祖师麻主要活性成分的口服吸收及代谢研究
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摘要
本篇论文研究来源于教育部科学技术研究重点项目“基于酶和转运蛋白的祖师麻口服吸收及代谢研究”(208051)和江苏省普通高校研究生科研创新计划项目“祖师麻主要活性成分的口服吸收及代谢研究”(CX07B_237Z)。
祖师麻为瑞香科植物黄瑞香Daphne giraldii NRsche、陕甘瑞香D.tanguticaMaxim或凹叶瑞香D.retusa Hemsl的茎皮和根皮,为西部地区民间用药。
采用紫外分光光度法(UV)测定了祖师麻中总香豆素的含量,高效液相色谱法(HPLC)测定了祖师麻中祖师麻甲素的含量,并分别进行了方法学考察。祖师麻药材中总香豆素和祖师麻甲素的含量分别为3%和0.2%左右。
采用弗式完全佐剂造成大鼠佐剂性关节炎模型,造模后第7天开始给药,持续21天后取关节滑膜组织做病理检查,观察滑膜细胞增生的情况;同时测定血清中巨噬细胞游走抑制因子(MIF)、肿瘤坏死因子(TNF-α)和白细胞介素1(IL-1)的含量。结果表明,祖师麻甲素能显著抑制膝关节滑液中炎细胞(主要是巨噬细胞)的过度增生,抑制滑膜组织增生和滑膜细胞下疏松结缔组织充血;能较显著降低血清中3种炎症细胞因子的含量。祖师麻总香豆素的作用比祖师麻甲素略强,但无显著性差异,因此可以确定祖师麻甲素是祖师麻治疗类风湿性关节炎的主要活性成分之一。
初步考察了祖师麻甲素的理化性质,如表观溶解度、表观油水分配系数等。结果表明,祖师麻甲素属于生物药剂学分类学中的第二类药物,即溶解性能较差,但渗透性能好,为祖师麻口服吸收机制及代谢研究奠定基础。
研究了祖师麻甲素在大鼠体内的药代动力学行为。祖师麻甲素经大鼠静脉注射(50 mg/kg)和灌胃给药(250 mg/kg)后的药-时曲线均符合单室开放模型;消除半衰期t_(1/2)分别为3.5和17.7 min,祖师麻甲素在大鼠体内能快速分布并消除,口服绝对生物利用度为24.6%。祖师麻甲素灌胃给药(5、25、125mg/kg)后2小时大鼠尿液内可检测到原形药物,48小时内经尿液排泄基本完全,原形药物的累积排泄量不超过给药量的6%。
采用大鼠在体单向肠灌流模型研究了不同剂量组、不同小肠区段(十二指肠、回肠、空肠、结肠)祖师麻甲素的吸收情况。结果表明:10μg/mL祖师麻甲素在大鼠各肠段的表观渗透系数按十二指肠、空肠、回肠、结肠的顺序依次为1.52±0.79、1.34±1.09、1.41±0.50、0.54±0.34;20μg/mL祖师麻甲素表观渗透系数按顺序依次为1.54±0.09、1.48±0.11、1.44±0.36、0.72±0.09;40μg/mL祖师麻甲素表观渗透系数按顺序依次为2.12±0.81、1.99±0.23、1.59±0.90、1.12±1.05。祖师麻甲素在大鼠肠段不同部位不存在吸收差异,其吸收以被动扩散为主。
采用Caco-2细胞单层模型研究了祖师麻甲素的摄取和跨膜转运,结果表明,祖师麻甲素摄取与时间、温度呈正相关,不受媒介pH值的影响。随着药物浓度的增加,祖师麻甲素P_(BA)/P_(AB)的比值呈降低趋势。在加入P-gp抑制剂维拉帕米后,祖师麻甲素P_(BA)/P_(AB)未发生明显变化;加入MRP2抑制剂环孢素后,P_(BA)/P_(AB)降低了10%;加入BCRP抑制剂潘生丁后,P_(BA)/P_(AB)降低了29%。祖师麻甲素跨膜转运的方式以被动扩散为主,其吸收不受P-gp的影响,但可能存在肠道转运蛋白MRP2、BCRP的外排。
祖师麻甲素在大鼠肠道和Caco-2细胞单层模型均发生了Ⅱ相代谢反应。在大鼠肠道灌流液中共检测到4个代谢产物,经色谱及质谱信息分析,推测这些代谢产物为祖师麻甲素-7-硫酸酯、祖师麻甲素-8-硫酸酯、祖师麻甲素-7-葡萄糖醛酸结合物和祖师麻甲素-8-葡萄糖醛酸结合物,祖师麻甲素在大鼠肠道的代谢途径主要是与葡萄糖醛酸、硫酸的结合反应;在Caco-2细胞单层模型接受液中共检测到2个代谢产物,经色谱及质谱信息分析,推测这2个代谢产物为祖师麻甲素-7-葡萄糖醛酸结合物和祖师麻甲素-8-葡萄糖醛酸结合物,祖师麻甲素在Caco-2细胞单层模型主要是与葡萄糖醛酸的结合反应。
研究了祖师麻甲素大鼠尿液、胆汁、血浆中的代谢情况。在大鼠尿液中发现祖师麻甲素的7个代谢产物,经色谱及质谱信息分析,推测这些代谢产物为祖师麻甲素-7-硫酸酯、祖师麻甲素-8-硫酸酯、祖师麻甲素-7-葡萄糖醛酸结合物、祖师麻甲素-8-葡萄糖醛酸结合物、祖师麻甲素-7-甲氧基-8-葡萄糖醛酸结合物、祖师麻甲素-8-甲氧基-7-葡萄糖醛酸结合物、祖师麻甲素甲基化产物、祖师麻甲素羟基化产物。在大鼠胆汁中发现2个代谢产物,经分析分别为祖师麻甲素-7-葡萄糖醛酸结合物、祖师麻甲素-8-葡萄糖醛酸结合物。在大鼠血浆中发现祖师麻甲素的1个代谢产物,经分析为祖师麻甲素甲基化产物。上述代谢产物均为首次发现。
