Anti-Aging Compound

Abstract

The invention relates to the use of amygdalin-containing compounds in delaying cellular senescence associated with genomic instability. The invention falls within the field of developing anti-aging agents and their in vitro and / or therapeutic use, particularly with applications aimed at regulating senescence dynamics in primary human fibroblast cells.

Description

1 TARIFF ANTI-AGING COMPOSITION Technical Area The invention suggests that amygdalin-containing compounds may be used to treat cellular senescence associated with genomic instability. The invention relates to its use in delaying the development of fibroblast cells, particularly in primary human fibroblast cells. 5 applications aimed at regulating aging dynamics and anti-aging agents This falls within the field of development and their in vitro and / or therapeutic uses. State of the Art Aging is the process of maintaining the integrity of biological systems and ensuring their continued existence over time. It can be understood as the gradual decrease in the capacity it possesses for life. Lifespan, It approaches its end with an inevitable aging process. This phenomenon is prevalent in today's growing and most advanced industries. More importantly, it has begun to play a larger role in the aging population. According to the World Health Organization... According to the WHO, the proportion of the population aged 60 and over will increase from 12% to 22% between 2015 and 2050. will almost double (1). This demographic increase will be accompanied by an increase in age-related diseases. They are seen together. These diseases include cardiovascular disorders, cancer, and neurodegenerative diseases. Diseases and diabetes are among the 15% of global deaths, and together they account for more than 70%. (2). This result indicates that increasing aging is equivalent to a healthy lifespan. This shows that it is not the case and opens the way for new interventions regarding longevity. Although Türkiye's total population nearly tripled from 1920 to 2018, the elderly... The adult (65 and older) population has increased almost sevenfold. In 2025, the adult population... It is predicted that they will become the largest individuals in Türkiye. In a very short time (25 to 50 years) 20 Türkiye, like other developed countries with aging populations such as the USA, Germany and Japan, has a socio- Turkey will face economic problems. Türkiye has the fastest aging population. Despite being one of the largest populations, there are very limited efforts to study aging in Türkiye. Life expectancy at birth is increasing in Türkiye, and the population continues to age. The median age, an important indicator of the population's age structure, and the 25+ age group, defined as the elderly population, are discussed here. The proportion of those aged 65 and over is expected to increase according to all scenarios. ADNKS 2023 According to the results of the year, the median age was 34, while in 2050, according to the main scenario, it will be 44.8, and in 2075... It is projected to reach 51.5 and 52.2 in 2100 (obtained from TUIK). According to López-Otín and colleagues (3), aging is defined by twelve distinct features. It is a complex process. These distinct characteristics cause cells to deteriorate and lose their function as they age. contributes to its loss (3,4). One of the main markers is the integrity of the genome. 2 It is related to the disruption of these distributions, mutations, and DNA repair mechanisms. This can lead to inhibition, which can cause the cells to age (5). Genome instability and consequent aging and longevity, life spans less than one week (C. elegans; the organism used as a sample to study aging and longevity) It is known that humans show differences of more than 100 years (40). Rats (Rattus 5 (norvegicus) and naked mole rats (Heterocephalus glaber) or chimpanzees (Pan troglodytes and humans (Homo sapiens) (39), although they live in almost similar environments, surprisingly They somehow show high variability in lifespan. This is in the evolution of lifespans. Despite striking differences, the molecular mechanisms of aging and longevity... Many 10 researchers use a comparative functional genomics approach to understand their mechanisms. There is little work. Due to the complex nature of aging and longevity, aging and / or longevity are important considerations. All mechanisms and molecular pathways involved in lifespan are not solely genetic (endogenous) or It is not possible to understand this by examining only environmental (exogenous) factors. Environmental factors... Together (around 70%), genetic factors (around 30%) are vital for a healthy, long life. plays a role (38). Amygdalin is a bioactive compound that occurs naturally in bitter almonds and apricot kernels. With its anticancer activity demonstrated by inhibiting cell proliferation and inducing apoptosis. It has received scientific attention (6). However, most of the research focuses on anti-tumor mechanisms. Because it is focused on the skin, its potential effect on aging is still unknown. Amygdalin, 20 Apricots have a long history of use in traditional medicine; for example, in Traditional Chinese Medicine. The kernels have been used for respiratory and general health purposes. Interestingly, apricots and almonds... In mythology, it has also been associated with health, rejuvenation, and eternal youth. This is a new This could reveal the mechanism and contribute to amygdalin-related research on aging and longevity. This could lead to a novel way of modulating genomic instability. 