Investigation On Active Components And Mechanism Of Mulberry Branch

As a by-product in the sericulture industry,mulberry branches are not currently utilized effectively.Jialing 20 is an artificial triploids mulberry that widely cultivated in southwest China.In this paper,we screened and characterized the activities of small-molecule and polysaccharides from the mulberry branch of Jialing 20,elucidated its active components and mechanism of action,provided a foundation for the development and utilization of mulberry branches.The following seven aspects were specifically carried out:（1）In order to fully understand the chemical composition of small molecules,the extract was analyzed by UPLC-MS/MS,and the antioxidation,inhibition of Alzheimer’s disease,hypoglycemia and cardiovascular protection of the extract were evaluated.The results showed that 42 components,including alkaloids,flavonoids,and coumarins,were tentatively identified from Jialing 20 mulberry branch extract（MBE）.The IC₅₀values for the scavenging DPPH and ABTS radicals were,respectively,31.23±0.57μg/m L and 8.88±0.36μg/m L(IC₅₀ values of positive control VC were,respectively,4.41±0.19μg/m L and 8.79±0.41μg/m L).The IC₅₀ value for inhibitingα-glucosidase was 1.90±0.05μg/m L(IC₅₀ value of positive control acarbose was 0.03μg/m L).The IC₅₀ values for inhibiting acetylcholinesterase and butyrylcholinesterase were,respectively,179.47±0.38μg/m L and 101.82±3.37μg/m L(IC₅₀ values of positive control berberine were,respectively,1.27±0.03μg/m L and 57.41±0.21μg/m L).MBE（10μg/m L and 40μg/m L）significantly increased the survival rate of oxidized low density lipoprotein（ox-LDL）induced human umbilical vein endothelial cells（HUVECs）and significantly decreased the intracellular reactive oxygen species.（2）In order to determine the material basis of the extract against Alzheimer’s disease,magnetic nanoparticles Fe3O4@BCh E were synthesized,combined with HPLC-SPE-HRMS-NMR technique,the BCh E inhibitors in mulberry branch extract were screened and identified quickly and accurately,and the activity evaluation and molecular docking of the target inhibitors were carried out.Three potential inhibitors of BCh E,kuwanon G,kuwanon H and morusin were isolated and identified from MBE.They all displayed potential inhibitory activity and the IC₅₀ values were 70.96±2.62μM,10.91±0.39μM and 78.65±2.61μM,respectively,（galanthamine,the positive control was 60.35±6.88μM）.Molecular docking showed that kuwanon H interacted mainly with the amino acid residues of Asp70,Ser72,Thr284,Leu286,and Ser287 in the BCh E protein through hydrogen bonding,and generated Hydrogen bond interactions with Pro285 through water molecular bridge bond,thereby exerting inhibitory activity.This provided theoretical support for mulberry branch research and development in dealing with Alzheimer’s disease.（3）In order to determine the potential anti-diabetic material basis of mulberry branch extract,Fe₃O₄@Si O₂@AG were synthesized and combined with UPLC-MS/MS to rapidly screen and identify non-alkaloidα-glucosidase inhibitors in the extract.The results showed that fourα-glucosidase-inhibiting active components were screened from MBE:kuwanon H,kuwanon G,kuwanon C and morusin.The IC₅₀ values of kuwanon H and kuwanon G onα-glucosidase inhibition were 2.82±0.68,2.83±0.31μM respectively,and the potent inhibitory activity was stronger than that of positive DNJ(IC₅₀=7.04±0.82μM).The results of molecular docking showed that kuwanon G interacted with Asp242,Ser311 and Glu411 residues on the target protein through hydrogen bond;kuwanon H not only forms hydrogen bond with Thr310,Glu411,Asn415 residues,but also forms cation-πinteraction between imidazole ring and Hip280 residues;Hydrogen bonding interaction between DNJ and Hip7,Pro8 and Glu11 residues.These two isopentenyl flavonoids and DNJ had differentα-glucosidase action sites.