大黄素肝肠代谢特征及性别差异研究
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摘要
大黄素(1,3,8-三羟基-6-甲基蒽醌)是天然的蒽醌类化合物中最重要的一种单体化合物。主要来自于大黄、芦荟、番泻叶等中药材。大黄素药理活性广泛,尤其是近年来研究发现大黄素对多种肿瘤细胞具有细胞毒性,其机制可能是抑制肿瘤细胞的扩散和粘黏,因此大黄素是一个具有开发价值前景的化合物,有着广泛的市场。但是,尽管大黄素有良好的药理活性和优越的市场前景,目前大黄素药理活性的研究仍以分子水平的较多,且基本集中于药理作用方面,药物动力学及代谢资料对其体内的相关研究尚未见深入报道,因此,研究大黄素的药物代谢动力学对于大黄素的开发利用有重要意义。
除此之外,大量的保健、减肥食品中都包含有大黄素,随着社会的进步,无论男女都有瘦身、健康的需求,服用含大黄素的减肥健身产品的人群越来越广泛,目前有关大黄素口服生物利用度的性别差异研究报道极少,因此大黄素的吸收和代谢是否存在性别差异性是值得深入探讨的问题,这也是本次实验研究的主要目的。
从各种保健品使用的结果反馈资料来看,由于许多中药减肥产品需要长期使用,已发现不少因出现肾衰竭现象报道后被FDA撤销的事件,因此,我们不可忽视长期服用含大黄素的中药而存在的毒性问题。目前,世界范围内正在关注大黄素的长期毒性、毒理情况。美国国家毒理研究中心大量毒理临床研究均显示大黄素等减肥中药会对全身不同器官或系统造成病理伤害,报道最多的是在大黄的长期用药过程中会引起肝、肾毒性。所以探讨大黄素的药物动力学特征,这对于弄清其药理作用机理及作用过程,对于指导新药设计,提高新药疗效和安全性,科学地制定药品质量标准均具有重要意义。
本文旨在对大黄素在不同种属、不同性别的动物体内、体外、在体的吸收代谢进行深入研究,以期从实验动物数据分别推至男性用药者和女性用药者,对临床合理用药提供依据。研究的主要内容包括有(1)大黄素的溶解度与稳定性的考察。(2)采用雄、雌性SD大鼠动物模型,分别口服(8mg/kg)、静脉(4mg/kg)给药大黄素,观察大鼠体内血药浓度的变化,考察生物利用度。(3)采用SD大鼠在体肠灌流模型,研究大黄素在雌雄SD大鼠的肠吸收动力学的差异。(4)考察大黄素在五种雄、雌动物肝微粒体中的代谢及其代谢酶动力学特征的种属差异和性别差异。(5)大黄素在雌雄大鼠肠微粒体中的代谢及其性别、肠道不同部位差异性。(6)考察大黄素在人不同的UGTs亚型中的代谢情况。
1.大黄素的稳定性和溶解度的考察
为了开展大黄素在药代动力学和大鼠在体肠灌流模型等实验,必须清楚了解大黄素的稳定性和溶解度。本实验用超高效液相色谱法对大黄素的稳定性进行考察,并同时测定其溶解度。结果表明大黄素制成包合物溶解度明显提高,能较好的溶解于HBSS溶液中,是能够进行肠灌流实验的,并且大黄素的HBSS溶液在pH值7.4下24h内稳定,结果表明,灌流实验用的大黄素最好新鲜配制或者实验前一天配制好。大黄素在pH为7.4到8.87之间比较稳定。另外本实验对采集、保存大黄素的最适条件进行了考察,发现酸化条件不利于大黄素的保存,适当的碱化和有机溶剂处理对大黄素样品的保存稳定有利。
2.大黄素及其代谢产物在大鼠体内的药物动力学研究
采用LC/MS法测定血清中大黄素的浓度,其血药浓度在0.001～20μg/ml范围内线性关系良好(R2=0.9979),并用同法测定了雌雄SD大鼠口服、静脉给药大黄素后的血药浓度及药物动力学参数,结果表明,无论在雌性SD大鼠还是雄性SD大鼠,大黄素的口服生物利用度都相当低,分别为7.5%(雄鼠)和5%(雌鼠)。
静脉给药(4mg／kg)后的大黄素在雌雄大鼠的体内均表现为二室模型,分布较快,消除较慢,t1/2α为13.26±6.28min(雄鼠)和13.52±7.28min(雌鼠),t1/2β为187.378±174.52min(雄鼠),118.5033±83.09min(雌鼠),血药浓度曲线下面积为AUC0-∞, i.v分别为430±160mg*μg/ml(雄鼠),283±98 mg*μg/ml(雌鼠)。口服给药8mg/kg大黄素后仅检测出少量原形药物,大黄素在体内的血药浓度变化无法用房室模型模拟,口服后大黄素的药代动力学参数分别为Cmax:0.31±0.094μg/ml(雄鼠)和0.039±0.011min(雌鼠)；Tmax:18±6.71 min(雄鼠),18.75±7.5min(雌鼠)；血药浓度曲线下面积AUC0-∞, p.o分别为65.76±34.77 mg*μg/ml(雄鼠),33.82±4.09 mg*μg/ml(雌鼠),大黄素的药代动力学参数存在一定的雌雄差异性。
静脉给药后的大黄素迅速代谢成大黄素葡萄糖醛酸化物,此代谢物在体内的血药浓度变化可用一室模型描述,t1／2Ke为167.4±50.9min(雄鼠)和251.3±114min(雌鼠),曲线下面积AUC0-∞, i.v分别为2210±980 mg*μg/ml(雄鼠),1540±290 mg*μg/ml(雌鼠)。口服后大黄素葡萄糖代谢产物的药代动力学参数分别为Cmax:6.69±1.058μg/ml(雄鼠)和1.807±0.576μg/ml(雌鼠),血药浓度曲线下面积AUC0-∞, p.o分别为2261.89±655.87 mg*μg/ml(雄鼠),458.50±373.29mg*μg/ml(雌鼠),大黄素葡萄糖醛酸结合物的药代动力学参数存在显著的性别差异性。
药代动力学研究表明大黄素在体内的主要代谢途径为UGT代谢,代谢速度快,而排泄速度较慢,这一结果是大黄素生物利用度低的原因之一。此研究内容充实了大黄素药物动力学方面的资料,为研究大黄素的吸收和代谢做了进一步的辅证,也为临床用药方面提供了参考依据。
3.大黄素的大鼠肠吸收动力学研究
为了进一步证明大黄素生物利用度低的原因,采用在体大鼠肠灌流法研究大黄素的吸收代谢情况。肠灌流液经UPLC法测定结果显示,大黄素被部分代谢为葡萄糖醛酸结合物。在雄性大鼠中,大黄素的吸收量存在着肠段依赖性,其中十二指肠与空肠的吸收量最高,其次是回肠,结肠部位吸收最少(p<0.05)；相反的,在雌鼠中,无肠段间的显著性差异(p>0.05)。在四段肠中大黄素的吸收量存在显著性性别差异(p<0.01)。
另一方面,雄性大鼠四段肠上大黄素代谢产物的细胞排出量也存在着显著性差异,代谢量最高的部位在十二指肠,其次在空肠,回肠和结肠部位吸收量最少(p<0.05)；而对于雌鼠,十二指肠与空肠中大黄素代谢产物的排出量最高,结肠中最少(p<0.05)。而四段肠代谢产物被排出细胞外的量也存在显著性性别差异(p<0.01),这一系列的结果证明肠道中的代谢酶会影响大黄素的代谢,大黄素在肠道细胞中有一定量的吸收,并存在明显的雌雄差异性和肠段差异性。
