Depression is a mental disorder with unclear pathological mechanism, which has become an important disease threatening people's health in contemporary society. Currently, antidepressants used in clinic are mainly single target drugs developed based on the "monoamine neurotransmitter hypothesis", including selective5-Serotonin（5-HT）Reuptake inhibitors, selective norepinephrine（NA）Reuptake inhibitors, selectivityNA/5-HTReuptake inhibitors, monoamine oxidase inhibitors（MAOI）And5-HTReceptor agonists, etc^[1]However, these drugs have low effective rate, many adverse reactions, and are easy to relapse after withdrawal, which limits their wide application in clinic^[1]With the development of traditional Chinese medicine, traditional Chinese medicine or traditional Chinese medicine monomer has brought new directions for the treatment of depression.

Acorus tatarinowii is a perennial herb of AraceaeAcorus tatarinowii SchottIt has the effects of inducing resuscitation, eliminating phlegm, awakening the mind and improving intelligence. It is mainly used in the treatment of nervous system diseases in clinic^[2]Acorus tatarinowii is one of the drugs that are frequently used in antidepressant traditional Chinese medicine compound. Different extraction parts of Acorus tatarinowii single drug can resistDepression and anxiety behaviors in mice^[3]However, the material basis and mechanism of antidepressant effect of Acorus tatarinowii are not clear. In this study, molecular docking technology was used toA（MAO-A）（DAT）、5-Serotonin transporter（SERT）H1Receptors（H1R）To study the material basis and mechanism of action of Acorus tatarinowii against depression, and provide references for its further development and utilization.

1  Materials and methods

1.1  Establishment of ligand library

Traditional Chinese medicine pharmacology database was used（TCMSP，http://lsp.nwu.edu.cn/tcmspsearch.php?）Establishment of oral easily absorbed compound library of Acorus tatarinowii^[4]. with Acorus tatarinowii as the key word105Compounds utilizing oral bioavailability（OB）≥30%Conduct secondary screening^[5], obtained65Compounds (including  Beta asarone、 Alpha asarone), accounting for% of the total compounds61.90%. withChemBio 3DThe three-dimensional structures of candidate compounds (ligands) were mapped and analyzed usingMM2The force field was used to optimize the conformation, and the processed small molecules were saved asMol2Format, and finally useAutoDock tools（ADT）Save asPDBQTAlternate format for.

1.2  Preparation of receptor proteins

Depression related drug target reference therapeutic target protein database（therapeutic target database TTD，http://bidd.nus. edu.sg/group/cjttd/）, combined with literature reports^[6-8], selecting those related to depression4Target proteins as receptor proteins, respectively, metabolize dopamine（DA）、5-HTandNAOfMAO-A; andDARelated to uptake and transportDAT; and5-HTUptake, transport relatedSERT; and those related to sedation and anti anxietyH1R. the target protein structures are all derived fromRCSBProtein database（http://www.rcsb.org/）, target code（PDB ID）And its protein bound protomers are shown in table1, withADTTo the originalPDBThe documents are processed as follows:（1）Removal of solvent molecules (i.e., water molecules); （2）Removal of ligand molecules; The（3）Polar hydrogenation of protein molecules.

1.3  Setting and verification of docking parameters

The molecular docking software used was Scripps Research InstituteOlsonDeveloped by the research groupAutoDockVina, and perform semi flexible molecular docking calculations.AutoDockVinaComplex gradient optimization algorithm is used to speed up the process of local minimization, and multithreading technology is adopted, compared toAutoDock 4The speed has increased by about2Orders of magnitude and better accuracy^[9]Semi flexible docking refers to that the conformation of small molecule ligands in docking is flexible and can be changed, while the receptor protein is rigid and unchanged. The grid point is set with the target protein protomer as the center, and the center coordinates and box size are shown in table2。

applyChimera^[10]The software extracts the conformation of the original ligand of the target protein and saves it asPDBFormat, and useADTProcessed as PDBQTAlternate format for. Then useAutodock VinaThe original ligand was docked back, and the conformation with the lowest energy was selected to calculate the root mean square deviation from the original ligand（RMSD）, affinity（K_i）To measure parametersRationality of the number setting. Using softwareVMDcalculateRMSD，K_iAccording to the following formula,K_i＝exp[(ΔG×1 000)/(Rcal×T)]，amongΔGThe unit iskcal/mol，T＝298 K，Rcal＝1.987 19 cal/(mol∙K)According to the minimum docking energyRMSD、K_iMeasure whether the parameter setting is reasonable. See table for docking parameters and results2。

The protomers of the above target proteins can be successfully docked to the active center of the target protein,RMSDAll less than0.20 nm, and itsK_iRespectively416.49、54.90、16.83、65.00 nmol/L, and literature reports^[11-14]The biological activity of was in the same order of magnitude, indicating that the docking parameters were set reasonably.

