目的 从夏枯草Prunella vulgaris（PV）多糖中挖掘结构相对清楚的活性寡糖组分，并评价其体外抗氧化与肠道菌群调节作用。方法 通过水提醇沉、Sevag法除蛋白制备夏枯草总多糖，分别在80 ℃与100 ℃、0.5 mol/L三氟乙酸（trifluoroacetic acid，TFA）条件下进行酸水解，并利用亲水相互作用色谱（hydrophilic interaction chromatography，HILIC）进行分级纯化。通过亲水相互作用色谱/离子淌度-四极杆飞行时间质谱（hydrophilic interaction chromatography-ion mobility-quadrupole time-of-flight-HDMS^E，HILIC/IM-QTOF-HDMS^E）、超高效液相色谱-紫外检测器（ultra-high performance liquid chromatography-ultraviolet detector，UHPLC-UV）及一维/二维核磁共振（¹H/¹³C-NMR、¹H-¹H COSY、HSQC、HMBC）对获得的寡糖组分进行结构解析；通过1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl，DPPH）自由基清除实验和体外小鼠粪便菌群厌氧共培养-实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）模型，评估其抗氧化活性及对肠道菌群丰度的调控作用。结果 获得聚合度（degrees of polymerization，DP）分别为2、3、5的寡糖组分DP2（得率5.60%）、DP3（1.19%）和DP5（1.70%），明确了各寡糖组分中的优势结构：DP2主要含由3-甲氧基-α-葡萄糖醛酸与木糖构成的二糖，其木糖端基存在α/β异构；DP3主要包含由木糖-阿拉伯糖-糖醛酸组成的三糖，其末端为4-甲氧基-β-葡萄糖醛酸或α-半乳糖醛酸；DP5主要由以2-甲氧基-α-葡萄糖醛酸起始、经3个木糖单元连接并以α/β 2种构象共存的3-甲氧基-葡萄糖醛酸结尾的线性五糖构成。抗氧化活性评价表明，DP2、DP3和DP5均具有显著的DPPH自由基清除能力，其中DP5的活性最优，半数抑制浓度（half maximal inhibitory concentration，IC₅₀）为0.36 μg/mL。在0.1 mg/mL质量浓度下，DP3和DP5清除率均超过84%，与阳性对照维生素C（VC，IC₅₀=0.11 μg/mL）相比无显著差异。体外肠道菌群分析显示，3种寡糖组分对肠道菌群表现出一定的调控作用，DP2可显著促进益生性乳杆菌属Lactobacillus的增殖；DP3能够上调有益菌门拟杆菌门（Bacteroidetes）的丰度，同时显著抑制致病菌门放线菌门（Actinobacteria）；DP5主要表现出对放线菌门的显著抑制作用。结论 从夏枯草多糖中分离富集得到聚合度分别为2、3、5的寡糖组分，并解析了其中优势结构特征，揭示了它们兼具抗氧化与肠道菌群调节活性。

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.

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天津市杰出青年科学基金项目（23JCJQJC00030）；现代中药新质生产力科技创新专项项目（24ZXZKSY00010）