| Objective1. To establish an animal model for loaded swimming and the metabolic changes during swimming were investigated so as to provide a basis for the study of anti-fatigue property of quercetin.2. To compare the anti-fatigue effect of quercetin with other common phytochemicals for the development of new anti-fatigue functional foods containing phytochemicals.3. To investigate the effects of quercetin on metabolism in swimming mice and the involved mechanisms related to energy metabolism, antioxidant capacity and mitochondrial functions.Methods1. The mice were fed an AIN-93 diet for 14 days, and forced to swim for 30,60 or 120 minutes, respectively, with a load on their tails equivalent to 2% body weight. The over all changes of serum metabolic profiles were analyzed using a1H NMR based metabolomic approach. Meanwhile, some biochemical measurements were also carried out so as to monitor the changes in energy metabolism. The optimal swimming time for further studies and new biochemical markers potentially for fatigue were investigated.2. The mice were fed a basic AIN-93M diet (normal control) or supplemented with quercetin, green tea polyphenols, soyasaponin, lycopene, or genistein at a dose of lg/kg for 14 days, and forced to swim to exhaust. The maximal swimming time was recorded to compare the antifatigue effect of quercetin with other common phytochemicals. Different doses of quercetin (0.5,1 or 2g/kg) were further supplemented to the diets and fed the mice for 14 days. The dose dependent effects of quercetin on swimming time were established.3. The mice were fed an AIN-93M diet supplemented with lg/kg quercetin for 14 days and then forced to swim for 60min. The effect of quercetin on serum metabolic profiles was analyzed metabolomically. The main components contributed significantly to the differences in serum metabolome between different groups were determined.4. The samples of blood, liver and muscles of hind legs were collected. The serum contents of glucose, lactate, nonprotein nitrogen, free fatty acids, VC, VE, FRAP, and hepatic or muscular contents of glycogen, GSH, MDA, activities of succinate dehydrogenase, ATPase were measured, respectively.5. The mitochondria were isolated from skeleton muscles by differential centrifugation. Mitochondrial respiratory chain complex, citrate synthase, membrane fluidity, membrane potential, mitochondrial respiratory function, permeability transition pore and antioxidant indices such as GSH, FRAP, MDA were measured.Results1. The serum metabolic profiles changed significantly after loaded swimming. Serum lactate, lipid were increased and glucose decreased. These changes were time dependent. The results of metabolomic analysis were consistent with that of biochemical analysis. Moreover, serum choline content was found to be decreased gradually during swimming, possibly related to enhanced lipid metabolism during swimming because choline plays a critical role in lipid oxidation. The metabolic changes turned to be maximal 60 min after swimming, which can be selected as the time point suitable for further studies.2. Quercetin exhibited the most potential anti-fatigue effect compared to other phytochemicals. It can be considered a new candidate for anti-fatigue components in functional foods. The supplementary dose of 1g/kg was demonstrated to be most effective in anti-fatigue test.3. The metabolic profiles were improved greatly after quercetin supplementation in swimming mice based on the results from loadings plot. This effect was possibly related to improved energy metabolism and antioxidant capacity, which provides a basis for further study on the mechanism of actions quercetin plays in swimming mice.4. The serum contents of lactate and NPN increased significantly after swimming and decreased after quercetin supplementation. The supplementation of quercetin increased serum content of free fatty acids, indicating that quercetin improves energy metabolism by decreasing protein oxidation and increasing lipid mobilization. Quercetin also improved TCA cycle by increasing SDH and ATPase activities. The MDA content was decreased and GSH content increased in muscle tissues after quercetin supplementation.5. Muscle mitochondrial function was protected significantly by quercetin. The activity of mitochondrial respiratory chain complex, mitochondrial membrane potential and membrane fluidity deceased after swimming and recovered after quercetin supplementation. Meanwhile, mitochondrial content of GSH and FRAP value increased and MDA content decreased, indicating that oxidative stress of mitochondria induced by swimming could be prevented by quercetin. The decrease of mitochondrial ATPase activity after swimming was also diminished partially after quercetin supplementation. The improved mitochondrial function contributes significantly to the anti-fatigue effect of quercetin.ConclusionOur study indicates that metabolomic technique is useful in providing systematical information for the nutritional intervention of fatigue. The animal swimming model used in this study has advantage in reflecting the status of energy metabolism. Quercetin is a powerful anti-fatigue and the optimal dose for supplementation is 1g/kg. The energy metabolism, antioxidant capacity, as well as mitochondrial function are improved significantly after quercetin supplementation in swimming mice and all of them contribute to the anti-fatigue effect of quercetin.
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