Study On Chemical Constituents And Hypoglycemic Activity Of Morus Nigra L.from Xinjiang

Morus Nigra L.is an ancient medicinal and food plant resource with Xinjiang characteristics.It is widely used in uygur folk hypoglycemic treatment,and it is a folk botanical medicine with regional characteristics.In our research group,α-glucosidase inhibition model was used in vitro to verify that the extract of leaves of Morus Nigra L.can effectively inhibitα-glucosidase activity.The polyhydroxy alkaloids,flavonoid glycosides and polysaccharide compounds in the extracts of leaves of Morus Nigra L.showed strong inhibitory activity againstα-glucosidase.However,what kind of active ingredients play a key role and its pharmacological effects need to be further studied.For this reason,the separation and structural identification of chemical constituents and the in vitro hypoglycemic activity of chemical constituents from Morus Nigra L.were studied in this study.In addition,the docking conformation ofα-glucosidase protein with small molecules was analyzed by molecular docking software,and the small molecules with hypoglycemic activity were screened.This study promoted the development of xinjiang Morus Nigra L.hypoglycemic medicine and laid a foundation for experimental data.The main research work and results are as follows:1.Separation of monomer compoundsBy solvent extraction,TLC,silica gel column chromatography,gel column chromatography,preparative liquid chromatography and other chromatographic separation techniques,eight monomer compounds were isolated from the n-butanol phase of leaves Morus Nigra L.The compounds were identified as Adenosine（1）,Benzyl-β-D-glucopyranoside（2）,Roseoside（3）,Episyringare-sinol-4-O-β-D-glucopyranoside（4）,Syringaresinol-4′-O-β-glucopyranoside（5）,Quercetin-3-O-β-D-glucopyranoside（6）,Dibutyl phthalate（7）,Quercetin-3,7-di-O-β-D-glucopyranoside（8）,by LC-MS and NMR.Among them,Episyringaresinol 4-O-β-D-glucopyranoside（4）and Syringaresinol-4′-O-β-D-glucopyranoside（5）were isolated from the leaves of Morus Nigra L.for the first time,which solved the problem that the diastereomers of bistetrahydrofuran lignans isolated had little difference in structure and were difficult to be obtained by synthesis or conventional separation methods.Compounds 1～5 and 8 were isolated for the first time,which enriched the chemical composition of monomer in Morus Nigra L..2.In vitro activity experimentDifferent polar extracts of 80%ethanol extract from dried leaves of Morus Nigra L.were used as test materials,acarbose was used as positive control,p-nitrophenol glucopyranoside（PNPG）was used as substrate to screen the extracts with strong inhibitory activity againstα-glucosidase.The results showed that all the five components had inhibitory effects onα-glucosidase,and the water extract from leaves of Morus Nigra L.had the strongest inhibitory effect with IC₅₀ of 0.394 mg/m L.In the experiment of enzymatic kinetics,K_II is 0.29 mg/m L while K_SI is 0.04 mg/m L,K_II>K_SI,by which it can be judged that the inhibition type of the residual aqueous phase is linear mixed reversible inhibition and the competitive effect is greater than that of non-competitive.Theα-glucosidase inhibitory activity of 8 monomers isolated from the n-butanol fractions of Morus Nigra L.leaves showed that:Compounds 1～8 showed high inhibitory activity at the concentration of 1 mg/m L,and the inhibitory activity was Quercetin-3,7-di-O-β-D glucopyranoside（85.20%）>Quercetin-3-O-β-D-glucopyranoside（55.33%）>Episyringaresinol-4-O-β-D-glucopyranoside（52.82%）>Adenosine（52.56%）>Benzyl-β-D-glucopyranoside（43.01%）>Dibutyl phthalate（38.39%）>Roseoside（37.51%）>Syringaresinol-4′-O-β-glucopy ranoside（31.44%）.Four monomer compounds with the highest inhibitory activity were selected for pairwise and pairwise compatibility.Four monomeric compounds with the highest inhibitory activity were selected for pairwise and pairwise combinations.Experimental results showed that the inhibitory rate ofα-glucosidase was 71.26%after the combination of Quercetin-3-O-β-D-glucopyranoside and Adenosine（1:1）,and the inhibitory effect ofα-glucosidase was increased by nearly 20%compared with that of single use,indicating a synergistic effect between the two.3.Molecular docking simulation experimentThe monomer separation experiment procedure is tedious,time-consuming and labor-intensive,and the amount of compounds obtained is very small,it can not meet the requirements of the later system activity experiment.Therefore,TCMSP database was used to screen reported components in mulberry leaves according to the medicinal principle,and molecular docking database of chemical components in Mulberry leaves was established to expand the screening range of hypoglycemic chemical components.The molecular docking computer simulation method was used to evaluate the docking energy and the number and strength of hydrogen bonds binding to key amino acids of hypoglycemic activity targets.From 269 mulberry leaf small molecules and 8 small molecule compounds isolated from Morus Nigra L.leaves,25 mulberry leaf small molecules and 8 small molecular compounds with strong inhibitory effect onα-glucosidase were screened.This study accelerated the research progress of active monomer ofα-glucosidase inhibitor.