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茉莉花渣中果胶型多糖的结构特征与免疫调节作用

来源：花匠小妙招时间：2025-01-27 02:05

摘要: 为探究茉莉花渣中果胶型多糖的结构特征与免疫调节作用，本研究通过水提醇沉、蛋白脱除、阴离子交换柱层析和凝胶柱层析分离得到两种多糖JSP-3和JSP-4。通过相对重均分子量测定、单糖组成测定、部分酸水解液-质联用测定以及核磁共振波谱分析了两种多糖结构特征，最后以小鼠巨噬细胞RAW 264.7为模型，通过探究其对小鼠巨噬细胞的增殖率、吞噬率、ROS产生量以及NO、TNF-α、IL-6分泌量的影响来研究免疫调节作用。结果表明，JSP-3和JSP-4为两种均一多糖，相对重均分子量分别为15.48 kDa和 44.75 kDa，主要由半乳糖醛酸、鼠李糖、半乳糖和阿拉伯糖构成，存在不同比例的半乳糖醛酸聚糖结构域（JSP-3：32.87%±3.53%；JSP-4：68.64%±0.67%）与鼠李糖半乳糖醛酸聚糖结构域（JSP-3：61.12%±3.37%；JSP-4：28.28%±0.46%），说明两者均为果胶型多糖。与对照组相比，JSP-3和JSP-4在12.5~100 μg/mL的浓度范围内无细胞毒性，在一定浓度下能够通过促进小鼠巨噬细胞的吞噬作用，显著增加ROS产生量以及NO、TNF-α和IL-6的分泌量（P<0.05）。在相同浓度下，JSP-4比JSP-3能够刺激细胞分泌更多的NO、TNF-α和IL-6，说明JSP-4具有更强的免疫调节作用，这可能与其更高的半乳糖醛酸结构域比例和相对重均分子量有关。上述研究成果初步证明了茉莉花渣果胶型多糖的免疫调节作用，为其在免疫调节剂方面的应用提供了初步依据。

Abstract: In order to explore the structural characteristics and immunomodulatory effects of pectic polysaccharides in Jasminum sambac flower waste, two polysaccharides, JSP-3 and JSP-4, were separated by water extraction, alcohol precipitation, deproteinization, anion exchange chromatography and gel chromatography. Its structural characteristics were analyzed with relative weight average molecular weight, monosaccharide composition, partial acid hydrolysis-LC-MS and NMR. Finally, the immunomodulatory effects of two polysaccharides were studied by using murine macrophage RAW 264.7 as a model, and measuring the effects on proliferation, phagocytosis, ROS production and secretion of NO, TNF-α and IL-6. The results showed that JSP-3 and JSP-4 were two homogeneous polysaccharides with relative molecular weights of 15.48 kDa and 44.75 kDa, respectively, mainly composed of galacturonic acid, rhamnose, galactose and arabinose, with different proportions of galacturonan domain (JSP-3: 32.87%±3.53%, JSP-4: 68.64%±0.67%) and rhamnogalacturonan domain (JSP-3: 61.12%±3.37%, JSP-4: 28.28%±0.46%), indicating that both are pectic polysaccharides. Compared with the control group, JSP-3 and JSP-4 have no cytotoxic effect within 12.5~100 μg/mL. At a certain concentration, it increased the production of ROS and the secretion of NO, TNF-α and IL-6, also promoted the phagocytosis of mouse macrophages significantly (P<0.05). At the same concentration, JSP-4 could stimulate cells to secrete more NO, TNF-α and IL-6 than JSP-3, indicating that JSP-4 had a stronger immunoregulatory effect, which might be associated with its higher proportion of galacturonan domain and relative molecular weight. The above research results preliminarily proved the immunomodulatory effect of pectic polysaccharide from Jasminum sambac flower waste, and would provide a basis for its potential application in immunomodulators.

