TWI823733B - Use of s-allylcysteine for the preparation of composition for delaying aging and preventing senile disease - Google Patents

Abstract

The present invention provides the use of S-allylcysteine for preparing a composition for delaying aging and preventing senile diseases. After the S-allylcysteine is administered, it has the potential to reduce body fat accumulation, delay muscle mass loss, and slow down anxiety behaviors caused by aging. From the aspect of serum biochemical values, it is shown that S-allylcysteine can reduce total cholesterol, and has liver and kidney protection benefits, so that the physiological state is closer to that of young subject. In addition, S-allylcysteine can prevent DNA damage, and can also dynamically regulate mitochondria to reduce oxidative stress and achieve the effect of delaying aging.

Description

Use of S-allylcysteine for preparing compositions for delaying aging and preventing age-related diseases

The present invention relates to the use of S-allylcysteine (SAC), in particular to S-allylcysteine and its use in preparing compositions for delaying aging and preventing senile diseases. the use of.

Declining fertility rates and longer life expectancy are the main causes of the aging of the global population. Although the life expectancy of the population has increased, the healthy lifespan has not increased accordingly, resulting in a huge medical burden. Therefore, rather than treating aging-related disease symptoms, treating "aging" as a disease and targeting intervention will have the effect of extending healthy lifespan. more significant.

Aging is one of the greatest risks for a variety of chronic and degenerative diseases. Therefore, exploring the causes and intervention strategies of aging is an urgent issue today. Studies have pointed out that mitochondrial dysfunction plays a key role in various aging indicators. Because mitochondria and other organelles have a complex and cross-linked interactive network, their dynamic imbalance will cause cellular energy metabolism disorders and increased oxidative stress. eventually leading to aging.

When mitochondrial dynamics are out of balance, it will lead to abnormal mitochondrial accumulation, mtDNA mutations, and reduced mitochondrial production and synthesis; because mitochondria and other organelles have a complex and cross-linked interactive network, the above-mentioned damage will Indirectly leading to abnormal performance of other organelles and causing the aging of the body.

S-allylcysteine (SAC) is the compound γ-Glutamylcysteine in raw garlic, which is converted into SAC through γ-Glutamyltranspeptidase. It is the most abundant organic sulfide in black garlic. Studies have proven that SAC can filter out reactive oxygen species, activate the Nrf2 transcription factor at the transcriptional level, promote the production of antioxidant enzymes, and achieve the effect of reducing oxidative stress. Oxidative stress is one of the causes of various neurodegenerative diseases. Research has found that SAC can effectively reduce brain oxidative damage or hippocampal nerve apoptosis caused by amyloid beta (Aβ), thereby improving cognitive ability.

Although SAC has been studied in the prior art and proven to be antioxidant, neuroprotective and prevents diabetes, the relevant mechanism of action of SAC and whether it can also have positive benefits in delaying aging in mammals are still unknown. Therefore, the present invention analyzes SAC It affects the individual's behavioral ability and biochemical values, explores the effectiveness of SAC in delaying the aging of individuals (especially mammals), and further explores whether it improves mitochondrial dynamics.

Therefore, the present invention provides a use of S-allylcysteine (SAC), which is used to prepare a composition for delaying aging and preventing age-related diseases.

According to an embodiment of the present invention, the composition is used to reduce body fat cholesterol, triglycerides or low-density lipoprotein of a user, and increase muscle mass of the user.

According to an embodiment of the present invention, the composition is used to alleviate anxiety behaviors caused by aging.

According to an embodiment of the present invention, the composition is used to slow down the functional decline of liver or kidney.

According to an embodiment of the present invention, the composition is used to prevent DNA damage or immunosenescence.

According to an embodiment of the present invention, the composition is used to reduce the synthesis of GLB1 and SA-βgal caused by cell senescence.

According to an embodiment of the present invention, the composition is used to enhance the mitochondrial synthesis efficiency of a user.

According to one embodiment of the present invention, the composition increases the mitochondrial biosynthetic efficiency by increasing the expression of SIRT1 and activating PGC-1α.

According to an embodiment of the present invention, the effective dose of S-allylcysteine is 12.2 to 48.6 mg/kg.

According to an embodiment of the present invention, the effective dose of S-allylcysteine is 30 to 48.6 mg/kg.

The use of S-allylcysteine of the present invention is used to prepare compositions for delaying aging and preventing senile diseases, and has the following advantages:

(1) After administration of SAC, it can reduce body fat accumulation and increase muscle mass. It can also maintain their vitality and slow down liver hypertrophy and anxiety behaviors caused by aging.

(2) Reduce the gluten transaminase and blood urea nitrogen in their serum biochemical values to protect the liver and kidneys. It can also dynamically regulate mitochondria to reduce oxidative stress by increasing the expression of OPA1 mRNA, SIRT1 and PGC-1α in the liver.

