TW202425962A - 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 a composition for delaying aging and preventing senile diseases

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

The decline in fertility and the increase in average life expectancy are the main causes of the global population aging. Although the population's expected life expectancy has increased, the healthy life expectancy has not increased accordingly, resulting in a huge medical burden. Therefore, rather than treating the symptoms of aging-related diseases, treating "aging" as a disease and intervening in it is more effective in extending healthy life expectancy.

Aging is one of the biggest risks leading to a variety of chronic and degenerative diseases, so exploring the causes of aging and intervention strategies is an urgent problem to be solved today. Studies have shown that mitochondrial dysfunction plays a key role in various aging indicators, because mitochondria and other organelles have a complex and interconnected interactive network, and their dynamic imbalance will cause cellular energy metabolism disorders and increased oxidative stress, ultimately leading to the occurrence of aging.

When mitochondrial dynamics are out of balance, it will lead to abnormal mitochondrial accumulation, mtDNA mutation and reduced mitochondrial biogenesis. Because mitochondria and other organelles have a complex and interconnected interactive network, the above damage will indirectly lead to abnormal performance of other organelles and cause aging of the body.

S-allylcysteine (SAC) is the compound γ-Glutamylcysteine in raw garlic, which is converted into SAC by γ-Glutamyltranspeptidase. It is the most abundant organic sulfide in black garlic. Studies have shown that SAC can filter reactive oxygen species, activate Nrf2 transcription factors 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 many neurodegenerative diseases. Studies have found that SAC can effectively reduce brain oxidative damage or hippocampal apoptosis caused by amyloid beta (Aβ), thereby improving cognitive ability.

Although SAC has been studied and confirmed in previous technologies to have antioxidant, neuroprotective and anti-diabetic effects, the relevant mechanism of action of SAC and whether it can also have positive effects on delaying aging in mammals are still unknown. Therefore, the present invention analyzes the impact of SAC on the behavioral ability and biochemical values of individuals, 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) for preparing a composition for delaying aging and preventing senile diseases.

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

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

According to one embodiment of the present invention, the composition is used to alleviate liver or kidney dysfunction.

According to one embodiment of the present invention, the composition is used to prevent DNA damage or immune senescence.

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

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

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

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

According to one 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 to prepare a composition for delaying aging and preventing senile diseases, which has the following advantages:

(1) Administration of SAC can reduce body fat accumulation and increase muscle mass. It can also maintain vitality and alleviate liver hypertrophy and anxiety caused by aging.

(2) Reduce serum biochemical values of alanine aminotransferase and blood urea nitrogen to protect the liver and kidneys. It can also dynamically regulate mitochondria to reduce oxidative stress by increasing the mRNA expression of OPA1, 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 reduce the p-p65/p65 ratio that mediates aging-related secretory expression.

The following embodiments should not be considered to unduly limit the present invention. A person having ordinary knowledge in the art to which the present invention belongs may modify and change the embodiments discussed herein without departing from the spirit or scope of the present invention, and still fall within the scope of the present invention.

In this document, unless the context otherwise states, the terms "comprising", "including", "having" or "containing" are inclusive or open, and do not exclude other unspecified elements or method steps; the terms "a" and "the" can be interpreted as singular or plural; the term "one or more" means "at least one", and thus can include a single feature or a mixture/combination. In addition, in this specification and the scope of the attached patent application, unless otherwise stated, "disposed on something" can be regarded as directly or indirectly contacting the surface of something by attachment or other forms, and the definition of the surface should be determined based on the context/paragraph meaning of the specification content and the common knowledge in the field to which this specification belongs.

The present invention provides a use of S-allylcysteine (SAC) for preparing a composition for delaying aging and preventing senile diseases. In the present invention, the composition is used in animals, and in one embodiment, the effects of SAC on the behavioral ability and biochemical values of aged mice are analyzed to explore the efficacy of SAC in delaying aging in mammals, and further explore whether it improves mitochondrial dynamics.

In one or more embodiments of the present invention, the animal experiment is conducted on male naturally aged mice. The experiment is conducted in two parts: an aging delay experiment and a mitochondrial dynamics experiment. The mice are divided into five groups, namely a young blank control group, an aging induction group, a low-dose SAC experimental group, a 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 are respectively mixed with 0.05% and 0.2% SAC in the mouse feed. Among them, in a preferred embodiment, the effective dose of S-allylcysteine converted to administration to a human individual is 12.2 to 48.6 mg/kg, such as 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-allylcysteine is 30 to 48.6 mg/kg, such as 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.

