摘要
中药委陵菜(Potentilla chinensis Ser.)为蔷薇科(Rosaceae)委陵菜属(PotentillaL)植物的干燥全草,在全国大部分地区都有分布,药材主产于山东、辽宁、安徽。其化学成分主要含有黄酮类、三萜类、甾体类、酚类及其衍生物,具有清热解毒,凉血止痢等功效,用于赤痢腹痛、久痢不止、痔疮出血、痈肿疮毒,是被中华人民共和国药典(2005版)收录的唯一一种委陵菜属植物。
目前,由于委陵菜与其近缘植物翻白草(Potentilla discolor Bge.)的植物形态特征非常相似,民间将这两种药用植物混用来治疗2型糖尿病。据文献报道翻白草具有较好的降血糖药理活性,且降血糖作用十分明确。本课题组在前期的研究中发现,从委陵菜中分离得到的化合物与翻白草中分离得到的化合物种类相似。为了探究民间将委陵菜与翻白草混用来治疗糖尿病是否有科学依据,本文对委陵菜进行了降血糖药理活性的筛选。
本文对降血糖活性较强的委陵菜乙酸乙酯萃取部位进行了分离、提取及结构鉴定。采用快速减压柱层析、硅胶开放柱色谱、ODS反相柱色谱及SephadexLH-20凝胶过滤等手段分离得到了3个化合物,鉴定结构为芹菜素、槲皮素和翻白叶苷A。
建立了高效液相色谱同时测定委陵菜中3中黄酮类成分(槲皮素、翻白叶苷A、芹菜素)的含量分析方法。以ZORBAX SB-C18(4.6 mm×150mm,5μm)为色谱柱,甲醇-0.3%磷酸水溶液(51:49)为流动相,0.9 mL/min的流速在360 nm检测波长下进行检测。结果,槲皮素、翻白叶苷A和芹菜素的进样量分别在0.72-10.08μg/mL(r=0.999 9)、0.86-12.04μg/mL(r=0.999 9)和0.54-7.56μg/mL(r=0.9999)范围内与其峰面积呈良好的线性关系,平均回收率分别为95.37%(RSD=1.79%)、96.87%(RSD=1.63%)和97.66%(RSD=2.08%),药材中三中黄酮的含量分别为100.0μg/g、323.0μg/g和55.0μg/g。建立起来的委陵菜黄酮类成分的含量测定方法为委陵菜的质量控制奠定基础。
槲皮素、翻白叶苷A和芹菜素为评价指标,考察乙醇浓度(A)、提取时间(B)、溶剂用量(C)、提取次数(D)四个因素,三个水平,应用L_9(3~4)正交设计实验方案优化了委陵菜总黄酮的提取工艺。
初步的抗糖尿病药效学研究表明,委陵菜的不同方法提取物均对四氧嘧啶诱导的糖尿病小鼠有不同程度的降血糖活性。其中,委陵菜水提液和乙酸乙酯萃取部分的降血糖活性具有显著性差异。
本科课题不仅丰富了对委陵菜降血糖有效部位的确定、黄酮类成分含量测定研究及总黄酮提取工艺的研究,而且为进一步研究开发委陵菜中药资源提供了科学依据。本课题为
基金项目:上海SK研究与发展基金(2003010-t)
Traditional Chinese medicine Potentilla chinensis Ser is the Rosaceae the Potentilla L plant's dry entire grass. It has the distribution in the national most districts, while mainly produces in Shandong, Liaoning and Anhui.Its chemical composition mainly contain flavonoids, triterpenes, steroid category, phenols and derivatives with Qingrejiedu, Liangxue Zhilishen such as effectiveness, for dysentery abdominal pain, more than Jiuli, bleeding hemorrhoids, sore swollen Carbuncle drugs. It is the only plant of Potentilla chinensis Ser which is included by the People's Republic of China pharmacopoeia(2005 editions).