(3) Prevent DNA damage or immune senescence, reduce GLB1 and SA-βgal in the liver caused by replicative senescence, and mediate the p-p65/p65 ratio of aging-related secretory symptoms.

The following embodiments should not be considered as unduly limiting the present invention. Those skilled in the art may make modifications and changes to the embodiments discussed herein without departing from the spirit or scope of the invention, while still falling within the scope of the invention.

In this article, unless the context indicates otherwise, the terms "comprising", "including", "having" or "containing" are inclusive or open-ended and do not exclude other unstated elements or method steps; the term "a ” and “the” may be interpreted in the singular or in the plural; the term “one or more” means “at least one” and may therefore include individual features or mixtures/combinations. In addition, in the scope of this specification and the appended patent application, unless otherwise specified, "disposed on something" can be regarded as directly or indirectly contacting the surface of something through attachment or other forms. The definition of the surface Judgment should be based on the meaning of the content before and after the description/paragraphs and the common knowledge in the field to which this description belongs.

The present invention provides a use of S-allylcysteine (SAC), which is used to prepare a composition for delaying aging and preventing senile diseases. In the present invention, the composition is used for animals, and in one embodiment, the effect of SAC on the behavioral ability and biochemical values of aging mice is analyzed, the effectiveness of SAC in delaying the aging of mammalian animals is explored, and its effect on granulation is further explored. Whether line dynamics are improved or not.

In one or more embodiments of the present invention, the animal experiment is carried out with male naturally aging mice. The experiment is carried out in two parts: an aging delay experiment and a mitochondrial dynamic experiment; the mice are divided into five groups, namely young Blank control group, aging induction group, low-dose SAC experimental group, high-dose SAC experimental group and a positive control group (resveratrol). Among them, the low-dose SAC experimental group and the high-dose SAC experimental group were mixed with 0.05% and 0.2% SAC into the mouse feed respectively. Among them, in a preferred embodiment, the effective dose of S-allylcysteine converted to a human subject is 12.2 to 48.6 mg/kg, for example, but not limited to: 13 mg/kg, 15.1 mg/kg, 17.3 mg/kg, 19.2 mg/kg, 23.5 mg/kg, 26.8 mg/kg, 30.3 mg/kg, 32.7 mg/kg, 35.1 mg/kg, 38.6 mg/kg, 40.3 mg/kg, 42.8 mg/kg, 45.4 mg/kg, 47.9 mg/kg, 48.4 mg/kg, more preferably, the effective dose of S-allyl cysteine is 30 to 48.6 mg/kg, for example, but not limited to: 31.3 , 32.7 mg/kg, 33.1 mg/kg, 35.1 mg/kg, 38.9 mg/kg, 39.6 mg/kg, 40.3 mg/kg, 41.8 mg/kg, 43.4 mg/kg, 45.3 mg/kg, 46.2 mg/kg , 47.5 mg/kg, 48.1 mg/kg, 48.5 mg/kg.

"Aging" as described in this article refers to the progressive decline of physiological functions, resulting in damage to tissue or organ functions. In addition, aging is also accompanied by a progressive decline in brain function, resulting in a decline in sensory, motor and cognitive abilities. Although aging The occurrence of aging is difficult to reverse, but it can be prevented or delayed to maintain a healthy state. The term "slowing aging" in this article refers to the ability to slow down aging indicators.

Aging indicators referred to in this article include behavioral manifestations of aging: body fat accumulation (including but not limited to reduction of body fat, triglycerides or low-density lipoprotein), loss of muscle mass and its function, and organs (including but not limited to liver (or kidney) function decline, progressive brain function decline, leading to sensory, motor and cognitive decline, etc.; in addition, this aging indicator also includes biochemical indicators of aging, such as DNA damage, aging-related lysosomes and SA-βgal (senescence-associated beta-galactosidase) activity or aging-associated secretory phenotype (SASP), etc.

In addition, most organelles require continuous regeneration and elimination of damaged parts to maintain health. Mitochondria are key organelles for energy production and prevention of endogenous oxidative stress, so their synthesis and degradation are even more important; therefore, Mitochondrial dysfunction also plays a key role in various aging indicators, and impaired mitochondrial function is a major cause of aging: when mitochondrial dynamics are imbalanced, abnormal mitochondrial accumulation, mtDNA mutations, and Reduce mitochondrial production and synthesis; and because mitochondria and other organelles have a complex and cross-linked interactive network, the above damage will indirectly lead to abnormal performance of other organelles and cause aging of the body. Therefore, another aspect of the present invention In this regard, SAC is provided to delay the occurrence of aging by balancing mitochondrial dynamics; specifically, SAC significantly increases the mRNA expression of the key mitochondrial fusion factor OPA1 in the liver, thereby leading to the synthesis of mitochondria-related proteins SIRT1 and PGC- The expression amount of 1α is also significantly increased, thereby effectively reducing oxidative stress, such as the content of MDA in the liver and 8-OHdG in the urine.

