海星皂甙的分离纯化及其对果蝇生长发育的影响
|
摘要
海星因为以海参、鲍鱼等海洋珍贵软体动物为食一直以来被视为影响海洋养殖业的有害动物,近些年来,国内外关于海星中活性成分的提取及其活性研究越来越多,尤其是海星皂甙的研究,但是关于其抑制昆虫生长发育的研究一直以来都是个空白。我国沿海的海星资源丰富,但一直以来的研究都无法使海星被人们大批量的利用,研究一种海星的生物活性使其可以大量的被开发利用应该引起人们更多的关注。本文主要研究了以下几个方面。
首先,将整体冻干的罗氏海盘车粉碎机粉碎后用改进的甲醇提取法进行浸提,先用甲醇浸提,再用水饱和的正丁醇进行提取,最终得到海星总皂甙,利用香草醛-硫酸检测法及其溶血活性对提取出的总皂甙进行初步鉴定。然后用甲醇复溶提取物后中压柱层析分离,最终得到了10个组分,其中F-2所得到的峰面积最大,对这十个组分进行高效液相色谱的分析,F-2组分的峰型最好,对F-2进行进一步分离纯化后得到较纯的一种化合物F-2B。
其次,对化合物F-2利用高效液相色谱-质谱联用等方法进行结构解析。
最后,对提取出的海星皂甙的活性进行研究。研究海星皂甙对果蝇生长发育的影响。用提取出的100μg/mL、50μg/mL、20μg/mL、10μg/mL的海星皂甙溶液以及对照组、100μg/mL的毛地黄皂甙溶液组、DMSO组配制果蝇培养基对同种条件果蝇进行饲养,研究其子代果蝇的蛹化和羽化情况。实验证明,与对照组相比,海星皂甙对果蝇子代的蛹化和羽化都有一定的抑制作用,其抑制活性随培养基中皂甙的浓度增加而增大,而DMSO组则没有抑制活性。其中100μg/ml在第七天的蛹化最大抑制率为48.9%,在第九天的羽化抑制率为68.75%,显示出比较高的抑制活性,而毛地黄皂甙的第七天蛹化抑制率为21.2%,第九天羽化抑制率为31.25%。
另外,利用MTT法检测500μg/mL、350μg/mL、250μg/mL、125μg/mL的海星总皂甙和毛地黄皂甙标准品对HepG2肝癌细胞的增殖抑制作用,结果表明,海星总皂甙和毛地黄皂甙对细胞都有比较高的抑制活性,并且随着标准品和样品浓度的增加,其抑制活性也增加,在终浓度500μg/mL浓度下对细胞有较大的抑制能力,抑制率分别为:49.89%和60.48%,而丝裂霉素的抑制率为63.55%。
本论文中提取出的海星总皂甙对果蝇的蛹化和羽化都有比较高的抑制活性,若在此基础上,进一步研究其抑制机理,并对其化学及其生理上进行研究,有望开发出一种新型的生物活性物质,可作为一种潜在的天然昆虫杀虫剂,同时,根据近些年的研究成果,亦可能开发成为一种新型的天然药物。这不仅可以解决昆虫杀虫剂抗性的问题,防止化学杀虫剂对环境的污染,更可以使海洋有害动物海星变害为宝。
Starfish has always been regarded as the pest affecting marine aquaculture because it eat sea cucumbers, abalone and other marine precious mollusks. In recent years, there are more and more researches on the extraction and activity of active ingredients from starfish , particularly on asterosaponins. But studies on its inhibition of insect growth has always been a blank. China is rich on the store of starfish in the east coast. Study on the biological activity of starfish to find a way to make a lot of utilization of starfish should be cause for more attention. This paper studies these following aspects.
Firstly, the lyophilized starfish is crushed by the mill. We use the improved extraction method and get the crud asterosaponin extracted with methanol and butanol saturated with water. Identify it use the vanillin-sulfuric acid detection and the hemolytic activity of the crude asterosaponin.Then redissove it with methanol to purify it by the medium pressure column chromatography and 10 fractions obtained at the end. Analysing these ten conmonents by HPLC, the F-2 peak area is the largest one. Through further purification of F-2,we obtained a pure compound F-2B.
Secondly, the structure of compounds F-2 was elucidated by LC-MS and other methods.
Finally, the activity of extracted astrosaponins impact on growth and development of drosophila was stedied. Feeding the fruit flies with the extracted at the dosage of 100μg/mL, 50μg/mL, 20μg/mL, 10μg/mL respectively, with 100μg/mL of digitonin, DMSO as control on the same preparation conditions to study the situation of drosophila pupae and eclosion. Compared with the DMSO asterosaponins have a certain inhibitory effect on the pupation of drosophila and the eclosion, the inhibitory activity increase with the concentration of asterosaponin increasing. The maximum inhibition rate of the group of 100μg/mL at the seventh day of the pupation is 48.9%, at the ninth day the eclosion inhibition rate is 68.75%, showing relatively high inhibitory activity compared with digitonin inhibitory rate of the seventh day of pupation is 21.2% and the ninth day of eclosion 31.25%.
In addition, using MTT method to find out the inhibition of 500μg/ml, 350μg/ml, 250μg/ml, 125μg/ml of asterosaponins and digitonin on the proliferation of HepG2 liver cancer cell, the results showed that the asterosaponins and digitonin has a relatively high inhibitory activity on the cell, and with the sample and standard concentration increases, the inhibitory rate also increased. In a final concentration of 500μg/mL have greater inhibition of cell capacity, the inhibitory rates were 49.89% and 60.48%, while the mitomycin C inhibition rate was 63.55%.
