Study On Effect Of Selenium And Chromium On Protein And Polysaccharide Content And Structure And Hypoglycaemic Activity Of Cordyceps Sinensis

As a food and medicinal fungus,Cordyceps Sinensis has shown high activity in anti-tumour,anti-oxidant and immune enhancement.Selenium and chromium are essential trace elements that cannot be synthesised in the human body and therefore must be obtained from food.It has been shown that Cordyceps can be used as a carrier for selenium and chromium enrichment,but there is less research on selenium and chromium enriched Cordyceps.In this paper,the distribution of selenium and chromium elements in Cordyceps sinensis was analysed by extracting selenium-chromium-containing proteins and polysaccharides from Cordyceps sinensis and their effects on the content and structure.The inhibition ofα-glucosidase by chromium selenium-rich Cordyceps polysaccharides was used to analyze the changes of hypoglycemia in vitro.A model of insulin resistance in Hep G2 cells was established to reveal the effect of selenium-rich chromium cordyceps polysaccharide on the activity of insulin-resistant cells and on glucose consumption.To investigate the hypoglycaemic mechanism of chromium selenium-rich Cordyceps at the cellular level.This study not only enriches the types of deep-processed Cordyceps products,but also provides a theoretical basis for its medicinal and health care aspects.The main findings are as follows;Effects of Cordyceps sinensis enrichment with selenium and chromium on protein content and structure.;The protein content of Cordyceps sinensis was 25.30 g/100 g and that of Cordyceps sinensis was 29.20 g/100 g.This indicates that the protein content of Cordyceps sinensis increased after the enrichment of selenium and chromium.According to the SDS-PAGE and Tricine-SDS-PAGE gel electrophoresis graphs,selenium and chromium did not affect the protein distribution and relative molecular weight of Cordyceps itself,but only changed the concentration of Cordyceps proteins.The graded proteins of Cordyceps were in the order of basic protein>water-soluble protein>salt-soluble protein>alcohol-soluble protein,according to the binding ability of selenium and chromium.The order of binding of chromium and selenium content of the graded proteins showed that the order of binding of selenium and chromium was consistent across the graded proteins;the ability to bind selenium and chromium in Cordyceps proteins was synergistic and there was no competitive inhibition.Structural analysis using circular dichroism and infrared spectroscopy revealed that the selenium-rich chromium Cordyceps and common Cordyceps proteins were structurally similar,both being dominated byβ-folding andβ-turning.Selenium and chromium caused a red shift in the-OH and a blue shift in the absorption peaks of the-CH,-NH and C-O functional groups in the protein,presumably involving selenium and chromium in the reaction.Effects of Cordyceps sinensis enrichment with selenium and chromium on polysaccharide content and structure;Cordyceps polysaccharide content is 4.20%,.The content of Cordyceps sinensis polysaccharides was 7.80%in chromium selenium rich.A total of three fractions of Cordyceps polysaccharides were determined by high performance gel permeation chromatography,with heavy average molecular weights of 26881,12749 and3185 Da;the ratios were 67.12%,24.85%and 8.03%,respectively.There were five fractions of chromium selenium-rich Cordyceps polysaccharides with heavy average molecular weights of 2979189,903802,287081,21968 and 2878 Da,respectively;the ratios were23.66%,21.24%,3.16%,46.41%and 5.53%,respectively.This indicates that the fraction and molecular weight of Cordyceps polysaccharides increased after the enrichment of selenium and chromium in Cordyceps.The polysaccharides of Cordyceps sinensis were determined by ion chromatography to be composed of six monosaccharides,namely arabinose,galactose,glucosamine hydrochloride,glucose,xylose and mannose,with a molar ratio of0.041:0.313:0.001:0.226:0.059:0.360.The results show that the enrichment of Cordyceps with selenium and chromium does not change the type of monosaccharides in Cordyceps polysaccharides but only their proportional composition.Round dichroism analysis of selenium and chromium enriched Cordyceps is similar to the structure of Cordyceps polysaccharides,but the addition of selenium and chromium affects the stereo-configuration of the polysaccharides and reduces the symmetry of the sugar chains.Infrared spectroscopic analysis revealed that selenium and chromium red-shifted the absorption peak of the C=O carbonyl group of the polysaccharide,presumably chromium reacted with the carbonyl oxygen atom.The redshift of the absorption peak of the pyranoside bond by the variable angle motion of CH₃and the redshift of the absorption peak of the pyranoside bond presumably involved selenium and chromium in the coordination reaction.The inhibition assay ofα-glucosidase in vitro with an IC₅₀value of 0.474 mg/m L demonstrated the high inhibitory activity of selenium-rich chromium Cordyceps polysaccharides.The hypoglycaemic activity of chromium selenium-rich Cordyceps polysaccharides was investigated at the cellular level.After experiments,human liver tumour cells Hep G2 cultured in high glucose were treated with insulin concentration of 10-6 mol/L for 24 h.The survival rate of Hep G2 cells was higher compared to the normal group and the consumption of glucose was significantly reduced compared to the normal group,and the insulin-resistant cell model was established with the cells cultured in this condition.The insulin-resistant cells were treated with different doses of polysaccharide fractions,which showed low toxicity.In addition,the viability of the cells increased with the increase of polysaccharide action time and concentration.At the same time,the glucose consumption of insulin-resistant cells was significantly increased by the selenium-rich chromium cordyceps polysaccharide compared with that of the damaged group（IR）.At the experimental concentrations,the consumption of glucose by cells was also significantly elevated（P<0.5）with increasing sample concentration and duration of action,while cell morphology recovered and apoptosis was reduced.It showed that selenium-rich chromium cordyceps polysaccharide could significantly promote the recovery of glucose consumption by IR-Hep G2 cells with good hypoglycaemic activity.At the protein level,the expression of P-PI3K,P-Akt,P-AMPK and GLUT4 proteins was up-regulated by selenium-rich chromium cordyceps polysaccharide compared to the injury group（IR）.Selenium chrome rich Cordyceps polysaccharide significantly improved the morphology of IR-Hep G2 cells,promoted the uptake of glucose and increased the cellular hepatic glycogen content,and showed positive regulation at the level of IRS-1/PI3K/Akt signalling pathway and AMPK signalling pathway,indicating that selenium chrome rich Cordyceps polysaccharide has certain hypoglycaemic activity.The selenium-and chromium-containing Cordyceps polysaccharides significantly improved the morphology of IR-Hep G2 cells,promoted glucose uptake and increased cellular hepatic glycogen content,and showed positive regulatory effects at the level of IRS-1/PI3K/Akt signalling pathway and AMPK signalling pathway,indicating that selenium-and chromium-rich Cordyceps polysaccharides have certain hypoglycaemic activity.In this study,the distribution of selenium and chromium in the proteins and polysaccharides and their effect on their structure were analysed.Selenium and chromium did not change the original structure of Cordyceps proteins and polysaccharides but only complexed with certain chemical bonds in Cordyceps.Cordyceps proteins bind selenium and chromium better than polysaccharides.By the same order of absorption of selenium and chromium by protein and polysaccharide,it is assumed that selenium and chromium are synergistically promoted in Cordyceps and there is no competitive inhibition relationship.The hypoglycaemic mechanisms of selenium-rich chromium Cordyceps polysaccharides are;inhibition ofα-glucosidase activity,improvement of IR-Hep G2 cell morphology,promotion of glucose uptake,increase in cellular hepatic glycogen content,and positive regulation in IRS-1/PI3K/Akt and AMPK signalling pathways.