中药藿香正气水质量评价与药动学研究

【作者】 卢建秋；

【导师】 乔延江；

【作者基本信息】 北京中医药大学 ， 中药学， 2007， 博士

【摘要】 目的:中药复方是中医临床用药的主要形式,是中医药学的特色与精髓。中药复方质量控制是中药现代化、国际化的关键问题之一。本文以中医经典名方藿香正气水为研究对象,分析藿香正气水中的主要有效成分,建立多指标含量测定方法,为建立其科学的质量评价方法,保证药品质量提供科学依据。方法:藿香正气水是由中医经典名方改制而来的酊剂,其处方组成苍术、陈皮、厚朴(姜制)、白芷、茯苓、大腹皮、生半夏、甘草浸膏、广藿香油、紫苏叶油等组成,临床疗效显著,应用广泛。本文采用LC-MS/MS和GC法,在藿香正气水中鉴定百秋李醇、厚朴酚、和厚朴酚、欧前胡素、异欧前胡素、橙皮苷、柚皮苷、甘草酸、甘草苷、川陈皮素、珊瑚菜素等11个成分。采用GC内标法测定藿香正气水中君药,广藿香中的有效成分百秋李醇的含量;采用HPLC法同时测定藿香正气水中甘草和陈皮的有效成分甘草苷、橙皮苷的含量;采用HPLC法同时测定藿香正气水中臣药、佐使药,厚朴中有效成分厚朴酚与和厚朴酚、白芷中欧前胡素和异欧前胡素、甘草中甘草酸的含量。采用LC-MS/MS法鉴别藿香正气提取液灌胃给药后,Beagle犬血浆中化学成分厚朴酚;采用HPLC内标法测定犬血浆中厚朴酚含量,应用药代动力学计算软件对实验动物的药时数据进行处理,得出藿香正气提取液中有效成分厚朴酚的药动学参数,研究厚朴酚在实验动物体内变化规律。结果:(1)通过GC法,以百秋李醇标准品对照确认藿香正气水中含有百秋李醇化学成分;通过质谱图解析,确认藿香正气水中含有厚朴酚、和厚朴酚、欧前胡素、异欧前胡素、橙皮苷、柚皮苷、甘草酸、甘草苷、川陈皮素、珊瑚菜素等化学成分,其中厚朴酚、和厚朴酚、欧前胡素、异欧前胡素、橙皮苷、柚皮苷、甘草酸、甘草苷等质谱图与其标准品质谱图对比确认。(2)藿香正气水中百秋李醇含量测定,用GC内标法,内标物正十八烷,百秋李醇在0.0411~0.4112mg·mL-1浓度范围内,峰面积与内标峰面积之比值与浓度呈良好的线性关系(r=0.9992);平均回收率为97.67%,RSD=1.48%(n=6)。(3)藿香正气水中同时测定橙皮苷和甘草苷含量,用HPLC外标法,甘草苷在13.35~267.0μg·mL-1浓度范围内,峰面积与浓度呈良好的线性关系(r=0.9999),平均回收率为101.3%,RSD=2.91%(n=6);橙皮苷在18.0~360μg·mL-1浓度范围内,峰面积与浓度呈良好的线性关系(r=0.9999);平均回收率为100.8%,RSD=1.47%(n=6)。(4)藿香正气水中同时厚朴酚等五个化学成分的含量测定,用HPLC外标法测定厚朴、白芷、甘草中有效成分,甘草酸、欧前胡素、异欧前胡素、厚朴酚、和厚朴酚的在进样量分别在93.50~467.5μg,41.30~206.5μg,18.15~90.75μg,235.0~1175μg和311.0~1555μg之间与峰面积呈良好的线性关系,平均回收率分别为98.31%,99.56%,98.57%,98.05%和99.57%。(5)藿香正气提取液中厚朴酚犬血药浓度测定及药代动力学研究,藿香正气提取液给犬灌胃给药,用LC-MS/MS鉴定犬血浆中厚朴酚和内标大黄素,用HPLC内标法测定犬血浆中厚朴酚浓度,厚朴酚在0.063~10.08μg·mL-1范围内线性关系良好,最低检测限为18ng·mL-1,样品的平均绝对回收率大于85%,日间、日内精密度及稳定性的RSD值均小于10%。藿香正气提取液中厚朴酚在犬体内代谢符合二室模型,权重为1/cc,其主要药动学参数Cmax为0.4870mg·mL-1,Tmax为2.0000h,AUC0~6为1.59mg·h·L-1,t1/2α为0.433h,t1/2β为1.256h,CL/F为3.937L·h-1·kg-1,k10为0.741h-1、k12为0.219h-1、k21为1.192h-1,MRT0~∞为8.7260h,MAT为11.8401h。结论:本文首次建立高效液相色谱内标法测定厚朴酚血药浓度方法,以中药复方藿香正气提取液中有效成分厚朴酚为指标,初步进行了藿香正气复方的药动学研究,阐述了藿香正气水的作用规律和特点。本文建立藿香正气水中主要成分的定性定量方法,对藿香正气水处方中50%的药材实现了控制,建立了中药复方多指标质量评价方法,其中GC法测定百秋李醇含量,实现对藿香正气水中君药有效成分的质量控制,对建立全面科学的藿香正气质量评价方法奠定了基础。更多还原