What is called "mitochondrial dynamics" in this article includes the changes in the shape of mitochondria, their movement along the cytoskeleton, and the process of forming an interactive network with other organelles. Dynamic changes require mitochondrial fusion and cleavage reactions to achieve them. If the mitochondrial dynamic regulatory protein is imbalanced, on the one hand, it will cause endogenous mitochondrial damage, thereby reducing mitochondrial biosynthetic activity; on the other hand, it will reduce the mitochondrial autophagy response, resulting in damaged mitochondria. Accumulation leads to aging and age-related diseases.

"Elderly diseases" as mentioned in this article refers to the decline in physiological functions and cell senescence caused by aging, which in turn leads to damage to the body's immune, tissue or organ functions and causes diseases. Common geriatric diseases include but are not limited to type 2 Diabetes, neurodegenerative diseases, cardiovascular diseases, osteoporosis, sarcopenic obesity and cancer. Therefore, the term "prevention of age-related diseases" in this article refers to the prevention of the decline in physiological functions and cellular senescence caused by aging, including but not excluding It is limited to preventing anxiety behaviors, DNA damage or immune senescence caused by aging.

The embodiments of the present invention are designed as follows: the experiment is divided into two parts, an aging delay experiment and a mitochondrial dynamic experiment. From the first stage of aging-delaying experiments, it is determined whether SAC can comprehensively delay the aging of aging mice from the outside to the inside, and at the same time maintain the health of mice, achieving the comprehensive effect of extending healthy life span. Then enter the second stage of mechanism exploration, trying to find out the key factors causing aging. The "free radical theory of aging" proposed by Harman extends to the connection between mitochondrial dysfunction and aging. The present invention explores the impact of oxidative stress on the aging process, and then detects whether there are corresponding changes in mitochondrial synthesis. Finally, it is discussed whether mitochondrial dynamics, a key factor that determines mitochondrial efficiency and quality, has a changing trend in the present invention. Example

Animal experimental group design

The present invention utilizes 49-week-old male mice and raises them in a natural aging mode for 11 weeks. When the mice reach the age of 60 weeks of reproductive senility, they are conducted for 12 years with reference to the "Method for Evaluation of the Anti-Aging Function of Healthy Foods" promulgated by the Ministry of Health and Welfare. Zhou's experiment reached the post-aging period of 72 weeks when various aging activity indicators begin to show, and tested whether the intervention of SAC can prevent aging. For young mice, 6-week-old mice were purchased 2 weeks before sacrifice, and sacrificed after 2 weeks of adaptation to ensure that the time of sacrifice coincided with the mice's adulthood, increasing their credibility as a blank control group.

During the feeding period of 49-week-old male rats, the daily food and water intake and weekly body weight changes were recorded. When the mice reach 60 weeks of age, the body composition of the mice will be analyzed, and the overall mobility of the mice will be tested using the open field test. The obtained body fat, body lean mass and weight results were then analyzed based on body composition, and the aging mice were divided into 4 groups on average (as shown in Table 1 below, namely the aging induction group (Actrl); low dose (SACL), high dose ( SACH) experimental group and positive control group (Res); and another one-year-old blank control group (Yctrl)). Twelve weeks after the intervention of the samples, the post-test of behavioral characteristics was analyzed together with the young rats that had been pre-reared for 2 weeks. Then sacrifice, and then measure the serum biochemical values as an indicator of whether the mouse's health status can be prolonged. Then measure aging-related biochemical indicators, and finally perform statistical analysis of the data.

Table 1 Group Number of animals feeding period Yctrl 10 Pellets feed; RO water 2 weeks Actrl 9 Powdered feed; RO water 12 weeks SACL 8 Powdered feed mixed with 0.05% SAC ; RO water 12 weeks SACH 8 Powdered feed mixed with 0.2% SAC ; RO water 12 weeks Res 8 Powdered feed mixed with 0.05% resveratrol; RO water 12 weeks

Example 1 : Body composition and athletic ability test

In the aging-delaying experiment, the effects of SAC on the body composition, exercise capacity, anxiety behavior, organ function, serum biochemical values and aging biochemical indicators of mice will be tested.

Please also refer to Figure 1 to test the effect of SAC on the body composition and exercise ability of mice. As age increases, the quality of human organs or tissues and their metabolic rate decrease, which will cause a decrease in resting metabolic rate; coupled with the decrease in sex hormones and growth hormone, as well as lack of exercise or insufficient protein intake, body composition will also decrease. Changes cause body lean mass to decrease and body fat to increase. The post-test results of body composition analysis in this animal experiment showed that the body fat of the aging-induced group (Actrl) was significantly higher than that of the blank control group (Yctrl) (Figure 1A); in contrast, body lean mass was significantly reduced (Figure 1C ). However, it can be found from the pre- and post-test changes that SAC intervention has the potential to reduce body fat and slow down the loss of body lean mass (Figure 1B, 1D).

