远志活性成分的吸收和代谢研究
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摘要
远志作为著名的益智药物,长期的临床实践表明对于失眠多梦、健忘惊悸有良好的改善作用,与其它中药配伍常用于学习记忆减退性疾病。但由于远志化学成份复杂、迄今为止远志益智作用的体内活性成分尚不清楚,因而无法有效地控制远志的质量。本论文采用具高灵敏度的液-质联用分析技术,以整体实验动物和体外细胞模型研究远志在口服给药后的体内药效成分,包括其原型药物成分和代谢产物的吸收及代谢。阐释远志益智作用活性成分的药物动力学和代谢过程和规律。
首先,利用HPLC和HPLC-MS方法对灌胃给药远志后的大鼠的血浆样品进行分析,通过比较给药前后血浆的色谱指纹图谱的差异,发现主要吸收入血的成分。采用各种层析方法和光谱技术从远志总苷中分离鉴定被吸收入血的成分,用SH-SY5Y细胞的H_2O_2、Aβ_(25-35)和谷氨酸损伤模型来考察其对神经细胞的作用。结果表明3,6′-二芥子酸蔗糖酯(3,6′-disinapoylsucrose,DSS)在三个模型中均具有显著的神经保护作用。
其次,本文建立了生物样品,包括大鼠血浆、尿液和胆汁中的DSS的LC-MS/MS分析方法,并以此方法研究了DSS在大鼠体内的药代动力学特性。结果显示,所建立的分析方法灵敏准确、稳定可靠、方便易行。体内药代动力学结果表明,DSS吸收迅速,在大鼠体内达峰时间约为(30.0±27.4)min;消除速度快,静脉和口服给药的t1/2,λz分别为(26.9±7.8)min和时间为(158.2±88.4)min;大鼠口服生物利用度低,仅为0.5%;DSS主要通过胆汁排泄,静脉给药后3h内累计排泄量可达到36.69%。DSS在机体稳定性研究的结果显示:大鼠胃和小肠的内容物对DSS稳定性没有明显影响,而大鼠结肠内容物对DSS具有较强的降解能力;这种降解很可能与结肠部位所生存的微生物或酶系统有关,而与其pH环境无关。DSS在新鲜血浆中稳定地以原型形式存在,血浆中的酯酶不能将其大量水解。
之后,Caco-2细胞模型对DSS的吸收机理研究结果显示:DSS透膜能力差,其表观渗透系数(Papp)仅为约1.1×10~(-7)cm/s,属于吸收较差的药物;DSS在Caco-2细胞模型上的转运不会明显地受药物浓度、pH环境、各种转运载体抑制剂、能量抑制剂的影响;而钙离子螯合剂EDTA-2Na和表面活性剂癸酸钠能明显提高DSS的Papp值。结果说明:DSS在Caco-2单层细胞膜上,主要是通过细胞胞旁通路转运的。
大鼠在体肠灌流模型的研究结果显示:DSS在大鼠小肠的平均Papp(blood)为3.97×10~(-5)cm/s,而当灌流液中同时含有EDTA-2Na和癸酸钠时,可使DSS的Papp(blood)分别提高至2.03×10~(-4)cm/s和1.46×10~(-4)cm/s。结果表明EDTA-2Na和癸酸钠能增强DSS在大鼠在体小肠的吸收,也进一步证实了DSS在小肠是通过细胞旁通路转运的。
最后,DSS的代谢途径研究结果发现:DSS在生物体内发生了异构化,在胆汁中,DSS大多数以异构体形式存在。HPLC-MS中采用MRM模式和MS和MS-MS自动切换扫描(IDA)模式综合分析,在给药后的血浆中鉴定了DSS的原型及二种代谢产物,分别是硫酸化DSS和葡萄糖醛酸化DSS;在静脉给药DSS后的胆汁中找到了DSS的原型及四种代谢产物,分别是硫酸化DSS、葡萄糖醛酸化DSS、甲基DSS和芥子酸。
本论文研究结果揭示了中药远志发挥体内益智作用的活性成分及其体内吸收代谢规律,为确立远志及其制剂的质量控制指标成分提供依据,为从远志中开发有效的益智药物提供了实验依据,为研究复杂的中药体系提供了新思路。
Polygala tenuifolia(Yuan Zhi) is one of the famous herbs to improve learning and memory in traditional Chinese medicine and has often been incorprated into many traditional compound formulas intended for the treatment of amnesia and dementia. However,due to its chemical complexity,the active ingredients of Yuan Zhi responsible for learning and memory improvement are largely unknown and uninvestigated.Therefore,in the present study,interdisciplinary technologies in chemistry,pharmacokinetics and pharmacology will be applied and aim to characterise and elucidate the chemical ingredients and metabolites in rat plasma after Yuan Zhi extract is intragastric administered,and to systemically investigate the effects of the identified compounds on learning and memory improvement in vitro. Our working hypothesis is that effects of oral extracts is elicited only by the ingredients and metabolites present in the plasma,and the identified pharmacologically active compounds may be useful as chemical markers for quality control of Yuan Zhi,together as lead compounds for the development of novel therapeutics.