The term "aging" as used in this article refers to the gradual decline in physiological functions, which leads to damage to tissue or organ functions. In addition, aging is also accompanied by a gradual decline in brain function, leading to a decline in sensory, motor and cognitive abilities. Although aging is difficult to reverse, it can be prevented or delayed, thereby maintaining a healthy state. The term "delaying aging" in this article refers to slowing down aging indicators.

The aging indicators referred to herein include behavioral signs 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 functional decline of organs (including but not limited to liver or kidney), progressive brain function decline, leading to decreased sensory, motor and cognitive abilities, etc. In addition, the aging indicators also include biochemical indicators of aging, such as DNA damage, aging-associated lysosomes and SA-βgal (senescence-associated beta-galactosidase) activity or aging-associated secretory phenotype (SASP), etc.

In addition, most organelles need to continuously regenerate and eliminate 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 mitochondrial dysfunction is a major cause of aging: when mitochondrial dynamics are disordered, it will lead to abnormal mitochondrial accumulation, mtDNA mutations and reduced mitochondrial synthesis; and because mitochondria and other organelles have complex and cross-linked The above damage will indirectly lead to abnormal expression of other organelles and cause aging of the body. Therefore, another aspect of the present invention is to provide SAC to delay the occurrence of aging by balancing mitochondrial dynamics; in detail, SAC significantly increases the mRNA expression of the key mitochondrial fusion factor OPA1 in the liver, so the expression of mitochondrial biogenesis-related proteins SIRT1 and PGC-1α also increases significantly, thereby effectively reducing oxidative stress, such as the content of MDA in the liver and 8-OHdG in the urine.

The so-called "mitochondrial dynamics" in this article include changes in mitochondrial shape, their movement along the cytoskeleton, and the process of forming an interactive network with other organelles. Dynamic changes can only be achieved through mitochondrial fusion and cleavage reactions. If the mitochondrial dynamics regulatory proteins are unbalanced, on the one hand, it will cause endogenous mitochondrial damage, thereby reducing mitochondrial biogenesis activity; on the other hand, it will reduce mitochondrial autophagy reactions, causing the accumulation of damaged mitochondria, leading to aging and aging-related diseases.

The "elderly diseases" mentioned in this article refer to the decline in physiological functions and cell aging caused by aging, which in turn lead to damage to the body's immune, tissue or organ functions and cause diseases. Common elderly diseases include but are not limited to type 2 diabetes, neurodegenerative diseases, cardiovascular diseases, osteoporosis, sarcopenia and cancer. Therefore, the term "prevention of elderly diseases" in this article refers to the prevention of the decline in physiological functions and cell aging caused by aging, including but not limited to the prevention of anxiety behaviors, DNA damage or immune aging caused by aging.

The embodiment of the present invention is designed as follows: the experiment is divided into two parts, an aging delay experiment and a mitochondrial dynamics experiment. The first stage of the aging delay experiment is used to determine whether SAC can delay the aging of aging mice from the outside to the inside, and at the same time maintain the health of the mice, thereby achieving the effect of extending the healthy life span. Then, the second stage of the mechanism is explored to try to find the key factors causing aging. The "free radical theory of aging" proposed by Harman is extended to the connection between mitochondrial dysfunction and aging, and the impact of oxidative stress on the aging process in the present invention is explored, and then the corresponding changes in mitochondrial synthesis are detected. Finally, we discuss whether the key factor that determines mitochondrial efficiency and quality, mitochondrial dynamics, has a trend of change in the present invention .

Animal Experiment Group Design

The present invention uses 49-week-old male mice, which are raised in a natural aging model for 11 weeks. When the mice reach the reproductive senescence of 60 weeks, a 12-week experiment is conducted with reference to the "Health Food Anti-aging Function Evaluation Method" issued by the Ministry of Health and Welfare. When the mice reach the aging stage after 72 weeks of age, when various aging activity indicators begin to appear, whether the intervention of SAC can prevent aging is detected. The young mice are 6-week-old mice purchased 2 weeks before the sacrifice, and the sacrifice is carried out 2 weeks after adaptation to ensure that the time of sacrifice is in the adult stage of the mice, increasing its credibility as a blank control group.

