乌头汤中苯甲酰新乌头胺及麻黄碱的药代动力学研究
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摘要
研究背景
类风湿性关节炎(Rheumatoid Arthritis, RA)是以关节组织慢性炎症性病变为主要临床表现的自身免疫性疾病,其患病率约为0.3%～1%。目前该病病因尚未完全明确,感染、遗传、雌激素水平、环境、劳累、外伤等因素均可能诱导RA发生。若治疗不及时、不规范,滑膜炎的持久、反复发作可导致关节内软骨和骨的破坏,引起严重关节畸形、关节功能障碍,从而严重影响RA患者的生活质量。西医对RA的治疗目标主要是缓解症状、控制病情发展,从而改善患者的生活质量。目前临床上用于治疗RA的药物主要有非甾体抗炎药、慢作用抗风湿药及糖皮质激素,这些药物可以快速起效,但是长期使用均可导致严重的不良反应。目前已有大量的临床研究显示一些传统中医药制剂以及中西医联合疗法对RA的治疗效果比单独使用西药治疗更让人满意。
乌头汤是一个应用历史悠久的经典方剂,由川乌、麻黄、白芍、甘草及黄芪组成,临床上主要用于RA、风湿性关节炎等疾病的治疗。乌头汤的疗效确切,已使广大的RA患者获益。然而,处方中的“君药”及“臣药”的川乌及麻黄均具有严重毒副作用,因此,必须通过科学手段阐明乌头汤产生疗效的物质基础和作用机制,以提高药物疗效和减轻毒副作用。
药代动力学着重研究研究药物产生临床疗效的物质基础及体内过程,药代动力学参数可用于指导临床用药、评价药品的安全性、促进新药的开发研究等。由于中药的化学成分比较复杂,目前,中药药代动力学研究常选取一种或几种代表成分作为研究对象,进而阐述中药或方剂的药代动力学特征。选取具有代表性的指标成分进行针对性的研究,是研究乌头汤药代动力学特征的关键。我们采用以下两个条件为基础选择具有代表性的指标成分：1)该物质的药理活性应当与乌头汤的药理作用一致；2)该物质在乌头汤提取液中应当具有相对较高的含量。
川乌为乌头汤的“君药”,是乌头汤产生临床疗效不可或缺的组分。众所周知,乌头类生物碱是川乌中的主要药理活性成分,包括双酯二萜类生物碱及单酯二萜类生物碱。双酯二萜类生物碱受热不稳定,煎煮过程中,易水解生成对应的单酯二萜类生物碱。已有文献报道附子水煎液加热30mmin,即有大部分双酯二萜类生物碱发生水解。乌头汤的制备需要较长时间的煎煮,可导致大部分双酯二萜类生物碱(乌头碱、次乌头碱、新乌头碱)发生水解,显然不符合我们对目标化合物选择的条件。苯甲酰新乌头胺(Benzoylmesaconine, BMA)是一种单酯二萜类生物碱,是新乌头碱(Mesaconitine, MA)的水解产物,也是川乌最主要的有效成分之一。BMA具有显著的镇痛作用,已有文献证实BMA对RA具有治疗作用；且其在乌头汤提取液中的含量显著高于其他乌头类生物碱。因此,本实验选择BMA作为乌头汤“君药”的代表成分,研究其药代动力学特征。
麻黄为乌头汤的“臣药”,麻黄碱是其主要有效成分。麻黄碱在乌头汤产生临床疗效的过程中发挥了重要的作用。麻黄碱具有类似于肾上腺素的化学结构,可与血管中肾上腺素受体结合。肾上腺素受体结合,产生舒张血管的作用,从而加快血流速度、增加皮肤血流量,从而辅助川乌以增强疗效。已有大量文献报道麻黄碱是麻黄含量最高的生物碱,其在乌头汤提取液中的含量也显著高于其他化学成分。因此,本实验选择麻黄碱作为乌头汤“臣药”的代表成分,研究它的药代动力学特征及肠道吸收特征。
然而,关于乌头汤有效成分的药代动力学研究有以下几个难点。首先,乌头汤由多种药材组成,其成分复杂；其次,生物样品中待测物质浓度低,对其提取、分离、纯化、检测均有较高难度：第三,中药药代动力学研究的生物样本量多,工作量大。因此,建立一个灵敏、准确、快速的生物样品定量分析方法是药代动力学研究的关键。超高效液相色谱-串联质谱(Ultra Performance Liquid Chromatography tandem Mass Spectrometry, UPLC-MS/MS)充分体现了色谱和质谱优势的互补。UPLC-MS/MS具有高灵敏度和较强的专属性,不需要使分析物之间实现完全的色谱分离,使得样品预处理过程简化,且大大缩短样品的分析时间；MRM模式还允许同时对多个成分进行定性、定量分析。采用UPLC-MS/MS可更加准确、快速的分析样品,有利于获得更精确的药代动力学参数。
研究目的
本实验旨在对乌头汤“君药”、“臣药”代表成分的药代动力学特征进行深入研究,以期为乌头汤更深入的研究及其临床应用提供物质基础和理论依据。主要研究内容包括以下几点：
1.研究口服乌头汤后BMA的药代动力学特征,考察乌头汤其他组分对“君药”代表成分的体内过程的影响作用。
2.研究口服乌头汤及麻黄单味药材提取液后麻黄碱的药代动力学特征,考察乌头汤其他组分对“臣药”代表成分的体内过程的影响作用。
3.采用大鼠在体肠灌流模型,研究乌头汤中麻黄碱在大鼠肠道不同部位的吸收特征,考察乌头汤其他组分对“臣药”代表成分在肠道吸收的影响作用。
实验方法及结果
1.“君药”代表成分苯甲酰新乌头胺药代动力学研究
我们建立了一个快速、灵敏的UPLC-MS/MS方法,用于定量测定样品中BMA的含量。冷冻干燥已制备的乌头汤提取液,制成冻干粉末,于实验前日使用UPLC-MS/MS测定其中BMA的含量。选用10只体重为230～280g的雄性SD大鼠,随机分为两组,按照5mg/kg (BMA)的剂量分别灌胃给予乌头汤提取液及BMA溶液。分别于给药后5、15、30、45、60、90、120、180、240、420、600min经大鼠眼眶静脉丛采集血样约0.3ml,所采集的血样在8000rpm离心8min,取上层血清至EP管,置于-80℃冰箱中保存备测。血浆样品处理如下：取80μl血浆样品,加入320μl甲醇(睾丸酮浓度为100nM),用于去除血浆的蛋白成分,在13000rpm离心30min,吸取适量上清液,室温下氮气吹干,所得残渣以100μl甲醇水溶液(50%)复溶,然后在13000rpm离心30min,吸取上清液进样分析。经UPLC-MS/MS测定样品中BMA的含量,并采用WinNonlin3.3软件的标准非室性模型计算BMA的药代动力学参数。
大鼠口服给药BMA溶液后,BMA的血药浓度于45min达峰；600min后,血浆样品中BMA浓度接近O。然而,口服给药等剂量BMA的乌头汤提取液,BMA的血药浓度于15min达峰；240min后,血浆中的BMA基本上完全被清除。以BMA溶液为参照组,口服给药乌头汤提取液后,BMA的相对生物利用度为19.9%。口服给药乌头汤提取液后,BMA在大鼠体内的AUC较低；其Tmax及MRT均显著缩短,经两独立样本t检验(Two Independent Samples t-test)进行分析,具有显著性差异(P<0.05)。
2.“臣药”代表成分麻黄碱药代动力学研究
