| Research objective:The prevalence of Alzheimer’s disease is increasing with the aging of the population.Finding drugs that can effectively alleviate and treat AD is the focus of research today.Some Studies have found that raspberries have anti AD effects,and the ethyl acetate site is the active site for anti AD.And the flavonoid component has a significant improvement on the memory ability of AD model rats,Phenolic components have pharmacological effects such as antioxidant and anti-tumor effects,However,there has been no systematic research on its anti AD effect.Therefore,based on the triple quadrupole liquid-mass spectrometry technology,this study explored the influence of the content of phenolic components on the anti-AD effect of different parts of raspberry through the inhibition of drugs on neuroinflammation,At the same time,the monomers with good anti-AD effect among phenolic components were screened out to provide theoretical basis for the development of anti-AD drugs.Research methods:1.The inflammatory reaction model produced by BV2 cells stimulated by LPS was used as the cell model of AD,and the pharmacodynamics of the extracts of dichloromethane,ethyl acetate and n-butanol from Raspberry palmate against AD was studied: The traditional ethanol reflux extraction method was used to extract the total extract of raspberry fruit,and then the extracts of dichloromethane,ethyl acetate and n-butanol were extracted in turn for the efficacy test.The inflammatory reaction model produced by BV2 cells stimulated by LPS was used as the cell model of AD.The above inflammatory reaction model was used to explore the concentration range of the extract by using the CCK8 method,and the drug was administered in different concentrations.According to the level of NO,IL-6,IL-1β,and TNF-α inflammatory factors in the cell supernatant,the anti-AD differences of different parts of the extract were reflected.The raspberries from Fuzhou,Fujian,Suqian,Jiangsu and Pan’ an,Zhejiang were compared and analyzed.2.A total of 9 samples from 3 parts of raspberry from different places were quantitatively analyzed by triple quadrupole liquid chromatography-mass spectrometry: A method for the simultaneous determination of EA,GA,TI,VA,FA,KA was established and conduct methodological validation by AB·Q-TRAP4500,The above 6 components in9 samples from 3 parts were quantitatively analyzed,and calculate the content of different components using the peak area results using the external standard method.Based on the results of anti AD activity of 9extracts,the influence of these 6 phenolic components in the extract on its anti AD effect was reflected through multiple linear regression and nonlinear fitting analysis.3.Predict the anti AD effect of six phenolic components based on molecular docking technology and study the inhibitory effect on neuroinflammatory response through BV2 cell inflammation model: Find AD-related receptor protein through literature and PDB database,and use Discovery Studio and Auto dock software to dock disease target with component target and inflammatory factor protein IL-6,IL-1β,TNF-α with component target respectively.According to binding fraction and binding,the anti-AD ability of six components can be effectively predicted.The inflammatory reaction model produced by BV2 cells stimulated by LPS was further used as the cell model of AD.After the concentration range of the components was found by CCK8 method,the models were made and administered with different concentrations respectively.According to the level of NO,IL-6,IL-1β,TNF-α inflammatory factors in the cell supernatant,the anti-AD differences of the six polyphenol components were reflected.Research results and conclusions:1.CCK8 test results showed that the concentration range of 9 parts of raspberry extract from three regions was 12.5-100g/ml,and there was no significant effect on the proliferation of BV2 cells.The optimal modeling concentration of LPS is 1μg/ml,and the extract of 9 parts can reduce the NO level of BV2 cells induced by LPS.At the same time,The raspberry extract produced in Pan’an,Zhejiang Province can more effectively inhibit the release of inflammatory factors,and its ethyl acetate fraction has the best inhibitory effect,indicating that this fraction has the best anti AD effect.2.A method for the simultaneous determination of EA,GA,TI,VA,FA,and KA by triple quadrupole liquid mass spectrometry was established.It has been verified that there is a good linear relationship within the measured concentration range,and the stability,precision,repeatability,and accuracy meet the requirements.Using external standard method,quantitative analysis showed that the content of compounds with significant anti AD activity such as EA,GA,TI,VA,FA,and KA in the ethyl acetate site was higher than that in other sites,and its content and anti-inflammatory factor effect showed a good linear relationship through multiple linear regression statistical analysis.The polynomial nonlinear fitting between the content of phenolic components in each extract and the anti AD efficacy was performed using python software,nonlinear equations and curves well reflect the impact of component content on drug efficacy,and can predict drug efficacy through the content of phenolic components.3.Auto dock molecular docking results showed that these six phenolic components can effectively bind to target proteins.The binding energy of EA,TI and KA is low,so it can be predicted that they can better inhibit the release of NO,TNF-α,IL-1β and IL-6 inflammatory factors.The docking results of Discovery Studio software show that these six compounds have high compatibility with the target protein of AD disease.The docking fraction of TI,EA,KA is significantly higher than that of FA,GA and VA,which also indicates that TI,EA,KA may have better anti-AD effects than that of FA,VA and GA.The results of cell experiment in vitro found that within the concentration range of 10-160 μM,these components have no significant effect on the growth of BV2 and HT22 cells,and these components can significantly inhibit the release of NO,TNF-α,IL-1β and IL-6 of BV2 cells induced by LPS.The inhibitory effects of EA,TI and KA are better,which is consistent with the results predicted by molecular docking,which further indicates that their anti-AD activity is stronger.The above research results have certain reference value for the treatment of neuritis with phenolic components,and provide a basis for the development of anti-AD drugs with phenolic components. |
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