Isolation,Screening,and Mechanism Of Hypoglycaemic Components From The Leaves Of Perilla Frutescens

P.frutescens(L.)Britt.is a traditional medicine-food herbs in China,and leaf is its most frequently used part.P.frutescens have important implications in various areas,such as food processing,medicine,and chemical industries,which due to its unique flavor,high nutritional value,and rich bioactive components,like phenolics,flavonoids,anthocyanins,tannins,and essential oil,thus exhibit a variety of activities,including antioxidant,antiallergy,antiinflammation,antitumor,and antibacterial activities.Several bioactive components in P.frutescens have been reported to be new potent?-glucosidase inhibitors.This underlies P.frutescens as a potential source of the hypoglycemic drug.At present,there are few reports of its effect on the prevention and control of diabetes.Moreover,the chemical composition,hypoglycemic effect in vivo,and the underlying mechanism remained unclear.In this study,the hypoglycemic activity of P.frutescens was evaluated by in vitro digestion model,diabetic rat and diabetic mouse model.The major active compounds were isolated and identified,the effect of P.frutescens on the gut microbiota of diabetic mice and its hypoglycemic mechanisms were also studied.The main research contents are as follows:The active compounds and functional activities of the Perilla frutescens leaf extract(PFLE)after the digestion process were measured.Results revealed that total phenolic content(TPC)and total flavonoid content(TFC)were significantly decreased after simulated digestion(ca.53%of phenolics and 40%of flavonoids).The IC₅₀ value of DPPH·scavenging activity and ABTS·⁺scavenging ability increased by 23%and56%,respectively,while ferric reducing antioxidant power reduced by 53%.For the inhibition ability on?-glucosidase,acetylcholinesterase and MCF-7 cell proliferation,their IC₅₀ values increased by 360%,197%,and 25%,respectively.Three phenolic acids and one flavonoid in PFLE were quantified by high?performance liquid chromatography,namely,(+)-catechin,clofibric acid,rosmarinic acid and Chlorogenic acid.Overall,although significant losses of the active components and biological activities occurred during in vitro gastrointestinal digestion,PFLE still exhibited certain antioxidant activity,?-glucosidase inhibitory ability,acetylcholinesterase inhibitory ability,and MCF-7 cell proliferation inhibitory ability,suggesting a potential for treatment and prevention of oxidative stress,cancer,diabetes,and Alzheimer's disease.Components with different polarities were obtained by PFLE extracted with different solvents,and the antioxidant activity,inhibition ability on?-glucosidase,acetylcholinesterase and tyrosinase were analyzed to assess the ability of PFLE in the prevention and treatment of oxidative stress relevant diseases.Components with the highest biological activity were quantified by high?performance liquid chromatography,and the diabetes a rat model was established.Among the samples,the ethyl acetate fraction possessed the high total phenolic(440.48?g GAE/mg DE)and flavonoid content(455.22?g RE/mg DE),the best antioxidant activity(the DPPH radical,ABTS radical,and superoxide anion scavenging activity,and ferric reducing antioxidant power were 1.71,1.14,2.40,1.29,and 2.4 times higher than that of control Vc,respectively),the most powerful?-glucosidase inhibitory ability with the IC₅₀ value of190.03?g/m L which was 2.2 folds higher than control acarbose.PFLEEA also showed high acetylcholinesterase,MCF-7 and Hep G2 cell proliferation inhibitory ability compared with other extracts.5 phenolic acids and 9 flavones in PFLEEA were detected by HPLC,luteolin,rosmarinic acid,rutin,and catechin were the dominant components.Animal experiments further demonstrated that the ethyl acetate fraction could significantly decrease the serum glucose level,food and water intake of streptozotocin-induced diabetic SD rats,increase the body weight,modulate their serum levels of TC,TG,HDL-C,and LDL-C,improve the histopathology and glycogen accumulation in liver and intestinal tissue.Moreover,the effect of PFLEEA on gut microbiota of C57BL/6 diabetic mice was also studied.In addition,we conducted high throughput RNA-seq and studied the transcriptome profiles of PFLEEA during diabetic treatment.After treatment with PFLEEA,a notable improvement in the blood sugar and lipids were observed in diabetic mice.PFLEEA alters the microbial ecosystem of the gut and its composition of diabetic mice,including reductions in the relative abundance of Bacillus,increases the relative abundance of beneficial bacteria,such as Clostridiales,Akkermansia,Enterobacteriaceae,and the relative abundance of bacteria which produce short chain fatty acids,like Bacteroidaceae,Lactobacillaceae.Transcriptomics analysis has shown that the treatment of PFLEEA upregulated 2 genes implicated in diabetes(Zfp422 and Ipmk),but downregulated 6 genes.Of these,Saa2,Atf3,and Gadd45?have been associated with insulin resistance and incident diabetes.The GO enrichment analysis results showed that the genes were mainly concentrated in biological process,and a total of 27 pathways were enriched using KEGG analysis,which Fox O signaling pathway was the most likely pathway.A total of 18 compounds in the ethyl acetate fraction of P.frutescens methanol crude extract(PFLEEA)were successfully isolated using thin-layer chromatography(TLC),silica gel column chromatography,and size exclusion chromatography(Sephadex LH-20),including three phenolic acids,methyl rosmarinate,and methyl gallate,and ursolic acid,respectively.seven flavonoids were also identified,such as baicalein-7-o-glucoside,luteolin,apigenin-7-o-glucuronic acid methyl ester,scutellarin(scutellarin b),5,7-dimethoxy-6-methyl-4'-methyl-dihydroflavone,p-hydroxycinnamic acid(trans cinnamic acid),etc.Three triterpenes(hawthorn acid,oleanolic acid,and oleanolane triterpene)and five other compounds(organic acids and fatty acids).In addition,32 compounds in PFLEEA were identified by means of UPLC-MS/MS analysis,including 13 phenolic acids,5 flavonoids,4 terpenoids,and 2 organic acids.These may be the main material basis for PFLEEA to play hypoglycemic effects.The influence of six probiotics on the biochemical compounds and biological functions of P.frutescens leaf were measured.Fermentation within 1-3 days could markedly enhance the content of active compounds and the activity of P.frutescens leaf.However,the biochemical compounds'contents and biological activities were significantly reduced after long-term fermentation.Among them,CICC 6038 and ATCC 334 could enhance the total phenolic content.For ATCC 8014,ATCC 53013,and CICC 6038,the flavonoid content increased significantly.The DPPH·scavenging activity and ABTS·⁺scavenging ability were significantly improved by ATCC 53013and CICC 6038,while the ferric reducing antioxidant power was significantly increased by CICC 6038.Surprisingly,ATCC 53013 could significantly improve the?-glucosidase inhibitory ability and acetylcholinesterase inhibitory ability of P.frutescens leaf.HPLC-DAD was further used to quantify the contents of these biochemical compounds,and it has been shown that the degradation and transformation were accrued during the fermentation process.Collectively,these results indicate that probiotic fermentation is an effective means to enhance active ingredient content and functional activity.These results provide valuable information for the diabetic's treatment by P.frutescens,and could provide the basis for further study of P.frutescens.