我们建立了一个快速、灵敏的UPLC-MS/MS方法,用于定量检测血浆中麻黄碱的浓度。取麻黄单味药材制备提取液,并制成冻干粉末。于实验前日使用HPLC测定乌头汤提取液和麻黄单味药材提取液冻干粉末中麻黄碱的含量。选用20只体重为230～280g的雄性SD大鼠,随机分为4组。其中三组按照10mg/kg(麻黄碱)的剂量分别灌胃给予盐酸麻黄碱溶液、麻黄单味药材提取液及乌头汤提取液,并于给药后5、15、30、45、60、90、120、180、240、420、600、1440min经大鼠眼眶静脉丛采集血样约0.3ml;另外一组按照4mg/kg：麻黄碱)的剂量静脉注射盐酸麻黄碱溶液,并于给药后1、5、12、18、30、45、72、100、150、240、480、600min经大鼠眼眶静脉丛采集血样约0.3ml。所采集血样在8000rpm离心8min,取上层血清至EP管,置于-80℃冰箱中保存。血浆样品处理如下：取80μl血浆样品,加入520μl乙酸乙酯(睾丸酮浓度为20μM),用于去除血浆的蛋白成分,在13000rpm离心30min,吸取上清液400μl,室温下氮气吹干,所得残渣以100μl甲醇水溶液(50%)复溶,然后在13000rpm离心30min,吸取上清液进样分析。经UPLC-MS/MS测定样品中麻黄碱的含量,并采用WinNonlin3.3软件的标准非室性模型计算BMA的药代动力学参数。
以10mg/kg(麻黄碱)的剂量灌胃给药盐酸麻黄碱溶液、麻黄单味药材提取液及乌头汤提取液后600min,血浆样品中麻黄碱的基本完全被清除；三组大鼠的绝对生物利用度分别为83.4%、71.4%、29.9%。以口服盐酸麻黄碱溶液为参照组,口服麻黄单味药材提取液及乌头汤提取液后,麻黄碱的相对生物利用度分别为85.6%、35.9%。口服乌头汤及麻黄单味药材提取液后,麻黄碱的Tmax显著缩短,经单因素方差分析(One Way ANOVA)进行分析,具有显著性差异(P<0.05)。
3“臣药”代表成分麻黄碱肠道吸收特征研究
冷冻干燥已制备的乌头汤提取液,制成冻干粉末,于实验前日使用HPLC测定其中麻黄碱的含量。精密称取适量盐酸麻黄碱及乌头汤冻干粉末,以HBSS溶液溶解稀释至麻黄碱浓度为40μg/ml。所用实验动物为230～280g的雄性SD大鼠。本实验采用大鼠在体肠灌流模型考察乌头汤中麻黄碱在肠道不同部位的吸收情况。以含有麻黄碱的HBSS溶液(盐酸麻黄碱溶液、乌头汤提取液)同时灌流大鼠的四个肠段(十二指肠、空肠上段、回肠下段、结肠),每隔30min收集一次样品。往收集到的灌流液样品中加入乙腈(v：v=2：1)去除蛋白,样品在13000rpm离心处理30min,吸取上清液。经UPLC系统检测分析,进样体积为10μl。
灌流盐酸麻黄碱溶液后,麻黄碱在十二指肠、空肠、回肠、结肠的渗透系数(Peff)分别为2.20、1.81、2.33、1.68；麻黄碱在四个肠段的吸收率依次为24.9%、19.5%、25.2%、17.0%。灌流乌头汤提取液后,麻黄碱在十二指肠、空肠、回肠、结肠的渗透系数(Peff)分别为2.33、2.31、3.35、2.68；麻黄碱在四个肠段的吸收率依次为27.7%、26.4%、37.4%、30.3%。乌头汤提取液中麻黄碱在空肠、回肠及结肠的吸收率较高,经两独立样本t检验(Two Independent Samples t-test)进行分析,具有显著性差异(P<0.05)。
结论
1.口服给药乌头汤后,BMA的相对生物利用度为19.9%：BMA在大鼠体内的Tmax显著缩短,表明乌头汤中存在可促进BMA吸收的成分,有利于乌头汤快速起效；BMA在大鼠体内的MRT显著缩短,表明乌头汤中存在可加速BMA清除的成分,有利于BMA的清除而避免发生蓄积。
2.口服给药乌头汤后,麻黄碱的绝对生物利用度为29.9%,低于口服给药含有相同剂量的盐酸麻黄碱溶液(83.4%)及麻黄单味药材提取液(71.4%)。口服给药乌头汤及麻黄单味药材提取液后,麻黄碱的大鼠体内的Tmax显著缩短,表明乌头汤中存在可促进麻黄碱吸收的成分。
3.使用乌头汤及盐酸麻黄碱溶液进行大鼠在体肠灌流,麻黄碱在大鼠全肠道吸收良好,无明显的特定吸收部位。乌头汤中的麻黄碱在大鼠空肠、回肠及结肠的吸收率显著升高,表明乌头汤中存在促进其肠道吸收的成分。
Background
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic arthritis, and it has an incidence of0.3%to1%. The etiology of this disease is still unclear. RA is associated with many factors, such as infection, heredity, estrogen levels, fatigue, trauma, and environment. Without timely treatment, the lasting recurrent synovial inflammation can lead to the destruction of joint cartilage and bone, causing severe joint deformity and joint dysfunction, which can seriously reduce the quality of life of patients. The Western medical treatment of RA relieves its symptoms and controls its progression, thereby improving the quality of life of patients. Non-steroidal anti-inflammatory drugs, slow-acting anti-inflammatory drugs, and glucocorticoids are used to treat RA. Although these drugs are fast acting, their long-term use can lead to serious adverse reactions. Numerous clinical studies shown that some Traditional Chinese Medicine (TCM) preparations and TCM combined with chemical drug therapy achieve more satisfactory therapeutic efficacy on RA than chemical drug therapy.
