【作者】 鲁鑫焱；

【导师】 曾苏； 蒋惠娣；

【作者基本信息】 浙江大学 ， 药物分析学， 2008， 博士

【摘要】 菊花为菊科植物菊[Chrysanthemum morifolium Ramat.)的干燥头状花序。具有疏风散热、明目、平肝和清热解毒之功效。现代药理学研究表明,菊花具有抗氧化和治疗心血管疾病等多种药理作用。进一步的研究表明,菊花的功效与其所含黄酮类化合物密切相关。而木犀草素(Luteolin)-7-O-β-D-葡萄糖苷和芹菜素(Apigenin)-7-O-β-D-葡萄糖苷为菊花中主要的黄酮类化合物,可在体内水解生成效应成分木犀草素和芹菜素。本课题组已对菊花提取物(CME)中主要黄酮类成分的药代动力学、排泄和组织分布等体内过程进行了一系列的研究,表明口服菊花提取物后,体内主要的效应成分为木犀草素和芹菜素,大鼠药动学结果显示,芹菜素的AUC显著高于木犀草素;木犀草素和芹菜素可经尿液、粪便和胆汁排泄;大鼠灌喂菊花提取物后,木犀草素和芹菜素广泛分布于各组织,二者在健康大鼠及心肌缺血大鼠中的分布存在一定差异(芹菜素在心肌缺血大鼠心肌中的分布显著高于健康大鼠)。但是,菊花中主要黄酮苷类化合物在大鼠体内的水解和吸收部位、水解部位环境的改变对其水解是否存在影响以及造成芹菜素AUC显著高于木犀草素的机制目前尚未阐明。此外,由于菊花药食同源,与许多药物存在伍用的可能,那么它是否会在吸收和代谢水平与伍用药物发生相互作用,也未见报道。为了阐明上述问题,本文采用多种模型,分别在体内、在体和体外水平,研究CME活性成分的吸收、代谢以及药物相互作用,以期为该类药物的体内过程研究提供新的思路和方法,并为临床合理用药和科学评价CME的药效学、药动学和毒理学提供依据。1.CME活性成分在大鼠体内的水解和吸收目的:评价CME活性成分在大鼠消化道内的脱糖代谢和吸收情况。方法:建立大鼠消化道内容物和组织中木犀草素和芹菜素的HPLC分析方法,研究CME效应成分——木犀草素和芹菜素在大鼠消化道的分布规律。结果:1)给药5min后,即可在血以及胃、十二指肠和空肠的内容物和组织中检测到木犀草素和芹菜素,表明CME在大鼠消化道内的水解和分布相当迅速。2)二者在胃、十二指肠、空肠、回肠、盲肠和结肠内容物中最高浓度分别出现在5min、5min、2h、5h和8h,消化道总滞留时间超过12h,特别是在大肠内可长时间保持较高浓度。3)木犀草素和芹菜素迅速出现在血液中,但消除相对缓慢,木犀草素和芹菜素的Tmax分别为30min和5h,表明木犀草素的吸收主要在消化道前段,而芹菜素的吸收部位可能更为广泛,芹菜素在12h时仍维持较高血药浓度。4)木犀草素和芹菜素在血、组织和内容物中的相对比例呈现一定特征,血中芹菜素的浓度远远高于木犀草素,在胃和小肠内容物中二者比例相当,但在盲肠和结肠内容物中木犀草素含量显著高于芹菜素。结论:CME在大鼠消化道内可发生迅速和广泛的水解代谢,生成效应成分木犀草素和芹菜素,这一过程存在明显的消化道和底物选择性。2.应用大鼠消化道原位结扎灌注技术研究CME活性成分的水解和吸收目的:分段评价各消化道的水解和吸收能力,以及影响水解和吸收的因素。方法:采用原位结扎灌注模型分别将CME与不同消化道进行孵化,同时采集血液、组织和内容物样品,评价不同吸收部位的水解和吸收差异,然后选择空肠测定了其水解和吸收动力学,并对影响其水解和吸收的因素(内容物、肠道菌、长期用药、性别和胃内酸性环境)进行了评价。结果:1)不同消化道组织均可水解CME活性成分,原位孵化10min后,各消化道内容物和组织中均可检测到木犀草素和芹菜素,盲肠对木犀草素苷的水解能力显著高于对芹菜素苷的水解能力,其他消化道对二者的水解能力相当,水解率均大于50%。木犀草素和芹菜素可被各消化道组织摄取,但仅在小肠段可以透过消化道进入血液。2)空肠原位孵化实验表明,CME活性成分的水解和吸收均很迅速,在内容物和组织中的最高浓度均为第一个时间点5min。其中芹菜素的吸收强于木犀草素,二者最高血药浓度分别为8.43和5.41μg/mL,AUC0-∞分别为999±100和339.4±49.9mg/L~*min,且水解和吸收过程呈现线性动力学特征。3)除去内容物、应用抗生素以及长期用药均使空肠内容物中木犀草素和芹菜素的含量出现一定程度降低,同时去除内容物使得组织摄取和吸收入血的量大大提高,但不同性别间未发现明显差异,其他因素对组织摄取和血药浓度影响也不大。结论:CME活性成分在各消化道均可发生迅速和广泛的水解代谢,但只有小肠段可有效的吸收木犀草素和芹菜素,这一水解和吸收过程具有消化道和底物选择性,并受多种因素影响。3.CME活性成分在大鼠组织提取物中的脱糖代谢目的:体外评价各消化道以及肝、肺和肾组织提取物对CME活性成分的水解代谢。方法:CME与各组织提取物共孵育,测定生成的木犀草素和芹菜素,通过考察底物浓度、孵育时间和酶浓度,确定最佳孵育条件,并在该条件下评价了不同组织提取物的水解代谢能力。采用LC-MS对其代谢产物进行了鉴定。结果:最佳孵育条件为:底物浓度18.75mg/mL,酶浓度0.50mg蛋白/mL,孵育时间30min。CME活性成分在各组织提取物中均可发生水解代谢,生成效应成分木犀草素和芹菜素。其中肾组织提取物的水解代谢能力最强,消化道组织中,空肠组织提取物的水解能力最强,胃水解能力最差,其他肠段组织提取物的水解能力介于空肠和胃之间。结论:CME活性成分可发生广泛的水解代谢,生成效应成分木犀草素和芹菜素,肾和空肠组织提取物的水解能力最强。4.木犀草素和芹菜素在大鼠原代肝细胞中的代谢目的:考察CME中效应成分木犀草素和芹菜素在肝脏发生的结合代谢反应。方法:采用原代肝细胞模型考察了二者的消除过程以及黄酮类化合物对其消除过程的影响。结果:1)木犀草素和芹菜素在原代肝细胞中的消除非常迅速,孵育120min,木犀草素和芹菜素的消除比率分别达到91.9%和86.7%,经二相结合物水解酶水解测得二者的二相代谢产物分别占给药量的54%和73%。2)性别和pH对其消除影响不大。3)少量木犀草素可转化为芹菜素(＜5%)。4)二者在消除过程中存在饱和现象,随着浓度增加,消除率下降;二者间同样存在相互抑制作用,木犀草素抑制芹菜素的IC50为17.3gmol/L,而芹菜素抑制木犀草素的IC50为38.2μmol/L,且二者在HCME中消除时所受到的抑制与二者间的相互抑制作用相近,说明二者在提取物中消除所受到的抑制主要来自它们自身的相互作用,与其他成分关系较小。5)多种黄酮苷对二者消除均无明显影响,黄酮苷元中黄酮醇和黄酮类成分对其抑制作用明显,异黄酮苷元和儿茶素类对其消除无明显影响。