海带多酚的分离提取及其抗肿瘤活性研究
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摘要
我国的海带资源十分丰富,海带养殖已成为中国海水养殖的支柱产业之一,产量居世界首位。越来越多的研究表明褐藻多酚具有很广泛的生物活性。为了充分利用海带,开发纯度较高的海带多酚,本论文以中国沿海常见的褐藻门海带为原料,依据药物高通量筛选理论,以抗肿瘤活性为目的,提取分离了海带中活性物质--海带多酚。
1、以单因素试验优化海带多酚的提取工艺,80%乙醇常温避光浸提海带粉末,12小时反复两次。氯仿脱色7小时,得到海带多酚粗提物。得率为24.25mg/kg海带。等体积乙酸乙酯进一步萃取,单因素试验优化萃取条件得到:pH值为3.4,萃取30分钟,两次。得率为10.3mg/kg海带。
2、比较四种主要的提取方法:浸提法、索氏回流法、微波法、超声波法。结果表明,微波法提取得率最高。比甲醇浸提法、索氏提取法高62.5%,比超声法高150.5%。通过4因素3水平正交试验优化了微波法提取海带多酚的条件,结果表明:PH=7,中火破碎,时间90s,料液比1:10为微波法提取海带多酚的最优条件。在该条件下得率为34.52mg/kg海带。
3、用葡聚糖凝胶柱G-25对海带多酚提纯物进一步分离纯化,以蒸馏水为流动相,流速为60mL/h洗脱。可以有效分离多酚提取物,未处理生海带提取物为3个组分,处理的熟海带提取物为2个组分。
5、采用MTT法测定海带提取液体外抗肿瘤活性。粗提液对HeLa细胞的IC_(50)为2.32μg·mL~(-1),使MCF-7和PC12细胞呈现出猝死现象,浓度达到6.91μg·mL~(-1)时可完全抑制三种肿瘤细胞的增殖;乙酸乙酯萃取物对HeLa细胞,MCF-7细胞和PC-12细胞表现为计量依赖型,IC_(50)分别是:0.211μg·mL~(-1)、0.174μg·mL~(-1)、0.158μg·mL~(-1)。当提取物浓度达到0.5μg·mL~(-1)时几乎没有存活细胞。凝胶柱层析得到未处理生海带组分一在浓度为1.45μg·mL~(-1)时抑制率达到72.41%,处理海带组分一在0.30μg/mL时抑制率达到90%以上,组分二浓度大于1.68μg·mL~(-1)时致死率达到90%以上。
Kelp is one common kind of brown alga along Dalian coast.They are abundant in bioactive constituents,which make them potential resources for advanced pharmaceutical utilization.In order to make the best use of kelp,the present reacher is focuse on the high throughput screening for the purpose of antitumor activities.The results are as follows:
1、Ethanol extraction method is optimised by single-factors test.The results showed that a high yeild of the crude phlorotannins of 24.25 mg·kg~(-1) was obtained under the conditions of pH 10,extracted twice with 80%ethanol-aqueous,then decloured by CHCl_3 by 7h.Then abstracted by ethyl acetate,the optimised condition determined by single-factors test is the equal volume ethyl acetate extract in pH 3.4 twice,30min once.The yeild is about 10.3 mg·kg~(-1).
2、Four methods including ethanol extraction,Soxhiet refluxing,ultrasound-assisted and microwave-assisted extractions are compared.Microwave-assisted extraction is proved to be the best method which yield is 52.5%higher than that of ethanol extraction or Soxhlet refluxing and higher than ultrasonic-assisted by 150.5%.The best conditions of microwave extraction optimized by orthogonal test are as follows:pH is 7,intermediate fire,90s,ratio of solid to liquor is 1:10.The yeild is 34.52 mg·kg~(-1).
4、The extract is further depurated by sephadex column chromatography G-25 which is eluted with deionized water.The flow rate is controlled at 60mL/h.Three groups can be seperated for untreated kelp and two groups for treated kelp.
