肝毒吡咯里西啶生物碱Adonifoline和Senecionine药物动力学研究
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摘要
目的:研究千里光总生物碱的富集工艺及Adonifoline的分离纯化工艺,建立生物样本中吡咯里西啶生物碱Adonifoline和Senecionine及其代谢产物的定量分析方法,并对Adonifoline和Senecionine及其代谢产物在大鼠体内的药物动力学进行研究,为吡咯里西啶生物碱的致毒和解毒机制研究提供依据。方法:采用正交实验设计法优化千里光总生物碱的富集工艺并用柱层析色谱法分离制备Adonifoline单体。在Adonifoline和Senecionine大鼠体内外代谢产物和代谢途径研究的基础上,结合固相萃取技术(SPE)处理血清样品,以Monocrotaline为内标,建立超高效液相色谱-质谱法(UPLC-MS)同时测定大鼠血清中Adonifoline和Senecionine及其代谢产物的含量,以获得各种目标分析物的血药浓度-时间曲线,利用PK Solutions 2药动软件计算Senecionine和Senecionine的氮氧化物(Senecionine-NO)、Adonifoline和Adonifoline的氮氧化物(Adonifoline-NO)药动学参数。利用SPSS 13.0统计软件对药动参数进行统计分析。结果:通过正交实验优化得到千里光总生物碱富集工艺为千里光乙醇提取物用3倍量的2%盐酸水溶液捏溶、氨水碱化pH值到10以上后用氯仿萃取5次,得到总生物碱,比色法测定总生物碱含量比提取物中生物碱含量提高417倍。总生物碱用硅胶拌样后进行硅胶柱层析,采用氯仿-甲醇系统分离纯化得到Adonifoline(700 mg),纯度达到98%以上。大鼠血清样品经SPE处理后,进行UPLC-MS法定量分析。色谱条件:流动相为乙腈(A)-0.1%甲酸水溶液(B)梯度洗脱;色谱柱:Waters ACQUITY UPLC BEH C18,2.1×50 mm,1.7μm;色谱柱温:45℃;进样量:2μL。质谱条件为:Capillary:4.2 kv,Cone:45 v,Extractor:2 v,RF Lens:0.1 v,Source Temprature:120℃,Desolution Temprature:300℃,Gas Flow: Desolution gas:600 L/hr,Cone gas:60 L/hr,LM Resolution:15,HResolution:15,Ion Energy:0.5,ESI+ Mode Scan。采用SIR模式监测Senecionine(m/z 336)、Senecionine-NO(m/z 352)、Adonifoline(m/z 366)、Adonifoline-NO(m/z 382) IS, , Monocrotaline (m/z 326)。Senecionine、Senecionine-NO、Adonifoline、Adonifoline-NO分别在0.556-8896 ng/mL,5.84-9344 ng/mL、0.5828-9324.8 ng/mL和24.8-9920 ng/mL范围内线性关系良好。最低定量限(LLOQ)分别为5.56 ng/mL、11.68 ng/mL、5.83 ng/mL和12.40 ng/mL,最低检测限(LOD)分别为1.390 ng/mL、2.920 ng/mL、1.470 ng/mL和3.10 ng/mL。平均回收率分别为109.8±4.52%,93.6±6.81%,92.1±2.69%,112.8±7.12%。分析方法符合生物样品分析的要求。大鼠静脉注射和口服Adonifoline和Senecionine之后,均可在血清中检测到原型药物和主要代谢产物Adonifoline-NO和Senecionine-NO。大鼠静脉注射给药后,Adonifoline和Adonifoline-NO的消除速度常数K分别为0.0033±0.0012 1/min和0.0038±0.0017 1/min;消除半衰期t1/2分别为241.2±90.0 min和181.9±60.4 min;血药浓度-时间曲线下面积AUC(0-t)分别为320.0323±77.2539μg·min/mL和173.0951±71.316μg·min/mL;体内平均注留时间MRT分别为128.4±46.5 min和177.6±64.2 min,表观分布容积Vd分别为4180.0916±1171.9706 mL/kg和6417.0678±2485.0695 mL/kg,清除率CL分别为12.7405±3.4207 mL/min/kg和24.9197±8.7771 mL/min/kg。大鼠口服Adonifoline低、中、高剂量后Adonifoline和Adonifoline-NO的AUC随剂量的提高而增大,具有剂量依赖性特征,而其消除半衰期等参数与剂量无关。大鼠静脉注射Senecionine后,Senecionine和Senecionine-NO的t1/2分别为306.33±177.28 min和163.99±105.18 min;AUC(0-t)分别为69.23±24.64μg?min/mL和302.39±82.55μg?min/mL;MRT分别为128.33±99.61 min和168..29±41.24 min;Vd分别为10864.55±8736.35 mL/kg和1250.52±850.13 mL/kg,CL为23.23±8.39 mL/min/kg和5.30±2.07 mL/min/kg。大鼠口服低、中、高剂量的Senecionine后,Senecionine和Senecionine-NO的半衰期等参数与剂量无关。结论:本研究建立的UPLC-MS法具有准确、分析速度快、灵敏度高、生物样品用量小等优点,适用于吡咯里西啶生物碱及其代谢产物的药物动力学研究。药物动力学研究结果表明Adonifoline和Senecionine代谢成氮氧化物后体内药动学过程发生了改变,为其毒性机制研究提供了依据。
Objective: To optimize enrichment technology of total alkaloids of Senecio scandens and purification of Adonifoline. To establish a method for the determination of the concentration of their metabolites and mother alkaloids in rat serum respectively and to study the pharmacokinetics of Senecionine and Adonifoline in order to provide evidence for explaining mechanism of their pharmacodynamic and toxicity. Methods: Orthogonal design was employed to optimize enrichment technology of total alkaloids and chromatography was used to isolate and purify Adonifoline. Serum samples were pre-treated by C18 solid phase extraction (SPE). An UPLC/ESIMS