柿果实齐墩果酸和熊果酸含量分析及其合成的分子生物学研究
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摘要
三萜类化合物广泛存在于多种植物中,种类繁多,其中最常见的是五环三萜中的齐墩果烷型和乌苏烷型,其代表化合物分别是齐墩果酸(OA)和熊果酸(UA)及其衍生物。目前在柿(Diospyros kaki L.)中关于OA和UA的研究主要集中在叶片和柿蒂中,而柿果皮和果肉中尚未有研究。
本论文以柿果实为试材,对32个品种(23个涩柿和9个甜柿)柿果肉OA和UA含量进行分析;选取代表品种(牛心柿和西村早生)测定柿果实不同发育阶段OA和UA的含量;并克隆柿子OA和UA等五环三萜合成相关基因(SQS、OSC)的全长或片段,研究柿果实不同发育阶段三萜合成相关基因的表达状况。主要研究结果如下:
1.对32个品种柿果肉OA和UA含量进行分析。结果表明,不同品种之间,其果肉OA和UA含量差异很大。涩柿品种OA和UA的含量分别为0～88.57μg.g-1 FW和0～27.64μg.g-1 FW,甜柿品种OA和UA的含量分别为1.24～26.43μg.g-1 FW和0～12.23μg.g-1 FW。
2.对代表品种(牛心柿和西村早生)柿果实不同部位、不同发育时期OA和UA含量进行分析。结果表明,柿果实不同部位中OA和UA的含量一般以果蒂最高,果皮次之,果肉最低。且OA和UA的含量随生长期不同而发生显著变化。一般果肉和果皮在果实发育初期,OA和UA的含量相对较高,而果蒂在果实成熟期OA和UA相对较高。而将涩柿和甜柿作比较,三个部位多数阶段OA和UA的含量都是甜柿高于涩柿。
3.根据植物鲨烯合成酶(SQS)基因的保守区序列,设计1对PCR引物,采用RT-PCR技术,以柿果肉RNA为模板克隆出鲨烯合成酶基因的片段,在此基础上设计引物进行3'和5'-RACE,最后拼接出cDNA全长1467 bp,经过序列分析,其编码415个氨基酸。在GenBank中进行同源性检索的结果表明,该序列编码的氨基酸与其他植物鲨烯合成酶编码的氨基酸同源性较高,注册号为FJ687954。
4.根据GenBank中其它植物2,3-氧化鲨烯环化酶(OSC)基因序列设计引物,以柿果肉RNA为模板模板,采用3'-RACE方法进行3'端片段的扩增得到cDNA 3'端部分序列。该cDNA3'端部分长522bp,包含一个298 bp的开放阅读框(ORF),3'端有224 bp的非翻译区(UTR)并具有完整的PolyA尾巴,编码98个氨基酸。
5.对柿果肉不同发育阶段SQS基因和OSC基因相对表达水平研究表明,SQS和OSC基因的表达水平与柿果肉中OA和UA的合成相关性不明显。
Triterpenoids, which include many varities, are widespread in plants. The most common compouds are oleanane and ursane-type pentacyclic triterpenoids. The representative compounds were oleanolic acid (OA) and ursolic acid (UA) and their derivatives. At present, the research of OA and UA in persimmon (Diospyros kaki L.) is mainly focused on leaves and pedicles, while OA and UA content in the peel and flesh of persimmon fruit has not been examined.
In this study, persimmon fruits of 32 cultivars (23 astringent and 9 non-astringent) were used as materials, and OA and UA content in these fruits were determined. OA and UA content in typically selected cultivars‘Niuxinshi’and‘Nishimurawase’at different developmental stages were also analysised. The full-length or fragment of SQS and OSC genes were cloned, and invesgated the expression levels of these genes. Research results are found as follows:
1. OA and UA contents in the flesh of 32 different cultivars were determined. Results showed that the contents of OA and UA in the fleshs of different persimmon cultivars were significantly different. The contents of OA and UA were 0~88.57μg.g-1 FW and 0 ~27.64μg.g-1 FW for astringent cultivars, respectively. While the contents of OA and UA were 1.24~26.43μg.g-1 FW and 0 ~12.23μg.g-1 FW for non-astringent cultivars, respectively.
