| 1. BackgroundFunctional foods have developed greatly and got more and more the market share in the past several decades. Now, Europe, USA and Japan have become the omni-directional centers contenting of production, consumption and research in the world. And the centers together push the related research and economy forward. The antioxidant food as the most part of functional food, have become the focus point of research. Especially the epidemiology data show that free radical is the main case and the antioxidant (nature, artificially synthesize) may balance the damage. For instance, the incidence rate of people will reduce after they have eaten the vast of vegetables and fruits a short time. Therefore, the preventive means which aim to the balance of oxidation have got the most health experts admission.To establish the excellent procedure, is an important project for us. From the area of global development we can see, free radical theory is deemed as the famous item, so it now become the main guideline of the antioxidant research. Despite there are more or less controversies currently from the research in vitro to research in vivo. It is more difficult for us to measure directly the antioxidant capacity, particular the operation and the cost. The testing research in vitro is prominent measuring method and gives the exact results of detection. The research in vitro in the several decades, we have established the DPPH, ABTS, FRAP, TRAP, ORAC and TOSC. The blood red cell tests, LDL-ox tests and cell or tissue oxidative damage in vitro induced. Also the research in vivo has developed enormously, and several animal models have established successfully induced by different methods, the effective biomarkers and the reliable detective methods have got the support from the most scientists.Animal model have been deem to be the one of the effective and reliable evolutional methods. In the terms of the evaluation we usually use the old rats in animal model, but lacking of the necessarily standards. In addition, we use the three kinds of animal models, for example, D-galactose,γ-radiation and bromo-benzen, especially D-galactose induced. There are three main strategies, first, induction of oxidative stress may be achieved by exposure to a toxic agent, such as carbon tetrachloride or paraquat or ethanol. Second, the dietary-induced oxidative stress or damage, caused by nutritional imbalance due to either an antioxidant deficient diet or a pro-oxidant overload diet. Third, other approaches to induce oxidative stress in experimental animals have been reported including exercise- or radiation-stress and transgenic animal model or senescence accelerated mouse, even the pathologic animal model. In order to give the damage evaluation of the tissue, applying for the effective biomarkers are the most department of procedure. The detection of the anti-oxidative system and oxidative damage biomarkers are the main point. There are different kinds of antioxidant substances, vitamin C, vitamin E, urea, and so on. The action and the damage of the tissues are the main detective targets. And the improved methods of MDA test, comet test and the other detective technology of protein oxidation products.The project of the research is based on the latest progress of antioxidant by the reliable and easy operation, in order to explore the more excellent evaluate technology.2. MethodsHarman had obviously advised the free radical and senescence theory at 1956. Following the theory, the abundant of research conferences and papers have been celebrated. The antioxidant capacity evaluation research in vitro is superior to the research in vivo. The project of research is divided into two departments, in vitro and in vivo.(1) The antioxidant capacity of research technology in vitroABTSABT response with potassium per sulfate to the base of the assessment—ABTS·+. When the bio-substance exists, ABTS·+ will become colorless solution. The extent of solution varies determined by the bioactive and the concentration of the existing substance in the system solution. The antioxidant capacity of substances adopts the equal grade of Trolox as the Unit.FRAPAt low PH environment, the bio-substances will use the Fe(Ⅲ)-2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ-Fe3+) deoxidize the blue TPTZ-Fe2+. The results of the response adopt the antioxidant capacity of equal grade of Fe2+ or standard of substance. But the assay can not use the -SH, because the kind of GSH can not response with Fe3+.According to the result of the test, there exist a series of filtering and evaluating methods, then rank, even give the final scientific conclusion and give the farther research in vivo (animal experiments, human interventional experiments)(2) The antioxidant capacity of research technology in vivoThe antioxidant capacity of research technology in vivo is also important department of the assessment technology. The assay divided into two segments, it is the animal model and the effective biomarkers. If we use the appropriate oxidative stress animal model and it is no doubt that it is the brief assay, and the effective biomarkers are also the premise of research.Animal modelEthanol-induced the oxidative stress mice model Ethanol-induced oxidative stress may relate with the cellar mitochondria damage and the metabolism of mitochondria. Due