In this thesis, the saponin extracted from starfish have a high inhibition activity on the fruit fly pupation and eclosion, on this basis, further study of its inhibition mechanism, and its chemical and physiological research is expected to develop a new types of biologically active substances can be used as a potential natural insect pesticides. According to recent research, it is also be developed as a new type of natural medicine. This can not only solve the problem of insect pesticides resistance, pollution of the environment by chemical pesticides, but also can change the marine pest starfish to treasure.
首先,将整体冻干的罗氏海盘车粉碎机粉碎后用改进的甲醇提取法进行浸提,先用甲醇浸提,再用水饱和的正丁醇进行提取,最终得到海星总皂甙,利用香草醛-硫酸检测法及其溶血活性对提取出的总皂甙进行初步鉴定。然后用甲醇复溶提取物后中压柱层析分离,最终得到了10个组分,其中F-2所得到的峰面积最大,对这十个组分进行高效液相色谱的分析,F-2组分的峰型最好,对F-2进行进一步分离纯化后得到较纯的一种化合物F-2B。
其次,对化合物F-2利用高效液相色谱-质谱联用等方法进行结构解析。
最后,对提取出的海星皂甙的活性进行研究。研究海星皂甙对果蝇生长发育的影响。用提取出的100μg/mL、50μg/mL、20μg/mL、10μg/mL的海星皂甙溶液以及对照组、100μg/mL的毛地黄皂甙溶液组、DMSO组配制果蝇培养基对同种条件果蝇进行饲养,研究其子代果蝇的蛹化和羽化情况。实验证明,与对照组相比,海星皂甙对果蝇子代的蛹化和羽化都有一定的抑制作用,其抑制活性随培养基中皂甙的浓度增加而增大,而DMSO组则没有抑制活性。其中100μg/ml在第七天的蛹化最大抑制率为48.9%,在第九天的羽化抑制率为68.75%,显示出比较高的抑制活性,而毛地黄皂甙的第七天蛹化抑制率为21.2%,第九天羽化抑制率为31.25%。
另外,利用MTT法检测500μg/mL、350μg/mL、250μg/mL、125μg/mL的海星总皂甙和毛地黄皂甙标准品对HepG2肝癌细胞的增殖抑制作用,结果表明,海星总皂甙和毛地黄皂甙对细胞都有比较高的抑制活性,并且随着标准品和样品浓度的增加,其抑制活性也增加,在终浓度500μg/mL浓度下对细胞有较大的抑制能力,抑制率分别为:49.89%和60.48%,而丝裂霉素的抑制率为63.55%。
本论文中提取出的海星总皂甙对果蝇的蛹化和羽化都有比较高的抑制活性,若在此基础上,进一步研究其抑制机理,并对其化学及其生理上进行研究,有望开发出一种新型的生物活性物质,可作为一种潜在的天然昆虫杀虫剂,同时,根据近些年的研究成果,亦可能开发成为一种新型的天然药物。这不仅可以解决昆虫杀虫剂抗性的问题,防止化学杀虫剂对环境的污染,更可以使海洋有害动物海星变害为宝。
Starfish has always been regarded as the pest affecting marine aquaculture because it eat sea cucumbers, abalone and other marine precious mollusks. In recent years, there are more and more researches on the extraction and activity of active ingredients from starfish , particularly on asterosaponins. But studies on its inhibition of insect growth has always been a blank. China is rich on the store of starfish in the east coast. Study on the biological activity of starfish to find a way to make a lot of utilization of starfish should be cause for more attention. This paper studies these following aspects.
Firstly, the lyophilized starfish is crushed by the mill. We use the improved extraction method and get the crud asterosaponin extracted with methanol and butanol saturated with water. Identify it use the vanillin-sulfuric acid detection and the hemolytic activity of the crude asterosaponin.Then redissove it with methanol to purify it by the medium pressure column chromatography and 10 fractions obtained at the end. Analysing these ten conmonents by HPLC, the F-2 peak area is the largest one. Through further purification of F-2,we obtained a pure compound F-2B.
Secondly, the structure of compounds F-2 was elucidated by LC-MS and other methods.
Finally, the activity of extracted astrosaponins impact on growth and development of drosophila was stedied. Feeding the fruit flies with the extracted at the dosage of 100μg/mL, 50μg/mL, 20μg/mL, 10μg/mL respectively, with 100μg/mL of digitonin, DMSO as control on the same preparation conditions to study the situation of drosophila pupae and eclosion. Compared with the DMSO asterosaponins have a certain inhibitory effect on the pupation of drosophila and the eclosion, the inhibitory activity increase with the concentration of asterosaponin increasing. The maximum inhibition rate of the group of 100μg/mL at the seventh day of the pupation is 48.9%, at the ninth day the eclosion inhibition rate is 68.75%, showing relatively high inhibitory activity compared with digitonin inhibitory rate of the seventh day of pupation is 21.2% and the ninth day of eclosion 31.25%.
In addition, using MTT method to find out the inhibition of 500μg/ml, 350μg/ml, 250μg/ml, 125μg/ml of asterosaponins and digitonin on the proliferation of HepG2 liver cancer cell, the results showed that the asterosaponins and digitonin has a relatively high inhibitory activity on the cell, and with the sample and standard concentration increases, the inhibitory rate also increased. In a final concentration of 500μg/mL have greater inhibition of cell capacity, the inhibitory rates were 49.89% and 60.48%, while the mitomycin C inhibition rate was 63.55%.