Changes in the mouse's body composition were also reflected in overall mobility. The overall activity is determined by the open field test, which detects the total moving distance of the mouse in the open field within 5 minutes. The longer the distance, the better the overall activity. Comparing the results of the post-test between the Actrl aging-induced group and the Yctrl blank control group (Figure 1E), the total moving distance of old mice was significantly lower than that of young mice, indicating a decreased motor behavior ability. Similarly, from the change diagram before and after the open field test, it can be seen that the overall activity level has a tendency to increase after SAC intervention (Figure 1F). This result is due to SAC maintaining the body lean mass content, which makes the mice have better exercise performance. .

It can be seen from the above characterization measurement results that SAC has the potential to reduce body fat accumulation, improve muscle mass and function, and make the body composition and overall activity of aging mice tend to be younger.

Example 2 : Anxiety Behavior Test

The above-mentioned "open field test" is based on the haptotaxis of rodents, which prefer to move around the periphery of facilities and are less likely to go to open central areas. Therefore, if the mouse spends more time exploring the central field or moves more frequently, it means that its anxiety level is lower.

In a preferred embodiment, the composition is used to alleviate anxiety behaviors caused by aging. Please refer to Figure 2 together. It can be seen from the post-test experimental results that compared with the Actrl aging induction group, the Yctrl blank control group stayed in the central field longer (Figure 2A) and the peripheral field time shorter (Figure 2A). 2C); and the pre- and post-test change diagram within the group shows that the Res positive control group and the SACL and SACH experimental groups of intervention samples significantly increased the time to explore the central field compared with the Actrl group (Figure 2B), while the relative Reduce its stay in the surrounding field (Figure 2D). It can be seen from this result that aging does tend to increase anxiety-like behavior in mice, but intervention with SAC can significantly improve it.

Therefore, SAC has a positive effect on alleviating anxiety-like behaviors, and it is speculated that it has a neuroprotective effect, allowing the hippocampus to function normally and reducing the occurrence of anxiety. The hippocampus described in this article is part of the limbic system of the brain, located below the cerebral cortex, and is responsible for short-term memory, long-term memory, and spatial positioning. The dentate gyrus region in the hippocampus plays a key role in regulating emotion, cognition, and memory. It will be damaged by aging, resulting in neurotransmission imbalance, reduced synaptic plasticity and neuroregeneration, and leading to cognitive or emotional disorders.

Example 3 : Serum biochemical value test

After confirming the effect of SAC on the appearance and behavioral characteristics of mice, their serum biochemical values were further tested to determine the health status of the mice. In a preferred embodiment, the composition is used to slow down the functional decline of the liver or kidneys.

The "liver" mentioned in this article not only sends nutrients absorbed by the intestines to the liver for storage or synthesis through the hepatic portal vein, it is also responsible for decomposing alcohol, drugs, toxic substances and metabolic waste, and is the body's main detoxification factory. The liver uses enzymes or cytochrome P450 to reduce the activity or toxicity of substances through oxidation, reduction, sulfation, or deamination; or it combines lipophilic metabolites with other substances and converts them into hydrophilic substances to facilitate elimination in the urine. Therefore, regardless of whether a substance provides nutrients to the body or is toxic, the liver is the organ most directly affected.

The "kidneys" mentioned in this article remove excess or harmful substances from the body through urine through filtration and reabsorption to regulate the body's electrolytes, pH and water content to maintain constant blood osmotic pressure and blood pressure. The kidneys are sensitive to toxic reactions and can effectively reflect fluctuations in physiological status and acute injuries. Therefore, studies often use the paired weight and appearance of the kidneys as indicators of whether a compound will cause toxic reactions in animals.

Please refer to Figure 3 together. The AST measurement results of the present invention have no significant difference from the Actrl group in the Yctrl blank control group, SACL and SACH experimental groups and Res positive control group (Table 1). It is inferred that AST is interfered by factors other than the liver and is relatively low. It is difficult to infer the actual condition of the liver. ALT, which is more specific to liver damage, has a tendency to be lower in the Yctrl group than in the Actrl group, indicating that aging will increase the degree of liver damage. Intervention of low-dose sample SAC can significantly reduce the value of ALT, and to the extent that there is no significant difference with the Yctrl group and Res positive control group (Table 1, Figure 3A), showing that SAC has the ability to protect the liver.

