Experimental Study On Anti-Breast Cancer Effect Of Forsythia Suspensa Guiwei Decoctio Study On Preparation Technology And Quality Standard Of Granules

Objective:This experiment was guided by the inhibition of the proliferation of human breast cancer MCF-7 cells and MDA-MB-231 cells.The optimal extracting solvent of Fructus Forsythiae Guiwei Decoction was selected according to the effective component transfer rate.The proliferation inhibition of human breast cancer MCF-7 cells and MDA-MB-231cells as well as the effects of Fructus Forsythiae Guiwei Decoction on cell migration,apoptosis and cycle were studied through cell level experiments.Anti-breast cancer pharmacodynamics;determine the extraction and moulding technology of Forsythia suspensa Guiwei decoction;establish the quality standard of Forsythia suspensa Guiwei granules.Methods:1.Eight concentrations of 0%,5%,20%,35%,50%,65%,80%and 95%ethanol were set up in this experiment.Guided by the inhibition of the proliferation of human breast cancer MCF-7 cells and MDA-MB-231 cells,the optimum extraction solvent for Fructus Forsythiae Guiwei decoction was selected.The content of effective components in different ethanol concentration extracts of Fructus Forsythiae Guiwei decoction was determined by HPLC,and the transfer rate of effective components was calculated.The best extraction solvent.2.The ethanol extract of Forsythia suspensa Guiwei decoction was diluted into different drug concentration,and blank group,negative control group,positive control group and drug adding group were established.The inhibition effect of ethanol extract of Forsythia suspensa Guiwei Decoction on the proliferation of breast cancer cells was determined after 24,48 and 72 hours of drug action,and IC₅₀0 was calculated.Cell scratch test was used to study the effect of ethanol extract of Forsythia suspensa Guiwei Decoction on cell migration.The effects of ethanol extract from Fructus Forsythiae and its tail Decoction on cell apoptosis were detected by Annexin V/PI double staining method,and the effects of ethanol extract from Fructus Forsythiae and its tail Decoction on cell cycle were detected by PI single staining method.3.With the extraction rate of Forsythia suspensa and the transfer rate of active ingredients as indexes,orthogonal experiments were used to study the effects of four factors,namely,the amount of solvent（8,10,12times）,decocting time（1h,2h,3h）,decocting times（1,2,3times）,soaking time（15min,30min,45min）,on Forsythia suspensa suspensa decoction,and to determine the optimal extraction process.The effects of different ratios of lactose,dextrin,sugar powder,starch and ethanol concentration on the forming process of Forsythia suspensa Guiwei granules were investigated.4.To identify the properties of Forsythia suspensa Guiwei granules,TLC was used to identify the individual medicines in the granules,and TLC was used to identify the individual medicines in the granules.According to the relevant provisions of the granules,the granularity,water content,solubility,arsenic salts and heavy metals of Forsythia suspensa Guiwei granules were examined.HPLC was used to establish forsythiaside and forsythiaside glycosides in Forsythia suspensa Guiwei granules.Content determination of chlorogenic acid and other indicators.Results:1.1.The results of cell proliferation inhibition test and effective component transfer rate in vitro showed that 20%ethanol extract had the best comprehensive effect in 8ethanol extract concentrations.After 24hours,48hours and 72hours,the inhibition rates of20%ethanol extract on MDA-MB-231 cells were 65.05%,78.03%,87.84%,70.61%,73.92%,75.20%and 46.25%respectively.2.Study on the pharmacodynamics of ethanol extract from Forsythia suspensa and Guiwei Decoction on breast cancer cells in vitro.（1）It was found that the inhibitory effect of ethanol extract of Forsythia suspensa Guiwei Decoction on human breast cancer MDA-MB-231 cells and MCF-7 cells increased with the prolongation of the action time,the greater the inhibitory effect,the greater the drug concentration and the greater the inhibitory effect.The IC₅₀0 of MDA-MB-231 cells were 0.7959 mg/mL,0.6989 mg/mL,0.5107 mg/mL,respectively,after 24,48 and 72hours of action.The IC₅₀0 of MCF-7 was 0.5642mg/mL,0.5576mg/mL and 0.5335mg/mL,respectively.The concentration of IC₅₀0 decreased with the increase of time,P<0.05.The data had statistical significance.（2）For MDA-MB-231 cells,the cell migration rate was 67.26%.The cell migration rates of low-dose group,middle-dose group and high-dose group were 15.96%,14.47%and 6.78%,respectively.For MCF-7 cells,the cell migration rate was 36.73%.After adding drugs,the cell migration rates of low-dose group,middle-dose group and high-dose group were 8.56%,3.66%and-1.65%,respectively,which indicated that Forsythia suspensa could be reunited.The greater the drug concentration,the greater the cell migration ability,P<0.05,the statistical significance of the data.