method was firstly developed and validated for the simultaneously quantitation of Pyrrolizidine Alkaloids (PAs) in rat serum. The pharmacokinetic parameters of Senecionine、Senecionine-NO, Adonifoline and Adonifoline-NO were researched by PK Solutions 2.0 Pharmacokinetic Software. Results: The factors of influencing enrichment of total alkaloids of Senecionine were volume of acid solution, pH value of alkalization and frequency of extraction. The content of total alkaloids was enhanced 417 fold by using optimization method compared with the traditional one. 700mg of Adonifoline with the purity of more than 98% were gained by purification in chloroform-methanol system after mixing total alkaloids samples with silica gel column chromatography. The condition of UPLC method was: mobile phase : gradient elution of acetonit-0.1% of formic acid, chromatographic column: Waters ACQUITY UPLC BEH C18,2.1×50 mm,1.7μm, column temperature: 45℃, sample size: 2μL. Mass-spectrum condition was as follows: Capillary:4.2 kv,Cone:45 v,Extractor:2 v,RF Lens:0.1 v,Source Temprature:120℃,Desolution Temprature:300℃,Gas Flow: Desolution gas:600 L/hr,Cone gas:60 L/hr,LM Resolution:15,HM Resolution:15,Ion Energy:0.5,ESI+ Mode Scan. Quantitation was performed using SIR of the m/z 336 for Senecionine, m/z 352 for Senecionine-NO, m/z 366 for Adonifoline, m/z 382 for Adonifoline-NO, and m/z 326 for IS, respectively. The linear range of Senecionine, Senecionine-NO, Adonifoline and Adonifoline-NO were 0.556-8896 ng/mL,5.84-9344 ng/mL,0.5828-9324.8 ng/mL and 24.8-9920 ng/mL respectively . The intra day and inter day precision were all less than 15 %. The extraction recoveries at high, medium and low concentration were between. 92.1±2.69% to 112.8±7.12%. The limit of detection (LOD) was 0.228 ng/mL for senecionine, 2.92 ng/mL for senecionine-NO, 0.291 ng/mL for adonoforline, and 3.100 ng/mL for adonifoline-NO. The lower limit of quantification (LLOQ) was 5.56 ng/mL for senecionine and 11.68 ng/mL for Senecionine-NO, 5.83 ng/mL for adonifoline and 12.40 ng/mL for adonifoline-NO. The pharmacokinetic parameters of adonifoline and adonifoline-NO after intravenous injection in rats were as follows respectively: K=0.0033±0.0012 1/min and 0.0038±0.0017 1/min;t1/2=241.2±90.0 min and 181.9±60.4 min , AUC(0-t)=320.0323±77.2539μg·min/mL and 173.0951±71.316μg·min/mL , MRT=128.4±46.5 min and 177.6±64.2 min , Vd=4180.0916±1171.9706 mL/kg and 6417.0678±2485.0695 mL/kg, CL=12.7405±3.4207 mL/min/kg and 24.9197±8.7771 mL/min/kg. Adonifoline and Adonifoline-NO showed linear pharmacokinetics after oral administration at low, medium and high dose because their AUC were increased proportionally with the dose respectively. But Senecionine didn’t show clearly linear pharmacokinetics after administration by the same way. The pharmacokinetic parameters of Senecionine and Senecion-NO after intravenous injection in rats were as follows respectively: t1/2=306.33±177.28 min and 163.99±105.18 min, AUC(0-t) =69.23±24.64μg?min/mL and ,02.39±82.55μg?min/mL;MRT=128.33±99.61 min and 168..29±41.24 min;Vd =10864.55±8736.35 mL/kg and 1250.52±850.13 mL/kg , CL=23.23±8.39 mL/min/kg and 5.30±2.07 mL/min/kg. Conclusion: UPLC-MS method is selective, sensitive and convenient, proved to be applicable to determine the concentration of Senecionine and Adonifoline in rat serum and for their pharmacokinetics study. It was suggested that the pharmacokinetics was difference between mother drug and their metabolites in rats. These results above clarified the pharmacokinetics process of PAs in vivo, and meanwhile, lay the foundation for the future research.
