水飞蓟宾亚微乳剂的研究
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摘要
水飞蓟宾是从植物药水飞蓟果实中提取的有效成分，是一种肝细胞膜稳定剂，能有效保护及增强肝细胞黏膜。临床上主要用于治疗急慢性肝炎、肝硬变、脂肪肝、酒精引起的肝损害、代谢中毒性肝损伤、胆结石和脾脏病等。由于药效高，毒性低，水飞蓟宾从发现至今一直备受关注。但其水溶性，脂溶性均很差，口服吸收差，有较大的首过效应，且体内的半衰期短等性质严重削弱了水飞蓟宾口服制剂药效的发挥。早年也有科研工作者研制了水飞蓟宾葡甲胺盐注射剂，但其水溶液不稳定，极易发生降解，制备成冻干粉针虽然可以解决其放置稳定性问题，但是水飞蓟宾葡甲胺盐水溶液的PH值在11左右，注射时局部刺激性较大，限制了临床上的应用。本实验以制备原形药物注射剂为目的，研制了水飞蓟宾亚微乳剂。
用单因素考察的方法对水飞蓟宾亚微乳剂的处方及制备工艺进行了筛选优化。对处方中油相的组成及比例、乳化剂的种类和用量、助乳化剂的使用、pH值等进行了考察，对工艺中药物的加入方法、磷脂的加入方法、均质压力和次数等主要影响因素优化筛选，实验中以所得乳剂的粒径分布、ζ-电位、离心稳定性常数及包封率为考察指标，结果得出水飞蓟宾亚微乳剂的最佳处方组成为：水飞蓟宾0.5％、大豆油10％、中链油10％、豆磷脂2.0％、Tween-80 0.2％、泊洛沙姆F-68 0.2％、油酸钠0.1％、甘油2.25％、PEG400 5％。最佳均质压力及均质次数分别为70MPa、8次，灌封氮气，100℃灭菌30 min。所制备的水飞蓟宾亚微乳剂为白色乳白液体，平均粒径为190nm，pH6．5-7，ζ-电位的绝对值大于20mV、包裹率可保持在80％以上。
建立了水飞蓟宾亚微乳剂中水飞蓟宾的含量测定方法，以乙醚、甲醇混合溶剂破乳，用HPLC法测定亚微乳剂中水飞蓟宾的含量，灵敏度较高，空白辅料、表面活性剂等辅料无干扰，适用于水飞蓟宾亚微乳剂的质量控制；振摇实验和血浆稳定性实验表明本品的稳定性良好；稳定试验研究表明本品在25±2℃及6±2℃条件下放置6个月物理化学性质几乎没有改变。
对水飞蓟宾亚微乳剂中药物的向分布进行了研究。分别用微渗析、超滤法、超速离心法确定了游离药物、水相中药物及油相中药物的百分含量，并间接计算出油水界面膜中药物的百分含量。研究表明约0.3％的药物游离于制剂中，约11.1％的药物存在于囊泡中，约3.9％的药物存在于油相中，约84.7％的药物存在于亚微乳剂的油水界面层中。可见大部分的药物存在于亚微乳剂的油水界面膜中。
对水飞蓟宾亚微乳剂在大鼠体内的药代动力学及组织分布进行了研究。用HPLC测定水飞蓟宾的血药浓度。水飞蓟宾亚微乳剂和普通溶液剂的大鼠体内药时过程均符合双隔室模型，药动学参数无显著性差异。非隔室模型分析结果表明，受试制剂药时曲线下面积(AUC)为1.91μg·mL~(-1)·h是参比制剂的99.48％，两制剂药时曲线形状相同。组织分布实验表明，与水飞蓟宾溶液剂相比，亚微乳剂静脉注射后更多的药物分布在肝、肺组织中，说明将水飞蓟宾以亚微乳剂的形式给药改变了其体内分布，水飞蓟宾亚微乳剂具有肝、肺靶向性。
Silybin, is isolated from the seeds of the milk thistle (Silibum marianum Gaertn). Well known as a hepatoprotector, silybin is found effective clinically to treat a variety of liver disorders, including acute and chronic viral hepatitis, toxin- and drug-induced hepatitis and cirrhosis, and alcoholic liver disease. Because silybin has the advantage of good therapy and little side-effect, it has been paid attention to since it was found, However, the effectiveness of silybin as liver disease remedy is discounted by its poor water solubility, big first pass effect and low bioavailability after oral administration. Although the formulations studied previously have been aqueous solutions of silybin in silybin-N-methylglucamine form, the stability is poor and easy to degrade. Powder for injection can solve this problem, but the pH of the solution is approx. 11, so that its aqueous injection produces severe irritation at the injection site, limiting its clinical application. To prepare silybin injection, we develop silybin submicron emulsion.