（3）For MDA-MB-231 cells,the concentration of Fructus Forsythiae Guiwei Decoction was 0.5 mg/mL,0.7 mg/mL and 0.9 mg/mL,respectively.The apoptotic rates were 10.88%,16.85%,17.92%for 12 h,13.08%,17.9%,28.51%for 24h,31.16%,35.94%and 37.04%for 48h,respectively.For MCF-7 cells,the concentration of Fructus Forsythiae Guiwei Decoction was 0.3 mg/mL,0.5 mg/mL,0.7 mg/mL,respectively.The apoptotic rates were 16.33%,19.84%and 20.52%at 12h,respectively,and 22.78%,32.63%and37.33%at 24 h,respectively.The apoptotic rates were 29.31%,36.52%and 42.73%at 48h.The effect of Fructus Forsythiae Guiwei Decoction on human breast cancer MDA-MB-231cells and MCF-7 cells could promote cell apoptosis in varying degrees,and the apoptotic rate increased with the increase of action time and drug concentration,P<0.05,the data have statistical significance.（4）For MDA-MB-231 cells,the positive control group inhibited cell proliferation by increasing the proportion of G₀/G₁ phase at 24h after treatment.With the increase of time,the proliferation of MDA-MB-231 cells was inhibited by increasing the G₂/M phase.At 24h,Forsythia suspensa Guiwei Decoction promoted cell proliferation,but with the increase of time,the proliferation of MDA-MB-231 cells was inhibited by increasing the proportion of G₀/G₁ phase.For MCF-7 cells,the positive control group,Forsythia suspensa and Guiwei Decoction for 24h,48h and 72h all inhibited cell proliferation by increasing the ratio of G₀/G₁ phase.The longer the drug concentration was,the stronger the inhibition was,P<0.05.The data had statistical significance.3.The results of orthogonal experiment showed that A₃B₁C₃D₃ was the best extracting technology.With the repose angle,bulk density,solubility and relative humidity as the indexes,the results of the molding process were 1.4 times as much as those of the additives.The proportion of the additives was sugar powder:dextrin（3:1）,and the concentration of ethanol was 70%.Three batches of Forsythia suspensa Guiwei granules were prepared by this process.4.The prepared Forsythia suspensa Guiwei granules are brown-yellow to brown granules with sweet taste and slight bitterness.TLC identification of Forsythia suspensa,honeysuckle,licorice,angelica and Daxuetong in Forsythia suspensa Guiwei granules was carried out.The identification results of these five medicines are negative,non-interference,clear spots and strong specificity.Three batches of Forsythia suspensa Guiwei granules were granulated according to the Chinese Pharmacopoeia of the 2015 edition.The determination methods of chlorogenic acid,forsythiaside A and forsythiaside in Forsythia suspensa Guiwei granules were established by gradient elution with acetonitrile and 0.1%formic acid as mobile phase.The accuracy,precision,repeatability,stability,linearity and range of the methods were investigated.The contents of chlorogenic acid,forsythiaside A and forsythiaside were determined.The mean values of chlorogenic acid,forsythiaside A and forsythiaside were 6.74 mg/g,18.20 mg/g and 2.03 mg/g,respectively.It was suggested that the limit should be set at 80%of the mean values.Conclusion:1.The optimum ethanol extracting concentration of Fructus Forsythiae GUIWEI decoction was 20%.2.Fructus Forsythiae Guiwei Decoction inhibits the proliferation of human breast cancer MDA-MB-231 cells and MCF-7 cells,affects cell migration ability,promotes cell apoptosis,and increases the proportion of G₀/G₁ phase,thus inhibiting cell DNA synthesis.3.The optimum extraction technology of Forsythia suspensa tail decoction was 12times of solvent,decocting three times,one hour each time,soaking for 45minutes;the optimum forming technology of Forsythia suspensa tail granules was 1.4times of supplementary materials,the proportion of supplementary materials was sugar powder:dextrin（3:1）,the ethanol concentration of wetting agent was 70%.4.The prepared Forsythia suspensa Guiwei granules are brown-yellow to brown granules with sweet taste and slight bitterness.The TLC identification of Forsythia suspensa,Honeysuckle,Licorice,Angelica sinensis and Daxuetong in Forsythia suspensa Guiwei granules has clear spots,strong specificity and no negative interference.According to the Chinese Pharmacopoeia of the 2015 edition,the granularity,solubility,moisture,heavy metals and arsenic of the three batches of granules have been studied.The method for determination of chlorogenic acid,forsythiaside A and forsythiaside in Forsythia suspensa Guiwei Granules was established,and the methodological investigation was carried out.The results showed that the method was simple,rapid and accurate,and suitable for quality control and evaluation of Forsythia suspensa Guiwei Granules.