1.4  Molecular docking of chemical constituents and target proteins of Acorus tatarinowii and its screening criteria

The processed candidate compounds and the above4Target usageAutoDockVinaPerform docking and calculateK_iAccording to the overall docking results of candidate compounds and positive drugs, avoid the shielding effect of high score (affinity) compounds, reduce the generation of false negative compounds, and rank the affinity of each target in the top10%(former7Compounds with the same affinity (in parallel if any) were identified as YangSexual outcomes^[15]. utilizationPymolWithLigplotAnalyze and observe the docking results of compounds and proteins^[16-17]。

2  result

2.1  The chemical constituents in Acorus tatarinowii and4Action of target proteins

through the use ofAutoDockVinaTo Acorus tatarinowiiOB≥30%Of65Compounds and4Target proteins were docked and found18Potential active compounds, and the results are shown in table3. can work simultaneously4The three targets were isopurple peucedanone, kaempferol2″-O-Methylisoliquiritigenin8-Isopentenyl kaempferol. Can act simultaneously2Bergamot lactone, nerolidol, and cycloartenol were targeted. the rest11Compounds can interact with1Targets interact. Therefore, the multiple components in Acorus tatarinowii may be through the inhibition ofDAMetabolism and reduction of neurotransmitters such asDA、5-HTUptake and inhibition ofH1R.

2.2  Affinity of potentially active compounds to each target、Hydrogen bonding and hydrophobic interaction

18Candidate compounds and4The docking results of the targets are shown in table4～7. vsMAO-ACompounds with strong affinity have7Species, and9-Aminoacridine, isopurple peucedanone, kaempferol2″-O-Methylisoliquiritigenin4The effect of these compounds was stronger than that of the original ligand carnitine (dehydrocarpine,harmine）; WithDATCompounds with strong affinity include10Species, cycloartenol8-The affinity of isopentenyl kaempferol exceeded that of the original ligand nortriptyline; WithH1RCompounds with strong affinity include9Species; WithSERTCompounds with strong affinity include7Species, including cycloartenol8-The affinity of isopentenyl kaempferol and kaempferol with target proteins is greater than or equal to that of positive drugsS-Citalopram.

9-Aminoacridine is a heterocyclic compound containing nitrogen atoms, which has beenTCMSPDatabase access is a unique species of Acorus tatarinowiiCompound, which is associated withMAO-AThe affinity of Acorus tatarinowii is very strong, which may be the main material basis for its antidepressant effect. Cycloartenol is a phytosterolClass compounds, with anti-inflammatory, anti-tumor, antioxidant, antibacterial, anti Alzheimer disease and other activities^[18]In this study, cycloartenol andDAT、SERTIt has a strong affinity, so cycloartenol may be developed into a traditional Chinese medicine monomer or lead compound with antidepressant activity, or promote the application of traditional Chinese medicine containing cycloartenol in antidepressant. 

Select the compound with the highest affinity to each target protein, and make a compound protein binding diagram, which can visually observe the binding mode of the active compound to the target and the interaction with the surrounding amino acid residues. The results are shown in Fig1。

9-The amino acridine occupies the position ofIle180、Asn181、Gln215、Ile335、Leu337、Met350、Tyr407、Tyr444andFad600The formed active cavity, whose amino group is related toAsn181It forms a hydrogen bond, and its ring structure is similar toTyr407formationπ-πAccumulation.Upadhyayetc.^[19]The results showed that whether it could be associated withPhe208、Asn181、Tyr407Interaction, which determines whether a compound can interact withMAO-AThe key of active site binding, which is consistent with the docking results of this study. In addition, the protogametophyte carnitine andTyr407There is also aπ-πAccumulation, which indicates thatTyr407Is whether the compound can interact withMAO-AA key residue for binding.

DATThe active cavity composition of theSer421andSer422All of them are hydrophobic residues, while cycloartenol has only1The triterpenoid structure of hydroxyl groups, i.e., the hydrophobic interaction is the interaction between cyclic atumol andDATThe main driving role of the combination.Penmatsaetc.^[20]Studies have shown that amino acid residues associated withPhe43、Val120Interaction is the key to antidepressant activity, which further verifies the reliability of the results of this study.

