感冒清热颗粒质量控制方法和活性研究

【作者】 田菁；

【导师】 李发美；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2001， 硕士

【摘要】 感冒清热颗粒由荆芥穗、防风、柴胡、葛根等11味中药制成，具有疏风散寒、清热解表的功效，是临床常用感冒药。然而目前缺少对感冒清热颗粒质量控制方法的研究。为了制订科学、合理、可行的质量控制方法，本文做了如下研究：1 感冒清热颗粒及其萃取部位的活性研究 本文将感冒清热颗粒这个复方药物看作为一个整体，按植物化学中对某一味中药进行系统成分分离的思路，从感冒清热颗粒清膏出发，采用有机溶剂萃取的方法，将药物分成氯仿层、乙酸乙酯层、正丁醇层、水层四个部位。依照对解表药现代药理学的研究，采用小鼠耳肿胀和试管法抑菌实验结果为指标，从这四个部位以及挥发油中寻找抗炎和抑菌有效部位、有效成分。发现乙酸乙酯层和水层是抗炎有效部位，水层是抑菌有效部位。2 质量控制方法研究 根据感冒清热颗粒整体药物的药效和上述活性研究结果以及已知的各味药中活性成分，选择主药防风中的升麻苷（prim-O-glucosylcimifugin）和5-O-甲基维斯阿米醇苷（4’-O-β-D-glucosyl-5-O-methylvisamminol）、抗炎活性成分大豆苷元（daidzein）、葛根的代表性成分葛根素（puerarin）、主药荆芥穗挥发油中的代表性成分胡薄荷酮（pulegone）以及薄荷挥发油中的代表性成分薄荷醇（menthol）作为指标成分，对感冒清热颗粒进行了定性鉴别和含量测定，实行质量控制。 分别以氯仿—甲醇（80:20）和环己烷—氯仿—乙酸乙酯（40:60:60）为展开剂，用自制的硅胶GF₂₅₄薄层板，在感冒清热颗粒中鉴别了来自于防风的5-O-甲基维斯阿米醇苷和来自于葛根的大豆苷元。萃取所得的感冒清热颗粒样品溶液在对照品斑点相应位置处有荧光斑点，防风阴性液和葛根阴性液在相应位置无干扰。采用GC-MS联用技术，鉴别出了感冒清热颗粒挥发性油中近90％的成分，其中主要来自于荆芥穗的胡薄荷酮和薄荷中的薄荷醇含量较大。 沈阳药科大学硕士学位论文 摘要 首次采用反相高效液相色谱法分别测定了感冒清热颗粒中的葛根 素、大豆苦元、升麻苦和5o－甲基维斯阿米醇苦的含量。 采用 APEX ODS色谱柱，以醋酸按缓冲液门 00mmol／L，PH 5刀)一 甲醇门：25)为流动相，检测波长为 250urn，流速为 0．Slllullllll。葛 根素在 2～20卜g／InL范围内峰面积与浓度线性关系良好什—0．999 9)；以 蔗糖型计算，测得其方法精密度为 RSD．l％，加样回收率为 98石％ （RSnl．8％）。感冒清热颗粒中葛根素含量分别为 1．52 mg／g（蔗糖型）、 3．50 m卵（无糖型）、0．288 m ＊（乳糖型）。 以醋酸铰缓冲液门 mmol几，pHS刀)－甲醇－四氢味哺“0：37： 3．5)为流动相，检测波长为 250 urn，流速为 0．8 InL／Inn。大豆昔元在口．2～1．8 卜ym上范围内峰面积与浓度线性关系良好什－0．999 7)，以蔗糖型计算， 测得其方法精密度为2刀％，加样回收率为95．5％（RSD＝l．8％）。测得感 冒清热颗粒屯大豆昔元的含量分别为 23．5 pg／g（蔗糖型）、78．2卜g／g（无 糖型）和 13石卜g／g湾糖型)， 以醋酸钠水溶液N，pH 6．8)一甲醇“5：35)为流动相， 检测波长为254run，流速为0．smL／min。升麻昔在0．8～7．2卜g／mL范围内 峰面积与浓度线性关系良好 r—0．999 9)，以蔗糖型计算，测得其方法 精密度为 1刀％，加样回收率为 101．4％（RSD＝．8％）。测得感冒清热颗 粒中升麻昔的含量分别为0．133m吮（蔗糖型）、0．317m眈（无糖型）、 0．549m吮（乳糖型）， 以水一甲醇一四氢吱哺（62：38：l）为流动相，检测波长为254urn， 流速为 0＊SInL／min。5－O－甲基维斯阿米醇苦在 0．8～7．2 pg／mL范围内峰面 积与浓度线性关系良好（r—0．999 9），以蔗糖型计算，测得其方法精密 度为1．8％，加样回收率为95刀％（RSD＝2．7％）。测得感冒清热颗粒中5－O－ 甲基维斯阿米醇苦的含量分别为 0．167mg／g（蔗糖型）、020ling／g（无 糖型）、0二32mg／g（乳糖型）。 本文对感冒清热颗粒的鉴别方法、含量测定方法及活性的研究为制 订感冒清热颗粒的质量标准提供了依据。更多还原

