Abstract: The aim of this study was to screen the best dosage of Radix Hedysari polysaccharide-1-1 (RHPS-1-1) regulating intestinal flora imbalance in mice. The single polysaccharide RHPS-1-1 was isolated and purified from the crude RHPS by DEAE-52 and Sephadex G-100, respectively. The monosaccharide composition of RHPS-1-1 was determined by high-performance anion-exchange chromatography (HPAEC), and the structure of the polysaccharide was identified by methylation and infrared spectroscopy. Ninety C57BL/6 mice were randomly divided into normal control group, model group, self-healing group, and different doses of RHPS-1-1 groups (12.5, 25, 50, 100, 200 and 400 mg·kg^-1) with 10 mice in each group. The intestinal flora imbalance mouse model was established by intragastric administration of antibiotic cocktail (ampicillin, neomycin, metronidazole and vancomycin) for 14 consecutive days. At the end of model establishing, the mice in model group were killed and sampled. Each drug administration group was gavaged with different doses of RHPS-1-1, and normal control and self-healing groups were gavaged with equal volume of normal saline for 14 consecutive days. Finally, 16S rDNA high-throughput sequencing method was used to analyze the changes of intestinal flora of cecal contents in each group, and the organ indices and histopathological changes of major organs in normal control, self-healing and 25 mg·kg^-1 RHPS-1-1 groups were calculated and observed. The results showed that the purified RHPS-1-1 was a single polysaccharide with a weight average molecular weight of 19.420 ku. RHPS-1-1 was mainly composed of glucose, the main chain of which was connected by 1,4-D-Glcp, and had a special absorption peak of plant polysaccharides. Gavaging with antibiotic cocktail caused a serious intestinal flora imbalance in mice. The abundance of Bacteroidetes, Firmicutes and Actinomycetes were extremely reduced, while Proteobacteria were relatively increased. And this alteration could not recover naturally. The diversity and richness of intestinal flora were changed after RHPS-1-1 administration, and the Chao 1 index of 25 mg·kg^-1 RHPS-1-1 group was significantly increased (P<0.05). The principal components analysis (PCA) and unweighted pair-group method with arithmetic averages (UPGMA) showed that the intestinal flora structure of 25 mg·kg^-1 RHPS-1-1 group was the closest to that of the normal control group. The abundance of harmful bacteria Muribaculaceae_unclassified was decreased, while the abundance of beneficial bacteria Ruminococcaceae_UCG-014 and Clostridiales_unclassified were increased. Compared with normal control group, liver index in self-healing group was significantly decreased (P<0.05), and significantly increased after 25 mg·kg^-1 RHPS-1-1 treatment (P<0.05). However, there were no obvious pathological changes in heart, liver, spleen, lung, kidney and brain in normal control group, self-healing group and 25 mg·kg^-1 RHPS-1-1 treatment group. The results demonstrated that RHPS-1-1 was a plant glucan with a molecular weight of 19.420 ku. At the dose of 25 mg·kg^-1, RHPS-1-1 had the best effect on promoting the recovery of intestinal flora disorder caused by antibiotics in mice, and could improve the decline of liver index. The present study provided evidences for the clinical application of RHPS regulating intestinal flora.

Key words: Radix Hedysari polysaccharide-1-1, isolation and purification, antibiotic cocktail, intestinal flora imbalance, dose screening