Analysis Of The Composition Of Mulberry Leaf Polyphenols And Its Effect On Disaccharide Digestion

As an important secondary metabolite of plants,polyphenols are widely found in plant foods such as fruits and vegetables.The active intervention of dietary polyphenols on glucose metabolism has been widely concerned at home and abroad.China’s sericulture industry ranks first in the world,the abundant mulberry leaf resources are mainly used for sericulture production,its important edible and medicinal value has not been fully utilized and developed.Modern pharmacological studies have confirmed thehypoglycemia effect of mulberry leaves,but most of the studies used crude extracts of mulberry leaves as raw materials,and the molecular mechanism of lowering blood glucose is not uniform.Therefore,based on the analysis of the composition of mulberry phenols from six representative mulberry varieties,the D101 macroporous adsorption resin was used to establish the enrichment process of mulberry leaf polyphenols,the extract of mulberry leaf polyphenols were prepared.The key enzyme-disaccharidase,which is important for digestion and absorptionof carbohydrates,was used as the research object to investigate the inhibitory effect of mulberry polyphenols on disaccharidase from different sources,Caco-2 cell model of intestinal absorption was used to research the effect of mulberry polyphenols on glucose transport and study the basic mechanism.1.Analysis of total polyphenols and monomeric phenolics in mulberry leaf of different mulberry varieties.The polyphenol monomers of six varieties of mulberry leaf(Da 10,Kangqing 10,A2,69,Nongsang 14 and Yuesang 11)were analyzed and compared through HPLC.The results showed that 12 phenolic monomers such as chlorogenic acid and vanillic acidwere detected in the mulberry leaves of six varieties.The gallic acid was only detected in A2.There were great differences in the composition and content of mulberry phenolic monomers in various varieties.According to the total content of monomer phenols,the content of polyphenols from high to low was in order: A2>69>Kangqing 10>Da 10>Nongsang 14>Yuesang 11.Since both A2 and 69 were not widely cultivated in the production,Kangqing 10 was selected for subsequent experiments.2.Optimization of enrichment process of mulberry leaf polyphenols.Taking the adsorption and resolution ability of mulberry polyphenols as the index,we choosed the best performance of D101 resin from 6 macroporous resins.The separation and purification conditions were optimized and the optimized adsorption conditions were as follows: polyphenol concentration in sample solution of 1 mg/mL,pH of 3.0,flow rate of 1mL/min and the volume of 11 BV.After the adsorption,10% ethanol of 1 BV was applied to the column to wipe off the impurities.Then polyphenols were collected with 3 BV 30% ethanol and 3 BV 50% ethanol at a flow rate of 1 mL/min.After purification,the purity of total phenol increased from 20.03% to 75.67%.3.Inhibition of disaccharidase by mulberry leaf polyphenols.The mulberry leaf polyphenols had a strong inhibitory effect on disaccharidase both from mouse and Caco-2 cell,the inhibitory effect is gradually enhanced with the increase of concentration.The order of IC50 value was: murine sucrase > murine maltase > Caco-2 cell Maltase>Caco-2 cell sucrase,mulberry leaf polyphenols had the strongest inhibitory effect on the sucrasefrom Caco-2 cells.For the mouse-derived enzyme,the mulberry leaf polyphenols had a stronger inhibitory effect on maltase,that was,the mulberry leaf polyphenol played a role in lowering blood sugar mainly by inhibiting the activity of maltase in mouse;For the enzyme from Caco-2 cells,mulberry leaf polyphenol had a stronger inhibitory effect on the sucrase,that was,the mulberry leaf polyphenol played a role in lowering blood sugar mainly by inhibiting the activity of the sucrase in Caco-2 cells.4.The effect of mulberry polyphenols on glucose transport.The Caco-2 monolayer cell model was established to simulate the glucose transport process in the human small intestine,it was found that within the concentration range of 0.5-2 mg/mL,the mulberry leaf polyphenols could significantly inhibit the transport of glucose,its inhibition rate increased with time and dose.The effect of phlorizin(SGLT1 inhibitor)control group on glucose transport also showed the same trend,which revealed that the mulberry leaf polyphenols may inhibit glucose transport mainly by inhibiting SGLT1.The results of real-time PCR showed that the mulberry leaf polyphenols inhibited the expression of SGLT1,GLUT2,phospholipase(PLC),protein kinase A(PKA)and protein kinase C(PKC).Based on mRNA level,the possible signaling pathways were: mulberry leaf polyphenols down-regulated the expression of PLC and PKA,PLC further down-regulated the expression of PKC,PKC and PKA jointly down-regulated the expression of SGLT1,The effect that PKC activates GLUT2 to transfer to the apical membrane also reduced,thereby reducing the body’s glucose Absorption.