Gene Expression of Antioxidant Metabolic Enzymes in Grape Extracts

Fruit can use its structural characteristics to stabilize the excess electrons of free radicals and prevent cell aging, which is the main source of antioxidants supplementation. In the current study, the expression of antioxidant metabolic genes was evaluated in fruit extract. Seventy mice were divided into one control group and six experimental groups. Six experimental groups were fed 0.1g of grape extract daily for 3, 7, 10, 14, 21, 24 and 30 days, respectively. The antioxidant activity of fruit extract was tested by antioxidant metabolic enzyme gene expression in the liver and kidney of different experimental groups. The results showed that grape extract could effectively enhance SOD activity, LPL relative expression and FAS relative expression in mice liver tissue; grape extract could effectively increase SOD relative expression, TrxR2 relative expression and CAT relative expression in mice kidney tissue; grape extract could effectively reduce MDA activity in mice liver tissue, and increase and decrease the extent of MDA activity and the duration of feeding in mice. The proportion of fruit extracts showed that fruit extracts could effectively improve the antioxidant performance of the body.


Introduction
Free radicals are intermediate products of biochemical reactions in the process of an organism's life activities. Under normal circumstances, the production and elimination of free radicals in the body are in a dynamic equilibrium. When it produces too much or eliminates too slowly in the body, it will cause damage to the body at the molecular level, cell level and organ level, attack the unsaturated fatty acids that make up the cells, leading to cell membrane aging, accelerate the aging process of the body, and induce cancer, cardiovascular disease and so on. Under the requirement of advocating nature and returning to nature, great progress has been made in the research and development of natural antioxidants (1). In addition, natural antioxidants have been developed from antioxidants for oil and fat-containing foods to scavengers for oxygen-free radicals in vivo. It has been proved that these natural compounds have physiological functions such as protecting human cell tissues, protecting cardiovascular and cerebrovascular circulation systems, anti-cancer and delaying aging. As we all know, fruits and vegetables not only provide some important nutrients such as vitamins and minerals but also have the functions of anti-oxidation, anti-mutation, cancer prevention, immunity enhancement, anti-allergy, blood pressure regulation and cholesterol (2). It also provides a variety of basic nutrients such as carbohydrates, minerals, proteins and so on.
Apple is called "all-round healthy fruit" by scientists. Eating apples regularly can reduce blood lipids, control blood pressure, prevent cancer, strengthen bones, maintain acid-base balance and lose weight. In addition to the traditional nutrition of pectin fiber and vitamin group, apple also contains a lot of antioxidants (3). These antioxidants can be hundreds of species and are considered to be the holy products of cancer prevention and aging. Strawberry, also known as red berry, asparagus, raspberry and so on, is the generic name of Rosacea strawberry plants, there are three "kinds in the world. Strawberry nutrients are easily digested and absorbed by the human body. It is healthy food for all ages. The traditional Chinese medicine circles believe that strawberry has the functions of moistening lung, nourishing the body, clearing heat and cooling blood, invigorating the spleen and eliminating alcohol, and has certain nourishing and regulating effects on the gastrointestinal tract and anemia (4,5). Strawberry is a plant rich in tannic acid, which can absorb and prevent the absorption of carcinogenic chemicals in the body and has the function of cancer prevention. Grapes are not only delicious but also of high nutritional value. The content of glucose in ripe berries is as high as 10%-30%. Glucose is the main component. Many kinds of fruit acids in grapes help digestion. Eating more grapes properly can strengthen the eyes and stomach. Grapes contain minerals such as calcium, potassium, phosphorus, iron, vitamin B1, vitamin B2, vitamin B6, vitamin C and vitamin P. They also contain a variety of amino acids needed by the human body. Frequent grapes are of great benefit to neurasthenia and fatigue (6). It was found that grapes could prevent thrombosis better than aspirin, reduce serum cholesterol level and platelet cohesion, and play a certain role in preventing cardiovascular and cerebrovascular diseases. Eating a moderate amount of fresh grapes every day not only reduces the risk of cardiovascular disease but also is particularly beneficial to the health of patients with ischemic heart disease and atherosclerotic heart disease (7). Flavonoids in fresh grapes can "clean" blood and prevent cholesterol plaque formation. The more black the grapes are, the more flavonoids they contain. But if grape skin and grape seeds are eaten together, the better the protection of the heart is.