2. OA and UA contents in different parts of persimmon fruits and at different developmental stages of two representative cultivars‘Niuxinshi’and‘Nishimurawase’were analyzed. The data showed that the contents of OA and UA varied according to different parts of persimmon fruits measured, pedicle ranking first, next peel and then flesh. Moreover, the contents of OA and UA differed significantly with their growing periods. The contents of OA and UA in flesh and peel were comparatively high in the initial growing period, while that of pedicle in mature period were higher than other periods. If comparisons were made between astringent and non-astringent persimmons, the latter’s contents of OA and UA were higher than that of the former regardless of the three different parts and various growing periods.
3. On the basis of conserved sequence of squalene synthase gene in other plants, a pair of PCR primers were designed to clone the fragment of squalene synthase gene in persimmon fruit with RNA as template by the support of RT-PCR techonology. Then, primers were designed for 3' and 5'-RACE, and finally the 1467 bp long full-length cDNA was spliced. The gene encoded 415 amino acids by sequence analysis. The result of homology research in GenBank showed that amino acids encoded by this sequence and other squalene synthase gene -encoded ones were much homologous, and the registration number was FJ687954.
4. On the basis of 2,3-oxidosqualene cyclase gene sequenc in other plants, primers were designed to amplied 3' partial sequence of 2,3-oxidosqualene cyclases gene cDNA in persimmon fruit with RNA as template through 3'-RACE method. This fragment was 522bp long and encoded with 98 amino acids, with an ORF 298 bp long, UTR 224 bp long and a complete PolyA tail.
5. The comparative expression researches of SQS and OSC genes in the flesh of persimmon fruit at different developmental stages indicated that SQS and OSC genes expressionswere not significantly correlated with the biosynthesis of OA and UA in persimmons flesh.
本论文以柿果实为试材,对32个品种(23个涩柿和9个甜柿)柿果肉OA和UA含量进行分析;选取代表品种(牛心柿和西村早生)测定柿果实不同发育阶段OA和UA的含量;并克隆柿子OA和UA等五环三萜合成相关基因(SQS、OSC)的全长或片段,研究柿果实不同发育阶段三萜合成相关基因的表达状况。主要研究结果如下:
1.对32个品种柿果肉OA和UA含量进行分析。结果表明,不同品种之间,其果肉OA和UA含量差异很大。涩柿品种OA和UA的含量分别为0～88.57μg.g-1 FW和0～27.64μg.g-1 FW,甜柿品种OA和UA的含量分别为1.24～26.43μg.g-1 FW和0～12.23μg.g-1 FW。
2.对代表品种(牛心柿和西村早生)柿果实不同部位、不同发育时期OA和UA含量进行分析。结果表明,柿果实不同部位中OA和UA的含量一般以果蒂最高,果皮次之,果肉最低。且OA和UA的含量随生长期不同而发生显著变化。一般果肉和果皮在果实发育初期,OA和UA的含量相对较高,而果蒂在果实成熟期OA和UA相对较高。而将涩柿和甜柿作比较,三个部位多数阶段OA和UA的含量都是甜柿高于涩柿。
3.根据植物鲨烯合成酶(SQS)基因的保守区序列,设计1对PCR引物,采用RT-PCR技术,以柿果肉RNA为模板克隆出鲨烯合成酶基因的片段,在此基础上设计引物进行3'和5'-RACE,最后拼接出cDNA全长1467 bp,经过序列分析,其编码415个氨基酸。在GenBank中进行同源性检索的结果表明,该序列编码的氨基酸与其他植物鲨烯合成酶编码的氨基酸同源性较高,注册号为FJ687954。
4.根据GenBank中其它植物2,3-氧化鲨烯环化酶(OSC)基因序列设计引物,以柿果肉RNA为模板模板,采用3'-RACE方法进行3'端片段的扩增得到cDNA 3'端部分序列。该cDNA3'端部分长522bp,包含一个298 bp的开放阅读框(ORF),3'端有224 bp的非翻译区(UTR)并具有完整的PolyA尾巴,编码98个氨基酸。
5.对柿果肉不同发育阶段SQS基因和OSC基因相对表达水平研究表明,SQS和OSC基因的表达水平与柿果肉中OA和UA的合成相关性不明显。
Triterpenoids, which include many varities, are widespread in plants. The most common compouds are oleanane and ursane-type pentacyclic triterpenoids. The representative compounds were oleanolic acid (OA) and ursolic acid (UA) and their derivatives. At present, the research of OA and UA in persimmon (Diospyros kaki L.) is mainly focused on leaves and pedicles, while OA and UA content in the peel and flesh of persimmon fruit has not been examined.