5、The effect of crude extract liquor on three cancer cells(HeLa cells,MCF-7 cells, PC-12 cells) is examined by MTT assay.The IC_(50) of crude phlorotannins on HeLa cells is 2.32μg·mL~(-1) and MCF-7 cells or PC12 cells is suddenly killed.IC_(50) of refined phlorotannins on HeLa cells,MCF-7 cells and PC12 cell are 0.21μg·mL~(-1)、0.174μg·mL~(-1)、0.158μg·mL~(-1) respectively.The exhibition on tumor cells of group 1 of untreated kelp is 72.41%at 1.45μg·mL~(-1).The exhibition on tumor cells of group 1 of treated kelp is more than 90%at 0.30μg·mL~(-1) and 90%for the group 2 at 1.68μg·mL~(-1).
1、以单因素试验优化海带多酚的提取工艺,80%乙醇常温避光浸提海带粉末,12小时反复两次。氯仿脱色7小时,得到海带多酚粗提物。得率为24.25mg/kg海带。等体积乙酸乙酯进一步萃取,单因素试验优化萃取条件得到:pH值为3.4,萃取30分钟,两次。得率为10.3mg/kg海带。
2、比较四种主要的提取方法:浸提法、索氏回流法、微波法、超声波法。结果表明,微波法提取得率最高。比甲醇浸提法、索氏提取法高62.5%,比超声法高150.5%。通过4因素3水平正交试验优化了微波法提取海带多酚的条件,结果表明:PH=7,中火破碎,时间90s,料液比1:10为微波法提取海带多酚的最优条件。在该条件下得率为34.52mg/kg海带。
3、用葡聚糖凝胶柱G-25对海带多酚提纯物进一步分离纯化,以蒸馏水为流动相,流速为60mL/h洗脱。可以有效分离多酚提取物,未处理生海带提取物为3个组分,处理的熟海带提取物为2个组分。
5、采用MTT法测定海带提取液体外抗肿瘤活性。粗提液对HeLa细胞的IC_(50)为2.32μg·mL~(-1),使MCF-7和PC12细胞呈现出猝死现象,浓度达到6.91μg·mL~(-1)时可完全抑制三种肿瘤细胞的增殖;乙酸乙酯萃取物对HeLa细胞,MCF-7细胞和PC-12细胞表现为计量依赖型,IC_(50)分别是:0.211μg·mL~(-1)、0.174μg·mL~(-1)、0.158μg·mL~(-1)。当提取物浓度达到0.5μg·mL~(-1)时几乎没有存活细胞。凝胶柱层析得到未处理生海带组分一在浓度为1.45μg·mL~(-1)时抑制率达到72.41%,处理海带组分一在0.30μg/mL时抑制率达到90%以上,组分二浓度大于1.68μg·mL~(-1)时致死率达到90%以上。
Kelp is one common kind of brown alga along Dalian coast.They are abundant in bioactive constituents,which make them potential resources for advanced pharmaceutical utilization.In order to make the best use of kelp,the present reacher is focuse on the high throughput screening for the purpose of antitumor activities.The results are as follows:
1、Ethanol extraction method is optimised by single-factors test.The results showed that a high yeild of the crude phlorotannins of 24.25 mg·kg~(-1) was obtained under the conditions of pH 10,extracted twice with 80%ethanol-aqueous,then decloured by CHCl_3 by 7h.Then abstracted by ethyl acetate,the optimised condition determined by single-factors test is the equal volume ethyl acetate extract in pH 3.4 twice,30min once.The yeild is about 10.3 mg·kg~(-1).
2、Four methods including ethanol extraction,Soxhiet refluxing,ultrasound-assisted and microwave-assisted extractions are compared.Microwave-assisted extraction is proved to be the best method which yield is 52.5%higher than that of ethanol extraction or Soxhlet refluxing and higher than ultrasonic-assisted by 150.5%.The best conditions of microwave extraction optimized by orthogonal test are as follows:pH is 7,intermediate fire,90s,ratio of solid to liquor is 1:10.The yeild is 34.52 mg·kg~(-1).
4、The extract is further depurated by sephadex column chromatography G-25 which is eluted with deionized water.The flow rate is controlled at 60mL/h.Three groups can be seperated for untreated kelp and two groups for treated kelp.
