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1、抗骨质增生口服液的半仿生模式研究 抗骨质增生口服液的半仿生模式研究 Study on the Preparation and Quality Standards of Anti-hyperostosis Oral Liquid【中文摘要】 骨质增生为现代医学病名,也称骨刺、骨赘,系中老年人的一种慢性疾病,其发病率与年龄的增长成正比,临床上又称增生性关节炎、退行性关节炎、骨性关节炎、老年性关节病等。随着我国开始进入老龄社会,骨质增生的发病率有逐年上升趋势,越来越受到医药专家的重视。抗骨质增生口服液处方是我院多位老中医历经多年的临床实践总结出来的经验方,由淫羊藿、骨碎补、赤芍、熟地黄、甘草、红花、
2、狗脊、鸡血藤、桃仁、莱菔子、鹿衔草、肉苁蓉十二味药组成,本品具有通经活络,舒筋止痛,补肾强筋,除风利湿之功效,临床用于骨质增生,瘀血肿痛,跌打损伤诸症。大量的临床观察证明其抗骨质增生疗效确切。我院制剂室已将其制成丸剂用于临床,但丸剂服用量大且以前的提取制备工艺欠合理。为尽可能多地保留有效成分、提高疗效,同时减小服用量,方便用药,本课题通过优选提取和制备工艺将该方制备成口服液并进行了质量标准的研究,主要内容如下:第一部分抗骨质增生口服液的制备工艺研究目的:用均匀设计优选抗骨质增生口服液半仿生提取法(简称SBE法)的工艺条件,并在半成品中加入适当辅料做成口服液。方法:采用均匀设计试验筛选制备工艺1
3、以第一煎水PH值、第二煎水PH值、第三煎水PH值和煎煮时间作为考察因素,以柚皮苷、甘草酸、淫羊藿苷、多糖和干膏得率为指标来优选工艺。2加入适当辅料做成口服液。结果:通过均匀设计试验筛选的最佳制备工艺1第一煎水PH值6.00、第二煎水PH值6.50、第三煎水PH值9.00和煎煮时间3.7h。2提取液高速离心后,上清液加适量蜂蜜,矫味,加苯甲酸钠3g,加水至1000ml,灌装,灭菌,即得。结论:通过均匀设计试验确定了抗骨质增生口服液的最佳提取工艺和最佳制备工艺,该工艺稳定、可行、有效成分含量高。第二部分质量标准的研究目的:1采用薄层色谱法,选择适当的展开剂,建立抗骨质增生口服液中熟地黄、赤芍、淫羊
4、藿和甘草的定性鉴别方法。2采用高效液相色谱法,建立淫羊藿苷和柚皮苷的含量测定方法。方法:1熟地黄的定性鉴别:使用硅胶G薄层板,以环己烷-乙酸乙酯(8:1)为展开剂,在紫外灯(365nm)下检视;赤芍的定性鉴别:使用硅胶G薄层板,以三氯甲烷-乙酸乙酯-甲醇-浓氨试液(8:1:3:0.5)为展开剂,展开,取出,晾干,喷以5%香草醛硫酸溶液,在105烘约5分钟。淫羊藿的定性鉴别:使用硅胶GF254板。以甲醇-三氯甲烷-甲酸(1:4:0.2)为展开剂,置紫外光灯(254nm)下检视。甘草的定性鉴别:使用1% NaOH溶液制备的硅胶G薄层板,以三氯甲烷-甲醇-水(6:2:0.3)为展开剂,喷以10%硫酸
5、乙醇溶液,在105加热至斑点清晰,置紫外光灯(365nm)下检视。2含量测定:(1)高效液相色谱法:参考相关文献选择合适的固定相,调整流动相的组成、配比、流速以及柱温,在最大吸收波长下测定,使指标峰分离完全、理论塔板数符合要求;紫外分光光度法:选择合适的显色方法和测定波长。(2)线性范围考察以色谱峰峰面积为纵坐标,以进样浓度为横坐标(紫外分光光度法以吸收度为纵坐标,以测定液浓度为横坐标),进行线性回归,得回归曲线并考察线性范围。(3)方法学考察:分别考察测定方法的精密度、重现性、稳定性和加样回收率。(4)样品含量测定:取3批抗骨质增生口服液分别制备供试品溶液,进样,测定指标成分的含量。结果:1
6、熟地黄、淫羊藿、赤芍和甘草的TCL图谱清晰,样品溶液在与对照药材溶液相应的位置上,均显相同颜色的斑点,阴性对照溶液显示无干扰。2柚皮苷的色谱条件为:Diamonsil(250m4.6mm,5um)色谱柱,流动相:甲醇-5%冰醋酸(33:67),流速1.0ml/min,柱温:30,检测波长283nm。回归曲线为y=1625.1x-9.6231,r=0.9997,柚皮苷在0.0664ug1.0624ug范围内线性关系良好。该方法精密度、重现性和在24小时内稳定稳定性的RSD分别为0.76%、0.43%和2.09%,平均加样回收率为100.12%,RSD为0.64%。3淫羊藿苷的色谱条件为:Diam
7、onsil(250m4.6mm,5um)色谱柱,流动相:乙腈-水(27:73),流速1.0ml/min,柱温:25,检测波长270nm。回归曲线为y=1617.4x+53.123,r=0.9976,淫羊藿苷在0.0948ug1.5168ug范围内线性关系良好。该方法精密度、重现性和在24小时内稳定稳定性的RSD分别为0.50%、1.75%和0.97%,平均加样回收率为100.03%,RSD为0.16%。结论:建立的薄层鉴别方法专属性强,斑点清晰,可对制剂中的中药进行准确鉴别;高效液相色谱法进行含量测定,准确度高、专属性强、重现性好,可对制剂质量进行有效控制。【英文摘要】 Hyperostosi
8、s as modern medicine name,also known as spur,osteophyte,it is a middle-aged and elderly chronic disease. As growth of age,its incidence is increasing,clinically also known as hypertrophic arthritis,degenerative arthritis,bone arthritis,joint diseases and so on.With entering the aging socie- ty,the i
9、ncidence of hyperostosis have an upward trend year after year,emphasizing by medical specialists.The prescription of anti-hyperostosis oral liquid was a empirical formula summarized by traditional Chinese physician in our hospital in years of clinical practice,which had been prooved effective by a g
10、reat quantity of clinical observations.The prescription was composed of Radix et Rhizoma Glycyrrhizae,Radix Rehman- niae,Herba Cistanches,Flos Carthami,Radix Paeoniae Rubra, Caulis Spatholobi,Rhizoma Cibotii,Rhizoma Drynariae, Semen Raphani,Semen Persicae,Herba Pyrolae and Herba Epimedii, which has