结论:CME效应成分木犀草素、芹菜素在大鼠原代肝细胞中可迅速发生二相代谢,这一过程可能发生药物.药物相互作用。5.CME活性成分对大鼠原代肝细胞药物代谢酶的调节目的:考察CME及其效应成分木犀草素、芹菜素对肝—相药物代谢酶的调节作用。方法:采用大鼠原代肝细胞模型,考察上述成分对各CYP3A探针底物睾酮和硝苯地平以及CYP2E探针底物氯唑沙宗消除的影响。结果:孵育1h后,睾酮的代谢分别被木犀草素、芹菜素、CME和HCME抑制了44.0±8.2%、48.5±4.7%、20.1±0.4%和70.5±0.8%;孵育2h后,硝苯地平的代谢分别被四者抑制了79.1±1.3%、80.1±0.2%、60.4±2.6%和92.2±0.4%;孵育2h后,氯唑沙宗的代谢分别被抑制了54.3±0.2%、42.6±0.8%、41.9±2.7%和69.9±1.8%。抑制能力为HCME物＞木犀草素≈芹菜素＞CME。结论:木犀草素、芹菜素、CME和HCME对原代肝细胞CYP3A和CYP2E均有一定抑制作用,水解代谢可激活抑制代谢酶的能力。6.CME活性成分对过表达人MDR1细胞中P-gp的调节目的:考察CME及其效应成分木犀草素、芹菜素对人P-gp的调节作用方法:分别采用Bcap-MDR1和MDCK-MDR1模型,考察木犀草素、芹菜素、CME和HCME对R123在细胞内积聚的影响。结果:木犀草素、芹菜素、CME和HCME对人P-gp有明显抑制作用,这一抑制作用呈现剂量依赖性,Bcap-MDR1中,四者的最高剂量可分别使罗丹明的摄取达对照组的2.79±0.063、1.68±0.078、1.15±0.051和5.73±0.073倍;MDCK-MDR1中可达对照组的2.07±0.039、1.44±0.065、1.67±0.091和7.23±0.65。抑制能力为HCME＞木犀草素＞芹菜素＞CME,两种模型得出的结论相似。结论:CME及其效应成分木犀草素和芹菜素对人P-gp具有调节作用,水解代谢可激活抑制人P-gp的能力。更多还原

【Abstract】 Flos Chrysanthemi is the flower of Chrysanthemum morifolium Ramat..It has many beneficial effects such as dispelling wind and heat,improving eyesight,calm the liver and suppress yang,heat-clearing and detoxicating.The pharmacological investigations indicated that CME has antioxidant activity and can be used in the treatment of cardiovascular diseases.And the further researches shows that the flavonoids in CME play an important role in these benefit.The main flavonoids in CM were luteolin-7-O-β-D-glucoside and apigenin-7-O-β-D-glucoside,which can be deglycosidated to their effective aglycone——luteolin and apigenin during absorption.The pharmacokinetic,absorption,distribution and excretion of main CM flavonoids have been systematically studied in our lab.After oral administration of CME,the main effective components were luteolin and apigenin.AUC of apigenin was higher than luteolin in pharmacokinetic study,and they can excrete by urine,stool and bile.They can distribute in various tissues and their distribution was different between normal and myocardial ischemia rats.While,the hydrolysis and absorption site of glucosides in CME,the effect of conditions on the hydrolysis and absorption process as well as the reason of the higher AUC of apigenin were still not well elucidated.In addition,CM can be use as food and drug,so there was more chance of drug-drug interactions,which was not reported too.In this paper,we will investigate the absorption,metabolism and drug-drug interactions of CME in vivo,in situ and in vitro level to explore new ideas and methods for seeking new drugs,and providing scientifical evidence to scientifically evaluate animal pharmacodynamics,pharmacokinetics and toxicology.1.Deglyeosidation and absorption of active flavonoids of CME in rats.Objective:To investigate the deglycosidation and absorption of active flavonoids of CME in