Study On The Discovery Of Terpenoids And Inhibitory Activities Of 11β-HSD1 From Euphorbia Sikkimensis

Natural products are characterized by structural diversity and easy binding to biological macromolecules and unique 3D structure,which determine they have incomparable advantages in participating in the physiological process of life.Therefore,natural products play an irreplaceable role in new drug research and development,and are an important source for the discovery of drug lead structures and drug candidates.According to reports,from 1981 to 2019,a total of 1881 new drugs have been approved worldwide,of which 23.5%are derived from Natural products(NP),Natural products Botanical,and Natural product derivative(ND).At present,plants are the most studied resources for natural pharmacologists,and they are also important sources of drug leads and drug candidates,which continue to receive extensive attention from pharmacologists.China has a vast territory with abundant resources and diversity of environment.And China has a wide variety of plants with complex secondary metabolites.The discovery and clinical application of drugs such as morphine,paclitaxel,artemisinin,and aspirin(acetylsalicylic acid)are the most famous successful cases of natural products in drug development.Therefore,continuous research on natural products of plants has broad development space and huge market value.11β-Hydroxysteroid dehydrogenase 1(11β-HSD1)is an enzyme that metabolizes glucocorticoids[1]in the body.It can catalyze the interconversion between active GC and inactive GC,which mainly acts as a reductase in metabolically active tissues.Increasing the level of GC induces the occurrence and development of a variety of metabolic diseases such as type 2 diabetes,obesity,hypertension,and dyslipidemia.Therefore,inhibiting the activity of 11β-HSD1 is a new strategy for the treatment of metabolic diseases.Based on the search for natural inhibitors of 11β-HSD1,we chose active and LC-MSdirected screening methods and selected Euphorbia sikkimensis Boiss.as the research object of this study.Euphorbia sikkimensis Boiss belongs to the Euphorbiaceae,which major secondary metabolites are terpenoids.However,there are few studies on the natural medicinal chemistry of Euphorbia sikkimensis Boiss.Since the ethanol extract of Euphorbia sikkimensis has significant 11β-HSD1 inhibitory activity,we study on the natural medicinal chemistry of it.On the one hand,it is beneficial to enrich the structural diversity of secondary metabolites.On the other hand,it is conductive to discovery of lead compounds with the inhibiyory activity of 11β-HSD1 and lays a structural foundation for the development of new drugs against metabolic diseases.In this thesis,the isolated,purified,chemical constituents and biological activities of Euphorbia sikkimensis Boiss.were studied by detail.A total of 28 pure compounds(1-28)were isolated and identified from the ethyl acetate fraction of the Euphorbia sikkimensis Boiss.Including 18 triterpenes(1-18),2 diterpenes(19,20),1 flavonoid(21),1 ester(22),1 fatty acid(23),and 5 phenolic compounds(24-28).Amongst,compounds 1-10 are ten novel compounds.A rearrangement of Me-30(14→8)in(23E)-25-methoxy-eupha-14,23-diene3β,7α-diol(1)and a degradation of Me-27 in(23E)-3β-dihydroxy-27-noreupha-7,23-diene25-one(2).Which are two rare cases in the euphane-type triterpenoid family.It is an interesting phenomenon that(23E)-3β-hydroxy-25-methoxy-eupha-8,23-diene-7-one(4)and(23E)-3β-hydroxy-25-methoxy-lanost-8,23-diene-7-one(5)coexist in the same plant,sharing the same planar structure but belonging to different structural types of triterpenoids.In this thesis,a LC-MS/MS based screening method for in vitro inhibitory activity was successfully established.Which included recombinant plasmid construction,plasmid transfection,protein expression,protein purification,biochemical reactions,and analytical methods.Compounds 1-17 were screened using the screening method described above.The results showed that compounds 3,4,5,13 and 16 had better activity,and their IC50 values were 6.50±0.22 μM,1.31±0.34μM,9.38±0.64μM,8.27±0.33μM,18.74±5.11μM,respectively.Compounds 4 and 5 display an approximately 8-fold difference in activity,which is in accord with the fact that 4 binds to the active pocket of 11β-HSD1 better than that of 5 in the molecular docking experiments.The molecular docking experiment shows that the euphanetype triterpenoids have great potential to be inhibitors of 11β-HSD1.