The Efficient Compounds Recognition Research Of Tibetan Medicine Rhodiola Crenulata Prevents Oxidative Stress

In recent years,the higher morbidity and mortality of cardiovascular diseases?CVD?have seriously threatened people's health."China Cardiovascular Diseases Report 2017"shows that the prevalence and mortality of CVD in China with increased frequency.CVD death accounts for the highest proportion of disease deaths in residents.Prevention and treatment of cardiovascular disease has been the focus of research consistently.In China,the Qinghai-Tibet Plateau has become a high-risk area for cardiovascular disease because of its geographical environment,high altitude polycythemia is the most common.Oxidative stress refers to the process of oxidative stress injury caused by accumulation of ROS in the body or cells as a result of the increase of oxygen free radical?or?scavenging capacity.It is an important influencing factor for the development of cardiovascular diseases.At present,it is an important way to prevent and treat these diseases by inhibiting excessive oxidative stress.Therefore,how to find effective drug to teat cardiovascular disease is imminent.Rhodiola crenulata?Hook.f.et Thoms.?H.Ohba is a perennial herb of Rhodiola L,which Tibetan medicine name“Suo Luo Ma Bao”,it is included in the“Chinese Pharmacopoeia”version 2015?Part 1?Variety,with tonifying Qi and invigorate the circulation of blood,Tongmai and preventing asthma effect.Modern pharmacological studies have shown that it has significant pharmacological functions suchasanti-aging,anti-oxidation,cardiovascularfunctionimprovement,anti-radiation,anti-tumor,anti-fatigue,and immunity enhancement.In this paper,the traditional Tibetan medicine Rhodiola crenulata,which is a commonly used Tibetan medicine for the prevention and treatment of high altitude cardiovascular disease.The research is about Rhodiola crenulata intervene the oxidative stress,a key influencing factor in the process of cardiovascular disease,and revealed the potential molecular mechanism of the antioxidative stress of the drug.Its provides a theoretical basis fou further research on the treatment of cardiovascular diseases.Objective1.Cell experiment was used to investigate the effect ofRhodiola crenulata absorption solution on against oxidative stress.On the basis of this effect,UHPLC-LTQ-Orbitrap tandem mass spectrometry was used to analyze the extract of Rhodiola crenulata and its intestinal absorption solution,and to identify the intestinal absorption compounds of Rhodiola crenulata.2.When got the analysis of intestinal absorption solution compouds,the molecular mechanism of Rhodiola crenulata intestinal absorption solution was predicted on against oxidative stress by network pharmacology,and the potential active components and target targets were obtained,and then the molecular docking technique was used to verify the predicted ingredients and targets in the network pharmacology.The possible antioxidative stress active substances and mechanisms of action were further elaborated.Methods1.The intestinal absorption solution of Rhodiola crenulata and the blank intestinal absorption solution were prepared by the method of ectropion enteric capsule.The H9c2 myocardial cells were used as the research object.The hydrogen peroxide?H₂O₂?solution was used as a mold making agent to establish the oxidative stress model.The survival rate,morphology and apoptosis of H9c2 myocardial cells induced by H₂O₂ were investigated by given intestinal absorption solution of Rhodiola crenulata.2.It is proved that the intestinal absorption of Rhodiola crenulata are effective on against oxidative stress,then the UHPLC-LTQ-Orbitrap high resolution mass spectrometer was applied to the rapid separation and detection of methanol extract,intestinal absorption liquid and blank intestinal absorption solution of Rhodiola crenulata.The molecular formula of the compound is derived,and the fragment ion is analyzed by some control products,literature and database,and the qualitative analysis of the compound is carried out,and intestinal absorption compounds was identified.3.Combine UHPLC-LTQ-Orbitrap high resolution mass spectrometry analysis to obtain the ingredients of Rhodiola crenulata intestinal absorption solution,use the OpenBabel GUI software to set the format conversion to get the InCHI number information,and the InCHI number to the BAT-MAN database platform to obtain the target information;the