This study aimed to analyze the structure of polysaccharides from eight different Dendrobium species and their protective effects on gastric mucosa. Ultraviolet (UV) analysis showed that the contents of eight polysaccharides ranged from 51.89 ± 6.91% to 80.57 ± 11.63%; the degree of acetylation ranged from 0.17 ± 0.03 to 0.48 ± 0.03. High-performance liquid chromatography (HPLC) results showed that these polysaccharides were mainly composed of mannose (Man) and glucose (Glc) with a small amount of galactose (Gal) and arabinose (Ara), and the monosaccharide ratios of different Dendrobium species were different. High-performance size exclusion chromatography—multi angle light scattering—refractive index detector (HPSEC-MALS-RID) showed that the molecular weight (Mw) of all Dendrobium polysaccharides was >1 × 10⁵ Da; D. huoshanense had the lowest molecular weight. Subsequently, an ethanol injured GES-1 cell model was constructed to evaluate the gastric mucosal protective potential of polysaccharides from eight different Dendrobium species. The results showed that the protective effect of the low concentration 50 μg/mL DHP treatment group was similar to that of the control group (p > 0.05), and the cell viability could reach 97.32% of that of the control group. Based on the polysaccharide composition, different kinds of Dendrobium have different degrees of migration and repair effects on GES-1 damaged cells, and the effect of DHP is slightly better than that of other varieties (83.13 ± 1.05%). Additionally, Dendrobium polysaccharides alleviated ethanol-induced oxidative stress and inflammatory response in gastric mucosal cells by enhancing the activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) and reducing the levels of malondialdehyde and reactive oxygen species. Overall, DHP can most effectively protect gastric mucosa. These findings enhance our understanding of the relationship between the structure and biological activity of Dendrobium polysaccharides, providing a foundation for the quality control of Dendrobium. Furthermore, these findings offer theoretical support for the development of Dendrobium polysaccharides as nutraceuticals to treat digestive system diseases.

Natural polysaccharides are important active biomolecules. However, the analysis and structural characterization of polysaccharides are challenging tasks that often require multiple techniques and maps to reflect their structural features. This study aimed to propose a new heart-cutting two-dimensional liquid chromatography (2D–LC) method for separating and analyzing polysaccharides to explore the multidimensional information of polysaccharide structure in a single map. That is, the first-dimension liquid chromatography (¹D–LC) presents molecular-weight information, and the second-dimension liquid chromatography (²D–LC) shows the fingerprints of polysaccharides. In this 2D–LC system, the size-exclusion chromatography–hydrophilic interaction chromatography (SEC–HILIC) model was established. Coupling with a charged aerosol detector (CAD) eliminated the need for the derivatization of the polysaccharide sample, allowing the whole process to be completed within 80 min. The methods were all validated in terms of precision, linearity, stability, and repeatability. The capability of the new 2D-LC method was demonstrated in determining various species of natural polysaccharides. Our experimental data demonstrated the feasibility of the whole systematic approach, opening the door for further applications in the field of natural polysaccharide analysis.