| Research Background:Qingke,as a kind of highland barley,is one of the most important staple foods among the agricultural regions in Qinghai-Tibet plateau.Compared to other cereals,the content of ?-glucan in qingke is the highest.As a kind of soluble fiber,?-glucan is composed of thousands of glucopyranose through ?-(1?3)or ?-(1?4)glycosidic bonds.It is reported that ?-glucan possesses multiple pharmacological properties,such as anti-hyperlipidemic,anti-diabetic,anti-oxidant,anti-inflammatory and anti-tumour effects.With the improvement of people's living standard,the incidence of hyperlipidemia or related cardiovascular disease is increasing,which becomes a large threaten to people's health.As we all know,the main source of ?-glucan is our principle food,which indicates that the natural substance can be absolutely safe and healthy,without any toxicities.When it comes to its pharmacological action,I think,it must have great potential to be developed as a new drug or health-care products.However,the complicated procedures of its preparation with low production or purity and the challenges on its sequence analysis have put an obstacle on its development and promotion.In addition,the mechanism of its effects is still ambiguous,which limits its application.Purposes:(1)To set a series of preparation procedures with simple operation,high production and high purity through optimizing several parameters.(2)To establish an effective method to analyze the structure and sequence of ?-glucan oligosaccharides rapidly and precisely.(3)To confirm its anti-hyperlipidemic effects and to investigate the possible mechanism of it.Methods:(1)We have optimized the concentration of precipitator by monitoring the existence of impurity polysaccharide through the analysis of monosaccharide composition.(2)The content of total saccharide in prepared samples was determined through phenol-sulfuric acid method and the configuration was confirmed through 13C-NMR.(3)The molecular weight distribution of ?-glucan was measured by SEC-MALLS-RI.(4)HPAEC-PAD coupled to Q-TOF was applied to analyze the sequences of ?-glucan oligosaccharides.(5)The cholic acid binding capacity of ?-glucan was investigated through the corresponding model in vitro.(6)We have attempted to confirm the anti-hyperlipidemic effects of highland barley ?-glucan and research the possible mechanism with western blot.Results:(1)We have discovered that the concentration of precipitator is the important factor that can influence the production and purity of final products.With the concentration of(NH4)2SO4 increasing,the production was increasing and the purity was decreasing.The solution with 25%(NH4)2SO4 was identified as the best condition to prepare when we took production and purity into considerations at the same time.And the major monosaccharide composition was glucose and the production was approximate 2.8%.(2)The content of total saccharide in the prepared sample was determined as higher than 99.6% through phenol-sulfuric acid method and the content of impurities was extremely low.From the result of 13C-NMR,we could confirm that the prepared polysaccharide was ?-configuration,without the existence of dextran,which indicated the feasibility of the preparing procedure.(3)From the results of SEC-MALLS-RI,we could find that the molecular weight of ?-glucan was approximate 794796.2 Da(±4.7%),and the index of its dispersion was 2.445(±6.772%).The molecular weight of ?-glucan was relatively high and concentrated.(4)We successfully applied HPAEC-PAD coupled to Q-TOF to analyze the sequence of ?-glucan oligosaccharides.The results showed that the peaks in the EIC and PAD pictures could be corresponding well with each other.(5)According to the results of HPAEC-PAD-MS/MS,we could discovered the characteristics of ?-glucan that there were repeating cycles among the whole carbohydrate chains.Different oligosaccharides could be described as dp3 n,dp3n+1or dp3n+2 respectively.Then,the oligosaccharides with dp3 n always had two configurations,[Glc1?4Glc1?4Glc]n or [Glc1?3Glc1?4Glc]n.The oligosaccharides with dp3n+1 were discovered to be the most abundant among one period and the linkage patterns are [Glc1?3Glc1?4Glc]n1?4Glc.Besides,the oligosaccharides with dp3n+2 always had relatively low abundance and were linked by single ?-(1?4)linkages.(6)We have found that ?-glucan possessed great bile acid binding capacity and the percentage of binding bile acid was increasing with the increasing concentration of ?-glucan.(7)Furthermore,The anti-hyperlipidemic effect of ?-glucan was confirmed by the experiments in vivo.The results showed that highland barley ?-glucan could decrease the concentrations of TC,TG and LDL-C and increase the level of HDL-C effectively.In addition,?-glucan could also protect the function of different organs through activating the activities of GSH-Px and SOD and decreasing the level of MDA.(8)We also discovered that ?-glucan could bind with bile acid to reduce the reabsorption of bile acid.To maintain a dynamic equilibrium of bile acid,the expression of CYP7A1 was increased.As a result,the metabolism of cholesterol in liver was promoted and the levels of TC and LDL-C were decreased.Conclusions: In this project,we have successfully set an extraction procedure of ?-glucan from highland barley with high purity and high production.Furthermore,the method with HPAEC-PAD-MS/MS,which was simple,rapid and effective,provided a large amount of information for the structural elucidation of ?-glucan.In addition,we have confirmed the anti-hyperlipdemic effects of prepared sample and have discovered that ?-glucan could bind with bile acid to reduce the reabsorption of bile acid to activate the activities of CYP7A1.The concept of “Homology of medicine and food” is becoming more and more popular.On the one hand,the results of this project could produce the evidence for the development,manufacturing and quality-control of ?-glucan in the future.On the other hand,for the reason that there is abundant source of ?-glucan in the cereals and that ?-glucan possesses great potential to pretreat and treat the occurrence of hyperlipidemia.The conclusions of this project could produce effective scientific guidance for normal dietary. |
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