The "Alanine transaminase (ALT)" and "Aspartate transaminase (AST)" described in this article are two enzymes in the liver. Their function is to catalyze the conversion of amine groups from Alanine or aspartic acid is transferred to α-ketoglutarate to form pyruvate and oxalacetic acid respectively, and then participate in the process of gluconeogenesis. Both are expressed in large amounts when the liver is damaged, and this abnormal increase is common in non-alcoholic fatty liver disease, alcoholic liver disease and hepatitis. ALT mainly exists in the cytoplasm, has the highest concentration in the liver, and has high specificity. Therefore, ALT is mainly used to confirm whether the lesion is located in the liver. When ALT and AST values are abnormal, clinically, the AST/ALT ratio will be used to judge different liver diseases. If the ratio is less than 1, it is usually non-alcoholic fatty liver; while if the ratio is greater than 2, it is related to alcoholic liver disease.

The "blood urea nitrogen (BUN)" mentioned in this article refers to the nitrogen contained in urea in the blood, which is called urea nitrogen; urea is the end product of protein metabolism and is excreted out of the body through the kidneys. If renal dysfunction leads to low excretion function, blood urea nitrogen will increase. Therefore, blood urea nitrogen is an important indicator to understand whether kidney function is normal.

Please refer to Figure 3 as well. The blood urea nitrogen concentration of the Actrl aging-induced group of the present invention is significantly higher than that of the Yctrl blank control group. It can be seen that aging will reduce the efficiency of the renal glomerulus in filtering blood urea nitrogen. After SAC intervention, regardless of low or high dose, blood urea nitrogen concentration could be significantly reduced, reaching the same level as the Yctrl group (Table 2, Figure 3B), showing that SAC can prevent the decline of mouse kidney function.

Table 2

It can be seen from the above results that neither SAC intervention nor the positive control compound resveratrol will cause toxic reactions or functional defects in various organs.

Example 4 : Blood lipid test

After confirming that SAC has the ability to protect the liver and kidneys, the effect of the composition on blood lipids was further tested. In a preferred embodiment, the composition is used to reduce a user's body fat cholesterol, triglycerides or low-density lipoprotein, and increase muscle mass in the user.

Dyslipidemia and hypertension are the greatest risks for cardiovascular disease, and blood lipids are mainly divided into cholesterol and triglycerides. In addition to regulating the strength and fluidity of cell membranes, cholesterol (total cholesterol, T-CHO) is also an important element of steroid hormones and bile acid. Since it is insoluble in water, it needs to be combined with lipoproteins to travel with the blood. Throughout the body, lipoproteins can be divided into low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Low-density lipoprotein transports cholesterol from the liver and small intestine to other body tissues; HDL transports cholesterol from tissues back to the liver. Triglycerides (TG) are the main components of very low-density lipoproteins and chylomicrons, and are used as an energy source through metabolism. When cholesterol, low-density lipoprotein, and triglycerides are too high, it will lead to dyslipidemia and damage vascular endothelial cells. Excess low-density lipoprotein can also easily accumulate on the inner walls of arteries, triggering local inflammatory reactions, attracting macrophages to engulf the accumulated fat, and gradually forming atherosclerosis.

Please refer to Figure 3 together. The present invention has measured that the cholesterol concentration in the Actrl aging-induced group is significantly higher than that in the Yctrl blank control group, showing that aging will increase the total cholesterol content, while SAC intervention will reduce its value, and has a dose effect. It shows the potential of SAC to prevent cholesterol accumulation (Table 1, Figure 3C). There is no significant difference in triglyceride between each group and the Actrl group. However, when studying the aging mice, it can also be found that intervention of SAC can reduce the value of triglyceride, towards the trend of the Res positive control group, and the effect is better than that of the positive control group. control group (Table 1). High-density lipoprotein was the highest in the Actrl group and was significantly higher than that of the Yctrl group. The experimental group and the positive control group also had a tendency to suppress high-density lipoprotein. It is speculated that this result is due to the fact that the Actrl group has higher cholesterol and therefore higher HDL. Although the HDL of other aging mouse groups is lower, there is no difference from the Yctrl group (Table 1). Therefore, It is inferred that they are not at higher risk of cardiovascular disease. There was no statistical significance between low-density lipoprotein in each group and the Actrl group, but after high-dose SAC intervention, its value dropped to no significant difference from the Yctrl group (Table 1, Figure 3D).

Based on the above experimental results, it is shown that SAC has the ability to protect the liver and kidneys. In addition, it has the tendency to reduce triglycerides. The SACH group that intervened in high-dose samples can reduce total cholesterol and low-density lipoprotein to the same level as the Yctrl group. There is no significant difference, indicating its ability to inhibit cholesterol accumulation, making the serum biochemical values of the mice more similar to those of young mice, and in a healthier state.

Example 5 : DNA Injury related tests

From the above experiments, it can be seen that the phenomenon of preventing or delaying aging can be observed in the groups that underwent SAC. The results of serum biochemical values also showed that the health status of the aging mice that underwent SAC was better than that of the aging-induced group. Therefore, we will next explore the internal Biochemical markers related to aging. In a preferred embodiment, the composition is used to prevent DNA damage or immune senescence. More preferably, the composition is used to reduce the synthesis of GLB1 and SA-βgal caused by cell senescence.

