WO2021091356A1 - Composition for preventing or treating stress-related diseases including methyl benzoate as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a functional food composition, and a cosmetic composition which include methyl benzoate or a derivative thereof as an active ingredient, and are for preventing or treating stress-related diseases. A composition according to the present invention significantly improves behavioral indicators of individuals exposed to stressful environments, significantly reduces blood cortisol concentration, and significantly suppresses cortisol concentration and stress-related gene expression in skin fibroblasts, and can thus be applied to neurological diseases and skin diseases caused by stress, as well as to various stress-related diseases accompanied by increased cortisol secretion. The present invention uses, as an active ingredient, a naturally-derived compound that is already being consumed as a food additive, and thus has few side effects even when administered for a long time. Thus, the present invention can be effectively used as an effective therapeutic composition for various stress-related diseases, most of which are chronic diseases.

Description

Composition for preventing or treating stress-related diseases comprising methyl benzoate as an active ingredient

The present invention relates to a composition for preventing or treating stress-related diseases comprising methyl benzoate and a derivative thereof as an active ingredient.

The World Health Organization defined mental health as'a well-being state in which an individual can recognize his or her abilities, cope with the daily stresses of life, work productively, and contribute to the community'. Stress here refers to changes in organisms caused by internal and external stimuli that break the balance of the living body. Selye, a pioneer of stress research, defined stress as a non-specific response of a living body to demand, and is one of the typical stress responses from the adrenal cortex to sugar. The secretion of glucocorticoid was considered as an important factor in the establishment of the stress response in vivo (Selye H., 1993, 1958, 1976).

Stimuli that cause stress are classified into physical, psychological, and physiological stimuli. Physical stimulation refers to temperature and ultraviolet rays that exist in nature, psychological stimulation refers to mental pain, anger, anxiety, tension, etc., and physiological stimulation refers to bacteria, viruses, and allergens. In particular, repeated symptoms such as depression, anxiety, insomnia, and loss of appetite caused by psychological stimulation cause diseases such as depression, anxiety, and sleep disorder. Depression is a disease that causes a variety of cognitive, mental, or physical symptoms, resulting in decreased motivation and depression as the main symptoms, leading to a decrease in daily function. Daily life is difficult due to a decrease in overall functions such as thought process, motivation, motivation, behavior, and sleep. , The prevalence rate, and suicide rate are also highly correlated. Anxiety is a mental illness that shows persistent and unrecoverable nervousness, tension, and anxiety over a period of more than 6 months, and causes extreme anxiety even in every minor event for no specific reason. Sleep disorders experience psychotic symptoms such as self-destruction, hallucinations, and delusions, and as a result of sleep deprivation experiments in rats, phenomena such as general weakness, skin diseases, weight loss, increased energy consumption and lower body temperature were found. It has been reported to lead to severe death (Dugovic et al., 1999 J. Neurosci . 19(19); 8656-8664).

In the case of excessive mental stress, hormones are secreted into the blood through the circulatory system leading to the hypothalamic-pituitary-adrenal axis (HPA axis). In the hypothalamus of the brain, a corticotropin releasing factor (CRF) is produced, which binds to the CRF receptor expressed in the pituitary gland to release adrenocorticotropic hormone (ACTH). ACTH reaches the adrenal glands through blood and lymph nodes, promoting the secretion of glucocorticoids such as cortisol, and glucocorticoids secreted into the blood are delivered to each organ of the body.

Cortisol is a stress hormone among steroid hormones that tensions muscles and makes sense organs sensitive, preparing the body to respond to mental stress. On the other hand, the continuous secretion of the hormone cortisol caused by repetitive mental stress increases its blood concentration, keeps the body in a tense state, and interferes with sleep, leading to sleep disturbances. This again causes mental stress, and the vicious cycle continues, leading to depressive symptoms. When cortisol secretion control function is lost due to extreme mental stress, cortisol secretion is excessively secreted, causing brain atrophy and damage throughout the nervous system, and thus, the possibility of serious depression and suicide has been reported.

