Regulatory Effect On The Gastrointestinal And Immune Function Of Spleen-deficiency Rats And Quality Control Of Qihuzha Granules

Objective:To study the effects of different fractions of Qihuzha Granules on the proliferation activity of macrophage RAW264.7 cells,and to screen the effective fraction;to study the regulation of the effective fraction on gastrointestinal and immune functions in spleen-deficiency model rats and explore its mechanism;to research the effective substances of Qihuzha Granules preliminary;establish methods to determinate the content of four components and total polysaccharide in Qihuzha Granules,and perform limits tset of heavy metals and pesticide residues to improve the quality control level of Qihuzha Granules.Method:The MTT method was used to detect the effects of the four fractions of Qihuzha granules on the proliferation activity of RAW264.7 cells for the screening of active fractions.(2)Rat were randomly divided into control(n=10)and modeling(n=70)groups.The method of combining bitter cold diarrhea,irregular diet and overfatigue was adopted to copy the spleen-deficiency model in rats.The established model rats were randomly divided into model,Buzhongyiqi granules,Qihuzha granules group as well as non-polysaccharide and low,medium and high doses of polysaccharides of Qihuzha granules(n=10).Evaluation the related indicators of the gastrointestinal function and immune function of rats in each group after administrated intervention,and the RT-PCR method was used to detect the m RNA expression levels of gastrin(GAS)and somatostatin(SS)in the small intestine tissue.(3)UPLC-Q-TOF was used to qualitatively analyze the major chemical constituents present in the Fr.C of Qihuzha granules.(4)UHPLC was used to determinate of 4 components in Qihuzha granules.(5)UV spectrophotometry was used to determinate of total polysaccharide in Qihuzha granules extract.(6)Atomic absorption spectrophotometry and cold vapor absorption method was used to detect content of lead,cadmium,arsenic,copper,mercury,and the GC was used to detect pesticide residues in 7 batches of Qihuzha granules.Result:(1)The proliferation of RAW264.7 cells can significantly promoted by both polysaccharide and non-polysaccharide 75% ethanol elution fraction of Qihuzha granules(P<0.05,P<0.01),and polysaccharide had a good promotion effect on the phagocytic function and proliferation cycle of the cell(P<0.05,P<0.01).(2)the thymus index,spleen index and serum TNF-α and IL-1 β contents of spleen-deficiency model rats can obviously increased by Qihuzha granules polysaccharides in varying degrees(P<0.05,P<0.01).The serum GAS and MTL levels and the expression level of GAS m RNA in the small intestine tissue of the spleen deficiency model rats were significantly increased,and the serum SS content and expression level of SS m RNA in the small intestine tissue were obviously reduced by the high,medium and low doses of Qihuzha granules polysaccharides(P<0.05,P<0.01).Both Qihuzha Granules and its non-polysaccharide of could regulated the level of above-mentioned indicators in spleen-deficiency model rats significantly(P<0.05).(3)UPLC-Q-TOF was used for qualitative analysis,and 8 chemical components were identified from non-polysaccharide 75% ethanol elution fraction,including six flavonoids,one triterpenoid,one iridoid.(4)Hesperidin,glycyrrhizin,isoliquiritigenin and glycyrrhizic acid in Qihuzha granules showed good linear relationship in concentration of 11.82 mg/L～378.11mg/L,4.61 mg/L～147.59 mg/L,1.27 mg/L ～ 40.69 mg/L,20.80 mg/L ～ 665.69 mg/L(r=0.9999),the average recoveries were from 98.21%～102.81%%,RSD 0.70%～2.1%.(5)D(+)-Glucose in Qihuzha granules extract showed good linear relationship in concentration of19.9～119.4μg/m L(r=0.9991),the average recovery was 97.44%(RSD=2.31%).(6)Organochlorine pesticide residues were not detected in the samples,and 5 species of heavy metals in 7 batches of Qihuzha granule and the contents of 5 heavy metals did not exceed the limits of Ch P.Conclusion:The polysaccharide of Qihuzha granules is the main effective fraction to promote the proliferation of RAW264.7 cells.The main medicinal substance of Qihuzha granules for treating spleen deficiency is polysaccharides,and the 8 compounds identified in the non-polysaccharide 75% ethanol elution fraction may be its minor active ingredients.The accuracy,sensitivity,and repeatability of Multi-index content and polysaccharides determination methods were suitable for the quality control of Qihuzha granules.The organochlorine pesticide residues and heavy metals did not exceed the limits of ChP.