摘要
本文的水飞蓟宾卵磷脂复合物的生物药剂学研究,从以下五方面获得了具有科学意义和应用价值的研究结果。
1.建立了用于制备水飞蓟宾A和水飞蓟宾B对照品的制备高效液相色谱法(pre-HPLC法),并建立了水飞蓟宾卵磷脂复合物(SPC)中水飞蓟宾A和B各自的高效液相测定法。此法简单,准确,可用于水飞蓟宾/素及其制剂中水飞蓟宾A和B的测定,为水飞蓟宾/素及其制剂更为严格的质量控制提供了技术保证。
2.分别建立了大鼠和人血浆中水飞蓟宾A和B测定的HPLC/MS/MS法,并用所建方法分别进行了SPC中水飞蓟宾A和B在大鼠和人体内的药代动力学研究。结果显示水飞蓟宾A和B在大鼠体内的绝对生物利用度分别为2.74%和1.82%;在大鼠实验的3个剂量组下,水飞蓟宾A和B均呈线性动力学特征;在大鼠实验中,药代动力学参数在水飞蓟宾A和B间均无显著性差异,但水飞蓟宾A和B在人体内有不同的药代动力学特征。
3.建立了大鼠组织匀浆中水飞蓟宾A和B测定的HPLC/MS/MS测定法,并对大鼠灌胃给予水飞蓟宾56mg/kg后,脑、心、肺、肝、脾、肾、胃、小肠、睾丸、子宫及卵巢各组织匀浆中水飞蓟宾A和B的浓度进行了测定。结果显示,在3个时间组(15min、1h和3h),胃,肠和肝中水飞蓟宾A和B的浓度均较高。
4.把水飞蓟宾作为一个整体,建立了人血浆中水飞蓟宾测定的HPLC-UV法,并用所建立的方法进行了SPC在人体内的药代动力学和相对生物利用度研究,结果显示在人体内水飞蓟宾呈线性动力学特征;SPC胶囊相对于水飞蓟素胶囊和水飞蓟宾葡甲胺片的生物利用度分别为270.4士139.6%和125.3±46.4%。
5.以小鼠和大鼠为实验动物,对SPC抗四氯化碳致急性肝损伤的保护作用进行了研究。结果显示SPC抗急性肝损伤的作用强于水飞蓟宾/素或其制剂,也强于卵磷脂;卵磷脂对水飞蓟宾的抗急性肝损伤具有协同作用,其不仅通过提高水飞蓟宾的生物利用度增强药效,而且其自身也具有保肝作用。
Through the biopharmaceutical studies of silybin– phosphatidylcholine complex (SPC), valuable results that are both academic and practical were achieved as follows:
1. A novel preparative HPLC method separating silybin was developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple and accurate HPLC method was developed to determine silybin A and silybin B, respectively, which supplied a strong support for the accurate determination of silybin A and silybin B in silybin/silymarin or their preparations
2. A simple,sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry(LC/MS/MS) method was developed for the quantification of silybin A and silybin B in rat and human plasma, and applied to the stereospecific analysis of silybin in plasma samples from pharmacokinetic studies in rat and human. The absolute bioavailability of silybin A and B in rat were 2.74% and 1.82%, respectively. The dose-independent pharmacokinetics was observed at three different administered doses to rat . In human, pharmacokinetic parameters between silybin A and B were different although those showed no significant difference between silybin A and B in rats.
3. A simple, sensitive, selective and reproducible LC/MS/MS method was developed for the quantification of silybin A and silybin B in different tissues of rat, such as brain, heart, lung, liver, spleen, kidney, stomach, intestine, testicle, uterus and ovary. It was found that the most concentrations of silybin A and B were detected in the stomach, intestine and liver at three different times (15min, 1h and 3h).
4. Taking silybin as a single entity, a new HPLC-UV method of determining silybin in human plasma was developed, and using the new developed method the pharmacokinetic of SPC was studied in healthy male Chinese volunteers. The dose-independent pharmacokinetics was observed after a single oral administration of SPC at three different doses. Through the study of relative bioavailability of SPC, it was showed that relative bioavailabilities of SPC were 270.4±139.6% and 125.3± 46.4% compared to silymarin and silybin meglumine, respectively.
5. The hepatoprotective potential of SPC was evaluated in rat and mice against CCl4-induced liver damage. Through the study, it was found that the hepatoprotective activity of SPC was better than silybin/silymarin or their preparations, and also phosphatidylcholine (PC). The hepatoprotective potential of SPC was improved not only by the improvement of bioavailability of silybin according to complex silybin with PC, also by the synergic action of PC.
