南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (10): 1227-.doi: 10.12122/j.issn.1673-4254.2019.10.15

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黑根霉胞外多糖硫酸酯的制备及其抗肿瘤活性

秦国正，邵太丽，李萍，周玉燕，李艳，洪旭晨，李璋，王国栋   

  • 出版日期:2019-10-20 发布日期:2019-10-20

Preparation and antitumor activity of sulfated exopolysaccharide from Rhizopus nigricans

  • Online:2019-10-20 Published:2019-10-20

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摘要： 目的分析EPS硫酸酯化修饰中配料比对硫酸基取代度的影响及其硫酸酯化修饰对EPS抗肿瘤活性的影响。方法采用 氯磺酸-吡啶法修饰EPS，氯化钡-明胶浊度法测定硫酸基取代度，分析酯化剂配料比对硫酸基取代度的影响；红外光谱分析硫酸 基团是否与EPS结合形成硫酸酯；CCK-8法检测EPS硫酸酯（SEPS）抑制人结肠癌HCT 116增殖活性；Annexin V-FITC/PI双染 法检测SEPS 诱导HCT 116 细胞凋亡活性。结果酯化剂和EPS 配料比1∶1 和2∶1 条件下产物硫酸基取代度分别为0.98% （SEPS-1）和1.18%（SEPS-2），增加酯化剂配料比可以提高产物取代度（P<0.05）。红外光谱分析发现1249 cm-1附近出现-OSO3 - 的S=O 伸缩振动吸收峰，表明硫酸基团与EPS 结合形成硫酸酯。CCK-8 实验表明，在0.02~0.10 mg/L 各浓度梯度范围内， SEPS-1对HCT 116细胞增殖的抑制率明显高于EPS（P<0.05）；在0.04、0.06、0.10 mg/mL浓度梯度水平时，SEPS-2活性高于EPS （P<0.05）；在0.04~0.08 mg/L 各浓度梯度水平，SEPS-1 活性高于SEPS-2（P<0.05）。流式细胞术检测结果显示SEPS-1 诱导 HCT 116细胞凋亡活性较EPS明显增强，随着SEPS-1浓度的增加，发生早期凋亡的细胞与坏死细胞相应增加，并呈现剂量依赖 性，0.06、0.08、0.10 mg/mL处理组细胞早期凋亡率分别为6.38%、11.8%与12.5%，晚期凋亡和坏死细胞率分别为5.26%、8.04%、 6.80%。结论黑根霉EPS硫酸酯化修饰中，酯化剂配料比对产物取代度有直接影响，增加酯化剂配料比可以提高产物取代度。 黑根霉EPS硫酸酯化修饰可以提高其抗肿瘤活性，但取代度高低与活性高低没有直接关系。

Abstract: Objective To explore the effect of the composition ratio on substitution of sulfate group in sulfated exopolysaccharide (EPS) from Rhizopus nigricans and how sulfate modification affects the anti-tumor activity of EPS. Methods We used a chlorosulfonic acid-pyridine method to modify EPS and analyzed the effect of esterification ratio on the degree of sulfate substitution using barium chloride turbidimetry. The sulfate groups binding with EPS were analyzed with infrared spectrum analysis. CCK-8 assay was used to evaluate the inhibitory effect of EPS sulfate (SEPS) on the proliferation of human colon cancer HCT 116 cells, and annexin V-FITC/PI double staining was used to assess the pro-apoptotic effect of SEPS in the cells. Results The esterifying agent and EPS at the composition ratios of 1∶1 and 2∶1 resulted in sulfate substitution of 0.98% (SEPS-1) and 1.18% (SEPS-2), respectively, and the substitution was improved by increasing the ratio of the esterifying agent (P<0.05). Infrared spectrum analysis showed that the S=O stretching vibration absorption peak of -OSO3 - appeared near 1249 cm-1, indicating that the sulfate group combined with EPS to form sulfate. CCK-8 assay showed that SEPS-1 produced stronger inhibitory effects on the proliferation of HCT 116 cells than EPS within the concentration range of 0.02-0.10 mg/L (P<0.05). At the concentrations of 0.04-0.08 mg/L, SEPS-2 showed a lower anti-tumor activity than SEPS-1 (P<0.05). SEPS-1 also showed stronger pro-apoptotic effect than EPS, and as its concentration increased, SEPS-1 dose-dependently increased the ratio of early apoptotic cells and necrotic cells; the cells treated with 0.06, 0.08 and 0.10 mg/mL SEPS-1 showed early apoptotic rates of 6.38%, 11.8% and 12.5%, and late apoptotic and necrotic rates of 5.26%, 8.04% and 6.80%, respectively. Conclusion The composition ratio of the esterifying agent has a direct impact on the degree of substitution of EPS, which can be improved by increasing the ratio of the esterifying agent. Sulfate modification of EPS can enhance its antitumor activity, which, however, is not directly related with the degree of substitution.