Cortex Daphnes is the stem cortex and root bark of Daphne giraldii Nitsche,D. tangutica Maxim or D.retusa Hemsl,a folk herb used in west of China.
The content of daphnetin and coumarins in raw material of Cortex Daphnes were determined about 0.2%and 3%by HPLC and UV method respectively.The adjuvant-arthritic(AA) rat model was developed to evaluate the anti-arthritic effects of Cortex Daphnes.The results showed that daphnetin significantly reduced paw swelling and erythrocyte sedimentation.The pathological examination demonstrated that articular cartilage degeneration with synovial hyperplasia and inflammatory cells infiltration in AA rats were suppressed by daphnetin.There was significant reduction in production of IL-1, TNF-αand MIF in serum of AA rats treated with daphnetin.In conclusion,daphnetin was the main active constituents in Cortex Daphnes for treating rheumatoid arthritis.
The physical and chemical parameters such as apparent solubility and n-octanol-water partition coefficient of daphnetin were measured.Daphnetin belongs to the classⅡdrug of Biopharmaceutics Classification System,which solubility is poor but membrane permeability is fine.
Daphnetin concentrations were investigated in blood plasma after ig or iv injection in rats by HPLC and pharmacokinetic parameters were calculated.Pharmacokinetic parameters of daphnetin after intravenous(50mg/kg) and oral administration(250mg/kg) in rats were all fit to the one-compartment model.Daphnetin was distributed and eliminated quickly.The absolute bioavailability was about 24.6%after oral administration. Daphnetin was detected in the urine collected within 2h after oral administration(5、25、125mg/kg) and almost completely excreted within 24h.Cumulative urinary excretion of daphnetin was not more than 6 percent of administration dosage.