In this study, persimmon fruits of 32 cultivars (23 astringent and 9 non-astringent) were used as materials, and OA and UA content in these fruits were determined. OA and UA content in typically selected cultivars‘Niuxinshi’and‘Nishimurawase’at different developmental stages were also analysised. The full-length or fragment of SQS and OSC genes were cloned, and invesgated the expression levels of these genes. Research results are found as follows:
1. OA and UA contents in the flesh of 32 different cultivars were determined. Results showed that the contents of OA and UA in the fleshs of different persimmon cultivars were significantly different. The contents of OA and UA were 0~88.57μg.g-1 FW and 0 ~27.64μg.g-1 FW for astringent cultivars, respectively. While the contents of OA and UA were 1.24~26.43μg.g-1 FW and 0 ~12.23μg.g-1 FW for non-astringent cultivars, respectively.
2. OA and UA contents in different parts of persimmon fruits and at different developmental stages of two representative cultivars‘Niuxinshi’and‘Nishimurawase’were analyzed. The data showed that the contents of OA and UA varied according to different parts of persimmon fruits measured, pedicle ranking first, next peel and then flesh. Moreover, the contents of OA and UA differed significantly with their growing periods. The contents of OA and UA in flesh and peel were comparatively high in the initial growing period, while that of pedicle in mature period were higher than other periods. If comparisons were made between astringent and non-astringent persimmons, the latter’s contents of OA and UA were higher than that of the former regardless of the three different parts and various growing periods.
3. On the basis of conserved sequence of squalene synthase gene in other plants, a pair of PCR primers were designed to clone the fragment of squalene synthase gene in persimmon fruit with RNA as template by the support of RT-PCR techonology. Then, primers were designed for 3' and 5'-RACE, and finally the 1467 bp long full-length cDNA was spliced. The gene encoded 415 amino acids by sequence analysis. The result of homology research in GenBank showed that amino acids encoded by this sequence and other squalene synthase gene -encoded ones were much homologous, and the registration number was FJ687954.
4. On the basis of 2,3-oxidosqualene cyclase gene sequenc in other plants, primers were designed to amplied 3' partial sequence of 2,3-oxidosqualene cyclases gene cDNA in persimmon fruit with RNA as template through 3'-RACE method. This fragment was 522bp long and encoded with 98 amino acids, with an ORF 298 bp long, UTR 224 bp long and a complete PolyA tail.
5. The comparative expression researches of SQS and OSC genes in the flesh of persimmon fruit at different developmental stages indicated that SQS and OSC genes expressionswere not significantly correlated with the biosynthesis of OA and UA in persimmons flesh.
引文
[1]张上隆,陈昆松.果实品质形成与调控的分子生理[M].北京:中国农业出版社, 2007: 233-238.
[2]杜瑜,李焕德.齐墩果酸的研究进展[J].中国药房, 2006, 17(4): 304-306..
[3]田丽婷,马龙,齐墩果酸的药理作用研究概况[J].中国中药杂志, 2002, 27(12): 884-886, 901.
[4]王立新,韩广轩,刘文庸,等.齐墩果酸的化学及药理研究[J].药学实践杂志, 2001, 19(2): 104-107.
[5]黄婉,杨耀芳.女贞子及其有效成分的药理及临床研究进展[J].现代中西医结合杂志, 2003, 12(7): 772-774.
[6] Kapil A, Sharma S. Effect of oleanolic acid on complement in adjurant and carrageenan-induced inflammation in rats[J]. Pharm Pharmacol, 1995, 47(7): 585.
[7]张东方,黄炜,黄济群,等.齐墩果酸抗人肺癌细胞增殖、侵袭和诱导细胞凋亡的研究[J].肿瘤防治研究, 2003, 30(3): 180-183.
[8] Huang Dan, Ding Yan, Li Yan, et al. Anti-tumor activity of a 3-oxo derivative of oleanolic acid[J]. Cancer Letters, 2006, 233: 289-296.
[9] Ovesna Z, Vachalkova A, Horvathova K, et al. Pentacyclic triterpenoic acids: new chemoprotective compounds[J]. Minireview Neoplasma, 2004, (5): 327-333.
[10] Martin-Aragon S, De las Heras B, Sanchez-Reus M I, et al. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride- induced liver damage in rats and primary cultures of rat hepatocytes[J]. Exp Toxicol Pathol, 2001, 53(2-3): 199-206.