During the breeding period, the daily food and water intake and weekly weight changes of 49-week-old male mice were recorded. When they reached 60 weeks of age, the body composition of the mice was analyzed, and the overall activity of the mice was tested using a field test. Based on the body fat, lean body mass and weight results obtained from the body composition analysis, the aged mice were evenly divided into 4 groups (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 a young blank control group (Yctrl)). After 12 weeks of intervention with the samples, the behavioral characteristics were analyzed together with the young mice that had been pre-bred for 2 weeks. Then they were sacrificed and the serum biochemical values were measured as an indicator of whether the health status of the mice could be prolonged. Then the aging-related biochemical indicators were measured, and finally the data were statistically analyzed.

Table 1 Group Number of animals Feeding Period Yctrl 10 Pellet 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

Embodiment 1 : Body composition and athletic ability test

In the aging delay experiment, the effects of SAC on the body composition, motor ability, anxiety behavior, organ function, serum biochemical values and biochemical indicators of aging in mice will be tested.

Please refer to Figure 1 to test the effect of SAC on the body composition and exercise capacity of mice. With the increase of age, the mass and metabolic rate of human organs or tissues decrease, which will cause a decrease in resting metabolic rate; coupled with the decrease of sex hormones and growth hormones, as well as lack of exercise or insufficient protein intake, the body composition will also change, resulting in a decrease in body lean meat and an increase in body fat. The post-test results of the body composition analysis in the 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, the body lean meat was significantly reduced (Figure 1C). However, from the before-after test change graph, it can be found that the intervention of SAC has the potential to reduce body fat and slow down the loss of body lean meat (Figure 1B, 1D).

Changes in the body composition of mice will also be reflected in their overall activity. The overall activity is determined by the open field test, which detects the total moving distance of mice in an open field within 5 minutes. The greater the distance, the better the overall activity. By comparing the results of the Actrl aging-induced group and the Yctrl blank control group (Figure 1E), the total moving distance of the old mice was significantly lower than that of the young mice, indicating a decrease in motor behavior ability. Similarly, the change graph before and after the open field test shows that the overall activity has a trend of recovery after the intervention of SAC (Figure 1F). This result is because SAC maintains the lean meat content in the body, which makes the mice have better motor performance.

The above characterization results show 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.

Embodiment 2 : Anxiety Behavior Test

The above-mentioned "open field test" is based on the tendency of rodents to move around in the periphery of the facility and are less likely to go to the open central area. Therefore, if mice spend more time exploring the central area or move more frequently, it means that their anxiety level is lower.

In a preferred embodiment, the composition is used to alleviate anxiety behaviors caused by aging. Please refer to Figure 2. From the post-test experimental results, it can be seen that the Yctrl blank control group spent a longer time in the central field (Figure 2A) and a shorter time in the peripheral field (Figure 2C) compared with the Actrl aging-induced group; and the pre- and post-test change graph within the group shows that the Res positive control group and the intervention sample SACL and SACH experimental groups, compared with the Actrl group, significantly increased the time spent exploring the central field (Figure 2B) and relatively reduced their stay in the peripheral field (Figure 2D). From the results, it can be seen that aging does have a tendency to increase the anxiety-like behavior of mice, but intervention with SAC can significantly improve it.

Therefore, the positive effect of SAC on reducing anxiety-like behaviors is speculated to have a neuroprotective effect, allowing the hippocampus to function normally and reduce 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 area in the hippocampus plays a key role in regulating emotions, cognition, and memory. It can be damaged by aging, causing imbalance in neurotransmission, reducing synaptic plasticity and neural regeneration, and leading to cognitive or emotional disorders.

Embodiment 3 ：Serum biochemistry test

After confirming the effects of SAC on the appearance and behavioral characteristics of mice, the 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 kidney.

The "liver" mentioned in this article not only delivers nutrients absorbed by the intestines to the liver for storage or synthesis through the portal vein, but is also responsible for breaking down alcohol, drugs, toxic substances and metabolic waste products, 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 to convert them into hydrophilic substances for easy excretion from urine. Therefore, regardless of whether the substance provides nutrition or toxicity to the body, the liver is the organ most directly affected.

