| Mulberry is a kind of medicinal and edible plant,and mulberry twig is the biggest waste in silkworm production,The extraction of mulberry bark and mulberry bark powder indicates the effects of hypoglycemia in vivo and in vitro.In recent years,sercin protein recovered in silk processing waste has been confirmed to have various biological activities,such as anti-ptraviolet activity,anti-oxidation,scavenging free radicals,and hypoglycemia activity.Ginkgo contains a variety of chemicals and therefore it has a variety of biologically active function.In order to develop a healthy food that has various functions of lowering blood sugar and antioxidantion,such as the popular high-sugar food-bread.It not only can diabetics eat,but also help to lower blood sugar.In the experiment,a single component bread is separately added into sercin powder or ginkgo powder,and in order to observe the contrast effect,the above mentioned mulberry branch powder,serein powder and ginkgo lyophilized powder are added into the flour to make a mixed bread,and then the activity of bread in vitro is detected.On this foundation,we further researched the hypoglycemia effects of bread on type 2 diabetic mice induced by the combination of streptozotocin(STZ)and high-fat diet.Furthermore,the physiological and biochemical function,the antioxidant function and the metabolic pathway were also elaborated.By sensory requirements,it can be concluded that the single sericin and ginkgo bread are higher than those of the mixed ternary bread,and the sensory requirements were slightly worse.DPPH free radicals and ABTS free radical scavenging ability were determined in vitro,indicating that the mixed bread had strong free radical scavenging ability with IC50 of 2.85±3.24 mg/mL and 156.31±9.31?g/mL,respectively It also has a strong function of inhibiting a-glucosidase activity with an IC50 of 0.31±2.94 mg/mL.The above results of in vitro activity assay indicate that the mixed bread has strong antioxidant capacity and ability to reduce oxidative stress.In this experiment,the mice were fasted for 12 hours,and each mouse was injected once with low dose STZ(90 mg/kg·BW).The diabetic mice were randomly divided into 4 groups:the model group,sericin bread group(SB),ginkgo bread group(GB),low-dose,middle-doseand high-dose ternary bread group(LTB,MTB and HTB).The final FBG level in SB was 8.83 mmol/L.The FBG level in 3 treated mixed groups in a state of fluctuation,but the tendency was decreased and in a dependent manner.The FBG level in HTB group went from 13.59±3.01 mmol/L to 9.26± 1.59 mmol/L,and the hypoglycemic rate was 31.84%,it significantly reduced the hypoglycemic of diabetic mice.The serum insulin levels in 5 treated groups were decreased in comparison with the model group,and the descent rate in HTB group was 53.26%higher than that SB group(43.41%)and GB group(40.72%),the results indicated that the functional bread may improve the insulin resistance.The body weight(BW)in SB and GB group went down and then went up,but the final BW(38.56±2.44 g,38.63±3.32 g)did not reach the initial BW(39.18±2.17 g,39.03±3.03 g).There was a tendency to lose BW first and then gain it slowly in HTB group and the final BW has reached to 40.69±2.86 g.On the 24th day,1.5 g/kg glucose were gavaged to measure glucose tolerance and insulin tolerance was measured by injection of 0.5 U/Kg insulin,it verified that bread can relieve abnormal glucose tolerance and insulin resistance.Bread also reduced the abnormally elevated glycosylated serum protein and serum liver functional enzyme activity(AST and ALT),reduce the TG and LDL-C levels and increased HDL-C level and regulated blood lipid metabolism.The results showed that bread also increased antioxidant activity in the liver of diabetic mice.In type 2 diabetic mice,the level of T-SOD decreased to 89.00±1.41 U/mgprot and was 78%of normal mice.In SB group and GB group,the level of T-SOD reached 1111.74±3.65 and 116.35±1.77 U/mgprot,respectively.In HTB group,the level of T-SOD reached 121.99±15.71 U/mgprot and was 7%higher than that the normal mice.The level of CAT in model group decrease to 83.65%in comparison with the normal mice.In SB group and GB group,the level of CAT in diabetic mice were 1.76%and 11.4%higher than that the normal mice.And in HTB group,the level of CAT reached 303.86±37.35 U/mgprot and was 13.37%higher that that the normal mice.MDA content in SB group and GB group decreased to 87.37%and 