Components Identification Of The Polysaccharide Extract Origined From Astragalus Fermentation And The Immunomodulation In PEDV Vaccine

Astragalus is widely used in the clinical practice of traditional Chinese medicine and its rhizome is the main part for medicine.Modern medical research has shown that Astragalus has a variety of components,including polysaccharides,saponins,brass,etc.,which have various biological activities.Studies of polysaccharides among these components have become hot topics in recent years.Polysaccharides have a variety of biological functions,such as immune regulation,anti-hyperglycemia,anti-inflammation,anti-oxidation,anti-virus,anti-radiation and so on.However,the biological activities of polysaccharides vary with different sources,extraction methods,or physical and chemical properties of polysaccharides.In this study,polysaccharide was extracted from radix astragali powder fermented by bacillus licheniformis,and the maximum polysaccharide content reached 5.417 g/100 g at 36h.In order to understand the monosaccharide composition of the achieved polysaccharide,the monosaccharide composition of astragalus polysaccharide fermentation and astragalus polysaccharide was identified by liquid chromatography.The results showed that the monosaccharides extracted by the two methods were composed of rhamnose,galacturonic acid,glucose,galactose and arabinose,mannose,xylose,arabinose and fucose.The contents of polysaccharide extracts from fermentation and water extraction were different compared with each other.The content of mannose in the polysaccharide extracted by fermentation is about 1.96 times what was extracted by wather extraction,the rhamnose was about 2.26 times,glucuronic acid was about 1 times,glucose was about 1.83 times,galacturonic acid was about2 times,arabinose was about 1.8 times.Molecular weight and its distribution affect the activity of polysaccharides.The polysaccharide products from water extraction and fermentation were monitored by laser scattering and gel chromatography.The results showed that the two methods had different molecular weights and ranges.The distribution of molecular weight spreaded followed by the increase of weight average molecular weight.The value of Mw/Mn in both polysaccharide products was higher than 1,which indicated that both extracted polysaccharides from water methodand fermentation were large molecular weight and had wide distribution.Through comparison,it was found that the molecular weight distribution of Peak 2 in the fermented astragalus polysaccharide was the narrowest and more stable than that of water extracted astragalus polysaccharide.To investigate the cytotoxicity of astragalus polysaccharide to Vero cells,the extracted astragalus polysaccharide was combined with different concentrations of 50,100,200,400and 800μg/mL respectively.In addition,no astragalus polysaccharide was added to the control group for cell culture.The results showed that the fermentation of stragalus polysaccharide had displayed no cytotoxicity to cells（P>0.05）.The effect of polysaccharide on PEDV titer was calculated by means of TCID₅₀.The experimental results showed that between the polysaccharide concentration of 100 and 400μg/mL,the TCID₅₀0 of the virus started to decrease following the increase of the dose,and there was no significant difference between the polysaccharide concentration of 100μg/mL and 200μg/mL（P>0.05）.When the polysaccharide concentration was 400μg/mL,the titer of the virus decreased,and it was significant（P<0.05）.The astragalus polysaccharide concentration of 400μg/mL could inhibit porcine epidemic diarrhea（PED）virus replication.Adding astragalus polysaccharide products to cells at different concentrations during different time periods could inhibit the replication of the virus in the polysaccharide in the early stage of virus proliferation from 24 h to 48 h,and the effect was best when the polysaccharide product concentration was 400μg/mL for 24 h.In order to explore different ways of inoculating piglets with the extracts of fermented astragalus polysaccharide to prevent PED.12 pigs were randomly divided into 4 groups,in which the control group was injected with normal saline.The first group in the other three experimental groups were inoculated with the PEDV vaccine,the second group inoculated with the vaccine and intramuscular injection of polysaccharide,and the third group received vaccine and intramuscular injection of polysaccharide at the houhai point.The pigs were injected with polysaccharide at a content of 400 mg/kg.Anterior venous blood was collected at 0,7,14,21 and 28 days post-inoculation（dpi）to monitor IL-6,IFN-γand sIgA levels.The results showed that the IL-6 level changed dynamically from 7 dpi to 28 dpi,and reached a peak on 14 dpi.On the 14 dpi and 21 dpi,there was a significant difference between the group of the injection with vaccine plus polysaccharide in houhai point and the group of vaccine alone（P<0.05）.While,on 14 dpi,there was no difference between the group of intramuscular polysaccharide extract injection and the group of vaccine alone（P>0.05）.On21 dpi,there was no significant difference between the group of intramuscular polysaccharide injection and that of houhai point（P>0.05）.Compared with the vaccine group,the level of IL-6 in the polysaccharide injection group was increased significantly.The IFN-γlevel had no significant difference between the different groups from 7dpi to 28 dpi（P>0.05）.On 14dpi and 21 dpi,there was no siginicant difference between the intramuscular injection of polysaccharide extract group and the injection of polysaccharide extract group at houhai point（P>0.05）.On 14 dpi,there was a significant difference between the group of polysaccharide injection at houhai point and the vaccine group（P<0.05）.The sIgA levels in the group of intramuscular injection polysaccharide and the group of muscle injection at houhai point showed an increasing trend during the 7 dpi to 21dpi period.On 14 dpi,there was a significant difference in sIgA content between the vaccine group and the polysaccharide product injection group（P<0.05）.On 21 dpi,the sIgA in the group of polysaccharide products injection at houhai point reached the highest level,which was significantly different to the vaccine group（P<0.05）.In this study,astragalus polysaccharide was extracted by the fermentation of Bacillus licheniformis,the yield of polysaccharides and monosaccharides obtained from fermentation was higher than that of water extraction.The astragalus polysaccharide products from fermentation showed significant enhancement effect on the immune response induced by PEDV vaccine.This study laid a good theoretical foundation for the popularization and application of natural polysaccharide plants such as Astragalus in animal husbandry.