The "kidney" mentioned in this article is the one that removes excess or harmful substances from the body through urine through filtration and reabsorption, in order to regulate the body's electrolytes, pH, and water content to maintain constant blood osmotic pressure and blood pressure. The kidney is sensitive to toxic reactions and can effectively respond to fluctuations in physiological states and acute injuries. Therefore, studies often use the weight and appearance of kidney components as indicators of whether a compound will cause toxic reactions in animals.

Please refer to Figure 3. The AST measurement results of the present invention in the Yctrl blank control group, SACL and SACH experimental groups and Res positive control group are not significantly different from the Actrl group (Table 1). It is inferred that AST is interfered by factors other than the liver and it is difficult to infer the actual condition of the liver. ALT, which is more specific to liver damage, has a lower trend in the Yctrl group than in the Actrl group, indicating that aging will increase the degree of liver damage. After the intervention of a low-dose sample SAC, the ALT value can be significantly reduced, and it has reached a level that is not significantly different from the Yctrl group and the 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 transfer of amino acids from alanine or aspartate to α-ketoglutarate, forming pyruvate and oxaloacetate respectively, and then participate in the process of gluconeogenesis. Both will be expressed in large quantities when the liver is damaged, and this abnormal increase is common in non-alcoholic fatty liver disease, alcoholic liver disease and hepatitis. ALT is mainly present in the cytoplasm, with the highest concentration and high specificity in the liver, so ALT is mainly used to confirm whether the lesion is located in the liver. When ALT and AST values are abnormal, the AST/ALT ratio is used clinically to diagnose different liver diseases. If the ratio is less than 1, it is usually non-alcoholic fatty liver disease; and a ratio greater than 2 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 final product of protein metabolism and is excreted to the outside of the body through the kidneys. If kidney dysfunction leads to low excretion function, it will lead to an increase in urea nitrogen in the blood. Therefore, blood urea nitrogen is an important indicator to understand whether the kidney function is normal.

Please refer to Figure 3. 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, indicating that aging reduces the efficiency of glomerular filtration of blood urea nitrogen. After the intervention of SAC, both low and high doses can significantly reduce the blood urea nitrogen concentration to the same level as the Yctrl group (Table 2, Figure 3B), indicating that SAC can prevent the functional decline of mouse kidneys.

Table 2

The above results show that intervention with SAC and the positive control compound resveratrol will not induce toxic reactions or functional defects in various organs.

Embodiment 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 is 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 the user's muscle mass.

Abnormal blood lipids and high blood pressure are the biggest risks for cardiovascular diseases, 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. Because it is insoluble in water, it needs to be combined with lipoproteins to circulate throughout the body with blood. Lipoproteins can be divided into low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Low-density lipoproteins transport cholesterol from the liver and small intestine to other tissues of the body, while high-density lipoproteins transport 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 lipoproteins and triglycerides are too high, it will lead to abnormal blood lipids, which will damage the endothelial cells of blood vessels. Excessive low-density lipoproteins are also prone to accumulate on the inner wall of arteries, triggering local inflammatory reactions, attracting macrophages to engulf the accumulated fat and gradually forming atherosclerosis.

Please refer to Figure 3. The cholesterol concentration of the Actrl aging-induced group measured by the present invention was significantly higher than that of the Yctrl blank control group, indicating that aging will increase the total cholesterol content, while intervention with SAC will reduce its value, and there is a dose effect, showing the potential of SAC to prevent cholesterol accumulation (Table 1, Figure 3C). Triglycerides in all groups were not significantly different from the Actrl group, but when investigating aging mice, it was also found that intervention with SAC could reduce the value of triglycerides, which tended to the trend presented by the Res positive control group, and the effect was better than the positive control group (Table 1). High-density lipoprotein was the highest in the Actrl group and significantly higher than the Yctrl group, and the experimental group and the positive control group also showed a trend of inhibiting high-density lipoprotein. This result is speculated to be due to the fact that the cholesterol in the Actrl group was higher, so the high-density lipoprotein was also relatively high. Although the high-density lipoprotein in other aged mouse groups was lower, it was not different from the Yctrl group (Table 1). Therefore, it is inferred that they are not at a higher risk of cardiovascular disease. The low-density lipoprotein in each group was not statistically significant compared with the Actrl group, but after the intervention of high-dose SAC, its value dropped to a level that was not significantly different from the Yctrl group (Table 1, Figure 3D).