[11] Saraswat B, Visen PK, Agarwal DP. Ursolic acid isolated from Eucalyptus tereticornis protect against ethanol toxicity in isolated rathepatocytes[J]. Phytother Res, 2000, 14(3): 163-166.
[12]熊筱娟,陈武,肖小华,等.乌索酸与齐墩果酸对小鼠实验性肝损伤保护作用的比较[J].江西师范大学学报(自然科学版), 2004, 28(6): 540-543.
[13] Chattopadhyay D, Arunachalam G, Mandal AB, et al. Antimicrobial and anti-inflammatory activity of folklore: Mallotus peltatus leaf ex-tract[J]. J Ethnopharmacol, 2002, 82(2-3): 229-237.
[14]王茜,樊明文,边专,等.熊果酸的提取及其对牙周病原菌的作用[J].中华口腔医学杂志, 2002, 37(5): 388-390.
[15]陈国宝,陈宝田.番石榴叶提取物体外抗轮状病毒的实验研究[J].中国医药学报, 2002, 17(8): 502-504.
[16] Kashiwada Y, Nagao T, Hashimoto A, et al. Anti-AIDS agents38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives[J]. J Nat Prod, 2000, 63(12): 1619-1622.
[17] Somova LO, Nadar A, Rammanan P, et al. Cardiovascular, antihyperlipidemic and antioxidant efiects of oleanolic and ursolic acids in experimental hypenension[J]. Phytomedicine, 2003, 10(2 -3): 115-121.
[18] Chung TK, Heo HJ, Kim EK, et al. Inhibitory effect of ursolic acid purined from Origanum majorana L on the acetylcholinesterase[J]. Mol Cells, 2001, 11(2): 137-143.
[19]张士勇,陈林红.薄层扫描法测定酒制木瓜中熊果酸的含量[J].中医药临床杂志, 2006, 18(4): 408-409.
[20]都述虎,饶金华,耿武松.薄层扫描法测定木瓜中齐墩果酸的含量[J].中草药, 2003, 34(1): 35-37.
[21]黄建林,张展霞. GC-MS同时测定番石榴叶中的齐墩果酸和熊果酸[J].分析测试技术与仪器, 2005, 11(3): 178-181.
[22]邓延倬,何金兰.高效毛细管电泳[M].北京:科学工业出版社, 1996:100-101.
[23] Hsieh YZ, Huang HY. Determination of Saikosaponins by Micellar Electrokinetic Capillary Chromatography [J]. Journal of Chromatography A, 1997, 759:193-201.
[24] Glockl I, Veit M, Blaschke G. Determination of Ginsenosides from Panax ginsengUsing Micellar Electrokinetic Chromatography [J]. Planta Medica, 2002, 68(2): 158-161.
[25]王瑞,王淑美,梁生旺,等.胶束电动毛细管色谱法分离分析山茱萸中齐墩果酸和熊果酸[J].中药材, 2007, 30(8): 946-950.
[26]刘春霞,朱培仪,宋粉云.白花蛇舌草中齐墩果酸和熊果酸的毛细管电泳高频电导法测定[J].中药材, 2008, 31(8): 1176-1178.
[27]王雨梅,杜瑞芳,刘卫东,等. HPLC法同时测定毛建草中齐墩果酸和熊果酸的含量[J].西北药学杂志, 2008, 23(2): 84-85.
[28]冯瑜娟,张艺. HPLC-ELSD法测定藏药翼首草中齐墩果酸和熊果酸的含量[J].成都中医药大学学报, 2007, 30(3): 54-56.
[29]陈武,熊筱娟,李开泉,等.乌索酸的化学、药理及临床研究[J].宜春医专学报, 2001, 13(2): 123-126.
[30]张登科. 35种植物的齐墩果酸含量比较[J].中药材, 1989, 12(12): 19.
[31] Atkinson CJ, Dodds PAA, Ford YY, et al. Effects of Cultivar, Fruit Number and Reflected Photosynthetically Active Radiation on Fragaria xananassa Productivity and Fruit Ellagic Acid and Ascorbic Acid Concentrations [J]. Annal of Botany, 2006, 97: 429-441.
[32] Pomar F, Novo M, Masa A. Varietal Differences among the Anthocyanin Profiles of 50 Red Table Grape Cultivars Studied by High Performance Liquid Chromatography [J]. Journal of Chromatography A, 2005, 1094: 34-41.
[33]王向红,刘孟军. HPLC法测定不同品种枣及酸枣中的齐墩果酸和熊果酸[J].食品科学, 2002, 23(6): 137-138.
