Study On Hypoglycemic Components Of Fenugreek And Its Hypoglycemic Mechanism

Fenugreek is the dried ripe seed of Trigonella foenum-graecum Linn. It’scultivated in some of the countries of Asia, Africa, and the Mediterranean region. Ithas been used for both medicinal qualities and as food ingredient for many years.Among the pharmacological properties of fenugreek, its hypoglycemic activity hasgenerated the greatest interest for a long time because of the public health concernsrelated to the increasing incidence of diabetes. However, the hypoglycemic activecomponents and the hypoglycemic mechanism of fenugreek are uncertain yet. In thisstudy, we aimed to clarify the hypoglycemic active components of fenugreek andelucidate their hypoglycemic mechanism. As reported, three kinds of the highestcontent of potential active components are polysaccharides (gum or soluble fiber),saponins and flavonoids. There contents were20%~25%,4%~8%,1%~2%,respectively. In this study, the component analysis, systematic study of hypoglycemicactivities and hypoglycemic mechanisms will be carried out in these three potentialactive components.1. Preparation and component analysis of fenugreek flavonoids components.The heating reflux extraction method and macroporous resin purification methodare designed to prepare the flavonoids components from defatted fenugreek seeds, andthe UPLC-LTQ/MS is used to analyze the component in it. As a result, by extractedwith70%ethanol solution via heating reflux, flavonoids containing extract wasprepared with the yield of flavonoids was1.45%. Through purified by polyamideresin and D101macroporous resin, the flavonoids components was obtained fromflavonoids containing extract, and the yield was1.42%and the purity was62.28%.Through analysis by UPLC-LTQ/MS,95%peaks were identified and they were19flavonoid compounds.2. Preparation and component analysis of fenugreek saponins components.The heating reflux extraction method and macroporous resin purification methodare designed to prepare the saponins components from defatted fenugreek seeds, andthe UPLC-QTOF/MS is used to analyze the component in it. As a result, by extractedwith70%ethanol solution via heating reflux, saponins containing extract was prepared with the yield of saponins was9.02%. Through purified by DM130macroporous resin, the saponins components was obtained from saponins containingextract, and the yield was6.05%and the purity was78.56%. Through analysis byUPLC-QTOF/MS,90%of the peaks were identified and they were55saponinscompounds.3. Preparation and monosaccharide composition analysis of fenugreekpolysaccharides components.The cold-soak extraction method is used to extract polysaccharides componentsfrom the remaining residue of fenugreek after the extraction of flavonoids containingextract or flavonoids containing extract. A two-objective (yield and purity) orthogonaltest was used to optimize the extraction of highly purified polysaccharides. Threeparameters were examined: solid-to-solvent ratio, extraction number, and extractiontime. The capillary zone electrophoresis method is used to analyze monosaccharidecomposition of fenugreek polysaccharides. The result showed, under the optimalextraction conditions (solid-to-solvent ratio of1:50[g/mL, water],3extractions, and1.0h at10°C with stirring), fenugreek galactomannan (F-GAL) was obtained with ayield of24.12%, purity of98.54%, and galactose/mannose ratio of1:1.08.4. Study on the hypoglycemic activities of three components and theirhypoglycemic mechanisms.Firstly, the STZ-induced diabetic rat is used to study the hypoglycemic activitiesof flavonoids components, saponins components and polysaccharides components.Four-objective including survival, fasting blood glucose (FBG), oral glucose tolerancetest (OGTT) and weight loss are used to evaluate the hypoglycemic activitiessystematically. The hypoglycemic activities experiments showed, all of the threecomponents having the significant hypoglycemic activities, and they were allhypoglycemic active components of fenugreek. However, compared topolysaccharides components, the hypoglycemic effects of flavonoids components andsaponins components were better. Such as, flavonoids components and saponinscomponents had better regulation on FBG and weight loss. Moreover, saponinscomponents could increase survival of the diabetic rats.Then, through analyzing pancreatic tissue morphology, blood insulin levels,hepatic glycogen content, muscle glycogen content, α-glucosidase inhibitory activityand HepG2insulin resistance, the hypoglycemic mechanisms of three components areelucidated. The hypoglycemic mechanisms experiments showed there are differences among the hypoglycemic mechanisms of the three components. Flavonoidscomponents corrects high blood glucose level by repairing damaged islet, promotinginsulin secretion, increasing hepatic glycogen content, inhibiting α-glucosidaseactivity and improving insulin resistance. Saponins components corrects high bloodglucose level by repairing damaged islet, increasing hepatic glycogen and muscleglycogen