5、The effect of crude extract liquor on three cancer cells(HeLa cells,MCF-7 cells, PC-12 cells) is examined by MTT assay.The IC_(50) of crude phlorotannins on HeLa cells is 2.32μg·mL~(-1) and MCF-7 cells or PC12 cells is suddenly killed.IC_(50) of refined phlorotannins on HeLa cells,MCF-7 cells and PC12 cell are 0.21μg·mL~(-1)、0.174μg·mL~(-1)、0.158μg·mL~(-1) respectively.The exhibition on tumor cells of group 1 of untreated kelp is 72.41%at 1.45μg·mL~(-1).The exhibition on tumor cells of group 1 of treated kelp is more than 90%at 0.30μg·mL~(-1) and 90%for the group 2 at 1.68μg·mL~(-1).
引文
[1]周杰,陈安国.海藻多糖的生物活性研究进展.饲料工业,2005,26(18):12-15.
[2]Nagasawa N,Mitomo H,Yoshii F,Kume T.Radiation-induced degradation of sodium alginate [J].Polymer Degradation and Stability.2000.69(3):279-285.
[3]杨志清.海藻酸钠在经纱上浆中的应用[J].现代纺织技术,2002,10(4):21-22.
[4]Pisani P,Bray F,Parkin DM.Estimates of the world-wide prevalence of cancer for 25 sites in the adult population[J].International Journal of Cancer,2002,97:72-81
[5]孙达旺.植物单宁化学.北京:中国林业出版社,1992
[6]石碧,狄莹.植物多酚.北京:科学出版社,2000.
[7]王长海.海洋生化工程概论[M].北京:化学工业出版社,2004:55.
[8]严小军,娄清香,吴真真.褐藻多酚连三羟基的测定[J].海洋科学,2000:24(12)
[9]魏玉西,郭道森,牛锡珍,等.褐藻中多酚化合物的研究进展[J].海洋科学,2002,26(10):18-20.
[10]石碧,狄莹著.植物多酚[M].北京:科学出版社,2000.
[11]Yan X.J.,Li X.C.,Zhou C.X.et al..Prevention of fish oil rancidity by phlorotannins from Sargassum kjellmanianum[J].Appl.Phycol.,1996,8:201-203.
[12]Yan X.J.,Li X.C.et al..Studies on extraction procedure and antioxidative activity of phlorotannins from Sargassum kjellmanianum.Chin.J.Oceanol.Limnol..1997,15(1):42-45.
[13]Yan X.J.,Fang G.M.et al.Studies on free radical scavenging activity in Chinese seaweeds.Chin.J.Oceanol.Limnol..1999,17(3):240-246.
[14]Koki Nagayama,Yoshitoshi Iwamura,Toshiyuki Shibata.Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome[J].Journal of Antimicrobial Chemotherapy.2002,50:889-893.
[15]师然新,徐祖洪.青岛沿海九种海藻的类脂及酚类抗菌活性的研究.中国海洋药物.1997,4:16-19.
[16]曾呈奎,王素娟,刘思俭.海藻栽培学.上海:上海科学技术出版社,1985.
[17]Glombitza K.W.,Vogels H.P..Antibiotics from algae.ⅩⅩⅩⅤ.Phlorotannins from Ecklonia maxima[J].Planta Medica,1985,51:308-312
[18]林超,于曙光,郭道森等.鼠尾藻中褐藻多酚化合物的抑菌活性研究.海洋科学。2006,30(3):94-97.
[19]Hudson J.B.,KimJ.H.,Lee M.K.et al..Antiviral compounds in extracts of Korean seaweeds:Evidence for multiple activities[J]..Appl.Phycol.,1999,10:427-434
[20]S.Ivaneheva,N.Manolova,J.Serkedjieva.Polyphenols from Bulgarian dedieinal plants with antiinfeetions aetivity.Plant polyphenols,R.Hemingway,P.Lakes(edu.),Plenum Press,NewYork,1992:621-638.
[21]Boettcher A.A.,Targett N.M.Role of polyphenolic molecular size in reduction of asssimilation efficiency in Xiphister mucosus.Ecology.1993,4:891-903.