The initial stage of the aging process is DNA damage, which triggers a series of aging changes. DNA damage and genomic instability are the main driving factors of the aging process. When DNA is exposed to external stimuli such as chemical agents, radiation or thermal damage, or is attacked by endogenous reactive oxygen species, there is a chance that damage such as modification of nucleotide bases and DNA single-strand or double-strand breaks will occur, among which Double-strand breaks are the most severe form of DNA damage because they are difficult to repair and can easily cause cytotoxicity. DNA double-strand breaks trigger a DNA damage response: the MRN complex, including MRe11, Rad50, and Nbs1, and ATM kinase are recruited to the damaged site, and then ATM kinase phosphorylates the histone variant H2AX into γ-H2AX. Then the DNA damage checkpoint protein MDC1 is recruited to promote BRCA1 and 53BP1 proteins to repair DNA. According to the characteristics of γ-H2AX combining with repair proteins to form nuclear foci in the double-stranded break area in the nucleus, its content can be used as an indicator of the degree of DNA double-stranded breaks, and can also be used to detect gene damage after the intervention of samples or compounds. changes in circumstances.

The present invention uses the Western blot method to detect the expression amount of γ-H2AX in mouse liver protein extract. The results show that the expression amount of γ-H2AX in the Actrl aging-induced group is higher than that in the Yctrl blank control group, verifying that aging is accompanied by an increase in DNA damage. After the intervention of resveratrol in aged mice, the expression level of γ-H2AX was significantly reduced to the level that was not significantly different from that in the Yctrl group. In the SACL and SACH experimental groups of intervention samples, γ-H2AX showed a downward trend (Figure 4A). It is concluded that SAC has the potential to prevent DNA damage.

When aging continues to occur, resulting in genome instability, telomere shortening, epigenetic changes, and loss of protein homeostasis, cells will enter the replicative senescence phase and irreversibly stop the cell cycle to avoid the spread of senescent cells with the risk of mutation. . And because cells stop metabolic renewal, old waste cells, damaged macromolecules or organelles will continue to accumulate, inducing a large number of lysosomes to be produced.

Studies have found that SA-βgal is significantly expressed in senescent cells, and its optimal pH value for detection is 6.0, which is different from lysosomes which usually detect pH value 4.5. Therefore, it is widely used as an indicator of cell senescence to determine whether the sample involved or Whether the condition has the potential to delay aging. The performance of SA-βgal is positively correlated with GLB1 mRNA, which mediates β-D-galactosidase production and synthesis. When the overall β-D-galactosidase concentration increases, SA-βgal will also increase together. Therefore, it is not the only Aging will cause an increase in SA-βgal; however, the proportion of SA-βgal upregulated in senescent cells is higher than that in normal cells.

The present invention also uses mouse liver protein extract for measurement, and uses the Western blot method to detect the expression of GLB1. The experimental results show that GLB1 in the Actrl aging induction group has a tendency to be higher than that in the Yctrl blank control group, indicating that with the occurrence of aging, The degree of cellular senescence also gradually increases. The positive control group that received resveratrol had a tendency to reduce GLB1, and the low-dose SACL experimental group could also be reduced to the same level as the Yctrl group, while the high-dose SACH experimental group could significantly reduce the expression of GLB1 (Figure 4B). The results of using a fluorescence spectrometer to detect SA-βgal are similar to those of GLB1. The SA-βgal in the Actrl aging-induced group is significantly higher than that in the Yctrl blank control group, while the Res positive control group has a tendency to delay the production and synthesis of SA-βgal. Intervention of high-dose samples The SACH group could also significantly reduce its content (Figure 4C). Based on the above results, it is shown that sample SAC can inhibit the expression of GLB1, thereby reducing the production and synthesis of SA-βgal, and achieving the effect of delaying replicative aging.

Based on the above experimental results, it is shown that SAC can slow down the possibility of DNA damage in the aging process, reduce the production and synthesis of lysosomal proteins GLB1 and SA-βgal induced by cellular aging, and delay replicative aging.

In the mitochondrial dynamics experiment, the effects of SAC on oxidative stress, mitochondrial biosynthesis, and mitochondrial dynamic genes in mice will be tested. In a preferred embodiment, the composition is used to enhance the mitochondrial biosynthesis efficiency of a user. More preferably, the composition is used to increase the expression of SIRT1 and activate PGC-1α, so that the Mitochondrial synthesis efficiency is increased.

Example 6 : Oxidation stress test

Regardless of whether an organism is undergoing normal physiological metabolism or is stimulated by external environment, such as air pollution, radiation or bacterial infection, it may produce reactive oxygen species and reactive nitrogen species (RNS), including independent unpaired ones. Electronic free radicals. When reactive oxygen free radicals attack cell membranes or polyunsaturated fatty acids, the lipid peroxidation metabolite malondialdehyde (MDA) will be produced. MDA can then be used to easily react with TBA (thiobarbituric acid) under low pH and high temperature conditions. The properties of nucleophilic addition reactions are examined.

