| The study on effectiveness, distribution, metabolism, toxic and side effect of pharmaceuticals possess important significance to the evaluation and DR of new medicine, for the various kinds of medicine or remedy are indispensable to human's life. Baclofen belongs to the neurotransmitter pharmaceutical, which is the fat-soluble derivate ofγ-aminobutyric acid and it always been adopted in the cure of relaxation of skeletal muscle, hyperkinesias and some traumatic injuries. Otherwise, this medicament is the inhibitory transmitter of nervus centrails (CNS) and also been used in alleviating the symptoms of cough and allergies in lungs. Few of the articles of study on baclofen andγ-aminobutyric acid focus on the binding interaction of proteins in human body and their side-effects in spite of the numerous achievements in the filed of effectiveness. In order to explore the unsolved problems mentioned above, with the use of spectroscopy, this thesis made a tentative study on the interactions with proteins, furthermore, investigated the interactions between the two and different proteins from a microscopic view, with the purpose of providing relative parameters to deepening the knowledge of the medicines. Specifically speaking, the preparation or experiments which had been done for this thesis are as following:1. The inclusion interaction ofβ-cyclodextrin and baclofen was studied by UV spectra and H1NMR.The stoichiometry ratio for the formation of the inclusion complexes is 1:1, which was determined by equation linear analysis and molar ratio method. The enthalpiy change (ΔH) and entropiy change (ΔS) were derived from the inclusion process by K at different temperatures. The results showed that the inclusion process is spontaneous, the hydrophobic force was main binding force ofβ-cyclodextrin inclusion complex. The interactions of BSA between baclofen or complex have been studied by ?uorescence spectroscopy. The results showed that the quenching effect of baclofen or complex on the BSA fluorescence is a static quenching process. The hydrophobic force of baclofen alone with BSA is mainly interaction force, and the electrostatic force of comlex with BSA is mainly interaction force, for the reason that baclofen is included by theβ-cyclodextrin.2. The binding interactions of BSA with baclofen orγ-aminobutyric acid were investigated, and the impact to these interaction from metal ions was observed. The agentγ-aminobutyric acid is capable to change theΔE ofπ→π* transition, which made a blue shift in UV spectroscopy. The reactants combined stably when the reaction lasted for about 32 seconds. Measured by the FT-IR spectroscopy, it was the result that both baclofen andγ-aminobutyric acid could change the secondary structure of BSA. Being improved by fluorescence spectra and UV spectra, we are assured that the binding interactions between baclofen and BSA were not belonging to the unradioactive resonate energy transfer. By observing the impact given by certain mental ions to the biding of baclofen with BSA, the study found that the proper density of mental ions guarantee the binding interaction of baclofen with BSA.3. The binding interaction between baclofen and pepsin was studied by ultraviolet absorption and fluorescence spectra. Baclofen caused the blue shift of the maximum of UV absorption peak of pepsin. Stern-Volmer equation indicated that the fluorescence intensity of pepsin was quenched by baclofen via a static quenching mechanism, and the associating constants were calculated. The thermodynamic parameters showed that the main sort of binding force was hydrophobic force. Meanwhile, according to the non-radiation energy transfer theory, there is a energy transferring between pepsin and baclofen, so the fluorescence intensity of pepsin was quenched. FT-IR spectroscopy was used to investigate the effect of baclofen on pepsin. The results showed that the secondary structure of pepsin has been changed by baclofen. Took advantage of the catalysis of pepsin, a study on the influence of baclofen to pepsin had been carried out, with its result showed that reactant baclofen restrained the activity of pepsin for about 46.50%.4. Explored the combine of baclofen with cytochrome C and found that reactant baclofen could make cytochrome C having a hyperchromic effect. It had been improved by the synchronous flourimetry that the fluorescence intensity of cytochrome C had been increased, for reason that there were combination between baclofen and tyrosine but weak bond with tryptophan. The secondary structure of cytochrome C have been influenced by both baclofen andγ-aminobutyric acid and then loosen its structure. These changes could be attested by FT-IR spectroscopy. Took UV spectrum as the method to investigate cytochrome C that has been combined with reagent, with the result suggested that both baclofen andγ-aminobutyric acid would restrain the activity of cytochrome C, which might cause a toxic and side effect after taking. |
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