[22]Toth G.,Pavia H..Lack of phlorotannin induction in the brown seaweed Ascophyllum nodosum in response to increased copper concentrations.MEPS.2000,192:119-126.
[23]Pavia H.,Cervin G.,Lindgren A.et al..The effect of UV2B radiation and herbivory on the production of phlorotannins in the brown seaweed Ascophyllum nodosum.MEPS.1997,157:139-146.
[24]Sundba ck K.,Odermark S.et al..Effects of enhanced UV2B radiation on a marine benthic diatom mat.Mar.Biol..1997,128:171-179.
[25]黄健、唐学玺、段德麟等.不同光照条件下海带体内各种化合物的含量及光合作用和呼吸作用的变化.海洋科学.2002,26(4):55-58.
[26]Tokita F.,Ishikawa M.et al..Deodorizing activity of some plant extracts against methyl mercaptan.Nippon Nogeikagaku Kaishi.1984,58:585-589.
[27]Kita N.,Fujimoto K.et al..Screening test for deodorizing substances from marine algae and bidentification of phlorotannins as the effective ingredients in Eisenia bicyclis[J].Appl.Phycol.1990,2:155-162
[28]Koki Nagayama,Toshiyuki Shibata,Ken Fujimoto.Algicidal effect of phlorotannins from the brown alga Ecklonia kurome on red tide microalgae[J].Aquaculture,2003,218:601-611
[29]Y.Wang,Z.Xu,S.J.Bach.Effects of phlorotannins from Ascophyllum nodosum(brown seaweed) on in vitro ruminal digestion of mixed forage or barley grain[J].Animal Feed Science and Technology,2007,1-27
[30]Koki Nagayama,Yoshitoshi Iwamura,Toshiyuki Shibata.Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome[J].Journal of Antimicrobial Chemotherapy.2002,50:889-893.
[31]Moon-Moo Kim,Quang Van Ta,Eresha Mendis.Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity[J].Life Sciences,2006,79:1436-1443
[32]范晓,严小军.海藻化学研究进展.海洋科学.1996,2:24-25.
[33]凌广庭.食品添加剂手册(第二版).北京:化学工业出版社,1997.
[34]韩丙军,彭黎旭.植物多酚提取技术及其开发应用现状.华南热带农业大学学报.2005,11(1):21-26.
[35]宋冠群,林金明.茶多酚分析方法研究与最新进展.世界科技研究与发展.2004,26(1):41-46.
[36]朱玉,张书胜,张西林等.薄层色谱分析葵花仁粕中的绿原酸.色谱.2001,19(1):82-84.
[37]王宝华,高增平,江佩芬.薄层扫描法测定杜仲茶中氯原酸的含量[J].北京中医药大学学报.2000,23(4):46-47.
[38]刘海兴,杨更亮,赵敬相,等.毛细管胶束电动色谱法测定夏枯草中两种有效成分.分析化学.2000,28(10):1275-1277.
[39]苗建中,邹汉法,袁倚盛,等.毛细管电泳法分离丹参水溶性有效成分--丹参素、原儿茶醛和原儿茶酸.中草药,2000,31(S):337-340.
[40]陈绩光,张敏生,莫金垣,等.毛细管电泳测定当归饮片中阿魏酸含量.中草药.2000,31(7):506-508.
[41]娄清香,严小军.褐藻多酚的毒理学研究.海洋科学.2000,24(9):53-55.
[42]杨世军,张会香.荔枝茶多酚保健饮料的研制[J].食品科技,2005,10:62-64.
[43]胡美忠,刘芸,郁建平等.苦丁茶茶多酚保健口香糖的研制[J].食品科学,2007,28(7):587-588
[44]谢异萍.天然抗氧化剂茶多酚的保健作用[J].中国临床保健杂志,2005,8(6):573-576
[45]杨官品,李晓捷,石媛螈等.海带属褐藻生物学研究成就与进展[J].中国海洋大学学报,2005,35(4):564-570
[46]吕惠敏,张侃.海藻的利用与开发.食品科技,1998,6:29-30
[47]张翼,李晓明,王斌贵.海藻生物活性物质研究的回顾与展望.科技前沿与学术评论世:世界科技研究与发展.2005:56-63
[48]严小军.中国常见褐藻的多酚含量测定[J].海洋科学集刊,1996.10:61-65
[49]Nakamura T,Nagayama K,Uchida K,et al.Antioxidant activity of phlorotannins isolated from the brown alga Eisenla bycyclis[J],fisheries Science,1996,62(6):923-926
[50]钱振明.海洋底栖硅藻生长条件及其理化成分的研究:(硕士学位论文).大连:大连理工大学,2006.8.