Various endogenous and exogenous factors will produce excess free radicals in the body, causing harm to the body. Some natural phytochemicals in fruits can protect, scavenge and repair excessive free radicals. These natural phytochemicals include carotenoids, phenolic compounds, phytic acid and so on. The antioxidants in grapes mainly include flavonoids such as flavanone and anthocyanin, catechin monomer and oligomeric protoplast anthocyanin, in addition to resveratrol and its glycosides. Catechin can significantly inhibit collagen-induced hydrogen peroxide production (8). Proanthocyanidins can also react directly with hydroxyl radicals and hydrogen peroxide.
The reactive oxygen species (ROS) cluster is an oxidative product produced by organisms during normal aerobic cell metabolism and respiratory burst.
It participates in many biological processes in vivo, including normal cell growth, programmed cell death and cell senescence. In reactive oxygen species, hydrogen peroxide is a peroxide that easily forms free radicals. The formation and action of singlet oxygenexcited molecular oxygen can also involve free radical reactions. Therefore, many free radical reactions in organisms are related to reactive oxygen species (9). Free radicals are substances with strong oxidation ability and active nature produced by redox reactions in the normal metabolism process of the body. Even a small amount of free radicals can cause great harm to the body. Complex systems of endogenous and exogenous antioxidant sources in vivo are used to alleviate the potential damage of free radicals (10). The production and removal of free radicals are in a dynamic balance. If the body's ability to scavenge free radicals decreases (11), resulting in excessive free radicals, structural abnormalities of cell membranes and mitochondrial membranes, dysfunction and aging may occur, and many chronic and degenerative diseases, including cancer. Atherosclerosis and neurodegenerative diseases are examples of free radical-mediated oxidative stress.
Anything that can oxidize stress all the time and damage it to the target molecule can become an antioxidant. The main function of antioxidants is to inhibit and eliminate free radicals. In order to protect itself from free radical-mediated oxidative stress, different cell antioxidants, such as glutathione, tocopherol and ascorbic acid, as well as peroxidase, superoxide dismutase and catalase, can interact with reactive oxygen species. The body can also alleviate oxidative stress in vivo by supplementing antioxidants. However, some synthetic antioxidants have shown potential adverse reactions such as liver injury, especially long-term use of synthetic antioxidants (12). Therefore, exploring safe and natural antioxidants to resist oxidative stress has become a research hotspot in recent years (13). Epidemiological studies have shown that the intake of fruits and vegetables is proportional to the prevention of diseases caused by oxidative stress. Flavonoids, anthocyanins, lignin and tannins in plant tissues all have good free radical scavenging capacity and belong to the category of natural antioxidants.
According to the relationship between antioxidants and health, Cömert et al. (14) draw the following three main conclusions: many epidemiological studies worldwide agree that a diet rich in vegetables containing antioxidants is an important component of several healthy behaviors related to health and longevity. Two large-scale dietary surveys in the United States have shown that the intake of betacarotene, VA and VE is associated with lower levels of ischemic heart disease and cancer risk. The concentration of some major antioxidants in plasma is a reliable indicator of the actual antioxidant capacity of the body. Blair et al. have shown that physical exercise and lifelong physical activity contribute to a slow decline in age-related functional capacity and prevent or delay the onset of some degenerative diseases (15). The effect of lifelong physical activity on life expectancy is an additional 2-7 years on the basis of average life expectancy. But it has never been determined whether physical activity can increase maximum life expectancy. Simon et al. have shown that physical exercise can reduce the balance of the body and cause a series of adaptive changes in the stress system. Regular exercise can promote the body to adapt to acute exercise stress and enthusiasm, thus restoring its balance. Zhang et al. (16) have shown that aerobic exercise can improve the level of endogenous antioxidant enzymes and enhance the ability of antioxidant defense. Its effect is the same as that of pure exogenous supplementation of antioxidants.
Exercise-induced increase in endogenous antioxidant levels and defensive capacity may be the first to resist the modification of LDL by ROS, MDA and endothelial cells without consuming the antioxidants contained in LDL. Studies on gene expression of Pre-oxidation and anti-oxidation states have shown that the regulation of protein and DNA can be promoted through physiological oxidationantioxidation balance. Many studies reviewed by Sen (17) have shown that exercise-induced changes in glutathione redox status contribute to protein synthesis and produce more antioxidant proteins. In order to effectively detect the antioxidant activity of fruits, grape extracts with high antioxidant activity were selected. The antioxidant activity of fruits was effectively monitored by studying the gene expression of antioxidant metabolic enzymes in 70 experimental mice.