引文
1 Kren V, Walterova D. Silybin and silymarin–new effects and applications. Biomed Papers, 2005, 149: 29~41
2朱铉,郑哲珠.水飞蓟素及其制剂的研究进展.中国野生植物资源, 2001, 20:47~49
3 Simanek V, Kren V, Ulrichova J, et al. Silymarin: what is in the name. Hepatology, 2000, 32: 442~443
4 Lee D Y W, Liu Y. Molecular structure and stereochemistry of silybin A, silybin B, isosilybin A, and isosilybin B, isolated from silybum marianum (milk thistle). J Nat Prod, 2003, 66, 1171~1174
5定天明,田颂九,张正行,等.胶束电动毛细管色谱法测定水飞蓟素及其制剂中主要有效成分含量.药学学报, 2000, 35:778~78l.
6中华人民共和国卫生部药典委员会编.中华人民共和国卫生部药品标准:第8册.北京:化学工业出版社, 1992. 154
7李凤前,胡晋红,朱全刚.水飞蓟素固体分散体中总黄酮的测定.中草药, 2002, 33: 31~33
8吴知行,张时行,徐丽蓉,等.水飞蓟宾及其水溶性衍生物和制剂含量测定方法的研究.南京药学院学报,1981,17:28~36
9定天明,田颂九,张正行,等.水飞蓟素制剂中有效成分HPLC分离测定.药物分析杂志,1999, 19: 304~307
10姚水宝,杨水新,施红敏,等.梯度高效液相色谱法分离测定利加隆片中水飞蓟宾的含量.中国药业, 2001,10:13~14
11杨晓云,吴爱英,张春辉. HPLC法测定利加隆片中多组分含量.中药新药与临床药理, 2004, 15: 176~178
12曹志胜,杨晓云,吴爱英,宿洁. HPLC法测定水飞蓟素胶囊中水飞蓟素异构体的含量.中药新药与临床药理, 2006, 17: 200~202
13陈黎,周继勇.水飞蓟宾葡甲胺质量标准研究.中草药, 2002: 1083~1084
14赵迎英,王进旗,杨彬彬,等.高效液相色谱法测定水飞蓟提取物中水飞蓟宾的含量.西北药学杂志, 2005, 20 63~64
15王伟,黄伟静,曾韶辉.水飞蓟宾非对映异构体的HPLC分离测定.药品评价, 2005, 2 280~282.
16傅静芝,上官一平. HPLC法测定益肝灵胶囊中水飞蓟宾的含量.中国药事, 2007, 21 428~429
17 Lee J I, Hsu B H, Wu D, et al. Separation and characterization of silybin, isosilybin, silydianin and silychristin in milk thistle extract by liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr A, 2006, 1116 57~68
18 Lee J I, Narayan M, Barrett J S. Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography–electrospray ionization mass spectrometry. J Chromatogr B, 2007, 845 95~103
19 Morazzoni P, Magistretti M J, Giachetti C, et al. Comparative bioavailability of silipide, a new flavanolignan complex, in rats. Euro J Drug Metab Pharmacokinet, 1992, 17: 39~44
20 Morazzoni P, Montalbetti A, Malandrino S, et al. Comparative pharmacokinetics of silipide and silymarin in rats. Euro J Drug Metab Pharmacokinet, 1993, 18: 289~297
21 Barzaghi N, Crema F, Gatti G, et al. Pharmacokinetic studies on IdB 1016, a silybin-phosphatidylcholine complex, in healthy human subjects. Euro J Drug Metab Pharmacokinet, 1990, 15: 333~338
22 Morazzoni P, Bombardelli E. Silybum marianum. Fitoterapia, 1995, 66: 3~42
23 Rickling B, Hans B, Kramarczyk R, et al. Two high-performance liquid chromatographic assays for the determination of free and total silibinin diastereomers in plasma using column switching with electrochemical detection and reversed-phase chromatography withultraviolet detection. J Chromatogr B, 1995, 670: 267~277
24肖衍宇,宋赘梅,陈志鹏,等.水飞蓟宾磷脂复合物的制备与大鼠生物利用度的研究.药学学报, 2005, 40: 611~617
25 Xiao Y Y, Song Y M, Chen Z P, et al. The preparation of silybin-phospholipid complex and the study on its pharmacokinetics in rats. Int J Pharm, 2006, 307: 77~82
26史向国,钟大放,张逸凡,等.反相高效液相色谱法测定大鼠血浆中水飞蓟宾的浓度.沈阳药科大学学报, 2001, 18: 113~115
27肖莉,翟所迪,赵荣生,等. RP-HPLC测定大鼠血浆中水飞蓟宾的浓度.华西药学杂志, 2002, 17: 428~429