Wutou decoction has been used to treat RA, constitutional hypotension, and hemicrania for hundreds of years. Wutou decoction consists of five medicinal herbs, Radix Aconiti(Chuan Wu), Herba Ephedrae (Ma Huang), Radix Paeoniae alba (Bai Shao), Radix Astragali (Huang Qi), and Radix Glycytthizae (Gan Cao). Chuan Wu is the monarch drug and Ma Huang is the ministerial drug of Wutou decoction, and they are the most important effective components. However, Chuan Wu and Ma Huang have serious side effects. Thus, clarifying the material basis and mechanism of the clinical efficacy of Wutou decoction is necessary.
Pharmacokinetic study has an important role in the research, development, and clinical application of drugs. Pharmacokinetic parameters can be used to guide clinical application, to assess drug safety, and to promote the development of new drugs. However, the effective components of TCM are much more complicated, and clarifying the pharmacokinetic characteristics of a TCM preparation is more difficult. At present, most of the pharmacokinetic studies on TCM preparations focus on one or several of the active components. Selecting representative compounds is the key to the pharmacokinetic study of Wutou decoction. The representative compounds were selected according to the following conditions. First, the pharmacologic activity of these compounds should be consistent with Wutou decoction. Second, these compounds should have higher content in Wutou decoction.
Considering Chuan Wu is the monarch drug of Wutou decoction, it plays the most important role in the decoction. Aconitum alkaloids are the active ingredients of Chuan Wu, including diester diterpenoid alkaloids (DDAs) and monoester diterpenoid alkaloids (MDAs). DDAs are easily hydrolyzed to MDAS during boiling. Most of the DDAs in aconite decoction are hydrolyzed by30min of boiling. Preparing Wutou decoction requires prolonged boiling; therefore, DDA is not an appropriate representative component of Wutou decoction. Benzoylmesaconine (BMA), a mesaconitine hydrolyzate, is one of the active ingredients of Chuan Wu. BMA has significant analgesic effects and a therapeutic effect on RA. In addition, the BMA content of Wutou decoction is significantly higher than other aconitum alkaloids. Thus, BMA was selected as the representative component of Chuan Wu. The pharmacokinetic characteristics of BMA in Wutou decoction were determined in our experiments.
Ephedrine exerts the most potent effect of Ma Huang, the ministerial drug of Wutou decoction. The chemical structure of ephedrine is similar to adrenaline, and it interacts with adrenaline receptors in vascular and diastolic blood vessels, thereby increasing blood flow. The ephedrine content of Wutou decoction is much higher than that of other compounds. Therefore, ephedrine was selected as the representative component of Ma Huang. The pharmacokinetic characteristics of ephedrine after oral administration of Wutou decoction and Ma Huang single herb decoction were investigated. A four-site perfusion model was also used to investigate the absorption of ephedrine in Wutou decoction at different intestinal segments.
The effective components of Wutou decoction are highly complicated, and the concentrations of target compounds, such as BMA and ephedrine, are quite low in plasma and perfusate samples. Furthermore, many groups and many time points are available for sample acquisition. Thus, a large sample size is needed for analysis. An efficient and sensitive detection method is required for sample analysis. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is sensitive and specific and it has a high throughput. Samples do not need to be separated completely, which simplifies the preparation process and greatly shortens the time for sample analysis. Multiple reaction monitoring (MRM) mode also allows simultaneous determination of several compounds. Using UPLC-MS/MS for pharmacokinetic study facilitates obtaining more accurate pharmacokinetic parameters.
Objectives
The objective of this study was to investigate the pharmacokinetic characteristic of BMA and ephedrine, the representative compounds of Wutou decoction. We believe that the pharmacokinetic study of the monarch drug (Chuan Wu) and ministerial drug (Ma Huang) of Wutou decoction can provide a material basis and theoretical foundation for the further study and clinical application of Wutou decoction. Our study could be divided into several parts as follows:
1. To determine the pharmacokinetic characteristics and bioavailability of BMA after oral administration of Wutou decoction.
2. To determine the pharmacokinetic characteristics and bioavailability of ephedrine after the oral administration of Wutou decoction and Mahuang single herb decoction.
