七叶皂苷四种异构体的药代动力学及相互转化研究
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摘要
七叶皂苷是从中华七叶树(Aesculus Chinensis Bunge)或天师栗(Aesculus Wilsonii Rehd)的干燥种子中提取得到的一组三萜类皂苷,具有抗水肿、消炎、抗渗出、提高静脉张力和改善微循环的作用,临床上广泛用于慢性静脉炎、痔及手术后水肿等疾病的治疗。药物化学及药理学研究表明,七叶皂苷Ⅰa,Ⅰb及异七叶皂苷Ⅰa、Ⅰb是七叶皂苷的四种主要活性成分,但迄今为止关于这四种单体组分的药代动力学研究尚未见报道。
本研究采用半制备高效液相色谱法从精氨酸七叶皂苷钠中分离制备了七叶皂苷Ia、Ib及异七叶皂苷Ia、Ib四种单体组分,并首次建立了同时测定大鼠生物样品中四种七叶皂苷异构体的LC/MS/MS分析方法。以此为基础,研究了七叶皂苷四种异构体单剂量给药在大鼠体内的药代动力学过程、七叶皂苷Ia多剂量给药的药代动力学过程、多组分同时给药后四种异构体药代动力学的相互影响、单剂量静脉注射给药后七叶皂苷Ia经尿、粪和胆汁的排泄情况；分析测定了四种异构体的血浆蛋白结合率；研究了四种异构体对大鼠肝微粒体CYP450酶的影响及四种异构体之间的体内、外转化机理。相关研究加深了对七叶皂苷的体内复杂药物代谢动力学过程及其分子机制的了解,为该类天然产物的结构改造、给药系统设计探求了新途径；为深入理解该类药物的作用机理、指导临床合理用药、预测药物间相互作用提供了理论依据；同时阐明其在体内发生重排与构型转化机制,对促进药物代谢基础理论的发展具有一定的科学意义。
Pharmacokinetic and isomerization study of four escin isomers
Escin is a natural mixture of saponins extracted from the seed of the horse chestnut tree (Aesculus. Hippocastanum L.) Because of its anti-oedematous, anti-inflammatory and venotonic properties, escin is largely used in the therapy of chronic venous insufficiency, haemorrhoids and post-operative oedema. A number of chemical and pharmacological studies have showed that escin la, escinⅠb (β-escin), isoescin la and isoescin Ib (α-escin) constitute the major bioactive constituents of escin. But up to now, no reference to pharmacokinetic and isomerization study in vivo for the single saponin can be found. In this study, the four isomers were isolated from Sodium Aescinate by semi-preparative HPLC method, and the LC/MS/MS methods were developed and validated for the simultaneous quantification of the four isomers in rat biosamples. Accordingly, the methods had been applied to the pharmacokinetic study of the four isomers in rats, and the isomerization of the four isomers in vitro was also investigated. The results were as followed:
1. The isolation of escin la, escinⅠb, isoescin la and isoescinⅠb
EscinⅠa, escinⅠb, isoescin la and isoescin Ib were isolated from Sodium Aescinate by semi-preparative HPLC method, and characterization by DAD, MS and 1H NMR. The purity of the four isomers was determined by HPLC, and the results showed that the purity of the four isomers was up to 99%, which met the requirements of reference standards.
2. Pharmacokinetic study of escin la, escinⅠb, isoescin la and isoescinⅠb in rats.
A sensitive, selective and rapid method for the simultaneous quantification of escin la, escin Ib, isoescin la and isoescin Ib in rat plasma was fully validated and successful applied for the pharmacokinetic study of the four isomers in rats. The results were as followed:
(1) After iv or ig administration, the dynamic process of the four isomers in rats fitted two-compartment model. The absolute bioavailabilities of the four isomers after ig administration were very low, which were not more than 2%. There was a structure-related pharmacokinetic behavior after either intravenous or oral administration:the anti-isomer (escinⅠa and isoescinⅠa) were more easily removed from the body than the cis-isomers (isoescinⅠa and isoescin Ib) after iv administration, and (3 isomers were more easily be removed than a-escin from body after ig administration.
(2) The dynamic process of escinⅠa in rats fitted two-compartment model after iv administration at the dose of 0.5,1.0 and 2.0 mg/kg. Non-linear pharmacokinetic characteristics in the dose range were found because the pharmacokinetic parameters (AUC0-t/dose and AUC0-∞/dose) were not linearly increased with the increase of dose, while the other pharmacokinetic parameters induing t1/2, CL, MRT and Vd were varied with the increase of the dose.
(3) Compared with single administration, the t1/2 was significantly longer after administration of the combination of four isomers, while the MRT extended and the CL slowed.