The formulation and preparation process of silybin submicron emulsion was determined by single factor investigation. Using size distribution, zeta potential, entrapment efficiency and stability as indexes, the effect of variable excipients of the formulation such as the amount of oil phase, the ration of medium chain fatty acid (MCT) and soybean oil (LCT), the amount of emulsifier, pH of the ultimate emulsion and the effect of processes such as the way of addition of silybin and soybean lecithin, the pressure and cycle of the homogenization process were investigated. The optimal formula based on the experimental data was silybin 0.05%, oil phase 20%(LCT:MCT=50:50), Tween-80 0.2%, soybean lecithin 2.0%, glcyrol 2.25%, sodium oleate 0.1%, poloxamer 188 0.2%, PEG400 5%. The best pressure was 70MPa and the optimal number of cycle was 8 during the process of homogenizeation. Filled in with nitrogen, the samples were water-bath sterilized under 100℃, 30 min. The final emulsion was white with mean particle size of about 190nm, pH of 6.5-7, the absolute value of zeta potential of above 20mV and theentrapment efficiency of above 80%.
A HPLC method was established for determination of silybin. The method was simple, rapid, acute, sensitive, reproducible and suitable for the quality control of silybin submicron emulsion. The results of shaking test and stability of emulsion mixed with plasma showed that the samples had a good stability; The stability test showed that silybin submicron emulsion showed no significant change after 6 months under 25+2℃and 6±2℃.
The drug distribution in submicron emulsion was studied. The amount of free drug, the percent of drug in aqueous phase and oil phase was determined separately by microdialysis, ultrafiltration and ultracentrifugation. Then the percent of drag in o/w interfacial layer was calculated. The results showed that the amount of the free drug was about 0.3%; 11.1% of the drug was entrapped in small unilamellar liposomes; 3.9% of the drug was in oil phase and 84.7% of the drug was in o/w interfacial layer of submicron emulsion. Therefore, most of the drag was in o/w interfacial layer of submicron emulsion..
The pharmacokinetics of silybin submicron emulsion and tissue distribution in rats was studied. The HPLC method was adopted for the determination of the concentration of silybin in plasma. The result of the plasma concentration showed that the silybin submicron emulsion and the reference silybin solution were of two compartments. The pharmacokinetics parameters showed no distinct difference. The AUC value of silybin submicron emulsion was 1.91μg·mL~(-1)·h, which was 99.48% relative to the AUC of the solution and the two c-t curves were alike. The results of tissue distribution study in the rats showed that compared with solution, after silybin submicron emulsion injection more drug distributed in liver and lung, which shows that the SB submicron emulsion changed the biodistribution of the drug. SB submicron emulsion had the characteristic of the liver and lung targeting.
用单因素考察的方法对水飞蓟宾亚微乳剂的处方及制备工艺进行了筛选优化。对处方中油相的组成及比例、乳化剂的种类和用量、助乳化剂的使用、pH值等进行了考察，对工艺中药物的加入方法、磷脂的加入方法、均质压力和次数等主要影响因素优化筛选，实验中以所得乳剂的粒径分布、ζ-电位、离心稳定性常数及包封率为考察指标，结果得出水飞蓟宾亚微乳剂的最佳处方组成为：水飞蓟宾0.5％、大豆油10％、中链油10％、豆磷脂2.0％、Tween-80 0.2％、泊洛沙姆F-68 0.2％、油酸钠0.1％、甘油2.25％、PEG400 5％。最佳均质压力及均质次数分别为70MPa、8次，灌封氮气，100℃灭菌30 min。所制备的水飞蓟宾亚微乳剂为白色乳白液体，平均粒径为190nm，pH6．5-7，ζ-电位的绝对值大于20mV、包裹率可保持在80％以上。
建立了水飞蓟宾亚微乳剂中水飞蓟宾的含量测定方法，以乙醚、甲醇混合溶剂破乳，用HPLC法测定亚微乳剂中水飞蓟宾的含量，灵敏度较高，空白辅料、表面活性剂等辅料无干扰，适用于水飞蓟宾亚微乳剂的质量控制；振摇实验和血浆稳定性实验表明本品的稳定性良好；稳定试验研究表明本品在25±2℃及6±2℃条件下放置6个月物理化学性质几乎没有改变。
对水飞蓟宾亚微乳剂中药物的向分布进行了研究。分别用微渗析、超滤法、超速离心法确定了游离药物、水相中药物及油相中药物的百分含量，并间接计算出油水界面膜中药物的百分含量。研究表明约0.3％的药物游离于制剂中，约11.1％的药物存在于囊泡中，约3.9％的药物存在于油相中，约84.7％的药物存在于亚微乳剂的油水界面层中。可见大部分的药物存在于亚微乳剂的油水界面膜中。
对水飞蓟宾亚微乳剂在大鼠体内的药代动力学及组织分布进行了研究。用HPLC测定水飞蓟宾的血药浓度。水飞蓟宾亚微乳剂和普通溶液剂的大鼠体内药时过程均符合双隔室模型，药动学参数无显著性差异。非隔室模型分析结果表明，受试制剂药时曲线下面积(AUC)为1.91μg·mL~(-1)·h是参比制剂的99.48％，两制剂药时曲线形状相同。组织分布实验表明，与水飞蓟宾溶液剂相比，亚微乳剂静脉注射后更多的药物分布在肝、肺组织中，说明将水飞蓟宾以亚微乳剂的形式给药改变了其体内分布，水飞蓟宾亚微乳剂具有肝、肺靶向性。