3. To investigate the absorption of ephedrine in Wutou decoction in different intestinal segments using a four-site perfusion model. Methods and Results
1. Pharmacokinetic study of BMA after oral administration of Wutou decoction
A rapid and sensitive UPLC-MS/MS method was developed for quantifying BMA in plasma samples. Wutou decoction was prepared and freeze-dried using a lyophilizer to obtain the lyophilized powder. The BMA content of the lyophilized powder was quantified by UPLC-MS/MS one day before the experiments. Male Sprague-Dawley (SD) rats weighing230g to280g were obtained for the experiments. Ten rats were divided into two groups, namely, BMA and Wutou decoction groups. BMA and Wutou decoction were administered orally at5mg of BMA per kilogram of body weight. Blood samples were collected at5,15,30,45,60,90,120,180,240,420, and600min after dosing. After centrifugation at8000rpm for8min, plasma fractions were transferred into a disposable tube and stored at -80℃until analysis. We mixed80μl of plasma with320μl of methanol containing100nM testosterone. The mixture was centrifuged at13,000rpm for30min. Approximately300μl of the supernatant was transferred into a disposable tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted with100μl of methanol-water (v:v=1:1), and injected into the UPLC-MS/MS system for analysis. The pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using WinNonlin3.3.
The Tmax of BMA after oral administration of BMA solution was about45min, and the plasma concentration of BMA was approximately0at600min after dosing. However, the Tmax of BMA after oral administration of Wutou decoction was15min, and BMA was completely eliminated at600min after dosing. The relative bioavailability of BMA after oral administration was19.9%. The Tmax and mean residence time (MRT) after administration were significantly shorter(P<0.05).
2. Pharmacokinetic study of ephedrine after oral administration of Wutou decoction and Ma Huang single herb decoction
A rapid and sensitive UPLC-MS/MS method was developed and validated for quantitative determination of ephedrine in plasma samples. Wutou decoction and Ma Huang single herb decoction were prepared and freeze-dried by a lyophilizer to obtain the lyophilized powder. The ephedrine content in lyophilized powders was quantified by HPLC the day before the experiments. Male SD rats weighing from230g to280g were obtained for the experiments. Ephedrine hydrochloride, Wutou decoction, and Ma Huang single herb decoction were administered orally at a dosage of10mg of ephedrine per kilogram of body weight. Ephedrine hydrochloride was also administered via intravenous injection at4mg/kg. Blood samples were collected at5,15,30,45,60,90,120,180,240,420,600, and1440min after oral administration; and at1,5,12,18,30,45,72,100,150,240,480, and600min after intravenous injection. After centrifugation at8000rpm for8min, plasma fractions were transferred into a disposable tube and stored at-80℃until analysis. We mixed80μl of plasma with520μl of ethyl acetate containing20μM testosterone. The mixture was centrifuged at13000rpm for30min. Approximately400μl of the supernatant was transferred to a disposable tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted with100μl of methanol-water (v:v=1:1), and injected into the UPLC-MS/MS system for analysis. The pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using WinNonlin3.3.
Ephedrine was completely eliminated at600min after oral administration of ephedrine hydrochloride, Wutou decoction, and Ma Huang single herb decoction. The absolute bioavailabilities of ephedrine in the rats after oral administration of ephedrine hydrochloride, Ma Huang single herb decoction, and Wutou decoction were83.4%,71.4%, and29.9%, respectively. The relative bioavailabilities of ephedrine in the rats after oral administration of Ma Huang single herb and Wutou decoctions were85.6%and35.9%, respectively. The Tma of ephedrine after oral administration of Wutou decoction and Mahuang single herb decoction were significantly shortened (P<0.05).
3. Absorption study of ephedrine in Wutou decoction at different intestinal segments
Wutou decoction was prepared and freeze-dried using a lyophilizer to obtain the lyophilized powder. The ephedrine content of the lyophilized powder was quantified by high-performance liquid chromatography (HPLC) the day before the experiments. Ephedrine hydrochloride and lyophilized powder were dissolved in Hank's balanced salt solution to obtain perfusate solutions containing40μg/ml ephedrine. Male SD rats weighing from230g to280g were obtained for the experiments. A four-site perfusion model was used to investigate the absorption of ephedrine at different intestinal segments. Four segments of the intestine (duodenum, upper jejunum, terminal ileum, and colon) were perfused simultaneously with the prepared perfusate. The perfusate samples were collected every30min. Acetonitrile was added into the perfusate (v:v=1:2) and centrifuged at13,000rpm for30min. We injected10μl of supernatant into the UPLC system for analysis.
After perfusion with ephedrine hydrochloride solution, the permeability coefficients (Peff) of ephedrine in the duodenum, jejunum, ileum, and colon were2.20,1.81,2.33, and1.68, respectively. Approximately24.9%,19.5%,25.2%, and17.0%of ephedrine was absorbed in the duodenum, jejunum, ileum, and colon, respectively. After perfusion with Wutou decoction, the Peff of ephedrine at duodenum, jejunum, ileum, and colon were2.33,2.31,3.35, and2.68, respectively. Approximately27.7%,26.4%,37.4%, and30.3%of ephedrine was absorbed at duodenum, jejunum, ileum, and colon, respectively. The absorption of ephedrine at jejunum, ileum, and colon following Wutou decoction perfusion was significantly higher than those with ephedrine hydrochloride (P<0.05).
Conclusions
1. The relative bioavailability of BMA after oral administration of Wutou decoction was19.9%. The Tmax. of BMA after oral administration was significantly shortened, which indicates that some compounds in the decoction accelerate BMA absorption. MRT was also significantly shortened, indicating that some compounds in the decoction promote BMA clearance.
2. The absolute bioavailabilities of ephedrine in the rats after oral administration of Wutou decoction was29.9%, which was much lower than the other groups. The Tmax of ephedrine after oral administration of Wutou decoction and Mahuang single herb decoction were significantly shortened, which indicates some compounds in the decoctions accelerate ephedrine absorption.
3. Ephedrine was well absorbed at different intestinal regions in rats perfused with ephedrine hydrochloride solution and Wutou decoction. The absorption of ephedrine in the jejunum, ileum, and colon after Wutou decoction perfusion was significantly higher than those with ephedrine hydrochloride, which suggests that some compounds in the decoction promote ephedrine absorption.