Objective: To optimize enrichment technology of total alkaloids of Senecio scandens and purification of Adonifoline. To establish a method for the determination of the concentration of their metabolites and mother alkaloids in rat serum respectively and to study the pharmacokinetics of Senecionine and Adonifoline in order to provide evidence for explaining mechanism of their pharmacodynamic and toxicity. Methods: Orthogonal design was employed to optimize enrichment technology of total alkaloids and chromatography was used to isolate and purify Adonifoline. Serum samples were pre-treated by C18 solid phase extraction (SPE). An UPLC/ESIMS method was firstly developed and validated for the simultaneously quantitation of Pyrrolizidine Alkaloids (PAs) in rat serum. The pharmacokinetic parameters of Senecionine、Senecionine-NO, Adonifoline and Adonifoline-NO were researched by PK Solutions 2.0 Pharmacokinetic Software. Results: The factors of influencing enrichment of total alkaloids of Senecionine were volume of acid solution, pH value of alkalization and frequency of extraction. The content of total alkaloids was enhanced 417 fold by using optimization method compared with the traditional one. 700mg of Adonifoline with the purity of more than 98% were gained by purification in chloroform-methanol system after mixing total alkaloids samples with silica gel column chromatography. The condition of UPLC method was: mobile phase : gradient elution of acetonit-0.1% of formic acid, chromatographic column: Waters ACQUITY UPLC BEH C18,2.1×50 mm,1.7μm, column temperature: 45℃, sample size: 2μL. Mass-spectrum condition was as follows: Capillary:4.2 kv,Cone:45 v,Extractor:2 v,RF Lens:0.1 v,Source Temprature:120℃,Desolution Temprature:300℃,Gas Flow: Desolution gas:600 L/hr,Cone gas:60 L/hr,LM Resolution:15,HM Resolution:15,Ion Energy:0.5,ESI+ Mode Scan. Quantitation was performed using SIR of the m/z 336 for Senecionine, m/z 352 for Senecionine-NO, m/z 366 for Adonifoline, m/z 382 for Adonifoline-NO, and m/z 326 for IS, respectively. The linear range of Senecionine, Senecionine-NO, Adonifoline and Adonifoline-NO were 0.556-8896 ng/mL,5.84-9344 ng/mL,0.5828-9324.8 ng/mL and 24.8-9920 ng/mL respectively . The intra day and inter day precision were all less than 15 %. The extraction recoveries at high, medium and low concentration were between. 92.1±2.69% to 112.8±7.12%. The limit of detection (LOD) was 0.228 ng/mL for senecionine, 2.92 ng/mL for senecionine-NO, 0.291 ng/mL for adonoforline, and 3.100 ng/mL for adonifoline-NO. The lower limit of quantification (LLOQ) was 5.56 ng/mL for senecionine and 11.68 ng/mL for Senecionine-NO, 5.83 ng/mL for adonifoline and 12.40 ng/mL for adonifoline-NO. The pharmacokinetic parameters of adonifoline and adonifoline-NO after intravenous injection in rats were as follows respectively: K=0.0033±0.0012 1/min and 0.0038±0.0017 