[51]王长海,孙颖颖.营养盐对球等鞭金藻生长及多糖等生化成分含量的影响.食品与发酵报告,2005,31(11):13-17.
[52]仇燕.菜芙蓉花中总黄酮微波提取的研究[J].中药材,2006,29(4):387-389.
[53]马成良,张海军,李素平.现代试验设计优化方法及应用.郑州:郑州大学出版社,2007.
[54]李雪梅,陈育如,骆跃军等.烟草废料中多酚类物质的浸提条件研究[J].高校化学工程学报,2006,20(2):315-318
[55]艾志录,郭娟,王育红等.微波辅助提取苹果渣中苹果多酚的工艺研究[J].农业工程学报,2006,22(6):188-191
[56]张力,孙秀利,王松青,等.微波法提取葡萄籽中原花青素的工艺研究[J].中草药Chinese Traditional and Herbal Drugs,2007,38(12):1808-1811.
[57]付为琳,杨立刚,孙桂菊.微波萃取在菊花黄酮提取工艺中的应用研究[J].食品研究与开发.2008.29(1):1-3.
[58]Yan XJ,Fang GM,et al.Studies on free radical scavening activity in Chinese seaweeds Part ⅠScreening results[J].Oceanol Limnol,1999,17(3):240-246
[59]魏玉西,于曙光.两种褐藻乙醇提取物的抗氧化活性研究[J].海洋科学Marine Sciences.2002,26(9):49-51.
[60]谭晓华,周殿元.茶多酚抗癌机理新进展[J].肿瘤,2000,20(2):138-140
[61]Liao S,Umekita Y,Guo J,et al.Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate[J].Cancer Letter,1995,96:239
[62]Okabe S,Suganuma M,Hayashi M,et al.Mechanisms of growth inhibition of human lung cancer cell line "PC-9" by tea polyphenols[J].Jpn J Cancer Res,1997,88:639
[63]Achiwa Y,Hibasami H,Katsuzaki H,et al.Inhibitory effects of persimmon(Diopyros kaki) extract and related polyphenol compounds on growth of human lymphoid leukemia cells[J].Biosci Biotechnol Biochem,1997,61:1099
[64]Sazuka M,Imazawa H,Shoji Y,et al.Inhibition of collagenases from mouse lung carcinoma cells by green tea catechins and black tea theaflavins[J].Biosci Biotechnol Biochem,1997,61:1504
[65]Naasani I,Seimiya H.Tsuruo T,et al.Telomerase inhibition,telomere shortening,and senescence of cancer cells by tea catechins[J].Biochem Biophys Res Commun,1998,249:391
[66]徐秀丽,范晓,宋福行.中国经济海藻提取物生物活性[J].海洋与湖沼,2004,35(1):55
[67]林超.两种褐藻中多酚化合物的生理活性研究[D].青岛:青岛大学化工学院,2005
[68]徐年军,范晓,韩丽君等.山东沿海海藻抗肿瘤活性的筛选[J].海洋与湖沼,2001,32(4):408
[69]Harada H.Selective Cytotoxicity of Marine Algae Extracts of Several Human Leukemic Cell Lines[J].Cyto-Technology,1997,25(1-3):213.
[70]魏玉西,孙峋,王长云等.鼠尾藻多酚的抗肿瘤活性研究[J].中草药Chinese Traditional and Herbal Drugs,2008,39(1):93-95.
[71]Vuatazl,Branden beraerh.Separation of the tea leaf polyphenols by cellulose column chromatography[J].Chromatography,1959(2):173-187.
[72]Amaro wiczr,Shahi dif.A rapid chromatographic method for separation of individual catechins from green tea[J].FoodResearch International,1996,29(1):71-76.
