Identification Of Phenolic Compound In Amomum Tsaoko Crevost Et Lemaire And Their Hypoglycemic Activity

Amomum tsaoko Crevost et Lemarie is a Zingiberaceae herb,mainly distributed in northern Vietnam and southwestern China,its main components are volatile oils,phenols,flavonoids,and so on.Polyphenols are widely found in plant foods and have antioxidant,anti-inflammatory,anti-tumor,and anti-diabetic activities.They are beneficial to human health,especially in the prevention of metabolic disorders and chronic diseases.The current research on Amomum tsaoko has mainly focused on volatile oils,and there is a lack of research on the analytical identification of Amomum tsaoko phenolics and their hypoglycemic activity.In this paper,the phenolic compounds in Amomum tsaoko were obtained by graded extraction,and the phenolic compounds were qualitatively and quantitatively analyzed by UHPLC-ESI-Q-TOF-MS/MS technique.The hypoglycemic activity of Amomum tsaoko phenolic compounds and their potential mechanisms were investigated by establishing a mouse model of type 2 diabetes mellitus（T2DM）.It provides the theoretical foundation for the development and exploitation of Amomum tsaoko phenolic compounds.The main research contents and results are as follows:（1）The phenolic compounds of Amomum tsaoko were extracted with ethanol,and then the petroleum ether fraction（PF）,chloroform fraction（CF）,ethyl acetate fraction（EF）,n-butanol fraction（BF）,and water fraction（WF）were obtained by fractional extraction with solvents of different polarity.The total phenolic content（TPC）and total flavonoid content（TFC）of each fraction of Amomum tsaoko were determined,in which the TPC of EF（10.26 mg GAE/g DW）and TFC（1.14 mg RE/g DW）were the highest,followed by BF,WF,CF,and PF.The phenolic compounds of Amomum tsaoko were mainly concentrated in EF.Using UHPLC-ESI-Q-TOF-MS/MS technique,a total of50 active compounds were identified from the five fractions of Amomum tsaoko,including 2 organic acids,11 phenolic acids and their derivatives,18 flavonoids and their derivatives,14 proanthocyanidins,and 5 other compounds.Among them,EF was enriched with the most types and contents of substances,and flavonoids,such as epicatechin and catechin,were the main phenolic compounds in EF.In addition,EF showed good antioxidant activity(DPPH and ABTS with IC₅₀=0.17 and 0.07 mg/m L,FRAP of 546.10 mg VCE/g DW)andα-glucosidase inhibitory activity(IC₅₀=20.14μg/m L)in vitro.Correlation analysis showed that kaempferol-3-O-glucoside and naringenin in the fractions were significantly and positively correlated with DPPH,ABTS,and FRAP.Epicatechin,isoquercitrin,and hypericin were significantly positively correlated with FRAP.Protocatechualdehyde,quercetin,kaempferol-3-O-glucoside,naringenin,and p-coumarin were positively correlated withα-glucosidase inhibitory activity.（2）The T2DM mouse model was induced by a high-fat diet combined with STZ.The EF with the highest TPC,TFC,in vitro antioxidant and hypoglycemic activities,was selected to intervene in T2DM mice to investigate the in vivo hypoglycemic activity.The results showed that EF,especially high doses of EF,could significantly reduce the food and water intake of T2DM mice,EF can decrease fasting blood glucose（FBG）by 24.12%,increase serum insulin（INS）content by 35.21%and improve oral glucose tolerance（OGTT）impairment.In addition,EF could significantly reduce serum total cholesterol（TC）,decrease triglyceride（TG）and low-density lipoprotein cholesterol（LDL-C）by 29.98%and 52.36%,increased the serum high-density lipoprotein cholesterol（HDL-C）by 111.24%in T2DM mice.The results suggest that EF may exert hypoglycemic effects by modulating the dysregulation of blood glucose and lipid metabolism in T2DM mice.（3）The protective effect of EF on liver and intestinal microbiome regulation was investigated in T2DM mice.The serum aspartate aminotransferase（AST）and alanine aminotransferase（ALT）activities,liver malondialdehyde（MDA）content,superoxide dismutase（SOD）,and glutathione peroxidase（GSH-Px）activities,tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）levels,16S r RNA gene sequencing were measured to analyze the effects of EF on liver function,oxidative stress,inflammatory factors,and intestinal microflora in T2DM mice.The results showed that EF significantly reduced serum ALT and AST activities and liver MDA content in T2DM mice,increased liver SOD and GSH-Px activities by 50.98%and 87.57%,decreased TNF-αlevels by 52.68%and IL-6 levels by 34.82%.It also improved the richness and species composition of the intestinal microbiota,increased the abundance of Bacteroidetes and Lactobacilli,decreased the abundance of Firmicutes and the ratio of Firmicutes to Bacteroides,indicating that EF can reduce liver tissue damage,improve oxidative stress and inflammatory response in the liver,and regulate the composition and abundance of the intestinal microflora.