[34] Zhou CH, Chen KS, Sun CD, et al. Determination of Oleanolic Acid, Ursolic Acid and Amygdalin in the Flower of Eriobotrya japonica Lindl. by HPLC[J]. Biomedical Chromatography, 2007, 21: 755-761.
[35]邹盛勤,陈武.高效液相色谱-光电二极管阵列检测器联用检测不同产地荆芥中乌索酸和齐墩果酸[J].光谱实验室, 2007, 24 (5): 972-975.
[36]刘名权,邹盛勤,陈武.超声辅助HPLC法测定毛泡桐中乌索酸和齐墩果酸含量[J].广东微量元素科学, 2007, 14(5): 19-22.
[37] Wang HH, Wang ZZ, Guo WB. Comparative Determination of Ursolic Acid and Oleanolic Acid of Macrocarpium officinalis (Sieb. et Zucc.) Nakai by RP-HPLC[J]. Industrial Crops and Products, 2008, 28: 328-332.
[38] Brown PD, Tokuhisa JG, Reichelt M, et al. Variation of Glucosinolate Accumulation among Different Organs and Developmental Stages of Arabopsis thaliana [J]. Phytochemistry, 2003, 62: 471-481.
[39]邹盛勤. RP-HPLC法同时测定不同采收期连翘中乌索酸和齐墩果酸的含量[J].沈阳药科大学学报, 2007, 24(3): 164-166,171.
[40]戚志华,王四旺. HPLC法测定陕西女贞子中齐墩果酸与熊果酸的含量[J].中国中医药信息杂志, 2007, 14(5):40-42.
[41]朱鹏程,于瑞涛,陶燕铎,等.藏药花锚中齐墩果酸和熊果酸含量测定[J].分析试验室, 2008, 27: 54-55.
[42]邹盛勤,黄宏伟.反相高效液相色谱-光电二极管阵列检测器法测定金樱子中乌索酸和齐墩果酸[J].食品研究与开发, 2007, 28(10): 130-133..
[43] Du SH, Chen LN, Ma KF, et al. Qualitative and Quantitative Analysis of Chinese Herb Fructus Chaenomelis [J]. Journal of Nanjing Mecical University, 2007, 21(1): 51-54.
[44]杨勇,阮小凤,王仁梓,李高潮.柿种质资源及育种研究进展[J].西北林学院学报, 2005, 20(2): 133-137.
[45]张雅利,郭辉,田忠民.柿子的药理作用研究及临床应用[J].中成药, 28(5): 720-722.
[46] Cui T, Li JZ, Kayahara H, Ma LG, Wu LX, Nakamura, K. Quantification of the polyphenols and triterpene acids in Chinese hawthorn fruit by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry, 2006, 54(13): 4574-81.
[47] Guo S, Duan J, Tang Y, Su S, Shang E, Ni S, Qian D. High-performance liquid chromatography-Two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis. Journal of Pharmaceutical and Biomedical Analysis, 2009, 49(5): 1296-1302.
[48]盛艳乐,赵大球,周春华,陶俊.植物熊果酸和齐墩果酸分析的研究进展[J].中国农学通报, 2009, 25(23): 347-350.
[49] Atkinson CJ, Dodds PAA, Ford YY, Le Mière J, Taylor JM,Blake PS and Paul N. Effects of cultivar, fruit number and reflected photosynthetically active radiation on Fragaria xananassa productivity and fruit ellagic acid and ascorbic acid concentrations.Annals of Botany, 2006,97: 429-441.
[50] Pomar F, Novo M and Masa A. Varietal differences among the anthocyanin profiles of 50 red table grape cultivars studied by high performance liquid chromatography. Journal of Chromatography A, 2005, 1094: 34-41.
[51] Brown PD, Tokuhisa JG, Reichelt M and Gershenzon J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry, 2003, 62: 471-481.
[52]郝宏蕾,朱旭芬,曾云中.类异戊二烯的生物合成及调控[J].浙江大学学报(农业与生命科学版), 2002, 28(2): 224-230.
[53]陈大华,叶和春,李国凤,等.植物类异戊二烯代谢途径的分子生物学研究进展[J].植物学报, 2000, 42(6): 551-558.
[54]黄志伟.人参(Panax japonicus C.A.Mey.)SQS基因cDNA的克隆及序列分析[D].福建农林大学, 2005, 43-44.
