淫羊藿总黄酮抗骨质疏松二类新药研究
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摘要
本文以淫羊藿二类新药开发为主题，进行了相关药效学、提取工艺、质量标准及其化学成分分离等方面的研究，内容共分四部分。
第一部分：淫羊藿总黄酮提取物抗骨质疏松相关药效学研究。本试验分别采用维甲酸致大鼠骨质疏松模型与切除卵巢大鼠骨质疏松模型，主要观察了淫羊藿总黄酮提取物对骨质疏松症的防治作用。结果显示，淫羊藿总黄酮提取物对维甲酸导致的雄性大鼠股骨密度降低有明显的抑制作用，对维甲酸导致的雄性大鼠尿钙含量及Ca/Cr比值的升高有显著的拮抗作用，提示淫羊藿总黄酮提取物对维甲酸致雄性大鼠骨质疏松有改善作用。淫羊藿总黄酮提取物对去卵巢大鼠股骨密度的降低有明显的抑制作用，对切除卵巢大鼠尿钙含量及Ca／Cr比值的升高有显著的拮抗作用，提示淫羊藿总黄酮提取物对去卵巢大鼠骨质疏松有改善作用。结论：淫羊藿总黄酮提取物对大鼠骨质疏松具有预防和治疗作用。
第二部分：淫羊藿提取工艺研究。本试验采用正交试验优选淫羊藿提取工艺，通过采用直观分析和方差分析，确定70％乙醇，12倍溶剂渗漉提取，流速3ml／min为最佳提取工艺。然后采用均匀设计优选淫羊藿纯化工艺研究，确定原料与树脂用量之比1.25；水与原料用量之比9；30％乙醇与原料用量之比4；60％乙醇与原料用量之比6，为淫羊藿药材大孔吸附树脂提纯的最佳条件。
第三部分：淫羊藿药材质量标准研究。本试验对淫羊藿药材中主要有效化学成分，如淫羊藿苷、绿原酸、金丝桃苷、木兰碱等进行了定量研究，制定了相关的质量标准。并对淫羊藿药材指纹图谱进行了相关研究，初步建立了以绿原酸为参照物的淫羊藿药材有机酸类成分的指纹图谱，以淫羊藿苷为参照物的淫羊藿药材黄酮类成分的指纹图谱。为鉴别淫羊藿药材真伪，控制淫羊藿药材质量，提供了切实可行的标准。
第四部分：淫羊藿药材化学成分分离与鉴定研究，本试验对淫羊藿药材化学成分的分离与鉴别进行了相关研究，采用高效液相色谱法与低压液相分离技术结合分离单体，结果得到了纯度90％以上的淫羊藿苷，证明以YWG-C_(18)H_(37)为固定相，低压分离淫羊藿黄酮具有可行性。
The article is the mainly part of "The researching of new drug from Epimedium Koreanum Nakai", which is belong to the science and technical title of liaoning Province. In the course of study, according to the technical claim of examination and approval method of new drug which was issued by State Drug Demonstration ,technological process, quality standard and pharmacodynamics related to valid part of material were studied in order.
Studies of pharmacodynamics: Rat osteoporosis models induced by 80mg/kg Retinoic Acid (RA) were used to evaluate the function of total flavonoids of Epimedium Koreanum Nakai (EKF) on osteoporosis. Three doses of (150, 300 and 600mg/kg) were given for four weeks by oral administration. EKF can reduce the bone loss and prevent osteoporosis in model rats.
Ovariectomized rat models were used to evaluate the effects of EKF. Female adult Wistar rats were divided randomly in to normal control, Ovariectomized, ovx plus Xianling Gubao, ovx plus 150ng/kg EKF, ovx plus 300mg/kg EKF, ovx plus 600mg/kg EKF, Compared with model group, 600mg/kg EKF decreased serum ALP in rats, and increased femur density (P<0. 05).
Studies of technological process: On the study of technological process, techno-logical process was optimized according to the result of the orthogonal experiment, fingerprinting was made deep studies in identification of Epimedium Koreanum Nakai. We defined HPLC identification method for Epimedium Koreanum Nakai quality standard and draft clarification of fingerprinting.
In short, through studies of technological process, quality standards and pharmacodynamics, it can be certificated that technique of the new drug is stable, quality standard is controllable, pharmacodynamics effect is reliable.