In this thesis, the saponin extracted from starfish have a high inhibition activity on the fruit fly pupation and eclosion, on this basis, further study of its inhibition mechanism, and its chemical and physiological research is expected to develop a new types of biologically active substances can be used as a potential natural insect pesticides. According to recent research, it is also be developed as a new type of natural medicine. This can not only solve the problem of insect pesticides resistance, pollution of the environment by chemical pesticides, but also can change the marine pest starfish to treasure.
引文
[1]张立新.山东沿海几种海洋无脊椎动物生物活性物质的研究[D].中国科学院海洋研究所,2003.
[2]王伟红.赤海星中生物活性化学成分的研究[D].沈阳:沈阳药科大学, 2003.
[3]王征,董平原,张天民,等.海洋抗肿瘤药物研究开发中的主要问题[J].食品与药品. 2006, 8(4A): 1-4.
[4]刘春亮,焦吉祥.抗肿瘤海洋生物的研究进展[J].安徽医药. 2009, 13(10): 1277-1279.
[5]汪开治(译).海洋生物药研发现状综述[J].生物技术通报. 2006(4): 133-138.
[6]董黎明,陈思迪,刘仲娜.海星活性成分研究进展[J].科技信息. 2008(25).
[7]汤海峰,易杨华,张淑瑜,等.海星皂苷的研究进展[J].中国海洋药物. 2004, 23(6): 48-57.
[8]周学家,刘信艺.山东近海海星种类及分布[J].齐鲁渔业. 1996, 13(1): 41-43.
[9]王伟红,李发美.海星化学成分及其活性的研究进展[J].中国海洋药物. 2002, 21(5): 46-50.
[10] Yadavalli S, Nadimpalli S K. Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate Asterias rubens (Echinodermate): biochemical and functional characterization of MPR 46 protein[J]. Glycoconj J. 2008, 25(9): 889-901.
[11]郭承华.海燕皂苷及其它活性物质的制备及其生物功能研究[D].中国海洋大学, 2005.
[12]吴鹏.海星化学成分及其生物活性的研究[D].吉林大学, 2006.
[13]李军,吴红梅,蒋碧蓉,等.海星多糖的分离纯化及鉴定[J].食品工业科技. 2010, 31(2): 122-125.
[14] Nam K S, Shon Y H. Chemopreventive effects of polysaccharides extract from Asterina pectinifera on HT-29 human colon adenocarcinoma cells[J]. BMB Rep. 2009, 42(5): 277-280.
[15]潘广昌,郭承华,刘传琳,等.砂海星酸性粘多糖的分离纯化[J].烟台大学学报:自然科学与工程版. 2007, 20(2): 108-111.
[16]周燕霞,赵丽华,邢翔,等.多棘海盘车多糖的提取及体外抑菌活性初探[J].中国海洋药物. 2009(3): 58-60.
[17]骆传环,黄荣清,等.海星多糖的分子量测定[J].军事医学科学院院刊. 2002, 26(2): 157-158.
[18]李军,蒋碧蓉,吴红梅,等.海星多糖的提取分离与抗氧化活性研究[J].广东化工. 2010(2): 188-189.
[19] Kannappan R, Ando M, Furuhata K, et al. Photoaffinity labeling of sialidase with a biotin-conjugated phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid[J]. Biol Pharm Bull. 2008, 31(3): 352-356.
[20] Mita M, Yoshikuni M, Ohno K, et al. A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera[J]. Proc Natl Acad Sci U S A. 2009, 106(23): 9507-9512.
[21] Harun-Or-Rashid M, Matsuzawa T, Satoh Y, et al. Purification and characterization of alpha-N-acetylgalactosaminidases I and II from the starfish Asterina amurensis[J]. Biosci Biotechnol Biochem. 2010, 74(2): 256-261.
[22]翟宗德,张虹,师彦平,等.用半制备高效液相色谱法制备少量的阿苯达唑亚砜单一对映体[J].分析化学. 2005, 33(1): 109-112.
[23]魏有良,陈志兴.土贝母皂苷原料及其注射液中土贝母总皂苷的含量测定[J].中国现代应用药学. 1999, 16(1): 41-43.
[24] Burt V T, Bee E, Pennock J F. The Formation of Menaquinone4 (Vitamin K) and its Oxide in some Marine Invertebrates[J]. Biochem.J. 1977, 162: 297-302.
[25] Smith A G, Goad L J. Sterol biosynthesis in the echinoderm Asterias rubens[J]. Biochem J. 1975, 146(1): 25-33.
[26] Smith A G, Goad L J. The Conversion of Cholest-5-en-3p-ol into Cholest-7-en-3p-ol by the Echinoderms Asterias rubens and Solasterpapposus[J]. Bolchem.J. 1975, 146: 35-40.
[27] Yasumoto T, Hashimoto Y. Properties of Asterosaponin B Isolated from a Starfish, Asterias amurensis[J]. Agr. Biol. Chem. 1967, 31(3): 368-372.
[28] Francis G, Kerem Z, Makkar H P, et al. The biological action of saponins in animal systems: a review[J]. Br J Nutr. 2002, 88(6): 587-605.
[29] Cheng G, Zhang X, Tang H F, et al. Asterosaponin 1, a cytostatic compound from the starfish Culcita novaeguineae, functions by inducing apoptosis in human glioblastoma U87MG cells[J]. J Neurooncol. 2006, 79(3): 235-241.
[30]程光,张赟,章翔,等.面包海星皂苷-1对恶性胶质瘤细胞系U87MG增殖抑制的体外研究[J].脑与神经疾病杂志. 2008(5).
[31] Kicha A A, Ivanchina N V, Kalinovsky A I, et al. Steroidal triglycosides, kurilensosides A, B, and C, and other polar steroids from the Far Eastern starfish Hippasteria kurilensis[J]. J Nat Prod. 2008, 71(5): 793-798.