28 Wu J W, Lin L C, Hung S C, et al. Analysis of silibinin in rat plasma and bile for hepatobiliary excretion and oral bioavailability application. J Pharmaceut Biomed, 2007, 45: 635~641
29缪海均,李志远,刘皋林.反相高效液相色谱法测定人血浆中水飞蓟宾的浓度.药物分析杂志, 2000, 20: 399~400
30王霄杨,尹莉芳,周建平,等. HPLC法测定犬血浆中的水飞蓟宾葡甲胺.中国天然药物, 2006, 4: 207~209
31陈玟,刘史佳,张军,等.水飞蓟宾片在犬体内的药动学研究.江苏药学与临床研究, 2005, 13: 1~3
32江波,印春华水飞蓟素磷脂复合物家兔体内生物利用度的研究.中国医药工业杂志, 2002,33: 598~599
33 Kvasnicka F, Biba B, Sevcik R, et al. Analysis of the active components of silymarin. J Chromatogr A, 2003, 990: 239~245
34 Hassan E M, Khamis E F, Kimary E I, et al. Development of a differential pulse voltammetric method for the determination of Silymarin/Vitamin E acetate mixture in pharmaceuticals. Talanta, 2008, 74: 773~778
35 Skottova N, Svagera Z, Vecera R, et al. Pharmacokinetic study of iodine-labeled silibinins in rat. Pharmacol Res, 2001, 44: 247~253
36肖莉,靳淑敏,张丽霞等.水飞蓟素制剂及其应用进展.中国药物与临床, 2005, 5: 205~208
37汤秀珍,侯世祥,贺英菊,王炳南.水飞蓟索增溶方法的比较研究.华西药学杂志, 2001, 16:193~195
38李范珠,康廷国,吕佳,颜凯新.水飞蓟素-β-环糊精包合物的研究.时珍国药研究, 1996, 7:146~147
39丁平田,吴雪梅.药物制剂的新型辅料2-羟丙基-β-环糊精.国外医药:合成药生化药制剂分册, 1996, 17: 107~111
40谢伯泰,杨国武,谢薇梅.羟丙基-β-环糊精特性及其在医药领域中的应用与安全性.国外医药:合成药生化药制剂分册, 2002, 23: 302~306
41陆斌.药物新剂型与新技术.北京:人民卫生出版社, 1998.31~33
42肖莉,翟所迪,赵荣生,等.水飞蓟素羟丙基-β-环糊精包合物在大鼠体内的生物利用度.中国医院药学杂志, 2006, 26: 140~142
43 EI-Samaligy M S, Afifi N N,Mahmoud E A. Evaluation of hybrid liposomes-encapsulated silymarin regarding physical stability and in vivo performance. Int J Pharm, 2006, 319: 121~129
44 EI-Samaligy M S,Afifi N N,Mahmoud E A. Increasing bioavailability of silymarin using a buccal liposomal delivery system: Preparation and experimental design investigation. Int J Pharm, 2006, 308: 140~148
45钟延强,刘善奎,陈国笋,等.水飞蓟素高生物利用度制剂的研究现状.药学实践杂志, 2003, 21: 139~140
46徐希明,李强,朱源,等.水飞蓟宾脂质纳米粒的制备与鼠体内分布研究.中国中药杂志,2005, 30: 1912~1914
47徐希明,朱源,李强,等.水飞蓟宾纳米胶束的制备及其鼠体内药物动力学研究.中国药学杂志, 2005, 40: 1874~1876
48欧阳净,黄华.自乳化释药系统的研究进展.中国药房, 2005, 16: 1499~1501
49 Kang B K, Lee J S, Chon S K, et a1.Development of self-microemulsifying drug delivery systems for oral bioavailabilityenhancement of simvastatin in beagle dogs.Int J Pharm,2004,274:65~73
50 Hong J Y, Kim J K, Song Y K, et a1.A new self-emulsifying formulation of itraconazole with improved dissolution and oral absorption.J Control Release,2006, 110: 332~338
51 Wu W, Wang Y, Que L.Enhanced bioavailability of silymarin by self-microemulsifying drug delivery system.Eur J Pharm Biopharm,2006, 63:288~294
52 Woo J S, Kim T S, Park J H, et a1.Formulation and biopharmaceutical evaluation of silymarin using SMEDDS. Arch Pharm Res, 2007, 30: 82~89
53平其能主编.现代药剂学.第1版.北京:中国医药科技出版社,1998. 599~600
54 Xiao Y Y, Song Y M, Chen Z P, et al. Preparation of silymarin proliposome: a new way to increase oral bioavailability of silymarin in beagle dogs. Int J Pharm, 2006, 319: 162~168
55于乐成,顾长海.水飞蓟素药理学效应研究进展.中国医院药学杂志,2001, 21: 493~494.