1/min;t1/2=241.2±90.0 min and 181.9±60.4 min , AUC(0-t)=320.0323±77.2539μg·min/mL and 173.0951±71.316μg·min/mL , MRT=128.4±46.5 min and 177.6±64.2 min , Vd=4180.0916±1171.9706 mL/kg and 6417.0678±2485.0695 mL/kg, CL=12.7405±3.4207 mL/min/kg and 24.9197±8.7771 mL/min/kg. Adonifoline and Adonifoline-NO showed linear pharmacokinetics after oral administration at low, medium and high dose because their AUC were increased proportionally with the dose respectively. But Senecionine didn’t show clearly linear pharmacokinetics after administration by the same way. The pharmacokinetic parameters of Senecionine and Senecion-NO after intravenous injection in rats were as follows respectively: t1/2=306.33±177.28 min and 163.99±105.18 min, AUC(0-t) =69.23±24.64μg?min/mL and ,02.39±82.55μg?min/mL;MRT=128.33±99.61 min and 168..29±41.24 min;Vd =10864.55±8736.35 mL/kg and 1250.52±850.13 mL/kg , CL=23.23±8.39 mL/min/kg and 5.30±2.07 mL/min/kg. Conclusion: UPLC-MS method is selective, sensitive and convenient, proved to be applicable to determine the concentration of Senecionine and Adonifoline in rat serum and for their pharmacokinetics study. It was suggested that the pharmacokinetics was difference between mother drug and their metabolites in rats. These results above clarified the pharmacokinetics process of PAs in vivo, and meanwhile, lay the foundation for the future research.
引文
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[29]徐晓彬,林红英,冯羽裳,等.千里光植物化学预试及抗菌有效部位化学成分检查[J].中兽医医药杂志, 2006; (3): 10-13.
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[31]杨秀东,冯乾坤,李婷婷,等.麻叶千里光化学成分及药理作用研究[J].长春中医药大学学报, 2006; 22(3):70-71.
[32] Pandey VB, Singh JP, Rao YV, et al. Isolation and pharmacological action of heliotrine, the major alkaloid of Helotropium indicum seeds [J]. Planta Med., 1982; 45(8): 229-233.
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[37] Jun Tang, Teruaki Akao, Norio Nakamura, et al. In vitro metabolism of isoline, a pyrrolizidine alkaloid from Ligularia duciformis, by rodent liver microsomal esteraseand enhanced hepatotoxicity by esterase inhibitors [J]. Drug. Metab. Dispos., 2007;35: 1832-1839.
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[41] Williams L, Chou MW, Yan J, et al. Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkaloid, and metabolites in rats and mice [J]. Toxicology and Applied Pharmacology, 2002;182:98–104.
[42] Brauchli J, Ltithy J, Zweifel U, and Schlatter C. Pyrrolizidine alkaloids from Symphytum officinale L. and their percutaneous absorption in rats [J]. Experientia, 1982; 38: 1085-1087.
[43] Aston N, Morris P,and Tanner S. Retrorsine in breast milk influences copper handling in suckling rat pups [J]. Journal of Hepatology, 1996; 25: 748-755.
[44] Reid MJ, Lame MW, Morin D, et al. Monocrotaline metabolism and distribution in Fisher 344 and Sprague-Dawley rats [J]. Comp. Biochem. Physiol., 1997; 117B(1):115-123.
[45] Estep JE, Lame MW, and Segall HJ. Excretion and blood radioactivity levels following [14C]senecionine administration in the rat [J]. Toxicology, 1990; 64: 179-189.
[46] Swick RA, Cheeke PR, Patton NM, et al. Absorption and excretion of pyrrolizidine (Senecio) alkaloids and their effects on mineral metabolism in rabbits [J]. Anim. Sci., 1982; 55: 1417-1424.