Sesquiterpenoids From Two Traditional Chinese Medicine And Interactions With Acetylcholinesterase

Alzheimer’s disease（AD）,commonly known as senile dementia,is a neurodegenerative disease,mainly manifested by irreversiblely degeneration of thinking,memory and cognitive ability.Acetylcholinase（Ach E）inhibitors are the most effective strategies currently for the treatment of AD,but most of them could cause adverse reactions including liver toxicity,gastrointestinal disorders,dizziness and others.Therefore,it is of great interest to find more safe and effective cholinesterase inhibitors to cure or modulate AD.Natural products have always been one of the most important sources for new drugs.In the past,studies on the search for cholinesterase inhibitors from natural products were mainly focused on alkaloids,flavones,coumarins and other compounds.But recently,terpenoids,especially some sesquiterpenoids,showed neuroprotective effects and have the potential to treat neurodegenerative diseases.Plants of Compositae family are rich in sesquiterpenoids,so it can be used as an important resource for potential Ach E inhibitors.Carpesii Fructus,the fruit of Carpesii abrotanoides L.,is widely used as insecticide.It’s also used to treat some eclampsias in clinical Chinese medicine.In recent years,a variety of sesquiterpenoids,especially sesquiterpenoid dimers with novel skeletons,have been isolated from C.abrotanoides L..However,there are few reports on the chemical constituents of C.Fructus.Aucklandia costus is a plant of Compositae family.Its roots were used to treat abdominal pain,vomiting and dysentery in folk medicine.There are also records for the treatment of epilepsy and convulsions.Its characteristic chemical constituents are sesquiterpenoids,and the two most active components are dehydrocostus lactone（DH）and costunolide（CL）,which have been reported depress effects on CNS.DH has been reported to show inhibitory activity on Ach E.In this study,we focused on sesquiterpenoids isolated from C.Fructus and A.costus,screened active compounds with inhibitory effect on Ach E,and studied the interaction between active sesquiterpenoids and Ach E at the molecular level.Preliminary toxicity evaluation was also performed.Objective:To investigate the interaction of active sesquiterpenoids with Ach E,providing experimental basis for the search of sesquiterpenoids as cholinesterase inhibitors to cure or modulate AD.Methods:1.C.Fructus was extracted with 95%Et OH and successive partition of the extract with petroleum ether,CH₂Cl₂ and Et OAc yielded three extracts.The CH₂Cl₂ extract were separated by silica gel column chromatography,Sephadex LH-20,p-TLC,semi-preparative HPLC,p-HPLC and other methods.Structure of the isolates were determined by HRESIMS,UV,CD,OR,¹H NMR,¹³C NMR,¹H-¹H COSY,HSQC,HMBC,NOESY,DEPT,ECD,calculated ¹³C NMR and modified Mosher’s method.The major active sesquiterpenoids of A.costus were isolated and enriched.2.The inhibitory effects of sesquiterpenoids on Ach E were evaluated by Ellman method.The interaction between the selected sesquiterpenoids and Ach E was investigated by UV-equilibrium dialysis,FTIR,CD,NMR and molecular dock.3.To study the antioxidant activity of the active sesquiterpenoids,hydroxyl radical scavenging ability was evaluated.Acute toxicity test was conducted to investigate the safety of the sesquiterpenoids.Results:1.Phytochemical study on C.Fructus and A.costus provided some samples for the follow-up activity screening.50 compounds were isolated and identified from C.Fructus and 17 were undescribed compounds,including 13sesquiterpene lactone dimers（SLDs）and 4 sesquiterpene lactones.The isolated compounds included 17 SLDs,28 sesquiterpene lactones,3flavonoids,1 lignan and 1 phenylpropyl compound.Among the 17 SLDs isolated,there were six different connecting modes of two sesquiterpene units and three were found for the first time:Compounds 1 and 2 were featured with a rare C-13—C-13′linkage;compounds 3 and 4 were SLDs with the two units connected by an O-ether linkage;compounds 15-17 were a group of SLDs with seco-lactone ring.In addition,6 sesquiterpene lactones,2 triterpenes and1 steroid were isolated from A.costus.2.Three sesquiterpenoids[DH,CL,santamarin（ST）]with different inhibitory activities to Ach E were selected based on the screening results using Ellman method.Equilibrium dialysis showed the binding rates of three siterpene lactones to Ach E were 49.4%,34.0%and 25.6%respectively,at37℃,p H 8.0.The results of CD and FTIR showed that the secondary structure of Ach E was changed by the interaction with DH and CL.NMR experiments showed that after binding with Ach E,NMR signals of sesquiterpenoids turned to be broader and the intensity of some proton signals decreased with a slight chemical shift changed.Binding mode of sesquiterpenes with Ach E was studied by molecular docking method,providing consistent results with those obtained from spectroscopy and NMR experiments.3.The three sesquiterpenes showed certain antioxidant capacity.In the acute toxicity test,compared with the control group,there were no obvious changes in hematological and biochemical indexes.No obvious histopathological changes of the three sesquiterpene administration groups were detected.Conclusions:1.In this study,17 SLDs（including 13 new compounds）and 28sesquiterpene lactones（including 4 new compounds）were isolated from C.Fructus.Studies on the chemical constituents of C.Fructus show that it is rich in sesquiterpene lactones,especially the SLDs with various skeletons.From the perspective of phytochemical constituents,this study provides a strong support for the study of the relationship between the related groups and the identification of confusers.2.In this study,the interaction between sesquiterpene and Ach E was studied here for the first time.The results showed that the binding of sesquiterpenoids with Ach E lead to the change of Ach E secondary structure.NMR results further confirmed the binding effect and it can be speculated that the binding site of DH was the unsaturated lactone ring.Binding mode of sesquiterpene and Ach E was further studied by molecular docking,and the effective binding was verified.3.The sesquiterpene lactones could increase the antioxidant capacity of nerve cells and have the potential to regulate AD in multi-targets.The acute toxicity experiments showed that the sesquiterpene molecules have certain safety.