[55]卢虹玉,刘敬梅,阳文龙,高山林.甘草鲨烯合成酶基因的分离及植物表达载体的构建.药物生物技术, 2007, 14(4): 225-258.
[56]黄志伟,林明,何水林,郑金贵.葡萄总RNA提取及SQS基因保守区域的克隆.武夷科学, 2006, 22(12): 169-171.
[57]钟家禹.灵芝鲨烯合成酶基因的克隆与表达特性初步分析.南京农业大学学位论文[D]. 2001.
[58]赵云生,万德光,陈新,李占林,田洪岭.五环三萜皂苷生物合成与调控的研究进展[J].中草药, 2009, 40(2): 327-330.
[59]徐昌杰,陈昆松,张波,等.柑橘组织RNA提取方法研究[J].果树学报, 2004, 21(2): 136-140.
[60] Lee MH, Jeong JH, Seo JW, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol, 2004, 45(8): 976-984.
[61]克丙申,黄象艳,陈世敏,等. PCR扩增试验的动力学数学模型[J].遗传,2002, 24(1): 55-59.
[62] Chio DW, Jung J, Ha YI, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairyroots to identify genes involvedinthe biosynthesis of ginsenosides and other secondary metabolites[J]. Plant Cell Rep, 2005, 23(8): 557-566.
[63] Suzuki H, Achnine L, Xu R, et al. A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula[J]. Plant J, 2002, 32(6):1033-1048.
[2]杜瑜,李焕德.齐墩果酸的研究进展[J].中国药房, 2006, 17(4): 304-306..
[3]田丽婷,马龙,齐墩果酸的药理作用研究概况[J].中国中药杂志, 2002, 27(12): 884-886, 901.
[4]王立新,韩广轩,刘文庸,等.齐墩果酸的化学及药理研究[J].药学实践杂志, 2001, 19(2): 104-107.
[5]黄婉,杨耀芳.女贞子及其有效成分的药理及临床研究进展[J].现代中西医结合杂志, 2003, 12(7): 772-774.
[6] Kapil A, Sharma S. Effect of oleanolic acid on complement in adjurant and carrageenan-induced inflammation in rats[J]. Pharm Pharmacol, 1995, 47(7): 585.
[7]张东方,黄炜,黄济群,等.齐墩果酸抗人肺癌细胞增殖、侵袭和诱导细胞凋亡的研究[J].肿瘤防治研究, 2003, 30(3): 180-183.
[8] Huang Dan, Ding Yan, Li Yan, et al. Anti-tumor activity of a 3-oxo derivative of oleanolic acid[J]. Cancer Letters, 2006, 233: 289-296.
[9] Ovesna Z, Vachalkova A, Horvathova K, et al. Pentacyclic triterpenoic acids: new chemoprotective compounds[J]. Minireview Neoplasma, 2004, (5): 327-333.
[10] Martin-Aragon S, De las Heras B, Sanchez-Reus M I, et al. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride- induced liver damage in rats and primary cultures of rat hepatocytes[J]. Exp Toxicol Pathol, 2001, 53(2-3): 199-206.
[11] Saraswat B, Visen PK, Agarwal DP. Ursolic acid isolated from Eucalyptus tereticornis protect against ethanol toxicity in isolated rathepatocytes[J]. Phytother Res, 2000, 14(3): 163-166.
[12]熊筱娟,陈武,肖小华,等.乌索酸与齐墩果酸对小鼠实验性肝损伤保护作用的比较[J].江西师范大学学报(自然科学版), 2004, 28(6): 540-543.
[13] Chattopadhyay D, Arunachalam G, Mandal AB, et al. Antimicrobial and anti-inflammatory activity of folklore: Mallotus peltatus leaf ex-tract[J]. J Ethnopharmacol, 2002, 82(2-3): 229-237.
[14]王茜,樊明文,边专,等.熊果酸的提取及其对牙周病原菌的作用[J].中华口腔医学杂志, 2002, 37(5): 388-390.
[15]陈国宝,陈宝田.番石榴叶提取物体外抗轮状病毒的实验研究[J].中国医药学报, 2002, 17(8): 502-504.
[16] Kashiwada Y, Nagao T, Hashimoto A, et al. Anti-AIDS agents38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives[J]. J Nat Prod, 2000, 63(12): 1619-1622.
[17] Somova LO, Nadar A, Rammanan P, et al. Cardiovascular, antihyperlipidemic and antioxidant efiects of oleanolic and ursolic acids in experimental hypenension[J]. Phytomedicine, 2003, 10(2 -3): 115-121.
