鸦胆子油微乳的处方研究及质量评价
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摘要
鸦胆子油是从苦木科植物鸦胆子的干燥成熟果实中提取而来,具有广泛的抗肿瘤活性,可以抑制多种癌细胞增殖,且毒副作用很低。微乳具有增加药物溶解度、延长药物释放时间、减小注射疼痛等优点,是一种应用前景良好的注射用载体。
本课题通过对鸦胆子油相关性质的考察,并借助伪三元相图,着重研究了微乳处方和制备工艺,得到了乳化剂用量较小、增溶效果较好、质量较稳定的鸦胆子油微乳;借助粒径/zeta电位测定仪、气相色谱仪等检测手段,对鸦胆子油微乳进行了质量研究。本课题,旨在为水难溶性中药成分制备成注射用微乳以及微乳走向生产和临床应用的可行性做出有益尝试。
在研究中我们选用了注射用大豆磷脂、Cremophor EL、Cremophor RH、吐温-80、波洛沙姆F68、Solutol HS等为乳化剂,无水乙醇、异丙醇、正丁醇、山梨醇、PEG200、PEG400、甘油等为助乳化剂,肉豆蔻酸异丙酯、中等链长脂肪酸三甘油酯(MCT)等为油相。分别选择不同的乳化剂、助乳化剂及油相组合,运用滴定法,初步考察能形成微乳的处方。
运用滴定法在绘制伪三元相图的基础上来考察乳化剂、助乳化剂及油相等对微乳区面积和油相增溶量能力的影响,进行乳化剂、助乳化剂和油相筛选。单一乳化剂体系中,Solutol HS15/山梨醇/肉豆蔻酸异丙酯/水体系能得到较大面积的微乳区域,且对油的增溶能力也较好。因此以Solutol HS15为乳化剂,山梨醇为助乳化剂,以肉豆蔻酸异丙酯为油相,对其配比Km值(乳化剂和助乳化剂的质量比)及乳化剂相与油相的比例对微乳区域面积的影响进行了研究,最终确定了微乳的处方为Solutol HS15:山梨醇:肉豆蔻酸异丙酯:鸦胆子油=27:9:9:5。随后按照微乳处方和制备工艺,进行了鸦胆子油微乳的制备,并对鸦胆子油微乳进行了质量研究,考察其外观形态、粒径及其分布、粘度、PH值、电导率等理化常数。所制鸦胆子油微乳为外观均一透明、淡蓝色液体,平均粘度为平均粘度为5.5mm~3·s~(-1),平均电导率为23.7μs·cm~(-1),pH为6.72。用激光粒度分析仪测定丹鸦胆子油微乳粒径及其分布情况,所制微乳平均粒径为23.8 nm。考察了鸦胆子油微乳的稀释稳定性,所制微乳用双蒸水、生理盐水和5%葡萄糖注射液稀释后未出现混浊,pH值几乎无变化。建立了气相色谱法测定鸦胆子油微乳中油酸的含量,在0.115～0.575mg/mL范围内线性关系良好,(R~2=0.9996,n=5)。用此法测得鸦胆子油微乳中油酸的平均含量为84.02mg/mL。运用经典恒温法,以外观、粒径及其分布、含量等为考察指标,对所制微乳的稳定性做了初步评价。40、50、60、70℃下放置8天,求出各温度下的反应速率常数,通过线性回归,求出25℃下的反应速率常数,得出其有效期约为568天,各指标无显著变化。初步稳定性实验结果显示,鸦胆子油微乳基本稳定。
然后对鸦胆子油微乳的药效学和一些毒副作用进行了研究。体外培养Hela细胞,给予不同浓度的鸦胆子油微乳及普通乳剂,作用不同的时间后,运用MTT法检测其抑癌率,并绘制抑癌率曲线。在光学显微镜下观察细胞形态,考察Hela细胞在鸦胆子油微乳及普通乳剂处理前后细胞形态的变化。结果表明鸦胆子油微乳比普通乳剂的抑癌效果好,随着药物浓度升高,作用时间增长,抑癌率升高。Hela细胞经鸦胆子油微乳处理后,细胞形态发生显著改变,随着作用时间的延长,细胞凋亡,在显微镜下可见细胞碎片。为新西兰大白兔耳静脉注射考察鸦胆子油微乳的血管刺激性,并与鸦胆子油乳比较,结果表明鸦胆子油微乳的血管刺激性较鸦胆子油乳的要小。豚鼠过敏性试验和体外溶血性实验都表明鸦胆子油微乳的安全性要比鸦胆子油乳的要好。
综上所述,本研究制得的鸦胆子油微乳,所用乳化剂、助乳化剂用量低、稳定性好,抑癌作用显著,副作用较小,对水难溶性中药成分制备成注射用微乳具有一定实用价值。
Brucea javanica oil is the extracts of Brucea javanica mature fruit, it has a broad anti-tumor activity. Brucea javanica oil can inhibit the proliferation of a variety of cancer cells and its toxicity is low. Microemulsion as a potential carrier for injectable drug with good application perspcetives,can improve the solubility of drugs,lengthen the drug`s acting time in vivo due to its slow release from microemulsion,and reduce pain caused by injection due to its low viscosity.
In this study, We seleeted microemulsion as drug carrier and Brucea javanica oil as the model drug to prepare Brucea javanica oil microemulsion.We investigated the charaeters of Brucea javanica oil correlated. Then by the pseudo-temary phase diagrams,we investigated the formulation and formula technology of microemulsion, obtained the formula of microemulsion with low content of surfaetants,good solubilization and stable quality,and prepared Brucea javanica oil microemulsion.The quality of Brucea javanica oil microemulsion was assessed by laser nano-sizer analyzer,GC and so on.This work aimed to supply valuable trials for the preparation of injectable microemulsion for Chinese medieinal ingredients with poor solubility.
