蜣螂抗良性前列腺增生症有效部位及其新型给药系统的研究
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摘要
良性前列腺增生症(benign prostatic hyperplasia,以下简称BPH)是中老年男性的常见病、多发病,属于中医学的“癃闭”、“精癃”范畴。据临床调查报道50岁以上的男性有50%患有该病症,随着我国老龄人口的不断增加,其发病率也越来越高,严重影响老年人的生活质量,防治迫在眉睫。
本项目根据前列腺增生症的病因病机,针对前列腺自身解剖特点、增生患者瘀血凝结的病理特点,结合中医辨证,选用临床疗效好、活血化瘀作用佳的蜣螂作为模型药物,对其治疗BPH的物质基础及作用机理进行研究,并在此基础上采用新技术、新工艺、新标准研制新剂型,以期为临床提供高效、优质、多样的用药选择。
1、物质基础研究
采用系统溶剂法,结合急性毒性试验和药效筛选(丙酸睾丸酮致小鼠前列腺增生模型与犬膀胱三角肌张力测定)试验,通过色谱法、透析技术等方法与手段对蜣螂治疗BPH的活性成分进行追踪分离,明确了蜣螂治疗BPH的物质基础主要为多肽与氨基酸,初步阐明了蜣螂治疗BPH的作用机理为抑制α受体而起效。
2、中间体的制备与质量控制
采用成分检测与理化参数表征相结合的过程控制模式进行渗漉法制备蜣螂有效部位的工艺研究,比较了泡沫分离与大孔树脂技术对提取液的纯化效果,结果后者较前者为优。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定了多肽的分子量,高效毛细管电泳法(HPCE)对有效部位进行了指纹图谱研究,氨基酸自动分析仪测定了氨基酸的种类与数量,可全面而有效地控制中间体的质量。
3、新型给药系统的研究
在药效物质基础清楚的基础上,以质量稳定可控的蜣螂有效部位中间体为原料,进行了两种新剂型给药系统的研究。在结肠定位释药系统(OCDDS)的研究中,将有效部位制成片芯,根据消化道pH值的变化,选择pH敏感型聚合物包衣材料Eudragit S100制备结肠定位释放片,总氨基酸和多肽的体外释放实验表明其达到了结肠定位释放的目的。采用分光光度法,以总氨基酸的含量为指标探讨了制剂的释药规律,结果符合Weibull模型。在生物粘附给药系统(BDDS)的研究中,选用生物粘附聚合物,以制剂的流变学特性、生物粘附性、体外释放度为指标筛选制剂处方与工艺条件,确定了较佳处方与成型工艺参数。对制剂的流变学特性、生物粘附机制、粘膜刺激性、稳定性、释药规律进行了系统评价,结果其为假塑性流体,流变学行为符合Ostwald模型；粘附机制可能为其含羟丙基甲基纤维素(HPMC),遇水后,分子链与组织表面的粘液缠结,分子中非离子化的羟基通过氢键与粘蛋白分子相连,产生粘附；稳定性研究中,对影响因素进行考察,结果强光照射、高温、低温条件下,制剂的稳定性均良好；在刺激性试验中,进行了蜣螂有效部位生物粘附凝胶剂对大鼠的急性刺激试验及多次给药刺激试验,结果经肉眼及光镜下观察粘膜均无充血、水肿及变性,表明该制剂基本无刺激性,安全性较好；在释药规律研究中,采用改进的Franz扩散池对制剂的释药规律进行研究,结果符合Weibull模型。采用老年犬自发前列腺增生模型对上述两种制剂的药效进行研究,结果均能抑制前列腺增生,疗效显著。
本研究明确了蜣螂治疗BPH的物质基础,初步阐明作用机理,完成了两种新型给药系统的研究,提出了理化参数表征与含量测定相结合的质量控制模式,可望为临床提供安全有效、质量稳定可控的治疗BPH的中药制剂。
Benign prostatic hyperplasia (BPH), which is a common and frequent disease to the elderly and wrinkly people, belongs to "uroschesis" and "retention of sperm" category in TCM. According to the clinical investigation, more than 50% of males(aged above 50) are suffering from the disease. With the increase of aging population, its incidence is also getting higher and higher, seriously affecting life quality of the elderly.Then the prevention and treatment is urgen.
According to etiology and pathogenesis of BPH, anatomical characteristics of prostate, pathological characteristics of congestion and dialectic in TCM, we selected Catharsius molossus Linnaeus, which has good clinical efficacy and satisfactory effect for blood circulation, as a model drug. We not only investigated material base and action mechanism, but also developed new drug delivery systems adopting new technologies new craft and new standards. Therefore, this reseach will provide effective,high quality and multiple drug selection for clinic.