At present, because of its relative Potentilla food plants Fanbai grass (Potentilla discolor Bge.) morphological characteristics of plants are very similar, some people mix these two medicinal plants to treat type 2 diabetes. There are many of the reported Fanbai grass hypoglycemic pharmacological activity of, and hypoglycemic effect is very clear. According to the study of the pre-discussion group, Potentilla dishes from the isolated compounds Fanbai grass and isolated the compounds, basically the same results. Therefore, this paper was commissioned Potentilla hypoglycemic pharmacological activity of the screening.
In this thesis, three compounds have been isolated from the ethyl acetate extract of Potentilla chinensis Ser by repeatedly chromatography on flash chromatography, Sephadex LH-20, normal and reversed-phase silica gel columns. On the basis of the chemical evidence and spectra analysis data, the structures of three compounds were apigenin, quercetin and potengriffioside A.
To develop a HPLC method to determine the content of 3 flavonoids in Potentilla chinensis Ser. The linear ranges of quercetin, potengriffioside A and apigenin were 0.72～10.08μg/mL, 0.86～12.04μg/mL and 0.54～7.56μg/mL, respectively (r= 0.999 9), the average recovery is 95.37%, 96.87% and 97.66%, the RSD is 1.79%, 1.63% and 2.08%, respectively; the content of 3 flavonoids in the herb is 100μg/g, 323μg/g and 55 μg/g, respectively. The methods established for determing total flavonoids of Potentilla chinensis Ser would lay a foundation for the quality control of Potentilla chinensis Ser。
We choosed main effective components quercetin, potengriffioside A and apigenin as standard substance and determined the quality of Potentilla chinensis Ser with RP-HPLC. We also reviewed the factors that influenced the extraction of the content of total flavonoids in Potentilla chinensis Ser by orthogonal test design, further established the optimum extracting and separating procedure of total flavones from Potentilla chinensis Ser.
Preliminary pharmacodynamical test showed that the compounds isolated from Potentilla chinensis Ser with different methods had lowering blood glycose bioactivities on Alloxan induced diabetics rats, the water extract from Potentilla chinensis Ser was significant, has statistics significance.
This thesis can help people to recognize the quality standard of Potentilla chinensisSer and also apply to the science research and development of Potentilla chinensis Ser.
引文
