Study on the Enzymatic Deproteinization Technology, Composition Analysis and Immunomodulatory Activity of Isatidis Radix Polysaccharides
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摘要:
目的 优化板蓝根多糖脱蛋白工艺, 并进一步探讨其免疫调节活性, 为板蓝根多糖的开发利用提供科学依据。 方法 通过单因素结合Box-Behnken响应面法优化酶法脱蛋白的最佳工艺条件; 利用紫外可见光谱、傅里叶变换红外光谱、高效凝胶渗透色谱、高效液相色谱和扫描电镜等方法对板蓝根多糖化学组成与结构特征进行分析; 采用斑马鱼免疫低下模型探讨脱蛋白板蓝根多糖对斑马鱼体内中性粒细胞、巨噬细胞、IL-1β和IL-6含量的影响。 结果 酶法脱蛋白最佳工艺为: 胰蛋白酶500 U·mL-1、pH 8.0、酶解时间5 h、酶解温度37 ℃, 脱蛋白率为(86.39±0.07)%, 综合评分(91.15±0.37)%。紫外、红外光谱扫描和电镜扫描显示酶法可以除去粗多糖中含有的蛋白质, 脱蛋白后相对分子量在5.82~60.26 kDa之间, 单糖摩尔组成为甘露糖∶鼠李糖∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=2.17∶0.96∶2.90∶83.25∶4.88∶5.84。免疫活性评价结果表明, 脱蛋白后的板蓝根多糖浓度在50~300 μg·mL-1时, 能显著增加斑马鱼免疫细胞密度, 增加巨噬细胞增殖, 降低免疫低下斑马鱼体内IL-1β和IL-6含量, 从而发挥免疫调节作用。 结论 酶法可以有效去除板蓝根粗多糖中的蛋白质, 脱蛋白后的板蓝根多糖具有一定的免疫调节作用。 Abstract:OBJECTIVE To optimize the deproteinization process of Isatidis Radix polysaccharides and further explore its immunomodulatory activity, and to provide a scientific basis for the development and utilization of it. METHODS The optimum conditions of enzymatic deproteinization were optimized by a single factor combined with the Box-Behnken response surface method. The chemical composition and structural characteristics of deproteinized Isatidis Radix polysaccharides were analyzed by UV-visible spectrum, Fourier transform-infrared spectroscopy, high-performance gel permeation chromatography, high-performance liquid chromatography and scanning electron microscopy. The effects of deproteinized Isatidis Radix Polysaccharide on neutrophils, macrophages, IL-1β and IL-6 in zebrafish were investigated by using a zebrafish immunocompromised model. RESULTS The optimal enzymatic deproteinization process was as follows: trypsin 500 U·mL-1, pH 8.0, enzymatic hydrolysis time 5 h, enzymatic hydrolysis temperature 37 ℃. The deproteinization rate was (86.39±0.07)%, and the comprehensive score was (91.15±0.37)%. Ultraviolet, infrared spectroscopy scanning and scanning electron microscopy showed that the protein contained in the crude polysaccharide could be removed by enzymatic method. The relative molecular weight of the polysaccharides were between 5.82 and 60.26 kDa. The monosaccharide mole composition was mannose∶ rhamnose∶galacturonic acid∶glucose∶galactose∶arabinose=2.17∶0.96∶2.90∶83.25∶4.88∶5.84. The results of immune activity evaluation showed that when the concentration of deproteinized Radix Isatidis polysaccharides was 50~300 μg·mL-1, it could significantly increase the density of zebrafish immune cells, increase the number of macrophages, and reduce the content of IL-1β and IL-6 in immunocompromised zebrafish, thus exerting immunomodulatory effects. CONCLUAION The enzymatic method can effectively remove the proteins contained in the crude polysaccharides of Isatidis Radix, and the deproteinized Isatidis Radix polysaccharides have certain immunomodulatory effects. -
图 1 单因素试验结果
注：A.单因素试验结果；B.pH考察；C.酶活考察；D.时间考察；E.温度考察