75.38%in comparison with model group,respectively.The level of MDA in MTB group dropped to 69.85%.The declining level of 8-OHdG in GB group and HTB group had statistically significant(P<0.05,P<0.001).Meanwhile,the HE stain in SB group and GB group had mild inflammation,but in other 3-treated mixed group,the liver cells arranged closely and inflammation was obviously reduced,the above results were related to liver index confirmed that bread can reduce oxidative stress and had a protective effects on the liver.In order to further explore the mechanism of hypoglycemic effects of functional bread,we researched the effects of bread on the insulin signaling pathway,glucose metabolism,lipid metabolism and inflammatory response in the liver of T2DM by western blot in detail.The experimental results showed that bread supplementation could increase insulin receptor(IR)and insulin receptor substrate(IRS)in the liver of mice in T2DM.By activating the impaired PI3K/AKT/AMPK pathway,phosphorylation of GSK3? was inactivated and the GS activity was increased,the level of G6pase and PCK were down-regulated,it increased glycogen synthesis and inhibited glycoeogenesis.Activation of GLUT4 translocation would accelerate the utilization of glucose.Activated AMPK increased the expression of enzymes related to glycolysis and promoted the occurrence of glycolysis,it also inhibited ACC production,thereby inhibiting lipid synthesis.In addition,the expression of several factors related to inflammation decreased,indicating that the inflammatory response was reduced.In addition,the pathological tissue sections and immunofluorescence staining of pancreas were determined.The results of pathological tissue sections showed that the pancreatic tissue of the SB group and the GB group had some improvement compared with the model group,and the structure was relatively complete.The other three mixed dose groups had some improvement in comparison with model group.And compared with the single-component bread group,the number of cells increased,the distribution was even and tight,and the structure was more complete,showing a certain dose effect,indicating that the degree of islet damage was greatly improved.The results of immunofluorescence staining showed that the sericin group showed strong green fluorescence and increased insulin secretion,indicating that the number of islet ? cells increased and the function was normal through the addition of sericin bread.The single GB group also had green fluorescence,but the effect was not obvious.Finally,through the addition of three mixed doses of bread,the green fluorescence intensity in the islets gradually became stronger,and the islet results were also continuously intact,indicating that the number of islet ? cells increased,and showed a certain dose effect.The level of T-SOD,T-AOC and GSH-PX in kidney were 18.19%,26.77%and 29.65%higher than that of normal mice.When the bread were added,these indexes were close to or exceed normal mice,but the dose effect was not obvious.At the same time,the level of TNF-? in GB group reduced significantly,the level of MCP-1 in HTB group markedly decrease in comparison with model group(P<0.01)and the level was 61.26±0.02 pg/mL.The level of p-P38? in model group was higher than that normal group,after the addition of bread,the MTB and HTB group had evident decline and the level of HTB group has reached to 14.96+0.03 pg/gprot.The above results showed that bread could improve the level of-antioxidant enzymes in the kidney and reduce the level of TNF-?,MCP-1 and p-P38a which related to diabetic nephropathyIn conclusion,mixed bread and a single ingredient bread can lower blood glucose to different degrees,enhance the antioxidant capacity,improve insulin resistance,regulate glucose metabolism and lipid metabolism,improve liver and kidney injury,and protect pancreatic islet cells of diabetic mice.This experiment is mainly to develop high-value-added multi-component mixed bread by using a large amount of waste or by-product mulberry powder and sericin powder in sericulture production,which not only enables diabetics or pre-diabetes to eat,but also help to lower blood sugar level and develope functional health food.It will also bring new benefits to the cultivation of mulberry sericulture farmers,the comprehensive utilization of silk waste,and the sustainable development of sericulture industry.. |
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