[1]Diabetes Mellitus, World Heslth Organization Fact Sheet, No. 138,1999,1-5
[2]Harris, M.I.; F legal, K.M.; C owie, C.C. et a 1. P revelenceo fd iabetes, im pairedf astingg lucose, and impaired glucose tolerance in U.S. adults. The third National Health and Nutrition Examinations urvey, 19 88-1994. D iabetesC are1998, 21,518-524.
[3]Bergstrom RW, Leonetti DL, Newell-Morris LL, et al. Association of plasma triglyceride and C-peptide with coronary heart disease in Japanese-American menwitha h ighp revalenceo fglucosein tolerance. Diabetologia, 1990; 33(8):489-496
[4]江苏新沂学院.中药大辞典[M].上海人民出版社,1977:1369
[5]张勇,李鹏,李彩霞,等.委陵菜属药用植物[J].中兽医医药杂质,2005,2(4):60-63
[6]国家药典委员会.中华人民共和国药典2005年版,一部[S].北京化学工业出版社,2005:149
[7]洪花,李善花,朴丽花,等.翻白草对实验性糖尿病大鼠治疗作用的研究[J].辽宁中医药大学学报,2007,9(1):155-157
[8]洪花.翻白草的降血糖作用及其机制的实验研究[D].延吉:延边大学医学部.2006
[9]王晓敏,王健红,徐冬平,等.翻白草水提液对小鼠降血糖作用[J].江西中医学院学报.2005,17(2):53-54
[10]王琦,周玲仙,罗晓东,等.翻白草不同方法提取物对小鼠降血糖作用[J].中国公共卫生,2007,23(2):225
[11]张萍.勿将委陵菜混作翻白草治疗糖尿病[J].中国中药杂志,2004,29(8):811-812
[12]沈阳,王庆贺,林厚文,等.委陵菜化学成分的研究[J].中药材,2006,29(3):237-239
[13]王庆贺,李志勇,沈阳,等.委陵菜三萜类化学成分研究[J].中国中药杂志,2006,31(17):1434-1436
[14]高雯,沈阳,张红军,等.委陵菜的化学成分研究[J].药学服务与研究, 2007,7(4):262-264
[15]薛培风,尹婷,梁鸿,等.翻白草化学成分研究[J].中国药学杂志,2005,40(14):1052-1054
[16]Xue, P. F; Lu, X. H; Wang, B; Liang, H; Zhao, Y. Y; The megastigman glycosides from herb of potentilla multifida. Zhongguo Zhong Yao Za Zhi,2005,30(13): 983-985
[17]张颖,张立木,赵雪梅,等.翻白草中化学成分的研究[J].泰山医学院,2007,28(3):168-169
[18]Potier, P. D, Bhupeshchandra; Bui, Anne M; Janot Maurice M; Pourrat, Aimee;pourrat, Henri.C.N.R.S. Gif-sur-Yvette, Fr, Structure of tormentic acid, a pentacyclic triterpene acid isolated from the roots of Potentilla multifida de la Societe Chimique de France, 1996, (11): 3458-2465
[19]邹忠杰,杨峻山,鞠建华.泥胡菜化学成分的研究[J].中草药,2006,37,(9):1303-1304
[20]张雁冰,王克让,刘宏民,等.马桑叶中黄酮类化合物的分离提取及结构鉴定[J].中草药,2006,37(3):341-343
[21]钟海军,陈继军,王惠英,等.翻白叶的化学成分[J].中草药,2000,31(7):488-490
[22]吴晓明、段斌志.银杏叶黄酮类化合物的测定方法[J].药物分析杂志,2001,21(2):138-141
[23]付玉杰,刘威,侯春莲,等.RP-HPLC/二极管阵列检测器同时测定飞机草中3中黄酮[J].应用化学,2007,24(12):1452-1453
[24]刘思曼,边清泉.RP-HPLC法测定翻白草中山奈酚、槲皮素的含量[J].绵阳师范学院学报,2006,25(5):37-39
[25]张敏,邓丽菊,垂文珍,等.RP-HPLC测定复方槲皮素片中槲皮素、芹菜素的含量[J].辽宁中医学院学报,2002,4(2):149-150
[26]张元桐,翟延君,曲静,等.HPLC测定水红花子中槲皮素的含量[J].中成药,2006,28(2):236-238
[27]王仁安.医学实验设计与统计分析[M].北京:北京医科大学出版社,1999,12
[28]D. Syiem, G S, P.Z. Khup, Hypoglucemic effects of Potentilla fulgens L. in normal and alloxaninduced diabetic mice. Journal of Ethnopharmacology 2002,(83): 55-61
[29]黄爱玲.黄酮类化合物药理作用研究进展[J].安徽农学通报,2007,13(10):71-72
[30]陈勇,张晴.五羟基黄酮的药理学研究近况[J].中草药,1998,29(8):569-570