rat.Methods:Established HPLC methods for analyzing luteolin and apigenin in rat gastrointestinal contents and tissues,and then evaluated the distribution of luteolin and apigenin in gastrointestine.Results:1 ) The hydrolysis and distribution of CME components in gastrointestine were very fast.Luteolin and apigenin emerged in plasma,the contents and tissues of stomach,duodenum and jejunum at 5min after administration.2) The highest concentration of contents were 5min,5min,2h,5h and 8h for stomach,duodenum, jejunum,ileum,cecum and colon,respectively.The total residence time of luteolin and apigenin in gastrointestinal tract exceeded 12h,and contained high concentration level in large intestine contents for long time.3 ) Luteolin and apigenin were absorbed fast,while the elimination process of them in plasma were relatively slow,Tmax of luteolin and apigenin in plasma were about 30min and 5h,respectively,which implied that luteolin was mainly absorbed in the upper instine while apigenin may be absorbed in wider scope,and the plasma concentration of apigenin was still high at 12h after administration.3 )The ratio of luteolin and apigenin in plasma,tissue and contents was different,such as the concentration of apigenin was far beyond luteolin in plasma,but concentration of luteolin was higher than apigenin in cecum and colon.Conclusion:CME components can be hydrolyzed fast in the whole gastrointestine and producted effective components——luteolin and apigenin.This process was site and substrate dependent.2.Deglycosylation and absorption of active components of CME in situ perfusing iigated intestinal segment.Objective:To assess the hydrolysis and absorption ability of different gastrointestinal parts independently and impact of factors on hydrolysis and absorption.Methods:An in situ perfusing ligated intestinal segment model was used.After incubated CME in various gastrointestinal segments,the plasma,tissues and contents sample were collected and evaluated to assess the discrepancy of different parts.In addition,jejunum was selected to investigate the pharmacokinetics of deglycosidation and absorption.Factors affecting hydrolysis and absorption（contents,bacteria,long term administration,gender and acid condition of stomach ） were also evaluated.Results:1 ) All parts of gastraintestine could hydrolyze CME,and luteolin and apigenin could be detected after 10min incubation with various parts.The hydrolysis ability of cecum on luteolin glucoside was higher than apigenin glucosides,while other parts had the fair ability of hydrolyzing luteolin and apigenin glucosides,and the hydrolysis rate were all beyond 50%.Luteolin and apigenin could be uptaked by various gastrointestinal tissues,but only small intestine could transport them to blood. 