Abstract：
During persistent hyperglycaemia, albumin, one of the major blood proteins, can undergo fast glycation. It can be expected that timely inhibition of protein glycation might be add quality years to diabetic patients\' life. Therefore, this study was designed to analyse the role of silibinin to reduced or delay amadori adduct formation at early glycation and its beneficial effect to improve the glycated albumin structure and conformation. We also analysed cytotoxic effect of amadori-albumin in the presence of silibinin on murine macrophage cell line RAW cells by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Formation of early glycated product (furosine) in all samples was confirmed by LCMS. Albumin incubated with glucose only showed presence of furosine like structure. Albumin treated with silibinin in the presence of glucose did not show such furosine like peak. This LCMS result showed the silibinin play a protective role in the formation of early glycated product. HMF contents were also reduced in the presence of silibinin, when albumin was incubated with increasing concentrations of silibinin (100 and 200 μM) in the presence of glucose. ANS binding fluorescence decrease by increasing silibinin concentrations with amadori-albumin. SDS-PAGE was also showed that no significant difference in the band mobility of albumin treated with silibinin as compared to native albumin. The secondary conformational alteration in amadori-albumin due to silibinin were confirmed by FTIR. This spectrum showed slight shift in amide I and Amide II band in albumin co-incubated with glucose and silibinin as compared to albumin incubated with glucose only. We further discussed about cytotoxic effect of amadori albumin and its prevention by silibinin. MTT assay results demonstrated that amadori-albumin showed cytotoxic effect on RAW cells but silibinin showed protective role and increased the cell viability. Moreover, the results showed that silibinin has anti-glycating potential and playing a role to prevent the formation of Amadori-albumin in-vitro. Silibinin possesses strong anti-glycating capacity and can improve albumin structure and function at early stage. It might be useful in delaying the progression of diabetes mellitus and its secondary complications at early stage.
摘要：
在持续性高血糖期间，白蛋白,主要的血液蛋白质之一，可以进行快速糖化。可以预期，及时抑制蛋白质糖基化可能会增加糖尿病患者的生活质量。因此,本研究旨在分析水飞蓟宾在早期糖基化过程中减少或延迟amadori加合物形成的作用及其对改善糖化白蛋白结构和构象的有益作用。我们还通过MTT（3-（4，5-二甲基噻唑基-2）-2，5-二苯基四唑溴化物）测定法分析了在水飞蓟宾存在下amadori-白蛋白对小鼠巨噬细胞系RAW细胞的细胞毒性作用。通过LCMS确认所有样品中早期糖化产物(糠氨酸)的形成。与葡萄糖一起孵育的白蛋白仅显示存在糠氨酸样结构。在葡萄糖存在下用水飞蓟宾处理的白蛋白没有显示出这样的糠氨酸样峰。该LCMS结果表明水飞蓟宾在早期糖基化产物的形成中起保护作用。HMF含量也在水飞蓟宾的存在下降低，当白蛋白与增加浓度的水飞蓟宾（100和200μM）在葡萄糖存在下孵育时。随着amadori-白蛋白增加水飞蓟宾浓度，ANS结合荧光会减少。SDS-PAGE还显示,与天然白蛋白相比,用水飞蓟宾处理的白蛋白的条带迁移率没有显著差异。由水飞蓟宾引起的amadori-白蛋白的次级构象改变通过FTIR进行了确认。该光谱显示,与仅与葡萄糖一起孵育的白蛋白相比,与葡萄糖和水飞蓟宾一起孵育的白蛋白中的酰胺I和酰胺II条带略有偏移。我们进一步讨论了amadori白蛋白的细胞毒性作用及其水飞蓟宾的预防作用。MTT实验结果表明，amadori-白蛋白对RAW细胞具有细胞毒性作用，而水飞蓟宾具有保护作用并增加细胞活力。此外，结果表明，水飞蓟宾具有抗糖基化潜力，并在体外发挥阻止Amadori-白蛋白形成的作用。水飞蓟宾具有较强的抗糖化能力，可早期改善白蛋白结构和功能。它可能有助于延缓早期糖尿病及其继发性并发症的进展。
