Composition and Immune Regulatory Effect of Polysaccharides Extracted from Different Parts of Urtica macrorrhiza Hand.-Mazz.
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摘要: 目的：研究粗根荨麻（Urtica macrorrhiza Hand.-Mazz.）不同部位粗多糖的含量及化学组成，初步评价其对巨噬细胞的调节作用。方法：利用热水提取、乙醇分级沉淀的方法分别制备粗根荨麻根、茎、叶粗多糖；通过高效液相色谱法分析其分子量及单糖组成；全波长扫描及红外光谱分析其结构特征；MTT法研究其细胞毒性；中性红染色法考察其对巨噬细胞吞噬活性的影响；Griess法和ELISA法分别检测其对巨噬细胞NO、TNF-α分泌的影响。结果：提取得到粗根荨麻叶（UML40、UML60），茎（UMS40、UMS60），根（UMR40、UMR60）共6种粗多糖，其中叶粗多糖得率最高为4.62%，UML40为占77.92%，茎粗多糖和根粗多糖得率分别为0.69%和1.13%；各粗多糖平均重均分子量从2.11 kDa到802.21 kDa不等，主要由D-Glc、D-Gal、D-Ara和D-GalA组成，但不同部位粗多糖的单糖组成摩尔比不同。此外，不同部位、不同分子量的粗根荨麻多糖免疫活性存在较大差异，其中UML40、UMS40、UMR40均能够显著活化巨噬细胞，促进其吞噬活性及NO、TNF-α分泌水平（P<0.05）；UMR60虽然不能促进巨噬细胞吞噬但能显著促进细胞因子的分泌（P<0.001）；UML60细胞毒性较大且活性较低；UMS60无激活巨噬细胞的活性。结论：粗根荨麻不同部位多糖在组成及免疫调节活性上存在一定差异，在对其进行研究以及开发利用时应当注意质量控制。Abstract: Objective: To study the content and chemical composition of crude polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz. and evaluate their regulatory effect on macrophage. Methods: The crude polysaccharides from the roots, stems and leaves of Urtica macrorrhiza Hand.-Mazz. were prepared by hot water extraction and ethanol fractional precipitation. Their molecular weight and monosaccharide composition of the polysaccharides were analyzed by high performance liquid chromatography (HPLC) and their structural characteristics were identified by full-wavelength scanning and infrared spectroscopy. The cytotoxicity of these polysaccharides was evaluated by MTT assay. The effect of polysaccharides on the phagocytic activity of macrophages was investigated by neutral red staining and their effect on the secretion of NO and TNF-α from macrophage was studied by Griess method and ELISA, respectively. Results: Six kinds of crude polysaccharides were extracted from leaves (UML40, UML60), stems (UMS40, UMS60) and roots (UMR40, UMR60), of which the highest yield of 4.62% was obtained from leaves, 77.92% from UML40, 0.69% from stems and 1.13% from roots. The average heavy average molecular weight of each crude polysaccharide ranged from 2.11 kDa to 802.21 kDa and consisted mainly composed of D-glucose, D-galactose, D-arabinose and D-galacturonic acid, while the molar ratio of monosaccharide components was various in different parts. In addition, the immune activities of polysaccharides from different parts with different molecular weights were obviously different, UML40, UMS40 and UMR40 could significantly activate macrophages, promote their phagocytosis activity, and increase their secretion of NO and TNF-α (P<0.05). Although UMR60 had no obvious effect on macrophage phagocytosis, it could significantly promote cytokine secretion (P<0.001). Whereas, UML60 showed higher cytotoxicity and lower activity, and UMS60 did not showed activity in activating macrophages. Conclusion: Polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz. varied in monosaccharide composition and immunomodulatory activity, thus quality control would be important for the research and development of these polysaccharides.
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图 1 粗根荨麻粗多糖提取分离流程图
Figure 1. Extraction and separation procedure of polysaccharides from Urtica macrorrhiza Hand.-Mazz.
