| OBJECTIVE:Polygonum divaricatum L.,a Mongolian medicinal herb,is a dry root or whole grass of Polygonum divaricatum L.And it was included in the monographs of Mongolian Medicine,such as"Wisdom of Wisdom","Pharmaceutical White Crystal"and"Innocent Mongolian Medicine".It is sour,bitter and astringent,as well as cool,thin,light,sputum and blunt.It has the effects of clearing away heat and stopping diarrhea.It can also treat Enteritis,dysentery,vomiting,osteoporosis,water and fire scald and various tumors after compatibility.And the clinical effect is very remarkable.Moreover,Polygonum divaricatum L.is widely distributed in China,and the resource reserves are very large.Therefore,in this study,Polygonum divaricatum L.was used as the research object,and the flavonoids were extracted from this herb by reflux extraction technique.The extraction and purification process was optimized,and the isolated flavonoids were identified.Reverse molecular docking and network pharmacology were used to predict the possible pharmacological activities and the mechanisms of total flavonoids of Polygonum divaricatum L.?TFP?.After clarifying its potential antitumor activity,we systematically study the anti-tumor effect of TFP-?in vitro and reveal its mechanism of action on human breast cancer MDA-MB-231 cells;then on this basis,the safety of TFP was evaluated in vivo.The result can provide experimental basis for the development and utilization of Polygonum divaricatum L.,additionally provide theoretical support for the development of new anti-tumor drugs for traditional Chinese medicine.METHODS:?1?TFP were extracted by reflux extraction technique.And the total flavonoids extraction process was optimized by using TFP yield as the index.The purity of TFP was taken as the index and the total surface area was optimized by column chromatography combined with response surface methodology to purified the flavonoids.Then the TFP were separated to obtain monomer compound,and the structure of the monomer compound is determined by UPLC-MS/MS technology.After clarifying the material basis of Polygonum divaricatum L.,the quality standards were established from the aspects of external form,microstructure,medicinal material quality and active ingredient content,and quality control.?2?Reverse virtual screening technology was used to determine the possible protein targets and to clarify the potential pharmacological activity of TFP.Then the mechanisms of this action were elucidated through network topology analysis.?3?MTT assay,scratch test and Transwell invasion assay were used to examined the proliferation,migration and invasion of human breast cancer MDA-MB-231cells,respectively.Then qPCR and Western-blot were used to detect the expression of apoptosis-related genes and proteins.In order to explore the potential mechanism of TFP-?induced tumor cell apoptosis.?4?The in vivo safety of TFP was evaluated by acute toxicity test and 28-day oral toxicity test in rats.RESULTS:?1?The optimal extraction process were:ethanolconcentration 70%,ratio of material to liquid 1:10,extraction time 2.2 h,extraction temperature 74°C,TPF yield was2.42±0.05%;optimal purification process were:22 mL of the extract,adjust the pH to 5,and elute with 65%ethanol at a flow rate of 2 mL/min to obtain a purity of 39.88%±0.19%of TFP.After that,the TFP was separated and the parent ion and the second fragment ion of the compound were obtained by UPLC-MS/MS technology.The structure of the monomer compound was determined by comprehensive software database and literature reports.As a result TFP mainly contained 7 flavonoids:Rutin,Isoquercitrin,Hyperoside,Avicularin,Quercetin,Kaempferol,and Myricetin.Afterwards,the quality standards of Polygonum divaricatum L.were studiedfrom the aspects of traits,powder microscopic identification,thin layer chromatography,moisture,total ash,acid-insoluble ash,heavy metals and harmful elements,extracts and content determination.?2?Firstly,the 7 flavonoids isolated from Polygonum divaricatum L.were drawn into a three-dimensional structure using ChemDraw software,and then reversed and docked with the 117423 pharmacophore models respectively contained in the Pharma DB database by the Discovery Studio 2016 client software.The Pharmacophores with a Fit Value of 0.8 or higher were used to identify the main active components of TFP and the