4.大黄素在不同雌雄动物的肝微粒体中的代谢研究
为了考察大黄素在肝中的代谢情况,以求进一步解释黄素生物利用度低的原因,本实验采用LC/MS法检测大黄素在五种动物(小鼠、大鼠、豚鼠、狗、人)的肝微粒体酶中经UGT途径代谢反应后的样品。结果证明,无论在哪种肝微粒体酶作用下,大黄素都会迅速代谢并生成葡萄糖醛酸结合物。对代谢产物进行收集提纯,经1HNMR法检测后,确定此结合物为大黄素-3-O-葡萄糖醛酸。同一性别的五种动物的肝微粒体代谢大黄素的速率有显著性种属差异(p<0.05),而同种属的动物的肝微粒体代谢大黄素的速率也存在着显著性的性别差异(p<0.05)。结果还显示大黄素的代谢在其高浓度的时候呈现饱和现象,大黄素在雄性豚鼠和男性的肝微粒体中的代谢符合典型的米氏方程(Michaelis-Menten equation),而其在雄性小鼠和狗的肝微粒体中的代谢符合双相抑制模式(Biaphasic pattern),只有在雄性大鼠中大黄素的代谢呈自身激活模式(Autoactivation pattern)。在雌性动物组,小鼠大鼠、豚鼠和女性的肝微粒体中的代谢均符合典型的米氏方程(Michaelis-Menten equation),只有雌性狗的肝微粒体中的代谢符合自身激活模式(Autoactivation pattern)。
Ⅰ相代谢反应中的大黄素代谢速度极慢,生成的代谢产物为羟基大黄素。将Ⅰ相反应和Ⅱ相反应的同一时间的大黄素剩余量进行比较,大黄素的Ⅱ相代谢反应明显快于Ⅰ相代谢反应,且Ⅰ相合并Ⅱ相代谢反应证明,Ⅱ相代谢占主导地位,并一定程度上抑制Ⅰ相代谢反应。
本实验结果说明大黄素在肝微粒体中极易发生葡萄糖醛酸结合反应,此代谢反应表现为显著的种属差异和性别差异。
5.对雌雄SD大鼠不同肠段的肠微粒体中大黄素的代谢研究
检测方法同上,我们分别考察了大黄素在雄性SD大鼠和雌性SD大鼠的空肠、回肠微粒体的代谢情况,不管是雄性SD大鼠还是雌性SD大鼠,大黄素在空肠微粒体中的葡萄糖醛酸化速率都大于在回肠微粒体中的速率(p<0.05)；并且无论空肠还是回肠微粒体,大黄素在雄性SD大鼠中的代谢速率远快于雌性SD大鼠(p<0.01)。大黄素在雄性SD大鼠的空肠微粒体中的代谢均符合典型的米氏方程(Michaelis-Menten equation),而其在雄鼠回肠微粒体中的代谢符合自身激活模式(Autoactivation pattern)。大黄素在雌性SD大鼠的空肠微粒体的代谢也符合典型的米氏方程,而在回肠中的代谢表现出双相抑制模式。
结果显示,大黄素的葡萄糖醛酸结合反应有显著性的性别差异性和肠段差异性。
6.对大黄素在12种人的UGT酶中的代谢研究
为了初步判断大黄素的主要代谢部位,本实验采用超高效液相色谱法考察了大黄素在12种不同人的UGT酶中的代谢情况,除了UGT1A4和UGT2B4中大黄素无代谢外,其他10种UGT酶均对大黄素的代谢有不同程度的影响,其中UGT1A家族占主要地位,大黄素在UGT2B家族的酶中代谢速率相对较慢。在UGT1A家族中,UGTIA10是大黄素的最主要代谢酶,其次是UGT1A8, UGT1A9, UGTIA1。大黄素在UGT1A9, UGTIA10, UGT2B15中的代谢均符合典型的米氏方程(Michaelis-Menten equation),而其在其他UGT酶中的代谢符合自身激活模式(Autoactivation pattern)。而大黄素在UGT1A3中的代谢不符合任何模式。从结果中可以看出UGT1A家族对大黄素的葡萄糖醛酸结合反应起着主要作用。特别是UGTIA10, UGT1A9, UGT1A8, UGT1A1。而这些UGT的亚型与两个最重要的代谢场所：肝、肠有关,UGT1A1在肝肠中都有分布,UGT1A9只在肝中表达,而UGTIA10, UGT1A8只在肠中表达。而UGTIA10对大黄素的代谢最快,和我们之前推断的肠道是大黄素的主要代谢场所相吻合。
综上所述,大黄素的生物利用度极低,存在性别差异性。主要原因可能是大黄素在体内易被UGT酶代谢成葡萄糖醛酸结合产物。大黄素在大鼠肠道中的吸收和代谢物的排泄量呈性别依赖性和肠段依赖性。大黄素在肝微粒体中的代谢存在性别、种属差异性,大黄素在肠微粒体中的代谢存在性别差异性,肠段差异性。从我们模拟的结果来看大黄素在人肝微粒体的葡萄糖醛酸结合反应可以用实验动物预测。综合我们的体外、体内、在体实验结果,可以判断小肠极有可能是大黄素的主要代谢场所。
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is one of the major active ingredients of rhubarb(rheuw officinale B.), aloe(aloe barbadensis M.), and leaf of senna(cassia angustifolia) plants. Decade ago, emodin was extensively studied for its traditional pharmacological activities. However, recent studies have put emodin back into limelight with its anti-cancer activities in several types of cancer cells with apoptosis as a possible mechanism of action.Emodin also has an inhibitory effect on cancer cell migration and invasion in in vitro studies and that is why emodin has good prospect and promising future in the market. Even though emodin has so many excellent application prospects as a chemical, little is known about its pharmacokinetics and metabolism in vivo. These studies are very important for development of emodin into a new anti-cancer pharmaceutical compound.