11、the function of anti-hyperostosis.It had being made into pellet by manufacturing laboratory of our hospital.Because the different active substance had the different physico- chemical property,the previous preparation was less reasonable. As to keep as many effective components as possible,and reduce
12、 the dosage and enhance the effectiveness and conv- enience of administration at the same time,the experiment was carried out to make it into oral liquid by optimizing the extraction and forming condition,and then make the quality standard of oral liquid.The principal elements of experiment were as
13、follows:Part one:Study on the optimal preparation condition of anti-hyperostosis oral liquidObjective:To optimize the extraction,impurity removal technique,and then add appropriate adjuvant to the intermediate product as oral liquid.Methods:uniform design was carried out for choosing the preparation
14、 process.With PH of first decoction,PH of second decoction,PH of third decoction and three decoction time as evaluation factors,and the content of Glycyrrhizin,glucose, naringin,icariin,and the yield of powdered extract as indexes, the optimal extracting condition for Radix et Rhizoma Glycy- rrhizae
15、,RadixRehmanniae,Herba Cistanches,Flos Carthami, Radix Paeoniae Rubra,Caulis Spatholobi,Rhizoma Cibotii, Rhizoma Drynariae,Semen Raphani,Semen Persicae,Herba Pyrolae and Herba Epimedii.Result:1The optimal extracting condition was PH of first decoction 6.0,PH of second decoction 6.5,PH of third decoc
16、tion 9.0 and three decoction time 3.7h.2After high-speed centrifugation,the supernatant approp- riately add honey, flavor,add sodium benzoate 3g,add water to 1000ml, filling,sterilization.Conclusion:Through the uniform design,the optimal extracting and forming process of anti-hyperostosis oral liqui
17、d was determined.The process was stabile with a high content of active substance.Part two:Studies on the quality standards of anti- hyperostosis oral liquidObjective:1To establish methods for identifying radix rehmann- iae,radix paeoniae rubra,herba epimedii and radix et rhizo- ma glycyrrhizae by TL
18、C.2To develop methods for determining the contents of Naringin and Icariin in Anti- hyperostosis oral liquid by HPLC. Methods:1Identification of Radix Rehmanniae preparata:silica gel G was used as the coating material and cyclohexane-ethyl acetate (8:1) as the mobile phase, examining under ultraviol
19、et light(365nm). Identification of radix paeoniae rubra:silica gel G was used as the coating material and the upper solution of chloroform-ethyl acetate-methanol- concentrated ammonia test solution (8:1:3:0.5) as the mobile phase,spray with 5% vanillin solution, heat at the temperature of 105for fiv
20、e minutes and examine in daylight. Identification of Radix Rehmanniae preparata:silica gel GF254 was used as the coating material and Methanol- chloroform-formic acid (1:4:0.2) as the mobile phase, examining under ultraviolet light(254nm).Identification of radix et rhizoma glycyrrhizae:silica gel G