related diseases through literature search and multiple databases such as TTD.A total of 14 diseases were involved,14targets related to anti-tumor effects,7 anti-tumor related targets,5 targets for treating neuro-degenerative diseases,and 13 functional targets such as biological enzymes.Therefore,it is speculated that TFP may have potential anti-tumor activity.Then a network based on the selected tumor-related targets was constructed and network topology analysis was performed:TFP is most likely to affect ERK/MAPK and PI3K/AKT signaling pathways,and affect cell cycle?CDK?,metabolism?PEPCK?,Migration?MMP2?and apoptosis?Bcl-2,Bcl-xl?to exert its anti-tumor effects.?3?MTT assay was used to detect the inhibition rate of cells in MDA-MB-231 cells treated with different concentrations of TFP-??50,120,200,250,300?g/mL?for 24 h and 48h,respectively.The TFP-?had a certain proliferation inhibition effect on breast cancer cells at 24 h and 48 h in a dose-and time-dependent manner,and its proliferation inhibition rate was 56.63%and IC500 was 243.7±5.6?g/mL at 48 h.As the concentration increases,the shape of the cells also changes significantly:the cells were mostly round,the number was reduced,the volume was reduced;and the adherence was not firm and the phenomenon of shedding occurred.MDA-MB-231 cells were treated with low,medium and high concentrations of TFP-??120,200,300?g/mL?for 48 h.The effects of TFP-?on tumor cell migration and invasion were investigated by scratch test and Transwell invasion assay:The low,medium and high concentration of TFP-?reduced the scratch closure by67.06%,81.24%and 92.81%respectively,and thecell invasion inhibition rates were23.91%,49.98%and 72.83%,respectively at 48 h.Western-blot experiments showed that the expression of ERK,p-ERK,AKT and p-AKT protein in cells was significantly lower than that in the control group after 48 hoursof TFP-?treatment in MDA-MB-231 cells,and PI3K and the expression level of p-PI3K protein was not significantly different from that of the control group?P>0.05?.The results of qPCR showed that the gene expression level of C-FOS,MMP2,CDK2,Bcl-2,Bcl-xl and PEPCK were significantly lower than that of the control group?P<0.05?in a dose-dependent manner.?4?Finally,the in vivo safety of Polygonum divaricatum L.was systematically evaluated.The acute toxicity and short term toxicity were observed by feeding different doses of the aqueous extract of Polygonum divaricatum L..The results showed that there was no obvious abnormality in behavior,activity,diet and excretion of mice in each dose group in the acute toxicity test.There was no significant difference between the blank group and the control group?P>0.05?.There were no abnormalities in the color,size and proportion of the important organs?heart,liver,Spleen,lung and kidney?of the mice.And no death of the mice occurred.According to the value of the acute toxicity dose classification table,it was determined that Polygonum divaricatum L.were not poisonous.In the 28-day oral toxicity test,no abnormalities were found in the mental activity,eating and drinking,and excretion of the rats.There was no significant difference in index change?P>0.05?.No death occurred in28 days,hematological parameters?hemoglobin,red blood cells,white blood cells,neutrophils,monocytes,lymphocytes?and blood biochemical indicators?Blood sugar,triglycerides,cholesterol,aspartate aminotransferase,alanine transoxygenase,urea nitrogen,creatinine,albumin and totalprotein?were not significantly different from the control group?P<0.05?.CONCLUSION:Polygonum divaricatum L.,the Mongolian herb,mainly contained flavonoids.And TFP-?can perform good anti-tumor effect and inhibit the proliferation,migration and invasion of human breast cancer MDA-MB-231 cells in vitro.Further exploring its mechanism,TFP may inactivate the transduction of ERK/MAPK,PI3K/AKT signaling pathways,thereby producing a role in inhibiting cell proliferation,migration and invasion.In addition,TFP-?reduced the gene expression of Bcl-2,Bcl-xl,CDK,PEPCK and MMP2 genes,thereby promoting apoptosis,inhibiting cell cycle progression,migration and glucose metabolism to exert its anti-tumor effect.Moreover,acute toxicity and subchronic toxicity were not observed in Polygonum divaricatum L.,indicating that they were basically safe. |
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