RESUMO
We previously reported that active Astragalus polysaccharides APS-Ⅱ generate strong immune activity. Here we establish the optimal method for APS-II acid degradation. After preliminary structural studies and separation and preparation of the degradation products, the oligosaccharide active center with the strongest immune activity was identified by in vitro immune cell culture experiments. The optimum acid degradation conditions for APS-II were determined by a single factor experiment and an orthogonal experiment. Astragalus oligosaccharides prepared under the optimal conditions were subjected to structural analysis by hydrophilic interaction chromatography - electrospray ionization source - high resolution time-of-flight mass spectrometry. The products were separated and oligosaccharide fragments with different degrees of polymerization were isolated by preparative purification chromatography. Finally, fragments of the immunologically active centers were identified by in vitro immune cell cultures from multiple perspectives. The results show that the optimal acid hydrolysis conditions for APS-Ⅱ are hydrolysis temperature 80 ℃, trifluoroacetic acid concentration 1.0 mol·L-1, hydrolysis time 1 h. The degradation conditions have good repeatability. The degradation product is a six-carbon aldehyde glycan structure with the main chain 1→4 connected. The immune activity screening experiment for six oligosaccharide fragments showed that larger molecular weight oligosaccharides have stronger immune-promoting effects. It is speculated that the immunologically active center of Astragalus oligosaccharide is located in the sugar chain of DP9-DP19. The animal welfare and the experimental process in this study follow the requirements of the Animal Ethics Committee of Shanxi University. This result suggests a foundation for the structural characterization and structure-activity relationship research of Astragalus oligosaccharides, and may promote the development of Astragalus oligosaccharide drugs.
RESUMO
Objective·To investigate the role of APS-Ⅱ-2 (akind of plant-derived natural drug)on amelioration of chorioamnionitis-induced alveolarization arrest and the underlying mechanism.Methods·Bronchopulmonary dysplasia (BPD) model was constructed by intra-amniotic injection of lipopolysaccharide (LPS) in SD rats (E16.5).The SD rats were randomly divided into control group (Saline group)、LPS model group (LPS+Saline group) and APS-Ⅱ-2administration group (LPS+APS-Ⅱ-2 group).Then neonatal rats in LPS+APS-Ⅱ-2 group were given an intraperitoneal injection with APS-Ⅱ-2 (50 mg/kg) for 3 consecutive days after birth,whereas rats in LPS+Saline group and Saline group were administrated with an equal amount of normal saline.To examine pathologic change of pulmonary in neonatal rats,hematoxylin-eosin (H-E) staining was performed at postnatal dayl and 3.Then bone marrowderived macrophages (BMDMs) from SD rats were detected by the technology of RNA-sequence to research the immunomodulation of APS-Ⅱ-2.Results·APS-Ⅱ-2 administration group had drastically higher terminal air spaces (P=0.033 at postnatal dayl) and secondary septa counts at postnatal dayl and 3,respectively (P=0.002,P=0.026) than LPS-induced model group,while mean linear intercept was the opposite situation at postnatal dayl and 3,respectively (P=0.006,P=0.004).The detection of RNA-sequence indicated that APS-Ⅱ-2 suppressed the expression of inflammatory cytokines such as Tlr3,Tlr7 and Tlr8 in BMDMs.Meanwhile,it also promoted some pleiotropic cytokines with anti-inflammatory effects such as Alox1 5 and Cd74.Conclusion·Administration of APS-Ⅱ-2 could improve the pathology of BPD,thereby supporting the ethnopharmacological uses of the plant.This effect may be directly caused by modulatory effects ofAPS-Ⅱ-2 on inflammation.