[32] Kicha A A, Ivanchina N V, Huong T T, et al. Two new asterosaponins, archasterosides A and B, from the Vietnamese starfish Archaster typicus and their anticancer properties[J]. Bioorg Med Chem Lett. 2010, 20(12): 3826-3830.
[33] Sandvoss M, Preiss A, Levsen K, et al. Two new asterosaponins from the starfish Asterias rubens: application of a cryogenic NMR probe head[J]. Magn. Reson. Chem. 2003, 41: 949-954.
[34] Ivanchina N V, Kicha A A, Kalinovsky A I, et al. Hemolytic polar steroidal constituents of the starfish Aphelasterias japonica[J]. J Nat Prod. 2000, 63(8): 1178-1181.
[35] N V I, A T V M. Asterosaponin Ophidianoside F from Gonads of the Far-Eastern Starfish Aphelasterias japonica [J]. Chemistry of Natural Compounds. 2005, 41(4): 481-482.
[36] Kawase O, Goo Y K, Jujo H, et al. Starfish, Asterias amurensis and Asterina pectinifera, as potential sources of Th1 immunity-stimulating adjuvants[J]. J Vet Med Sci. 2011, 73(2): 227-229.
[37] Haifengtang,Yanghuayi,Lingli,等. A New Asterosaponin from the Starfish Culcita novaeguineae[J].中国化学快报:英文版. 2005, 16(5): 619-622.
[38] Tang H F, Yi Y H, Li L, et al. Asterosaponins from the starfish Culcita novaeguineae and their bioactivities[J]. Fitoterapia. 2006, 77(1): 28-34.
[39] Tang H F, Yi Y H, Li L, et al. Asterosaponins from the starfish Culcita novaeguineae and their bioactivities[J]. Fitoterapia. 2006, 77(1): 28-34.
[40] Tang H F, Cheng G, Wu J, et al. Cytotoxic asterosaponins capable of promotingpolymerization of tubulin from the starfish Culcita novaeguineae[J]. J Nat Prod. 2009, 72(2): 284-289.
[41] Tang H F, Yun J, Lin H W, et al. Two new triterpenoid saponins cytotoxic to human glioblastoma U251MG cells from Ardisia pusilla[J]. Chem Biodivers. 2009, 6(9): 1443-1452.
[42] Guo C, Tang X, Dong X, et al. Studies on the expectorant, antitussive and antiasthmatic properties of asterosaponin extracted from Luidia quinaria[J]. African Journal of Biotechnology. 2009, 8(23): 6694-6696.
[43] Peng Y, Zheng J, Huang R, et al. Polyhydroxy steroids and saponins from China Sea starfish Asterina pectinifera and their biological activities[J]. Chem Pharm Bull (Tokyo). 2010, 58(6): 856-858.
[44]樊廷俊,张铮,袁文鹏,等.水溶性海星皂苷的分离纯化及其抗真菌活性研究[J].山东大学学报:理学版. 2008, 43(9): 1-5.
[45] Fedorov S N, Makarieva T N, Guzii A G, et al. Marine two-headed sphingolipid-like compound rhizochalin inhibits EGF-induced transformation of JB6 P+ Cl41 cells[J]. Lipids. 2009, 44(9): 777-785.
[46] Inagaki M, Shiizaki M, Hiwatashi T, et al. Constituents of Crinoidea. 5. Isolation and structure of a new glycosyl inositolphosphoceramide-type ganglioside from the feather star Comanthina schlegeli[J]. Chem Pharm Bull (Tokyo). 2007, 55(11): 1649-1651.
[47] Shah A K, Kinoshita M, Kurihara H, et al. Glycosylceramides obtain from the starfish Asterias amurensis Lutken[J]. J Oleo Sci. 2008, 57(9): 477-484.
[48] Inagaki M, Saito T, Miyamoto T, et al. Isolation and structure of hematoside-type ganglioside from the starfish Linckia laevigata[J]. Chem Pharm Bull (Tokyo). 2009, 57(2): 204-206.
[49] Pan K, Inagaki M, Ohno N, et al. Identification of sixteen new galactocerebrosides from the starfish Protoreaster nodosus[J]. Chem Pharm Bull (Tokyo). 2010, 58(4): 470-474.
[50] Ohta E, Uy M M, Ohta S, et al. Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and relatedcompounds[J]. Biosci Biotechnol Biochem. 2008, 72(7): 1764-1771.
[51]李厚金,李淑青,王丰,等.长棘海星的体内脂肪酸成分及其生物学意义分析[J].中山大学学报:自然科学版. 2009(5): 55-60.
[52] Canty M N, Hutchinson T H, Brown R J, et al. Linking genotoxic responses with cytotoxic and behavioural or physiological consequences: differential sensitivity of echinoderms (Asterias rubens) and marine molluscs (Mytilus edulis)[J]. Aquat Toxicol. 2009, 94(1): 68-76.
[53]高玫梅,许东晖,许实波.海星甾烯AST对异丙肾上腺素致大鼠心肌缺血的保护作用[J].解剖学研究. 2009, 31(6): 428-430.
[54]高玫梅,许东晖,许实波.海星甾烯AST对离体豚鼠心房肌的作用[J].临床医学工程. 2010(1): 5-7.
[55]李玉珠,王芳,王金辉,等.海星醇提取物对疾病引起的胃肠动力障碍的改善作用[J].沈阳药科大学学报. 2009, 26(11): 911-915.
[56]赵松岩,杨静玉,王金辉,等.海星醇提物促小鼠胃排空作用考察及有效部位确定[J].沈阳药科大学学报. 2007, 24(10): 636-638.