[31]徐向进,张家庆,黄庆玲.槲皮素对糖尿病大鼠肾脏非酶糖化及氧化的抑制作用[J].中华内分泌代谢杂志,1998,14(1):34
[1]中国植物志编委会.中国植物志(第三十七卷)[M].北京:科学出版社,1985,223-331
[2]张勇,王一峰,国产委陵菜属植物资源[J].西北师范大学学报,1998,34(1):59-62
[3]张勇,李鹏,李彩霞,等.委陵菜属药用植物[J].中国兽医医药杂志,2005,(2):60-63
[4]Xue, P. F; Lu, X. H; Wang, B; Liang, H; Zhao, Y. Y; The megastigman glycosides from herb of potentilla multifida. Zhongguo Zhong Yao Za Zhi, 2005,30(13):983-985
[5]Dicke, M. V. B, T. A; Posthumus, M. A; Dom, N. Ben; Van Bokhoven, H; De Groot, A. E, Isolation and identification of volatile kairomone that affects acarine predato-prey interactions: involvement of host plant in its production. Journal of Chemical Ecology, 1990,16 (2): 381-396
[6]陈芳群,委陵菜化学成分的研究[J].华西药学杂志,1989,4(1):6-8
[7]王庆贺,李志勇,沈阳,等.委陵菜三萜类化学成分研究[J].中国中药杂志,2006,31(17):1434-1436
[8]沈阳,王庆贺,林厚文,等委陵菜化学成分的研究[J].中药材.2006,29(3):237-239
[9]高雯,林后文,张红军,等.委陵菜的化学成分研究[J].药学服务与研究,2007,7(4):262-264
[10]Ganenko, T. V. S, A. A. Tashkent. Gos. Univ. Tashkent, USSR, Chemical composition of potentilla fruticosa.Ⅱ. Triterpenoids. Khimiya Prirodnykh Soedine nii, 1989, (6): 856
[11]Bilia, A. R. P, E; Catalano, S; Flamini, G; Morelli, I. Dip. Chim. Bioorg. Univ.Stud. Pisapisa, Italy, New trierpenoid saponins from the roots of roots df Potentilla tormentilla. Jourmal of Natural Products, 1994, 57(3): 333-338
[12]钟海军,陆继军,王惠英,等.翻白叶的化学成分[J].中草药,2003,31,(7): 488-490
[13]Li, Q. H,Jing; Shang, Dejing; Wu, Lijun; Ma, Xiaochi, Investigarion of the chemical constituents of the roots of Potentilla anserine L. in Tibet. Chinese Pharmaceutical Journal(Taipei, Taiwan) 2003,55(3): 179-184
[14]薛培凤,桥梁,梁鸿,等.翻白草化学成分研究[J].中国药学杂志,2005,40(14):1052-1054
[15]薛培凤,桥梁,梁鸿,等.多裂委陵菜化学成分研究[J].北京大学学报(医学报),2004,36(1):21-23
[16]Li, p. L. Z, Zhan Xin; Jia, Zhong Jian, A new taraxastane-type tritepenoid from Potentilla multicaulis. Chinese Chemical Letters, 2006,17(12): 1573-1575
[17]Li, P. L; Lin, C. j; Zhang, Z. X; Jia, Z. J, Three new tritepenoids from Potentilla multicaulis Chemistry & Biodiversity, 2007,4(1): 17-24
[18]Potier, P. D, Bhupeshchandra; Bui, Anne M; Janot Maurice M; Pourrat, Aimee;pourrat, Henri.C.N.R.S. Gif-sur-Yvette, Fr, Structure of tormentic acid, a pentacyclic triterpene acid isolated from the roots of Potentilla.Bulletin de la Societe Chimique de France, 1996, (11): 3458-2465
[19]张彦文,姚智,刘段.委陵菜三萜成分研究[J].中国中药杂志,2006,31(22):1875-1879
[20]Stachurski, L; Bednarek, E; Dobrowolski, J. C; Srezelecka, H; Mazurek, A. P,Tormentoside and 2 of Its Isomers Obtained from the Rhizomes of Porentilla-Erecta. Planta Medica, 1995, (1): 94-95
