INTERACTIONS OF THYMOQUINONE PRINCIPAL COMPONENT OF NIGELLA SATIVA IN MOLECULAR AND CELLULAR MODELS

In presents years ,increased attention has been focused on the TQ and its reduced form as an antioxidant but in spite of extensive clinical experimental evidence , the moelecular mechanisms responsible for he antioxidant activity or their potential biological target are still debated . Thymoquinone(TQ) is the bioactive constituent of the volatile oil of nigella sativa and has been shown to exert antioxidant , antineoplastic , and anti-inflammatory effects. We investigated the interaction of TQ with different endogenous non- enzymatic redox systems and found that TQ reacts chemically (i.e. non-enzymatically) with glutathione (GSH) ,NADH and NADPH.A combination of liquid chromatography/UV-Vis spectrophotometry/Mass spectrometry analyses was used to identify the products of these reactions. The reaction that occur in physiological conditions indicates the formation of only two products, glutathionyl-dihydrothymoquinone (DHTQ -GS) after rapid reaction with GSH, and dihydrothymoquinone (DHTQ) after slow reaction time with NADH and NADPH. Measurement of the antioxidant activity of reduced compounds against organic radicals such as 2,2¢-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) also revealed a potential scavenging activity for glutathionyl-dihydrothymoquinone similar to that of dihydrothymoquinone. The reactivity of thymoquinone towards different redox states of haemoglobin and myoglobin in the presence of endogenous reducing compounds as GSH, NADH, and NADPH was evaluated by optical spectral analysis. In the presence of reducing compounds, thymoquinone is able to convert met-hemoglobin and met-myoglobin to their oxo-derivatives in physiological conditions. Conversely, thymoquinone in the presence of the same compounds increases the rate of conversion of FeIV to FeIII and then rapidly to FeII form. The reaction is mediated by the intermediate quinone forms of TQ as glutathionyl-dihydrothymoquinone (DHTQ-GS) and dihydrothymoquinone (DHTQ), formed from direct interaction of TQ with GSH or NADH (NADPH). The ability of TQ to recycle oxidation states of haem protein provides a novel antioxidant mechanism of TQ and may represent an important defence mechanism against oxidative tissue injury . Thymoquinone and its reduced derivative dihydrothymoquinone(DHTQ) were examined also for their protective effects against peroxynitrite oxidation of dihydrorhodamine (DHR) 123 , dihydrodichlorofluoresceine (DCFH) as well as cytochrome c2+ and hemoglobin oxidation. All these phenolics, were fairly active in preventing the oxidation of DHR 123 and DCFH and in the cytochrome c test. DHTQ shows the highest antioxidant activity against peroxynitrite comparable with that shown from trolox used as control in all experiments. DHTQ also protect oxy-hemoglobin oxidation, mediated by peroxynitrite ; the influence of peroxynitrite concentration on the pseudo †"first order rate constants was studied. However the interaction of thymoquinone to Human Serum Albumin(HSA) has been studied for the first time by spectroscopic methods including Furier transform infrared(FTIR) spectroscopy, circular dichroism, UV-absorption spectroscopy and fluorescence quenching under physiological conditions. The results of thermodynamic parameters obtained for the interaction of TQ with HSA (?H? and ?S changes were calculated according to the van't Hoff equation) suggest that hydrophobic interaction is the predominant intermolecular forces stabilizing the complex which is also in good agreement with the results of the molecular modelling study. Minor alteration of protein secondary structure in the presence of thymoquinone in aqueous solution was shown by CD and FTIR spectroscopes. The quenching mechanism were obtained by fluorescence titration data. Fluorescence displacement measurements confirmed that thymoquinone binds HSA on site I. The potential antioxidant effects of the hydrophobic TQ and its reduced form DHTQ on the peroxidation of human non-HDL fraction catalyzed by radicals and transition metals were also investigated. Iron-mediated lipid peroxidation and auto-oxidation of Fe2+ ion were inhibited markedly by DHTQ , and less effectively by TQ. Similar behaviour was shown for Copper-dependent oxidation of low density lipoprotein (LDL). Inhibition of LDL oxidation by DHTQ was closely related to its activities on copper reducing and scavenging a stable radical, 1,10-diphenyl-2-picrylhydrazyl (DPPH). Antioxidant properties of DHTQ against copper oxidation can be explained by direct interaction with this metal. Potent inhibitory effect of DHTQ on lipid peroxidation by Fe2+ ion probably may be related to the decreased formation of perferryl ion or the iron†"oxygen chelate complex as the initiating factor of lipid peroxidation by keeping iron at a reduced state. Inhibition of LDL oxidation by DHTQ is due to the suppression of free radical cascade of lipid peroxidation in LDL by reducing copper ion.Inhibition of oxidative activity by DHTQ could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects . Our results indicate that the some biological effects observed for TQ can be attributed , at least partialy ,to its ability to function as antioxidant in biology redox cycling between quinine and hydroquinone and this properties may shield different biological components against the harmful effects of different reactive radical species .