[57]白淑萍,张咏梅,李今越.家蝇对常用杀虫剂的抗性及其对策[J].中华卫生杀虫药械. 2005, 11(1): 39-41.
[58]邱星辉.杀虫剂抗性:遗传学、基因组学及应用启示[J].昆虫学报. 2005, 48(6): 960-967.
[59]晓岚.杀虫剂抗性机制[J].农药译丛. 1999, 21(2): 21-25.
[60]任太军,戚晓娴,程功,等.印楝素可能通过干扰细胞凋亡抑制果蝇生长发育[J].湖南农业大学学报:自然科学版. 2009, 35(1): 92-95.
[61]许珊,姚珧.黑腹果蝇单胚培养技术的建立[J].遗传. 1994, 16(2): 25-28.
[62]郭承华,张芳,刘迅,等.黄海产海燕(Asterina pectinifira)海星皂甙的制备[J].海洋通报. 2000, 19(2): 93-96.
[63]郭承华,贺凤丽.罗氏海盘车海星皂甙的提取分离及其分析鉴定[J].烟台大学学报:自然科学与工程版. 1995(4): 13-17.
[64]郭承华,高学良.罗氏海盘车组织中海星皂甙的分布及活性研究[J].黄渤海海洋. 2000, 18(2): 56-60.
[65]张立新,薛峰,赵爱云,等.海星中皂苷总含量测定方法的研究[J].食品科学.2007, 28(1): 243-246.
[66]魏有良,陈志兴.土贝母皂苷原料及其注射液中土贝母总皂苷的含量测定[J].中国现代应用药学. 1999, 16(1): 41-43.
[67]陈彦,袁艺.粉菝葜皂甙的溶血作用研究[J].生物学杂志. 1997(1): 26-27.
[68]文震,党志,宗敏华,等.不同方法制备的海星皂甙细胞毒性评价[J].海洋通报. 2006, 25(6): 26-29.
[69] Gibbs A, Green C, Doctor V M. Isolation and anticoagulant properties of polysaccharides of Typha Augustata and Daemonorops species[J]. Thromb Res. 1983, 32(2): 97-108.
[70]孙光,侯明晓.人参总皂甙溶血作用的研究[J].大连医学院学报. 1994, 16(2): 107-110.
[71]文震,党志,朱志鑫,等.海星甾醇的分离精制与测定[J].海洋科学. 2003, 27(9): 61-64.
[72]聂传朋.果蝇的生活史及其人工饲养[J].生物学通报. 2002, 37(2): 21.
[73]刘卫东,吕小东.果蝇培养过程中各环节的优化[J].生物学教学. 2009(9): 54.
[74]陈宗礼,延志莲.果蝇培养基的选择研究[J].生物学杂志. 1994(3): 31-33.
[75]宋文贞,夏曦中,朱丽华.实验用果蝇培养技术探索[J].氨基酸和生物资源. 2007, 29(3): 36-37.
[76]陈晓芸.果蝇的饲养[J].生物学通报. 2006, 41(2): 51.
[77]刘红英. NaCl对果蝇生长发育的影响[J].安徽农业科学. 2009, 37(22): 10545-10546.
[78]王彩丽.实验用果蝇饲养及保种注意事项[J].生物学通报. 2009, 44(11): 49-50.
[79]刘梅.枸杞提取物对果蝇寿命及SOD和MDA的影响[J].安徽农业科学. 2010(4): 1860-1861.
[80]周鹏,顾谦群.海星皂甙及其他活性成分研究概况[J].海洋科学. 2000, 24(2): 35-37.
[81]胡虹.海洋生物中天然产物的主要化学类型[J].青海科技. 2004, 11(5): 31-34.
[82]梁健成,王秀梅.抗病毒海洋天然产物研究进展[J].中药材. 2008, 31(7): 1104-1106.
[83]潘敏祥.疣海星生物活性成分研究[D].第二军医大学, 2007.
[84]王艳,刘文杰,周培根,等.一种海星皂甙的分离纯化[J].食品科技. 2006, 31(11): 145-147.
[85]郭承华,刘传琳.砂海星海星皂甙的制备[J].中国海洋药物. 1997, 16(1): 36-39.
[86]李丽华,陈锦屏.皂甙的提取纯化方法及其生物活性的研究进展[J].农产品加工. 2008(1): 60-62.
[87]潘广昌.砂海星主要活性物质的制备及其皂苷的生物功能研究[D].烟台大学, 2007.
[88]宁德生,梁小燕,方宏,等.半制备高压液相色谱法制备罗汉果苷Ⅴ标准品[J].食品科学. 2010, 31(12): 137-140.
[89]赵可星.高效液相色谱法分析并半制备党参化学成分的方法学探索[D].北京中医药大学, 2007.
[90]甘肃省药品检验所.关于皂甙的溶血作用的试验[S]. 2008.
[91]郑乃绮.咖啡因对果蝇生长的影响[R].台湾.
[92]凤严,陈贵才,何国声.昆虫抑制素Y_14对果蝇羽化的抑制效果[J].河北科技师范学院学报. 2004, 18(1): 79-80.
[93]徐曼妮,张欣文,徐思红.邻苯二甲酸二丁酯对雌性果蝇生育力的影响[J].同济大学学报:医学版. 2008, 29(1): 53-55.
[94]张欣文,范鸿杰,徐曼妮,等.邻苯二甲酸二丁酯对雌性果蝇生殖能力的双重作用[J].生殖与避孕. 2008, 28(6): 329-333.
[95]吴霞,陈志萍.由日本萍蓬草提取的抗醋霉菌果蝇的杀虫生物碱[J].农药译丛. 1998, 20(6): 25-30.