Kaempferol3、7、1The oxygen atom of NoSer111、Thr194、Tyr431Hydrogen bonds are formed, and its ring structure is similar to that ofTyr108、Trp428、Phe432、Phe435、Ile454There is hydrophobic interaction, and the interaction withTyr431、Phe432existenceTTypalπ-πAccumulation.Heifetzetc.^[21]The results also showed that the compounds with amino acid residuesAsp107、Tyr108、Ser111、Thr112、Asn198、Phe199、Trp428、Tyr431、Phe432、Tyr458yesH1RKey residues that interact with compounds.

SERTOf the active cavity is mainly composed ofTyr95、Ile172、Ala173、Tyr176、Phe335、Gly338、Phe341、Val343、Gly338、Ser439、Glu493Composition, cycloartenol well occupiedSERTOf the active cavity, and withTyr95、Ile172、Tyr176、Phe335andPhe341There is a good hydrophobic effect, which may be the reason for its better score.Henryetc.^[22]The site directed mutagenesis experiments showed that,Tyr95andIle172yesSERT, which is consistent with the results of this study.

3  discuss

In this study, a total of18Compounds interact with the target proteins of antidepressant action, among which flavonoids3Species, coumarins2Species, sterols1Species and volatile oils9Species, lignans1Species, other categories2Species, and volatile oil accounts for the majority, which is consistent with the main material basis of the pharmacological effect of Acorus tatarinowii is volatile oil^[23-24]。However, the main volatile oil components of Acorus tatarinowiiβ-Asarone was molecularly docked, and it was found that its inhibition of monoamine oxidase and monoamine transporters was poor, indicating thatβ-Asarone may exert antidepressant effects through other mechanisms, such as significantly inhibiting the apoptosis of cerebral cortical neurons and hippocampal neurons in rats^[25]。

In this study4These compounds can simultaneously act on the above4Three targets were identified, namely, imperatoride, kaempferol2″-O-Methylisoliquiritigenin and8-Isopentenyl kaempferol, post3Species are flavonoids. Flavonoids have been reported to have antidepressant activity^[26], Wang Jia et al^[27]The experiment verified that kaempferol has the ability to resist largeThe effect of depression in rats can improve the brain function of depression ratsNA、DA、5-HTIt is consistent with the results of virtual screening in this study.8-The fat solubility of isopentenyl kaempferol is greater than that of kaempferol, which is more conducive to penetrate the blood-brain barrier and exert its antidepressant effect, which needs to be verified by subsequent pharmacological experiments. . Coumarins are a class of active substances widely existing in medicinal plants, which have a wide range of pharmacological activities, such as antiviral, anti-inflammatory, anti-tumoretc.^[28]However, the antidepressant effect of coumarin is rarely reported in the literature. This study provides a theoretical guidance for the development of coumarin compounds as antidepressant drugs.

There have been some experimental reports on the antidepressant effect and mechanism of action of Acorus tatarinowii. Wu quan'e et al^[29]Studies have shown that pseudogentiana acutissima combined with Acorus tatarinowii can significantly improve the brain5-HTThe level of; Zhou Tian et al^[30]The results showed that Acorus tatarinowii extract and its low polarity parts could significantly improve the depressive symptoms of depression model mice, and could significantly increase the brain5-HTandACTH; Liu Mei et al^[31]The results showed that Lihuan Jieyu capsules containing Acorus tatarinowii could significantly increase the serum of depressed miceNA、DA、5-HTThe content of anti depression; The above experimental results can prove that the target selection of virtual screening in this study is reasonable.

Acorus tatarinowii in this study18Among the active compounds are9Species andH1RIt has good affinity, and the anti anxiety and depression drug doxepin is similar toH1RHowever, whether the antidepressant effect of Acorus tatarinowii is related to its antihistamine effect needs further study.

In this study, molecular docking technology was used to screen out18Among the antidepressant active ingredients in Acorus tatarinowii, the compounds with strong activity in interacting with target proteins are3Species, respectively9-Aminoacridine, kaempferol and cycloartenol. .

References (omitted)  

Come   Source: liuyongjie, Xiang Huilong, Chen   Hao, xulingyun Screening of antidepressant active ingredients in Acorus tatarinowii based on molecular docking technology [j]. Chinese herbal medicine, 2019, 50 (11): 2612-2619