Studies On The Separation,Antiepileptic Activity And Mechanism Of α-asaronol In Acorus Tatarinowii

Epilepsy is a chronic recurrent cerebral dysfunction syndrome featured by abnormal discharge of brain neurons,which is characterized by a large number of patients,high incidence,high treatment cost and little research on new drugs.It is listed as the second largest burden of neurological diseases in the world.Traditional Chinese medicine（TCM）has a long history in the treatment of epilepsy.There is a great potential to research and development of antiepileptic drugs derived from traditional Chinese medicine.In this paper,Acorus tatarinowii that is commonly used in antiepileptic traditional Chinese medicine and its typical herb pair of Arisaema cum bile-Acorus tatarinowii are conducted a study followed by high performance liquid chromatography（HPLC）and liquid chromatography-mass spectrometry（HPLC-Q-TOF-MS）technology.It is found for the first time thatα-asaronol is an inherent natural product in Acorus tatarinowii.Adding Arisaema cum bile to Acorus tatarinowii can increase the content ofα-asaronol in the extracting solution.Next,the antiepileptic activity,mechanism and toxicity ofα-asaronol are studied in vivo and in vitro.This thesis is divided into the following three parts:1.The components of Acorus tatarinowii and herb pair of Arisaema cum bile-Acorus tatarinowii are analyzed by HPLC and HPLC-Q-TOF-MS.The active moleculeα-asaronol is found to be the original component of Acorus tatarinowii.Among them,Arisaema cum bile is helpful to increase the content ofα-asaronol in Acorus tatarinowii,which indicate that the compatibility of Arisaema cum bile and Acorus tatarinowii is reasonable.Meanwhile,the extraction,separation and structure identification ofα-asaronol and its related compounds in Acorus tatarinowii are studied.Eight compounds were separated successfully from Acorus tatarinowii.Among them,α-asaronol and（E）-3-（2,4,5-Trimethoxyphenyl）acrylaldehyde were first found in this plant.2.The SH-SY5Y cell injury model induced by H₂O₂ is used to evaluate cell protection effects of compounds isolated from Acorus tatarinowii.Among them,the protective effect ofα-asaronol is the most significant.The antiepileptic activity and toxicity ofα-asaronol are evaluated by zebrafish model.It is found thatα-asaronol has significant anti-epileptic activity.Under the experimental dosage,it can significantly prolong the time of zebrafish latent epilepsy,shorten the swimming distance of zebrafish in epileptic model,reduce the expression of c-fos caused by epilepsy and decrease the spike frequency and amplitude of EEG.In addition,α-asaronol exhibit less toxic to zebrafish,with a lethal concentration(LC₅₀)of 723μM.3.Through whole-cell patch clamp technique,α-asaronol is found to act on HEK293T cell lines stably expressing the GABA_A receptor（α1β2γ2 subtype）.It can significantly promote chloride influx,and its half effective concentration(EC₅₀)is 43.78μM.The activity inhibition of lactic dehydrogenase（LDH1 and LDH5）by ultraviolet and mass spectrometry show that the activity of LDH1 and LDH5 is significantly inhibited byα-asaronol,which is stronger than that of the positive drug stiripentol,and the inhibition type is non-competitive.In addition,RNA-seq analysis,KEGG pathway enrichment analysis,RT-PCR analysis and zebrafish behavioral study indicate thatα-asaronol can play an antiepileptic role by promoting the expression of PPAR-γ.