[96]刘金双,白利峰,周颖,等.毛蚶中天然牛磺酸的提取及其对果蝇寿命的影响[J].食品研究与开发. 2006, 27(7): 182-184.
[2]王伟红.赤海星中生物活性化学成分的研究[D].沈阳:沈阳药科大学, 2003.
[3]王征,董平原,张天民,等.海洋抗肿瘤药物研究开发中的主要问题[J].食品与药品. 2006, 8(4A): 1-4.
[4]刘春亮,焦吉祥.抗肿瘤海洋生物的研究进展[J].安徽医药. 2009, 13(10): 1277-1279.
[5]汪开治(译).海洋生物药研发现状综述[J].生物技术通报. 2006(4): 133-138.
[6]董黎明,陈思迪,刘仲娜.海星活性成分研究进展[J].科技信息. 2008(25).
[7]汤海峰,易杨华,张淑瑜,等.海星皂苷的研究进展[J].中国海洋药物. 2004, 23(6): 48-57.
[8]周学家,刘信艺.山东近海海星种类及分布[J].齐鲁渔业. 1996, 13(1): 41-43.
[9]王伟红,李发美.海星化学成分及其活性的研究进展[J].中国海洋药物. 2002, 21(5): 46-50.
[10] Yadavalli S, Nadimpalli S K. Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate Asterias rubens (Echinodermate): biochemical and functional characterization of MPR 46 protein[J]. Glycoconj J. 2008, 25(9): 889-901.
[11]郭承华.海燕皂苷及其它活性物质的制备及其生物功能研究[D].中国海洋大学, 2005.
[12]吴鹏.海星化学成分及其生物活性的研究[D].吉林大学, 2006.
[13]李军,吴红梅,蒋碧蓉,等.海星多糖的分离纯化及鉴定[J].食品工业科技. 2010, 31(2): 122-125.
[14] Nam K S, Shon Y H. Chemopreventive effects of polysaccharides extract from Asterina pectinifera on HT-29 human colon adenocarcinoma cells[J]. BMB Rep. 2009, 42(5): 277-280.
[15]潘广昌,郭承华,刘传琳,等.砂海星酸性粘多糖的分离纯化[J].烟台大学学报:自然科学与工程版. 2007, 20(2): 108-111.
[16]周燕霞,赵丽华,邢翔,等.多棘海盘车多糖的提取及体外抑菌活性初探[J].中国海洋药物. 2009(3): 58-60.
[17]骆传环,黄荣清,等.海星多糖的分子量测定[J].军事医学科学院院刊. 2002, 26(2): 157-158.
[18]李军,蒋碧蓉,吴红梅,等.海星多糖的提取分离与抗氧化活性研究[J].广东化工. 2010(2): 188-189.
[19] Kannappan R, Ando M, Furuhata K, et al. Photoaffinity labeling of sialidase with a biotin-conjugated phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid[J]. Biol Pharm Bull. 2008, 31(3): 352-356.
[20] Mita M, Yoshikuni M, Ohno K, et al. A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera[J]. Proc Natl Acad Sci U S A. 2009, 106(23): 9507-9512.
[21] Harun-Or-Rashid M, Matsuzawa T, Satoh Y, et al. Purification and characterization of alpha-N-acetylgalactosaminidases I and II from the starfish Asterina amurensis[J]. Biosci Biotechnol Biochem. 2010, 74(2): 256-261.
[22]翟宗德,张虹,师彦平,等.用半制备高效液相色谱法制备少量的阿苯达唑亚砜单一对映体[J].分析化学. 2005, 33(1): 109-112.
[23]魏有良,陈志兴.土贝母皂苷原料及其注射液中土贝母总皂苷的含量测定[J].中国现代应用药学. 1999, 16(1): 41-43.
[24] Burt V T, Bee E, Pennock J F. The Formation of Menaquinone4 (Vitamin K) and its Oxide in some Marine Invertebrates[J]. Biochem.J. 1977, 162: 297-302.
[25] Smith A G, Goad L J. Sterol biosynthesis in the echinoderm Asterias rubens[J]. Biochem J. 1975, 146(1): 25-33.
[26] Smith A G, Goad L J. The Conversion of Cholest-5-en-3p-ol into Cholest-7-en-3p-ol by the Echinoderms Asterias rubens and Solasterpapposus[J]. Bolchem.J. 1975, 146: 35-40.
[27] Yasumoto T, Hashimoto Y. Properties of Asterosaponin B Isolated from a Starfish, Asterias amurensis[J]. Agr. Biol. Chem. 1967, 31(3): 368-372.
[28] Francis G, Kerem Z, Makkar H P, et al. The biological action of saponins in animal systems: a review[J]. Br J Nutr. 2002, 88(6): 587-605.
[29] Cheng G, Zhang X, Tang H F, et al. Asterosaponin 1, a cytostatic compound from the starfish Culcita novaeguineae, functions by inducing apoptosis in human glioblastoma U87MG cells[J]. J Neurooncol. 2006, 79(3): 235-241.
[30]程光,张赟,章翔,等.面包海星皂苷-1对恶性胶质瘤细胞系U87MG增殖抑制的体外研究[J].脑与神经疾病杂志. 2008(5).
[31] Kicha A A, Ivanchina N V, Kalinovsky A I, et al. Steroidal triglycosides, kurilensosides A, B, and C, and other polar steroids from the Far Eastern starfish Hippasteria kurilensis[J]. J Nat Prod. 2008, 71(5): 793-798.
[32] Kicha A A, Ivanchina N V, Huong T T, et al. Two new asterosaponins, archasterosides A and B, from the Vietnamese starfish Archaster typicus and their anticancer properties[J]. Bioorg Med Chem Lett. 2010, 20(12): 3826-3830.