The "free radicals" mentioned in this article refer to substances produced by the body after metabolism. They are extremely reactive and can easily react with other substances. When tissues and organs are injured, a large amount of free radicals will accumulate. It is an unstable factor that attacks healthy cells, robs them of electrons, triggers cell apoptosis and leads to aging.

The present invention uses mouse liver protein extract to detect MDA concentration. The results showed that the MDA content in the Actrl aging-induced group was significantly higher than that in the Yctrl blank control group, indicating that the oxidative stress in aging mice was significantly higher than that in young mice, and the intervention of high-dose sample SAC could significantly reduce the MDA content (Figure 5A) , it can be seen that SAC has the ability to reduce lipid oxidative stress.

When reactive oxygen radicals attack nucleotides, there is a chance that the 8th carbon of Guanine will be connected to a hydroxyl group to form 8-OHdG, resulting in the replacement of nucleic acid GC:TA. Therefore, the content of 8-OHdG is often regarded as DNA oxidative damage. When 8-OHdG is cleaved by DNA damage repair enzymes, it will be released into saliva, urine or plasma. Most of the substances measured use urine, because the urine sample volume is sufficient and it is a non-invasive method. In addition, research also pointed out that the urinary excretion of 8-OHdG is equivalent to the oxidation rate of Guanine.

Therefore, the present invention also enables mice to naturally excrete without external stimulation and collects urine as samples. The measurement results showed that the urinary 8-OHdG in the Actrl aging-induced group was significantly higher than that in the Yctrl blank control group, indicating that in addition to lipids being susceptible to oxidative stress, nucleic acids are also susceptible to attacks by reactive oxygen free radicals, which increases the proportion of DNA damage. This situation tended to slow down in the positive control group that received resveratrol. However, in the low-dose SACL experimental group, the content of 8-OHdG was significantly reduced and reached the same level as that of young mice (Figure 5B). SAC has been shown to be effective in preventing aging-related increases in DNA oxidative damage.

Based on the above experimental results, it is shown that SAC has the potential to activate the antioxidant mechanism of organisms and effectively reduce the oxidative stress on lipids and DNA. At the same time, the reduction of oxidative stress also delays the aging process of organisms.

Example 7 : Mitochondrial synthesis efficiency test

Due to the process of evolution, mitochondrial proteins are not transcribed and translated through genes encoded in the cell nucleus. Instead, their own circular mitochondrial DNA is responsible for encoding the 13 proteins required for aerobic respiration. And because the replication process of mtDNA is different from that of the nucleus, the production and synthesis of mitochondria must simultaneously coordinate the gene sequences encoded by both. PGC-1α plays an important role in mitochondrial biogenesis and synthesis. It is a nuclear receptor that, after being deacetylated by SIRT1, can regulate the expression of NRF-1 (nuclear respiratory factor-1) and NRF-2 or By increasing the activity of ERRs (estrogen-related receptors), these transcription factors trigger TFAM in the cytoplasm and transfer it to the D-loop of mitochondria to activate the transcription and replication of mitochondrial genes.

The present invention uses the Western blot method to detect the expression levels of SIRT1 and PGC-1α in mouse liver protein extracts. The results showed that the relative expression levels of both SIRT1 and PGC-1α in the Actrl aging-induced group tended to be lower than those in the Yctrl blank control group, indicating that mitochondrial production and synthesis are reduced in aging individuals. Past literature pointed out that resveratrol is a promoter of SIRT1, so in the positive control group of this experiment, it did significantly increase the expression of SIRT1 and PGC-1α. The SACL and SACH groups of interventional samples also significantly increased the content of SIRT1 (Figure 6A); in PGC-1α, the low-dose SACL group significantly increased its expression; a significant increase in value was also observed in the SACH group (Figure 6B ). In addition, RT-qPCR was used to measure the expression level of TFAM mRNA in mouse livers, and the results showed that both the SACL and SACH experimental groups showed an upward trend compared with the Actrl aging-induced group (Figure 6C).

Previous literature has pointed out that in addition to activating PGC-1α, SIRT1 can also regulate p53, FOXO (forkhead box O), NF-κB and other factors by deacetylating histones, thereby increasing the organism's resistance to environmental stress and anti-inflammation. and balancing nutritional metabolic pathways. Mice overexpressing SIRT1 significantly increase their lifespan and exhibit signs of delayed aging such as increased activity and oxygen intake. Therefore, in the present invention, SAC can increase the expression of SIRT1, thereby activating one of its downstream PGC-1α, increasing the production and synthesis of mitochondria, allowing cells to maintain normal oxidative metabolism, and reduce the amount of proteins such as MDA and 8-OHdG. and other oxidative damage (Figure 5A, 5B).