2 ) CME was hydrolyzed and absorpted fast in jejunum,and the highest concentration emerged was 5min after administration.The absorption of apigenin was higher than luteolin with Cmax value 8.43 and 5.41μg/mL,respectively,while AUC_0-∞were 999±100 and 339.4±49.9mg/L*min,respectively.The hydrolysis and absorption potential depended on the dosage.3 ) Without contents,using antibiotics as well as long-time administration were all decreased the concentration of luteolin and apigenin in jejunum contents,while gender had no significant effect on it.Without contents,the tissue uptake and plasma concentration of luteolin and apigenin were increased,but other factors have no significant effect on their tissue and plasma concentration.Conclusion:CME components can be hydrolyzed fast in the whole gastrointestine and producted effective components——luteolin and apigenin.They can be absorbed in small intestine.This process was site and substrate dependent,and can be influenced by various factors.3.Deglycosidation of CME in cell free fractions of different tissues.Objective:To investigate the hydroysis ability of cell-free fractions of various tissues in vitro.Methods:Determinated the concentration of luteolin and apigenin after incubated CME with various cell free fractions.Investigated the best concentration of substrates, incubation time and protein contents,and compare the hydrolysis ability of various cell free fractions.LC-MS was also used to identify the metabolites.Results:The incubation condition selected was:18.75mg/mL of substrates,0.5mg protein/mL,and incubate for 30min.CME can deglycosidated in all investigated cell-free fractions,and produced effective luteolin and apigenin.Kidney cell free fraction can hydrolyze more CME than other tissues during the same time.Jejunum can hydrolyze more CME than other gastrointrestinal tissues,while stomach was the weakest one.Conclusion:CME can be hydrolyzed in many tissues,and produce effective lutelin and apigenin.Kidney and jejunum cell free fractions can hydrolyze CME more efficently. 4.Metabolism of luteolin and apigenin in primary cultured hepatocytes.Objective:Investigated the conjugation metabolism of luteolin and apigenin in liver after absorption.Methods:Primary cultured rat hepatocytes model was selected.The elimination process of them was evaluated and the effect of other flavonoids on the elimination rate of luteolin and apigenin was also studied.Results:1 ) The eliminaton of luteolin and apigenin was very fast.91.9%of luteolin and 86.7%of apigenin disappeared after 120min incubation.The percentage of phaseⅡmetabolites reached 54%and 73%of dose for luteolin and apigenin,respectively. 