We chose the soybean phospholipids, Cremophor EL,Cremophor RH,tween-80, span-80,poloxamer F68, Solutol HS15 as surfactants.ethanol,iso-propyl alcohol, n-butanol,sorbitol,Polyethylene glycol 200, Polyethylene glycol 400 and glycerine as co-surfactants.isopropyl myristate,MCT and injectable soybean oil as oil phase.On the basis of the Pseud-triangular phase diagrams obtained from titration method,the effects on the area of microemulsion and the solubilization amounts of oil of various elements(surfactants,co-surfactants and oils)were investigated to search rational microemulsion formula. We found that the area of microemulsion and the solubilization amounts of oil was small with various surfactants single used.The system composed of Solutol HS15/ sorbitol / isopropyl myristate / water showed the best effect.After further research we found that the area of microemulsion and the solubilization amounts of oil was better than other microemulsion formula.Using the Solutol HS15 as surfactants,the effeets of Km(the mass ratio of surfaetant / co-surfaetant) and the mass ratio of surfaetant/oil on the area of microemulsion were further investigated.Finally we aseertained the Brucea javanica oil mieroemulsion formula,the optimized one was the system of Solutol HS15/ sorbitol / isopropyl myristate/ Brucea javanica oil(27:9:9:5).
The Brucea javanica oil mieroemulsion prepared,The quality of Brucea javanica oil microemulsion was assessed. Brucea javanica oil microemulsion was uniform and transparent fluid with light blue,its viscosity was 5.5mm~3·s~(-1),with the conductivity 23.7μs·cm~(-1). The particle diameter and its distribution of Brucea javanica oil microemusion were determined by Nano-ZS 90 grainsize analyzer,its mean diameter was 23.8 nm.We established an accurate method for the determination of oleic acid of Brucea javanica oil in the microemulsion. oleic acid of Brucea javanica oil was determined by GC,the line rang was 0.115～0.575mg/mL,(R~2=0.9996,n=5).Its average recovery rate was 100.01% and the RSD 0.39%.The method was convenient, rapid,accurate and suitable for assay of oleic acid of Brucea javanica oil in the microemulsion,and the mean content of oleic acid in Brucea javanica oil microemulsion was 84.02mg/mL.Using the classical constant temperature method, taken appearance, size and distribution, content as indicators, the preliminary evaluation of the stability of the microemulsion system has been done. The sample bene placed under 40,50,60,70℃for eight days, The reaction rate constant at every temerature bene calculated.The reaction rate constant at 25℃been calculated by linear regression. Finally, a validity period about 568 days been obtained. The experimental results showed that the Brucea javanica oil microemulsion is basic stable.
