Studies On The Mechanism Of Improving Learning And Memory And Pharmacokinetics Of Deoxyvasicine

1.Background and objectives:Peganum harmala Linn is is a well-known perennial herbaceous plant and it has been long used to treat epilepsy,neuralgia,hypertension,asthma,subcutaneous tumors,pruritus and several chronic pain in China,Middle East and South Asia.In our previous studies,two kinds of alkaloids（mainlyβ-carboline alkaloids and quinazoline alkaloids）were found in the extracts of Peganum harmala.Because of its strong reversible inhibitory effects on monoamine oxidase（MAO）,acetylcholinesterase（ACh E）and butyrylcholinesterase（BCh E）,β-carboline alkaloids can significantly improve learning and memory,and have good potential in the treatment of mental disorders and neurodegenerative diseases.However,β-carboline alkaloids have certain toxicity to the central nervous system,and cause side effects such as tremor and hallucination.Deoxyvasicine（DVAS）is an active quinazoline alkaloid in the plant of Peganum harmala,along with various pharmacological activities,such as antimicrobial,antitussive,antiasthmatic and insecticidal effects.It was also found that DVAS is a strong cholinesterase inhibitor,and its in vitro cholinesterase inhibitory activity is stronger than that of harmaline,harmine and vasicine（VAS）,which is a potential drug to treat Alzheimer’s disease.However,there is no report on the pharmacodynamics to treat Alzheimer’s disease,in vivo and in vitro metabolism,and pharmacokinetics of DVAS.Therefore,the pharmacology,pharmacodynamics and pharmacokinetics of DVAS were studied by using the principles and techniques of pharmacology,pharmacokinetics and analytical chemistry of traditional Chinese medicine.It can help to elucidate the mechanism of DVAS in improving learning and memory and its in vivo process,which laid a foundation for its new drug research and development.2.methods:（1）Study on the mechanism of DVAS in improving learning and memory:AD model of acquired memory impairment was established by intraperitoneal injection of scopolamine into C57BL/6 mice.Morris water maze was applied to investigate the effect of DVAS on behavior of memory impaired mice,and to evaluate the effect of DVAS on improving learning and memory.Animals were divided into six groups:control group,model group,positive control group（Huperzine A,0.2 mg/kg）,DVAS low dose group（5mg/kg）,DVAS medium dose group（15 mg/kg）,DVAS high dose group（45 mg/kg）.After7 days of continuous administration of the corresponding drugs,except for the control group（saline）,the remaining 5 groups were fixed daily 30 minutes after intragastric administration from the 8th day,then injected 1 mg/kg scopolamine intraperitoneally for7 days.On the 15th day,the spatial probe trial was carried out.The next day,after 30minutes of intraperitoneal injection of scopolamine,the mice were treated and the hippocampus and cortex tissues were taken.After that,enzyme-linked immunosorbent assay,histopathological section,protein imprinting and other pharmacological techniques were used to preliminarily study the activity of related enzymes,inflammatory factors,oxidative stress factors and protein expression.In addition,the content of neurotransmitters of C57BL/6 mice was quantified by previous method in our research group,and the effect of DVAS on important neurotransmitters in the brain was preliminarily discussed.（2）In vivo and in vitro metabolism study of DVAS:DVAS was incubated in phase I and II co-incubation system of rat liver microsomes in vitro;plasma,bile,urine and feces samples were acquired after oral administration of 40 mg/kg DVAS.The metabolites were identified by UPLC-QTOF-MS/MS,and the metabolic profiles and metabolic characteristics of DVAS in vivo and in vitro were investigated.（3）Pharmacokinetic study of DVAS and its metabolites in rats:A UPLC-MS/MS method for determination of DVAS and its metabolites in rat plasma was established and full-validated.Pharmacokinetics of DVAS and its metabolites in rat plasma after intravenous and oral administration of DVAS were studied to obtain the oral bioavailability.The correlation between cholinesterase inhibitive activity with plasma drug concentrations were acquired to analyze the main inhibitory active component.3.Results:（1）Study on the mechanism of DVAS in improving learning and memory:Scopolamine was used to induce learning and memory impairment in mice,and morris water maze experiment showed DVAS can significantly improve scopolamine-induced memory acquired impairment.The results showed that after 7 days of intraperitoneal injection of scopolamine（1 mg/kg）,the swimming distance and onstage latency of mice in the model group increased significantly compared with those in the control group on the 4th to 5th days of orientation navigation tests（P<0.01）.When scopolamine was injected intraperitoneally,the swimming distance and latency to platform of mice in DVAS low,medium and high dose groups were gradually decreased in hidden platform tests,and decreased significantly on the 4th to 5th days of orientation navigation tests compared with the model group（P<0.05,P<0.01 or P<0.01）.In spatial probe trials,the number of mice in the model group was significantly lower than that in the control group（P<0.01）.Compared with the model