In modern era, more and more people, no matter men or women aspire to be fit and healthy. Therefore, in addition to the above mentioned effects, emodin's health food and slimming product intake has increased due to its weight loss property through its traditionally known stimulant laxative activity. However, again, no study has been done to show the gender differences in oral bioavailability of emodin in rodents as well as humans. Hence, we propose to study gender-dependent difference in absorption and metabolism of emodin as the main aim of our study.
Toxic effects of emodin such as renal and liver toxicity have received significant attention from all over the world. Due to renal failure issues, number of weight loss products containing emodin was recalled back from the market by FDA. Recently the toxicity of emodin was assessed by National Toxicology Program. The acute and chronic toxicity of emodin is investigated in liver and kidney of rodents. This proposed study investigates the gender-dependent differences in absorption and disposition of emodin which might help in understanding the pharmacological mechanism, guiding drug design, improving efficacy and safety and development of scientific standards of the quality of emodin.
The proposed study delineates gender-dependent differences in absorption and metabolism of emodin in different animal species using in vitro, in situ as well as in vivo study models. Our intentions were to put forth experimental data in different animal models to make an informative decision about the emodin dosage regimen in men and women. Our research has been divided into several parts as follows:1) to study stability and solubility of emodin in various buffer solutions; 2) to determine gender differences in absorption and disposition of emodin in SD rats using in vivo single dose (8mg/kg) oral and intravenous (4mg/kg) PK model; 3) to study gender difference in absorption and metabolism of emodin by using in situ four site rat intestinal perfusion model; 4) to determine the species and gender differences in glucuronidation of emodin by using male and female dog, guinea pig, mouse, rat and human liver microsomes; 5) to study gender-dependent and regioselective intestinal emodin glucuronidation difference by using female and male SD rat intestinal and liver microsome model; 6) to determine the major human uridine diphospho-glucuronosyl transferase (UGT) isoforms (s) responsible for glucuronidation of emodin using various human UGT isoforms available in the market.
1. Stability and solubility of emodin
Before conducting in vivo and in situ studies to understand the gender-dependent differences in bioavailability as well as absorption and disposition of emodin in rats, we needed clear understanding of stability and solubility of emodin. We determined emodin's stability and solubility by using UPLC method. Emodin stock solution was prepared in HPβCD solution to improve the solubility and stability of poorly soluble emodin in HBSS. We used emodin-HPβCD-HBSS (pH 7.4) as the perfusate, which was stable for 24hr under room temperature. Therefore, the perfusate was prepared fresh every time right before the start of the experiment. Emodin was stable in pH7.4～8.87, so Ph 7.4 HBSS buffer was used in intestinal perfusion model. Furthermore we also found out that not acidic but alkaline treatment was good for the stability of emodin in aqueous media.
2. Pharmacokinetics of emodin in female and male SD rats
To determine the plasma concentrations of emodin and its metabolites from in vivo PK plasma samples, we used LC/MS instrument. Emodin and emodin glucuronide showed a good linear plasma standard curve in the range of 0.01～20μg/ml. Pharmacokinetics studies to determine bioavailability of emodin were done using a single dose oral and intravenous administration in female and male SD rats. The oral bioavailability of emodin is extremely low whether in male rats (5%) and female rats (7.5%).
Following a single intravenous injection of 4 mg/kg emodin, emodin was distributed rapidly and eliminated slowly. The results showed a good two-compartment model fit for emodin plasma concentration-time data for male and female SD rats. The t1/2αwere 13.26±6.28min (male rats) and 13.52±7.28min (female rats). The t1/2βwere 187.38±0.16min (male rats) and 118.5033±83.09min (female rats). Emodin showed significant gender differences in iv PK profiles with higher AUC values in male (430±160 mg*μg/ml) than female (283±98mg*μg/ml) SD rats (n=6).
A little amount of emodin was detected using LC/MS after the oral administration of dose of 8mg/kg emodin. However, there was no good fit for any PK compartmental model for the plasma concentration-time data for single dose intravenous administration of emodin (8mg/kg). Analyzing the intravenous PK data using non-compartmental model, Cmax, Tmax and AUC0-∞, p.o of emodin in male rats were:0.31±0.094 wereμg/ml,18.0±6.71min and 65.76±34.77 mg*μg/ml respectively; whereas Cmax, Tmax and AUC0-∞，P.o of emodin in female rats were:0.039±0.011μg/ml, 18.75±7.51min and 33.82±4.09 mg*μg/ml respectively.
Emodin was metabolized very fast after the intravenous administration, suggesting a good fit for single compartmental model for the plasma emodin metabolite concentrations. t1/2Ke were 167.4±50.9min(male rats) and 251.3±114min (female rats), the area under the curve (AUC0-∞，i.v) were 2210±980 mg*μg/ml and 1540±290 mg*μg/ml (female rats) (n=6).
