羊栖菜的研究述评，海洋水产研究，2003，23(3)：67～74