group,the number of perforations in DVAS low,medium and high dose groups were increased significantly（P<0.05,P<0.01 or P<0.001）.In conclusion,DVAS can significantly improve the spatial memory ability of scopolamine-induced memory impairment model mice in a dose-dependent manner.Compared with the control group,scopolamine significantly increased ACh E activity in the cortex and hippocampus of mice（P<0.01 or P<0.001）,while Ch AT and BDNF activity were decreased significantly（P<0.01）.The activity of ACh E in cortex and hippocampus were decreased significantly after DVAS treatment（P<0.05,P<0.01or P<0.001）,and the activity of Ch AT and BDNF in hippocampus were increased significantly（P<0.05,P<0.01 or P<0.001）,especially in DVAS high dose group.The activity of Ch AT and BDNF in cortex were increased significantly in DVAS high dose group（P<0.05）.Secondly,compared with the control group,the activity of TNF-αand the production of NO in the cortex of the model group mice were significantly increased（P<0.01）.The content of TNF-αin the cortex of the mice was significantly decreased（P<0.05,P<0.01 or P<0.001）after oral administration of 5,15 and 45 mg/kg DVAS,while the contents of NO in the cortex of DVAS low and medium dose group were significantly decreased（P<0.01 or P<0.05）.In addition,compared with the control group,the activities of SOD and GSH-px in the cortex of the model group mice were significantly decreased（P<0.01）.The activity of GSH-px in the cerebral cortex of mice was increased significantly after DVAS treatment（P<0.05,P<0.01 or P<0.001）,but the content of SOD in the cortex of mice treated with DVAS had no statistical difference compared with the model group.Compared with the control group,the contents of ACh,5-HT andγ-GABA in the cortex of mice in the model group were significantly decreased（P<0.05 or P<0.01）,while the contents of 5-HIAA and Glu were significantly increased（P<0.05 or P<0.01）,but DVAS had no significant effect on the contents of Ch,L-Asp and L-Trp in cortex（P>0.05）.The levels ofγ-GABA in the cortex of mice were increased significantly after DVAS treatment（P<0.05,P<0.01 or P<0.001）.In addition,the levels of ACh and 5-HT in other dosage groups were increased significantly（P<0.05,P<0.01 or P<0.001）,and the levels of 5-HIAA in the cortex of mice in all dosage groups of DVAS were decreased significantly（P<0.05,P<0.01 or P<0.001）.Except for low dose of DVAS,Glu levels in cortex of all groups were significantly decreased（P<0.01or P<0.001）,and the above neurotransmitters regulation fucntion showed a dose-dependent manner.（2）In vivo and in vitro metabolism study of DVAS:In the study of metabolism of DVAS in vivo and in vitro,23 metabolites were identified,23 and 22 metabolites were found in urine and bile,6 metabolites in rat liver microsomes,13 metabolites in feces,8metabolites in plasma and 13 metabolites in feces.The results showed that most metabolites of DVAS were excreted mainly through urine and bile.In addition,hydroxylation and carbonylation were the main phase I metabolic pathways,acetylation,methylation,glucuronidation and sulfation were the main phase II metabolic pathways.（3）Pharmacokinetic study of DVAS and its metabolites in rats:Based on the identification of metabolites,an efficient and sensitive UPLC-ESI-MS/MS method was developed and validated to simultaneously determine DVAS and its four metabolites in rat plasma and relatively determine other four metabolites,and the pharmacokinetics of rats after oral and intravenous administration of DVAS were studied.The oral bioavailability of DVAS was about 47.46%.The AUC ratios of DVAO and prototype were31.91%and 30.66%respectively at 5 and 15 mg/kg DVAS,15.8%and 16.40%respectively at 5 and 15 mg/kg DVAS,but 22.17%and 3.23%at 45 mg/kg DVAS,which were significantly lower than those at other doses,In addition,when the dose was 45mg/kg or higher,the metabolic enzymes of DVAS in rats would be saturated or there exsit other further metabolic processes.In vitro experiments showed that plasma anticholinesterase activity was mainly related to DVAS,but less related to other metabolites.4.Conclusion:DVAS can improve learning and memory mainly by enhancing choline system（inhibiting ACh E and increasing Ch AT enzyme activity and protein expression）,anti-inflammatory effect（inhibiting TNF-αactivity,regulating NO content）,anti-oxidative stress（increasing GSH-px activity）,neuroprotective effect（increasing BDNF and decreasing Glu content）,neurotransmitter regulation,and has a dose-dependent effect.There are many metabolites in DVAS in vivo and in vitro,including hydroxylation,carbonylation,acetylation,methylation,glucuronidation and sulfation.Pharmacokinetic studies have found that DVAS has good oral bioavailability,and its peak and elimination time in vivo are quick(T_max is about 0.46 h,T_1/2ke is about 1.47 h).In addition,the plasma concentrations of DVAO and VAO,the major metabolites,were decreased significantly at the dose of 45 mg/kg,which implied drug-metabolizing enzyme saturation or their further metabolisms at this dosage in rats.In terms of cholinesterase inhibitory activity,its in vivo activity is mainly attributed to the prototype DVAS.The metabolism of DVAS in plamsa may undergo metabolic inactivation process in consideration of BCh E inhibitive activity.