类风湿性关节炎(Rheumatoid Arthritis, RA)是以关节组织慢性炎症性病变为主要临床表现的自身免疫性疾病,其患病率约为0.3%～1%。目前该病病因尚未完全明确,感染、遗传、雌激素水平、环境、劳累、外伤等因素均可能诱导RA发生。若治疗不及时、不规范,滑膜炎的持久、反复发作可导致关节内软骨和骨的破坏,引起严重关节畸形、关节功能障碍,从而严重影响RA患者的生活质量。西医对RA的治疗目标主要是缓解症状、控制病情发展,从而改善患者的生活质量。目前临床上用于治疗RA的药物主要有非甾体抗炎药、慢作用抗风湿药及糖皮质激素,这些药物可以快速起效,但是长期使用均可导致严重的不良反应。目前已有大量的临床研究显示一些传统中医药制剂以及中西医联合疗法对RA的治疗效果比单独使用西药治疗更让人满意。
乌头汤是一个应用历史悠久的经典方剂,由川乌、麻黄、白芍、甘草及黄芪组成,临床上主要用于RA、风湿性关节炎等疾病的治疗。乌头汤的疗效确切,已使广大的RA患者获益。然而,处方中的“君药”及“臣药”的川乌及麻黄均具有严重毒副作用,因此,必须通过科学手段阐明乌头汤产生疗效的物质基础和作用机制,以提高药物疗效和减轻毒副作用。
药代动力学着重研究研究药物产生临床疗效的物质基础及体内过程,药代动力学参数可用于指导临床用药、评价药品的安全性、促进新药的开发研究等。由于中药的化学成分比较复杂,目前,中药药代动力学研究常选取一种或几种代表成分作为研究对象,进而阐述中药或方剂的药代动力学特征。选取具有代表性的指标成分进行针对性的研究,是研究乌头汤药代动力学特征的关键。我们采用以下两个条件为基础选择具有代表性的指标成分：1)该物质的药理活性应当与乌头汤的药理作用一致；2)该物质在乌头汤提取液中应当具有相对较高的含量。
川乌为乌头汤的“君药”,是乌头汤产生临床疗效不可或缺的组分。众所周知,乌头类生物碱是川乌中的主要药理活性成分,包括双酯二萜类生物碱及单酯二萜类生物碱。双酯二萜类生物碱受热不稳定,煎煮过程中,易水解生成对应的单酯二萜类生物碱。已有文献报道附子水煎液加热30mmin,即有大部分双酯二萜类生物碱发生水解。乌头汤的制备需要较长时间的煎煮,可导致大部分双酯二萜类生物碱(乌头碱、次乌头碱、新乌头碱)发生水解,显然不符合我们对目标化合物选择的条件。苯甲酰新乌头胺(Benzoylmesaconine, BMA)是一种单酯二萜类生物碱,是新乌头碱(Mesaconitine, MA)的水解产物,也是川乌最主要的有效成分之一。BMA具有显著的镇痛作用,已有文献证实BMA对RA具有治疗作用；且其在乌头汤提取液中的含量显著高于其他乌头类生物碱。因此,本实验选择BMA作为乌头汤“君药”的代表成分,研究其药代动力学特征。
麻黄为乌头汤的“臣药”,麻黄碱是其主要有效成分。麻黄碱在乌头汤产生临床疗效的过程中发挥了重要的作用。麻黄碱具有类似于肾上腺素的化学结构,可与血管中肾上腺素受体结合。肾上腺素受体结合,产生舒张血管的作用,从而加快血流速度、增加皮肤血流量,从而辅助川乌以增强疗效。已有大量文献报道麻黄碱是麻黄含量最高的生物碱,其在乌头汤提取液中的含量也显著高于其他化学成分。因此,本实验选择麻黄碱作为乌头汤“臣药”的代表成分,研究它的药代动力学特征及肠道吸收特征。
然而,关于乌头汤有效成分的药代动力学研究有以下几个难点。首先,乌头汤由多种药材组成,其成分复杂；其次,生物样品中待测物质浓度低,对其提取、分离、纯化、检测均有较高难度：第三,中药药代动力学研究的生物样本量多,工作量大。因此,建立一个灵敏、准确、快速的生物样品定量分析方法是药代动力学研究的关键。超高效液相色谱-串联质谱(Ultra Performance Liquid Chromatography tandem Mass Spectrometry, UPLC-MS/MS)充分体现了色谱和质谱优势的互补。UPLC-MS/MS具有高灵敏度和较强的专属性,不需要使分析物之间实现完全的色谱分离,使得样品预处理过程简化,且大大缩短样品的分析时间；MRM模式还允许同时对多个成分进行定性、定量分析。采用UPLC-MS/MS可更加准确、快速的分析样品,有利于获得更精确的药代动力学参数。
研究目的
本实验旨在对乌头汤“君药”、“臣药”代表成分的药代动力学特征进行深入研究,以期为乌头汤更深入的研究及其临床应用提供物质基础和理论依据。主要研究内容包括以下几点：
1.研究口服乌头汤后BMA的药代动力学特征,考察乌头汤其他组分对“君药”代表成分的体内过程的影响作用。
2.研究口服乌头汤及麻黄单味药材提取液后麻黄碱的药代动力学特征,考察乌头汤其他组分对“臣药”代表成分的体内过程的影响作用。
3.采用大鼠在体肠灌流模型,研究乌头汤中麻黄碱在大鼠肠道不同部位的吸收特征,考察乌头汤其他组分对“臣药”代表成分在肠道吸收的影响作用。
实验方法及结果
1.“君药”代表成分苯甲酰新乌头胺药代动力学研究
我们建立了一个快速、灵敏的UPLC-MS/MS方法,用于定量测定样品中BMA的含量。冷冻干燥已制备的乌头汤提取液,制成冻干粉末,于实验前日使用UPLC-MS/MS测定其中BMA的含量。选用10只体重为230～280g的雄性SD大鼠,随机分为两组,按照5mg/kg (BMA)的剂量分别灌胃给予乌头汤提取液及BMA溶液。分别于给药后5、15、30、45、60、90、120、180、240、420、600min经大鼠眼眶静脉丛采集血样约0.3ml,所采集的血样在8000rpm离心8min,取上层血清至EP管,置于-80℃冰箱中保存备测。血浆样品处理如下：取80μl血浆样品,加入320μl甲醇(睾丸酮浓度为100nM),用于去除血浆的蛋白成分,在13000rpm离心30min,吸取适量上清液,室温下氮气吹干,所得残渣以100μl甲醇水溶液(50%)复溶,然后在13000rpm离心30min,吸取上清液进样分析。经UPLC-MS/MS测定样品中BMA的含量,并采用WinNonlin3.3软件的标准非室性模型计算BMA的药代动力学参数。
大鼠口服给药BMA溶液后,BMA的血药浓度于45min达峰；600min后,血浆样品中BMA浓度接近O。然而,口服给药等剂量BMA的乌头汤提取液,BMA的血药浓度于15min达峰；240min后,血浆中的BMA基本上完全被清除。以BMA溶液为参照组,口服给药乌头汤提取液后,BMA的相对生物利用度为19.9%。口服给药乌头汤提取液后,BMA在大鼠体内的AUC较低；其Tmax及MRT均显著缩短,经两独立样本t检验(Two Independent Samples t-test)进行分析,具有显著性差异(P<0.05)。
2.“臣药”代表成分麻黄碱药代动力学研究
