Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China; The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicine, 1200 Cailun Rood, Shanghai 201210, China.
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China; The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicine, 1200 Cailun Rood, Shanghai 201210, China; Key Laboratory of Liver and Kidney Diseases (Ministry of Education), Institute of Liver Diseases, Shuguang Hospital Affiliated with Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai 201203, China.
J Ethnopharmacol. 2019 May 23;236:288-301. doi: 10.1016/j.jep.2019.03.020. Epub 2019 Mar 11.
Aerial parts of Peganum harmala Linn are a Uighur traditional medicinal herb in China used to treat amnesia, bronchial asthma, and cough. Deoxyvasicine (DVAS), a potent cholinesterase inhibitor exhibiting anti-senile dementia activity, is one of the chief active ingredients in aerial parts of P. harmala and plays a key role in mediating the pharmacological effects of P. harmala. However, the metabolic profiling and in vivo pharmacokinetic characteristics of DVAS still remain unknown.
The aim of this present study was to investigate the metabolism and pharmacokinetic properties of DVAS in rats by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method.
The metabolic profiling of DVAS was evaluated in vitro and in vivo by rat liver microsomes (RLMs) incubation and by rat bio-specimens, such as urine, feces, plasma, and bile, after the oral administration of 45 mg/kg DVAS. An efficient and sensitive UPLC-ESI-MS/MS method was developed and validated to simultaneously determine DVAS and its major four metabolites, namely, vasicine, deoxyvasicinone, vasicinone, and 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-β-D-glucuronide in rat plasma. For pharmacokinetic studies, 32 Sprague-Dawley rats were randomly divided into four groups, namely, intravenous dosage group (2 mg/kg DVAS) and three oral dosage groups (5, 15, and 45 mg/kg DVAS). In addition, the activity of the components in plasma after intravenous administration of DVAS was evaluated by in vitro anti-butyrylcholinesterase (BChE) assays.
A total of 23 metabolites were found in RLMs, plasma, urine, feces, and bile by UPLC-ESI-QTOF-MS. The metabolic pathway of DVAS in vivo and in vitro mainly involved hydroxylation, dehydrogenation, acetylation, methylation, glucuronidation, and O-sulphate conjugation, and the C-3 and C-9 sites were the main metabolic soft spots. All 23 metabolites were detected in the urine sample, and 13, 8, 22, and 6 metabolites were identified from rat feces, plasma, bile, and RLMs, respectively. The standard curves of DVAS and four metabolites in rat plasma showed good linearity in the concentration range of 0.82-524.00 ng/mL with acceptable selectivity, precision, accuracy, recovery, and stability. DVAS exhibited linear dose-proportional pharmacokinetics at doses of 5, 15, and 45 mg/kg after oral administration, and the average oral absolute bioavailability of DVAS was 47.46%. The in vitro anti-BChE assays implied that the inhibitive activities were mainly due to the different concentrations of prototype DVAS.
DVAS can be rapidly absorbed and excreted by blood, and it is also extensively metabolized in vivo, and the anti-BChE activity in blood is mainly attributed to DVAS. These findings can lay a foundation for new drug development for DVAS.
骆驼蓬（Peganum harmala Linn）的地上部分是中国维吾尔族传统药用植物，用于治疗健忘症、支气管哮喘和咳嗽。脱氧育亨宾（DVAS）是一种具有抗老年痴呆活性的强效胆碱酯酶抑制剂，是骆驼蓬地上部分的主要活性成分之一，在介导骆驼蓬的药理作用方面发挥着关键作用。然而，脱氧育亨宾的代谢特征和体内药代动力学特征仍然未知。
本研究旨在采用超高效液相色谱-电喷雾串联四极杆飞行时间质谱（UPLC-ESI-QTOF-MS）和超高效液相色谱-串联质谱（UPLC-ESI-MS/MS）方法，通过大鼠肝微粒体（RLMs）孵育和大鼠生物样本（如尿液、粪便、血浆和胆汁），研究 DVAS 的代谢和药代动力学特性。
通过大鼠肝微粒体孵育和大鼠生物样本（如尿液、粪便、血浆和胆汁），评价 DVAS 的体外和体内代谢特征。在口服给予 45mg/kg DVAS 后，采用高效灵敏的 UPLC-ESI-MS/MS 方法同时测定大鼠血浆中的 DVAS 及其主要的 4 种代谢物，即育亨宾、脱氧育亨宾酮、育亨宾酮和 1,2,3,9-四氢吡咯[2,1-b]喹唑啉-3-β-D-葡萄糖醛酸苷。为了进行药代动力学研究，将 32 只 Sprague-Dawley 大鼠随机分为 4 组，即静脉注射组（2mg/kg DVAS）和 3 个口服剂量组（5、15 和 45mg/kg DVAS）。此外，通过体外抗丁酰胆碱酯酶（BChE）测定评估静脉注射 DVAS 后血浆中各成分的活性。
通过 UPLC-ESI-QTOF-MS 在 RLMs、血浆、尿液、粪便和胆汁中发现了 23 种代谢物。DVAS 体内和体外的代谢途径主要涉及羟化、脱氢、乙酰化、甲基化、葡萄糖醛酸化和 O-硫酸酯化结合，C-3 和 C-9 位点是主要的代谢弱点。所有 23 种代谢物均在尿液样本中检测到，在大鼠粪便、血浆、胆汁和 RLMs 中分别鉴定出 13、8、22 和 6 种代谢物。大鼠血浆中 DVAS 和 4 种代谢物的标准曲线在 0.82-524.00ng/mL 的浓度范围内具有良好的线性关系，具有可接受的选择性、精密度、准确度、回收率和稳定性。口服给予 5、15 和 45mg/kg DVAS 后，DVAS 表现出线性剂量比例药代动力学特征，DVAS 的口服绝对生物利用度平均为 47.46%。体外抗 BChE 测定表明，抑制活性主要归因于不同浓度的原型 DVAS。
DVAS 可以被血液迅速吸收和排泄，并在体内广泛代谢，血液中的抗 BChE 活性主要归因于 DVAS。这些发现为 DVAS 的新药开发奠定了基础。