Materials and methods Laboratory Animals
Seventy C57BL/6 mice aged 7 weeks, half male and half female, average weight 16.9g, SPF grade, certificate number: SCXK (Shanghai) 2007-0005. After being purchased from Shanghai Laboratory Animal Center, the IVC system in the laboratory was routinely kept in cages for one week. All cages, food, water and cushions contacted by mice were sterilized under high pressure.

Reagents
SOD and MDA kits were purchased from Nanjing Jiancheng Bioengineering Company; Molecular biology reagents were purchased from Promega and Sigma Company, nylon membrane was purchased from Amershan Company, and recombinant plasmid PUC13-RCS containing mouse SOD gene (DNA size0.6kb, Cloningsite: EcoRI) was purchased from Wcyne State University. E. coli JM-109 was provided by the Microbiology Teaching and Research Department of the Academy of Life Sciences, Nankai University. The others are domestic and imported analytical pure. A-32P-dCTP was purchased from Beijing Yahui Biomedical Engineering Company.

Preparation of fruit extract
The fresh grapes were washed and dried. The skinned grapes were cut into thin slices about 2 mm thick. The grape pulp was centrifuged at 40,000 rpm for 20 minutes. The volume of sampling liquid was measured and the sediment was discarded. Because there was a lot of pectin in fruits, it is necessary to remove pectin: add absolute ethanol of the same volume as the sample solution, gently stir (18) with a glass rod, and then pour it into the centrifugal cup after 30 minutes, centrifuge for 10 minutes at the speed of 4000 revolutions per minute, and take the supernatant. If a small amount of flocculent precipitation is found in the supernatant, the extract can be obtained by filtration.

Experimental group
70 mice were divided into 1 control group and 6 experimental groups. The mice in 6 experimental groups were fed 0.1g fruit extract at the same time every day. The duration of each group was different. The specific feeding time and groups were shown in Table 1.

Primer design and synthesis
Primers were designed and synthesized according to the gene sequence of mice published by Genbank and references. The primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. Primer information is shown in Table 2.

Experimental steps
Animal treatment: The experiment was carried out according to the above animal groups. Animals were decapitated and killed. Livers and kidneys were quickly taken from ice tables to prepare tissue homogenate and supernatant was taken for detection (19).
Total RNA extraction: Guanidine isothiocyanate-phenol-chloroform one-step extraction method was used to extract and preserve at -80°C.

Determination of antioxidant components in fruit extracts
Determination of total phenol content: Folin phenol method was used. A 25 µL gallic acid (GAE) standard or fruit extract was added to a 96-well plate, and a 125 µL Folin phenol reagent was added to the plate. After 10 minutes of reaction at room temperature, a 125 µL saturated sodium carbonate solution was added. The absorbency was determined at 765 nm by an enzymelabeled instrument after 30 minutes at room temperature. The total phenol content is expressed in mgGAE/g. All samples were determined three times.
The content of total flavonoids was determined by the improved colorimetric method by Chun. The extract was prepared into 200 mg/mL solution for reserve. Mix 2 mL solution with 0.2 mL 5 g/100 mL sodium nitrite solution. After 6 minutes, add 0.2 mL10g/100 mLAlCl3·6H2O solution, mix and shake well (20). After 5 minutes, 2 mL1mol/L sodium hydroxide solution was added, and the reaction solution was fully mixed. The absorbance was determined at 510 nm after 15 minutes. The content of total flavonoids was calculated according to the standard curve of rutin. The total flavonoid content was expressed by rutin (RT) equivalent milligram per gram of dry quality sample material (mgRT/g). All samples were determined three times.
Quantitative fluorescence polymerase chain reaction to detect the relative expression of gene mRNA QRT-PCR was performed according to SYBR Premix Ex Taq II Kit instructions. The reaction system was 25 μL: SYBR Premix Ex Taq II 12.5 μL, DNA 2 μL, upstream and downstream primers 1 μL, and sterilized water was added to 25 μL.
The reaction conditions were as follows: predenaturation at 95℃ for 30 seconds, denaturation at 95℃ for 5 seconds, annealing at 60℃ for 30 seconds, extension at 60 (?) for 30 seconds, 40 cycles, dissolution at 55℃ for 10 seconds and 81 cycles (18). ActB was used as an internal reference and 2 -ΔΔCt was used to calculate the relative expression of each gene.