【Abstract】 Ganmao qingre keli is an effective Chinese traditional formula against common cold,~-which is composed of 11 medicinal herbs: Herba Schizonepetae, Herba Menthae, Radix Saposhnikaviae, Radix Bupleuri, Herba Perillae, Radix Puerariae, Radix Plaiycodon. Semen Prunus, Radix Angelica, Herba Co~ydal is, Herba Pragmites.1 BI0AcTIvITY STUDYFour fractions were obtained from successive extraction of the crude drug with chloroform, ethyl acetate and n-butanol. According to the modem pharmacological methods for traditional diaphoretic medicines, antiinflaminative and bacteriostatic effects were used as the criteria to evaluate the bioactivities of those four fractions and volatile components of Ganmao qingre keli. It was found that ethyl acetate layer and water layer were antiinflammative while water layer was bacteriostatic. Daidzein. a compound from ethyl acetate fraction showed anti-inilammative activity.2 STUDY ON QUALITY CONTROL METHODBased on the bioactivity of Ganmao qingre keli and the known components of its composed herbs, pulegone, menthol, puerarin, daidzein, prim-O-glucosylciniifugin and 4?O- ~ -D-glucosyl-5-O-methylvisanminol were chosen as indexes of quality control.P -D-glucosyl-5-O-methylvisamminol from Saposhnikavia divaricata (Turcz.) Schischk and daidzein from Pueraria lobata (Willd.) Ohwi were identified by inC. and pulegone from Schizonepeta tenu~folia Briq., and3menthol from Mentha haplocalyx were identified by GC-MS.Four HPLC methods were developed to determine puerarin, daidzein, prim-O-glucosylcimifiigin and 4?Q- ~ -D-glucosyl-5-O-methylvisanuninol in Ganmao qingre keli.Ganmao qingre keli (with sucrose, without sugar, with lactose) were extracted with water and ethanol, centrifuged and filtered. An APEX ODS column (25OnimX4.6mm, 5pm) was used in this study.To determine puerarin, the mobile phase was 1 OOmmoIIL ammonium acetate (pH5.0)- methanol (75: 25). The flow rate was 0.8 mL/min, and the detection was set at 250 nm. The calibration curve was linear in the range of 2?0 pg/mL puerarin (r=0.999 9). The relative standard derivation was 1.1% and the average recovery was 98.6% (RSD1 .8%) in Ganmao qingre keli (with sucrose). The average contents of puerarin in Ganmao qingre keli (with sucrose, withGut sugar, with lactose) were 1.52 mg/g (RSD1.1%), 3.48 mglg (RSDO. 8%), 0.288mg1g (RSThZ3 .6%) respectively.To determine daidzein, the mobile phase was I OOmmolIL ammonium acetate pH5.0-methanol-THF (60:37:3.5). The flow rate was 0.8 mL/min, and the detection was set at 250 nm. The calibration curve was linear in the range of 0.2? .8 pg/mL daidzein (r=O.999 7). The relative standard derivation was 2.0% and the average recovery was 95.5% (RSD=l .8%) in Ganmao qingre keli (with sucrose). The average contents of daidzein in Ganmao qingre keli (with sucrose, without sugar, with lactose) were 23.5 pg/g (RSD=2.0%), 78.2 pg/g (RSD=l.4%), 13.6 pglg (RSD=3.5%) respectively.To determine prim-O-glucosylcitnifiigin, a mobile phase of methanol-40 mniol/L sodium acetate pH6.8 (35:65) was used with a flow rate of 0.8 mlimin and a detection set at 254 nm. The calibration curve was linear in the range of 0.8p.g/mL ?.2 pg/mL for prim-O-glucosylcimifugin (r=0.999 9). The relative standard derivation was 1.0% and the average recovery was 101.4% (RSD=l.8%) in Ganmao qingre keli (with sucrose). The average contents of prim-O-glucosylcimifugin in Ganmao qingre keli (with sucrose, without sugar, with lactose) were 0.1 32mgIg (RSD=1 .0%), 0.31 7mg/g (RSD 1.7%), 0.549mg1g (RSD=2.2%) respectively.To determine 4?O- P -D-glucosyl-5-O-methylvisamminol, a mobile phase of L120-methanol-THF (62:38:1) was employed with a flow rate of 0.8 mL/min, and a detection set at 254 nm. The calibration curve was linear in the4range of 0.8p.gImL ?.2 ~ig/mL for 4?O- P -D-glucosyl-5-Omethylvisamrninol (r=0.999 9). The relative standard derivation was 1.8% and the average recovery was 95.0% (RSD=1 .8%) in Ganmao qingre keli (with sucrose). The average contents of 4?O-更多还原