李志鹏, 谭露露, 邓尧, 等. 大黄酸衍生物的合成及其抗炎活性评价[J]. 华南农业大学学报, 2024, 45(0): 1-11. DOI: 10.7671/j.issn.1001-411X.202405012
    引用本文: 李志鹏, 谭露露, 邓尧, 等. 大黄酸衍生物的合成及其抗炎活性评价[J]. 华南农业大学学报, 2024, 45(0): 1-11. DOI: 10.7671/j.issn.1001-411X.202405012
    LI Zhipeng, TAN Lulu, DENG Yao, et al. Synthesis of rhein derivatives and evaluation of their anti-inflammatory activity[J]. Journal of South China Agricultural University, 2024, 45(0): 1-11. DOI: 10.7671/j.issn.1001-411X.202405012
    Citation: LI Zhipeng, TAN Lulu, DENG Yao, et al. Synthesis of rhein derivatives and evaluation of their anti-inflammatory activity[J]. Journal of South China Agricultural University, 2024, 45(0): 1-11. DOI: 10.7671/j.issn.1001-411X.202405012

    大黄酸衍生物的合成及其抗炎活性评价

    Synthesis of rhein derivatives and evaluation of their anti-inflammatory activity

    • 摘要:
      目的 大黄酸具有抗癌、抗菌、抗炎等多种生物活性,但其溶解性差、毒性作用强、生物利用度低,限制了临床应用;本研究选择羟基癸酸和癸醇作为修饰物对大黄酸进行结构修饰,以提高脂溶性、降低毒副作用、提高抗炎抗氧化活性,促进临床应用。
      方法 首先,基于大黄酸构效关系,选择大黄酸的羧基与十碳羟基癸酸和癸醇进行酯化反应,合成4种大黄酸衍生物——大黄酸-5-癸醇(R-5HD)、大黄酸-癸醇(R-DA)、大黄酸-10-羟基癸酸(R-10HA)、大黄酸-10-羟基-2-癸烯酸(R-10HDA),通过核磁共振氢谱(1H nuclear magnetic resonance,1H NMR)、傅里叶变换红外光谱(Fourier transfrom infrared spectroscopy,FTIR)确定大黄酸衍生物的化学结构特征。其次,采用CCK-8试剂盒法测定大黄酸及其衍生物对小鼠单核巨噬细胞白血病细胞RAW 264.7存活率的影响,确定大黄酸及其衍生物的适用浓度范围。最后,为了验证大黄酸衍生物的抗炎活性,构建脂多糖(Lipopolysaccharide,LPS)诱导的RAW 264.7细胞模型,测定其对活性氧(Reactive oxygen species,ROS)、一氧化氮(NO)、肿瘤坏死因子(Tumor necrosis factor-α,TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)表达量的影响。通过ROS荧光探针双氯荧光黄乙酸乙酯(2, 7-dichlorofluorescin diacetate,DCFH-DA)分析大黄酸衍生物的抗氧化活性。
      结果 通过1H NMR和FTIR方法证明4种大黄酸衍生物成功制备。与大黄酸相比,大黄酸衍生物显著提高了RAW 264.7细胞存活率。通过LPS诱导的RAW 264.7细胞模型发现,4种大黄酸衍生物均能显著降低NO的释放量,R-DA显著降低了TNF-α和IL-6以及IL-1β的表达量。与大黄酸相比,R-5HD和R-DA显著抑制ROS的产生。
      结论 大黄酸与十碳羟基癸酸或者癸醇发生酯化反应,经过体外抗炎模型验证,大黄酸经修饰后能显著降低大黄酸的细胞毒性,在LPS处理的RAW 264.7细胞模型中更好地发挥抗炎抗氧化活性。本研究聚焦于十碳羟基癸酸或者癸醇对大黄酸进行结构修饰,通过体外实验证实,R-DA具有较强的抗炎、抗氧化活性,为后续大黄酸衍生物的研究和开发提供理论依据。

       

      Abstract:
      Objective Rhein possesses a variety of biological activities, including anticancer, antibacterial and anti-inflammatory effects. However, its poor solubility, toxic effects and low bioavailability limit its clinical application. In this study, hydroxydecanoic acid and decanol were chosen as modifiers for the structural modification of rhein. This study aims to increase the lipid solubility of rhein, reduce the toxic effects, and improve the anti-inflammatory and antioxidant effects for potential clinical application.
      Method Firstly, based on the structure-activity relationship of rhein, the carboxyl group in rhein was selected for esterification with ten-carbon hydroxydecanoic acid and decanol to synthesize four rhein derivatives including 5-decanol rhubarbate (R-5HD), rhubarbic acid decanol (R-DA), 10-hydroxydecanoic acid rhubarbic acid (R-10HA), 10-hydroxydec-2-denoic acid (R-10HDA). The chemical structural characteristic of the rhein derivatives were investigated by 1H nuclear magnetic resonance hydrogen spectroscopy (1H NMR) and Fourier transform infrared spectroscopy (FTIR). Secondly, the CCK-8 kit was used to determine the effects of rhein derivatives on cell viability of mouse mononuclear macrophage leukemia cells RAW 264.7. The most suitable concentration range of rhein and its derivatives was determined according to the cell viability. Finally, to further verify the anti-inflammatory activity of rhein derivatives, a lipopolysaccharide (LPS)-induced RAW 264.7 cell model was constructed, and the reactive oxygen species (ROS), nitric oxide (NO), tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) expression were determined. The antioxidant activity of the rhein derivatives were analyzed by the ROS fluorescent probe ethyl dichlorofluorescin diacetate (DCFH-DA).
      Result In this study, four rhein derivatives were prepared successfully proved by 1H NMR and FTIR methods. Four rhein derivatives significantly increased the cell viability of RAW 264.7 compared with rhein. The LPS-induced RAW 264.7 cell model revealed that all four rhein derivatives significantly reduced NO release, and R-DA significantly reduced the expression of TNF-α and IL-6. R-5HD and R-DA significantly inhibited ROS production compared to rhein.
      Conclusion Rhein was esterified with decahydroxy decanoic acid or decanol. The experimental results showed that rhein significantly reduced the cytotoxicity of rhein after modification, and showed better anti-inflammatory and antioxidant activity in the LPS-treated RAW 264.7 cell model. This study focused on the structural modification of rhein by decahydroxycapric acid or decanol. In vitro experiments confirmed that R-DA had strong anti-inflammatory and antioxidant activities, which provided theoretical basis for the subsequent research and development of rhein derivatives.

       

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