Silybin, is isolated from the seeds of the milk thistle (Silibum marianum Gaertn). Well known as a hepatoprotector, silybin is found effective clinically to treat a variety of liver disorders, including acute and chronic viral hepatitis, toxin- and drug-induced hepatitis and cirrhosis, and alcoholic liver disease. Because silybin has the advantage of good therapy and little side-effect, it has been paid attention to since it was found, However, the effectiveness of silybin as liver disease remedy is discounted by its poor water solubility, big first pass effect and low bioavailability after oral administration. Although the formulations studied previously have been aqueous solutions of silybin in silybin-N-methylglucamine form, the stability is poor and easy to degrade. Powder for injection can solve this problem, but the pH of the solution is approx. 11, so that its aqueous injection produces severe irritation at the injection site, limiting its clinical application. To prepare silybin injection, we develop silybin submicron emulsion.
The formulation and preparation process of silybin submicron emulsion was determined by single factor investigation. Using size distribution, zeta potential, entrapment efficiency and stability as indexes, the effect of variable excipients of the formulation such as the amount of oil phase, the ration of medium chain fatty acid (MCT) and soybean oil (LCT), the amount of emulsifier, pH of the ultimate emulsion and the effect of processes such as the way of addition of silybin and soybean lecithin, the pressure and cycle of the homogenization process were investigated. The optimal formula based on the experimental data was silybin 0.05%, oil phase 20%(LCT:MCT=50:50), Tween-80 0.2%, soybean lecithin 2.0%, glcyrol 2.25%, sodium oleate 0.1%, poloxamer 188 0.2%, PEG400 5%. The best pressure was 70MPa and the optimal number of cycle was 8 during the process of homogenizeation. Filled in with nitrogen, the samples were water-bath sterilized under 100℃, 30 min. The final emulsion was white with mean particle size of about 190nm, pH of 6.5-7, the absolute value of zeta potential of above 20mV and theentrapment efficiency of above 80%.
A HPLC method was established for determination of silybin. The method was simple, rapid, acute, sensitive, reproducible and suitable for the quality control of silybin submicron emulsion. The results of shaking test and stability of emulsion mixed with plasma showed that the samples had a good stability; The stability test showed that silybin submicron emulsion showed no significant change after 6 months under 25+2℃and 6±2℃.
The drug distribution in submicron emulsion was studied. The amount of free drug, the percent of drug in aqueous phase and oil phase was determined separately by microdialysis, ultrafiltration and ultracentrifugation. Then the percent of drag in o/w interfacial layer was calculated. The results showed that the amount of the free drug was about 0.3%; 11.1% of the drug was entrapped in small unilamellar liposomes; 3.9% of the drug was in oil phase and 84.7% of the drug was in o/w interfacial layer of submicron emulsion. Therefore, most of the drag was in o/w interfacial layer of submicron emulsion..
The pharmacokinetics of silybin submicron emulsion and tissue distribution in rats was studied. The HPLC method was adopted for the determination of the concentration of silybin in plasma. The result of the plasma concentration showed that the silybin submicron emulsion and the reference silybin solution were of two compartments. The pharmacokinetics parameters showed no distinct difference. The AUC value of silybin submicron emulsion was 1.91μg·mL~(-1)·h, which was 99.48% relative to the AUC of the solution and the two c-t curves were alike. The results of tissue distribution study in the rats showed that compared with solution, after silybin submicron emulsion injection more drug distributed in liver and lung, which shows that the SB submicron emulsion changed the biodistribution of the drug. SB submicron emulsion had the characteristic of the liver and lung targeting.