【Abstract】 Objective: The Formula usually used in clinic is the characteristic and soul of TCM(Traditional Chinese Medicine). Quality control of the TCM Formula is focused by researchers, and it is difficult for modernization and internationalization of TCM. In this study Huoxiangzhengqi Liquid(HXZQL) was selected as an example to analyze the effective components and establish multi-components determination method. These will provide scientific evidence for fingerprint and quality control of drugs.Methods: The Formula of HXZQL is composed of Atractylodes lancea(Thunb.)DC., Citrus reticulata Blanco, Magnolia of’ficinalis Rehd . et Wils, Angelica dahurica.(Fisch. Ex Hoffm.)Benth. et Hook. f, Poria cocos(Schw.)Wolf, Areca catechu L. Pinellia ternate(Thunb.)Breit, Extractum Glycyrrhizae, Oleum Pogostemonis, oil of Folium Perillae. HXZQL is used extensively in clinic because of its remarkable effect. In the study, LC-MS/MS(Liquid Chromatography-Mass Spectrum/Mass Spectrum) and GC(Gas Chromatography) were used for constituents identification of HXZQL. The constituents included Patchouli alcohol, Magnolol, Honokiol, Imperatorin, Isoimperatorin, Hesperidin, Narrigin, Glycyrrhizic acid, Liquiritin, Nobiletin and Phellopterin were identified. GC with the internal standard method was used to determine the content of Patchouli alcohol of Pogostemonis which plays the part of kingship in the HXZQL Formula. HPLC(High Performance Liquid Chromatography) was used to determine the content of Liquiritin and Hesperidin at one time. The contents of Magnolol, Honokiol, Imperatorin, Isoimperatorin and Glycyrrhizic acid were determined at the same time by HPLC. LC-MS/MS was used to identify Magnolol in the plasma samples after oral administration with the extraction of HXZQL. A method with the internal standard for the determination of the Magnolol concentrations in the dog plasma samples was performanced with HPLC. The primary Pharmacokinetic parameters and the regularity of Magnolol distribution were estimated with the Pharmacokinetic calculating software. The relationship between analgesic effect and time was observed by the experiments of writhing induced by acetic acid in mice.Results: (1) Patchouli alcohol of HXZQL was identified with its contrast by GC; Magnolol, Honokiol, Imperatorin, Isoimperatorin, Hesperidin, Narrigin, Glycyrrhizic acid, Liquiritin, Nobiletin and Phellopterin were identified by analyzing the data of Mass Spectrum, thereinto, Magnolol, Honokiol, Imperatorin, Isoimperatorin, Hesperidin, Narrigin, Glycyrrhizic acid and Liquiritin were also identified with their contrasts.(2) GC was used to determine the content of Patchouli alcohol of HXZQL, and n-octadecand was used as internal standard. A good linearity was obtained in the range of 0.0411~0.4112mg·mL-1, r=0.9992. The average recovery was 97.67% with a RSD of 1.48%(n=6).(3) HPLC was used to determine the content of Liquiritin and Hesperidin at one time. A good linearity of Liquiritin was obtained in the range of 13.35~267μg·mL-1, r=0.9999. The average recovery was 101.3% with a RSD of 2.91%(n=6). A good linearity of Hesperidin was obtained in the range of 18.00~360.0μg·mL-1, r=0.9999. The average recovery was 100.8% with a RSD of 1.47%(n=6).(4) HPLC was used to determine the content of Magnolol, Honokiol, Imperatorin, Isoimperatorin, Glycyrrhizic acid at the same time. The good linearity was obtained in the ranges of 93.50~467.5μg, 41.30~206.5μg, 18.15~90.75μg, 235.0~1175μg, 311.0~1555μg, respectively. The average recoveries were 98.31%, 99.56%, 98.57%, 98.05%, 99.57%, respectively.(5) Magnolol and Emodin were identified by LC-MS/MS. HPLC was used to determine the concentrations of Magnolol in the dog plasma samples and Emodin was used as internal standard. A good linear relationship was obtained between 0.063~10.08μg·mL-1. The limit of detection was 18ng·mL-1. The average absolute recovery was more than 85%. The RSD values of within-day and between-day precision and stability were both below 10%. The metabolism of Magnolol in dogs was confirmed to two-compartment model with weight 1/C2.The main parameters were as follows: Cmax0.4870mg·mL-1, Tmax2.0000h, AUC0~61.59mg·h·L-1, t1/2α0.433h, t1/2β1.256h, CL/F3.937L·h-1·kg-1, k100.741h-1, k120.219h-1, k211.192h-1, MRT0~∞8.7260h, MAT11.8401h, respectively.Conclusion: The method with internal standard for determination of the magnolol concentration in dog plasma was established for the first time. Through the determination of magnolol which is a active chemical from extraction of HXZQL, the pharmacokinetic study of the formula was accomplished, and the functionary regularity and feature of it were also illustrated.The qualitative and quantitative methods of the main constituents were found. .It provided multi-components determination method which can provide quality control for 50% of material medecas in HXZQL. The GC method for the determination of patchouli alcohol implemented the quality control of the king part in the HXZQL formula. All of these were the foundations of establishing scientific and comprehensive methods to appraise the quality of HXZQL in further study.更多还原