[18] Chung TK, Heo HJ, Kim EK, et al. Inhibitory effect of ursolic acid purined from Origanum majorana L on the acetylcholinesterase[J]. Mol Cells, 2001, 11(2): 137-143.
[19]张士勇,陈林红.薄层扫描法测定酒制木瓜中熊果酸的含量[J].中医药临床杂志, 2006, 18(4): 408-409.
[20]都述虎,饶金华,耿武松.薄层扫描法测定木瓜中齐墩果酸的含量[J].中草药, 2003, 34(1): 35-37.
[21]黄建林,张展霞. GC-MS同时测定番石榴叶中的齐墩果酸和熊果酸[J].分析测试技术与仪器, 2005, 11(3): 178-181.
[22]邓延倬,何金兰.高效毛细管电泳[M].北京:科学工业出版社, 1996:100-101.
[23] Hsieh YZ, Huang HY. Determination of Saikosaponins by Micellar Electrokinetic Capillary Chromatography [J]. Journal of Chromatography A, 1997, 759:193-201.
[24] Glockl I, Veit M, Blaschke G. Determination of Ginsenosides from Panax ginsengUsing Micellar Electrokinetic Chromatography [J]. Planta Medica, 2002, 68(2): 158-161.
[25]王瑞,王淑美,梁生旺,等.胶束电动毛细管色谱法分离分析山茱萸中齐墩果酸和熊果酸[J].中药材, 2007, 30(8): 946-950.
[26]刘春霞,朱培仪,宋粉云.白花蛇舌草中齐墩果酸和熊果酸的毛细管电泳高频电导法测定[J].中药材, 2008, 31(8): 1176-1178.
[27]王雨梅,杜瑞芳,刘卫东,等. HPLC法同时测定毛建草中齐墩果酸和熊果酸的含量[J].西北药学杂志, 2008, 23(2): 84-85.
[28]冯瑜娟,张艺. HPLC-ELSD法测定藏药翼首草中齐墩果酸和熊果酸的含量[J].成都中医药大学学报, 2007, 30(3): 54-56.
[29]陈武,熊筱娟,李开泉,等.乌索酸的化学、药理及临床研究[J].宜春医专学报, 2001, 13(2): 123-126.
[30]张登科. 35种植物的齐墩果酸含量比较[J].中药材, 1989, 12(12): 19.
[31] Atkinson CJ, Dodds PAA, Ford YY, et al. Effects of Cultivar, Fruit Number and Reflected Photosynthetically Active Radiation on Fragaria xananassa Productivity and Fruit Ellagic Acid and Ascorbic Acid Concentrations [J]. Annal of Botany, 2006, 97: 429-441.
[32] Pomar F, Novo M, Masa A. Varietal Differences among the Anthocyanin Profiles of 50 Red Table Grape Cultivars Studied by High Performance Liquid Chromatography [J]. Journal of Chromatography A, 2005, 1094: 34-41.
[33]王向红,刘孟军. HPLC法测定不同品种枣及酸枣中的齐墩果酸和熊果酸[J].食品科学, 2002, 23(6): 137-138.
[34] Zhou CH, Chen KS, Sun CD, et al. Determination of Oleanolic Acid, Ursolic Acid and Amygdalin in the Flower of Eriobotrya japonica Lindl. by HPLC[J]. Biomedical Chromatography, 2007, 21: 755-761.
[35]邹盛勤,陈武.高效液相色谱-光电二极管阵列检测器联用检测不同产地荆芥中乌索酸和齐墩果酸[J].光谱实验室, 2007, 24 (5): 972-975.
[36]刘名权,邹盛勤,陈武.超声辅助HPLC法测定毛泡桐中乌索酸和齐墩果酸含量[J].广东微量元素科学, 2007, 14(5): 19-22.
[37] Wang HH, Wang ZZ, Guo WB. Comparative Determination of Ursolic Acid and Oleanolic Acid of Macrocarpium officinalis (Sieb. et Zucc.) Nakai by RP-HPLC[J]. Industrial Crops and Products, 2008, 28: 328-332.
[38] Brown PD, Tokuhisa JG, Reichelt M, et al. Variation of Glucosinolate Accumulation among Different Organs and Developmental Stages of Arabopsis thaliana [J]. Phytochemistry, 2003, 62: 471-481.
[39]邹盛勤. RP-HPLC法同时测定不同采收期连翘中乌索酸和齐墩果酸的含量[J].沈阳药科大学学报, 2007, 24(3): 164-166,171.
