Study On The Inhibitory Effect Of Miao Medicine Periplocae Radix Et Rhizoma On The Growth Of Lung Cancer Cell A549

In recent years,lung cancer has become a major disease endangering life and health.According to global cancer statistics 2018 released by WHO,there will be 2.1million new lung cancer cases and 1.8 million deaths in 2018.Seriously,the 5-year survival rate of lung cancer in China is less than 20%.Therefore,it is imminent to find effective drugs to prevent and treat lung cancer.Miao medicine is a kind of medicine used to prevent and cure diseases under the guidance of the theory of Miao medicine or used by the Miao people.It is an important part of traditional Chinese medicine.Miao medicine is characterized by more single prescription,fewer compound medicines,more fresh medicines and fewer dry medicines.The processing of Miao medicine is very peculiar and has a very fancy function.And,Miao medicine has good curative and quick effect.Periploca forrestii Schltr.belongs to the genus of Periploca in Asclepiadancae.It is called Hvofa mongb dleib“Wa Man Sai”.It is recorded in the 2003 edition of"Quality Standards for Traditional Chinese Medicines and Ethnic Medicines in Guizhou Province".According to the principles of"cold disease treated with heat"of Miao medicine theory,and the experience of regional medication,Hei Gu Teng was used to treat rheumatism,arthralgia,fall injury and other diseases.Modern pharmacological studies have shown that Hei Gu Teng has inhibitory effects on the growth of various lung cancer cells,such as NCI H460,A549.However,its pharmacodynamic substances and molecular mechanism are still unclear.In this thesis,taking the traditional Miao medicine Periplocae Radix et Rhizoma as the research object,and Study on the intervention process of Periplocae Radix et Rhizoma in lung cancer cell A549.Preliminarily revealed the potential molecular mechanism of anti-lung cancer effect of Periplocae Radix et Rhizoma.And,provided a theoretical basis for further research on its treatment of lung cancer.Objective1.The inhibitory effects of petroleum ether,chloroform,ethyl acetate,n-butanol and water extracts on the growth of lung cancer cells A549 were investigated.On the basis of the pharmacodynamics,the strong phytochemical fractions of the extracts were separated.2.Predict the pharmacodynamic material and molecular mechanism of anti-lung cancer,and obtain its potential active ingredients and targets and pathways.And,the molecular docking technique was used to validate the predicted active ingredients,chemical constituents isolated for the first time from Periplocae Radix et Rhizoma,and target.Further elaborate its anti-lung cancer active substances and mechanisms.Methods1.SRB method was used to investigate the inhibition of the extracts from Periplocae Radix et Rhizoma on the growth of lung cancer cell A549.2.Extract and isolate the effective parts of Periplocae Radix et Rhizoma,and identify the structure of the isolated monomer compounds by spectroscopy(¹H-NMR,¹³C-NMR,IR,COSY,HMBC).3.Predict the active ingredients,molecular functions,biological processes and pathways of the anti-lung cancer of Periplocae Radix et Rhizoma by network pharmacology technology,and construct the“component-target”network.Combining with literature,analyze the key targets and pathways of the anti-lung cancer of Periplocae Radix et Rhizoma.4.Molecular docking technology was used to predict the binding patterns and affinities of 42 active components in the network and compounds isolated from Periplocae Radix et Rhizoma.to 4 key target proteins,which were further validated by literature analysis.Results1.In addition to petroleum ether fraction,the extract fractions of Periplocae Radix et Rhizoma did inhibit the groeth of lung cancer A549 cells,and the order of activity was n-butanol,water,chloroform and ethyl acetate fraction.2.Six compounds were isolated from the n-butanol fraction of Periplocae Radix et Rhizoma,including 2,6-Dimethoxy-1,4-benzoqquinone?1?,Periplogin?2?,2-Thiophenecarboxylic acid?3?,Periforgenin A?4?,21-O-methyl-5-pregnene-3?,14?,17?,20,21-pentaol?5?,?3?,14?,17??-3,14,17-trihydroxy-21-methoxypregn-5-en-20-one?6?,?3?,14??-3,14-dihydroxy-21-methoxypregn-5-en-20-one?7?,5?-pregnane-3?,4?,21-trihydroxy-20-one?8?.Compounds 1,3 were isolated for the first time from Asclepiadancae.Compounds 5,6,7 were isolated for the first time from Periplocae Radix et Rhizoma.Compounds 8 is a new natural product.3."component-target" network was successfully constructed,including 42 active compounds related to lung cancer,53 key nodes of active components and disease.The main molecular functions include regulating the binding of NF-?B and the activity of ubiquitin ligase;biological processes include oxidative stress,regulating transcription and growth;signal pathways are mainly concentrated in cell proliferation,apoptosis,stress and ubiquitin-mediated water.There are 16 signaling pathways such as solution and MAPK.The key targets of anti-lung cancer are EGFR,GRB2,TP53,and NF-?B,which mainly play a role through PI3K-AKT or MAPK signaling pathway.4.Molecular docking results showed that 48 components could be docked effectively with key targets,mainly based on the hydrogen bond and van der Waals interaction between hydroxyl and hydroxyl groups in their mother nucleus.The docking scores of compounds with NF-?B and TP53 were higher than those of EGFR and GRB2.The higher docking scores were Protocatechuic aldehyde,Emodin,3-O-Caffeoyl quinic acid,2-Thiophenecarboxylic acid,2,6-Dimethoxy-1,4-benzoquinone.ConclusionThis thesis integrates cell experiment,chemical composition separation,network pharmacology and molecular docking multidisciplinary techniques and methods to clarify the growth inhibition effect of Miao medicine Periplocae Radix et Rhizoma.on lung cancer cell A549,revealing that the main regulatory signaling pathway of its anti-lung cancer effect is PI3K-AKT pathway.The n-butanol site with the strongest growth inhibition activity of lung cancer cell A549 contains Cardiac glycoside,C₂₁ steroids,Quinones and so on.Among them,Protocatechuic aldehyde,Emodin,3-O-caffeoyl quinic acid,2-Thiophenecarboxylic acid,2,6-Dimethoxy-1,4-benzoquinone were the main candidate substances for the anti-lung cancer effect.