引文
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[2] 孙铁民，李铣．水飞蓟素药理研究进展[J]．中草药，2000，31(3)：229-231．
[3] 闫玉峰，于健东．水飞蓟的化学成分及药理研究进展[J]．中国药事，2000，14(5)：335-337．
[4] 缪春平，林信章．水飞蓟的临床新用途[J]．中国社区医师，2005，11：21
[5] 肖莉，靳淑敏，张丽霞，王永利，翟所迪，贾淑琴．水飞蓟素制剂及其应用进展[J]．中国药物与临床，2005，5(3)：205-208
[6] 曹伟新．脂肪乳剂的特点和应用[J]．腹部外科，2000，13(4)：206-208．
[7] Collins-Gold LC, Lyons, RT, Bartholow LC. Parenteral emulsions for drug delivery. Adv. Drug. Deliv. Rew. 1990, 5:189-208.
[8] Yutaka M, Lipid microsphere (lipid emulsion) as a drug carder- An overview, Adv. Drug. Deliv. Rew. 1996, 20:113-115
[9] R.H. M, Solemuls-novel technology for the formulation of i.v. emulsions with poorly soluble drugs. Int. J .pharm. 2004,269:293-302.
[10] Akkar, A., Müller, RH. Formulation of intravenous Carbamazepine emulsions by SolEmuls(?) technology. Eur. J. Pharm.Biopharm. 2003, 55:305-312
[11] Kidd, PM. Phosphatidylcholine (Monograph). In: Czap K, Miller AL, Head KA, et al, eds. Alternative Medicine Review Monographs Volume One. Dover, ID: Thorne Research, Inc. 2002,310-315.
[12] 宋梅，平其能，吴正红．水飞蓟宾纳米乳的制备及家兔体内药动学[J]．中国药科大学学报，2005，36(5)：427-431
[13] 韩继洪，抗癌药物FT-207脂肪乳剂物理稳定性质的研究[J]．沈阳药学院学报，1991，8(1)：14-7
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[2] 孙铁民，李铣．水飞蓟素药理研究进展[J]．中草药，2000，31(3)：229-231．
[3] 闫玉峰，于健东．水飞蓟的化学成分及药理研究进展[J]．中国药事，2000，14(5)：335-337．
[4] 缪春平，林信章．水飞蓟的临床新用途[J]．中国社区医师，2005，11：21
[5] 肖莉，靳淑敏，张丽霞，王永利，翟所迪，贾淑琴．水飞蓟素制剂及其应用进展[J]．中国药物与临床，2005，5(3)：205-208
[6] 曹伟新．脂肪乳剂的特点和应用[J]．腹部外科，2000，13(4)：206-208．
[7] Collins-Gold LC, Lyons, RT, Bartholow LC. Parenteral emulsions for drug delivery. Adv. Drug. Deliv. Rew. 1990, 5:189-208.
[8] Yutaka M, Lipid microsphere (lipid emulsion) as a drug carder- An overview, Adv. Drug. Deliv. Rew. 1996, 20:113-115
[9] R.H. M, Solemuls-novel technology for the formulation of i.v. emulsions with poorly soluble drugs. Int. J .pharm. 2004,269:293-302.
[10] Akkar, A., Müller, RH. Formulation of intravenous Carbamazepine emulsions by SolEmuls(?) technology. Eur. J. Pharm.Biopharm. 2003, 55:305-312
[11] Kidd, PM. Phosphatidylcholine (Monograph). In: Czap K, Miller AL, Head KA, et al, eds. Alternative Medicine Review Monographs Volume One. Dover, ID: Thorne Research, Inc. 2002,310-315.
[12] 宋梅，平其能，吴正红．水飞蓟宾纳米乳的制备及家兔体内药动学[J]．中国药科大学学报，2005，36(5)：427-431
[13] 韩继洪，抗癌药物FT-207脂肪乳剂物理稳定性质的研究[J]．沈阳药学院学报，1991，8(1)：14-7
[14] Davis, S. S.; Hadgraft, J.; Palin, K. J. Medical and pharmaceutical applications of emulsions" in "Encyclopedia of Emulsion Technology, Becher, P. (Ed.), Marcel Dekker Inc., New York, 1985, 2:159-238
[15] Chambrier C, Guriraud M, Gibault JP, et al Medium and long-chain triacglycerols in postoperatice patients: structured lipid versus a physical mixture, Nutrition. 1999, 15:274-277
[16] Tomasits E, Rischak K, et al, Effects of administration of different intravenous lipid emulsion on plasma LP-X concentrations in the rats.JPEN, 1995,19:369
[17] Eccleston, G. M. Emulsions in Encyclopedia of Pharmaceutical Technology, Swarbrick, J.; Boylan, J. C. (Eds.), 1992, 5:137-188
[18] Simon B, Doron F, Marta W. Physostigmine emulsion: a new injectable controlled releasee delivery system. Int. J. Pharm. 1986, 30:47-55
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