[73]Pcinelli A,et al.Analysis of polyphenols in apple products[J].Z Lebensm Unters A.1997,204:48-51
[74]Yinrong Lu,Yeap Foo.Identication and quantification of major polyphenols in apple pomace[J].Food Chemistry,1997,59(2):187-194
[75]杜冠华.高通量药物筛选.北京:化学工业出版社.2002.
[2]Nagasawa N,Mitomo H,Yoshii F,Kume T.Radiation-induced degradation of sodium alginate [J].Polymer Degradation and Stability.2000.69(3):279-285.
[3]杨志清.海藻酸钠在经纱上浆中的应用[J].现代纺织技术,2002,10(4):21-22.
[4]Pisani P,Bray F,Parkin DM.Estimates of the world-wide prevalence of cancer for 25 sites in the adult population[J].International Journal of Cancer,2002,97:72-81
[5]孙达旺.植物单宁化学.北京:中国林业出版社,1992
[6]石碧,狄莹.植物多酚.北京:科学出版社,2000.
[7]王长海.海洋生化工程概论[M].北京:化学工业出版社,2004:55.
[8]严小军,娄清香,吴真真.褐藻多酚连三羟基的测定[J].海洋科学,2000:24(12)
[9]魏玉西,郭道森,牛锡珍,等.褐藻中多酚化合物的研究进展[J].海洋科学,2002,26(10):18-20.
[10]石碧,狄莹著.植物多酚[M].北京:科学出版社,2000.
[11]Yan X.J.,Li X.C.,Zhou C.X.et al..Prevention of fish oil rancidity by phlorotannins from Sargassum kjellmanianum[J].Appl.Phycol.,1996,8:201-203.
[12]Yan X.J.,Li X.C.et al..Studies on extraction procedure and antioxidative activity of phlorotannins from Sargassum kjellmanianum.Chin.J.Oceanol.Limnol..1997,15(1):42-45.
[13]Yan X.J.,Fang G.M.et al.Studies on free radical scavenging activity in Chinese seaweeds.Chin.J.Oceanol.Limnol..1999,17(3):240-246.
[14]Koki Nagayama,Yoshitoshi Iwamura,Toshiyuki Shibata.Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome[J].Journal of Antimicrobial Chemotherapy.2002,50:889-893.
[15]师然新,徐祖洪.青岛沿海九种海藻的类脂及酚类抗菌活性的研究.中国海洋药物.1997,4:16-19.
[16]曾呈奎,王素娟,刘思俭.海藻栽培学.上海:上海科学技术出版社,1985.
[17]Glombitza K.W.,Vogels H.P..Antibiotics from algae.ⅩⅩⅩⅤ.Phlorotannins from Ecklonia maxima[J].Planta Medica,1985,51:308-312
[18]林超,于曙光,郭道森等.鼠尾藻中褐藻多酚化合物的抑菌活性研究.海洋科学。2006,30(3):94-97.
[19]Hudson J.B.,KimJ.H.,Lee M.K.et al..Antiviral compounds in extracts of Korean seaweeds:Evidence for multiple activities[J]..Appl.Phycol.,1999,10:427-434
[20]S.Ivaneheva,N.Manolova,J.Serkedjieva.Polyphenols from Bulgarian dedieinal plants with antiinfeetions aetivity.Plant polyphenols,R.Hemingway,P.Lakes(edu.),Plenum Press,NewYork,1992:621-638.
[21]Boettcher A.A.,Targett N.M.Role of polyphenolic molecular size in reduction of asssimilation efficiency in Xiphister mucosus.Ecology.1993,4:891-903.
[22]Toth G.,Pavia H..Lack of phlorotannin induction in the brown seaweed Ascophyllum nodosum in response to increased copper concentrations.MEPS.2000,192:119-126.
[23]Pavia H.,Cervin G.,Lindgren A.et al..The effect of UV2B radiation and herbivory on the production of phlorotannins in the brown seaweed Ascophyllum nodosum.MEPS.1997,157:139-146.
