Optimization Of The Purification Process Of Chelidonium Majus L Alba Polysaccharide And Its Effect On The Immune And Antioxidant Functions Of Weaned Piglets

Chelidonium majus L,a perennial herb,is a dried whole plant of the genus Chelidonium in the family Popaceae.Current studies have shown that Chelidonium contains a large number of bioactive components with great medicinal value and has great prospects for development as a new drug.In this experiment,the crude polysaccharide was extracted from C.leucocephala as raw material,and the process of purification of C.leucocephala polysaccharide by macroporous adsorption resin was optimized by combining single-factor experiment and response surface experimental design,and the effects of C.leucocephala polysaccharide on immune function and antioxidant function of weaning piglets were also investigated to provide scientific basis for the addition of C.leucocephala polysaccharide to the feed of weaning piglets.Methods:(1)In this experiment,crude polysaccharide was extracted from the dried whole herb of C.alba by aqueous extraction and alcoholic precipitation,and the polysaccharide standard curve and protein standard curve were established using glucose and bovine serum protein as standards,and the protein removal of crude polysaccharide from C.alba was performed by Sevag method.(2)The macroporous adsorption resin was applied to further purify the polysaccharide of C.alba,and the comprehensive scores of decolorization and polysaccharide retention of the resin were investigated by weighted summation method.(3)The range of the Box-Behnken experimental design was examined in terms of four factors,including sample loading concentration,pH,diameter-to-height ratio and flow rate,respectively,and the optimal adsorption process parameters of the macroporous adsorption resin were optimized using the response surface method,and then the optimal elution process was determined based on the known parameters.(4)Infrared spectroscopy was performed on the leucovorin polysaccharide samples before and after deproteinization and purification,respectively.(5)In order to investigate the effects of leucovorin on the immune function and antioxidant function of weaned piglets,30 healthy 21-day-old weaned piglets were divided into control group and Chinese medicine group with 15 piglets in each group using one-way randomized group experimental design.The immunological indexes such as IgA,IgM,IgG and oxidation indexes such as CAT,T-COA,T-SOD,GSH-Px,MDA,GSH,GSSG were measured in the piglets’serum according to the corresponding reagent kit method.Results:(1)The equation of the standard curve of C.alba polysaccharide was Y=0.0211x-0.1988,R~2=0.9956,and the standard curve of protein was Y=0.0112x+0.1561,R~2=0.9917;after three parallel experiments,the mean value of C.alba polysaccharide extraction was 11.52±1.91%and the protein removal rate was 77.36±1.59%.(2)After screening,the D900 type macroporous adsorbent resin was the best,with the decolorization rate as high as90.35%and the polysaccharide retention rate of 67.47%,and the overall score was 78.91;the adsorption kinetic results showed that the adsorption time was 3 h.(3)The results of the single-factor experiments showed that the best experimental diameter-to-height ratio was 1:8,and the range of the Box-Behnken experimental design:the sample loading concentration of 1mg/mL mL～3 mg/mL,pH 6～8,and flow rate 1 mL/min～3 mL/min.The results of response surface method optimization showed that the overall score was 78.27 at the sample loading concentration of 2 mg/mL,pH 7,and flow rate of 2 mL/min;the results of leakage curve examination showed that the optimal sample loading volume was 3BV;the elution volume was 3BV with 30%ethanol as the eluent.(4)The infrared scanning spectrum of C.alba polysaccharide showed that the pattern showed several obvious polysaccharide features.(5)At10 d,20 d and 30 d of piglet administration,the serum levels of IgA,IgG and IgM in the weaned piglets of the Chelidonium polysaccharide group were significantly higher than those in the control group(P<0.05),and the serum levels of IgA,IgG and IgM in the piglets after 30 d of administration were significantly higher than those at 10 d and 20 d of administration;the serum levels of T-AOC activity,T-SOD,GSH-Px and GSH-Px in the weaned piglets of the Chelidonium polysaccharide group were significantly higher than those in the control group.T-AOC activity,T-SOD,GSH-Px activity and GSH content were significantly higher in the serum of piglets weaned in the C.alba group than in the control group(P<0.05),while CAT,MDA and GSSG content were significantly lower than in the control group(P<0.05).Conclusion:(1)The best purification process conditions for C.alba polysaccharide were as follows:the column was wet loaded with D900 type macroporous resin at a diameter to height ratio of 1:8,the mass concentration of C.alba polysaccharide solution was 2 mg/mL,the pH was adjusted to 7,the flow rate of the sample was 2 mL/min,the volume of the sample was 3 BV,and the adsorption time was 3 h.The elution volume was 3 BV with 30%ethanol.The content of the polysaccharide of C.leucocephala was stabilized above 50%.(2)Chelidonium polysaccharide can improve the immunity and antioxidant function of weaned piglets.