21、prepared 1% NaOH solution was used as the coating material and chloroform-methanol-water (6:2:0.3)as the mobile spray phase,with 10% sulphuric acidalcohol solution,heat at the temperature of 105for several minutes,examining under ultraviolet light(365nm).2Determination of the contents of active subs
22、tance2.1Chromatogram condition:choose appropriate stationary phase and adjust the composition,proportion and flow rate of mobile phase to separate the peak of Naringin,or Aspero- saponin well and meet the requirements of number of theoretical plates.UV spectrophotometry:choose a suitable coloration
23、method and the wavelengths of maximal absorption.2.2The investigation of linear range:a series of the reference solutions were prepared and the regression equation was obtained with the contents of reference as abscissa and the peak area(absorbability in UV absorption spectrophotometry)as ordinate.2
24、.3The investigation of technology:investigate the precision,reproducibility,stability and recovery.2.4Determination of the sample:take three batch of samples for six portion and prepare for the test solution, determine the content of active substance in samples.Results:1Radix rehmanniae,radix paeoni
25、ae rubra,herba epimedii and radix et rhizoma glycyrrhizae all had clear map of TLC.Sample solution showed the same color of dots in the corresponding position with reference solution,and negative control solution showed no interference.2Naringin chromatographic conditions Diamonsil (250m- x4.6mm,5um
26、) column. mobile phase:methanol-5% acetic acid (33:67),flow rate 1.0ml/min,column temperature: 30C,The detection wavelength was set at 283nm.Regression equation: y=1625.1x-9.6231,r=0.9997.Naringin showed good linear relationship in 0.0664ug1.0624ug.The RSD of the precision, reproducibility and stabi
27、lity in twenty four-hour period were 0.76%,0.43% and 2.09% respectively.The average recovery and RSD were 100.12% and 0.64%.3Icariin chromatographic conditions Diamonsil (250m- x4.6mm,5um) column.mobile phase:acetonitrile-water (27:73), flow rate 1.0ml/min,column temperature: 25C,The detection wavel
28、ength was set at 270nm. Regression equation: y=1617.4x+53.123,r=0.9976.Icariin showed good linear relationship in 0.0948ug1.5168ug.The RSD of the prcis- ion,reproducibility and stability in twenty four-hour period were 0.50%,1.75% and 0.97% respectively. The average recovery and RSD were 100.03% and
29、 0.16%.Conclusion:The TLC methods had good specificity and clear spots which can be use for the accurate identification of the traditional Chinese medicine in preparation;HPLC were stable,accurate,specific.They can effectively control the quality of the preparation.【中文关键词】 抗骨质增生口服液; 制备工艺; 质量标准; 柚皮苷;
30、 淫羊藿苷; 甘草次酸; 多糖; 骨质增生 【英文关键词】 Anti-hyperostosis oral liquid; Preparation; Quality standards; Naringin; Icariin; Glycyrrhizin; Glucose; Hyperostosis 【毕业论文目录】中文摘要 4-8 英文摘要 8-12 研究论文 抗骨质增生口服液的半仿生模式研究 14-68 第一部分 抗骨质增生口服液制备工艺的研究 14-51 前言 14 材料与方法 14-28 结果 28-29 附图 29-34 附表 34-47 讨论 47-48 参考文献 48-51 第二部分 抗骨质增生口服液质量标准的研究 51-68 前言 51-52 材料与方法 52-57 结果 57-60 附图 60-63 附表 63-67 讨论 67 结论 67 参考文献 67-68 综述 骨质增生的中医药研究进展 68-74 致谢 74-75 个人简历 75