[24]Sundba ck K.,Odermark S.et al..Effects of enhanced UV2B radiation on a marine benthic diatom mat.Mar.Biol..1997,128:171-179.
[25]黄健、唐学玺、段德麟等.不同光照条件下海带体内各种化合物的含量及光合作用和呼吸作用的变化.海洋科学.2002,26(4):55-58.
[26]Tokita F.,Ishikawa M.et al..Deodorizing activity of some plant extracts against methyl mercaptan.Nippon Nogeikagaku Kaishi.1984,58:585-589.
[27]Kita N.,Fujimoto K.et al..Screening test for deodorizing substances from marine algae and bidentification of phlorotannins as the effective ingredients in Eisenia bicyclis[J].Appl.Phycol.1990,2:155-162
[28]Koki Nagayama,Toshiyuki Shibata,Ken Fujimoto.Algicidal effect of phlorotannins from the brown alga Ecklonia kurome on red tide microalgae[J].Aquaculture,2003,218:601-611
[29]Y.Wang,Z.Xu,S.J.Bach.Effects of phlorotannins from Ascophyllum nodosum(brown seaweed) on in vitro ruminal digestion of mixed forage or barley grain[J].Animal Feed Science and Technology,2007,1-27
[30]Koki Nagayama,Yoshitoshi Iwamura,Toshiyuki Shibata.Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome[J].Journal of Antimicrobial Chemotherapy.2002,50:889-893.
[31]Moon-Moo Kim,Quang Van Ta,Eresha Mendis.Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity[J].Life Sciences,2006,79:1436-1443
[32]范晓,严小军.海藻化学研究进展.海洋科学.1996,2:24-25.
[33]凌广庭.食品添加剂手册(第二版).北京:化学工业出版社,1997.
[34]韩丙军,彭黎旭.植物多酚提取技术及其开发应用现状.华南热带农业大学学报.2005,11(1):21-26.
[35]宋冠群,林金明.茶多酚分析方法研究与最新进展.世界科技研究与发展.2004,26(1):41-46.
[36]朱玉,张书胜,张西林等.薄层色谱分析葵花仁粕中的绿原酸.色谱.2001,19(1):82-84.
[37]王宝华,高增平,江佩芬.薄层扫描法测定杜仲茶中氯原酸的含量[J].北京中医药大学学报.2000,23(4):46-47.
[38]刘海兴,杨更亮,赵敬相,等.毛细管胶束电动色谱法测定夏枯草中两种有效成分.分析化学.2000,28(10):1275-1277.
[39]苗建中,邹汉法,袁倚盛,等.毛细管电泳法分离丹参水溶性有效成分--丹参素、原儿茶醛和原儿茶酸.中草药,2000,31(S):337-340.
[40]陈绩光,张敏生,莫金垣,等.毛细管电泳测定当归饮片中阿魏酸含量.中草药.2000,31(7):506-508.
[41]娄清香,严小军.褐藻多酚的毒理学研究.海洋科学.2000,24(9):53-55.
[42]杨世军,张会香.荔枝茶多酚保健饮料的研制[J].食品科技,2005,10:62-64.
[43]胡美忠,刘芸,郁建平等.苦丁茶茶多酚保健口香糖的研制[J].食品科学,2007,28(7):587-588
[44]谢异萍.天然抗氧化剂茶多酚的保健作用[J].中国临床保健杂志,2005,8(6):573-576
[45]杨官品,李晓捷,石媛螈等.海带属褐藻生物学研究成就与进展[J].中国海洋大学学报,2005,35(4):564-570
[46]吕惠敏,张侃.海藻的利用与开发.食品科技,1998,6:29-30
[47]张翼,李晓明,王斌贵.海藻生物活性物质研究的回顾与展望.科技前沿与学术评论世:世界科技研究与发展.2005:56-63
[48]严小军.中国常见褐藻的多酚含量测定[J].海洋科学集刊,1996.10:61-65
[49]Nakamura T,Nagayama K,Uchida K,et al.Antioxidant activity of phlorotannins isolated from the brown alga Eisenla bycyclis[J],fisheries Science,1996,62(6):923-926
[50]钱振明.海洋底栖硅藻生长条件及其理化成分的研究:(硕士学位论文).大连:大连理工大学,2006.8.