In the case of mice overexpressing the CRF gene, the behavior of anxiety and depression increases, and the search behavior decreases. It is reported that it may be implicated in emotional disorders and cause cushing syndrome. It also shows physical changes such as excessive fat accumulation, muscle atrophy, thin skin and hair loss, and increases plasma levels of adrenal cortical stimulating hormone (ACTH) and cortisol. It has been reported that these symptoms are alleviated by administration of a CRF antagonist (Stenzel-poore et al., 1994).

The early stress recovery dietary supplement market is St. John's wort (capsicum sprouts: improving depression and anxiety disorders) and Valerian (valier grass: sedation, sleep improvement) have been leading, but as the anti-stress market is rapidly expanding, the development of new anti-stress substances is required. However, only a small number of counterparts such as GABA, Theanine, and Phosphatdyl serine have been proposed, and new products are being developed by combining at least one of the five anti-stress substances.

Depression treatments are drugs that induce therapeutic effects through temporary relaxation effects by inhibiting the reabsorption of serotonin secreted from nerve cell extremities into pre-synaptic cells, thereby increasing the concentration and activity of serotonin in the blood. Prozac and celexa are sold under approval. However, in the case of such a serotonin reuptake inhibitor, only about half of the patients show symptomatic improvement and require a dose period of at least 4 months, and depending on the patient, there is a problem in that it must be continuously taken for 2 to 3 years. In addition, in the case of the serotonin reuptake inhibitor, most symptoms recur within 6 to 12 months were observed when the drug was discontinued, and side effects were also known to be at a serious level. As anti-anxiety drugs, benzodiazepines such as dizepam, lorazepam, clonazepam, and alprazolam are mainly used. Drugs of the imidazopyridine family are also used to treat short-term insomnia caused by anxiety. However, drugs used for anxiety disorders and mental disorders act directly on GABA (gamma-aminobutyric acid) receptors in the central nervous system to suppress the central nervous system, resulting in excessive sedation, and central inhibitory effects such as depression, muscle relaxation, and hypnosis. May appear reinforced. In addition, drugs used for anxiety disorders and mental disorders pose a risk of being psychologically and physically dependent, causing concern about drug abuse.

Methyl benzoate is an ester-based compound, also known as benzoic acid methyl ester, and the like, its molecular formula is C ₈ H ₈ O ₂ and its molecular weight is 136.15 g/mol, and the structural formula is as follows. same:

Methyl benzoate is a pale yellow transparent liquid with a melting point of -12℃ and a boiling point of 199℃. As a fat-soluble substance, it is not well soluble in water, but is known to be well soluble in most organic solvents.

Methyl benzoate is a fragrance component contained in Allspice and various flower oils (banana, cherry, pimento berry, clove, Monstera deliciosa, etc.). It is mainly a perfume and food flavoring agent. , It is used as a component such as air purifier. It is also listed as a flavoring agent in the FDA as a food additive, and has been approved as a flavoring agent and food additive by FEMA (Flavor and Extract Manufacturers Association) and JECFA (Joint FAO/WHO Expert Committe on Food Additives), respectively. However, there have been no reports on the physiological activity of methyl benzoate other than the flavoring agent function.

When methyl benzoate is chronically administered orally to rats, it has been reported that the NOEL (No Observed Effect Level) value is more than 500 mg/kg bw, which is relatively safe (Drug Research, 112(5): 394-403, 1960).

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have conducted extensive research to develop an effective natural-derived therapeutic composition suitable for the treatment of chronic diseases, as it has excellent therapeutic activity for various stress-related diseases, including stress neurological diseases and stress skin diseases, and has few side effects even when administered for a long period of time. I tried. As a result, methyl benzoate and its derivatives significantly improved the behavioral indicators of experimental animals exposed to an artificial stress environment and significantly reduced the plasma corticosterone concentration, as well as cortisol concentration and stress in skin fibroblasts. By remarkably suppressing the expression of the gene, the present invention has been completed by finding that it can be used as an effective therapeutic composition for various diseases caused by stress, including neurological diseases and skin diseases.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating stress-related diseases.