Statistical analysis
The experimental data were expressed by mean±standard deviation). The results of LSD multiple comparisons were analyzed by the single factor variance method using SPSS 17.0. When P < 0.05, there is a significant difference in the experimental results.When P < 0.01, there is a significant difference in the experimental results. The above two cases show that the experimental results have statistical significance.

Results and Discussions Component identification of grape extract
The high-performance liquid chromatography (HPLC) image of grape extract at 280 nm is shown in Figure 1. The results of HPLC-MS analysis of grape extracts are shown in Table 3.
The results of Figure 1 and Table 3 show that the main components of the grape extract absorbed at 280 nm are trans-resveratrol, hexose protocatechuate, quercetin-3-O-rhamnoside, hexose vanillin, dimethylanthocyanidin-3,5-diglucoside and anthocyanin-3,5-diglucoside, which contain a large number of antioxidants, which effectively illustrates the effectiveness of the grape extract and can be applied to anti-oxidation. Then, studying on gene expression of oxidative metabolic enzymes is useful.

Antioxidant activity of fruit extracts
The antioxidant activity, total phenolic content and total flavonoid content of grape extract are shown in Table 4. According to the relevant research results, the total phenolic content of 1 g mango extract is about 1.0 mg; 1 g strawberry extract is about 2.8 mg; 1 g mango extract is about 5.3 mg; and 1 g strawberry extract is about 7.1 mg. The results of Table 4 showed that the total phenolic content of the grape extract was (19.64±0.99) mg GAE/g) and total flavonoid content was (33.12±2.35) mg RT/g. The total phenolic content and total flavonoid content of grape extract were much higher than those of mango and strawberry. Phenolic compounds were the main antioxidant components of fruit extract. The experimental results showed that the antioxidant activity of the grape extract was obvious. The antioxidant activity of the grape extract was higher than that of mango and strawberry, which verified the validity of selecting grape extract to study the gene expression of antioxidant metabolic enzymes.

Effect of fruit extract on SOD activity in liver tissue of mice
The comparison of SOD activity in liver tissues of experimental mice in each group is shown in Table 5. Superoxide Dismutase Orgotein (SOD), alias liver protein. SOD is an active substance derived from living organisms, which can eliminate harmful substances produced by organisms in the process of metabolism. Continuous supplementation of SOD has a special anti-aging effect on the human body. The experimental results of each group in Table 5 were all P < 0.05, with statistical significance. The results of Table 5 showed that the SOD activity in liver tissue of mice fed with grape extract was significantly higher than that of control mice, and the duration of feeding was proportional to the SOD activity. The experimental results showed that the SOD activity of mice fed with fruit extract could be effectively enhanced.

Effect of a grape extract on MDA activity in liver tissue of mice
The comparison of MDA activity in liver tissues of experimental mice in each group is shown in Table 6. Malondialdehyde (MDA), an end product of lipid oxidation, affects the activity of mitochondrial respiratory chain complexes and key enzymes in mitochondria in vitro. MDA is one of the most important products of membrane lipid peroxidation, and its production can also aggravate membrane damage. Therefore, the content of MDA is a commonly used index in plant senescence physiology and resistance physiology research. The degree of membrane lipid peroxidation can be understood by MDA to indirectly measure the degree of membrane system damage and plant stress resistance. The experimental results of each group in Table 6 were all P < 0.05, with statistical significance. The results of Table 6 showed that the activity of MDA in liver tissue of mice fed with grape extract was significantly lower than that of control mice, indicating that grape extract could slow down the aging rate of mice, and the duration of feeding was inversely proportional to the activity of MDA. The results showed that the activity of MDA in mice fed with fruit extract could be effectively reduced.