[51]王长海,孙颖颖.营养盐对球等鞭金藻生长及多糖等生化成分含量的影响.食品与发酵报告,2005,31(11):13-17.
[52]仇燕.菜芙蓉花中总黄酮微波提取的研究[J].中药材,2006,29(4):387-389.
[53]马成良,张海军,李素平.现代试验设计优化方法及应用.郑州:郑州大学出版社,2007.
[54]李雪梅,陈育如,骆跃军等.烟草废料中多酚类物质的浸提条件研究[J].高校化学工程学报,2006,20(2):315-318
[55]艾志录,郭娟,王育红等.微波辅助提取苹果渣中苹果多酚的工艺研究[J].农业工程学报,2006,22(6):188-191
[56]张力,孙秀利,王松青,等.微波法提取葡萄籽中原花青素的工艺研究[J].中草药Chinese Traditional and Herbal Drugs,2007,38(12):1808-1811.
[57]付为琳,杨立刚,孙桂菊.微波萃取在菊花黄酮提取工艺中的应用研究[J].食品研究与开发.2008.29(1):1-3.
[58]Yan XJ,Fang GM,et al.Studies on free radical scavening activity in Chinese seaweeds Part ⅠScreening results[J].Oceanol Limnol,1999,17(3):240-246
[59]魏玉西,于曙光.两种褐藻乙醇提取物的抗氧化活性研究[J].海洋科学Marine Sciences.2002,26(9):49-51.
[60]谭晓华,周殿元.茶多酚抗癌机理新进展[J].肿瘤,2000,20(2):138-140
[61]Liao S,Umekita Y,Guo J,et al.Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate[J].Cancer Letter,1995,96:239
[62]Okabe S,Suganuma M,Hayashi M,et al.Mechanisms of growth inhibition of human lung cancer cell line "PC-9" by tea polyphenols[J].Jpn J Cancer Res,1997,88:639
[63]Achiwa Y,Hibasami H,Katsuzaki H,et al.Inhibitory effects of persimmon(Diopyros kaki) extract and related polyphenol compounds on growth of human lymphoid leukemia cells[J].Biosci Biotechnol Biochem,1997,61:1099
[64]Sazuka M,Imazawa H,Shoji Y,et al.Inhibition of collagenases from mouse lung carcinoma cells by green tea catechins and black tea theaflavins[J].Biosci Biotechnol Biochem,1997,61:1504
[65]Naasani I,Seimiya H.Tsuruo T,et al.Telomerase inhibition,telomere shortening,and senescence of cancer cells by tea catechins[J].Biochem Biophys Res Commun,1998,249:391
[66]徐秀丽,范晓,宋福行.中国经济海藻提取物生物活性[J].海洋与湖沼,2004,35(1):55
[67]林超.两种褐藻中多酚化合物的生理活性研究[D].青岛:青岛大学化工学院,2005
[68]徐年军,范晓,韩丽君等.山东沿海海藻抗肿瘤活性的筛选[J].海洋与湖沼,2001,32(4):408
[69]Harada H.Selective Cytotoxicity of Marine Algae Extracts of Several Human Leukemic Cell Lines[J].Cyto-Technology,1997,25(1-3):213.
[70]魏玉西,孙峋,王长云等.鼠尾藻多酚的抗肿瘤活性研究[J].中草药Chinese Traditional and Herbal Drugs,2008,39(1):93-95.
[71]Vuatazl,Branden beraerh.Separation of the tea leaf polyphenols by cellulose column chromatography[J].Chromatography,1959(2):173-187.
[72]Amaro wiczr,Shahi dif.A rapid chromatographic method for separation of individual catechins from green tea[J].FoodResearch International,1996,29(1):71-76.
[73]Pcinelli A,et al.Analysis of polyphenols in apple products[J].Z Lebensm Unters A.1997,204:48-51
[74]Yinrong Lu,Yeap Foo.Identication and quantification of major polyphenols in apple pomace[J].Food Chemistry,1997,59(2):187-194
[75]杜冠华.高通量药物筛选.北京:化学工业出版社.2002.