A novel polysaccharide (NAP-3) was isolated and purified from Naematelia aurantialba after water extraction. The structure of NAP-3, which was determined by FT-IR, HPLC, GC-MS, and NMR, indicated that NAP-3 was a homogeneous polysaccharide with the molecular weight of 428 kDa, mainly consisted of β-1, 3-D-Manp, β-1, 2, 3-D-Manp, β-D-Xylp, β-1, 4-D-Glcp, β-1, 4-D-Rhap in a molar ratio of 6.49: 1.11: 2.4: 0.13: 0.83. In vitro α-glucosidase and α-amylase inhibitory assay showed that NAP-3 had a low IC value, which exhibited similar enzyme inhibitory activity as acarbose. NAP-3 was evaluated as an adjuvant with metformin for antidiabetic therapy in HFD/STZ-induced diabetic mice and insulin resistance HepG2 cells. The combination of NAP-3 and metformin in diabetic mice exhibited significant hypoglycemic activity, reducing body weight, serum insulin levels, glucose tolerance, insulin tolerance, and increasing antioxidant levels compared to metformin alone. The combination of NAP-3 and metformin improved oxidative stress by increasing ROS clearance, thereby enhancing glucose uptake in HepG2 cells. This study provided new data for the study of Naematelia aurantialba polysaccharides and offers a new adjuvant therapy for the treatment of diabetes.
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