3. The plasma protein binding ratio study of escinⅠa, escinⅠb, isoescinⅠa and isoescinⅠb in rats.
The plasma protein binding ratio was tested using equilibrium dialysis method. A relative high plasma binding ratio (>90%) was found for the four isomers after equilibrium, and there was no significant difference among them.
4. The excretion studies of escinⅠa in rats.
The LC/MS/MS methods for the simultaneous determination of escinⅠa and its metabolites isoescinⅠa in bile, urine and feces were developed to study the excretion of escinⅠa in rats. After an iv administration of 1.0 mg/kg escinⅠa, only less than 13%of the dose was excreted in urine and feces in the form of prototype, while less than 10%of the dose was excreted in urine and feces in the form of metabolite isoescinⅠa. Additional, after iv administration, the unchanged cumulative plus the isoescinⅠa amount in bile within 24 h reached to 16.5% of the dose, which was much more than the amount cumulated in feces, suggesting some form of redistribution or enterohepatic recycling of escin.
5. The influence of escinⅠa, escinⅠb, isoescinⅠa and isoescin Ib on CYP450 enzymes in rat liver microsomes.
The influence of escinⅠa, escin Ib, isoescinⅠa and isoescin Ib on the livermicrosome proteins, CYP450 enzymes and the activity of the main CYP450 subfamilies (CYP1A2, CYP2D6, CYP3A4, CYP2C9 and CYP2C19) in rat liver microsomes was investigated after continuously intragastric administration at the dose of 0.5 mg/kg/day (for iv administration) or 4.0 mg/kg/day (for ig administration) in a week. The determination of the main CYP450 subfamilies probe substrates and their respective metabolic products were processed by LC/MS/MS methods. The results showed that CYP1A2 activity had been obvious induced by isoescin la after ig administration and escin la after iv administration; CYP3A4 activity had been obvious inhibited by isoescin la after ig administration, while CYP2C19 had been obvious induced by isoescin la after iv administration.
6. The isomerzation study of escin la, escinⅠb, isoescin la and isoescinⅠb.
The mechanism of the four isomers isomerization was clarified by using rat by gastrointestinal local, ig and iv administration. The results of local administration experiments suggested that the translation ofβ-escins and a-escins happened in both stomach and intestinal tract. It was showed the isomerization was interconvertible after ig or iv administration, and the extent of isomerization fromβ-escins to a-escins was much higher than that from a-escins toβ-escins. Route of administration and dose had no significant effect on the conversion. The isomerizaiton of the four isomers was pH dependent but pepsin independent. The isomerizaiton happened only when pH<2 or pH>3. The higher pH value, the higher isomerzation was happened. The above results proved that the isomerization of the four isomers was mainly a physical and chemical processes which was pH dependent, and the enzyme was not directly involved in the translation of process.
本研究采用半制备高效液相色谱法从精氨酸七叶皂苷钠中分离制备了七叶皂苷Ia、Ib及异七叶皂苷Ia、Ib四种单体组分,并首次建立了同时测定大鼠生物样品中四种七叶皂苷异构体的LC/MS/MS分析方法。以此为基础,研究了七叶皂苷四种异构体单剂量给药在大鼠体内的药代动力学过程、七叶皂苷Ia多剂量给药的药代动力学过程、多组分同时给药后四种异构体药代动力学的相互影响、单剂量静脉注射给药后七叶皂苷Ia经尿、粪和胆汁的排泄情况；分析测定了四种异构体的血浆蛋白结合率；研究了四种异构体对大鼠肝微粒体CYP450酶的影响及四种异构体之间的体内、外转化机理。相关研究加深了对七叶皂苷的体内复杂药物代谢动力学过程及其分子机制的了解,为该类天然产物的结构改造、给药系统设计探求了新途径；为深入理解该类药物的作用机理、指导临床合理用药、预测药物间相互作用提供了理论依据；同时阐明其在体内发生重排与构型转化机制,对促进药物代谢基础理论的发展具有一定的科学意义。
Pharmacokinetic and isomerization study of four escin isomers
Escin is a natural mixture of saponins extracted from the seed of the horse chestnut tree (Aesculus. Hippocastanum L.) Because of its anti-oedematous, anti-inflammatory and venotonic properties, escin is largely used in the therapy of chronic venous insufficiency, haemorrhoids and post-operative oedema. A number of chemical and pharmacological studies have showed that escin la, escinⅠb (β-escin), isoescin la and isoescin Ib (α-escin) constitute the major bioactive constituents of escin. But up to now, no reference to pharmacokinetic and isomerization study in vivo for the single saponin can be found. In this study, the four isomers were isolated from Sodium Aescinate by semi-preparative HPLC method, and the LC/MS/MS methods were developed and validated for the simultaneous quantification of the four isomers in rat biosamples. Accordingly, the methods had been applied to the pharmacokinetic study of the four isomers in rats, and the isomerization of the four isomers in vitro was also investigated. The results were as followed:
1. The isolation of escin la, escinⅠb, isoescin la and isoescinⅠb
EscinⅠa, escinⅠb, isoescin la and isoescin Ib were isolated from Sodium Aescinate by semi-preparative HPLC method, and characterization by DAD, MS and 1H NMR. The purity of the four isomers was determined by HPLC, and the results showed that the purity of the four isomers was up to 99%, which met the requirements of reference standards.
