| 引用本文: |
毛林夕,王梦云,杨皓宇,覃艳,王炜.谷胱甘肽响应型探针的制备及其在天然药物筛选中的应用[J].湖南中医药大学学报,2023,43(5):838-846[点击复制] |
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| 谷胱甘肽响应型探针的制备及其在天然药物筛选中的应用 |
| 毛林夕,王梦云,杨皓宇,覃艳,王炜 |
| (湖南中医药大学药学院, 湖南 长沙 410208) |
| 摘要: |
| 目的 制备谷胱甘肽(glutathione, GSH)响应的香豆素类荧光探针并应用于中药GSH调节剂的筛选。方法 以香豆素为荧光团,2-氯-5-硝基嘧啶为淬灭基团,构建GSH响应的荧光探针(HCN),通过紫外、高效液相色谱、荧光光谱和核磁等表征手段确定HCN的结构及其对GSH响应的可行性、特异性、灵敏性,以确定最佳响应条件。通过MTT实验及细胞成像考察HCN的细胞毒性及胞内荧光信号。以GSH商品化消耗剂(NEM)为阳性对照,选取民族药血筒中16种化合物为筛选对象,通过HCN检测所选化合物对GSH水平的调控能力,并进一步采用分子对接手段验证筛选结果的可靠性。结果 核磁谱表明HCN合成成功,能与GSH特异性响应并呈线性关系,且HCN的最佳反应温度和反应时间分别为37℃和100 min。MTT实验表明,在HepG2细胞中,与0 μmol/mL相比,80 μmol/mL和100 μmol/mL的HCN降低了细胞活力(P<0.05),说明HCN对HepG2细胞增殖有影响;在HL-7702细胞中,与0 μmol/mL相比,各浓度HCN对细胞活力影响微弱(P>0.05),说明HCN对HL-7702细胞增殖无影响。在细胞成像实验中,与0 μmol/mL相比,各浓度HCN的荧光强度增强(P<0.05)。在药物筛选中,与control相比,血筒中的化合物五内酯B、南五内酯和异南内酯A下调了GSH的水平(P<0.001,P<0.05)。分子对接结果表明,血筒中的化合物五内酯B、南五内酯和异南内酯A与GSH形成了2个及以上的氢键,结合能分别为-3.94、-3.64、-4.87 kal/mol。结论 本文设计合成了一种GSH响应的香豆素类探针HCN,HCN具有良好的GSH响应特异性、灵敏性、光稳定性及低细胞毒性等特点,能应用于中药GSH调节剂的筛选,该研究可为荧光探针技术服务与中药活性成分筛选提供参考。 |
| 关键词: 荧光探针 香豆素 谷胱甘肽 生物硫醇 天然产物 血筒 |
| DOI：10.3969/j.issn.1674-070X.2023.05.012 |
| 投稿时间：2023-03-06 |
| 基金项目:国家自然科学基金项目（82174078，82003931）；湖湘青年科技人才创新项目（2021RC3100）。 |
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| Preparation of GSH-responsive probe and its application in screening natural medicine |
| MAO Linxi,WANG Mengyun,YANG Haoyu,QIN Yan,WANG Wei |
| (College of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To prepare the glutathione (GSH)-responsive coumarin fluorescent probe and apply it to screen GSH regulators in Chinese medicine. Methods A GSH-responsive fluorescent probe (HCN) was constructed by coumarin as fluorophore and 2-chloro-5-nitropyrimidine as quench group. The structure of HCN and the feasibility, specificity, and sensitivity of its response to GSH were determined by ultraviolet-visible (UV-Vis), high performance liquid chromatography (HPLC), fluorescence spectrum and nuclear magnetic resonance (NMR) spectrum, so as to ascertain the optimal response conditions. The cytotoxicity and intracellular fluorescence signal of HCN were examined by MTT assay and cell imaging, respectively. Using GSH commercial consumable (NEM) as the positive control, 16 compounds of Xuetong in the ethnic medicine were selected as screening objects. The regulatory ability of the selected compounds on GSH level was checked by HCN, and the reliability of the screening results was further verified by molecular docking method. Results The NMR spectrum showed that HCN was synthesized successfully and could respond to GSH in a linear relationship. The optional temperature and time of reaction of HCN were 37 ℃ and 100 min, respectively. MTT assay showed that, in HepG2 cells, HCN of 80 μmol/mL and 100 μmol/mL lowered the cell viability compared with HCN of 0 μmol/mL (P<0.05), which indicated the influence of HCN on HepG2 cell proliferation. While in HL-7702 cells, HCN with various concentrations has mild effects on cell viability compared with HCN of 0 μmol/mL (P>0.05), which indicated that HCN had no effects on HL-7702 cell proliferation. Cell imaging experiment demonstrated that HCN of various concentrations possessed higher fluorescence intensity compared with HCN of 0 μmol/mL (P<0.05). Schisanlactone B, kadsudilactone, and heteroclitalactone A of Xuetong down-regulated GSH level compared with control in drug screening (P<0.001, P<0.05). Molecular docking results showed that two or more hydrogen bonds were formed by Schisanlactone B, kadsudilactone, and heteroclitalactone A of Xuetong, and GSH, with the binding energies of -3.94, -3.64, and -4.87 kal/mol. Conclusion In this paper, HCN, a GSH-responsive coumarin fluorescent probe, was designed and synthesized. It is featured by good GSH response specificity, sensitivity, light stability and low cytotoxicity, which could help screen GSH regulators of Chinese medicines. This study aimed to provide reference for fluorescence probe technology to screen active ingredients in Chinese medicines. |
| Key words: fluorescent probe coumarin glutathione biological thiols natural products Xuetong |
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