After oral administration of emodin, the metabolite of emodin appeared in the plasma rapidly. Non-compartmental model analysis was done for emodin and its metabolite plasma concentrations after single dose oral administration of emodin (20mg/kg). The parameters of emodin glucuronide were significant different with emodin, the Cmax, Tmax and AUC0-∞, p.o of emodin glucuronide in male rats were 6.69±1.058μg/ml,240min and 2261.89±655.87 mg*μg/ml respectively, in female rats, the Cmax, Tmax and AUC0-∞, P.o were 1.807±0.578μg/ml,60min and 458.5±373.39 mg*μg/ml respectively. The oral absolute bioavailability of emodin was less than 7.5% and 5% in male and female SD rats respectively. Emodin glucuronidation michaelis menten kinetics parameters showed significant gender-dependent differences.
The pharmacokinetics results of emodin suggested emodin glucuronide as a major metabolite in vivo. Rapid glucuronidation with slow metabolite elimination was one of the reasons of low bioavailability of emodin. Our research had enriched the pharmacokinetics aspects of emodin and provided the information for clinical use of emodin.
3. Gender-differences in intestinal absorption and disposition of emodin in rats
To confirm the reason of low bioavailability of emodin, gender-dependent intestinal absorption and disposition of emodin were investigated in rats by using the in situ intestinal perfusion model. The perfusate was analyzed for emodin and emodin glucuronide. Absorption and glucuronide excretion of emodin displayed regioselectivity (p<0.05) in male rats. The rank order for amounts of absorption of emodin in male rats was duodenum≥jejunum>ileum>colon (p<0.05). On the contrary, in female rats, the amounts of absorption showed no regioselectivity (p>0.05). Furthermore, gender-dependent higher intestinal emodin absorption was observed in female than in male SD rats in all 4 regions of intestine (p<0.01).
The amount of emodin glucuronide in duodenum was significant higher (p<0.05) than in jejunum, followed (p<0.05) by ileum and colon in male rats. In female rats group, the rank order of amount of metabolite excreted was jejunum≈duodenum> ileum>colon (p<0.05). In contrast to intestinal emodin absorption, emodin glucuronide excreted lower in all four regions of the male rat intestine than the female rat intestine (p<0.01). No biliary excretion of emodin or emodin glucuronide was detected in both male and female rats. The results suggested that not liver metabolism, but intestine metabolism, played an important role in disposition of emodin in rats.
4. Species and gender-dependent hepatic glucuronidation of emodin by using liver microsomes of different animal species.
To find out the main reason of low bioavailability of emodin, ten kinds of liver microsomal (male mouse, rat, guinea pig, dog, human and female mouse, rat, guinea pig, dog, human) were used, UGT metabolite formation was determined by LC/MS method. The results showed that emodin was glucuronidated fast in all ten kinds of liver microsomes.1H-NMR spectra of the metabolite displayed that the metabolite was emodin 3-O-β-D-glucuronide. The results also showed species-dependent different in glucuronidation rates of emodin among the five species (p<0.05). Significant gender difference in glucuronidation rates was shown in all test species. The results also indicated that metabolism of emodin was saturable at higher concentrations. Among the five species of males, guinea pig and human followed simple Michaelis-Menten kinetics whereas mouse and dog followed a biaphasic kinetics, and only rat followed autoactivation kinetics. Among the five species, mouse, rat, guinea pig and human all followed simple Michaelis-Menten kinetics whereas dog followed Autoactivation kinetics. In liver microsomes, the glucuronidation rates were faster in the female rats, especially at lower concentration. In the liver of other 4 species, rates were faster in male mice, but were slower in female guinea pigs and dogs. There was very little difference in emodin glucuronidation rates between human male and female liver microsomes, although, hepatic rate of glucuronidation showed significant gender differences in certain species such as mice.
Significant lower amount of hydroxyemodin was detected after phaseⅠmetabolism of emodin by LC/MS. There was no detectable PhaseⅠmetabolites but the glucuronide emodin in the mixed reaction system, which suggested that PhaseⅡmetabolism (glucuronidation) was major route of disposition of emodin in rats.
The resulted indicated that glucuronidation was the major pathway for emodin metabolism which was significant gender and species dependent.
5. Region and gender-dependent glucuronidation of emodin by intestinal microsomes
We measured glucuronidation rates of emodin in jejunal and ileal microsomes of male and female rats, using by UPLC-MS/MS method. The result showed that emodin was glucuronidated faster in rat jejunal microsomes than in ileal microsomes regardless of gender (p<0.05). Furthermore, emodin was metabolized faster in male than in female rats at all tested concentrations (p<0.01). Emodin glucuronidation in jejunal microsomes showed simple Michaelis-Menten kinetics, whereas glucuronidation in ileal microsomes followed autoactivation kinetics. In female rat intestine, glucuronidation in jejunal microsomes also showed simple Michaelis-Menten kinetics, whereas glucuronidation in ileal microsomes followed biaphasic kinetics.
The results indicated that glucuronidation of emodin showed significant gender and regioselective intestinal difference.
6. Fingerprinting of UGT glucuronidation of emodin by 12 expressed human UGTs isoforms.
To identify the main UGT isoform responsible for metabolism of emodin, we used 12 expressed human UGTs isoforms. Except UGT1A4 and UGT2B4, the other 10 UGTs metabolized emodin at different rates. The average glucuronidation rate of emodin in UGT2A familiy was faster than UGT2B familiy. Amongst the 10 UGT enzymes isoforms, glucuronidation of emodin by UGT 1A9, UGT1A10 and UGT2B15 followed classical Michaelis-menten kinetics, while others followed autoactivation kinetics, and UGT1A3 didn't fit any pattern.
In the UGTs study, the four major isoforms, responsible were UGT1A1,1A8, 1A9 and especially 1A10. Qualitative and quantitative expression of these UGT isoforms in two main metabolic organ, liver and intestine were quite different. UGT1A1 was expressed in all organs, although its expression was higher in the liver than in intestine. UGT1A9 was only expressed in liver whereas UGT1A8 and UGT1A10 were predominantly expressed in the small intestine.