2. Pharmacokinetic study of escin la, escinⅠb, isoescin la and isoescinⅠb in rats.
A sensitive, selective and rapid method for the simultaneous quantification of escin la, escin Ib, isoescin la and isoescin Ib in rat plasma was fully validated and successful applied for the pharmacokinetic study of the four isomers in rats. The results were as followed:
(1) After iv or ig administration, the dynamic process of the four isomers in rats fitted two-compartment model. The absolute bioavailabilities of the four isomers after ig administration were very low, which were not more than 2%. There was a structure-related pharmacokinetic behavior after either intravenous or oral administration:the anti-isomer (escinⅠa and isoescinⅠa) were more easily removed from the body than the cis-isomers (isoescinⅠa and isoescin Ib) after iv administration, and (3 isomers were more easily be removed than a-escin from body after ig administration.
(2) The dynamic process of escinⅠa in rats fitted two-compartment model after iv administration at the dose of 0.5,1.0 and 2.0 mg/kg. Non-linear pharmacokinetic characteristics in the dose range were found because the pharmacokinetic parameters (AUC0-t/dose and AUC0-∞/dose) were not linearly increased with the increase of dose, while the other pharmacokinetic parameters induing t1/2, CL, MRT and Vd were varied with the increase of the dose.
(3) Compared with single administration, the t1/2 was significantly longer after administration of the combination of four isomers, while the MRT extended and the CL slowed.
3. The plasma protein binding ratio study of escinⅠa, escinⅠb, isoescinⅠa and isoescinⅠb in rats.
The plasma protein binding ratio was tested using equilibrium dialysis method. A relative high plasma binding ratio (>90%) was found for the four isomers after equilibrium, and there was no significant difference among them.
4. The excretion studies of escinⅠa in rats.
The LC/MS/MS methods for the simultaneous determination of escinⅠa and its metabolites isoescinⅠa in bile, urine and feces were developed to study the excretion of escinⅠa in rats. After an iv administration of 1.0 mg/kg escinⅠa, only less than 13%of the dose was excreted in urine and feces in the form of prototype, while less than 10%of the dose was excreted in urine and feces in the form of metabolite isoescinⅠa. Additional, after iv administration, the unchanged cumulative plus the isoescinⅠa amount in bile within 24 h reached to 16.5% of the dose, which was much more than the amount cumulated in feces, suggesting some form of redistribution or enterohepatic recycling of escin.
5. The influence of escinⅠa, escinⅠb, isoescinⅠa and isoescin Ib on CYP450 enzymes in rat liver microsomes.
The influence of escinⅠa, escin Ib, isoescinⅠa and isoescin Ib on the livermicrosome proteins, CYP450 enzymes and the activity of the main CYP450 subfamilies (CYP1A2, CYP2D6, CYP3A4, CYP2C9 and CYP2C19) in rat liver microsomes was investigated after continuously intragastric administration at the dose of 0.5 mg/kg/day (for iv administration) or 4.0 mg/kg/day (for ig administration) in a week. The determination of the main CYP450 subfamilies probe substrates and their respective metabolic products were processed by LC/MS/MS methods. The results showed that CYP1A2 activity had been obvious induced by isoescin la after ig administration and escin la after iv administration; CYP3A4 activity had been obvious inhibited by isoescin la after ig administration, while CYP2C19 had been obvious induced by isoescin la after iv administration.
6. The isomerzation study of escin la, escinⅠb, isoescin la and isoescinⅠb.
The mechanism of the four isomers isomerization was clarified by using rat by gastrointestinal local, ig and iv administration. The results of local administration experiments suggested that the translation ofβ-escins and a-escins happened in both stomach and intestinal tract. It was showed the isomerization was interconvertible after ig or iv administration, and the extent of isomerization fromβ-escins to a-escins was much higher than that from a-escins toβ-escins. Route of administration and dose had no significant effect on the conversion. The isomerizaiton of the four isomers was pH dependent but pepsin independent. The isomerizaiton happened only when pH<2 or pH>3. The higher pH value, the higher isomerzation was happened. The above results proved that the isomerization of the four isomers was mainly a physical and chemical processes which was pH dependent, and the enzyme was not directly involved in the translation of process.
引文
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