The above experimental results show that SAC has the ability to protect the liver and kidneys. In addition, it has a tendency to lower triglycerides. The SACH group that intervened with a high dose of samples was able to reduce total cholesterol and low-density lipoprotein to a level that was not significantly different from the Yctrl group, showing its ability to inhibit cholesterol accumulation, making the serum biochemical values of mice more similar to those of young mice, and in a healthier state.

Embodiment 5 : DNA Damage related tests

From the above experiments, it can be seen that the phenomenon of preventing or delaying aging can be observed in the groups intervened with SAC. The results of serum biochemical values also show that the health status of aging mice intervened with SAC is better than that of the aging-induced group. Therefore, the internal aging-related biochemical indicators are then explored. In a preferred embodiment, the composition is used to prevent DNA damage or immune aging, and more preferably, the composition is used to reduce the synthesis of GLB1 and SA-βgal caused by cell aging.

The aging process begins with DNA damage, which in turn triggers a series of aging changes. Among DNA damage, genomic instability is the main driving factor of the aging process. When DNA is exposed to exogenous stimuli such as chemical agents, radiation or heat damage, or attacked by endogenous reactive oxygen species, there is a chance of nucleotide base modification, DNA single-strand break or double-strand break and other damages. Among them, double-strand break is the most serious DNA damage because it is difficult to repair and easily causes 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 the ATM kinase phosphorylates the histone variant H2AX to become γ-H2AX. Subsequently, the DNA damage checkpoint protein MDC1 is recruited to promote BRCA1 and 53BP1 proteins to repair DNA. Based on the characteristic that γ-H2AX and repair proteins form nuclear foci in the double-strand break area in the cell nucleus, its content can be used as an indicator of the degree of DNA double-strand breaks, and can also detect changes in gene damage after intervention with samples or compounds.

The present invention uses Western blot to detect the expression of γ-H2AX in mouse liver protein extracts. The results show that the expression 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 increased DNA damage. After the aged mice were treated with resveratrol, the expression of γ-H2AX was significantly reduced to a level that was not significantly different from the Yctrl group; in the SACL and SACH experimental groups of the intervention samples, γ-H2AX showed a downward trend (Figure 4A), and it is inferred that SAC has the potential to prevent DNA damage.

When aging continues, the genome of the organism's cells becomes unstable, telomeres shorten, epigenetics change, and protein homeostasis is lost. The cells will enter a period of replicative aging and irreversibly stop the cell cycle, preventing the spread of aging cells with mutation risks. Because the cells stop metabolic renewal, old and dead cells, damaged macromolecules or organelles will continue to accumulate, inducing the mass production of lysosomes.

Studies have found that SA-βgal is significantly expressed in aging cells, and its optimal detection pH is 6.0, which is different from the lysosomes that usually detect pH 4.5. Therefore, it is widely used as an indicator of cell aging to determine whether the samples or conditions involved have the potential to delay aging. The expression of SA-βgal is positively correlated with GLB1 mRNA, which mediates the synthesis of β-D-galactosidase. When the overall β-D-galactosidase concentration increases, SA-βgal will also increase. Therefore, it is not only aging that causes an increase in SA-βgal; but the proportion of SA-βgal upregulation in aging cells is higher than that in normal cells.

The present invention also used mouse liver protein extract for determination, and used Western blot to detect the expression of GLB1. The experimental results showed that the GLB1 in the Actrl aging-induced group was higher than that in the Yctrl blank control group, indicating that with the occurrence of aging, the degree of cell senescence will gradually increase. The positive control group with the intervention of resveratrol had a trend of reducing GLB1, and it was reduced to the same level as the Yctrl group, just like the low-dose SACL experimental group, while the high-dose SACH experimental group could significantly reduce the expression of GLB1 (Figure 4B). The results of detecting SA-βgal using fluorescence spectrometer were similar to those of GLB1. The SA-βgal in the Actrl aging-induced group was significantly higher than that in the Yctrl blank control group, while the Res positive control group showed a trend of delaying the synthesis of SA-βgal. The SACH group with high-dose samples also significantly reduced its content (Figure 4C). The above results show that the sample SAC can inhibit the expression of GLB1, thereby reducing the synthesis of SA-βgal and achieving the effect of delaying replicative aging.

The above experimental results show that SAC can slow down the possibility of DNA damage and reduce the synthesis of lysosomal proteins GLB1 and SA-βgal induced by cell aging, thereby delaying replicative aging.