The mechanism of daphnetin intestinal absorption was first investigated systematically at in vivo and cellular level.The absorption of daphnetin was studied by the rat intestinal perfusion model.The permeability of daphnetin(10μg/mL) in duodenum, jejunum,ileum and colon was 1.52±0.79、1.34±1.09、1.41±0.50、0.54±0.34 respectively; the permeability of daphnetin(20μg/mL) in duodenum,jejunum,ileum and colon was 1.54±0.09、1.48±0.11、1.44±0.36、0.72±0.09 respectively;the permeability of daphnetin (20μg/mL) in duodenum,jejunum,ileum and colon was 2.12±0.81、1.99±0.23、1.59±0.90、1.12±1.05 respectively.The results indicated that the absorption of daphnetin was passive diffusion and no deference in different intestinal segment of rats.
The absorption mechanism of daphnetin was investigated by Caco-2 monolayer model.The uptake of daphnetin was positive correlation to time and temperature,but not affected by pH.The ration of P_(BA)/P_(AB) was reduced along with the increase of daphnetin concentration.The absorption of daphnetin was passive diffusion at the dominating process.Daphnetin may be a substrate of BCRP and MRP2,but not P-gp substrate.
The phaseⅡmetabolic reactions of daphnetin were found in intestine of rats and Caco-2 monolayer model.Four metabolites of daphnetin were detected in the intestine of rat.These metabolites were daphnetin-7-sulfate,daphnetin-8-sulfate,daphnetin-7--glucuronide,daphnetin-7-glucuronide respectively by analyzed LC and MS data.The metabolic pathways of daphnetin in the intestine of rat were mainly glucuronidation and sulfation.Two metabolites of daphnetin detected in Caco-2 monolayer model were daphnetin-7-glucuronide,daphnetin-7-glucuronide respectively by analyzed LC and MS data.The metabolic pathways of daphnetin in the intestine of rat were mainly glucuronidation.
The metabolism of daphnetin in urine,bile and plasma were also investigated.Eight metabolites of daphnetin were detected in urine of rat.These metabolites were daphnetin-7-sulfate,daphnetin-8-sulfate,daphnetin-7-glucuronide,daphnetin-7--glucuronide,daphnetin-7-methoxyl-8-glucuronide,daphnetin-7-methoxyl-8-glucuronide, daphnetin methylate and daphnetin hydroxylate respectively by analyzed LC and MS data. Two metabolites of daphnetin detected in bile of rat were daphnetin-7-glucuronide, daphnetin-7-glucuronide.One metabolite of daphnetin detected in plasma of rat was daphnetin methylate.These metabolites of daphnetin were all found at the first time.