Firstly,chemical ingredients and metabolites in the rat plasma after the Yuan Zhi extract is orally administered are to be identified using HPLC,LC-MS techniques based on our previous phytochemical data,which have determined the mass fragment profiles of various chemicals in the Yuan Zhi extract.Following identification,these bio-available components will be isolated and purified using various chromatographic techniques,and subjected to in vitro screening assay. Neuronprotective effects of bioavailable components should be evaluated by in vitro cell models using hydrogen peroxide,Aβ_(25-35) and glutamic acid-induced SH-SY5Y cells.3,6'-disinapoylsucrose(DSS) was demonstrated to be the bioavailble active components of Yuanzhi.
The developed LC-MS/MS method has been applied to a pharmacokinetic study for DSS in rats.The results indicated that DSS was absorbed rapidly into the body with peak time averaged 30 min after i.g.administration.However,its plasma concentration levels were very low with peak concentration averaged 16.22 ng/mL even given with the relative high dose of 50 mg/kg.The data from i.v.administration shows that DSS was eliminated rapidly from the body with half-life approximate 27 min.The absolute bioavailability of DSS after i.g.administration was found only 0.5%.After i.v.administration to rats,DSS was mainly excreted from bile 36.69%in 3 hours.
Therefore,its fate in gastric intestinal tract serve as further investigation. Stability of DSS by peptidases was investigated by incubation of the drug with the contents of stomach,small intestine and colon at 37℃.Results from the experiments show the content of DSS decreased 50%at four hours in colon juice.These results indicate that the stability of DSS in GI can be affected by the Microorganism and enzyme in colon.
In the caco-2 cell model,the absorption transport of DSS was fairly poor with Papp values of 1.1×10~(-7) cm/s.Kinetic permeability parameters were provided by bidirectional Caco-2 experiment and pH and concentracion dependency measurements.The transport route and putative transporters involved in DSS were studied using EDTA-2Na and several inhibitors.The bidirectional studies were performed under non-gradient conditions at a concentration of 5～33μM.Opening up the paracellular tight junctions by EDTA-2Na and sodium caprate did increase the apparent permeability coefficients(Papp) of DSS.Transport of DSS was not reduced by others inhibitions.In the study of DSS in situ rat intestinal perfusion model,the Papp(blood) of DSS was 3.97×10~(-5) cm/s for perfusing of DSS,which did significantly differ from that obtained for perfusion of DSS with EDTA-2Na (2.03×10~(-4) cm/s) and sodium caprate(1.46×10~(-4) cm/s).So,transport route of DSS was the passive paracellular.
Finally,LC-MS was used to determine DSS and its metabolites in rat plasma and bile samples.DSS and three and six metabilites were detected and identified in biles collected after i.v.and oral administration by using MRM and IDA model.
首先,利用HPLC和HPLC-MS方法对灌胃给药远志后的大鼠的血浆样品进行分析,通过比较给药前后血浆的色谱指纹图谱的差异,发现主要吸收入血的成分。采用各种层析方法和光谱技术从远志总苷中分离鉴定被吸收入血的成分,用SH-SY5Y细胞的H_2O_2、Aβ_(25-35)和谷氨酸损伤模型来考察其对神经细胞的作用。结果表明3,6′-二芥子酸蔗糖酯(3,6′-disinapoylsucrose,DSS)在三个模型中均具有显著的神经保护作用。
其次,本文建立了生物样品,包括大鼠血浆、尿液和胆汁中的DSS的LC-MS/MS分析方法,并以此方法研究了DSS在大鼠体内的药代动力学特性。结果显示,所建立的分析方法灵敏准确、稳定可靠、方便易行。体内药代动力学结果表明,DSS吸收迅速,在大鼠体内达峰时间约为(30.0±27.4)min;消除速度快,静脉和口服给药的t1/2,λz分别为(26.9±7.8)min和时间为(158.2±88.4)min;大鼠口服生物利用度低,仅为0.5%;DSS主要通过胆汁排泄,静脉给药后3h内累计排泄量可达到36.69%。DSS在机体稳定性研究的结果显示:大鼠胃和小肠的内容物对DSS稳定性没有明显影响,而大鼠结肠内容物对DSS具有较强的降解能力;这种降解很可能与结肠部位所生存的微生物或酶系统有关,而与其pH环境无关。DSS在新鲜血浆中稳定地以原型形式存在,血浆中的酯酶不能将其大量水解。
之后,Caco-2细胞模型对DSS的吸收机理研究结果显示:DSS透膜能力差,其表观渗透系数(Papp)仅为约1.1×10~(-7)cm/s,属于吸收较差的药物;DSS在Caco-2细胞模型上的转运不会明显地受药物浓度、pH环境、各种转运载体抑制剂、能量抑制剂的影响;而钙离子螯合剂EDTA-2Na和表面活性剂癸酸钠能明显提高DSS的Papp值。结果说明:DSS在Caco-2单层细胞膜上,主要是通过细胞胞旁通路转运的。
大鼠在体肠灌流模型的研究结果显示:DSS在大鼠小肠的平均Papp(blood)为3.97×10~(-5)cm/s,而当灌流液中同时含有EDTA-2Na和癸酸钠时,可使DSS的Papp(blood)分别提高至2.03×10~(-4)cm/s和1.46×10~(-4)cm/s。结果表明EDTA-2Na和癸酸钠能增强DSS在大鼠在体小肠的吸收,也进一步证实了DSS在小肠是通过细胞旁通路转运的。
最后,DSS的代谢途径研究结果发现:DSS在生物体内发生了异构化,在胆汁中,DSS大多数以异构体形式存在。HPLC-MS中采用MRM模式和MS和MS-MS自动切换扫描(IDA)模式综合分析,在给药后的血浆中鉴定了DSS的原型及二种代谢产物,分别是硫酸化DSS和葡萄糖醛酸化DSS;在静脉给药DSS后的胆汁中找到了DSS的原型及四种代谢产物,分别是硫酸化DSS、葡萄糖醛酸化DSS、甲基DSS和芥子酸。