第一部分：淫羊藿总黄酮提取物抗骨质疏松相关药效学研究。本试验分别采用维甲酸致大鼠骨质疏松模型与切除卵巢大鼠骨质疏松模型，主要观察了淫羊藿总黄酮提取物对骨质疏松症的防治作用。结果显示，淫羊藿总黄酮提取物对维甲酸导致的雄性大鼠股骨密度降低有明显的抑制作用，对维甲酸导致的雄性大鼠尿钙含量及Ca/Cr比值的升高有显著的拮抗作用，提示淫羊藿总黄酮提取物对维甲酸致雄性大鼠骨质疏松有改善作用。淫羊藿总黄酮提取物对去卵巢大鼠股骨密度的降低有明显的抑制作用，对切除卵巢大鼠尿钙含量及Ca／Cr比值的升高有显著的拮抗作用，提示淫羊藿总黄酮提取物对去卵巢大鼠骨质疏松有改善作用。结论：淫羊藿总黄酮提取物对大鼠骨质疏松具有预防和治疗作用。
第二部分：淫羊藿提取工艺研究。本试验采用正交试验优选淫羊藿提取工艺，通过采用直观分析和方差分析，确定70％乙醇，12倍溶剂渗漉提取，流速3ml／min为最佳提取工艺。然后采用均匀设计优选淫羊藿纯化工艺研究，确定原料与树脂用量之比1.25；水与原料用量之比9；30％乙醇与原料用量之比4；60％乙醇与原料用量之比6，为淫羊藿药材大孔吸附树脂提纯的最佳条件。
第三部分：淫羊藿药材质量标准研究。本试验对淫羊藿药材中主要有效化学成分，如淫羊藿苷、绿原酸、金丝桃苷、木兰碱等进行了定量研究，制定了相关的质量标准。并对淫羊藿药材指纹图谱进行了相关研究，初步建立了以绿原酸为参照物的淫羊藿药材有机酸类成分的指纹图谱，以淫羊藿苷为参照物的淫羊藿药材黄酮类成分的指纹图谱。为鉴别淫羊藿药材真伪，控制淫羊藿药材质量，提供了切实可行的标准。
第四部分：淫羊藿药材化学成分分离与鉴定研究，本试验对淫羊藿药材化学成分的分离与鉴别进行了相关研究，采用高效液相色谱法与低压液相分离技术结合分离单体，结果得到了纯度90％以上的淫羊藿苷，证明以YWG-C_(18)H_(37)为固定相，低压分离淫羊藿黄酮具有可行性。
The article is the mainly part of "The researching of new drug from Epimedium Koreanum Nakai", which is belong to the science and technical title of liaoning Province. In the course of study, according to the technical claim of examination and approval method of new drug which was issued by State Drug Demonstration ,technological process, quality standard and pharmacodynamics related to valid part of material were studied in order.
Studies of pharmacodynamics: Rat osteoporosis models induced by 80mg/kg Retinoic Acid (RA) were used to evaluate the function of total flavonoids of Epimedium Koreanum Nakai (EKF) on osteoporosis. Three doses of (150, 300 and 600mg/kg) were given for four weeks by oral administration. EKF can reduce the bone loss and prevent osteoporosis in model rats.
Ovariectomized rat models were used to evaluate the effects of EKF. Female adult Wistar rats were divided randomly in to normal control, Ovariectomized, ovx plus Xianling Gubao, ovx plus 150ng/kg EKF, ovx plus 300mg/kg EKF, ovx plus 600mg/kg EKF, Compared with model group, 600mg/kg EKF decreased serum ALP in rats, and increased femur density (P<0. 05).
Studies of technological process: On the study of technological process, techno-logical process was optimized according to the result of the orthogonal experiment, fingerprinting was made deep studies in identification of Epimedium Koreanum Nakai. We defined HPLC identification method for Epimedium Koreanum Nakai quality standard and draft clarification of fingerprinting.
In short, through studies of technological process, quality standards and pharmacodynamics, it can be certificated that technique of the new drug is stable, quality standard is controllable, pharmacodynamics effect is reliable.
引文
1．刘忠厚，等．骨质疏松研究与防治．北京．化学工业出版社，1994．
2．刘忠厚，等．骨质疏松学．科学出版社，1998．
3．梁立，等．补肾中药治疗骨质疏松症临床观察．中医杂志．1992，33(11)：36．
4．李青南，吴铁，等．淫羊霍提取液对去睾丸大鼠骨代谢的影响．中草药1993，2(12)：637．
5．吴普．神农本草经·卷三·下经·草药·中部．中华医典光盘数据库．
6．李青南，黄莲芳，谢华，等．糖皮质激素所致大鼠骨质疏松症的定量研究．中国药理学通报1993，9：475．
7．中华人民共和国卫生部药典委员会．中华人民共和国药典一部．北京．化学工业出版社1995．
8．沙明，曹爱民，孟宪生，等．高效液相色谱法在中药规范研究中的应用．辽宁科学技术出版社2000．
2．刘忠厚，等．骨质疏松学．科学出版社，1998．
3．梁立，等．补肾中药治疗骨质疏松症临床观察．中医杂志．1992，33(11)：36．
4．李青南，吴铁，等．淫羊霍提取液对去睾丸大鼠骨代谢的影响．中草药1993，2(12)：637．
5．吴普．神农本草经·卷三·下经·草药·中部．中华医典光盘数据库．
6．李青南，黄莲芳，谢华，等．糖皮质激素所致大鼠骨质疏松症的定量研究．中国药理学通报1993，9：475．
7．中华人民共和国卫生部药典委员会．中华人民共和国药典一部．北京．化学工业出版社1995．
8．沙明，曹爱民，孟宪生，等．高效液相色谱法在中药规范研究中的应用．辽宁科学技术出版社2000．