25 Maintaining the stability of the genome is a time-dependent process, as it is susceptible to damage (11). Therefore, all organisms have a powerful mechanism to detect damage and take action accordingly. develop repair mechanisms (12). However, over time, cells develop repair mechanisms It begins to accumulate damage, including mutations it cannot resist. This situation affects the tissue. degeneration, age-related diseases and increased susceptibility to them, and aging at 30 may contribute to triggering (13). Therefore, there is a relationship between genome stability and aging. A linear relationship emerges that develops over time. 3 Amygdalin is a naturally occurring chemical compound with a molecular weight of 457.42 g / mol and the chemical formula C20H27O11. It is a raw Chinese medicine compound containing cyanide, a plant-based glycoside, and is used in the pharmaceutical industry as laetrile. Also known as (18, 19). Amygdalin is used in cancer treatments to target cancer cells. effective in delaying its progression, halting the cell cycle, or inducing apoptosis. These methods have been reported (20,21). 5 However, after hydrolysis, HCN is a cytotoxic byproduct for both cancer and normal cells. It is produced and generally causes poisoning (22). The β-glucosidase enzyme, It cleaves glucose from amygdalin and releases HCN, which is highly toxic to cells. This leads to the production of HCN. This cytotoxicity kills cancer cells (23). The released HCN enters the cell. It enters and disrupts mitochondrial respiration by inhibiting cytochrome oxidase C, resulting in ATP 10. suspends synthesis and leads to cell death (24). On the other hand, when apoptosis is triggered, the tumor Anti-apoptotic gene expression to promote cytoprotection in non-apoptotic cells It applies (25). In kidney cancer cells, it leads to inhibition of the cell cycle in the G1 / S phase. The increase in p19 protein expression that triggers this is another factor that prevents cell proliferation. is a mechanism (9). 15 The genome of fibroblast cells obtained from human primary foreskin was manipulated. It has not undergone this process, and these types of fibroblasts are suitable for human-specific aging (cellular aging) applications. These are the most common and suitable cells for (26, 27). Although it varies from person to person, each The aging cycle of human skin cells is limited. These primary cell lines undergo a specific division. After its cycle, it exhibits the cellular senescence phenotype, dividing into 20... It stops it. For example, even if a primary human skin cell undergoes 34 divisions in cell culture. after a primary cell obtained from another person undergoes 60 cyclical divisions It stops dividing and enters the cellular senescence cycle. In primary cell lines... Cyclical lifespan, relatively speaking (many other factors must be considered) compared to humans. It shows a linear proportion with lifespan. Therefore, these 25 of human primary cell lines... Factors affecting aging are used as models for therapeutic agents (especially Human-specific aging phenotypes can be identified. Brief Description of the Invention The discovery suggests that amygdalin delays cellular aging by regulating genomic instability, and this This shows that it can be used as a new anti-aging agent. For this, 30 Primary human foreskin fibroblast cells in cell culture as an in vitro model. Primary fibroblast cells were used, both incubated and unincubated with amygdalin. 4 The cultures were divided into two different experimental groups, one for the control group and one for the other. These cultures were used to treat senescent cells. to selectively target and determine the change in the rate of cellular aging It was monitored in terms of morphological changes. As a result, amygdalin has an anti-aging effect. it can be used as a therapeutic agent and cultured primary fibroblast cells It has been observed that it can delay aging. 5 Shapes Figure 1: Cell growth of amygdalin group versus control group at weeks 2, 4, and 6. comparison Figure 2. Morphological changes in cells. Photographs were taken with 10X microscope lenses. Detailed Description of the Invention 10 Isolation and Culture of Human Foreskin Fibroblasts HFF cell lines were obtained from foreskin samples. They were then grouped into 3 categories in IMDM. The samples were grown and separated into subcultures. The medium was mixed with 1% Pen Strep antibiotic and 1% Non-Essential It contained 10% FBS with amino acids. To isolate the cells, the samples were mixed with DPBS. The blood was removed by washing. Then the samples were placed in 100 mm petri dishes and bistouri 15 It was divided into 1-2 mm pieces. The torn tissues were mixed with 2 ml of 0.45 to further digest the tissue. It was treated with a concentrated commercial collagenase solution of U / ml (29, 30). For enzyme digestion, it was kept at 37 °C. It was incubated for 90 minutes. After incubation, it was centrifuged at 400 g for 5 minutes. Then the enzyme was added. The cells were absorbed and the resulting cells were cultured in their own vials. 