本论文研究结果揭示了中药远志发挥体内益智作用的活性成分及其体内吸收代谢规律,为确立远志及其制剂的质量控制指标成分提供依据,为从远志中开发有效的益智药物提供了实验依据,为研究复杂的中药体系提供了新思路。
Polygala tenuifolia(Yuan Zhi) is one of the famous herbs to improve learning and memory in traditional Chinese medicine and has often been incorprated into many traditional compound formulas intended for the treatment of amnesia and dementia. However,due to its chemical complexity,the active ingredients of Yuan Zhi responsible for learning and memory improvement are largely unknown and uninvestigated.Therefore,in the present study,interdisciplinary technologies in chemistry,pharmacokinetics and pharmacology will be applied and aim to characterise and elucidate the chemical ingredients and metabolites in rat plasma after Yuan Zhi extract is intragastric administered,and to systemically investigate the effects of the identified compounds on learning and memory improvement in vitro. Our working hypothesis is that effects of oral extracts is elicited only by the ingredients and metabolites present in the plasma,and the identified pharmacologically active compounds may be useful as chemical markers for quality control of Yuan Zhi,together as lead compounds for the development of novel therapeutics.
Firstly,chemical ingredients and metabolites in the rat plasma after the Yuan Zhi extract is orally administered are to be identified using HPLC,LC-MS techniques based on our previous phytochemical data,which have determined the mass fragment profiles of various chemicals in the Yuan Zhi extract.Following identification,these bio-available components will be isolated and purified using various chromatographic techniques,and subjected to in vitro screening assay. Neuronprotective effects of bioavailable components should be evaluated by in vitro cell models using hydrogen peroxide,Aβ_(25-35) and glutamic acid-induced SH-SY5Y cells.3,6'-disinapoylsucrose(DSS) was demonstrated to be the bioavailble active components of Yuanzhi.
The developed LC-MS/MS method has been applied to a pharmacokinetic study for DSS in rats.The results indicated that DSS was absorbed rapidly into the body with peak time averaged 30 min after i.g.administration.However,its plasma concentration levels were very low with peak concentration averaged 16.22 ng/mL even given with the relative high dose of 50 mg/kg.The data from i.v.administration shows that DSS was eliminated rapidly from the body with half-life approximate 27 min.The absolute bioavailability of DSS after i.g.administration was found only 0.5%.After i.v.administration to rats,DSS was mainly excreted from bile 36.69%in 3 hours.
Therefore,its fate in gastric intestinal tract serve as further investigation. Stability of DSS by peptidases was investigated by incubation of the drug with the contents of stomach,small intestine and colon at 37℃.Results from the experiments show the content of DSS decreased 50%at four hours in colon juice.These results indicate that the stability of DSS in GI can be affected by the Microorganism and enzyme in colon.
In the caco-2 cell model,the absorption transport of DSS was fairly poor with Papp values of 1.1×10~(-7) cm/s.Kinetic permeability parameters were provided by bidirectional Caco-2 experiment and pH and concentracion dependency measurements.The transport route and putative transporters involved in DSS were studied using EDTA-2Na and several inhibitors.The bidirectional studies were performed under non-gradient conditions at a concentration of 5～33μM.Opening up the paracellular tight junctions by EDTA-2Na and sodium caprate did increase the apparent permeability coefficients(Papp) of DSS.Transport of DSS was not reduced by others inhibitions.In the study of DSS in situ rat intestinal perfusion model,the Papp(blood) of DSS was 3.97×10~(-5) cm/s for perfusing of DSS,which did significantly differ from that obtained for perfusion of DSS with EDTA-2Na (2.03×10~(-4) cm/s) and sodium caprate(1.46×10~(-4) cm/s).So,transport route of DSS was the passive paracellular.
Finally,LC-MS was used to determine DSS and its metabolites in rat plasma and bile samples.DSS and three and six metabilites were detected and identified in biles collected after i.v.and oral administration by using MRM and IDA model.
引文
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