我们建立了一个快速、灵敏的UPLC-MS/MS方法,用于定量检测血浆中麻黄碱的浓度。取麻黄单味药材制备提取液,并制成冻干粉末。于实验前日使用HPLC测定乌头汤提取液和麻黄单味药材提取液冻干粉末中麻黄碱的含量。选用20只体重为230～280g的雄性SD大鼠,随机分为4组。其中三组按照10mg/kg(麻黄碱)的剂量分别灌胃给予盐酸麻黄碱溶液、麻黄单味药材提取液及乌头汤提取液,并于给药后5、15、30、45、60、90、120、180、240、420、600、1440min经大鼠眼眶静脉丛采集血样约0.3ml;另外一组按照4mg/kg：麻黄碱)的剂量静脉注射盐酸麻黄碱溶液,并于给药后1、5、12、18、30、45、72、100、150、240、480、600min经大鼠眼眶静脉丛采集血样约0.3ml。所采集血样在8000rpm离心8min,取上层血清至EP管,置于-80℃冰箱中保存。血浆样品处理如下：取80μl血浆样品,加入520μl乙酸乙酯(睾丸酮浓度为20μM),用于去除血浆的蛋白成分,在13000rpm离心30min,吸取上清液400μl,室温下氮气吹干,所得残渣以100μl甲醇水溶液(50%)复溶,然后在13000rpm离心30min,吸取上清液进样分析。经UPLC-MS/MS测定样品中麻黄碱的含量,并采用WinNonlin3.3软件的标准非室性模型计算BMA的药代动力学参数。
以10mg/kg(麻黄碱)的剂量灌胃给药盐酸麻黄碱溶液、麻黄单味药材提取液及乌头汤提取液后600min,血浆样品中麻黄碱的基本完全被清除；三组大鼠的绝对生物利用度分别为83.4%、71.4%、29.9%。以口服盐酸麻黄碱溶液为参照组,口服麻黄单味药材提取液及乌头汤提取液后,麻黄碱的相对生物利用度分别为85.6%、35.9%。口服乌头汤及麻黄单味药材提取液后,麻黄碱的Tmax显著缩短,经单因素方差分析(One Way ANOVA)进行分析,具有显著性差异(P<0.05)。
3“臣药”代表成分麻黄碱肠道吸收特征研究
冷冻干燥已制备的乌头汤提取液,制成冻干粉末,于实验前日使用HPLC测定其中麻黄碱的含量。精密称取适量盐酸麻黄碱及乌头汤冻干粉末,以HBSS溶液溶解稀释至麻黄碱浓度为40μg/ml。所用实验动物为230～280g的雄性SD大鼠。本实验采用大鼠在体肠灌流模型考察乌头汤中麻黄碱在肠道不同部位的吸收情况。以含有麻黄碱的HBSS溶液(盐酸麻黄碱溶液、乌头汤提取液)同时灌流大鼠的四个肠段(十二指肠、空肠上段、回肠下段、结肠),每隔30min收集一次样品。往收集到的灌流液样品中加入乙腈(v：v=2：1)去除蛋白,样品在13000rpm离心处理30min,吸取上清液。经UPLC系统检测分析,进样体积为10μl。
灌流盐酸麻黄碱溶液后,麻黄碱在十二指肠、空肠、回肠、结肠的渗透系数(Peff)分别为2.20、1.81、2.33、1.68；麻黄碱在四个肠段的吸收率依次为24.9%、19.5%、25.2%、17.0%。灌流乌头汤提取液后,麻黄碱在十二指肠、空肠、回肠、结肠的渗透系数(Peff)分别为2.33、2.31、3.35、2.68；麻黄碱在四个肠段的吸收率依次为27.7%、26.4%、37.4%、30.3%。乌头汤提取液中麻黄碱在空肠、回肠及结肠的吸收率较高,经两独立样本t检验(Two Independent Samples t-test)进行分析,具有显著性差异(P<0.05)。
结论
1.口服给药乌头汤后,BMA的相对生物利用度为19.9%：BMA在大鼠体内的Tmax显著缩短,表明乌头汤中存在可促进BMA吸收的成分,有利于乌头汤快速起效；BMA在大鼠体内的MRT显著缩短,表明乌头汤中存在可加速BMA清除的成分,有利于BMA的清除而避免发生蓄积。
2.口服给药乌头汤后,麻黄碱的绝对生物利用度为29.9%,低于口服给药含有相同剂量的盐酸麻黄碱溶液(83.4%)及麻黄单味药材提取液(71.4%)。口服给药乌头汤及麻黄单味药材提取液后,麻黄碱的大鼠体内的Tmax显著缩短,表明乌头汤中存在可促进麻黄碱吸收的成分。
3.使用乌头汤及盐酸麻黄碱溶液进行大鼠在体肠灌流,麻黄碱在大鼠全肠道吸收良好,无明显的特定吸收部位。乌头汤中的麻黄碱在大鼠空肠、回肠及结肠的吸收率显著升高,表明乌头汤中存在促进其肠道吸收的成分。
Background
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic arthritis, and it has an incidence of0.3%to1%. The etiology of this disease is still unclear. RA is associated with many factors, such as infection, heredity, estrogen levels, fatigue, trauma, and environment. Without timely treatment, the lasting recurrent synovial inflammation can lead to the destruction of joint cartilage and bone, causing severe joint deformity and joint dysfunction, which can seriously reduce the quality of life of patients. The Western medical treatment of RA relieves its symptoms and controls its progression, thereby improving the quality of life of patients. Non-steroidal anti-inflammatory drugs, slow-acting anti-inflammatory drugs, and glucocorticoids are used to treat RA. Although these drugs are fast acting, their long-term use can lead to serious adverse reactions. Numerous clinical studies shown that some Traditional Chinese Medicine (TCM) preparations and TCM combined with chemical drug therapy achieve more satisfactory therapeutic efficacy on RA than chemical drug therapy.
