Study On Chemical Constituents Of Codonopsis Pilosula(Franch.) Nannf. From Yunnan

Codonopsis pilosula（Franch.）Nannf.,is a plant of the genus Codonopsis,It is widely recognized and applied in Yunnan province.It is widely used in the high altitude areas in Yunnan Province,western Yunnan and southern Yunnan.The folk people are mostly used for tonic and meat stew.Yi medicine is used to treat the asthenia of body and weak body,emaciation and so on.The Tibetan medicine is used to cure the spleen deficiency,food and loose stool,limb weakness,palpitation,and shortness of breath.The traditional Chinese medicine has the effect of passing through the meridian,nourishing the gas and producing blood,supplementing the lungs and promoting the cough and helping digestion,and the modern research shows that the national drugis in the immune system,The digestive system and blood system all play a regulatory role.Objective:1.the chemical constituents were extracted and separated from the dried root of the stinky ginseng in Xundian County of Kunming,Yunnan province.The structure of the compound was identified by modern spectroscopy,and the chemical components of the root of the ginseng were revealed.2.the molecular docking activity of cholesterol metabolism was predicted and verified by the cholesterol metabolism activity test,in order to obtain the chemical constituents of the anti tumor bioactivity in the root of the ginseng,and provide the theoretical basis and experimental basis for the medicinal herbs of the national drug.Method:1.the extract was extracted by 85%ethanol（80 L x 4 x 24 h）by extracting the root of dried stinky ginseng.The extract was extracted after extraction.The extract was dissolved by water suspension and extracted with ethyl acetate in turn.The extract was extracted by the extraction solution.2.the columns of ethyl acetate,MCI gel CHP 20P,RP-18 and Sephadex LH-20were used respectively,and all kinds of monomers were finally purified by HPLC and TLC.The structures of various compounds were identified by spectroscopic methods,including 1H-NMR,13C-NMR,COSY,HSQC,HMBC,IR,ROESY,MS and UV.3.the Maestro molecular docking technique was used to analyze the interaction between the new chemical constituents isolated from the stinky ginseng and the target of cholesterol metabolism,and to predict the molecular mechanism of the new compounds in the metabolism of cholesterol.the CCK-8 detection method was used to detect the effects of three compounds with high,medium and low concentrations on the proliferation of HepG2 cells.Real-time fluorescence quantitative PCR was used to detect the effect of three concentrations of high,medium and low levels of new compounds on the key genes of cholesterol synthase in HepG2 cells.Result:1.through the separation of ethyl acetate,31 compounds were obtained,o f which 24 known compounds were（1）trandirin（2）4-Caffeoylquinic Acid（3）polycarbonate（4）（+）-Catechin 7-O-B-D-Glucopyranoside（5）（-）secoisolariciresi nol（6）pilosulyne C（7）7R,8S-2-（4-hydroxy-3-methoxyphenyl）-3-hydroxymethyl-5-（3-methoxyallyl）-7-methoxycoumaran（8）Burselignan（9）（+）-Diasyringaresinol（10）fragransin B₃（11）（7S,8R）-dihydrodehydrodiconiferyl alcohol（12）5-Meth oxy-（+）-isolariciresinol（13）（7S,8S）-1-（4-Hydroxy-3-methoxyphenyl）-2-O-（6-hydro xy-2-methoxy-4-omegahydroxypropylphenyl）propane-1,3-diol（14）salicifoliol（15）Tetvadeca-trans,trans-6,12-diene-8,l0-diyne-1,5,14-tril（16）7R,8S-2-（4-hydroxy-3-methoxyphenyl）-3-hydroxymethyl-5-（3-methoxyallyl）-7-methoxycoumaran（17）car uilignan D（18）Syringaldehyde（19）gentisic acid（20）Protocatechuic acid（21）Eugenic acid（22）Isovanillin（23）Vanillic acid（24）Phenylacetic acid。2.new compounds were separated from 7 new compounds,namely,new compounds 1（E）-tetradeca-12-en-8,10-diyne-1,6,14-triol,new compound 2（5E,7S,8R,13E）-7,8-dihydroxypentadeca-5,13-dien-9,11-diynoic acid,new compound 3（4E,12E）-tetradeca-4,12-dien-8,10-diyne-1,6-diol,new compound 4（4E）-dien-8,10-diyn-1-ylacetate,newcompound5（2E,4E,12E）-2-ethylidene-6,7-dihydroxytetradeca-4,12-dien-8,10-diynal,new compound 6 4-（1R,2R）-1,3-dihydroxy-2-（2-hydroxy-4-（2-hydroxy-4-））,new natural product 7.3.the new constituents of the stinky ginseng have better binding activity with the relative receptor protein.The better targets for docking with the 1 molecules of the new compounds are SREBP-2,HMGCR,SQLE,and the better targets for docking with the new compounds 2 molecules are HMGCR and SQLE.new compound 1-5was used to determine the activity of cholesterol metabolism.The results showed that5 new compounds had no obvious toxicity to HepG2 cells,and the new compound 1of 20 mu M could significantly reduce the RNA level of sterol regulator element binding protein,three hydroxyl three methyl coenzyme A reductase and squalene epoxide（P<0.05）.The mRNA level of the lysozyme 9 had no obvious effect.20 mu M new compound 2 significantly reduced the mRNA level of three hydroxyl three methyl coenzyme A reductase and squalene epoxide（P<0.05）,and had no significant effect on the mRNA level of sterol regulator element binding protein and preprotein invertase（prealbumin）lysozyme 9.20 mu M new compound 3,new compound 4,and new compound 5 have no obvious inhibitory effect on sterol regulator element binding protein 2,three hydroxyl three methyl coenzyme A reductase,squalene epoxide,and preprotein invertase wit lysozyme 9.