Another object of the present invention is to provide a food composition and a cosmetic composition for improving or relieving stress.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating stress-related diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In the above formula, R is C ₁ -C ₃ alkyl.

The present inventors have conducted extensive research to develop an effective natural-derived therapeutic composition suitable for the treatment of chronic diseases, as it has excellent therapeutic activity for various stress-related diseases, including stress neurological diseases and stress skin diseases, and has few side effects even when administered for a long period of time. I tried. As a result, methyl benzoate and its derivatives significantly improved the behavioral indicators of experimental animals exposed to an artificial stress environment and significantly reduced the plasma corticosterone concentration, as well as cortisol concentration and stress in skin fibroblasts. By remarkably suppressing the expression of the gene, it was found that it can be used as an effective therapeutic composition for various diseases caused by stress, including neurological diseases and skin diseases.

According to a specific embodiment of the present invention, the stressful disease that can be prevented or treated with the composition of the present invention is selected from the group consisting of a stress neurological disease, a stress skin disease, a stress cardiovascular disease, and a stress infectious disease.

As used herein, the term “stress-related neurological disorder” refers to any disease that causes dysfunction in the brain, spine, and the nervous system that connects them by being exposed to a stressful situation. For example, depression , Sleep disorders, anxiety disorders, panic disorders, fatigue syndrome, headaches, neurodegenerative diseases, Alzheimer's, eating disorders, anorexia nervosa, alcohol withdrawal syndrome and stress-related mental disorders. More specifically, the stress neurological disease that can be prevented or treated with the composition of the present invention is a group consisting of depressive disorder, sleep disturbance, anxiety disorder, and panic disorder. Is selected from

In the present specification, the term “stress-related skin neurological disorder” is a disease in which various damages are applied to skin tissues by exposure of an individual to a stressful situation, and not only when psychological factors are the main cause of skin tissue damage. As a secondary cause, it is meant to encompass all pathological conditions that worsen or accelerate the progression of skin diseases caused by other causes. It is reported that about 40% of all skin diseases are directly related to stress, and since continuous stress also decreases the therapeutic effect, suppression of the stress response in stress-related skin diseases is the key to the underlying treatment of chronic diseases. . Skin diseases that cause stress include psoriasis, acne, scaly glands, rash, stress dermatitis, atopic dermatitis, lichen simplex chronicus, alopecia areata, purigo, stress skin aging, stress. Acne and acute vesiculobullous hand eczema.

More specifically, stress skin diseases that can be prevented or treated with the composition of the present invention include stress dermatitis, atopic dermatitis, lichen simplex chronicus, alopecia areata, prurigo, stress skin aging, Stress acne and acute vesiculobullous hand eczema.

In addition, it is reported that those who are easily stressed have a three times higher incidence of cardiovascular disease than those who do not, and those who do not have specific cardiovascular lesions and have normal blood lipid levels or inflammatory indicators are also exposed to psychological stress for a long period of time. It can cause infarction, which is called stress cardiomyopathy. In addition, when cortisol, the stress hormone, decreases the number of lymphocytes produced in the thymus and lymph glands, weakens the immune function, it is easily infected with various pathogens such as viruses and bacteria.

Accordingly, the composition of the present invention, which significantly reduces cortisol in various tissues including plasma and skin, can be used as an effective therapeutic composition for cardiovascular diseases and infectious diseases caused by stress.

In the present specification, the term "alkyl" refers to a linear or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, and the like. C ₁ -C ₃ alkyl means an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, the number of carbon atoms of the substituent is not included.

According to a specific embodiment of the present invention, R ₁ in Formula 1 is C ₁ alkyl, that is, methyl. According to the present invention, the _{compound of formula 1 wherein R 1} is methyl is methyl benzoate. Methyl benzoic acid is a fragrance component contained in plants such as Salvinia molesta , pimento berry, clove, and Monstera delicios a. There is no known pharmacological effect on stress disorders such as anxiety disorder and stress dermatitis. In the present invention, there is little risk of side effects even when long-term administration as a pharmaceutical composition or long-term feeding as a food composition by using a natural product-derived compound or a derivative thereof.