Wutou decoction has been used to treat RA, constitutional hypotension, and hemicrania for hundreds of years. Wutou decoction consists of five medicinal herbs, Radix Aconiti(Chuan Wu), Herba Ephedrae (Ma Huang), Radix Paeoniae alba (Bai Shao), Radix Astragali (Huang Qi), and Radix Glycytthizae (Gan Cao). Chuan Wu is the monarch drug and Ma Huang is the ministerial drug of Wutou decoction, and they are the most important effective components. However, Chuan Wu and Ma Huang have serious side effects. Thus, clarifying the material basis and mechanism of the clinical efficacy of Wutou decoction is necessary.
Pharmacokinetic study has an important role in the research, development, and clinical application of drugs. Pharmacokinetic parameters can be used to guide clinical application, to assess drug safety, and to promote the development of new drugs. However, the effective components of TCM are much more complicated, and clarifying the pharmacokinetic characteristics of a TCM preparation is more difficult. At present, most of the pharmacokinetic studies on TCM preparations focus on one or several of the active components. Selecting representative compounds is the key to the pharmacokinetic study of Wutou decoction. The representative compounds were selected according to the following conditions. First, the pharmacologic activity of these compounds should be consistent with Wutou decoction. Second, these compounds should have higher content in Wutou decoction.
Considering Chuan Wu is the monarch drug of Wutou decoction, it plays the most important role in the decoction. Aconitum alkaloids are the active ingredients of Chuan Wu, including diester diterpenoid alkaloids (DDAs) and monoester diterpenoid alkaloids (MDAs). DDAs are easily hydrolyzed to MDAS during boiling. Most of the DDAs in aconite decoction are hydrolyzed by30min of boiling. Preparing Wutou decoction requires prolonged boiling; therefore, DDA is not an appropriate representative component of Wutou decoction. Benzoylmesaconine (BMA), a mesaconitine hydrolyzate, is one of the active ingredients of Chuan Wu. BMA has significant analgesic effects and a therapeutic effect on RA. In addition, the BMA content of Wutou decoction is significantly higher than other aconitum alkaloids. Thus, BMA was selected as the representative component of Chuan Wu. The pharmacokinetic characteristics of BMA in Wutou decoction were determined in our experiments.
Ephedrine exerts the most potent effect of Ma Huang, the ministerial drug of Wutou decoction. The chemical structure of ephedrine is similar to adrenaline, and it interacts with adrenaline receptors in vascular and diastolic blood vessels, thereby increasing blood flow. The ephedrine content of Wutou decoction is much higher than that of other compounds. Therefore, ephedrine was selected as the representative component of Ma Huang. The pharmacokinetic characteristics of ephedrine after oral administration of Wutou decoction and Ma Huang single herb decoction were investigated. A four-site perfusion model was also used to investigate the absorption of ephedrine in Wutou decoction at different intestinal segments.
The effective components of Wutou decoction are highly complicated, and the concentrations of target compounds, such as BMA and ephedrine, are quite low in plasma and perfusate samples. Furthermore, many groups and many time points are available for sample acquisition. Thus, a large sample size is needed for analysis. An efficient and sensitive detection method is required for sample analysis. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is sensitive and specific and it has a high throughput. Samples do not need to be separated completely, which simplifies the preparation process and greatly shortens the time for sample analysis. Multiple reaction monitoring (MRM) mode also allows simultaneous determination of several compounds. Using UPLC-MS/MS for pharmacokinetic study facilitates obtaining more accurate pharmacokinetic parameters.
Objectives
The objective of this study was to investigate the pharmacokinetic characteristic of BMA and ephedrine, the representative compounds of Wutou decoction. We believe that the pharmacokinetic study of the monarch drug (Chuan Wu) and ministerial drug (Ma Huang) of Wutou decoction can provide a material basis and theoretical foundation for the further study and clinical application of Wutou decoction. Our study could be divided into several parts as follows:
1. To determine the pharmacokinetic characteristics and bioavailability of BMA after oral administration of Wutou decoction.
2. To determine the pharmacokinetic characteristics and bioavailability of ephedrine after the oral administration of Wutou decoction and Mahuang single herb decoction.
