Investigations On The Detection Techniques And Molecular Mechanism Of The Interaction Between Plant Active Ingredients And DNA In Vitro

Active ingredients extracted from plants such as flavonoid, coumarins, saponins, alkaloids, polysaccharides, phenolic acids and etc. possess a variety of pharmacological activities. Because nucleic acid is physical basis of gene expression, the target of most of durgs with anticancer and antiviral activities in living organisms is DNA. They interfere the duplication of DNA by binding with it, thus causing destruction to tumour and virus cells.The study has constructed a method system, which combing multiple spectroscopy methods, containting ultraviolet-visible(UV–vis) absorption, fluorescence, circular dichroism(CD) and fourier transform infrared spectroscopy(FT-IR) with chemometrics and molecular docking. The hyphenated technique was used to investigate the binding properties and molecular mechanism of interacation between several plant active ingredients like psoralen, etc, and calf thymus DNA(ct DNA) in physiological buffer(pH 7.4). These investigations will provide new research approach and technical platform for detecting interaction between drug and DNA, and provide theoretical basis for understanding interaction mechanism and pharmacological activity from the molecular level and screening of drugs in vitro.The main contents of these investigations were summarized as follows:1. A brief introduction about the structural and physiological properties of DNA was presented in the first chapter. The investigating methods and current situation on the interaction between DNA and small molecules were summarized in this chapter.2. A method system was constructed by integrating multivariate curve resolution–alternating least squares(MCR–ALS) with spectroscopic techniques including UV–vis absorption, fluorescence, CD and FT-IR spectroscopy and molecular docking, and applied to explore the binding properties of psoralen(PSO) or 8–MOP to ct DNA and their molecular interaction mechanism. An extended data matrix with rich information was built by several UV–vis spectral data matrix of PSO or 8–MOP interacting with ct DNA obtained from titration experiments under different modes. The data matrix obtained from UV–vis spectra was resolved by MCR–ALS approach. The pure spectra and the equilibrium concentration profiles for the components(PSO, ct DNA and PSO–ct DN or 8–MOP, ct DNA and 8–MOP–ct DNA) were extracted from the highly overlapping composite response, so the quantitative determination of the interaction between PSO or 8–MOP and ct DNA was realized. It was been found during the exploration process on the binding modes of PSO or 8–MOP to ct DNA that, the existence of PSO or 8–MOP could cause decrease in iodide quenching effect, ct DNA melting temperature and increase in ct DNA viscosity, which supported the result that the two drug interacted with bases of ct DNA via intercalative mode. FT-IR spectra studies showed that PSO preferentially bound to adenine bases, while 8–MOP mainly interacted with A, T bases. CD spectra analysis found that PSO and 8–MOP could perturb the base stacking and double helix of ct DNA, however, the B–form of ct DNA was not been changed. The molecular docking confirmed and the experimental and intuitively displayed the binding mode and gesture of PSO or 8–MOP to ct DNA.3. The interaction and binding characteristics of daphnetin with ct DNA were detected by the integrated technique united UV–vis absorption spectroscopy with MCR–ALS and three-way synchronous fluorescence spectroscopy with parallel factor analysis modeling(PARAFAC). The expanded UV–vis spectral data matrix was processed by MCR–ALS to obtain the pure spectra and concentration profiles of the three main components(daphnetin, ct DNA and daphnetin–ct DNA complex), which quantitatively monitored the daphnetin-ct DNA interaction and the formation of complex. The daphnetin could cause little change in melting temperature and viscosity of ct DNA and iodide quenching effect, and lead to an increase in single-stranded DNA quenching effect, which indicated that daphnetin bound to the groove of ct DNA. And three-way synchronous fluorescence spectra data for the competitive binding between daphnetin and Hoechst 33258 for ct DNA was resolved by PARAFAC, which further ascertained the groove binding of daphnetin to ct DNA. The molecular docking vividly visualized the interaction of daphentin with adenine and thymine bases in the minor groove of ct DNA, which was in accordance with the results of the FT-IR analysis. The circular dichroism spectra showed that daphnetin led to the conformational change of ct DNA from B–form to A–form.4. The combined method system including MCR–ALS, multiple spectroscopies, ct DNA thermal denaturation studies, viscosity measurements and DNA cleavage was exerted to investigate the coordination between daphnetin and Cu2+ and determine the interaction properties of daphnetin–Cu(II) complex with ct DNA. The results indicated that daphnetin–Cu(II) complex was formed at molar ratio of 1:1, and the intercalation mode of daphnetin–Cu(II) complex to ct DNA was supported by the increase in DNA viscosity and melting temperature and the competitive binding between daphnetin–Cu(II) complex and ethidium bromide(EB) for ct DNA. The interaction also induced changes in CD spectra of ct DNA due to the reduction of helicity and bases stacking. The analysis of FT-IR showed that the binding sites of daphnetin–Cu(II) to ct DNA were between A and T bases, and the B-form of DNA had not been changed. Moreover, DNA cleavage experiment suggested that daphnetin–Cu(II) could cause cleavage of plasmid DNA due to its strong binding ability to DNA. The calculated enthalpy and entropy changes suggested that the driving force in the binding of daphnetin–Cu(II) to ct DNA were hydrogen bonds and hydrophobic forces.5. The interaction between asiaticoside(AC) and ct DNA in physiological buffer(pH 7.4) was investigated by fluorescence, CD and FT–IR spectroscopy coupled with ct DNA melting studies and viscosity measurements. The results showed that the fluorescence of AC could be remarkably quenched by ct DNA, and the probable quenching mechanism was static quenching. The binding of AC to ct DNA was able to increase the melting temperature and relative viscosity of DNA, which suggested the binding mode was an intercalation binding, and hydrogen bonds and van der Waals forces were main driving forces. The changes in CD and FT–IR spectra of ct DNA indicated the adenine and thymine base pair was the mainly binding site and the B conformation of ct DNA was stabilized by AC.