Then,pharmacodynamics and side effects of Brucea javanica oil microemulsion have been studied. Hela cells was cultured in vitro, Different concentrations of Brucea javanica oil microemulsion and emulsion been given, affecting different time, Using MTT method the rate of the tumor suppressor was assayed and tumor suppressor mapping curve was drawn.The cell morphology was observed under the optical microscope to study the change of Hela cells` morphology before and after Brucea javanica oil microemulsion and general emulsion. The results show that the Brucea javanica oil microemulsion better than its emulsion in the tumor suppressor effect. As the drug concentration and affacting time increased, the rate of tumor suppressor increased. Hela cells treated with Brucea javanica oil microemulsion, cell morphogenesis changed significantly, with the extension of time, cell appear apoptosis, cell debris can be seen under the microscope. Using New Zealand white rabbits for study of Brucea javanica oil microemulsion vascular irritation, and compared with the Brucea javanica oil emulsion, results showed that the blood vessels irritatant of Brucea javanica oil Brucea javanica oil microemulsion smaller than its emulsion. Allergic test and in vitro hemolytic experiments indicate that Brucea javanica oil microemulsion`s more safe than its emulsion.
In conclusion, The brucea javanica oil microemulsion were obtained through this study with low contents of emulsifier and good stability,which significantly improved the solubility of brucea javanica oil . Its tumor suppressor effects is significant,its side-effects is small. This study supply valuable trials for the preparation of injectable microemulsion for Chinese medicinal ingredients with poor solubility.
本课题通过对鸦胆子油相关性质的考察,并借助伪三元相图,着重研究了微乳处方和制备工艺,得到了乳化剂用量较小、增溶效果较好、质量较稳定的鸦胆子油微乳;借助粒径/zeta电位测定仪、气相色谱仪等检测手段,对鸦胆子油微乳进行了质量研究。本课题,旨在为水难溶性中药成分制备成注射用微乳以及微乳走向生产和临床应用的可行性做出有益尝试。
在研究中我们选用了注射用大豆磷脂、Cremophor EL、Cremophor RH、吐温-80、波洛沙姆F68、Solutol HS等为乳化剂,无水乙醇、异丙醇、正丁醇、山梨醇、PEG200、PEG400、甘油等为助乳化剂,肉豆蔻酸异丙酯、中等链长脂肪酸三甘油酯(MCT)等为油相。分别选择不同的乳化剂、助乳化剂及油相组合,运用滴定法,初步考察能形成微乳的处方。