15 ml of IMDM medium was placed in the incubator. They were cultured in T75 flasks containing. These steps were performed separately for each tissue sample, and 20 Samples were stored under standard conditions at 37°C and 5% CO2. D2 cell line He was elected. Cell Subculture and Counting Cells that reached 70% confluence were transferred as subcultures into three T25 flasks for both groups. Control group (not incubated) and group treated with amygdalin. 25 for subculture. First, the culture medium in T75 was removed and 5 ml of trypsin was added. Trypsin draws the cells from the bottom of the vial. It is used to remove. The bottle was left for about 5 minutes while gently shaking it. All cells After removal, add 10 ml of IMDM medium to the bottle to slow down the reaction while washing it. The mixed solution containing the cells was added to a Falcon and centrifuge at 330 g for 5 minutes. transferred to a tube. Then the supernatant was discarded and the cells were resuspended for 30 minutes. 10 ml of culture medium was added for (31). From here, the cell counting part begins. The cells are counted to determine the total number, and then ten... T25 is divided into twelve thousand five hundred cells evenly in the flask. Also, the proliferation rate, It is important to determine and evaluate the health of the cell and the viability of the cell culture (32). Our sample cells were separated by trypsinization and then thoroughly resuspended in the medium. After being brought in, 2.5 μl of Trypan Blue is placed on parafilm. 5 μl of blue dye from 10 μl of the sample... It is added and pipetted until the sample is stained. A total of 12.5 μl is collected in the hemocytometer. and placed under a microscope for counting. Blue dye (Trypan Blue) coats the membrane of living cells. It is used to detect dead cells because it cannot pass through (33). Under the 10X lens of the microscope The number of cells should be evenly distributed among the squares. If the number is too high, the sample should be diluted. The cells in the large central square were considered. Two rooms were counted and their average was taken. 10 This process was carried out at least twice. After the second count, the average of all counts was taken. The resulting number was then multiplied by 104 to find the cell concentration in 1 ml. This The number is then multiplied by the amount of medium in which the cells are resuspended. The total number of cells in each fals was obtained. In this case, the cells were in 10 ml of medium. They were multiplied by 10 because they were resuspended (34). Twelve thousand five hundred cells 15 The cells were diluted and cultured in T25 flasks. The remaining cells contained approximately one million cells. They were cultured or frozen as a backup. Cell Freezing This procedure was performed after the cells were counted, so that approximately the frozen cells were counted. The number could be determined. Samples were initially subjected to 330 g for 5 minutes, then again to 20... Centrifuged. IMDM was discarded when Freezing Medium containing 10% DMSO was added. Solution 9 ml It contains FBS and 1 ml DMSO. The cells are resuspended before being transferred to cryotubes. It was brought to this state. It was kept at -20°C for two hours until it froze. Then it was placed at -80°C. (35). Amygdalin Stock Solution 25 In a preferred application of the invention, amygdalin is prepared, preferably in DMSO. and a final concentration of approximately 0.5 micromolar to 5 millimolar in cell culture medium. It can be applied within this concentration range. This concentration range refers to the concentration of amygdala in cells. It offers a suitable study range in terms of observing its effect in the environment. In experiments, amygdalin was used at a dose of 30 to observe the effect more clearly and in a shorter time. It was used at a final concentration of 2.18 millimolar in cell culture medium. 6 Amygdalin was ensured to be 98% pure. For the main solution, 0.1 g of amygdalin was dissolved in 1 ml of DMSO. It was resolved. The main solution concentration was 0.218 M, and consequently, the final solution concentration was... The concentration was 2.18 mM. For the experiment, the stock solution (diluted at a ratio of 1 / 100) was used. 150 μl of amygdalin stock solution was added to 15 ml of IMDM medium. The 15 ml medium was divided into three 5 ml portions. It was divided into T25 vials. Amygalin stock solution can be stored at 25°C. 5 However, higher concentrations may have undesirable effects in long-term applications. It has been observed that it can give birth. In particular, the last one, at a level of approximately 21.8 millimolar. At these concentrations and under long-term incubation conditions of approximately 3 weeks, amygdalin It was found that toxic effects became apparent and the beneficial effect could be suppressed by adverse effects. Therefore, within the scope of this invention, amygdalin concentration is considered an anti-aging 10 It is preferable to select within appropriate ranges that will limit toxicity while ensuring the desired effect is achieved. is being done. In terms of incubation time, amygdalin is associated with genomic instability and cellular senescence. In order to demonstrate its effect in a meaningful and reliable way, the applied Depending on the concentration, an incubation period of at least 3 weeks is preferred. More 15 While certain positive effects can be observed in short-term applications, below these periods... limited or insufficient effect on genomic instability and cellular senescence parameters It has been assessed that it remains at this level. Therefore, within the scope of the invention, especially in the long term... application regimens are preferred in terms of achieving an anti-aging effect. It constitutes the application methods. 