Anxiolytic Effect Of Natural Small Molecule Phenol And Its Structure - Activity Relationship

Our previous studies indicated that phenanthrene compounds have anxiolytic effects, which were also found in other small molecule natural phenols (SMNPs), such as kaempferol, quecetin and paeonol. By this view, we propose a hypothesis that SMNPs, in generation, have anxiolytic effects. The working hypothesis has been verified with experiments in this dissertation study. Firstly, the anxiolytic effects of orcinol monohydrate (OM) and orcinol glucoside (OG), two natural phenols, were revealed comprehensively by three behavioral tests and LC-MS qualitative detection, demonstrating that SMNPs might have anxiolytic effect. Secondly, the anxiolytic activity of seven SMNPs, including eugenol, ferulic acid, caffeic acid, vanillin, protocatechuic aldehyde, thymol, and phloroglucinol, were tested with the classic model of animal anxiety-elevated plus maze (EPM), further validating the hypothesis. Based on the previous two parts of the experimental work, a pilot study on relationships between structure and the anxiolytic effect of SMNPs was finally launched.This dissertation consists of six chapters:Chapter 1. Literature reviewFive sections are included in this chapter, the pathogenesis of anxiety, anxiolytic drugs, animal models of anxiety, the research advances of anxiolytic effects of SMNPs, and the anxiolytic mechanism.Chapter 2. IntroductionThe research rationale, experimental design and methods, and new findings are briefly described in this chapter.Chapter3. The anxiolytic effects of orcinol monohydrate (OM) and orcinol glucoside (OG) in miceAnxiolytic effects of OM and OG in mice were tested with the elevated plus-maze (EPM) and hole-board tests. OM (2.5 and 5mg/kg) and OG (5,10, and 20mg/kg) increased the time spent in open arms and the number of entries into open arms in EPM test, indicating the anxiolytic effects of OM and OG. In the hole-board test, OM (2.5 and 5mg/kg) and OG (5 and10mg/kg) increased the number of head-dips, which verified the anxiolytic effects of OG and OM found in EPM test.Because changes in general motor activity might affect the behaviors of open arm entering and head-dipping of mice, to exclude the possible false-positive/-negative findings in the plus-maze and hole-board tests, the open-field test was adopted to determine whether OM and OG affect general locomotion of mice. The results demonstrated that OM and OG at the doses showing anxiolytic had no effect on general locomotion of mice.In order to verify whether OM (5mg/kg) enter the blood-brain barrier and affect the central nervous system, liquid chromatography coupled with mass spectrometry (LC-MS) was used to detect orcinol in homogenized brain tissue. Results showed that orcinol could be detected in the mouse brain homogenates 30min after oral OM administration, indicating that OM was centrally active.Chapter 4. The anxiolytic effects of eugenol, ferulic acid, caffeic acid, vanillin, protocatechuic aldehyde, thymol, and phloroglucinol in miceEugenol and other six representative SMNPs were selected under the consideration of our previous studies and the SciFinder database exploration. Their anxiolytic effects were measured with EPM test.The results showed that eugenol (5mg/kg), vanillin (5mg/kg), protocatechuic aldehyde (10mg/kg), thymol (20mg/kg) significantly increased the time spent in the open arms and the enter numbers into the open arms. Ferulic acid (5,10,20mg/kg), caffeic acid (10,20mg/kg), phloroglucinol (5mg/kg) significantly increased the time spent in the open arms. Phloroglucinol(2.5,10mg/kg) significantly increased the enter numbers into the open arms. The above results demonstrated that the seven representative SMNPs possessed anxiolytic activity, which further verified the hypothesis in the perspective of structural diversity.Chapter 5. Pilot research on the structure-activity relationship of SMNPsIn order to investigate the role of phenyl ring of SMNPs in anxiolytic activity, the anxiolytic effects of gallic acid and shikimic acid were evaluated by EPM test in mice. Gallic acid (40mg/kg) with phenyl ring showed a significant anxiolytic effect, while shikimic acid without phenyl ring had no effect. Results revealed that phenyl ring was an essential substructure of SMNPs for anxiolytic effect.The anxiolytic effect of 3,4-Dimethoxycinnamic acid,4-hydroxycinnamic acid (4-OH CA) and 4-chlorocinnamic acid (4-Cl CA), methyleugenol and methylvanillin were evaluated with EPM test in mice to explore the role of phenolic hydroxyl group in the activity of SMNPs. 4-OH CA(20,40mg/kg), but not 4-Cl CA and 3,4-dimethoxycinnamic acid, showed an obvious anti-anxiety effect, which provided the evidence that phenolic hydroxyl group was the essential substructure of SMNPs. Furthermore, compared with the vehicle control, methyleugenol and methylvanillin had anxiogenic effect. These results further demonstrated that phenolic hydroxyl group might greatly contribute to the anxiolytic activity of SMNPs.To our knowledge, this is the first study documenting the anxiolytic activity of OG and OM at dosage of no sedation. The results of EPM tests show that eugenol, ferulic acid, caffeic acid, vanillin, protocatechuic aldehyde, phloroglucinol and thymol have anxiolytic effect. Therefore, it can be presumed that SMNPs might have anxiolytic effect in general. The results from the pharmacodynamic studies of gallic acid and shikimic acid,3,4-dimethoxycinnamic acid; 4-OH CA and 4-Cl CA, methyleugenol and methylvanillin have verified that phenyl ring and phenolic hydroxyl group are the essential substructures of SMNPs for anxiolytic effect.Chapter 6. Summary and prospectionThe results and imperfections of the research were summarized, and a further prospection was made based on this research.