[21]Omurkamzinova, V. B; Erzhanova, M. S, Flavans of Ptentilla-Erecta Rootstocks.Khimiya Prirodnykh Soedinenii 1986, (3): 374-375
[22]Zhang, B; Huang, Q; Zhang, H. Y; Li, C. S; Lu, L; Nonaka, G I; Nishioka,I,Studies on Tannin Constituents of Root from Potentilla-Viscosa. Chinese Science Bulletin, 1990, 35(3): 239-243
[23]Herrmann,K. U. H., Tannin and flavones from the leaves of Fragatia vesca.Pharmazeutische Zentralhalle fuer Deutschland, 1949, (88): 374-378
[24]Miliauskas,G; van Beek, T. A.; Venskutonis, P. R.; Linssen, J. P. H.; de Waard, P.;Sudholter, E. J., Antioxidant activity of Potentilla fruticosa. Journal of the Science of Food and Agriculture, 2004, 84(15): 1997-2009
[25] Kombal, R; Glasl, H., Flavan-3-OIs and Flayonoids from Potentilla-Anserina.Planta Medica, 1995, 61(5): 484-485
[26] Goncharov, N. F; Stupakova, E. P; Komissarenko, N. F, Polyphenol Composition of the Epigeal Part of Porentilla-Erecta. Khimiya Prirodnykh Soedinenii, 1989,(3): 431-432
[27] Goncharov, N. F; Stupakova, E. P; Komissarenko, N. F, Flavonoids of Potentilla-Erecta.Khimiya prirodnykh Soedinenii, 1989, (2): 286-287
[28] Geiger, C; Scholz, E; Rimpler, H, Ellagitannins from Alchenilla-Xanthochlora and Porentilla-Erecta. Planta Medica, 1994, 60(4): 384-385
[29] Tomczyk, M, Secondary metabolites from Potentilla argentea. Biochemical Systematics and Ecology, 2006, 34(10): 770-773
[30] Choi, Y-H.K, Myong-Jo;Lee Haeng-Soon; Yun, Bong-Sik; Hu, Changxu;Kwak,Sang-Soo, Antioxidatiye compounda in aerial parts of Potentilla fragarioides, Ssengyak Hakhoechi, 1998, 29(2): 79-85
[31] Terashima, S; Shimizu, M; Nakayama, H; Ishikura, M; Ueda, Y; Imai, K; Suzui,A; Morita, N, Studies on Aldose Redutase Inhibitors from Natural-Products. 3.Studies on Aldose Peductase Inhibitors from Medicinal Plant of Sinfito Potentilla-Candicans, and Further Synthesis of Their Related-Compounds,Chemical&Pharmaceutical Bulletin, 1990, 38(10): 2733-2736
[32] Lund, K R, Horst. Freibug, Fed. Rep, Ger, Tormentilla, root, Isolation of an ellagitannin and pharmacological screening, Deutsche Apotheker Zeitung, 1985,125 (3): 105-108
[33] Okuda, T. Y, Takashi; Kuwahara, Memon, M. Usman; Shingu, Tetsuro,Tannins of rosaceous medicinal plants. I. Structures of potentillin, agrimonic acids A and agrimoniin a dimeric ellagitannin. Chenical & Pharmaceutical Bulletin, 1984,32(6): 2165-2173
[34] Huang, Q; Zhang, B; Zhang, H. Y; Li, C.S; Lu, L; Ninaka, G; Nishioka, I,Studies on Constiruents of Tannin from Potentilla-Viscosa J Don. Acta Chimica Sinica, 1990, 48(8): 825-829