[40]戚志华,王四旺. HPLC法测定陕西女贞子中齐墩果酸与熊果酸的含量[J].中国中医药信息杂志, 2007, 14(5):40-42.
[41]朱鹏程,于瑞涛,陶燕铎,等.藏药花锚中齐墩果酸和熊果酸含量测定[J].分析试验室, 2008, 27: 54-55.
[42]邹盛勤,黄宏伟.反相高效液相色谱-光电二极管阵列检测器法测定金樱子中乌索酸和齐墩果酸[J].食品研究与开发, 2007, 28(10): 130-133..
[43] Du SH, Chen LN, Ma KF, et al. Qualitative and Quantitative Analysis of Chinese Herb Fructus Chaenomelis [J]. Journal of Nanjing Mecical University, 2007, 21(1): 51-54.
[44]杨勇,阮小凤,王仁梓,李高潮.柿种质资源及育种研究进展[J].西北林学院学报, 2005, 20(2): 133-137.
[45]张雅利,郭辉,田忠民.柿子的药理作用研究及临床应用[J].中成药, 28(5): 720-722.
[46] Cui T, Li JZ, Kayahara H, Ma LG, Wu LX, Nakamura, K. Quantification of the polyphenols and triterpene acids in Chinese hawthorn fruit by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry, 2006, 54(13): 4574-81.
[47] Guo S, Duan J, Tang Y, Su S, Shang E, Ni S, Qian D. High-performance liquid chromatography-Two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis. Journal of Pharmaceutical and Biomedical Analysis, 2009, 49(5): 1296-1302.
[48]盛艳乐,赵大球,周春华,陶俊.植物熊果酸和齐墩果酸分析的研究进展[J].中国农学通报, 2009, 25(23): 347-350.
[49] Atkinson CJ, Dodds PAA, Ford YY, Le Mière J, Taylor JM,Blake PS and Paul N. Effects of cultivar, fruit number and reflected photosynthetically active radiation on Fragaria xananassa productivity and fruit ellagic acid and ascorbic acid concentrations.Annals of Botany, 2006,97: 429-441.
[50] Pomar F, Novo M and Masa A. Varietal differences among the anthocyanin profiles of 50 red table grape cultivars studied by high performance liquid chromatography. Journal of Chromatography A, 2005, 1094: 34-41.
[51] Brown PD, Tokuhisa JG, Reichelt M and Gershenzon J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry, 2003, 62: 471-481.
[52]郝宏蕾,朱旭芬,曾云中.类异戊二烯的生物合成及调控[J].浙江大学学报(农业与生命科学版), 2002, 28(2): 224-230.
[53]陈大华,叶和春,李国凤,等.植物类异戊二烯代谢途径的分子生物学研究进展[J].植物学报, 2000, 42(6): 551-558.
[54]黄志伟.人参(Panax japonicus C.A.Mey.)SQS基因cDNA的克隆及序列分析[D].福建农林大学, 2005, 43-44.
[55]卢虹玉,刘敬梅,阳文龙,高山林.甘草鲨烯合成酶基因的分离及植物表达载体的构建.药物生物技术, 2007, 14(4): 225-258.
[56]黄志伟,林明,何水林,郑金贵.葡萄总RNA提取及SQS基因保守区域的克隆.武夷科学, 2006, 22(12): 169-171.
[57]钟家禹.灵芝鲨烯合成酶基因的克隆与表达特性初步分析.南京农业大学学位论文[D]. 2001.
[58]赵云生,万德光,陈新,李占林,田洪岭.五环三萜皂苷生物合成与调控的研究进展[J].中草药, 2009, 40(2): 327-330.
[59]徐昌杰,陈昆松,张波,等.柑橘组织RNA提取方法研究[J].果树学报, 2004, 21(2): 136-140.
[60] Lee MH, Jeong JH, Seo JW, et al. Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol, 2004, 45(8): 976-984.
[61]克丙申,黄象艳,陈世敏,等. PCR扩增试验的动力学数学模型[J].遗传,2002, 24(1): 55-59.
[62] Chio DW, Jung J, Ha YI, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairyroots to identify genes involvedinthe biosynthesis of ginsenosides and other secondary metabolites[J]. Plant Cell Rep, 2005, 23(8): 557-566.
[63] Suzuki H, Achnine L, Xu R, et al. A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula[J]. Plant J, 2002, 32(6):1033-1048.
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