Study On Mechanism Of Quantitative Research On Multi-component Fraxini Cortex Based On Overall Quality Control And The Effect Of Chalcone Against The New Tumor Target

Objective:This project intends to carry out research on the quality control and anti-tumor activity of Fraxini Cortex.On the one hand,the total coumarin content of Fraxini Cortex is determined by single standard for the determination of multiple components,and the overall quality control method based on the multi-component quantitative analysis of Fraxini Cortex is established.The ingredients and their analogues carry out activity studies against new anti-tumor targets.Methods:This subject takes Ash Bark as the target to investigate the extraction method of the test solution and the chromatographic conditions.The multi-component quantitative analysis of Ash Bark is carried out by SSDMC.With Esculin as the reference substance,three index components are selected,and the conversion factor is calculated respectively.The content calculations are performed using the correction factors.Finally,single standard for determination of multiple components is used to determine the total coumarin content in 17 batches of Ash Bark,and the acceptance criteria is established to construct the overall quality control standard for Fraxini Cortex.On the basis of establishing the quality control of Fraxini Cortex,we conduct research on the activity of new anti-tumor targets on the main coumarins and flavonoids in Fraxini Cortex,and found that flavonoids have a certain inhibitory effect on the new target.Further expanding the structure,it is found that chalcone which has the flavonoid analogue has stronger inhibitory activity,especially the compound 119 extracted from Catharanthus roseus has an inhibitory effect on the new target of non-small cell lung cancer A549.However,the content of chalcone compounds in plants is low.In order to effectively obtain chalcone compounds,we have established the condensation reaction of acetophenone and benzaldehyde under the action of PPh₃/I₂ to obtain chalcone compounds.Results:1)The optimized preparation of sample solution:Transfer about 500 mg of Ash Bark,moderately powdered and accurately weighed,to a 100-m L round-bottom flask.Add25.0 m L of extraction solvent（a mixture of ethanol and water 75:25,v/v）,and weigh.Heat on a water-bath under a reflux condenser for 90 min,extraction is performed once.Cool and weigh again.Compensate for the loss of solvent and mix.Filter through a membrane（nominal pore size 0.45μm）.When the injection volume is 2μL,the solvent effect is not obvious.When the injection volume is 5μL,the solvent effect is more obvious in the chromatogram.Therefore,a step of dilution with water is added after preparing the test solution.Finally,transfer accurately 1.00 m L filtrate to a 2-m L volumetric flask,adjust with water to volume,mix.2)The optimized chromatographic conditions:UV wavelength is334nm;chromatographic column is Agilent ZORBAX SB C18（4.6×100mm,3.5μm）;the solution A is phosphoric acid and water（1:999,v/v）,and the solution B is methanol;mobile phase acidity is 0.1%aqueous solution of phosphoric acid;the methanol ratio in the mobile phase is 17%,and the gradient is as follows:0-10 min,17%-17%methanol;10-20 min,17%-50%methanol;the flow rate is 1.5 m L/min;the column temperature is 35°C;when the injection volume is 5μL,the best chromatographic separation conditions are reached.3)The SSDMC method is used to determine the total coumarin content in 17 batches of Ash Bark and then establish the acceptance criteria,of which the total coumarin content reaches2.50%as the qualified standard.The results show that the total coumarin content of 13batches of Ash Bark meet the requirements,and the pass rate is 76.5%.The value of SSDMC/ESM calculated shows that there is no significant difference between the two methods.Through the structural analysis and structural modification of the compound 119,16analogues which are synthesized and separated are subjected to in vitro activity screening for anti-non-small cell lung cancer A549 activity evaluation,and compounds with improved anti-tumor activity are obtained.The in vitro activity evaluation results show that the newly synthesized compounds have better inhibitory activity.Using acetophenone and benzaldehyde as a model reaction,PPh₃/I₂ as a catalyst and Me CN as a solvent,the target compound chalcone is synthesized at a temperature of 80℃with a yield of 26%,which proves the feasibility of this method.Next,by screening the reaction conditions,the influence of the solvent,the amount of catalyst and the reaction temperature on the reaction yield are discussed,and the optimal reaction conditions are summarized:1,4-dioxane is the solvent,and PPh₃/I₂ is 1.0 equiv,the reaction occurs under reflux conditions,and the yield is60%.In addition,we also investigate the reaction under reflux conditions with 1,4-dioxane as the solvent without adding PPh₃/I₂,and found that the yield of the desired product chalcone is only 20%.Conclusion:This subject uses SSDMC to calculate the total coumarin content of Fraxini Cortex,to establish a multi-component quantitative analysis method for Fraxini Cortex,and builds an overall quality control system for Fraxini Cortex.The established single standard for determination of multiple components is applied to the determination of17 batches of Ash Bark.The value of SSDMC/ESM shows that there is no significant difference between the two methods.It shows that it is feasible to use the SSDMC method for multi-component quantitative analysis of Fraxini Cortex.At the same time,the acceptance criteria is established,and the total coumarin content reaches 2.50%as the qualified standard.This method can provide a demonstration study for the establishment of a unified overall quality control system for similar Chinese medicines.Through the optimization of the system conditions,a method for the condensation reaction of acetophenone and benzaldehyde under the action of PPh₃/I₂ is established.We report this synthesis method for the first time,which can avoid the condensation of acetophenone and benzaldehyde under non-strong alkali conditions.The condensation has good resistance to common chemical groups.At the same time,a series of chalcone compounds are evaluated for their inhibitory effects on new targets,and several compounds with significantly improved activities are found.