In summary, systemic metabolic characterization study suggested rapid metabolite (glucuronide) formation might be the major reason for emodin's low bioavailability in rats. The elimination of emodin and emodin glucuronide might follow UGT enzyme. In liver microsomes, the gender-dependent metabolism of emodin was also species-dependent. In intestinal microsomes, the gender-dependent metabolism of emodin also showed regioselectivity. Therefore, informative conclusion can be drawn from our results about glucuronidation of emodin in humans. Based on in vivo, in situ and invitro studies of emodin, we predicted that the main organ of metabolism for emodin to be intestine.
除此之外,大量的保健、减肥食品中都包含有大黄素,随着社会的进步,无论男女都有瘦身、健康的需求,服用含大黄素的减肥健身产品的人群越来越广泛,目前有关大黄素口服生物利用度的性别差异研究报道极少,因此大黄素的吸收和代谢是否存在性别差异性是值得深入探讨的问题,这也是本次实验研究的主要目的。
从各种保健品使用的结果反馈资料来看,由于许多中药减肥产品需要长期使用,已发现不少因出现肾衰竭现象报道后被FDA撤销的事件,因此,我们不可忽视长期服用含大黄素的中药而存在的毒性问题。目前,世界范围内正在关注大黄素的长期毒性、毒理情况。美国国家毒理研究中心大量毒理临床研究均显示大黄素等减肥中药会对全身不同器官或系统造成病理伤害,报道最多的是在大黄的长期用药过程中会引起肝、肾毒性。所以探讨大黄素的药物动力学特征,这对于弄清其药理作用机理及作用过程,对于指导新药设计,提高新药疗效和安全性,科学地制定药品质量标准均具有重要意义。
本文旨在对大黄素在不同种属、不同性别的动物体内、体外、在体的吸收代谢进行深入研究,以期从实验动物数据分别推至男性用药者和女性用药者,对临床合理用药提供依据。研究的主要内容包括有(1)大黄素的溶解度与稳定性的考察。(2)采用雄、雌性SD大鼠动物模型,分别口服(8mg/kg)、静脉(4mg/kg)给药大黄素,观察大鼠体内血药浓度的变化,考察生物利用度。(3)采用SD大鼠在体肠灌流模型,研究大黄素在雌雄SD大鼠的肠吸收动力学的差异。(4)考察大黄素在五种雄、雌动物肝微粒体中的代谢及其代谢酶动力学特征的种属差异和性别差异。(5)大黄素在雌雄大鼠肠微粒体中的代谢及其性别、肠道不同部位差异性。(6)考察大黄素在人不同的UGTs亚型中的代谢情况。
1.大黄素的稳定性和溶解度的考察
为了开展大黄素在药代动力学和大鼠在体肠灌流模型等实验,必须清楚了解大黄素的稳定性和溶解度。本实验用超高效液相色谱法对大黄素的稳定性进行考察,并同时测定其溶解度。结果表明大黄素制成包合物溶解度明显提高,能较好的溶解于HBSS溶液中,是能够进行肠灌流实验的,并且大黄素的HBSS溶液在pH值7.4下24h内稳定,结果表明,灌流实验用的大黄素最好新鲜配制或者实验前一天配制好。大黄素在pH为7.4到8.87之间比较稳定。另外本实验对采集、保存大黄素的最适条件进行了考察,发现酸化条件不利于大黄素的保存,适当的碱化和有机溶剂处理对大黄素样品的保存稳定有利。
2.大黄素及其代谢产物在大鼠体内的药物动力学研究
采用LC/MS法测定血清中大黄素的浓度,其血药浓度在0.001～20μg/ml范围内线性关系良好(R2=0.9979),并用同法测定了雌雄SD大鼠口服、静脉给药大黄素后的血药浓度及药物动力学参数,结果表明,无论在雌性SD大鼠还是雄性SD大鼠,大黄素的口服生物利用度都相当低,分别为7.5%(雄鼠)和5%(雌鼠)。
静脉给药(4mg／kg)后的大黄素在雌雄大鼠的体内均表现为二室模型,分布较快,消除较慢,t1/2α为13.26±6.28min(雄鼠)和13.52±7.28min(雌鼠),t1/2β为187.378±174.52min(雄鼠),118.5033±83.09min(雌鼠),血药浓度曲线下面积为AUC0-∞, i.v分别为430±160mg*μg/ml(雄鼠),283±98 mg*μg/ml(雌鼠)。口服给药8mg/kg大黄素后仅检测出少量原形药物,大黄素在体内的血药浓度变化无法用房室模型模拟,口服后大黄素的药代动力学参数分别为Cmax:0.31±0.094μg/ml(雄鼠)和0.039±0.011min(雌鼠)；Tmax:18±6.71 min(雄鼠),18.75±7.5min(雌鼠)；血药浓度曲线下面积AUC0-∞, p.o分别为65.76±34.77 mg*μg/ml(雄鼠),33.82±4.09 mg*μg/ml(雌鼠),大黄素的药代动力学参数存在一定的雌雄差异性。
静脉给药后的大黄素迅速代谢成大黄素葡萄糖醛酸化物,此代谢物在体内的血药浓度变化可用一室模型描述,t1／2Ke为167.4±50.9min(雄鼠)和251.3±114min(雌鼠),曲线下面积AUC0-∞, i.v分别为2210±980 mg*μg/ml(雄鼠),1540±290 mg*μg/ml(雌鼠)。口服后大黄素葡萄糖代谢产物的药代动力学参数分别为Cmax:6.69±1.058μg/ml(雄鼠)和1.807±0.576μg/ml(雌鼠),血药浓度曲线下面积AUC0-∞, p.o分别为2261.89±655.87 mg*μg/ml(雄鼠),458.50±373.29mg*μg/ml(雌鼠),大黄素葡萄糖醛酸结合物的药代动力学参数存在显著的性别差异性。
药代动力学研究表明大黄素在体内的主要代谢途径为UGT代谢,代谢速度快,而排泄速度较慢,这一结果是大黄素生物利用度低的原因之一。此研究内容充实了大黄素药物动力学方面的资料,为研究大黄素的吸收和代谢做了进一步的辅证,也为临床用药方面提供了参考依据。
3.大黄素的大鼠肠吸收动力学研究
为了进一步证明大黄素生物利用度低的原因,采用在体大鼠肠灌流法研究大黄素的吸收代谢情况。肠灌流液经UPLC法测定结果显示,大黄素被部分代谢为葡萄糖醛酸结合物。在雄性大鼠中,大黄素的吸收量存在着肠段依赖性,其中十二指肠与空肠的吸收量最高,其次是回肠,结肠部位吸收最少(p<0.05)；相反的,在雌鼠中,无肠段间的显著性差异(p>0.05)。在四段肠中大黄素的吸收量存在显著性性别差异(p<0.01)。