The present invention extracts mouse liver mRNA, and then measures the expression of mitochondrial dynamic genes through RT-qPCR. Experimental results on mitochondrial fusion-related genes showed that MFN1 had an upward trend in the Res positive control group compared with the Actrl aging-induced group; and the SAC-involved experimental group also developed in this trend (Figure 7A). In MFN2, there is no statistical significance between each group (Figure 7B). However, due to the high similarity of the two fusion proteins, they can complement each other. The key gene OPA1, which fuses the mitochondrial inner membrane and maintains wrinkle morphology, has a higher trend in the Yctrl blank control group than the Actrl aging-induced group, and both the intervention sample SAC and resveratrol can increase the relative content of OPA1. Especially in the sample high-dose SACH group, its expression level can be significantly increased (Figure 7C).

To sum up, the physiological phenomena related to delaying aging generally tend to enhance mitochondrial fusion. SAC can increase the efficiency of mitochondrial production and synthesis by activating the fusion reaction. Therefore, an increase in SIRT1 and PGC-1α was observed (Figure 6A, 6B) and a reduction in oxidative stress (Figure 5). At the same time, the overall activity increased (Figure 1F), showing that SAC increases ATP production by promoting mitochondrial fusion, ultimately achieving the effect of delaying aging.

In summary, male naturally aging mice have the potential to reduce body fat accumulation and delay muscle mass loss after SAC intervention, thus preventing the decline of overall activity. In terms of perceived emotions, it can significantly improve aging-related anxiety-like behaviors. In addition, serum biochemical values showed that SAC intervention could reduce total cholesterol and have protective effects on the liver and kidneys, making the physiological state closer to that of young mice. Biochemical values show that SAC intervention can reduce liver DNA damage indicator γ-H2AX, replication aging indicators GLB1 and SA-βgal, achieving the effect of delaying aging.

Then we further explored the relevant molecular mechanisms and found that SAC intervention could significantly increase the expression of mitochondrial fusion-related gene OPA1 mRNA in the liver, thereby promoting the levels of mitochondrial synthesis-related proteins SIRT1 and PGC-1α, thus improving mitochondrial fusion. Due to body dynamics, the antioxidant enzyme SOD has an upward trend; the oxidative stress MDA and 8-OHdG significantly decrease, thereby achieving the effect of extending healthy life span.

All ranges provided herein are intended to include each specific range within the given range and every combination of subranges between the given ranges. Furthermore, unless otherwise indicated, all ranges provided herein include the endpoints of the ranges. Thus, the range 1-5 specifically includes 1, 2, 3, 4, and 5, as well as subranges such as 2-5, 3-5, 2-3, 2-4, 1-4, and the like.

All publications and patent applications cited in this specification are hereby incorporated by reference, and each individual publication or patent application is expressly and individually indicated to be incorporated by reference for any and all purposes. In the event of any inconsistency between this document and any publication or patent application incorporated by reference, this document shall control.

The present invention has been described in detail above. However, the above descriptions are only preferred embodiments of the present invention. They should not be used to limit the scope of the present invention, that is, any equivalent changes made in accordance with the patent scope of the present invention. and modifications should still fall within the scope of the patent of the present invention.

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Implementations of the present technology will now be described by way of example only with reference to the accompanying drawings, in which:

Figure 1 is a graph illustrating the effect of SAC on body composition and exercise capacity of mice according to one embodiment of the present invention.

Figure 2 is a graph showing the effect of SAC on anxiety behavior in mice according to one embodiment of the present invention.

Figure 3 is a graph showing the effect of SAC on mouse serum biochemical values according to one embodiment of the present invention.

Figure 4 is a chart showing the effect of SAC on aging biochemical indicators in mice according to one embodiment of the present invention.

Figure 5 is a graph showing the effect of SAC on oxidative stress in mice according to one embodiment of the present invention.

Figure 6 is a chart showing the effect of SAC on genes and proteins related to mitochondrial production and synthesis in mice according to one embodiment of the present invention.

Figure 7 is a schematic diagram of the effect of SAC on mouse mitochondrial fusion genes according to one embodiment of the present invention.

It should be understood that aspects of the present invention are not limited to the arrangements, means and characteristics shown in the drawings.

without

Claims (4)

A use of S-allylcysteine (SAC) to prepare a composition that enhances the mitochondrial synthesis efficiency of a user. The use of claim 1, wherein the composition increases the mitochondrial production and synthesis efficiency by increasing the expression of SIRT1 and activating PGC-1α. Such as the use of claim 1 or 2, wherein the effective dose of the SAC is 12.2 to 48.6 mg/kg. Such as the use of claim 3, wherein the effective dose of the SAC is 30 to 48.6 mg/kg.