[33] Sandvoss M, Preiss A, Levsen K, et al. Two new asterosaponins from the starfish Asterias rubens: application of a cryogenic NMR probe head[J]. Magn. Reson. Chem. 2003, 41: 949-954.
[34] Ivanchina N V, Kicha A A, Kalinovsky A I, et al. Hemolytic polar steroidal constituents of the starfish Aphelasterias japonica[J]. J Nat Prod. 2000, 63(8): 1178-1181.
[35] N V I, A T V M. Asterosaponin Ophidianoside F from Gonads of the Far-Eastern Starfish Aphelasterias japonica [J]. Chemistry of Natural Compounds. 2005, 41(4): 481-482.
[36] Kawase O, Goo Y K, Jujo H, et al. Starfish, Asterias amurensis and Asterina pectinifera, as potential sources of Th1 immunity-stimulating adjuvants[J]. J Vet Med Sci. 2011, 73(2): 227-229.
[37] Haifengtang,Yanghuayi,Lingli,等. A New Asterosaponin from the Starfish Culcita novaeguineae[J].中国化学快报:英文版. 2005, 16(5): 619-622.
[38] Tang H F, Yi Y H, Li L, et al. Asterosaponins from the starfish Culcita novaeguineae and their bioactivities[J]. Fitoterapia. 2006, 77(1): 28-34.
[39] Tang H F, Yi Y H, Li L, et al. Asterosaponins from the starfish Culcita novaeguineae and their bioactivities[J]. Fitoterapia. 2006, 77(1): 28-34.
[40] Tang H F, Cheng G, Wu J, et al. Cytotoxic asterosaponins capable of promotingpolymerization of tubulin from the starfish Culcita novaeguineae[J]. J Nat Prod. 2009, 72(2): 284-289.
[41] Tang H F, Yun J, Lin H W, et al. Two new triterpenoid saponins cytotoxic to human glioblastoma U251MG cells from Ardisia pusilla[J]. Chem Biodivers. 2009, 6(9): 1443-1452.
[42] Guo C, Tang X, Dong X, et al. Studies on the expectorant, antitussive and antiasthmatic properties of asterosaponin extracted from Luidia quinaria[J]. African Journal of Biotechnology. 2009, 8(23): 6694-6696.
[43] Peng Y, Zheng J, Huang R, et al. Polyhydroxy steroids and saponins from China Sea starfish Asterina pectinifera and their biological activities[J]. Chem Pharm Bull (Tokyo). 2010, 58(6): 856-858.
[44]樊廷俊,张铮,袁文鹏,等.水溶性海星皂苷的分离纯化及其抗真菌活性研究[J].山东大学学报:理学版. 2008, 43(9): 1-5.
[45] Fedorov S N, Makarieva T N, Guzii A G, et al. Marine two-headed sphingolipid-like compound rhizochalin inhibits EGF-induced transformation of JB6 P+ Cl41 cells[J]. Lipids. 2009, 44(9): 777-785.
[46] Inagaki M, Shiizaki M, Hiwatashi T, et al. Constituents of Crinoidea. 5. Isolation and structure of a new glycosyl inositolphosphoceramide-type ganglioside from the feather star Comanthina schlegeli[J]. Chem Pharm Bull (Tokyo). 2007, 55(11): 1649-1651.
[47] Shah A K, Kinoshita M, Kurihara H, et al. Glycosylceramides obtain from the starfish Asterias amurensis Lutken[J]. J Oleo Sci. 2008, 57(9): 477-484.
[48] Inagaki M, Saito T, Miyamoto T, et al. Isolation and structure of hematoside-type ganglioside from the starfish Linckia laevigata[J]. Chem Pharm Bull (Tokyo). 2009, 57(2): 204-206.
[49] Pan K, Inagaki M, Ohno N, et al. Identification of sixteen new galactocerebrosides from the starfish Protoreaster nodosus[J]. Chem Pharm Bull (Tokyo). 2010, 58(4): 470-474.
[50] Ohta E, Uy M M, Ohta S, et al. Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and relatedcompounds[J]. Biosci Biotechnol Biochem. 2008, 72(7): 1764-1771.
[51]李厚金,李淑青,王丰,等.长棘海星的体内脂肪酸成分及其生物学意义分析[J].中山大学学报:自然科学版. 2009(5): 55-60.
[52] Canty M N, Hutchinson T H, Brown R J, et al. Linking genotoxic responses with cytotoxic and behavioural or physiological consequences: differential sensitivity of echinoderms (Asterias rubens) and marine molluscs (Mytilus edulis)[J]. Aquat Toxicol. 2009, 94(1): 68-76.
[53]高玫梅,许东晖,许实波.海星甾烯AST对异丙肾上腺素致大鼠心肌缺血的保护作用[J].解剖学研究. 2009, 31(6): 428-430.
[54]高玫梅,许东晖,许实波.海星甾烯AST对离体豚鼠心房肌的作用[J].临床医学工程. 2010(1): 5-7.
[55]李玉珠,王芳,王金辉,等.海星醇提取物对疾病引起的胃肠动力障碍的改善作用[J].沈阳药科大学学报. 2009, 26(11): 911-915.
[56]赵松岩,杨静玉,王金辉,等.海星醇提物促小鼠胃排空作用考察及有效部位确定[J].沈阳药科大学学报. 2007, 24(10): 636-638.
[57]白淑萍,张咏梅,李今越.家蝇对常用杀虫剂的抗性及其对策[J].中华卫生杀虫药械. 2005, 11(1): 39-41.