Effect of a grape extract on LPL expression in liver tissue of mice
The relative expression of LPL in liver tissues of experimental mice in each group was compared as shown in Table 7. LPL is a key enzyme for lipid deposition in animal tissues. It is a rate-limiting enzyme for the degradation of triglycerides to glycerol and free fatty acid (FFA), and plays an important role in lipid metabolism and transport. By controlling the expression of LPL in adipose tissue and other tissues and organs, it directly determines the relative amount of lipid substrates in adipose tissue and other tissues and organs, thus indirectly deciding the metabolic future of lipid intake from food: stored in the form of body fat or consumed as energy substrates, and ultimately has a decisive impact on body fat accumulation. The experimental results of each group in Table 7 were all P < 0.05, with statistical significance. The results of Table 7 showed that the relative expression of LPL in liver tissue of mice fed with grape extract was significantly higher than that of control mice, and the duration of feeding was proportional to the relative expression of LPL. The experimental results showed that the relative expression of LPL in mice fed with fruit extract could be effectively increased.

Effect of a grape extract on the expression level of FAS in liver tissue of mice
The relative expression of FAS in liver tissues of experimental mice in each group was compared as shown in Table 8.  Table 8 were all P < 0.05, with statistical significance. The results of Table 8 showed that the relative expression of FAS in liver tissue of mice fed with grape extract was significantly higher than that of control mice, and the duration of feeding was proportional to the relative expression of FAS. The experimental results showed that the relative expression of FAS in mice fed with fruit extract could be effectively increased.

Effect of a grape extract on SOD expression in kidney tissue of mice
The relative expression of SOD in kidney tissue of experimental mice in each group was compared as shown in Table 9. In the SOD-catalyzed disproportionation of O2 -, H2O2 was produced, and the accumulation of H2O2 inhibited SOD. Normally, H2O2 produced by SOD disproportionation can be transformed into a non-toxic substance by CAT in time, thus regulating the balance of O2and H2O in the body. The experimental results of each group in Table 9 were all P < 0.05, with statistical significance. The results of Table 9 showed that the relative expression of SOD1 and SOD2 in kidney tissue of mice fed with grape extract was significantly higher than that of control mice, and the duration of feeding was proportional to the relative expression of SOD. The experimental results showed that the relative expression of SOD in kidney tissue of mice fed with fruit extract could be effectively increased.

Effect of grape extract on TrxR2 expression in kidney tissue of mice
The relative expression of TrxR2 in kidney tissues of mice in each group was compared as shown in Table 10. TrxR2 is an oxidoreductase that mainly exists in mitochondria. It can maintain the reductive form of Trx and regulate oxidative stress in mitochondria. Therefore, it is important to detect the relative expression of TrxR2 in kidney tissues of mice in each group for studying the gene expression of antioxidant metabolic enzymes in fruit extracts. The experimental results of each group in Table 10 were all P < 0.05, with statistical significance. The results of table 10 showed that the relative expression of TrxR2 in kidney tissue of mice fed with grape extract was significantly higher than that of control mice, and the duration of feeding was proportional to the relative expression of TrxR2. The experimental results showed that the relative expression of TrxR2 in kidney tissue of mice fed with fruit extract could be effectively increased.