（4）In order to determine the basis of potential anti-atherosclerotic substances in the extract,living cell extraction combined with HPLC-MS analysis was used to quickly screen the target active components from MBE to protect against ox-LDL induced HUVECs damage,and the activity and mechanism of the target substances were studied.The results showed that kuwanon G,kuwanon H and morusin in MBE could not only be captured by cells,but also decreased in the model group compared with the blank control group,it shows that it may be a potential active component.All the three compounds had good cytoprotective activity at the concentration of 20μM.According to the results of cell survival,MDA and SOD,it was found that kuwanon H had the best activity.Western Blot and RT-q PCR showed that kuwanon H might exert its antioxidant activity by activating Nrf-2/HO-1 signal pathway.RNAi showed that the effect of kuwanon H was reversed when the expression of Nrf-2 was inhibited.The above results provided theoretical support for the application of MBE and isopentenyl flavonoids in cardiovascular diseases.（5）In order to determine its main active part,Jialing 20 mulberry branch polysaccharide（MBP）was divided into three components（MBP30,MBP60 and MBP90）by ethanol fractional precipitation,and their physical and chemical properties,antioxidant and hypoglycemic activities were evaluated.The results showed that with the increase of ethanol concentration,the contents of total sugar,total protein and total phenol in different mulberry branch polysaccharides（MBPs）were different,and the molecular weight distribution decreased gradually.FT-IR showed that it has similar functional group composition,and SEM showed that its morphology is quite different.The composition of monosaccharides showed that MBPs was composed of glucose,galacturonic acid,galactose,arabinose,rhamnose and mannose,but their relative proportion was different.All MBPs groups had good scavenging activities of DPPH/ABTS/OH free radical and inhibitα-glucosidase activity,and their activity was MBP90>MBP60>MBP30.In addition,MBPs could protect Caco-2 cells from damage induced by H₂O₂,and its activity was MBP90>MBP60>MBP30.The results laid foundations for the separation,purification and structure identification of active polysaccharides.（6）In order to reveal the main active substances of hypoglycemic and scavenging free radicals in MBP90,under the guidance of activity,the sugars of mulberry branches inhibitingα-glucosidase and scavenging DPPH free radicals were separated and identified by column chromatography and identified by DEAE-52 and Sephadex G-100,respectively,and the activities of polysaccharides withα-glucosidase inhibitory activity were evaluated at animal level.The results showed that the Fr1-2 part of MBP showed the best hypoglycemic activity,and its hypoglycemic components were aminoglycosides with low molecular weight,including DNJ,fagomine and its glycosides,which were mainly glucosides（92.5%）and a small amount of galactosides（6.4%）.These compounds could reduce the levels of blood lipids and fasting blood glucose in T2DM mice induced by HFD/STZ,and protected their liver function.The MBP-3 showed the best antioxidant activity,and the content of uronic acid in the antioxidant polysaccharide was as high as 65.6%,and the most important residue wasα-1,4-Galp A.（7）The glucan with high content in MBP was isolated and purified by Sephadex G-100,and the structure and immunomodulatory activity of the purified polysaccharide was determined.After several times of purification,the polysaccharide MBP-2 part with high content in MBP was obtained,and its molecular weight,monosaccharide composition,FT-IR,methylation and NMR were analyzed and determined,meanwhile,the immunomodulatory activity of the polysaccharide was evaluated by macrophage RAW 264.7.The results showed that MBP-2 was a neutral sugar with a molecular weight of of about 29.1 k Da,which is mainly composed of the main chain of→4)-α-D-Glcp(1→and contains the glucan structure ofα-D-Glcp（1→and→4,6）-α-D-Glcp(1→.The polysaccharide can significantly enhance the release of NO from RAW264.7 cells,and play an immunomodulatory activity by activating the expression of related inflammatory cytokines m RNA,and then promoting the release of related cytokines（IL-6 and TNF-α）.Its immunomodulatory activity may be achieved by activating TRIF-dependent signaling pathways by TLR4 receptors.The above results provided a new idea for the application of mulberry branch polysaccharides.