祖师麻为瑞香科植物黄瑞香Daphne giraldii NRsche、陕甘瑞香D.tanguticaMaxim或凹叶瑞香D.retusa Hemsl的茎皮和根皮,为西部地区民间用药。
采用紫外分光光度法(UV)测定了祖师麻中总香豆素的含量,高效液相色谱法(HPLC)测定了祖师麻中祖师麻甲素的含量,并分别进行了方法学考察。祖师麻药材中总香豆素和祖师麻甲素的含量分别为3%和0.2%左右。
采用弗式完全佐剂造成大鼠佐剂性关节炎模型,造模后第7天开始给药,持续21天后取关节滑膜组织做病理检查,观察滑膜细胞增生的情况;同时测定血清中巨噬细胞游走抑制因子(MIF)、肿瘤坏死因子(TNF-α)和白细胞介素1(IL-1)的含量。结果表明,祖师麻甲素能显著抑制膝关节滑液中炎细胞(主要是巨噬细胞)的过度增生,抑制滑膜组织增生和滑膜细胞下疏松结缔组织充血;能较显著降低血清中3种炎症细胞因子的含量。祖师麻总香豆素的作用比祖师麻甲素略强,但无显著性差异,因此可以确定祖师麻甲素是祖师麻治疗类风湿性关节炎的主要活性成分之一。
初步考察了祖师麻甲素的理化性质,如表观溶解度、表观油水分配系数等。结果表明,祖师麻甲素属于生物药剂学分类学中的第二类药物,即溶解性能较差,但渗透性能好,为祖师麻口服吸收机制及代谢研究奠定基础。
研究了祖师麻甲素在大鼠体内的药代动力学行为。祖师麻甲素经大鼠静脉注射(50 mg/kg)和灌胃给药(250 mg/kg)后的药-时曲线均符合单室开放模型;消除半衰期t_(1/2)分别为3.5和17.7 min,祖师麻甲素在大鼠体内能快速分布并消除,口服绝对生物利用度为24.6%。祖师麻甲素灌胃给药(5、25、125mg/kg)后2小时大鼠尿液内可检测到原形药物,48小时内经尿液排泄基本完全,原形药物的累积排泄量不超过给药量的6%。
采用大鼠在体单向肠灌流模型研究了不同剂量组、不同小肠区段(十二指肠、回肠、空肠、结肠)祖师麻甲素的吸收情况。结果表明:10μg/mL祖师麻甲素在大鼠各肠段的表观渗透系数按十二指肠、空肠、回肠、结肠的顺序依次为1.52±0.79、1.34±1.09、1.41±0.50、0.54±0.34;20μg/mL祖师麻甲素表观渗透系数按顺序依次为1.54±0.09、1.48±0.11、1.44±0.36、0.72±0.09;40μg/mL祖师麻甲素表观渗透系数按顺序依次为2.12±0.81、1.99±0.23、1.59±0.90、1.12±1.05。祖师麻甲素在大鼠肠段不同部位不存在吸收差异,其吸收以被动扩散为主。
采用Caco-2细胞单层模型研究了祖师麻甲素的摄取和跨膜转运,结果表明,祖师麻甲素摄取与时间、温度呈正相关,不受媒介pH值的影响。随着药物浓度的增加,祖师麻甲素P_(BA)/P_(AB)的比值呈降低趋势。在加入P-gp抑制剂维拉帕米后,祖师麻甲素P_(BA)/P_(AB)未发生明显变化;加入MRP2抑制剂环孢素后,P_(BA)/P_(AB)降低了10%;加入BCRP抑制剂潘生丁后,P_(BA)/P_(AB)降低了29%。祖师麻甲素跨膜转运的方式以被动扩散为主,其吸收不受P-gp的影响,但可能存在肠道转运蛋白MRP2、BCRP的外排。
祖师麻甲素在大鼠肠道和Caco-2细胞单层模型均发生了Ⅱ相代谢反应。在大鼠肠道灌流液中共检测到4个代谢产物,经色谱及质谱信息分析,推测这些代谢产物为祖师麻甲素-7-硫酸酯、祖师麻甲素-8-硫酸酯、祖师麻甲素-7-葡萄糖醛酸结合物和祖师麻甲素-8-葡萄糖醛酸结合物,祖师麻甲素在大鼠肠道的代谢途径主要是与葡萄糖醛酸、硫酸的结合反应;在Caco-2细胞单层模型接受液中共检测到2个代谢产物,经色谱及质谱信息分析,推测这2个代谢产物为祖师麻甲素-7-葡萄糖醛酸结合物和祖师麻甲素-8-葡萄糖醛酸结合物,祖师麻甲素在Caco-2细胞单层模型主要是与葡萄糖醛酸的结合反应。
研究了祖师麻甲素大鼠尿液、胆汁、血浆中的代谢情况。在大鼠尿液中发现祖师麻甲素的7个代谢产物,经色谱及质谱信息分析,推测这些代谢产物为祖师麻甲素-7-硫酸酯、祖师麻甲素-8-硫酸酯、祖师麻甲素-7-葡萄糖醛酸结合物、祖师麻甲素-8-葡萄糖醛酸结合物、祖师麻甲素-7-甲氧基-8-葡萄糖醛酸结合物、祖师麻甲素-8-甲氧基-7-葡萄糖醛酸结合物、祖师麻甲素甲基化产物、祖师麻甲素羟基化产物。在大鼠胆汁中发现2个代谢产物,经分析分别为祖师麻甲素-7-葡萄糖醛酸结合物、祖师麻甲素-8-葡萄糖醛酸结合物。在大鼠血浆中发现祖师麻甲素的1个代谢产物,经分析为祖师麻甲素甲基化产物。上述代谢产物均为首次发现。
Cortex Daphnes is the stem cortex and root bark of Daphne giraldii Nitsche,D. tangutica Maxim or D.retusa Hemsl,a folk herb used in west of China.