20 Experimental Setup The two main groups consist of five T25 vials containing twelve thousand five hundred cells in 5 ml of medium. For the control group, 150 μl of DMSO was added to 15 ml of medium. The media of both groups were then mixed with the added solution. It is parallel to the solutions. Data Analysis 25 To determine if there is a difference between amygdalin-incubated cells and control cells. Cell counts from weeks 2, 4, and 6 were used. Standard deviation of the data and Student's coefficient were used. A T-test was conducted to compare and find significant changes caused by amygdala. ImageJ software was also used to compare morphological and numerical changes. 7 Growth Curve in Weeks 2, 4, and 6 Amygdalin affects the growth and cellular senescence cycle of human primary cell lines. To examine its effect, cells were counted at weeks 2, 4, and 6. Cell growth curves were created and analyzed. The number of cells measured in the amygdalin group was three. It was also significantly higher at the time point compared to the control group (week 2: p = 0.0128 5 < 0.05; Week 4: p = 0.0050 < 0.05; Week 6: p = 0.0085 < 0.05). However, in both groups A decrease in cell count has been observed over time. These results suggest that amygdalin treatment... This shows that it is associated with a higher cell count compared to the control group (Figure 1). Morphological change Photographs were taken every 4 days to observe changes in cell morphology. 10 Changes in the control group and the group incubated with amygdalin were observed together randomly. observed (Figure 2). Evaluation of Results There is significant research supporting the role of amygdalin as an anti-cancer agent. There was a difference of 15 between the group incubated with amygdalin and the control group at all time points examined. A statistically significant difference in cell number was observed in both groups over time. Although a decrease in cell number occurred, this decrease was observed in cells incubated with amygdalin. It was significantly less pronounced in the group. This observation suggests that amygdala is only involved in proliferation. It can affect not only the rates but also the dynamics of cellular aging. This is thought-provoking. A possible explanation is that amygdalin selectively targets aging cells. It may affect; alternatively, the initiation or progression of cellular senescence. By delaying it, it can keep the proportion of living cells higher over time. This second possibility, Morphological differences observed between the control group and the groups incubated with amygdalin This is consistent with the differences. Aging cells typically exhibit increased spreading on the culture surface. They exhibit an expanded and flattened morphology (36), this feature is more prevalent in the control group 25 It has been clearly observed. Within the scope of this invention, amygdalin is particularly involved in cellular senescence associated with genomic instability. It is used to delay its onset and / or slow its progress. (Word) The subject of use is to reduce the rate at which age-related cellular changes occur, over time, maintaining a higher level of live cell ratio and 30 associated with senescence This can manifest as a delay or reduction in morphological changes. in this context, the enlarged, flattened, and diluted appearance typically observed in aging cells. 8 The morphological feature appearing later or becoming less pronounced, invention It is considered among the indicators of the impact within its scope. Aging is an inevitable and unavoidable part of human life, as well as a necessity. It is a broad field of research as it increases. This research aims to regulate and improve health duration. This could pave a new way to slow down aging and combat genomic instability. It could be used as a novel way of protection. 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Claims

11 REQUESTS 1. An anti-aging compound containing amygdalin, whose characteristic feature is to reduce genomic instability. Its use is to delay cellular aging by regulating it.

2. It is a composition according to claim 1, and its characteristic feature is that it marks the onset of cellular senescence in question. It is used to delay. 5 3. It is a composition according to claim 1, and its characteristic feature is that it inhibits the progression of the cellular senescence in question. It is used to slow things down.

4. It is a composition according to claim 1, and its characteristic feature is that the cellular senescence in question is replicative. It is the occurrence of cellular aging.

5. It is a composition according to claim 1, and its characteristic is that the cellular senescence in question is DNA damage 10 It is cellular senescence associated with accumulation.

6. Compound according to claim 1, its characteristic is to delay cellular senescence. It is the use of.

7. Usage according to claim 6, and its characteristic is cellular senescence, the division of cells. stopping, showing enlarged morphology and / or flattened cell morphology 15 It is characterized by its development. 25

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