另一方面,雄性大鼠四段肠上大黄素代谢产物的细胞排出量也存在着显著性差异,代谢量最高的部位在十二指肠,其次在空肠,回肠和结肠部位吸收量最少(p<0.05)；而对于雌鼠,十二指肠与空肠中大黄素代谢产物的排出量最高,结肠中最少(p<0.05)。而四段肠代谢产物被排出细胞外的量也存在显著性性别差异(p<0.01),这一系列的结果证明肠道中的代谢酶会影响大黄素的代谢,大黄素在肠道细胞中有一定量的吸收,并存在明显的雌雄差异性和肠段差异性。
4.大黄素在不同雌雄动物的肝微粒体中的代谢研究
为了考察大黄素在肝中的代谢情况,以求进一步解释黄素生物利用度低的原因,本实验采用LC/MS法检测大黄素在五种动物(小鼠、大鼠、豚鼠、狗、人)的肝微粒体酶中经UGT途径代谢反应后的样品。结果证明,无论在哪种肝微粒体酶作用下,大黄素都会迅速代谢并生成葡萄糖醛酸结合物。对代谢产物进行收集提纯,经1HNMR法检测后,确定此结合物为大黄素-3-O-葡萄糖醛酸。同一性别的五种动物的肝微粒体代谢大黄素的速率有显著性种属差异(p<0.05),而同种属的动物的肝微粒体代谢大黄素的速率也存在着显著性的性别差异(p<0.05)。结果还显示大黄素的代谢在其高浓度的时候呈现饱和现象,大黄素在雄性豚鼠和男性的肝微粒体中的代谢符合典型的米氏方程(Michaelis-Menten equation),而其在雄性小鼠和狗的肝微粒体中的代谢符合双相抑制模式(Biaphasic pattern),只有在雄性大鼠中大黄素的代谢呈自身激活模式(Autoactivation pattern)。在雌性动物组,小鼠大鼠、豚鼠和女性的肝微粒体中的代谢均符合典型的米氏方程(Michaelis-Menten equation),只有雌性狗的肝微粒体中的代谢符合自身激活模式(Autoactivation pattern)。
Ⅰ相代谢反应中的大黄素代谢速度极慢,生成的代谢产物为羟基大黄素。将Ⅰ相反应和Ⅱ相反应的同一时间的大黄素剩余量进行比较,大黄素的Ⅱ相代谢反应明显快于Ⅰ相代谢反应,且Ⅰ相合并Ⅱ相代谢反应证明,Ⅱ相代谢占主导地位,并一定程度上抑制Ⅰ相代谢反应。
本实验结果说明大黄素在肝微粒体中极易发生葡萄糖醛酸结合反应,此代谢反应表现为显著的种属差异和性别差异。
5.对雌雄SD大鼠不同肠段的肠微粒体中大黄素的代谢研究
检测方法同上,我们分别考察了大黄素在雄性SD大鼠和雌性SD大鼠的空肠、回肠微粒体的代谢情况,不管是雄性SD大鼠还是雌性SD大鼠,大黄素在空肠微粒体中的葡萄糖醛酸化速率都大于在回肠微粒体中的速率(p<0.05)；并且无论空肠还是回肠微粒体,大黄素在雄性SD大鼠中的代谢速率远快于雌性SD大鼠(p<0.01)。大黄素在雄性SD大鼠的空肠微粒体中的代谢均符合典型的米氏方程(Michaelis-Menten equation),而其在雄鼠回肠微粒体中的代谢符合自身激活模式(Autoactivation pattern)。大黄素在雌性SD大鼠的空肠微粒体的代谢也符合典型的米氏方程,而在回肠中的代谢表现出双相抑制模式。
结果显示,大黄素的葡萄糖醛酸结合反应有显著性的性别差异性和肠段差异性。
6.对大黄素在12种人的UGT酶中的代谢研究
为了初步判断大黄素的主要代谢部位,本实验采用超高效液相色谱法考察了大黄素在12种不同人的UGT酶中的代谢情况,除了UGT1A4和UGT2B4中大黄素无代谢外,其他10种UGT酶均对大黄素的代谢有不同程度的影响,其中UGT1A家族占主要地位,大黄素在UGT2B家族的酶中代谢速率相对较慢。在UGT1A家族中,UGTIA10是大黄素的最主要代谢酶,其次是UGT1A8, UGT1A9, UGTIA1。大黄素在UGT1A9, UGTIA10, UGT2B15中的代谢均符合典型的米氏方程(Michaelis-Menten equation),而其在其他UGT酶中的代谢符合自身激活模式(Autoactivation pattern)。而大黄素在UGT1A3中的代谢不符合任何模式。从结果中可以看出UGT1A家族对大黄素的葡萄糖醛酸结合反应起着主要作用。特别是UGTIA10, UGT1A9, UGT1A8, UGT1A1。而这些UGT的亚型与两个最重要的代谢场所：肝、肠有关,UGT1A1在肝肠中都有分布,UGT1A9只在肝中表达,而UGTIA10, UGT1A8只在肠中表达。而UGTIA10对大黄素的代谢最快,和我们之前推断的肠道是大黄素的主要代谢场所相吻合。
综上所述,大黄素的生物利用度极低,存在性别差异性。主要原因可能是大黄素在体内易被UGT酶代谢成葡萄糖醛酸结合产物。大黄素在大鼠肠道中的吸收和代谢物的排泄量呈性别依赖性和肠段依赖性。大黄素在肝微粒体中的代谢存在性别、种属差异性,大黄素在肠微粒体中的代谢存在性别差异性,肠段差异性。从我们模拟的结果来看大黄素在人肝微粒体的葡萄糖醛酸结合反应可以用实验动物预测。综合我们的体外、体内、在体实验结果,可以判断小肠极有可能是大黄素的主要代谢场所。
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is one of the major active ingredients of rhubarb(rheuw officinale B.), aloe(aloe barbadensis M.), and leaf of senna(cassia angustifolia) plants. Decade ago, emodin was extensively studied for its traditional pharmacological activities. However, recent studies have put emodin back into limelight with its anti-cancer activities in several types of cancer cells with apoptosis as a possible mechanism of action.Emodin also has an inhibitory effect on cancer cell migration and invasion in in vitro studies and that is why emodin has good prospect and promising future in the market. Even though emodin has so many excellent application prospects as a chemical, little is known about its pharmacokinetics and metabolism in vivo. These studies are very important for development of emodin into a new anti-cancer pharmaceutical compound.