运用滴定法在绘制伪三元相图的基础上来考察乳化剂、助乳化剂及油相等对微乳区面积和油相增溶量能力的影响,进行乳化剂、助乳化剂和油相筛选。单一乳化剂体系中,Solutol HS15/山梨醇/肉豆蔻酸异丙酯/水体系能得到较大面积的微乳区域,且对油的增溶能力也较好。因此以Solutol HS15为乳化剂,山梨醇为助乳化剂,以肉豆蔻酸异丙酯为油相,对其配比Km值(乳化剂和助乳化剂的质量比)及乳化剂相与油相的比例对微乳区域面积的影响进行了研究,最终确定了微乳的处方为Solutol HS15:山梨醇:肉豆蔻酸异丙酯:鸦胆子油=27:9:9:5。随后按照微乳处方和制备工艺,进行了鸦胆子油微乳的制备,并对鸦胆子油微乳进行了质量研究,考察其外观形态、粒径及其分布、粘度、PH值、电导率等理化常数。所制鸦胆子油微乳为外观均一透明、淡蓝色液体,平均粘度为平均粘度为5.5mm~3·s~(-1),平均电导率为23.7μs·cm~(-1),pH为6.72。用激光粒度分析仪测定丹鸦胆子油微乳粒径及其分布情况,所制微乳平均粒径为23.8 nm。考察了鸦胆子油微乳的稀释稳定性,所制微乳用双蒸水、生理盐水和5%葡萄糖注射液稀释后未出现混浊,pH值几乎无变化。建立了气相色谱法测定鸦胆子油微乳中油酸的含量,在0.115～0.575mg/mL范围内线性关系良好,(R~2=0.9996,n=5)。用此法测得鸦胆子油微乳中油酸的平均含量为84.02mg/mL。运用经典恒温法,以外观、粒径及其分布、含量等为考察指标,对所制微乳的稳定性做了初步评价。40、50、60、70℃下放置8天,求出各温度下的反应速率常数,通过线性回归,求出25℃下的反应速率常数,得出其有效期约为568天,各指标无显著变化。初步稳定性实验结果显示,鸦胆子油微乳基本稳定。
然后对鸦胆子油微乳的药效学和一些毒副作用进行了研究。体外培养Hela细胞,给予不同浓度的鸦胆子油微乳及普通乳剂,作用不同的时间后,运用MTT法检测其抑癌率,并绘制抑癌率曲线。在光学显微镜下观察细胞形态,考察Hela细胞在鸦胆子油微乳及普通乳剂处理前后细胞形态的变化。结果表明鸦胆子油微乳比普通乳剂的抑癌效果好,随着药物浓度升高,作用时间增长,抑癌率升高。Hela细胞经鸦胆子油微乳处理后,细胞形态发生显著改变,随着作用时间的延长,细胞凋亡,在显微镜下可见细胞碎片。为新西兰大白兔耳静脉注射考察鸦胆子油微乳的血管刺激性,并与鸦胆子油乳比较,结果表明鸦胆子油微乳的血管刺激性较鸦胆子油乳的要小。豚鼠过敏性试验和体外溶血性实验都表明鸦胆子油微乳的安全性要比鸦胆子油乳的要好。
综上所述,本研究制得的鸦胆子油微乳,所用乳化剂、助乳化剂用量低、稳定性好,抑癌作用显著,副作用较小,对水难溶性中药成分制备成注射用微乳具有一定实用价值。
Brucea javanica oil is the extracts of Brucea javanica mature fruit, it has a broad anti-tumor activity. Brucea javanica oil can inhibit the proliferation of a variety of cancer cells and its toxicity is low. Microemulsion as a potential carrier for injectable drug with good application perspcetives,can improve the solubility of drugs,lengthen the drug`s acting time in vivo due to its slow release from microemulsion,and reduce pain caused by injection due to its low viscosity.
In this study, We seleeted microemulsion as drug carrier and Brucea javanica oil as the model drug to prepare Brucea javanica oil microemulsion.We investigated the charaeters of Brucea javanica oil correlated. Then by the pseudo-temary phase diagrams,we investigated the formulation and formula technology of microemulsion, obtained the formula of microemulsion with low content of surfaetants,good solubilization and stable quality,and prepared Brucea javanica oil microemulsion.The quality of Brucea javanica oil microemulsion was assessed by laser nano-sizer analyzer,GC and so on.This work aimed to supply valuable trials for the preparation of injectable microemulsion for Chinese medieinal ingredients with poor solubility.
