Objective To isolate structurally defined bioactive oligosaccharide fractions from Prunella vulgaris (PV) polysaccharides and evaluate their in vitro antioxidant activity and intestinal microbiota-modulating effects. Methods Total polysaccharides were extracted via water extraction and alcohol precipitation, followed by deproteinization using the Sevag method. Partial acid hydrolysis was performed under two conditions (80 ℃ and 100 ℃, 0.5 mol/L trifluoroacetic acid). The hydrolysates were fractionated and purified by hydrophilic interaction chromatography (HILIC). Structural characterization of the dominant components in each fraction was performed using HILIC/IM-QTOF-HDMSE, UHPLC-UV and 1D/2D-NMR (1H/13C-NMR, COSY, HSQC, HMBC). Antioxidant activity of three oligosaccharide components was assessed via a DPPH free radical scavenging assay, while their modulatory effects on gut microbial abundance were evaluated using an in vitro anaerobic co-culture model of mouse fecal microbiota coupled with real-time quantitative PCR. Results Three oligosaccharide fractions with degrees of polymerization (DP) of 2, 3, and 5 were obtained, namely DP2 (yield: 5.60%), DP3 (1.19%), and DP5 (1.70%). The dominant structural features of three fractions were elucidated as follows: DP2 is mainly composed of a disaccharide composed of 3-O-methyl-α-glucuronic acid linked to xylose, with α/β anomerism at the xylose terminus; DP3 mainly contains a trisaccharide consisting of xylose-arabinose-uronic acid, terminating in either 4-O-methyl-β-glucuronic acid or α-galacturonic acid; DP5 is primarily a linear pentasaccharide starting with 2-O-methyl-α-glucuronic acid, linked through three xylose units, and ending with 3-O-methyl-glucuronic acid existing in both α- and β-forms. Antioxidant assessment revealed that all three fractions exhibited significant DPPH radical-scavenging activity, with DP5 being the most potent (IC₅₀ = 0.36 μg/mL). At 0.1 mg/mL, both DP3 and DP5 showed scavenging rates exceeding 84%, comparable to the positive control VC (IC₅₀ = 0.11 μg/mL, P > 0.05). Microbiota analysis indicated that all three fractions modulated the intestinal flora: DP2 significantly promoted the growth of the probiotic genus Lactobacillus; DP3 increased the abundance of the beneficial phylum Bacteroidetes while suppressing the potentially pathogenic phylum Actinobacteria; DP5 primarily exerted inhibitory effects on Actinobacteria. Conclusion This study successfully isolated three oligosaccharide fractions (DP2, DP3, and DP5) from the PV polysaccharides and characterized their predominant structural features, revealing the antioxidant and gut microbiota-regulating activities.

Prunella vulgaris L.  /  oligosaccharide component  /  structural characterization  /  antioxidant activity  /  gut microbiota-regulating activity