[58]邱星辉.杀虫剂抗性:遗传学、基因组学及应用启示[J].昆虫学报. 2005, 48(6): 960-967.
[59]晓岚.杀虫剂抗性机制[J].农药译丛. 1999, 21(2): 21-25.
[60]任太军,戚晓娴,程功,等.印楝素可能通过干扰细胞凋亡抑制果蝇生长发育[J].湖南农业大学学报:自然科学版. 2009, 35(1): 92-95.
[61]许珊,姚珧.黑腹果蝇单胚培养技术的建立[J].遗传. 1994, 16(2): 25-28.
[62]郭承华,张芳,刘迅,等.黄海产海燕(Asterina pectinifira)海星皂甙的制备[J].海洋通报. 2000, 19(2): 93-96.
[63]郭承华,贺凤丽.罗氏海盘车海星皂甙的提取分离及其分析鉴定[J].烟台大学学报:自然科学与工程版. 1995(4): 13-17.
[64]郭承华,高学良.罗氏海盘车组织中海星皂甙的分布及活性研究[J].黄渤海海洋. 2000, 18(2): 56-60.
[65]张立新,薛峰,赵爱云,等.海星中皂苷总含量测定方法的研究[J].食品科学.2007, 28(1): 243-246.
[66]魏有良,陈志兴.土贝母皂苷原料及其注射液中土贝母总皂苷的含量测定[J].中国现代应用药学. 1999, 16(1): 41-43.
[67]陈彦,袁艺.粉菝葜皂甙的溶血作用研究[J].生物学杂志. 1997(1): 26-27.
[68]文震,党志,宗敏华,等.不同方法制备的海星皂甙细胞毒性评价[J].海洋通报. 2006, 25(6): 26-29.
[69] Gibbs A, Green C, Doctor V M. Isolation and anticoagulant properties of polysaccharides of Typha Augustata and Daemonorops species[J]. Thromb Res. 1983, 32(2): 97-108.
[70]孙光,侯明晓.人参总皂甙溶血作用的研究[J].大连医学院学报. 1994, 16(2): 107-110.
[71]文震,党志,朱志鑫,等.海星甾醇的分离精制与测定[J].海洋科学. 2003, 27(9): 61-64.
[72]聂传朋.果蝇的生活史及其人工饲养[J].生物学通报. 2002, 37(2): 21.
[73]刘卫东,吕小东.果蝇培养过程中各环节的优化[J].生物学教学. 2009(9): 54.
[74]陈宗礼,延志莲.果蝇培养基的选择研究[J].生物学杂志. 1994(3): 31-33.
[75]宋文贞,夏曦中,朱丽华.实验用果蝇培养技术探索[J].氨基酸和生物资源. 2007, 29(3): 36-37.
[76]陈晓芸.果蝇的饲养[J].生物学通报. 2006, 41(2): 51.
[77]刘红英. NaCl对果蝇生长发育的影响[J].安徽农业科学. 2009, 37(22): 10545-10546.
[78]王彩丽.实验用果蝇饲养及保种注意事项[J].生物学通报. 2009, 44(11): 49-50.
[79]刘梅.枸杞提取物对果蝇寿命及SOD和MDA的影响[J].安徽农业科学. 2010(4): 1860-1861.
[80]周鹏,顾谦群.海星皂甙及其他活性成分研究概况[J].海洋科学. 2000, 24(2): 35-37.
[81]胡虹.海洋生物中天然产物的主要化学类型[J].青海科技. 2004, 11(5): 31-34.
[82]梁健成,王秀梅.抗病毒海洋天然产物研究进展[J].中药材. 2008, 31(7): 1104-1106.
[83]潘敏祥.疣海星生物活性成分研究[D].第二军医大学, 2007.
[84]王艳,刘文杰,周培根,等.一种海星皂甙的分离纯化[J].食品科技. 2006, 31(11): 145-147.
[85]郭承华,刘传琳.砂海星海星皂甙的制备[J].中国海洋药物. 1997, 16(1): 36-39.
[86]李丽华,陈锦屏.皂甙的提取纯化方法及其生物活性的研究进展[J].农产品加工. 2008(1): 60-62.
[87]潘广昌.砂海星主要活性物质的制备及其皂苷的生物功能研究[D].烟台大学, 2007.
[88]宁德生,梁小燕,方宏,等.半制备高压液相色谱法制备罗汉果苷Ⅴ标准品[J].食品科学. 2010, 31(12): 137-140.
[89]赵可星.高效液相色谱法分析并半制备党参化学成分的方法学探索[D].北京中医药大学, 2007.
[90]甘肃省药品检验所.关于皂甙的溶血作用的试验[S]. 2008.
[91]郑乃绮.咖啡因对果蝇生长的影响[R].台湾.
[92]凤严,陈贵才,何国声.昆虫抑制素Y_14对果蝇羽化的抑制效果[J].河北科技师范学院学报. 2004, 18(1): 79-80.
[93]徐曼妮,张欣文,徐思红.邻苯二甲酸二丁酯对雌性果蝇生育力的影响[J].同济大学学报:医学版. 2008, 29(1): 53-55.
[94]张欣文,范鸿杰,徐曼妮,等.邻苯二甲酸二丁酯对雌性果蝇生殖能力的双重作用[J].生殖与避孕. 2008, 28(6): 329-333.
[95]吴霞,陈志萍.由日本萍蓬草提取的抗醋霉菌果蝇的杀虫生物碱[J].农药译丛. 1998, 20(6): 25-30.
[96]刘金双,白利峰,周颖,等.毛蚶中天然牛磺酸的提取及其对果蝇寿命的影响[J].食品研究与开发. 2006, 27(7): 182-184.