[35]Fedoseeva, G M, Flauonoid Compounds of Potentilla Fruticosa. Khimiya Prirodnykh Soedinenii, 1979, (4):'575-576
[36]蔡玉华,刘梁,韩丁献,等.三叶委陵菜根化学成分的研究[J].武汉理工大学学报,2005,27(9):32-33
[37]娄宁,李亚,李瑜.茅银露梅挥发性化学成分研究[J].兰州大学学报,2004,40(4):58-61
[38]Ovodova, R. G; Bushneva O. A; Shashkoy, A. S; Chizhov, A. O; Ovodov, Y. S,Structural studies on pectin from marshi cinquefoil Comarum palustre L.Biochemistry (Mosc), 2005,70(8): 867-877
[39]D. Syiem, G S, P.Z. Khup, Hypoglucemic effects of Potentilla fulgens L. in normal and alloxaninduced diabetic mice. Journal of Ethnopharmacology, 2002,(83): 55-61
[40]王琦,周玲仙,罗晓东,等.翻白草不同方法提取物对小鼠降血糖作用[J].中国公共卫生,2007,23(2):225
[41]郭新民,崔荣军.翻白草对2型糖尿病血清胰岛素和胸腺指数的影响[J].牡丹江医学院学报,2004,25(4):1-3
[42]郭新民,崔荣军,申梅淑,等.翻白草对2型糖尿病大鼠血脂代谢紊乱的影响[J].牡丹江医学院学报,2005,26(2):4-6
[43]崔荣军,李怀慧,郭新民.翻白草对2型糖尿病大鼠血糖的影响[J].中国临床康复,2005,9(2):84-85
[44]孟令云,朱黎霞,杜慧.翻白草对家兔血糖影响的研究[J].中医药学报.,2001,29(4):35
[45]洪花,李善花,朴丽花,等.翻白草对实验性糖尿病大鼠治疗作用的研究[J].辽宁中医药大学学报,2007,9(1):155-157
[46]韩永明,段妍君,袁芳,等.翻白草对糖尿病大鼠胰岛形态结构的影响[J].湖北中医学院学报,2005,7(3):28-29
[47]韩永明,段妍君,袁芳,等.翻白草对糖尿病大鼠血管内皮细胞一氧化氮合酶表达的影响[J].中国中医药信息杂志,2006,13(6):41-42
[48]边可君,徐辉碧,黄开勋,等.三叶委陵菜乙醇提取物阵痛作用的研究[J]. 江苏临床医学杂志,2002,6(3):200-203
[49]Ilhan Gurbuz, A. M. O, Erdem Yesilad, Anti-ulcerogenic activity of some plants used in folk medicine of Pinarbasi(Kayseri, Turkey). Journal of Ethnopharmacology, 2005,101,313-318
[50]万永红,周向宁.八种中草药抑菌试验报告[J].生物学杂志,1999,16(2):31-32
[51]张立新,杭瑚,王宗花,等.21种野生植物抗氧化活性的研究[J].中草药,2000,31(8):609-610
[52]G Miliauskas, P. R. V, T. A. van Beek Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry, 2004, (85):231-237
[53]辽宁省北镇县中安地区医院.委陵菜防治细菌性痢疾的初步观察[J].中国临床医生.1973,(2):65-67
[54]万孝臣,王永发,李玉海.委陵菜治疗急性肠炎和菌痢[J].现代医学,1979,(4):247
[55]苏广双,苏文盛,苏世文,等.委陵菜抗菌活性成分的研究[J].沈阳药学院学报,1986,3(28):195-196
[56]宋宇.委陵菜提取液的抑菌作用[J].安徽农业科学,2007,35(8):2207-2256
[57]向红.民间草药西南委陵菜抗菌作用的实验[J].贵州师范大学学报(自然科学版),2003,21(2):55-57
[58]陈孙能,黄龙飞委陵菜灭滴灵治疗肠道鞭毛虫病疗效对比观察[J].福建中医药,1995,26(01):5-6
[59]刘梁,韩丁献,周军,等.三叶委陵菜根中三萜类化合物抗病毒作用研究[J].时珍国医药,2006,17(8):1484-1485
[60]张巧玲,杨占秋,陈科力,等.4种药用植物提取物体外抗柯萨奇病毒B3作用的研究[J].武汉大学学报(医学板),2005,26(2):157-160
[61]蔡光明,赵艳玲,袁海龙,等.鹅绒委陵菜有效部位(总皂苷)的分离与抗鸭乙肝病毒(DHBV-DNA)作用[J].中南药学,2003,1(1):17-21
[62]A. R. McCutcheon, T. E. R., E. Gibbons, et al.,Antiviral screening of British Columbian medicinal plants. Journal of Ethnopharmacology, 1995, (49):101-110
[63]边可君,黄开勋,徐辉碧.三叶委陵菜对四氯化碳致小鼠肝损伤保护作用[J].时珍国医药,2001,14(4):294-295
[64]李贞,张铁权,叶量,等.委陵菜对四氯化碳致小鼠肝损伤保护作用[J].辽宁中医药杂志,2004,31(5):422-423
[65]姜秀莲,曲淑岩,毋英杰,等.粘液委陵菜对小鼠肝损伤的保护作用[J].吉林中医药,1987,(4):34
[66]琳娜,李建荣,杨滨,等.蕨麻对免疫功能低下小鼠免疫功能的影响[J].中国中医药信息杂志,1999,6(2):35-36