1. Studies of material base
Using methods,such as systematic solvent extraction,combined acute toxicity test and pharmacodynamic screening tests (benign prostatic hyperplasia model of mouse induced by testosterone and the tensity test of dog's bladder deltoid muscle), chromatographic method, as well as dialysis technique, we traced and separated active ingredient of Catharsius molossus Linnaeus.The results indicated that material base were mainly polypeptide and amino acid. Morover, Beside that, we initially clarified that the action mechanism of Catharsius molossus Linnaeus for BPH treatment, which could inhibit a receptor.
2. Preparation and quality control of intermediate
Combining determination with physical and chemical parameters, we prepared effective fraction of Catharsius molossus Linnaeus by percolation. When it comes to purified effect for extract, macroporous resin was superior to foam separation. we determined the molecular weight of polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). By means of high performance capillary electrophoresis (HPCE), we conducted the fingerprint research. Finally, we determined the type and quantity of amino acids by automatic amino acid analyzer.All of those could control quality of intermediate comprehensively and effectively.
3. Studies of new drug delivery system
With the clear material base of drug efficacy, we studied two new drug delivery systems for stable and controllable intermediate. One is colon drug delivery system(OCDDS), the effective fraction was made into tablet. Then according to the pH changes in digestive tract, we choosed the pH-sensitive polymer Eudragit S100 for preparation of colon-released tablet. And released tests of amino acids and polypeptide in vitro showed that the tablet reached purpose of colon specific delivery. Taking total amino acid content as index, we discussed the release law of the preparation by spectrophotometric method. The result demonstrated that it was in accordance with Weibull model. The other one was bioadhesive drug delivery system (BDDS). We selected bioadhesive polymers, screened prescription by indices of rheology characteristic, bioadhesive character and release rate in vitro, and choosed suitable prescription and parameters of craft. The systematic evaluation showed that the preparation was pseudoplastic fluid and its rheological behavior was correspondent with Ostwald model. The adhesive mechanism was supposed to be that the molecular chain of HPMC, tangled with mucus of tissue surface when it met water, and non-ionized hydroxy linked up with mucin through hydrogen bonds. The stability of preparation was good under highlight, high temperature and low temperature condition.We also did acute and multiple dosing stimulation test of the bioadhesive gel to rats. It was observed there was no hyperemia, edema and degeneration by naked eyes and light microscope, which indicated the preparation were safe and non-irritative.With improved Franz diffusion cell, we investigated release law, which was in accordance with Weibull model. Adopting spontaneous BPH model of old dogs, the pharmacodynamics comparison of two drug delivery system was studied. The result demonstrated that they all could inhibit prostatic hyperplasia and they had significant effect.
The study identified the material base of Catharsius molossus Linnaeus for treatment of BPH, clarified action mechanism initially, completed research of two new drug delivery system, proposed the quality control model which associated physico-chemical parameters with determination of component. Finally, we expected to provide a safe, effective, stable and controllable preparation of TCM for clinical treatment of BPH.
本项目根据前列腺增生症的病因病机,针对前列腺自身解剖特点、增生患者瘀血凝结的病理特点,结合中医辨证,选用临床疗效好、活血化瘀作用佳的蜣螂作为模型药物,对其治疗BPH的物质基础及作用机理进行研究,并在此基础上采用新技术、新工艺、新标准研制新剂型,以期为临床提供高效、优质、多样的用药选择。
1、物质基础研究
采用系统溶剂法,结合急性毒性试验和药效筛选(丙酸睾丸酮致小鼠前列腺增生模型与犬膀胱三角肌张力测定)试验,通过色谱法、透析技术等方法与手段对蜣螂治疗BPH的活性成分进行追踪分离,明确了蜣螂治疗BPH的物质基础主要为多肽与氨基酸,初步阐明了蜣螂治疗BPH的作用机理为抑制α受体而起效。
2、中间体的制备与质量控制
采用成分检测与理化参数表征相结合的过程控制模式进行渗漉法制备蜣螂有效部位的工艺研究,比较了泡沫分离与大孔树脂技术对提取液的纯化效果,结果后者较前者为优。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定了多肽的分子量,高效毛细管电泳法(HPCE)对有效部位进行了指纹图谱研究,氨基酸自动分析仪测定了氨基酸的种类与数量,可全面而有效地控制中间体的质量。
3、新型给药系统的研究
在药效物质基础清楚的基础上,以质量稳定可控的蜣螂有效部位中间体为原料,进行了两种新剂型给药系统的研究。在结肠定位释药系统(OCDDS)的研究中,将有效部位制成片芯,根据消化道pH值的变化,选择pH敏感型聚合物包衣材料Eudragit S100制备结肠定位释放片,总氨基酸和多肽的体外释放实验表明其达到了结肠定位释放的目的。采用分光光度法,以总氨基酸的含量为指标探讨了制剂的释药规律,结果符合Weibull模型。在生物粘附给药系统(BDDS)的研究中,选用生物粘附聚合物,以制剂的流变学特性、生物粘附性、体外释放度为指标筛选制剂处方与工艺条件,确定了较佳处方与成型工艺参数。对制剂的流变学特性、生物粘附机制、粘膜刺激性、稳定性、释药规律进行了系统评价,结果其为假塑性流体,流变学行为符合Ostwald模型；粘附机制可能为其含羟丙基甲基纤维素(HPMC),遇水后,分子链与组织表面的粘液缠结,分子中非离子化的羟基通过氢键与粘蛋白分子相连,产生粘附；稳定性研究中,对影响因素进行考察,结果强光照射、高温、低温条件下,制剂的稳定性均良好；在刺激性试验中,进行了蜣螂有效部位生物粘附凝胶剂对大鼠的急性刺激试验及多次给药刺激试验,结果经肉眼及光镜下观察粘膜均无充血、水肿及变性,表明该制剂基本无刺激性,安全性较好；在释药规律研究中,采用改进的Franz扩散池对制剂的释药规律进行研究,结果符合Weibull模型。采用老年犬自发前列腺增生模型对上述两种制剂的药效进行研究,结果均能抑制前列腺增生,疗效显著。