contents, inhibiting α-glucosidase activity and improving insulin resistance.Polysaccharides components corrects high blood glucose level by increasing hepaticglycogen content, inhibiting α-glucosidase activity, improving insulin resistance anddelaying glucose absorption (according to the OGTT). As noted above, onlyflavonoids components can promote insulin secretion and has the strongest α-glucosidase inhibitory activity among the three components. In addition, compared toflavonoids components and polysaccharides components, saponins components canincrease hepatic glycogen and muscle glycogen contents more, and improve insulinresistance more effectively. While only polysaccharides components has the ability todelay glucose absorption for its high viscosity characteristics.Lastly, the ultrafiltration mass spectrometer (ultrafiltration MS) method is usedto screen the α-glucosidase inhibitors from the flavonoids components which has thestrong α-glucosidase inhibitory activity. Through analysis, five compounds areidentified as active compounds. They are apigenin6,8-di C-hexoside, orientin,isoorientin, vitexin and isovitexin. In addition, the ultrafiltration MS is also used toscreen the α-glucosidase inhibitors from the Radix Astragali extract. Through analysis,seven compounds are identified as active compounds. They are calycosin-7-O-β-D-glucoside, biochanin A, calycosin-7-O-β-D-glycoside-6″-O-malonate, ononin,calycosin, formononetin-7-O-β-D-glycoside-6″-O-malonate and formononetin.5. Metabolomic applied to study the diabetes mellitus (DM) treated mechanismsof three components.The metabolomic analysis will be used to study the DM treated mechanisms ofthe flavonoids components, saponins components and polysaccharides components.The UPLC-QTOF/MS detection method, principal component analysis (PCA) methodand orthogonal partial least squares-discriiminate analysis (OPLS-DA) method areused to analyze the metabolic changes in urine and blood of components treateddiabetic rats, untreated diabetic rats and normal rats to find out the biomarkers. Theresult showed there are differences among the regulated pathways and biomarkers totreat DM of the three components. The regulated pathways and potential biomarkers of flavonoids components arehistidine metabolism (biomarker is urocanic acid), tryptophan metabolism (biomarkeris picolinic acid), phenylalanine metabolism (biomarker is hippuric acid), the Krebscycle (biomarker is citric acid), hepatic enzyme activity of glucose metabolism(biomarker is D-glucuronic acid), lysoPCs metabolism (biomarkers are lysoPC (20:2),lysoPC (15:0) and lysoPC (16:0)), sphingolipid metabolism (biomarker issphinganine), arachidonic acid metabolism (biomarker is arachidonic acid) anddiabetes complicated kidney damage (biomarkers are creatinine and2-phenylethanolglucuronide).The regulated pathways and potential biomarkers of saponins components arehistidine metabolism (biomarker is urocanic acid), tryptophan metabolism(biomarkers are picolinic acid and L-tryptophan), phenylalanine metabolism(biomarker is hippuric acid), the Krebs cycle (biomarker is citric acid), hepaticenzyme activity of glucose metabolism (biomarker is D-glucuronic acid), fatty acidmetabolism (biomarker is valerylglycine), lysoPCs metabolism (biomarkers arelysoPC (20:2), lysoPC (18:2), lysoPC (15:0)), lysoPEs metabolism (biomarkers arelysoPE (20:4), lysoPE (20:2) and lysoPE (18:2)), arachidonic acid metabolism(biomarker is arachidonic acid), diabetes complicated kidney damage (biomarkers arecreatinine, indoxyl sulfate and2-phenylethanol glucuronide) and liver damage(biomarkers are cholic acid and deoxycholic acid).The regulated pathways and potential biomarkers of polysaccharides componentsare histidine metabolism (biomarker is urocanic acid), tryptophan metabolism(biomarker is picolinic acid), phenylalanine metabolism (biomarker is hippuric acid),the Krebs cycle (biomarker is citric acid), hepatic enzyme activity of glucosemetabolism (biomarker is D-glucuronic acid), lysoPCs metabolism (biomarkers arelysoPC (20:2), lysoPC (18:2), lysoPC (15:0) and lysoPC (16:0)), lysoPEs metabolism(biomarkers are lysoPE (20:4), lysoPE (20:2) and lysoPE (18:2)), sphingolipidmetabolism (biomarker is sphinganine), arachidonic acid metabolism (biomarker isarachidonic acid) and diabetes complicated kidney damage (biomarkers are indoxylsulfate and2-phenylethanol glucuronide).In conclusion, flavonoids components, saponins components andpolysaccharides components are hypoglycemic active components in fenugreek, andall of them having significant hypoglycemic activities. Under the same oral dose, thehypoglycemic effects of flavonoids components and saponins components are better than polysaccharides components. The hypoglycemic mechanisms and the DM treatedmechanisms of the three components are illustrated, and we can see there are somedifferences among the mechanisms of them. In a word, this study confirmed themedicinal qualities of flavonoids components, saponins components andpolysaccharides components of fenugreek in the treatment of DM.