In the mitochondrial dynamics experiment, the effects of SAC on oxidative stress, mitochondrial biogenesis and mitochondrial dynamics genes in mice will be tested. In a preferred embodiment, the composition is used to enhance the biogenesis efficiency of a user's mitochondria, and more preferably, the composition enhances the biogenesis efficiency of the mitochondria by enhancing the expression of SIRT1 and activating PGC-1α.

Embodiment 6 ：Oxidation stress test

Whether the organism is undergoing normal physiological metabolism or being stimulated by the external environment, such as air pollution, radiation or bacterial infection, it is possible to produce active oxygen and reactive nitrogen species (RNS), including free radicals with independent unpaired electrons. When reactive oxygen free radicals attack cell membranes or polyunsaturated fatty acids, lipid peroxidation metabolites malondialdehyde (MDA) are produced. MDA is then detected by using its characteristic of easily undergoing nucleophilic addition reaction with TBA (thiobarbituric acid) under low pH and high temperature conditions.

The "free radicals" mentioned in this article refer to substances produced by the body after metabolism. They are extremely active and can easily react with other substances. When tissues and organs are injured, a large number of free radicals will accumulate. It is an unstable factor that will attack healthy cells, rob healthy cells of their electrons, induce cell apoptosis and lead to aging.

The present invention uses mouse liver protein extract to detect MDA concentration. The results show that the MDA content in the Actrl aging-induced group is significantly higher than that in the Yctrl blank control group, indicating that the oxidative stress in the aged mice is significantly higher than that in the young mice. The intervention of a high-dose sample SAC can significantly reduce the MDA content (Figure 5A), which shows the ability of SAC to reduce lipid oxidative stress.

When active oxygen free 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 a replacement of nucleic acid GC:TA. Therefore, the content of 8-OHdG is often used as an indicator of DNA oxidative damage. When 8-OHdG is removed by DNA damage repair enzymes, it will be released into saliva, urine or plasma. Most of the substances measured use urine because the amount of urine samples is more sufficient and it is a non-invasive method. In addition, studies have also pointed out that the urine excretion of 8-OHdG is equivalent to the oxidation rate of Guanine.

Therefore, the present invention also allows mice to excrete naturally without external stimulation, and collects urine as a sample. The measurement results show that the urine 8-OHdG in the Actrl aging-induced group is 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 during aging, which increases the proportion of DNA damage. This situation has a tendency to slow down in the positive control group that intervenes with resveratrol. However, in the low-dose SACL experimental group, the content of 8-OHdG can be significantly reduced to the same level as that of young mice (Figure 5B). It shows that SAC can effectively prevent the increase in aging-related DNA oxidative damage.

The above experimental results show that SAC has the potential to activate the antioxidant mechanism of organisms and can 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.

Embodiment 7 ：Mitochondrial biosynthesis efficiency test

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

The present invention uses Western blotting to detect the expression of SIRT1 and PGC-1α in mouse liver protein extracts. The results show that the relative expression of SIRT1 or PGC-1α in the Actrl aging-induced group tends to be lower than that in the Yctrl blank control group, indicating that mitochondrial synthesis is reduced in aging individuals. Previous literature has indicated that resveratrol is a promoter of SIRT1, so in this experiment, the positive control group did significantly increase the expression of SIRT1 and PGC-1α. The SACL and SACH groups of the intervention samples also significantly increased the content of SIRT1 (Figure 6A); in PGC-1α, the low-dose SACL group significantly upregulated its expression; a significant increase in the SACH group was also observed (Figure 6B). In addition, RT-qPCR was used to measure the expression of TFAM mRNA in mouse liver. The results showed that the SACL and SACH experimental groups showed an increasing 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 factors such as p53, FOXO (forkhead box O), and NF-κB by deacetylation of histones, thereby increasing the body's resistance to environmental stress, anti-inflammation, and balancing nutrient metabolism pathways. Mice expressing SIRT1 significantly increase their lifespan and show 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 PGC-1α, one of its downstream factors, to increase mitochondrial synthesis, allowing cells to maintain normal oxidative metabolism and reduce oxidative damage such as MDA and 8-OHdG (Figures 5A, 5B).