The content of daphnetin and coumarins in raw material of Cortex Daphnes were determined about 0.2%and 3%by HPLC and UV method respectively.The adjuvant-arthritic(AA) rat model was developed to evaluate the anti-arthritic effects of Cortex Daphnes.The results showed that daphnetin significantly reduced paw swelling and erythrocyte sedimentation.The pathological examination demonstrated that articular cartilage degeneration with synovial hyperplasia and inflammatory cells infiltration in AA rats were suppressed by daphnetin.There was significant reduction in production of IL-1, TNF-αand MIF in serum of AA rats treated with daphnetin.In conclusion,daphnetin was the main active constituents in Cortex Daphnes for treating rheumatoid arthritis.
The physical and chemical parameters such as apparent solubility and n-octanol-water partition coefficient of daphnetin were measured.Daphnetin belongs to the classⅡdrug of Biopharmaceutics Classification System,which solubility is poor but membrane permeability is fine.
Daphnetin concentrations were investigated in blood plasma after ig or iv injection in rats by HPLC and pharmacokinetic parameters were calculated.Pharmacokinetic parameters of daphnetin after intravenous(50mg/kg) and oral administration(250mg/kg) in rats were all fit to the one-compartment model.Daphnetin was distributed and eliminated quickly.The absolute bioavailability was about 24.6%after oral administration. Daphnetin was detected in the urine collected within 2h after oral administration(5、25、125mg/kg) and almost completely excreted within 24h.Cumulative urinary excretion of daphnetin was not more than 6 percent of administration dosage.
The mechanism of daphnetin intestinal absorption was first investigated systematically at in vivo and cellular level.The absorption of daphnetin was studied by the rat intestinal perfusion model.The permeability of daphnetin(10μg/mL) in duodenum, jejunum,ileum and colon was 1.52±0.79、1.34±1.09、1.41±0.50、0.54±0.34 respectively; the permeability of daphnetin(20μg/mL) in duodenum,jejunum,ileum and colon was 1.54±0.09、1.48±0.11、1.44±0.36、0.72±0.09 respectively;the permeability of daphnetin (20μg/mL) in duodenum,jejunum,ileum and colon was 2.12±0.81、1.99±0.23、1.59±0.90、1.12±1.05 respectively.The results indicated that the absorption of daphnetin was passive diffusion and no deference in different intestinal segment of rats.
The absorption mechanism of daphnetin was investigated by Caco-2 monolayer model.The uptake of daphnetin was positive correlation to time and temperature,but not affected by pH.The ration of P_(BA)/P_(AB) was reduced along with the increase of daphnetin concentration.The absorption of daphnetin was passive diffusion at the dominating process.Daphnetin may be a substrate of BCRP and MRP2,but not P-gp substrate.
The phaseⅡmetabolic reactions of daphnetin were found in intestine of rats and Caco-2 monolayer model.Four metabolites of daphnetin were detected in the intestine of rat.These metabolites were daphnetin-7-sulfate,daphnetin-8-sulfate,daphnetin-7--glucuronide,daphnetin-7-glucuronide respectively by analyzed LC and MS data.The metabolic pathways of daphnetin in the intestine of rat were mainly glucuronidation and sulfation.Two metabolites of daphnetin detected in Caco-2 monolayer model were daphnetin-7-glucuronide,daphnetin-7-glucuronide respectively by analyzed LC and MS data.The metabolic pathways of daphnetin in the intestine of rat were mainly glucuronidation.
The metabolism of daphnetin in urine,bile and plasma were also investigated.Eight metabolites of daphnetin were detected in urine of rat.These metabolites were daphnetin-7-sulfate,daphnetin-8-sulfate,daphnetin-7-glucuronide,daphnetin-7--glucuronide,daphnetin-7-methoxyl-8-glucuronide,daphnetin-7-methoxyl-8-glucuronide, daphnetin methylate and daphnetin hydroxylate respectively by analyzed LC and MS data. Two metabolites of daphnetin detected in bile of rat were daphnetin-7-glucuronide, daphnetin-7-glucuronide.One metabolite of daphnetin detected in plasma of rat was daphnetin methylate.These metabolites of daphnetin were all found at the first time.
引文
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