Figure 1. The results of single factor test
图 2 各因素对除蛋白影响的响应面图和等高线图
Figure 2. Response surface and contour maps of the effects of various factors on protein removal
图 4 脱蛋白前后板蓝根多糖紫外光谱图
注：A.酶解前; B.酶解后
Figure 4. Ultraviolet spectra of Isatidis Radix polysaccharides before and after deproteinization
图 6 脱蛋白前后板蓝根多糖扫描电镜图
注：a~c.酶解前; d~f.酶解后。从左到右依次为×100、×500、×5 000。
Figure 6. SEM images of Isatidis Radix polysaccharides before and after deproteinization
图 7 脱蛋白前后板蓝根多糖分子质量图
注：A.酶解前; B.酶解后
Figure 7. Molecular weight map of Isatidis Radix polysaccharides before and after deproteinization
图 8 IRPS的单糖组成HPLC谱图
注：A.标准品; B.IRPS；1.甘露糖; 2.鼠李糖; 3.葡萄糖醛酸; 4.半乳糖醛酸; 5.葡萄糖; 6.半乳糖; 7.阿拉伯糖
Figure 8. HPLC chromatogram of monosaccharide composition of IRPS
图 10 IRPS对斑马鱼免疫细胞密度的影响
注：A.荧光斑马鱼显微图像; B.荧光强度统计图。a.空白对照(Control)组; b.模型(CAP)组; c.阳性药(LH)组; d~g.50、100、200、300 μg·mL-1IRPS组。与空白对照组比较，##P＜0.01;与模型组比较，* *P＜0.01。x±s, n=10。
Figure 10. Effect of IRPS on the density of zebrafish immune cells
图 11 IRPS对斑马鱼巨噬细胞影响
注：A.斑马鱼头部显微图像; B.巨噬细胞数目统计图。a.未染色正常斑马鱼头部；b.空白对照(Control)组; c.模型(CAP)组; d.阳性药(LH)组; e~h. 50、100、200、300 μg·mL-1IRPS组。与空白对照组比较，##P＜0.01;与模型组比较，*P＜0.05, * *P＜0.01。x±s, n=10。
Figure 11. Effect of RIPS on the number of macrophages of zebrafish
图 12 IRPS对斑马鱼IL-6、IL-1β含量的影响
注：与空白对照(Control)组比较，##P＜0.01;与模型(CAP)组比较，*P＜0.05, * *P＜0.01。x±s, n=6。
Figure 12. Effects of IRPS on the contents of IL-6 and IL-1β in zebrafish
表 1 Box-Behnken实验设计
Table 1. Box-Behnken experimental design
因素 水平 -1 0 1 A(酶活/U) 400 500 600 B(酶解时间/h) 3 4 5 C(pH) 7.5 8.0 8.5 
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表 2 Box-Behnken响应面实验结果
Table 2. Box-Behnken response surface experiment result
实验号 A(酶活/U·mL-1) B(时间/h) C(pH) 蛋白清除率/% 多糖保留率/% 综合评分 1 400 5 8.0 82.44 76.72 80.15 2 500 5 8.5 67.41 83.15 64.68 3 500 4 8.0 96.79 87.44 93.05 4 500 4 8.0 89.20 81.59 86.15 5 400 4 8.5 34.02 72.34 49.35 6 500 4 8.0 85.82 93.77 89.00 7 500 4 8.0 80.18 93.57 85.54 8 600 4 8.5 44.18 73.02 55.71 9 600 3 8.0 49.95 85.10 78.92 10 600 3 8.0 32.92 79.55 70.95 11 400 4 7.5 62.36 83.83 51.57 12 500 3 7.5 75.13 81.79 77.79 13 600 4 7.5 66.59 97.42 64.01 14 500 3 8.5 76.53 93.18 59.79 15 500 5 7.5 45.58 81.10 83.19 16 600 5 8.0 55.68 78.18 73.71 17 500 4 8.0 87.51 98.54 91.92
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表 3 方差分析结果
Table 3. Variance analysis results
变异源 平方和 自由度 均方 F值 P值 显著性 模型 3 021.91 9 335.77 13.70 0.001 2 significant A(酶活) 51.66 1 51.66 2.11 0.189 9 B(时间) 25.49 1 25.49 1.04 0.341 8 C(pH) 276.48 1 276.48 11.28 0.012 1 * AB 51.91 1 51.91 2.12 0.189 0 AC 9.24 1 9.24 0.38 0.558 7 BC 0.065 1 0.065 0.002 6 0.960 4 A2 909.98 1 909.98 37.12 0.000 5 ** B2 9.49 1 9.49 0.39 0.553 5 C2 1 563.67 1 1563.67 63.78 ＜0.000 1 ** 残差 171.61 7 24.52 拟失项 126.68 3 42.23 3.76 0.1167 not significant 纯误差 44.94 4 11.23 总和 3 193.52 16 Std.Dev. 4.92 R2 0.946 3 Mean 73.85 Adj R2 0.877 2 C.V.% 6.70 Pred R2 0.343 3 PRESS 2 097.06 信噪比 10.763 0 注:*P＜0.05，* *P＜0.01。
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