3. To investigate the absorption of ephedrine in Wutou decoction in different intestinal segments using a four-site perfusion model. Methods and Results
1. Pharmacokinetic study of BMA after oral administration of Wutou decoction
A rapid and sensitive UPLC-MS/MS method was developed for quantifying BMA in plasma samples. Wutou decoction was prepared and freeze-dried using a lyophilizer to obtain the lyophilized powder. The BMA content of the lyophilized powder was quantified by UPLC-MS/MS one day before the experiments. Male Sprague-Dawley (SD) rats weighing230g to280g were obtained for the experiments. Ten rats were divided into two groups, namely, BMA and Wutou decoction groups. BMA and Wutou decoction were administered orally at5mg of BMA per kilogram of body weight. Blood samples were collected at5,15,30,45,60,90,120,180,240,420, and600min after dosing. After centrifugation at8000rpm for8min, plasma fractions were transferred into a disposable tube and stored at -80℃until analysis. We mixed80μl of plasma with320μl of methanol containing100nM testosterone. The mixture was centrifuged at13,000rpm for30min. Approximately300μl of the supernatant was transferred into a disposable tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted with100μl of methanol-water (v:v=1:1), and injected into the UPLC-MS/MS system for analysis. The pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using WinNonlin3.3.
The Tmax of BMA after oral administration of BMA solution was about45min, and the plasma concentration of BMA was approximately0at600min after dosing. However, the Tmax of BMA after oral administration of Wutou decoction was15min, and BMA was completely eliminated at600min after dosing. The relative bioavailability of BMA after oral administration was19.9%. The Tmax and mean residence time (MRT) after administration were significantly shorter(P<0.05).
2. Pharmacokinetic study of ephedrine after oral administration of Wutou decoction and Ma Huang single herb decoction
A rapid and sensitive UPLC-MS/MS method was developed and validated for quantitative determination of ephedrine in plasma samples. Wutou decoction and Ma Huang single herb decoction were prepared and freeze-dried by a lyophilizer to obtain the lyophilized powder. The ephedrine content in lyophilized powders was quantified by HPLC the day before the experiments. Male SD rats weighing from230g to280g were obtained for the experiments. Ephedrine hydrochloride, Wutou decoction, and Ma Huang single herb decoction were administered orally at a dosage of10mg of ephedrine per kilogram of body weight. Ephedrine hydrochloride was also administered via intravenous injection at4mg/kg. Blood samples were collected at5,15,30,45,60,90,120,180,240,420,600, and1440min after oral administration; and at1,5,12,18,30,45,72,100,150,240,480, and600min after intravenous injection. After centrifugation at8000rpm for8min, plasma fractions were transferred into a disposable tube and stored at-80℃until analysis. We mixed80μl of plasma with520μl of ethyl acetate containing20μM testosterone. The mixture was centrifuged at13000rpm for30min. Approximately400μl of the supernatant was transferred to a disposable tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted with100μl of methanol-water (v:v=1:1), and injected into the UPLC-MS/MS system for analysis. The pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using WinNonlin3.3.
Ephedrine was completely eliminated at600min after oral administration of ephedrine hydrochloride, Wutou decoction, and Ma Huang single herb decoction. The absolute bioavailabilities of ephedrine in the rats after oral administration of ephedrine hydrochloride, Ma Huang single herb decoction, and Wutou decoction were83.4%,71.4%, and29.9%, respectively. The relative bioavailabilities of ephedrine in the rats after oral administration of Ma Huang single herb and Wutou decoctions were85.6%and35.9%, respectively. The Tma of ephedrine after oral administration of Wutou decoction and Mahuang single herb decoction were significantly shortened (P<0.05).
3. Absorption study of ephedrine in Wutou decoction at different intestinal segments
Wutou decoction was prepared and freeze-dried using a lyophilizer to obtain the lyophilized powder. The ephedrine content of the lyophilized powder was quantified by high-performance liquid chromatography (HPLC) the day before the experiments. Ephedrine hydrochloride and lyophilized powder were dissolved in Hank's balanced salt solution to obtain perfusate solutions containing40μg/ml ephedrine. Male SD rats weighing from230g to280g were obtained for the experiments. A four-site perfusion model was used to investigate the absorption of ephedrine at different intestinal segments. Four segments of the intestine (duodenum, upper jejunum, terminal ileum, and colon) were perfused simultaneously with the prepared perfusate. The perfusate samples were collected every30min. Acetonitrile was added into the perfusate (v:v=1:2) and centrifuged at13,000rpm for30min. We injected10μl of supernatant into the UPLC system for analysis.
After perfusion with ephedrine hydrochloride solution, the permeability coefficients (Peff) of ephedrine in the duodenum, jejunum, ileum, and colon were2.20,1.81,2.33, and1.68, respectively. Approximately24.9%,19.5%,25.2%, and17.0%of ephedrine was absorbed in the duodenum, jejunum, ileum, and colon, respectively. After perfusion with Wutou decoction, the Peff of ephedrine at duodenum, jejunum, ileum, and colon were2.33,2.31,3.35, and2.68, respectively. Approximately27.7%,26.4%,37.4%, and30.3%of ephedrine was absorbed at duodenum, jejunum, ileum, and colon, respectively. The absorption of ephedrine at jejunum, ileum, and colon following Wutou decoction perfusion was significantly higher than those with ephedrine hydrochloride (P<0.05).
Conclusions
1. The relative bioavailability of BMA after oral administration of Wutou decoction was19.9%. The Tmax. of BMA after oral administration was significantly shortened, which indicates that some compounds in the decoction accelerate BMA absorption. MRT was also significantly shortened, indicating that some compounds in the decoction promote BMA clearance.
2. The absolute bioavailabilities of ephedrine in the rats after oral administration of Wutou decoction was29.9%, which was much lower than the other groups. The Tmax of ephedrine after oral administration of Wutou decoction and Mahuang single herb decoction were significantly shortened, which indicates some compounds in the decoctions accelerate ephedrine absorption.
3. Ephedrine was well absorbed at different intestinal regions in rats perfused with ephedrine hydrochloride solution and Wutou decoction. The absorption of ephedrine in the jejunum, ileum, and colon after Wutou decoction perfusion was significantly higher than those with ephedrine hydrochloride, which suggests that some compounds in the decoction promote ephedrine absorption.
引文
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