2 ) Gender and pH have no significant effect on the elimination rate of luteolin and apigenin.3 ) Luteolin can transformed to apigenin to mimor extent（＜5%）.4 ) When monolayers were exposed to increasing amounts of luteolin and apigenin,a concentration-dependent inhibition were observed;They can inhibit the elimination of each other with the IC₅₀ value of 17.3μmol/L and 38.2μmol/L,respectively. Elimination of luteolin and apigenin were also inhibited in HCME.5 ) Other flavonoids glycosides didn’t make any difference in elimination rate of luteolin and apigenin.As to flavonoids aglycone,flavonol and flavone can inhibit their elimination effectively,while isoflavone and catechin have no significant effect.Conclusion:Metabolism of luteolin and apigenin in hepatocytes was very fast.Drug interactions occured in this process.5.Modulation effect of CM flavonoids on metabolizing enzymes of hepatocytes.Objective:To investigate the Modulation effect of CM flavonoids on metabolizing enzymes.Methods:A rat primary cultured hepatocytes was used.The modulation effect was valued by the elimination of probes.The probes of CYP3A were testosterone and nifedipine and probe of CYP2E was chlorzoxazone.Results:Metabolism of testosterone after 1h incubation were inhibited to 44.0±8.2%, 48.5±4.7%,20.1±0.4%and 70.5±0.8%by luteolin,apignein,CME and HCME, respectively.Metabolism of nifedipine after 2h incubation were inhibited to 79.1±1.3%,80.1±0.2%,60.4±2.6%and 92.2±0.4%,respectively.Metabolism of chlorzoxazone after 2h incubation were inhibited to 54.3±0.2%,42.6±0.8%, 41.9±2.7%and 69.9±1.8%,respectively.The inhibition of elimination of probes was HCME＞luteolin≈apignein＞CME.Conclusion:CM fiavonids can inhibit CYP3A and CYP2E enzymes of hepatocytes. Hydrolysis process may bioactivate this inhibition effect.6.Modulation effect of CM flavonoids on human P-gp.Objective:Investigated the modulation effect of CM flavonoids on human P-gp.Methods:Overexpressed MDR1 cell line——MDR1-Bcap and MDR1-MDCK were selected to investigate the inhibition effect of CM flavonoids.The accumulation of rodamine 123 in above cell lines was used to value this inhibition effect.The inhibition of rodamine accumulation was HCME＞luteolin＞apignein＞CME,and the results from different models was equol.Results:Luteolin,apignein,CME and HCME can inhibit human P-gp significantly, and this inhibition was dose dependent.The accumulation of rodamine in Bcap-MDR1 can reach 2.79±0.063,1.68±0.078,1.15±0.051 and 5.73±0.073 fold of control for the highest dose of luteolin,apignein,CME and HCME,respectively;The accumulation of R123 in MDCK-MDR1 can reach 2.07±0.039,1.44±0.065, 1.67±0.091 and 7.23±0.65 fold of control for the highest dose of luteolin,apignein, CME and HCME,respectively.Conclusion:CM flavonoids can mudulate human P-gp.Hydrolysis process may bioactivate this inhibition effect.更多还原