Proanthocyanidin B₂ Stabilizes Postprandial Blood Glucose And Its Distribution In Buckwheat

Proanthocyanidins?PC?is a mixture of bioflavonoids made up of different amounts of?+?-catechin?C?or?-?-epicatechin?EC?.As the most important proanthocyanidin dimer in food,proanthocyanidin B₂?PB₂?has received extensive attention due to its various physiological functions.Previous studies have found that proanthocyanidins have a-glucosidase?AGH?inhibitory activity,but there are few corresponding research on PB₂ in terms of its inhibitory activity and mechanism of AGH.On the other hand,the number of patients with diabetes?Diabetes mellitus,DM?is increasing year by year,and the threat to human health is increasing.In addition to the treatment of existing hypoglycemic and sugar-control drugs,it is urgent to explore the natural sources of phytochemicals to prevent and adjuvant therapy diabetes.As the preferred staple food source for diabetic patients,buckwheat is rich in flavonoids that can lower blood sugar,and the glycemic index?GI?of buckwheat is low.Previous studies on the distribution of phytochemicals such as quercetin and rutin in buckwheat have been studied,but the contribution of proanthocyanidins to the low GI characteristics of buckwheat remains to be further explored,and the distribution of PB₂ and proanthocyanidin monomer?C and EC?in different buckwheat kernels is still lacking systematic analysis.This study demonstrated the inhibitory effect of PB₂ and proanthocyanidin monomer on AGH and its inhibitory mechanism by in vitro chemical experiments,UV spectroscopy,fluorescence spectroscopy,atomic force microscopy and molecular simulation docking techniques.The effects of three proanthocyanidins on the digestion and absorption of glucose in the small intestine were studied in vitro.The sucrose load of mice was used to further verify the regulation of PB₂ and proanthocyanidin monomer in vivo.Finally,the selection of the Shaanxi Yulin planting experiment was conducted under the same conditions.The 14 tartary buckwheat varieties and 4 common buckwheat varieties collected and stored were used as research materials.The distribution of C,EC and PB₂ in different buckwheat kernels was analyzed by HPLC.This study explored the inhibitory mechanism of three proanthocyanidins on AGH in vitro,and analyzed the effects of PB₂ and proanthocyanidin monomer on glucose digestion and absorption by in vitro tissue and in vivo experiments,and identified three proanthocyanidins in different varieties of buckwheat grains.The distribution law in order to provide a theoretical basis for revealing the mechanism of PB₂ and proanthocyanidin monomer regulating blood sugar and the material basis of low GI characteristics of buckwheat,and at the same time,it provides a theoretical basis for screening buckwheat varieties suitable for new hypoglycemic food development materials and diabetic staple food sources.The main findings of this paper are as follows:?1?Study on the inhibition and mechanism of PB₂ on AGH:Under the experimental conditions,PB₂ has the strongest inhibitory activity against AGH,about twice as much as C and EC,and belongs to the mixed inhibition type.The semi-inhibitory concentrations(IC₅₀)of C,EC and PB₂ inhibiting AGH were 1.12±0.03?M,0.95±0.02?M and 0.40±0.02?M?n=3?,respectively.The inhibitory effect is reversible.Fluorescence and thermodynamic analysis show that all three inhibitors can quench the endogenous fluorescence of the enzyme and pass electrostatic attraction and Van der Waals forces and hydrogen bonding interact with enzymes.Atomic force microscopy and molecular simulation docking studies have shown that the combination of inhibitors and enzymes alters the microenvironment around the enzyme and,in combination with the amino acid residues in the active center of the enzyme,reduces AGH activity and thus hinders glucose production.?2?PB₂ inhibits glucose digestion and absorption in the small intestine and stabilizes postprandial blood glucose:The in vitro tissue of the animal was used to simulate glucose absorption in the small intestine,and the inhibitory effect of PB₂ and proanthocyanidin monomer on AGH and its effects on the sugar transporters SGLT1 and GLUT2 are studied.The effect of PB₂ on postprandial blood glucose in mice was studied by in vivo animal experiments.The results showed that in the intestinal everted model,the IC₅₀0 for inhibiting glucose production by C,EC and PB₂ were?1.49±0.02?×10^-44 M,?0.67±0.01?×10^-44 M,?2.07±0.06?×10^-5M?n=3?,PB₂ inhibitory activity was superior to its structural monomer.PB₂ mainly inhibits glucose transmembrane transport by GLUT2 as carrier,and the concentration of glucose entering the blood is reduced by 56%after adding inhibitor.The largest influence on SGLT1 is C,the blood glucose concentration was reduced by 35%.the mouse sucrose load test results showed that C,EC and PB₂ still have the effect of inhibiting sugar absorption in vivo,and PB₂ inhibition is superior to its structural monomer C and EC.At the same time,the blood glucose level of the mice in which sucrose and PB₂ were ingested decreased by 3.7 mmol/L compared with the control group.?3?The distribution of PB₂ and its structural monomers in buckwheat kernels:The contents of C,EC and PB₂ in 18 kinds of buckwheat kernel powder and shell powder were determined by RP-HPLC.Within the scope of this study,PB₂ was mainly distributed in the kernel powder of tartary buckwheat seeds,which was 6¹0 times higher than that of common buckwheat,and Fengku No.2 has the highest content of PB₂?21.48±0.06 mg/100g DW?.In the tatary buckwheat kernel,the average level of PB₂ was 5.2 times than that of shell powder,and the distribution of PB₂ was more uniform in common buckwheat shell powder and kernel powder.In addition,the distribution of proanthocyanidin monomer in buckwheat kernels was studied.It was found that C were mainly distributed in tartary buckwheat shell powder,and the highest content of tartary buckwheat was Qianku No.5?39.31±0.28 mg/100g DW?.The content of C in common buckwheat kernel powder was 1.8times than tatary buckwheat.The EC was mainly found in the common buckwheat kernel powder,and the Longtianqiao No.1?13.68±0.18 mg/100g DW?had the highest content.The above results indicate that PB₂ has a significantly lower AGH inhibitory activity than its structural monomer.Meanwhile,PB₂ and proanthocyanidin monomer exert blood glucose regulating effects by inhibiting the production of glucose in the small intestine and inhibiting the transport of glucose in intestinal epithelial cells.PB₂ is distributed in both common and tatary buckwheat,and is mainly enriched in tartary buckwheat kernel,suggesting that buckwheat is the preferred staple food source for diabetic patients.Its low GI properties and stable postprandial blood glucose effects may be partly due to proanthocyanidins in buckwheat.Therefore,this study has a further understanding of active substances that can stabilize blood sugar,and also provides a new perspective for the development of buckwheat,especially tatary buckwheat,stable postprandial blood glucose food.