As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids are hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluroacetic acid, citric acid, methane And sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium and the like.

In the present specification, the term "prevention" has not been diagnosed as having a disease or disease, but refers to suppressing the occurrence of a disease or disease in a subject who is likely to have such a disease or disease.

As used herein, the term “treatment” refers to (a) inhibition of the development of a disease, disease or condition; (b) alleviation of the disease, disease or condition; Or (c) to eliminate the disease, disease or condition. Administration of the composition of the present invention to a subject significantly improves behavioral indicators in a stressful environment, significantly reduces plasma corticosterone concentration, and reduces the expression of stress-related genes, thereby inhibiting or eliminating the development of symptoms of stress-related diseases. Or it serves as a relief. Accordingly, the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological ingredients and applied as a therapeutic adjuvant for the disease. Accordingly, the term “treatment” or “therapeutic agent” in the present specification includes the meaning of “treatment aid” or “treatment aid”.

In the present specification, the term “administration” or “administer” refers to a therapeutically effective amount of the composition of the present invention being directly administered to a subject so that the same amount is formed in the subject's body.

In the present invention, the term "therapeutically effective amount" refers to the amount of the composition contained in a sufficient degree to provide a therapeutic or prophylactic effect to the individual who intends to administer the pharmaceutical composition of the present invention. It is meant to include “prophylactically effective amount”.

As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of the present invention is a human.

According to a specific embodiment of the present invention, the composition of the present invention reduces the concentration of cortisol in the blood.

Cortisol is a glucocorticoid hormone secreted from the adrenal cortex and is involved in the regulation of the individual's response to stressors and homeostasis, in the form of cortisol in humans and primates, and in the form of corticosterone in rodents. Secreted into Cortisol (or corticosterone) increases in concentration when exposed to stress, and its decrease means that the nerve damage inflicted on the subject due to the stressful environment has been alleviated or eliminated.

According to the present invention, the composition of the present invention can be predicted to significantly reduce the concentration of plasma corticosterone in mice, which are experimental animals, thereby significantly reducing cortisol, which is the same glucocorticoid secretion form in humans. It can be seen that it induces a stress response.

In the present specification, the term “reduction in blood corticosterone concentration” means that the concentration of cortisol in blood is significantly increased to a measurable level compared to the control group not administered the composition of the present invention, and specifically, it is reduced by 10% or more. It means, and more specifically, it means that it is reduced by 13% or more.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and specifically, may be administered orally, intravenously, subcutaneously or intraperitoneally.

The appropriate dosage of the pharmaceutical composition of the present invention is prescribed in various ways depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

According to another aspect of the present invention, the present invention provides a food composition for improving or relieving stress, comprising the compound of Formula 1 or a food pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In the above formula, R is C ₁ -C ₃ alkyl.

Since the compound of Formula 1 used in the present invention and the stress state or stress-related disease that can be improved or alleviated by using the same have been described above, description thereof will be omitted to avoid excessive redundancy.

In the present specification, the term “food wise acceptable salt” refers to a salt in a form that can be used in food compositions among salts in which cations and anions are bound by electrostatic attraction, and specific examples thereof are “pharmaceutical And the example of “acceptable salt”.

When the composition of the present invention is prepared as a food composition, it may include not only the compound of the present invention as an active ingredient, but also carbohydrates, flavoring agents, and flavoring agents that are commonly added during food production. Examples of carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol are not limited thereto. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glysirhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, cephalic extract, jujube extract, licorice extract, etc. are added in addition to the pine bark extract, which is the active ingredient of the present invention. Can be included as.

According to another aspect of the present invention, the present invention provides a cosmetic composition for improving or relieving stress comprising the compound of Formula 1 or a cosmetically acceptable salt thereof as an active ingredient:

Formula 1

In the above formula, R is C ₁ -C ₃ alkyl.