56 Radi R, Becman J S, Bush K M, et a1. Peroxynitrite induced membrane lipid peroxidation: the cytotoxic potential of superoxide andnitric oxide. Arch Biochem Biophys, 1991, 288: 481~487
57 DehmLow C, Erhard J, Groot H. Inhibition of Kupffer cell functions as an explanation for the hepatoprotective properties of silibinin. Hepatology, 1996, 23: 749~754
58 Videla L, Valenzuela A. Alcohol ingestion, liver glutathione and lipoperoxidation,metabolic interrelations and pathological implications. Life Sci, l982, 3l:2395~2407
59 Valenzuela A, Aspillaga M, Vial S, et al. Selectivity of silymarin on the increase of the glutathione content in different tissues of the rat. Planta Med, 1989, 55: 420~422
60张俊平,胡振林,冯增辉,等.水飞蓟宾对小鼠肝脏炎症损伤和肿瘤坏死因子的产生及活性的影响.药学学报, 1996, 31: 577~580
61 Wang Y K, Hong Y J, Huang Z Q. Protective effects of silybin on human umbilical vein endothelial cell injury induced by H2O2 in vitro. Vasc Pharmacol, 2005, 43: 198~206
62 Schumann J, Prockl J, Alexandra K, Angelika M, et al. Silibinin protects mice from T cell-dependent liver injury. J Hepatol, 2003, 39: 333~340
63田静.水飞蓟素抗肝损药理及其复合物的研究进展.海峡药学, 2004, 16: 7~9
64孙毓庆主编.分析化学:下册.第3版.北京:人民卫生出版社, 1995
65刘文英主编.药物分析.第5版.北京:人民卫生出版社出版, 2003
66王伟,黄燨乐,利红宇,陈一岳.水飞蓟宾对四氯化碳所致肝损伤的治疗与预防作用.广东药学院学报, 2005, 21: 565~567
67钱茜.水飞蓟宾-磷脂酰胆碱对肝损伤的保护作用.中国误诊学杂志, 2005, 5: 1242~1243
68陈正跃,吴子钊,张光军,许建文,等.水飞蓟宾-磷脂酰胆碱复合物对四氯化碳致小鼠急性肝损伤的保护作用.中国新药杂志, 2004, 13: 319~321
69贾俊清,程钢,陈正跃,吴子钊.水飞蓟宾-磷脂酰胆碱复合物对小鼠急性肝损伤保护作用的量效关系.新乡医学院学报, 2003, 20: 234~236
70 Conti M, Malandrino S, Magistretti M J. Protective activity of silipide on liver damage in rodents. Jpn J Pharmacol, 1992, 60: 315~321
71 Enjalbert F, Rapior S, Nouguier-Soule J, Guillon S, et al. Treatment of amatoxin poisoning: 20-year retrospective analysis. J Toxicol Clin Toxicol, 2002, 40: 715~757
72 Ferenci P, Dragosics B, Dittrich H, Frank H, et al. Randomized controlled trial of silymarin in patients with cirrhosis of the liver. J Hepatol, 1989, 9: 105~113
73陈正跃,吴子钊,许建文,张光军,等.水飞蓟宾-磷脂酰胆碱复合物与水飞蓟宾对四氯化碳所致小鼠急性肝损伤保护作用的对比.中国生化药物杂志, 2004, 25: 221~223
74池莉平,赵敏,谭剑斌,等.王凤岩大豆卵磷脂对酒精性肝损伤保护作用效果观察.海峡预防医学杂志, 2007, 13: 51~52
75刘媛,张小东,刘久红.卵磷脂治疗脂肪肝142例效果观察.华北国防医药, 2005, 17: 358~359
76国家食品药品监督管理局. [H]GPT5-1化学药物非临床药代动力学研究技术指导原则. 2005
77钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题.药物分析杂志, 1996,16: 343-346.
78 Shah V P, Midha K K, Hulse J D, et al. Bioanalytical methods validation-a revisit with a decade of progress. Pharm Res, 2000, 17: 1551~1557
79国家食品药品监督管理局. [H]GCL1-2化学药物临床药代动力学研究技术指导原则. 2005
80国家食品药品监督管理局. [H]GCL2-1化学药物制剂人体生物利用度和生物等效性研究技术指导原则. 2005
81李仪奎.中药药理实验方法学.上海:上海科学技术出版社,1989.458~459