本研究明确了蜣螂治疗BPH的物质基础,初步阐明作用机理,完成了两种新型给药系统的研究,提出了理化参数表征与含量测定相结合的质量控制模式,可望为临床提供安全有效、质量稳定可控的治疗BPH的中药制剂。
Benign prostatic hyperplasia (BPH), which is a common and frequent disease to the elderly and wrinkly people, belongs to "uroschesis" and "retention of sperm" category in TCM. According to the clinical investigation, more than 50% of males(aged above 50) are suffering from the disease. With the increase of aging population, its incidence is also getting higher and higher, seriously affecting life quality of the elderly.Then the prevention and treatment is urgen.
According to etiology and pathogenesis of BPH, anatomical characteristics of prostate, pathological characteristics of congestion and dialectic in TCM, we selected Catharsius molossus Linnaeus, which has good clinical efficacy and satisfactory effect for blood circulation, as a model drug. We not only investigated material base and action mechanism, but also developed new drug delivery systems adopting new technologies new craft and new standards. Therefore, this reseach will provide effective,high quality and multiple drug selection for clinic.
1. Studies of material base
Using methods,such as systematic solvent extraction,combined acute toxicity test and pharmacodynamic screening tests (benign prostatic hyperplasia model of mouse induced by testosterone and the tensity test of dog's bladder deltoid muscle), chromatographic method, as well as dialysis technique, we traced and separated active ingredient of Catharsius molossus Linnaeus.The results indicated that material base were mainly polypeptide and amino acid. Morover, Beside that, we initially clarified that the action mechanism of Catharsius molossus Linnaeus for BPH treatment, which could inhibit a receptor.
2. Preparation and quality control of intermediate
Combining determination with physical and chemical parameters, we prepared effective fraction of Catharsius molossus Linnaeus by percolation. When it comes to purified effect for extract, macroporous resin was superior to foam separation. we determined the molecular weight of polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). By means of high performance capillary electrophoresis (HPCE), we conducted the fingerprint research. Finally, we determined the type and quantity of amino acids by automatic amino acid analyzer.All of those could control quality of intermediate comprehensively and effectively.
3. Studies of new drug delivery system
With the clear material base of drug efficacy, we studied two new drug delivery systems for stable and controllable intermediate. One is colon drug delivery system(OCDDS), the effective fraction was made into tablet. Then according to the pH changes in digestive tract, we choosed the pH-sensitive polymer Eudragit S100 for preparation of colon-released tablet. And released tests of amino acids and polypeptide in vitro showed that the tablet reached purpose of colon specific delivery. Taking total amino acid content as index, we discussed the release law of the preparation by spectrophotometric method. The result demonstrated that it was in accordance with Weibull model. The other one was bioadhesive drug delivery system (BDDS). We selected bioadhesive polymers, screened prescription by indices of rheology characteristic, bioadhesive character and release rate in vitro, and choosed suitable prescription and parameters of craft. The systematic evaluation showed that the preparation was pseudoplastic fluid and its rheological behavior was correspondent with Ostwald model. The adhesive mechanism was supposed to be that the molecular chain of HPMC, tangled with mucus of tissue surface when it met water, and non-ionized hydroxy linked up with mucin through hydrogen bonds. The stability of preparation was good under highlight, high temperature and low temperature condition.We also did acute and multiple dosing stimulation test of the bioadhesive gel to rats. It was observed there was no hyperemia, edema and degeneration by naked eyes and light microscope, which indicated the preparation were safe and non-irritative.With improved Franz diffusion cell, we investigated release law, which was in accordance with Weibull model. Adopting spontaneous BPH model of old dogs, the pharmacodynamics comparison of two drug delivery system was studied. The result demonstrated that they all could inhibit prostatic hyperplasia and they had significant effect.
The study identified the material base of Catharsius molossus Linnaeus for treatment of BPH, clarified action mechanism initially, completed research of two new drug delivery system, proposed the quality control model which associated physico-chemical parameters with determination of component. Finally, we expected to provide a safe, effective, stable and controllable preparation of TCM for clinical treatment of BPH.
引文
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