Since the compound of Formula 1 used in the present invention and the stress state or stress-related disease that can be improved or alleviated by using the same have been described above, description thereof will be omitted to avoid excessive redundancy.

According to a specific embodiment of the present invention, the stress state that can be improved or alleviated by the composition of the present invention is a stress neurological disease, a stress-related skin injury, or a stress-related infectious disease. In the present specification, the term "improvement or alleviation of stress-related skin damage" is used in the same meaning as "improvement of stress-related skin conditions". Therefore, the composition of the present invention can be expressed as a "composition for improving skin condition".

In the present specification, the term “cosmetically acceptable salt” refers to a salt in a form that can be used cosmetically among salts, which are substances in which cations and anions are bound by electrostatic attraction, and specific examples of the kind are Examples of the above-described "pharmaceutically acceptable salt" are included.

Ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the compound of Formula 1 or a hydrolysis product thereof as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. It includes conventional adjuvants, such as, and a carrier.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, and may be formulated as a spray, but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

According to another aspect of the present invention, the present invention provides a method for preventing or treating stress-related diseases comprising administering to a subject a composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof:

Formula 1

Since the chemical formula 1 compound used in the present invention and stress diseases that can be prevented or treated using the same have been described above, descriptions thereof will be omitted in order to avoid excessive redundancy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition, a functional food composition, and a cosmetic composition for preventing or treating stress-related diseases comprising methyl benzoate or a derivative thereof as an active ingredient.

(b) The composition of the present invention significantly improves behavioral indicators of individuals exposed to a stress environment, significantly reduces blood cortisol concentration, and significantly suppresses cortisol concentration and stress-related gene expression in skin fibroblasts, thereby reducing stress. It can be applied as an effective therapeutic agent for various stress-related diseases accompanied by increased cortisol secretion, including neurological diseases and skin diseases as the cause.

(c) The present invention is also useful as an effective therapeutic composition for various stress-related diseases, most of which are chronic diseases, because the present invention uses natural-derived compounds that have already been eaten as food additives as an active ingredient and has few side effects even when administered for a long period of time. Can be.

FIG. 1 is a diagram showing the change in behavioral indicators of mice to which methyl benzoate was administered, and shows the results of a tail hanging experiment (FIG. 1a ), a forced swimming experiment (FIG. 1b ), and an open field experiment (FIG. 1c ), respectively. Each value was expressed as mean±standard error (n=4), and if there was a significant difference between groups, it was indicated with an asterisk (*P <0.05).

FIG. 2 is a diagram showing changes in plasma corticosterone indices of mice administered with methyl benzoate. Each value was expressed as mean±standard error (n=4), and if there was a significant difference between groups, it was indicated with an asterisk (*P <0.05).

3 is a diagram showing the change in the amount of cortisol secretion in human skin fibroblasts treated with methyl benzoate. Each value was expressed as mean±standard error (n=4), and if there was a significant difference between groups, it was indicated with an asterisk (*P <0.05).

FIG. 4 is a diagram showing changes in the expression of stress indicator genes in human skin fibroblasts treated with methyl benzoate, and shows the expression patterns of GILZ (FIG. 4A), THBD (FIG. 4B) and SDPR (FIG. 4C), respectively. Each value was expressed as mean±standard error (n=4), and if there was a significant difference between groups, it was indicated with an asterisk (*P <0.05).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method

Experimental animals and experimental diet

8-week-old male C57BL/6 mice used as experimental animals were supplied from Orient Bio Co., Ltd. (143-1, Daesangwon-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do, Korea). Experimental animals were divided into a control group (CON, n=4) and a methyl benzoate administration group (Methyl benzoate, n=4) after a 1-week adaptation period in the breeding room. Olive oil as a control group and methyl benzoate (50 mg/kg body weight) were dissolved in olive oil for the methyl benzoate-administered group and administered intraperitoneally 30 minutes before the stress behavioral indicator experiment. Used olive oil and methyl benzoate were purchased from Sigma-Aldrich (Missouri, USA). Experimental animals were reared in a space maintained at a constant temperature of 23±1℃, humidity of 50±10%, and a 12-hour light and dark cycle, and freely ingested Chow (Orient Bio) and drinking water.