We chose the soybean phospholipids, Cremophor EL,Cremophor RH,tween-80, span-80,poloxamer F68, Solutol HS15 as surfactants.ethanol,iso-propyl alcohol, n-butanol,sorbitol,Polyethylene glycol 200, Polyethylene glycol 400 and glycerine as co-surfactants.isopropyl myristate,MCT and injectable soybean oil as oil phase.On the basis of the Pseud-triangular phase diagrams obtained from titration method,the effects on the area of microemulsion and the solubilization amounts of oil of various elements(surfactants,co-surfactants and oils)were investigated to search rational microemulsion formula. We found that the area of microemulsion and the solubilization amounts of oil was small with various surfactants single used.The system composed of Solutol HS15/ sorbitol / isopropyl myristate / water showed the best effect.After further research we found that the area of microemulsion and the solubilization amounts of oil was better than other microemulsion formula.Using the Solutol HS15 as surfactants,the effeets of Km(the mass ratio of surfaetant / co-surfaetant) and the mass ratio of surfaetant/oil on the area of microemulsion were further investigated.Finally we aseertained the Brucea javanica oil mieroemulsion formula,the optimized one was the system of Solutol HS15/ sorbitol / isopropyl myristate/ Brucea javanica oil(27:9:9:5).
The Brucea javanica oil mieroemulsion prepared,The quality of Brucea javanica oil microemulsion was assessed. Brucea javanica oil microemulsion was uniform and transparent fluid with light blue,its viscosity was 5.5mm~3·s~(-1),with the conductivity 23.7μs·cm~(-1). The particle diameter and its distribution of Brucea javanica oil microemusion were determined by Nano-ZS 90 grainsize analyzer,its mean diameter was 23.8 nm.We established an accurate method for the determination of oleic acid of Brucea javanica oil in the microemulsion. oleic acid of Brucea javanica oil was determined by GC,the line rang was 0.115～0.575mg/mL,(R~2=0.9996,n=5).Its average recovery rate was 100.01% and the RSD 0.39%.The method was convenient, rapid,accurate and suitable for assay of oleic acid of Brucea javanica oil in the microemulsion,and the mean content of oleic acid in Brucea javanica oil microemulsion was 84.02mg/mL.Using the classical constant temperature method, taken appearance, size and distribution, content as indicators, the preliminary evaluation of the stability of the microemulsion system has been done. The sample bene placed under 40,50,60,70℃for eight days, The reaction rate constant at every temerature bene calculated.The reaction rate constant at 25℃been calculated by linear regression. Finally, a validity period about 568 days been obtained. The experimental results showed that the Brucea javanica oil microemulsion is basic stable.
Then,pharmacodynamics and side effects of Brucea javanica oil microemulsion have been studied. Hela cells was cultured in vitro, Different concentrations of Brucea javanica oil microemulsion and emulsion been given, affecting different time, Using MTT method the rate of the tumor suppressor was assayed and tumor suppressor mapping curve was drawn.The cell morphology was observed under the optical microscope to study the change of Hela cells` morphology before and after Brucea javanica oil microemulsion and general emulsion. The results show that the Brucea javanica oil microemulsion better than its emulsion in the tumor suppressor effect. As the drug concentration and affacting time increased, the rate of tumor suppressor increased. Hela cells treated with Brucea javanica oil microemulsion, cell morphogenesis changed significantly, with the extension of time, cell appear apoptosis, cell debris can be seen under the microscope. Using New Zealand white rabbits for study of Brucea javanica oil microemulsion vascular irritation, and compared with the Brucea javanica oil emulsion, results showed that the blood vessels irritatant of Brucea javanica oil Brucea javanica oil microemulsion smaller than its emulsion. Allergic test and in vitro hemolytic experiments indicate that Brucea javanica oil microemulsion`s more safe than its emulsion.
In conclusion, The brucea javanica oil microemulsion were obtained through this study with low contents of emulsifier and good stability,which significantly improved the solubility of brucea javanica oil . Its tumor suppressor effects is significant,its side-effects is small. This study supply valuable trials for the preparation of injectable microemulsion for Chinese medicinal ingredients with poor solubility.
引文
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