to the huge of ROS/RNS may lead to the damage of tissue. The cycle of experiment is lasting for 5 weeks and corresponding to the test substance by gavage at 1th week, from the 2nd week give the experimental mice the concentration of ethanol (25%, w/v) at the later 4 weeks. At the end period of the experiment kill all the experimental mice and take the related tissues in order to test the status of antioxidant system.Cadmium-induced the oxidative damage mice model cadmium, a widely used heavy metal in modern industry and human life. The target of the attack is the lung, liver, kidney and testicles. Because cadmium has only single redox-valence state, no direct produce ROS/RNS. The test uses the cadmium chloride and is administered subcutaneously at a single dose of 7 mg/kg body*weight at 1 hour after the last dose of corresponding the antioxidants and take the related tissues in order to test the status of antioxidant system.The effective biomarkersThere are several different kinds of antioxidants in plasm, consisting of vitamin E, coenzyme Q, vitamin C, GSH, eura and TAC. In addition, the oxidative damage of the lipid, protein and DNA in blood and urine. For example, 8-OHdG, Thymine glyeol, Comet assay and Protein carbonyls, Lipid peroxidation. The experiment based on the former regulation, have established the new fragment.3. Results and conclusion3.1 The antioxidant capacity of evaluation result in vitro3.1.1 The result of detection by ABTSApplying for the ABTS, respectively detect the activity of quercetin, curcumin, vitamin E and procyanidine. The experiment progress three times and the results are more stable, and there is no statistic difference (P < 0.05). The equation of equation is Y=92.661x+2.29, R2=0.999. And according to the equation, the equal grade of quercetin and curcumin is 2.02, 0.50. 1gram of vitamin E and procyanidine which eliminate free radical is 2.06mmol, 2.897mmol the equal grade of Trolox.3.1.2 The result of detection by FRAPApplying for the FRAP, respectively detect the activity of quercetin, curcumin, vitamin E, procyanidine and Trolox. The experiment progress three times and the results are more stable, and there is no statistic difference (P < 0.05). First, we adopt the FeSO4 standard curve, and the equal is Y=1.095x+0.002, R2=0.999. According to the curve, the results of the experiment use 1.0mmol/l FeSO4 as the standard reference, quercetin, curcumin and Trolox respectively is 5.73, 1.18 and 2.09. And the antioxidant capacity of vitamin E and procyanidine respectively is 207.7mg, 156.36mg equal to 1.0mmol/l FeSO4.3.2 The antioxidant capacity of evaluation result in vivo3.2.1 The analysis result of ethanol-induced oxidative stress mice model(1) The general condition of growAll the mice adapt to the environment of animal room for 1 week, the weight of mice in different groups no change. From the 4th day, the experiment process and note the weight in every 3 days. And there is no difference a little.(2) The weight of viscera of mice in different groupThe weight of brain between the 2nd group and 1th group, there is no difference between them. And the weight of kidney is different, there is significance (P<0.05). The weight of liver in 2nd group is lower than 1th group, and there is no difference.(3) The biochemical index of experimental mice in different groupThe activity of GPx and SOD between 2nd group and 1th group, it is lower in GPX and higher in SOD.The biochemical index of liver, the 2nd group contrasting to the 1th group, the activity of GPx is lower and there is a statistical significance (P<0.05). But there is no significance between SOD and MDA in different groups.3.2.2 The analysis result of cadmium-induced oxidative damage mice model (1) The weight of mice viscera in different groupsThe weight of mice viscera in different group, such as brain, kidney and liver, especially the liver in the 2nd group is lower than other groups, and there is a statistical significance (P<0.05).(2) The MDA content of the blood in different groupsContrasting to the 1th group, the MDA content of the plasm is higher than other groups, there is a statistical significance (P<0.05). And the MDA content of the plasm in 3rd group is also a statistical significance (P<0.05).(3) The MDA and PCO content of brain and the PCO content of kidneyContrasting to the MDA and PCO between the 2nd, 3th, 4th, 5th group and the 1th group, the content is higher and there is a statistical significance (P<0.05). And the PCO content of kidney higher than other groups.(4) The activity of SOD and CAT and the content of MDA and PCO in liverContrasting to the activity of SOD and CAT between the 2nd and 1th group, the activity of SOD and CAT is lower and a statistical significance (P<0.05). The MDA and PCO content is higher than others.(5) The oxide-product of DNA content (8-OHdG) of liverFrom the results of 8-OHdG there is higher than the 1th group and there is a statistical significance (P<0.05).4. SuggestionThe project has been established the methods in vitro, such as ABTS and FRAP in physical and chemic environment, but lacking of the structural obstacle. Therefore, we suggest that add the related red blood cell to the procedure and LDL-ox tests, and we also suggest that add the biomarker of PCO and 8-OHdG, 8-iso-PGF2a. |
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