Measurement of anti-stress, anti-depressant, and anti-anxiety effects by administration of methyl benzoate

1. Tail suspension test (TST)

The tail hanging experiment was performed using the method of Steru et al. (1985). After attaching a fixing device to the end of the tail of the experimental mouse about 1cm, hang it 50cm from the ground, and measure the immobility time of the experimental animal using a video tracking system (smart v.2.5.21). I did. A case in which the mouse was suspended completely without any movement was regarded as an immobile state, and the condition for 4 minutes was measured after a 2 minute adaptation time. In general, when a mouse is under stress, anxiety increases and depression increases, resulting in less activity. Therefore, it was determined that the shorter the immobility time of the mouse, the more anti-stress, antidepressant, and anti-anxiety effects were found.

2. Forced swimming test (FST)

For the forced swimming experiment, the method of Porsolt et al. (1997) was used. After filling a water tank with a height of 40cm and a diameter of 20cm with 30cm of water at a temperature of 25±1℃, place the mice one by one into the water tank and use the video tracking system (smart v.2.5.21) to move the experimental animals. Immobility time was measured. A case in which the mouse stands upright with only its face on the surface of the water and floats without moving was regarded as an immobile state, and it was measured through a state for 4 minutes after passing through an acclimatization time of 2 minutes. In general, when a mouse is under stress, anxiety increases and depression increases, resulting in less activity. Therefore, it was determined that the shorter the immobility time of the mouse, the more anti-stress, antidepressant, and anti-anxiety effects were found.

3. Open field test (OFT)

In the open field experiment, the method of Noldus et al. (2001) was used. The mice to which stress was added through the forced swimming experiment were placed in a white acrylic box of 50×50×50cm. After that, the 30×30cm area in the center of the field is set as the central zone, and then the time that the experimental animal stayed in the center is 2 minutes using a video tracking system (smart v.2.5.21). It was measured for 8 minutes after passing through the adaptation time. In general, when a mouse is under stress, anxiety increases, so the activity time in the center of the open field decreases and the time to stay at the edge increases. Therefore, the longer the time the mouse stays in the center, the more anti-stress, anti-depressant and anti-anxiety effects are found. saw.

Measurement of plasma corticosterone concentration

After the stress behavior indicator experiment was over, blood was collected under anesthesia with avertin. Blood collected from the abdominal aorta was centrifuged at 2000×g for 15 minutes to separate plasma. Plasma corticosterone concentration was measured using a commonly used EIA kit (Corticosterone EIA kit, Enzo Life Sciences, NY, USA). The basic experimental method was performed according to the manufacturer's instructions. Increased corticosterone secretion is a typical symptom of stress, so the lower the plasma corticosterone concentration was, the more anti-stress, anti-depressant, and anti-anxiety effects were considered.

Measurement of skin stress relief efficacy

1. Cell culture and treatment materials

Human skin fibroblasts HS68 were purchased and used from ATCC (Manassas, VA, USA). The purchased cells were cultured in an incubator at _{37° C. and 5% CO 2} using DMEM (Dulbecco's Modified Eagle's Media) containing 10% fetal bovine serum and used in the experiment.

2. Cortisol concentration measurement

In order to measure the cortisol concentration in human skin fibroblasts, cells were dispensed into 12 well-plates at 0.2×10 ⁶ cells/well, and then adrenocorticotropic hormone releasing factor (CRF) (Sigma-Aldrich, Missouri, USA) and Methyl benzoate was added at concentrations of 100 nM and 5 μM, respectively, and incubated in a _{CO 2 incubator for 24 hours.} After incubation for 24 hours, the concentration of cortisol secreted into the medium was measured using a cortisol ELISA kit (ENZO, USA).