Effect of a grape extract on CAT expression in kidney tissue of mice
The expression of CAT in kidney tissues of experimental mice in each group was compared as shown in Table 11. CAT is also an important antioxidant enzyme, which is indispensable in the regulation of superoxide anion free radicals and hydrogen peroxide. SOD and CAT play a protective role through mutual regulation.
In Table 11, the experimental results of each group were all P < 0.05, with statistical significance. The results of Table 11 showed that the relative expression of CAT in kidney tissue of mice fed with grape extract was significantly higher than that of control mice, and the duration of feeding was proportional to the relative expression of CAT. The experimental results showed that continuous feeding of fruit extract could effectively increase the relative expression of CAT in the kidney tissue of mice.
The results showed that fruit extract could effectively improve the function of the antioxidant system and inhibit lipid peroxidation in mice kidneys. Too little antioxidant leads to the decrease of TrxR2 synthesis, the increase of oxidative stress in mice kidneys, and the oxidative damage of mitochondria. Mitochondrial damage leads to the disorder of cellular aerobic metabolism and the increase of O 2production, which affects the activity and expression of SOD and CAT. The supplementation of fruit extracts can protect animal kidneys and organisms better by inhibiting lipid peroxidation, reducing mitochondrial damage and scavenging oxygen free radicals.
Antioxidants are any substance that prevents oxidation of lipids, carbohydrates, proteins, DNA and other oxidizable substrates. Many enzymes such as superoxide dismutase, catalase, and some nonenzymatic substances such as vitamin E, vitamin C, beta-carotene and glutathione have antioxidant effects (21). Among them, the antioxidant enzymes with important physiological functions are SOD, CAT and GSH. Antioxidants, such as VE, VC and GSH, can synergize with various compounds to scavenge free radicals or interrupt free radical reactions. The role of antioxidant enzymes in health has attracted close attention in part because of the anti-aging effect of the mass antioxidant market.
Free radicals are atoms, clusters or molecules with unpaired electrons in outer orbits. According to statistics, in every 25 oxygen molecules used for metabolism, one oxygen can produce unstable forms of oxygen, called superoxide anion free radicals. During the oxidative phosphorylation of mitochondria, most of the oxygen is reduced to water, but at the same time a small amount of oxygen (2-5%) is transformed into an intermediate state substance with active and unpaired electrons, called reactive oxygen groups (ROS). When oxygen is not completely reduced to water through the transfer of four hydrogens and four electrons, a superoxide anion radical or some other ROS will be produced. To restore atomic and molecular stability, superoxide anion radicals actively seek to strip an electron from various cell sources containing one hydrogen. In order to pair an electron with the lone electron in its outer orbit, superoxide anion radicals attack sugar, protein, DNA, polyunsaturated fatty acids and other biological molecules. ROS is a general name used to describe oxygen-centered free radicals and some non-free radical oxygen derivatives. ROS is also a relative term for atoms or molecules that have more activity than other atoms or molecules in the search for electrons. There is a lot of evidence that ROS products can cause cell and tissue damage through a series of physiological and biochemical reactions, as well as some diseases and even aging. Usually, whatever ROS attacks, it will be changed in some way (22). In this way, it will no longer play an appropriate role. However, recent evidence suggests that some types of ROS can act as signal transduction messengers. In fact, this may be an important adaptive signal in muscle tissue that promotes enzyme repair and protein synthesis.
While superoxide anion radicals and ROS are produced, the body also has a complete antioxidant system to scavenge superoxide anion radicals and ROS. The antioxidant system protects the body against the harmful effects of superoxide anion radicals and ROS by providing electrons that stabilize free radical molecules or by regulating the transformation of superoxide anion radicals and ROS into lower harmful forms. Antioxidants play an independent role in cells and tissues and cooperate with each other to some extent to scavenge superoxide anion free radicals and ROS. The three main antioxidant enzymes are SOD, CAT and glutathione Superoxidase (GPX). SOD scavenges superoxide anion radicals by converting two oxygen radicals and two hydrogens into hydrogen peroxide. The hydrogen peroxide produced by SOD has potential toxicity to cells (23). Previous studies have shown that H2O2 can indirectly induce HIV-1 gene expression in the human immunodeficiency virus. Although the transformation of oxygen free radicals into H2O2 is temporarily beneficial to the body, the removal of H2O2 is essential for the survival of cells. Cat helps to remove H2O2 and generates harmless water in the process. GPX can also remove H2O2 from cells by converting GSH to oxidized glutathione using H2O2. When the body is in a quiet state or slightly exercising, the oxidation and antioxidant systems are in a balanced state, but with the increase of exercise intensity, the oxidation and antioxidant state will be unbalanced. If the unbalanced relationship between superoxide anion free radicals and ROS production and antioxidants is beneficial to the former, oxygen stress will occur, which is related to many chronic diseases (such as cancer, diabetes mellitus and hypertension) related toxicity (5,24).