图 1 JSP在弱阴离子交换层析上的洗脱曲线

Figure 1. Elution curve of JSP on weak anion exchange chromatography

图 2 JSP-3和JSP-4的尺寸排阻色谱图（A）和葡聚糖标准曲线（B）

Figure 2. SEC-ELSD chromatogram of JSP-3 and JSP-4 (A), and standards curve of dextran (B)

图 3 JSP-3和JSP-4的核磁共振氢谱（A, C）和核磁共振碳谱（B,D）

Figure 3. 1H NMR spectra of JSP-3(A) and JSP-4(C) and 13C NMR spectra of JSP-3(B) and JSP-4(D)

图 4 JSP-3和JSP-4对RAW 264.7的细胞增殖率（A）和吞噬作用（B）的影响

注：与对照相比，ns：不显著；*：具有显著性P<0.05；**：具有极显著性P<0.01；图5同。

Figure 4. Effects of JSP-3 and JSP-4 on RAW 264.7 cell viability(A) and phagocytos(B)

图 5 JSP-3和JSP-4对RAW 264.7的ROS产生量（A）、NO分泌量（B）、TNF-α分泌量（C）和IL-6分泌量（D）的影响

Figure 5. Effects of JSP-3 and JSP-4 on ROS production（A）, NO（B）, TNF-α（C） and IL-6（D） secretions of RAW 264.7 macrophages

图 6 JSP-3和JSP-4对TAK-242和C29预处理后的RAW 264.7的NO分泌量（A）、TNF-α分泌量（B）和IL-6分泌量（C）的影响

注：ns：不显著；与对照相比，*：具有显著性P<0.05；**：具有极显著性 P<0.01；与每组无抑制剂数据相比，##：具有极显著性 P<0.01。

Figure 6. Effects of JSP-3 and JSP-4 on NO(A), TNF-α(B) and IL-6(C) secretion pretreated by TAK-242 and C29

表 1 JSP-3和JSP-4的主要化学组成

Table 1 Major chemical composition of JSP-3 and JSP-4

多糖名称中性糖含量（%）糖醛酸含量（%）蛋白含量（%） JSP-333.72±1.6451.25±2.530.84±0.09JSP-427.14±1.5277.43±3.120.89±0.08

表 2 JSP-3和JSP-4的单糖组成摩尔比和结构域占比

Table 2 Monosaccharide molecular ratio and structural domain ratio of JSP-3 and JSP-4

摩尔比（%）JSP-3JSP-4 Man0.53±0.021.29±0.16Rha13.86±1.145.56±0.30GlcA2.74±0.460.77±0.10GalA46.83±2.5074.31±0.37Gal14.22±0.2611.20±0.60Xyl2.75±0.640.80±0.30Ara18.98±1.256.17±0.28HGa32.87±3.5268.64±0.67RG-Ib61.12±3.3728.29±0.46 注：a: HG=GalA-Rha; b: RG-I=2（Rha）+Ara+Gal。

表 3 部分酸水解后的JSP-3和JSP-4 的LC-MS解析结果

Table 3 The LC-MS identification results of partial acid hydrolyzed JSP-3 and JSP-4

JSP-3 JSP-4推测结构保留时间（min）[M+H]+保留时间（min）[M+H]+ 120.037525.2003 19.546525.19962PMP-GalA215.568701.230815.161701.23022PMP-（GalA）2312.870877.264312.463877.26122PMP-（GalA）3411.6891053.294311.4511053.29402PMP-（GalA）4510.7621229.332910.5231229.3262PMP-（GalA）5610.0871405.35089.8481405.3512PMP-（GalA）6718.857495.224818.366495.22132PMP-Rha819.953671.252019.462671.25612PMP-Rha-GalA917.092817.3171ndnd2PMP-Rha-GalA-Rha1014.725993.3444ndnd2PMP-（Rha-GalA）21112.2071315.4335ndnd2PMP-（Rha-GalA）3 注：nd: 未检出。 [1] 安会敏, 欧行畅, 熊一帆, 等. 茉莉花茶挥发性成分在窨制过程中的变化研究[J]. 茶叶通讯,2020,47(1):67−74. [AN H M, OU X C, XIONG Y F, et al. Study on the change of volatile components in jasmine tea during the scenting process[J]. Journal of Tea Communication,2020,47(1):67−74. doi: 10.3969/j.issn.1009-525X.2020.01.013

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