3. PCR analysis

To confirm whether methyl benzoate relieves the expression pattern of the gene expressed by the stress hormone, human fibroblasts ^{were dispensed into a 90 mm dish at 2.5×10 6} cells/well, followed by CRF and methyl benzoate at 100 nM and 5 μM, respectively. It was added at a concentration of and incubated in a CO ₂ incubator for 24 hours. After culturing for 24 hours, human fibroblasts ^{were harvested by adding 500 µl of a Trizol solution per 1 × 10 7} cells, and then centrifuged at 4° C. and 12,000 × g for 10 minutes. After transferring the supernatant to a new tube, 100 µl of chloroform was added, followed by vortexing. Again, the supernatant was transferred to a new tube, and isopropanol was added so that the ratio of the supernatant to isopropanol was 1:1. After shaking vigorously 10 times, let stand at room temperature for 15 minutes, centrifuge at 12,000 × g, 4°C for 10 minutes, remove the supernatant, add 1 mL of 70% ethanol to the remaining precipitate, and add 7,500 × g, Centrifuged at 4° C. for 5 minutes. After removing the ethanol, the tube containing the RNA precipitate was dried at room temperature for 15 minutes, and the RNA pellet was dissolved using nuclease free water. Integrity of RNA samples by measuring the concentration of RNA samples extracted at 260 nm and 280 nm wavelengths using a UV/VIS spectrophotometer (Beckman coulter, DU730), and performing agarose gel electrophoresis. (integrity) was confirmed. CDNA is synthesized by performing reverse transcription using oligo dT primer and superscript reverse transcriptase (GIBCO BRL, Gaithersburg, MD, USA) targeting RNA samples extracted from human fibroblasts. I did. The synthesized cDNA is used as a template, and the 5'and 3'flanking sequences of the cDNA to be amplified are used as primers, iQ SYBR green supermix (Bio-Rad) and CFX Connect™ Real-Time PCR Quantitative PCR was performed using the Detection System (Bio-Rad). The primer sequences used at this time are shown in Table 1 below.

gene primer Sequence (5'→3') Annealing temperature (℃) Glucocorticoid-induced leucine zipper (GILZ) Forward direction GCTGTGAGAGAGGAGGTGGA 55 Reverse CTTCAGGGCTCAGACAGGAC Thrombomodulin (THBD) Forward direction GACCTTCCTCAATGCCAGT 55 Reverse CCGTTCAGTAGCAAGGAAATG Serum deprivation-response protein (SDPR) Forward direction AGTCACGGTGCTCACGCTCC 55 Reverse GTTGCTGGTGGAGGCCTGGT Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Forward direction ggagattgttgccatcaacg 55 Reverse tgacaagcttcccattctcg

Statistical processing

Statistical analysis of all data was performed using the SPSS (statistical package for the social sciences, version 21.0, IBM, Armonk, NY, USA) PC package, and the analysis values were expressed as mean ± standard error. Significant differences between groups were verified by performing an independent sample T- test (*P <0.05).

Experiment result

Antistress, antidepressant, and anti-anxiety effects by administration of methyl benzoate

1. Tail Suspension Test (TST)

Compared to the control group (CON), it was observed that the immobility time significantly decreased (-22%) in the methyl benzoate group, and through this, the administration of methyl benzoate showed anti-stress, anti-depressant and anti-anxiety effects. It was confirmed (Fig. 1a).

2. Forced Swimming Test (FST)

It was observed that the immobility time significantly decreased (-17%) in the methyl benzoate-treated group compared to the control group, and through this, it was confirmed that the administration of methyl benzoate exhibited anti-stress, anti-depressant and anti-anxiety effects (Fig. 1b).

3. Open field test (OFT)

Compared to the control group, it was observed that the active time in the center significantly increased (+19%) in the methyl benzoate-treated group, and through this, it was confirmed that the administration of methyl benzoate exhibited anti-stress, anti-depressant and anti-anxiety effects ( Fig. 1c).

Plasma Corticosterone Concentration

It was observed that the plasma corticosterone concentration was significantly decreased (-14%) in the methyl benzoate-treated group compared to the control group, and through this, it was confirmed that the administration of methyl benzoate exhibited anti-stress, anti-depressant and anti-anxiety effects (FIG. 2). ).

