Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran.
Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
DNA Cell Biol. 2024 Jan;43(1):26-38. doi: 10.1089/dna.2023.0269. Epub 2023 Dec 11.
In this study, we determined the interaction of piperine and calf thymus DNA (ct DNA) in Tris-HCl buffer solution at pH = 6.8 and also evaluated the binding mechanism through the data of multi-spectroscopic techniques along with thermal melting and viscosity measurements. The outcomes of fluorescence quenching confirmed the occurrence of interactions between piperine and ctDNA and pointed out the role of piperine as the quencher. In addition, the K values were measured at three different temperatures of 298, 303, and 308 K to be 4.5 × 10 M, 5.65 × 10 M, and 9.36 × 10 M, respectively, which suggested the dominance of dynamic mechanism as the fluorescence quenching of piperine-ctDNA. The thermodynamic parameters revealed the predominance of hydrophobic forces in the interaction of ctDNA with piperine. According to the resonance light scattering data, the formation of a complex between piperine and ctDNA led to the creation of a larger particle. Ethidium bromide (EB) and acridine orange (AO) displacement studies, along with the ionic effects of NaCl and KI assessments, confirmed the interaction of piperine-ctDNA through a groove binding mode. The melting temperature assay of ctDNA upon the addition of piperine concentration indicated the probable groove binding of piperine to ctDNA, which was affirmed by relative viscosity measurement as well. The lack of detecting any alterations in the circular dichroism (CD) spectrum of CD investigation verified as a characteristic sign of groove binding mechanism and also confirmed all the experimental results with regard to the binding of piperine-ctDNA complex. Next to observing a concentration and time-dependent cytotoxicity in MDA-MB-231 cells, the impact of piperine on increasing lipid peroxidation and decreasing the activity of superoxide dismutase was also noticed. Apparently, piperine is capable of inducing caspase-3 activity as well.
在这项研究中，我们在 pH = 6.8 的 Tris-HCl 缓冲溶液中确定了胡椒碱和小牛胸腺 DNA（ctDNA）的相互作用，并且还通过多光谱技术以及热融和粘度测量的数据评估了结合机制。荧光猝灭的结果证实了胡椒碱和 ctDNA 之间存在相互作用，并指出胡椒碱是猝灭剂。此外，在 298、303 和 308 K 三个不同温度下测量了 K 值，分别为 4.5×10^-4、5.65×10^-4 和 9.36×10^-4 M，这表明动态机制是胡椒碱-ctDNA 荧光猝灭的主要机制。热力学参数表明，在 ctDNA 与胡椒碱的相互作用中，疏水作用力占主导地位。根据共振光散射数据，胡椒碱和 ctDNA 之间形成复合物导致形成更大的颗粒。吖啶橙（AO）和溴化乙锭（EB）的置换研究以及 NaCl 和 KI 的离子效应评估，证实了胡椒碱-ctDNA 通过沟结合模式相互作用。加入胡椒碱浓度后 ctDNA 的熔点测定表明，胡椒碱可能与 ctDNA 以沟结合方式结合，相对粘度测量也证实了这一点。圆二色性（CD）研究中 CD 谱的变化表明，这是沟结合机制的特征标志，也证实了所有关于胡椒碱-ctDNA 复合物结合的实验结果。胡椒碱在 MDA-MB-231 细胞中观察到浓度和时间依赖性细胞毒性作用，同时还注意到胡椒碱对脂质过氧化的增加和超氧化物歧化酶活性的降低的影响。显然，胡椒碱也能够诱导 caspase-3 活性。