In modern era, more and more people, no matter men or women aspire to be fit and healthy. Therefore, in addition to the above mentioned effects, emodin's health food and slimming product intake has increased due to its weight loss property through its traditionally known stimulant laxative activity. However, again, no study has been done to show the gender differences in oral bioavailability of emodin in rodents as well as humans. Hence, we propose to study gender-dependent difference in absorption and metabolism of emodin as the main aim of our study.
Toxic effects of emodin such as renal and liver toxicity have received significant attention from all over the world. Due to renal failure issues, number of weight loss products containing emodin was recalled back from the market by FDA. Recently the toxicity of emodin was assessed by National Toxicology Program. The acute and chronic toxicity of emodin is investigated in liver and kidney of rodents. This proposed study investigates the gender-dependent differences in absorption and disposition of emodin which might help in understanding the pharmacological mechanism, guiding drug design, improving efficacy and safety and development of scientific standards of the quality of emodin.
The proposed study delineates gender-dependent differences in absorption and metabolism of emodin in different animal species using in vitro, in situ as well as in vivo study models. Our intentions were to put forth experimental data in different animal models to make an informative decision about the emodin dosage regimen in men and women. Our research has been divided into several parts as follows:1) to study stability and solubility of emodin in various buffer solutions; 2) to determine gender differences in absorption and disposition of emodin in SD rats using in vivo single dose (8mg/kg) oral and intravenous (4mg/kg) PK model; 3) to study gender difference in absorption and metabolism of emodin by using in situ four site rat intestinal perfusion model; 4) to determine the species and gender differences in glucuronidation of emodin by using male and female dog, guinea pig, mouse, rat and human liver microsomes; 5) to study gender-dependent and regioselective intestinal emodin glucuronidation difference by using female and male SD rat intestinal and liver microsome model; 6) to determine the major human uridine diphospho-glucuronosyl transferase (UGT) isoforms (s) responsible for glucuronidation of emodin using various human UGT isoforms available in the market.
1. Stability and solubility of emodin
Before conducting in vivo and in situ studies to understand the gender-dependent differences in bioavailability as well as absorption and disposition of emodin in rats, we needed clear understanding of stability and solubility of emodin. We determined emodin's stability and solubility by using UPLC method. Emodin stock solution was prepared in HPβCD solution to improve the solubility and stability of poorly soluble emodin in HBSS. We used emodin-HPβCD-HBSS (pH 7.4) as the perfusate, which was stable for 24hr under room temperature. Therefore, the perfusate was prepared fresh every time right before the start of the experiment. Emodin was stable in pH7.4～8.87, so Ph 7.4 HBSS buffer was used in intestinal perfusion model. Furthermore we also found out that not acidic but alkaline treatment was good for the stability of emodin in aqueous media.
2. Pharmacokinetics of emodin in female and male SD rats
To determine the plasma concentrations of emodin and its metabolites from in vivo PK plasma samples, we used LC/MS instrument. Emodin and emodin glucuronide showed a good linear plasma standard curve in the range of 0.01～20μg/ml. Pharmacokinetics studies to determine bioavailability of emodin were done using a single dose oral and intravenous administration in female and male SD rats. The oral bioavailability of emodin is extremely low whether in male rats (5%) and female rats (7.5%).
Following a single intravenous injection of 4 mg/kg emodin, emodin was distributed rapidly and eliminated slowly. The results showed a good two-compartment model fit for emodin plasma concentration-time data for male and female SD rats. The t1/2αwere 13.26±6.28min (male rats) and 13.52±7.28min (female rats). The t1/2βwere 187.38±0.16min (male rats) and 118.5033±83.09min (female rats). Emodin showed significant gender differences in iv PK profiles with higher AUC values in male (430±160 mg*μg/ml) than female (283±98mg*μg/ml) SD rats (n=6).
A little amount of emodin was detected using LC/MS after the oral administration of dose of 8mg/kg emodin. However, there was no good fit for any PK compartmental model for the plasma concentration-time data for single dose intravenous administration of emodin (8mg/kg). Analyzing the intravenous PK data using non-compartmental model, Cmax, Tmax and AUC0-∞, p.o of emodin in male rats were:0.31±0.094 wereμg/ml,18.0±6.71min and 65.76±34.77 mg*μg/ml respectively; whereas Cmax, Tmax and AUC0-∞，P.o of emodin in female rats were:0.039±0.011μg/ml, 18.75±7.51min and 33.82±4.09 mg*μg/ml respectively.
Emodin was metabolized very fast after the intravenous administration, suggesting a good fit for single compartmental model for the plasma emodin metabolite concentrations. t1/2Ke were 167.4±50.9min(male rats) and 251.3±114min (female rats), the area under the curve (AUC0-∞，i.v) were 2210±980 mg*μg/ml and 1540±290 mg*μg/ml (female rats) (n=6).
After oral administration of emodin, the metabolite of emodin appeared in the plasma rapidly. Non-compartmental model analysis was done for emodin and its metabolite plasma concentrations after single dose oral administration of emodin (20mg/kg). The parameters of emodin glucuronide were significant different with emodin, the Cmax, Tmax and AUC0-∞, p.o of emodin glucuronide in male rats were 6.69±1.058μg/ml,240min and 2261.89±655.87 mg*μg/ml respectively, in female rats, the Cmax, Tmax and AUC0-∞, P.o were 1.807±0.578μg/ml,60min and 458.5±373.39 mg*μg/ml respectively. The oral absolute bioavailability of emodin was less than 7.5% and 5% in male and female SD rats respectively. Emodin glucuronidation michaelis menten kinetics parameters showed significant gender-dependent differences.