Changes in Cortisol Concentration in Human Skin Fibroblasts

In a stressful situation, skin cells also secrete corticotropin releasing factor (CRF) and cause a stress response, so treating CRF directly into skin cells is a widely used method for constructing a stress model (slominski). et at, 2013). It is known that the CRF receptor is expressed in human skin fibroblasts and can accept the stress signal.When the CRF receptor receives the signal, it generates cortisol, the stress hormone through sub-signal, which adversely affects the skin such as skin elasticity and skin barrier reduction. (Cohen et al., 1977; Kao et al., 2003; Slominski et al., 2007).

As a result of investigating the direct effect of the stress regulating effect of methyl benzoate on the skin, the amount of cortisol secretion significantly increased in the control cells (+CRF) treated with CRF compared to the normal cells (-CRF), and the group treated with benzoic acid along with CRF In (Methyl benzoate), cortisol concentration was significantly decreased (-46%) compared to cells treated with CRF only (+CRF), and through this, methyl benzoate treatment showed anti-stress effect in human skin fibroblasts. Was confirmed (Fig. 3).

Changes in the expression of stress-related genes in human skin fibroblasts

It is known that the increase in the amount of cortisol secretion in the skin binds to the cortisol receptor in the cytoplasm, enters the nucleus and acts as a transcription factor, making various stress-related genes. Among these cortisol target genes, GILZ (Glucocorticoid-induced leucine zipper), THBD (Thrombomodulin), and SDPR (Serum deprivation-response protein) are known representatively, so changes in the expression levels of these target genes are used as reliable stress indicators (Wang et al., 2004, Oakley et al., 2014).

In the control cells treated with CRF only (+CRF), the expression of stress-related genes was significantly increased compared to that of normal cells (-CRF), and in the group treated with CRF and benzoic acid, the stress-related genes were significantly decreased compared to +CRF cells (GILZ:-38%; THBD:-22%; SDPR:-26%), and through this, it was confirmed that the treatment of methyl benzoate has a remarkable anti-stress effect in human skin fibroblasts ( Figures 4a-4c).

As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (12)

A composition for preventing or treating stress-related diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: Formula 1

In the above formula, R is C ₁ -C ₃ alkyl. The composition of claim 1, wherein R ₁ in Formula 1 is C ₁ alkyl. The composition of claim 1, wherein the stressful disease is selected from the group consisting of a stress neurological disease, a stress skin disease, a stress cardiovascular disease, and a stress infectious disease. The composition of claim 3, wherein the stress neurological disorder is selected from the group consisting of depressive disorder, sleep disturbance, anxiety disorder, and panic disorder. The method of claim 3, wherein the stress skin disease is stress dermatitis, atopic dermatitis, lichen simplex chronicus, alopecia areata, prurigo, stress skin aging, and acute vesiculobullous hand eczema. eczema) composition, characterized in that selected from the group consisting of. The composition of claim 1, wherein the composition decreases the concentration of cortisol in the blood. A food composition for improving or alleviating stress comprising the compound of Formula 1 or a food pharmaceutically acceptable salt thereof as an active ingredient: Formula 1

In the above formula, R is C ₁ -C ₃ alkyl. The composition of claim 7, wherein R ₁ in Formula 1 is C ₁ alkyl. The composition according to claim 7, wherein the composition has an improvement or alleviation effect on stress neurological disease, stress skin disease, stress cardiovascular disease, or stress infectious disease. A cosmetic composition for improving or relieving stress comprising the compound of Formula 1 or a cosmetically acceptable salt thereof as an active ingredient: Formula 1

In the above formula, R is C ₁ -C ₃ alkyl. 11. The composition of claim 10, wherein R ₁ in Formula 1 is C ₁ alkyl. 11. The composition of claim 10, wherein the composition has an improvement or alleviation effect on stress neurological disease, stress skin damage, or stress infectious disease.

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