图 2 粗根荨麻各部位粗多糖的分子量分布曲线
Figure 2. Molecular weight distribution curves of crude polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz.
图 3 经PMP衍生化后的HPLC图UML40（A）、UML60（B）、UMS40（C）、UMS60（D）、UMR40（E）、UMR60（F）、标准混合单糖（G）
Figure 3. HPLC chromatogram of PMP derivatives of UML40 (A), UML60 (B), UMS40 (C), UMS60 (D), UMR40 (E), UMR60 (F) and standard mixed monosaccharides (G)
注：1：D-Man，2：L-Rha，3：GlcNAc，4：D-GlcA，5：D-GalA，6：D-Glc，7：D-Gal，8：D-Ara。
图 4 粗根荨麻各部位多糖全波长扫描图（A），红外光谱图（B）与二阶求导红外光谱图（C）
Figure 4. Spectroscopic analysis of polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz. full wavelength scanning (A), infrared spectrum (B) and second derivative infrared spectrum (C)
图 5 粗根荨麻不同部位粗多糖对巨噬细胞的毒性
Figure 5. Toxicity of crude polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz. to macrophages
注：**：与空白组相比P<0.01；L：低浓度，0.1 μg/mL；M：中浓度，1 μg/mL；H：高浓度，10 μg/mL。
图 7 粗根荨麻不同部位粗多糖对巨噬细胞吞噬活性的影响
Figure 7. The phagocytic activity of macrophages of polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz.
注：与空白组相比，*P<0.05，**P<0.01,***P<0.001；图8同。
图 8 粗根荨麻不同部位粗多糖对巨噬细胞细胞因子释放的影响
Figure 8. Effects of polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz. on cytokine release from macrophages
注：A. NO释放量；B. TNF-α分泌量
表 1 各单糖PMP衍生物的回归方程和线性范围
Table 1. Regression equations and linear ranges of PMP derivatives of each monosaccharides
单糖 回归方程 相关系数 线性范围（mg/mL） D-Man y=19222x−224.05 R2=0.9997 0.05~0.83 L-Rha y=31004x−447.73 R2=0.9996 0.05~0.83 D-GlcNAc y=5384.1x−140.62 R2=0.9995 0.10~1.67 D-GlcA y=5671.7x+277.36 R2=0.9923 0.10~1.67 D-GalA y=4396.9x+123.75 R2=0.996 0.10~1.67 D-Glc y=14616x−216.91 R2=0.9995 0.05~0.83 D-Gal y=22096x−299.78 R2=0.9996 0.05~0.83 D-Ara y=19315x−292.13 R2=0.9996 0.05~0.83 
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表 2 粗根荨麻不同部位粗多糖得率及占比
Table 2. The yield and proportion of crude polysaccharide in different parts of Urtica macrorrhiza Hand.-Mazz.
样品名称 UML40 UML60 UMS40 UMS60 UMR40 UMR60 产率（%） 3.60 1.02 0.35 0.34 0.81 0.32 占比（%） 55.90 15.84 5.43 5.28 12.58 4.97
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表 3 粗根荨麻不同部位粗多糖的单糖比例
Table 3. Characteristics of monosaccharide composition of crude polysaccharides from different parts of Urtica macrorrhiza Hand.-Mazz.
样品 摩尔比 Man Rha GlcA GalA Glc Gal Ara UML40 1.00 0.30 0.18 0.75 1.56 3.36 2.23 UML60 1.00 0.45 0.18 1.43 1.07 4.84 1.93 UMS40 1.00 − 7.97 31.11 4.94 21.38 5.89 UMS60 1.00 1.98 3.75 13.02 78.37 25.22 16.03 UMR40 1.00 0.46 0.73 1.26 4.31 6.59 3.62 UMR60 1.00 1.98 2.97 5.39 18.77 28.86 15.87 注：“−”表示未检出。
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