Study On Chemical Constituents Of Sophora Davidii (Franch.) Skeels

For thousands of years,medicinal plants and their extracts have been used to treat various diseases through unique theories and therapeutic systems.Many evidences showed that the herbal medicine and its active ingredients have the advantages of low toxicity,few side effects,and easy availability.Sophora davidii（Franch.）Skeels,fabaceae family,is shrubs or dungarungas,growing in river valley dunes and bushes on hillside roads below 2500 m.It is mainly distributed in Guizhou,Ningxia,Yunnan and Sichuan provinces of China.Its flowers,roots,leaves and fruits are traditional medicines used for the treatment of sore throat,hematochezia,cough and dysentery,etc.The previous research of our research team showed that the flavonoid-rich compound from Sophora davidii（Franch.）Skeels exhibited good antidiabetic activity in vitro and in vivo.The flavonoid-rich extract from S.davidii can significantly promote the transport of glucose transporter 4（GLUT-4）in L6 cells,and increase the uptake of glucose through the AMPK pathway,thereby improving insulin resistance in KKAy mice.In order to further study the chemical composition of Sophora davidii（Franch.）Skeels,determine its active substance basis,and discover more novel compounds with anti-diabetic potential,we systematically carried out the isolation,purification and structural elucidation of chemical substances.Therefore,in the present research,based on biological activity guiding,we used various techniques to carry out a large number of purification,separation,and structural identification of the chemical substances of the active ethyl acetate fraction of S.davidii,and used a GLUT-4 translocation assay system based on L6 cells stably expressed IRAP-m Orange to screen the in vitro pharmacological effects of the isolated pure compounds.Finally,the specific chemical components of Sophora davidii（Franch.）Skeels with good anti-diabetic activity were discovered.In this study,59 compounds were separated and identified through various spectroscopic methods,of which 18 were new compounds.The isolated compounds have many types,mainly including flavonoids,stilbenes,benzofurans,lignans and others.Moreover,In addition,the anti-diabetic activity of some selected compounds in vitro was tested.The flavonoid-rich extract of Sophora davidii（Franch.）Skeels has shown good anti-diabetic activity in the pharmacological activity test in vivo and in vitro.A total of 59 compounds were obtained from this plant,18 new compounds.There are 34 flavonoids,of which 8 new flavonoids1-5,8,23,and 34 were identified as davidone A（1）,davidone B（2）,davidone C（3）,davidone D（4）,davidone E（5）,cyclolicoisoflavones A₃（8）,davidone F（23）and davidone G（34）by 1D and 2D NMR and MS.The remaining compounds are identified as leachianone A（6）,brosimacutin C（7）,crotalarin（9）,gerontoisoflavone A（10）,griffonianone H（11）,7,2’-dihydroxy-4’,5’-methylenedioxyisoflavone（12）,genistein（13）,7-methoxy-daidzein（14）,7-hydroxy-3-（4-hydroxyphenyl）-1-benzopyran-4-one（15）,2’,4’,5,7-tetrahydroxy-isoflavone（16）,isoluteolin（17）,calycosin（18）,7,4’-dihydroxy-3’,5’-dimethoxy-isoflavone（19）,7,6’-dihydroxy-3’-methoxy-isoflavone（20）,3’-methoxydaidzein（21）,acacetin（22）,naringenin（24）,liquiritigenin（25）,8-demethylfarrerol（26）,6-methylgenistein（27）,dihydrokaempferol（28）,3,4’,7-trihydroxyflavanone（29）,kaempferol（30）,6-methyl-kaempferol（31）,4’-methylisoliquiritigenin（32）and isoliquiritigenin（33）.There are 11 stilbene compounds,of which 8 new compounds 35-42 were identified as davidiol E（35）,davidiol F（36）,davidinin B（37）,davidinin C（38）,davidinin D（39）,davidinin E（40）,davidinin F（41）and davidinin G（42）by 1D and 2D nuclear magnetic resonance spectroscopy and mass spectrometry.The remaining compounds are identified as pterostilbene（43）,pinostilbene（44）and 3,4’,5-trihydroxystilbene（45）;There are 5benzofuran compounds,of which compounds 46 and 49 are new compounds,identified as shandougenine C（46）and davidinin A（49）.The remaining compounds are identified as shandougenine B（47）,shandougenine A（48）,and 2-（2’,4’-dihydroxy）-5,6-dioxomethylene benzofuran（50）;5 lignans were identified as davidinol A（51）,（+）-lirioresinol-A（52）,（+）-syringaresinol（53）,burselignan（54）and3,3’-bis（3,4-dihydro-6-methoxy-2H-1-benzopyran）（55）;5 other compounds,identified as HNEA（56）,dibutyl phthalate（57）,isovanillic acid（58）and p-hydroxybenzoic acid（59）.The absolute configurations of compounds 1-5,23,34-36 and 49 were determined by NMR chemical shift value DP4+analysis and electronic circular dichroism（ECD）calculation.The purified and separated 23 compounds（1-11,16-17,23,35-36,43,46-50 and 52）were screened for the transmembrane activity of GLUT-4,and the fluorescence intensity was increased by 1.28-3.00 times respectively.Among them,compounds 1-7,17,23,35-36,47,50 and 52showed good GLUT-4 transfer membrane activity,especially the compound lachianone A（6）and compound isoluteolin（17）have the best effects,respectively increasing 2.00 and 1.60 times.Subsequently,the effect of compounds 6 and 17 on the translocation of GLUT-4 was observed with a laser confocal microscope,and it was found that the membrane transfer activity was obvious when the concentration was10μg/m L.