The present invention extracts mouse liver mRNA and then measures the expression of mitochondrial dynamic genes by RT-qPCR. The experimental results of mitochondrial fusion-related genes show that MFN1 has an upward trend in the Res positive control group compared to the Actrl aging-induced group; and the experimental group with SAC intervention also develops in the same direction (Figure 7A). For MFN2, there is no statistical significance between the groups (Figure 7B), but because the two fusion proteins are highly similar, they can complement each other. The expression of OPA1, a key gene for fusing mitochondrial membranes and maintaining wrinkled morphology, was higher in the Yctrl blank control group than in the Actrl aging-induced group. Both SAC and resveratrol intervention increased the relative content of OPA1, especially in the high-dose SACH group, which significantly increased its expression (Figure 7C).

In summary, the physiological phenomena related to delaying aging tend to promote mitochondrial fusion. SAC can increase the efficiency of mitochondrial biogenesis by activating fusion reactions, thereby observing an increase in SIRT1 and PGC-1α (Figure 6A, 6B) and a decrease in oxidative stress (Figure 5), while increasing overall activity (Figure 1F), indicating that SAC increases ATP production by promoting mitochondrial fusion, ultimately achieving the effect of delaying aging.

In summary, after SAC intervention, male naturally aged mice have the potential to reduce body fat accumulation and delay muscle loss, thereby preventing the decline of overall activity. In terms of perception and emotion, it can significantly improve aging-related anxiety-like behaviors. In addition, serum biochemical values show that SAC intervention can reduce total cholesterol and has liver and kidney protection benefits, 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.

Further exploration of the relevant molecular mechanisms revealed that intervention with SAC significantly increased the expression of OPA1 mRNA, a gene associated with liver mitochondrial fusion, and promoted the levels of SIRT1 and PGC-1α, proteins associated with mitochondrial synthesis. The antioxidant enzyme SOD showed an upward trend due to improved mitochondrial dynamics, while oxidative stress MDA and 8-OHdG decreased significantly, thereby achieving the effect of prolonging healthy lifespan.

Herein, all ranges provided are intended to include each specific range within the given range and the combination of sub-ranges between the given ranges. In addition, unless otherwise specified, 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 sub-ranges such as 2-5, 3-5, 2-3, 2-4, 1-4, etc.

All publications and patent applications cited in this specification are incorporated herein by reference, and each individual publication or patent application is specifically and individually indicated as incorporated herein 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 controls.

The present invention has been described in detail above. However, what has been described above is only the preferred embodiment of the present invention and should not be used to limit the scope of implementation of the present invention. That is, all equivalent changes and modifications made according to the scope of the patent application of the present invention should still fall within the scope of the patent of the present invention.

without

The implementation of the present invention is now described by way of example only with reference to the accompanying drawings, in which:

FIG. 1 is a graph showing the effects of SAC on body composition and motor ability of mice according to an embodiment of the present invention.

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

FIG. 3 is a graph showing the effect of SAC on serum biochemical values in mice according to an embodiment of the present invention.

FIG. 4 is a graph showing the effects of SAC on biochemical indicators of aging in mice according to an embodiment of the present invention.

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

FIG. 6 is a graph showing the effects of SAC on genes and proteins related to mitochondrial biogenesis in mice according to an embodiment of the present invention.

FIG. 7 is a schematic diagram showing the effect of SAC on mouse mitochondrial fusion gene according to an embodiment of the present invention.

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

without

Claims (10)

A use of S-allylcysteine (SCA) is to prepare a composition for delaying aging and preventing senile diseases. The use of claim 1, wherein the composition is used to reduce a user's body fat cholesterol, triglyceride or low-density lipoprotein, and increase the user's muscle mass. The use of claim 1, wherein the composition is used to alleviate anxiety behaviors caused by aging. The use of claim 1, wherein the composition is used to alleviate liver or kidney function decline. The use of claim 1, wherein the composition is used to prevent DNA damage or immune senescence. The use of claim 5, wherein the composition is used to reduce the synthesis of GLB1 and SA-βgal induced by cell senescence. The use of claim 1, wherein the composition is used to enhance the mitochondrial biosynthesis efficiency of a user. The use of claim 6, wherein the composition enhances the mitochondrial biogenesis efficiency by increasing the expression of SIRT1 and activating PGC-1α. The use of any one of claims 1 to 8, wherein the effective dose of the SCA is 12.2 to 48.6 mg/kg. The use of claim 9, wherein the effective dose of SCA is 30 to 48.6 mg/kg.