A large number of convincing epidemiological studies support the relationship between antioxidants, health and disease status. Epidemiological studies from many countries have shown a negative correlation between increased risk of heart disease and stroke and reduced antioxidant intake. These studies have shown that some of the main antioxidant concentrations in the body depend mainly on the dietary supply. The WHO cardiovascular disease surveillance project and vitamin sub-project studies show that among comparable risk factors for heart disease, some of the major antioxidants measured show a statistically significant negative correlation between the age of cardiac death and the absolute concentration of vitamin E. In addition to the fruit studied in this paper has certain antioxidant properties, physical exercise is also an important way to improve the body's antioxidant properties.
Studies have shown that moderate exercise can increase some antioxidant concentrations, while physical activity restriction can reduce antioxidant activity. Guo Lin and others have shown that endurance exercise can improve the activity of antioxidant enzymes and the ability to fight free radicals in tissues, and reduce the damage caused by free radicals. Endurance training can lead to the doubling of mitochondrial components such as cytochrome c and a. Cytochrome in the electron transfer system helps to completely convert four electrons and hydrogen into oxygen to form water. If no electrons are leaked from the formation of ROS from ETS, the body will weaken its need for antioxidants. Therefore, the decreased regulation of antioxidant activity during quiet time in exercise-trained people may be due to the more smooth ETS function (25). The effects of plant and fruit extracts on gene expression have been investigated in many studies (26)(27)(28)(29)(30)(31)(32).
During exercise, it is easy to increase the total body oxygen consumption by more than 10 times. Increased energy demand and calorific output related to exercise can make ETS strive to completely reduce oxygen to water, resulting in the formation of hydrogen peroxide due to the leakage of some electrons. Studies have shown that the increase in antioxidant levels is proportional to the demand for exercise. Participation in regular physical activity usually results in a proportional change in antioxidants and oxygen stress. That is to say, when the current oxidation activity is low (e.g., when the oxygen consumption is at baseline level at rest), the antioxidant level is also low. When the current oxidation activity is high (e.g. during exercise), the antioxidant level increases correspondingly with the pre-oxidation activity. Many studies have reported that long-term physical activity can improve antioxidant levels after exercise. Longterm training did not significantly affect antioxidant levels at rest, perhaps due to the increase in mitochondrial number or volume, and the rare occurrence of interruptive reactions during oxygen reduction to water, resulting in an increase in the body's natural resistance to oxygen stress. There is a close relationship between the level of cardiovascular quality and the ability of oxidation and antioxidation at rest. The higher a person's cardiovascular quality level is, the higher the activity of oxidase (e.g., cytochrome a and c), and the lower the level of antioxidants at rest. This suggests that less oxygen stress may be due to the reduction of ROS products by promoting the ability of electrons to convert to oxygen. Relative to the percentage of maximum aerobic capacity, exercise-induced oxygen stress can affect oxidation and antioxidant status. Untrained people have less antioxidant and oxidative capacity than trained people. It is speculated that individuals who are physically active may be better trained to deal with acute oxygen stress associated with exercise, or some other metabolic stressors. Regular physical activity is essential to naturally improve the ability of endogenous antioxidants to resist superoxide anion radicals and ROS, as well as to reduce the risk of some aging-related diseases or delay the onset of these diseases. If health and longevity are significantly affected by oxygen stress, then exogenous and endogenous antioxidants can affect health and longevity. If endogenous antioxidants cannot effectively eliminate superoxide anion radicals and ROS, antioxidant supplements can provide a second line of defense for scavenging superoxide anion radicals and ROS. Regular physical activity can not only increase muscle quality and oxidative capacity, but also enhance immune function, enhance the ability of anti-oxidation to resist oxygen free radicals, and promote the ability of tissues to resist oxygen stress.

Conclusions
More and more studies have shown that antioxidant is an important step to prevent aging because free radicals or oxidants can decompose cells and tissues, affect metabolic function, and cause different health problems. If excessive oxidative free radicals can be eliminated, many free radical-induced and agingrelated diseases can be prevented. For example, common cancers, arteriosclerosis, diabetes, cataracts, cardiovascular diseases, Alzheimer's disease, arthritis and so on, these diseases are considered to be associated with free radicals. Adequate antioxidants should be ingested to slow down the speed of body degradation, prevent skin aging, and keep a youthful spirit at all times. Fruit has more antioxidants, which is the main source of antioxidants supplementation. 70 mice were fed with grape extract. The antioxidant activity of fruit extract was tested by detecting the gene expression level of antioxidant metabolic enzymes in liver and kidney of experimental mice. The results showed that the fruit extract could enhance the antioxidant level of the body, and had a certain anti-aging effect.