Bioactivity Evaluation,isolation,identification And Fermentation Promotion Of Polysaccharides From Guava Leaves

Psidium guajava Linn.?guava?belongs to the family of Myrtaceae.Guava leaves have been used as folk medicine to treat or control various diseases such as diarrhea,indigestion and abdominal distention,gum pain,eczema and diabetes.In this work,the extraction,purification,structure and digestive characteristics of polysaccharide from guava leaves were investigated.Furthermore,the antioxidant and hypoglycemic activities of guava leaf polysaccharide were evaluated by in vitro and vivo.Moreover,the yield and biological activities of guava leaf polysaccharide were improved by solid-state fermentation technology.The main results are as follows:Response surface methodology was applied to optimize the ultrasonic-assisted extraction parameters of polysaccharides from guava leaves?GLP?.Under the optimum conditions?ultrasonic power of 404 W,extraction temperature of 62?,ultrasonic time of 20 min?,the yield of GLP reached 1.00±0.04%,which was consistent with the maximum predicted value of 0.96%.The GLP extraction efficiency was increased by nearly 1.5 times.The total sugar content of GLP was 64.42%,and uronic acid content was 9.13%.GLP did not contain protein.The results of antioxidant activity in vitro showed that GLP had strong scavenging capacities of DPPH,ABTS and hydroxyl radical,and their IC₅₀ values were 46.49?g/m L,102.82?g/m L and 175.52?g/m L,respectively.The potential hypoglycemic effect of GLP was evaluated by inhibiting?-amylase and?-glucosidase in vitro.GLP exhibited a remarkable inhibitory effect on?-glucosidase with an IC₅₀of 16.28?g/m L and was in a dose-dependent manner but had less of an inhibitory effect on?-amylase with an IC₅₀of 49.13 mg/m L.The diabetic mice model was established by high-fat diet combined with streptozotocin.Diabetic mice were given different dosages of GLP?50 mg/kg and 100 mg/kg body weight?by gavage for 4 weeks.Results demonstrated that GLP could effectively enhance the condition of diabetic mice.On the one hand,GLP evidently reduced the fasting blood glucose,improved glucose tolerance and ameliorated weight loss of diabetic mice.GLP also markedly decreased triglyceride,total cholesterol,glycosylated serum protein and serum creatinine level of diabetic mice.On the other hand,GLP significantly improved the antioxidant system of diabetic mice through increasing the total antioxidant capacity,superoxide dismutase activity and glutathione peroxidase activity and reducing malondialdehyde content.Moreover,GLP exerted a protective effect on the liver,kidney and pancreas of diabetic mice.A new heteropolysaccharide GLP-1 was obtained from the crude polysaccharides of guava leaves by Sephadex G-200 column chromatography.The sugar content of GLP-1 was89.69%and the average molecular weight was 812.83 k Da.GLP-1 was composed of glucose?59.37%?,arabinose?14.49%?,galactose?14.25%?,galacturonic acid?4.58%?,rhamnose?4.19%?,mannose?1.73%?and glucuronic acid?1.39%?.The main linkage types of GLP-1were proved to be?1?4?and?1?6?linked glucose,?1?6?linked galactose,?1?3?linked rhamnose,?1?5?linked arabinose,?1?3,6?linked mannose and?1??linked glucose terminal.GLP-1 has a complex structure and may have triple helix conformations.GLP-1exhibited strong free radical scavenging ability and?-glucosidase inhibitory activity.The simulated gastrointestinal digestion experiments showed that GLP-1 was flocculated in the simulated gastric juice,and the average molecular weight was reduced from 812.44k Da to 802.83 k Da.GLP-1 was re-dissolved in the simulated small intestinal juice and its average molecular weight decreased slightly.The increase of reducing sugar content in digestive juice and the absence of free monosaccharide indicated that the reduction of GLP-1molecular weight was caused by the break of glycosidic bond between molecular chains.After gastrointestinal digestion,GLP-1 still maintained certain antioxidant capacity(IC₅₀=904.89?g/m L)and strong?-glucosidase inhibition ability(IC₅₀=37.89?g/m L).The effects of fermentation degree on the composition and bioactivities of polysaccharides in guava leaves were studied.Results showed that solid-state fermentation could significantly improve the yield,uronic acid and conjugated protein content,antioxidant and?-glucosidase inhibitory activity of polysaccharides from guava leaves.The composition and bioactivity of polysaccharides in guava leaves changed dynamically,and there was the best turning point during fermentation.The highest yield of polysaccharides was 3.53%after 3-day Bacillus megaterium fermentation,which was 1.30 times of that of unfermented guava leaves.Meanwhile,their antioxidant and?-glucosidase inhibition activities were the best.The average molecular of the main fraction was decreased from 697.61 k Da to 633.28 k Da during guava leaves fermented by Bacillus megaterium.The yield of polysaccharides from guava leaves reached the maximum?2.54%?after 7-day Monascus fermentation,and their antioxidant activity and?-glucosidase inhibition were the strongest.The average molecular weight of the main fraction decreased from 893.88 k Da to 672.76 k Da during guava leaves fermented by Monascus anka.The cellulase secreted in the process of microbial fermentation was closely related to the release of polysaccharides from guava leaves,the degradation of high molecular weight polysaccharides and the formation of low molecular weight polysaccharides.Guava leaf polysaccharide could be developed into potential functional food or complementary medicine for hyperglycemia.Solid-state fermentation technology can be applied to the efficient production,extraction and modification of polysaccharides.