interaction between targets and targets was constructed based on the STRING database;target information was imported into the DAVID database andfunctionalpathwayenrichmentanalysiswasperformedtoobtain GOTERM_BP_ALL and KEGG_PATHWAY information data related to oxidative stress.By extracting core targets associated with oxidative stress function and pathways,combined with BATMAN-TCM target prediction results,the chemical components that may act on the core target were screened from the components of the drug-containing intestinal absorption fluid,and"component-target-function-pathway"network diagram were drawn using Cytoscape 3.5.1 software.4.In order to further verify the results of pharmacological prediction of antioxidant stress network of Rhodiola crenulata,select the main active components and target targets in the network.Through the corresponding structure treatment,Maestro 11.1 software is used to optimize the energy of its compounds and optimize its force field as OPLS₂005.The corresponding target protein modifications,dehydration molecules,hydrogenation and other optimization,and the original ligand as the center to generate docking active sites,and finally begining the molecular docking.Results1.Rhodiola crenulata intestinal absorption solution can increase the survival rate of H9c2 cardiomyocytes,maintain cell morphology and reduce apoptosis.It indicates that it has a good antioxidant stress damage effect,which indirectly indicateswell antioxidant stress effect of Rhodiola crenulata,and to a certain extent,it shows that Rhodiola crenulata is mediated oxidative stress reaction to achieve the effect of treating cardiovascular disease.2.In the positive and negative ion mode,the ingredients of Rhodiola crenulata,intestinal absorption liquid and blank intestinal absorption liquid were identified.56components of the extract were identified through the database of Rhodiola and the public database.A preliminary identification of 47 prototype compounds through intestinal absorption was identified,and compared with 12 control products,10components were identified from Rhodiola crenulata,includingSalidroside,Tyrol,Gallic acid,Ethyl gallate,Catechin,Kaempferide,Kaempferol,Caffeic acid,Protocatechuic acid and Vanilla acid.3.Network pharmacology analysis results shows the"compounds-targets-pathway-function"network has been successfully established.Results 43 compounds related to oxidative stress were selected from the ingredients of intestinal absorption solution,including Salidroside,Ethyl gallate,Catechin and so on.58 core targets related to oxidative stress were enriched,and three actions were mainly intervened in cell apoptosis,calcium signaling pathway and nerve activity ligand receptor interaction.It involves biological processes such as cell death,regulation of oxidoreductase activity,positive regulation of oxidoreductase activity,oxidative stress reaction and nitric oxide metabolic process,which play a protective role in Rhodiola crenulata previent oxidative stress injury.4.Molecular docking results showed that there were 42 effective docking ingredients and 40 targets in the antioxidation stress network of Rhodiola crenulata,and the absolute value of the Glide gscore score of the ligand and target receptor was mostly greater than 5,indicating that the ligands of each compound could be well combined with the target receptor,and the combination of some ligands and receptors has been reported in the literature,indicating the accuracy and reliability of the method,thus improving the accuracy and reliability of the results of network pharmacology prediction analysis,and further validating the potential molecular mechanism of the antioxidation stress of Rhodiola crenulata.ConclusionInthisstudy,Combinedwiththestudyofcellexperiments,UHPLC-LTQ-Orbitrap high resolution mass spectrometry,network pharmacology,and molecular docking techniques,this study found that the Tibetan medicine Rhodiola crenulata has a good effictive on pevent oxidative stress,which effectively reveals the potential material basis and molecular role of antioxidation stress in Rhodiola crenulata.The molecular mechanism of prevention and treatment the cardiovascular and cerebrovascular diseases is preliminarily elucidated.This study integrates multi-disciplinary technology research ideas and methods to provide some scientific references for Tibetan medicine to prevent and treat cardiovascular diseases such as high altitude polycythemia.