Study On The Anti-anxiety Mechanism Of Natural Small Molecule Phenol

Objective:According to our previous studies and using the SciFinder database for the research status of natural small molecule phenols(NSMPs),it has been found that NSMPs may be a class of natural compounds whose importance is underestimated,anti-anxiety may be a biological activity shared by natural small molecule phenols(NSMPs).In this study,the structure-activity relationships of the anxiolytic effects of NSMPs were first explored.On this basis,cerebral microinjection,in vivo multi-channel simultaneous recording and whole-cell patch clamp were used to study the target brain region and cellular target of NSMPs.Methods:1)The classic anxiety-like behavior model,the elevated plus maze,were first used to evaluate the anxiolytic effects of ten NSMPs in mice and our previous working hypothesis is further verified that the NSMPs may all have an anxiolytic effect.On this basis,elevated plus maze model also was used to evaluate the anxiogenic activity of ten phenylethylamines that were structurally similar to NSMPs but whose phenolic hydroxyl groups were substituted.By comparing the structure-activity relationships of NSMPs and phenylethylamines,the importance of phenolic hydroxyl group for the expression of their activities was further verified.2)The brain nucleus microinjection was used to externally administer small molecule phenol,Homovanillic acid(HVA),to the mPFC and observed its effects on the anxiety-like behavior of rats.In order to explore the target brain regions of NSMPs;then,High performance liquid chromatography with electrochemical detector(HPLC-ECD)was used to test the monoamine neurotransmitters and their metabolites in the medial prefrontal cortex(mPFC)of rats with different levels of anxiety,which further verified the aforementioned results.Rats’ in vivo multi-channel simultaneous recording technology was used to monitor the spikes of hippocampal CA1 neurons of after administration of NSMPs in order to clearly identified whether CA1 of Brain hippocampus,closely related to anxiety regulation,was involved in the anxiolytic effect of NSMPs.3)In order to identify molecular targets of NSMPs,whole-cell patch clamp technique was used to evaluate the effect of HVA on L-type calcium channels in the pyramidal neurons of mPFC.In addition,the effects of compounds such as 4-OH CA,4-Cl CA,3,4dimethoxycinnamic acid,and dehydroepthritol on L-type calcium currents were preliminarily investigated.Results:1)Ten NSMPs significantly increased the time spent in the open arms or entered numbers to the open arms.The results showed that ten NSMPs had significant anxiolytic effects.Ten phenylethylamine derivatives had the tendency to decrease the number of entering the open arms and the time spent in open arms,and had certain anxiogenic biological activity.Compared with the anxiolytic effect of NSMPs and the anxiogenic effects of the phenylethylamines,our working hypothesis is further verified that the NSMPs may all have an anxiolytic effect,and the phenolic hydroxyl group is crucial for the expression of their activities.2)The results of brain nuclear micro injection showed that the anxiety level in rats was indeed related to the HVA content in mPFC.The results showed that mPFC was involved in the anxiety-like behavior of rats,and HVA may be involved in regulating anxiety levels.HPLC-ECD results showed that compared with the low anxiety group,the HVA content in the mPFC in the high anxiety group rats was significantly increased which verified the aforementioned experimental results.3)In vivo multi-channel simultaneous recording monitoring results showed that 4-OH CA(14 mg/kg)significantly increased the number of spikes in the rat hippocampal CA1 region;4-Cl CA had no significant effects.This study validated the role of phenolic hydroxyls in the anxiolytic effect,and also suggested that neurons in the hippocampal CA1 region are involved in the anxiolytic effect of NSMPs.4)Whole-cell patch clamp results showed that homo vanillic acid(80 μM)significantly inhibited L-type calcium current(P<0.05).4-OH CA and dehydroeffusol also had inhibitory effects on L-type calcium current.The results suggest that the L-type calcium channel in the pyramidal neurons of mPFC is one of molecular targets of NSMPs.Conclusion:1)The structure-activity relationship of natural small-molecule phenols shows that the anxiolytic effect is a common biological activity of NSMPs but with different efficacy,and phenolic hydroxyl group is its essential group;2)The results of HPLC-ECD,cerebral micro injection,and in vivo multi-channel simultaneous recording suggest that neuronal activities in hippocampal CA1 region and mPFC are two brain target nuclei;3)Whole-cell patch clamp results indicate that L-type calcium channel is one of molecular targets of NSMPs and HVA may a mediator of NSMPs on their modulation on anxiety-like behaviors.