The pharmacokinetics results of emodin suggested emodin glucuronide as a major metabolite in vivo. Rapid glucuronidation with slow metabolite elimination was one of the reasons of low bioavailability of emodin. Our research had enriched the pharmacokinetics aspects of emodin and provided the information for clinical use of emodin.
3. Gender-differences in intestinal absorption and disposition of emodin in rats
To confirm the reason of low bioavailability of emodin, gender-dependent intestinal absorption and disposition of emodin were investigated in rats by using the in situ intestinal perfusion model. The perfusate was analyzed for emodin and emodin glucuronide. Absorption and glucuronide excretion of emodin displayed regioselectivity (p<0.05) in male rats. The rank order for amounts of absorption of emodin in male rats was duodenum≥jejunum>ileum>colon (p<0.05). On the contrary, in female rats, the amounts of absorption showed no regioselectivity (p>0.05). Furthermore, gender-dependent higher intestinal emodin absorption was observed in female than in male SD rats in all 4 regions of intestine (p<0.01).
The amount of emodin glucuronide in duodenum was significant higher (p<0.05) than in jejunum, followed (p<0.05) by ileum and colon in male rats. In female rats group, the rank order of amount of metabolite excreted was jejunum≈duodenum> ileum>colon (p<0.05). In contrast to intestinal emodin absorption, emodin glucuronide excreted lower in all four regions of the male rat intestine than the female rat intestine (p<0.01). No biliary excretion of emodin or emodin glucuronide was detected in both male and female rats. The results suggested that not liver metabolism, but intestine metabolism, played an important role in disposition of emodin in rats.
4. Species and gender-dependent hepatic glucuronidation of emodin by using liver microsomes of different animal species.
To find out the main reason of low bioavailability of emodin, ten kinds of liver microsomal (male mouse, rat, guinea pig, dog, human and female mouse, rat, guinea pig, dog, human) were used, UGT metabolite formation was determined by LC/MS method. The results showed that emodin was glucuronidated fast in all ten kinds of liver microsomes.1H-NMR spectra of the metabolite displayed that the metabolite was emodin 3-O-β-D-glucuronide. The results also showed species-dependent different in glucuronidation rates of emodin among the five species (p<0.05). Significant gender difference in glucuronidation rates was shown in all test species. The results also indicated that metabolism of emodin was saturable at higher concentrations. Among the five species of males, guinea pig and human followed simple Michaelis-Menten kinetics whereas mouse and dog followed a biaphasic kinetics, and only rat followed autoactivation kinetics. Among the five species, mouse, rat, guinea pig and human all followed simple Michaelis-Menten kinetics whereas dog followed Autoactivation kinetics. In liver microsomes, the glucuronidation rates were faster in the female rats, especially at lower concentration. In the liver of other 4 species, rates were faster in male mice, but were slower in female guinea pigs and dogs. There was very little difference in emodin glucuronidation rates between human male and female liver microsomes, although, hepatic rate of glucuronidation showed significant gender differences in certain species such as mice.
Significant lower amount of hydroxyemodin was detected after phaseⅠmetabolism of emodin by LC/MS. There was no detectable PhaseⅠmetabolites but the glucuronide emodin in the mixed reaction system, which suggested that PhaseⅡmetabolism (glucuronidation) was major route of disposition of emodin in rats.
The resulted indicated that glucuronidation was the major pathway for emodin metabolism which was significant gender and species dependent.
5. Region and gender-dependent glucuronidation of emodin by intestinal microsomes
We measured glucuronidation rates of emodin in jejunal and ileal microsomes of male and female rats, using by UPLC-MS/MS method. The result showed that emodin was glucuronidated faster in rat jejunal microsomes than in ileal microsomes regardless of gender (p<0.05). Furthermore, emodin was metabolized faster in male than in female rats at all tested concentrations (p<0.01). Emodin glucuronidation in jejunal microsomes showed simple Michaelis-Menten kinetics, whereas glucuronidation in ileal microsomes followed autoactivation kinetics. In female rat intestine, glucuronidation in jejunal microsomes also showed simple Michaelis-Menten kinetics, whereas glucuronidation in ileal microsomes followed biaphasic kinetics.
The results indicated that glucuronidation of emodin showed significant gender and regioselective intestinal difference.
6. Fingerprinting of UGT glucuronidation of emodin by 12 expressed human UGTs isoforms.
To identify the main UGT isoform responsible for metabolism of emodin, we used 12 expressed human UGTs isoforms. Except UGT1A4 and UGT2B4, the other 10 UGTs metabolized emodin at different rates. The average glucuronidation rate of emodin in UGT2A familiy was faster than UGT2B familiy. Amongst the 10 UGT enzymes isoforms, glucuronidation of emodin by UGT 1A9, UGT1A10 and UGT2B15 followed classical Michaelis-menten kinetics, while others followed autoactivation kinetics, and UGT1A3 didn't fit any pattern.
In the UGTs study, the four major isoforms, responsible were UGT1A1,1A8, 1A9 and especially 1A10. Qualitative and quantitative expression of these UGT isoforms in two main metabolic organ, liver and intestine were quite different. UGT1A1 was expressed in all organs, although its expression was higher in the liver than in intestine. UGT1A9 was only expressed in liver whereas UGT1A8 and UGT1A10 were predominantly expressed in the small intestine.
In summary, systemic metabolic characterization study suggested rapid metabolite (glucuronide) formation might be the major reason for emodin's low bioavailability in rats. The elimination of emodin and emodin glucuronide might follow UGT enzyme. In liver microsomes, the gender-dependent metabolism of emodin was also species-dependent. In intestinal microsomes, the gender-dependent metabolism of emodin also showed regioselectivity. Therefore, informative conclusion can be drawn from our results about glucuronidation of emodin in humans. Based on in vivo, in situ and invitro studies of emodin, we predicted that the main organ of metabolism for emodin to be intestine.
引文
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