KR20210135834A - Composition for preventing or treating post traumatic stress disorder comprising hesperidin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a food composition, and a feed composition for the prevention or treatment of post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active component, and a method for preventing or treating post-traumatic stress disorder comprising a step of administering the pharmaceutical composition. Since hesperidin of the present invention exhibits an excellent PTSD treatment effect, hesperidin can be included as the active component in the pharmaceutical composition or food and used to develop a therapeutic agent or an alleviating agent for PTSD.

Description

A composition for preventing or treating post traumatic stress disorder comprising hesperidin comprising hesperidin as an active ingredient

The present invention relates to a composition for preventing or treating post-traumatic stress disorder comprising hesperidin (HSD). Specifically, the present invention relates to post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to a method for preventing or treating post-traumatic stress disorder comprising administering a pharmaceutical composition, a food composition, a feed composition, and the pharmaceutical composition for the prevention or treatment of

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder that exhibits symptoms such as arousal, escapism, and explicit memory. Characteristics of this disorder include re-experience symptoms of shock, symptoms of avoidance, and recall of memories of fear. Reexperience symptoms are caused by the memory of an aversive traumatic event that appears during the day or night, or suddenly, and 10% of the group with PTSD shows symptoms such as memory dysfunction and cognitive impairment.

Accordingly, there has been a continuous demand for a treatment method for post-traumatic stress disorder, and related research has been actively conducted. For example, International Patent Publication WO 2015-134133 discloses the treatment of patients suffering from post-traumatic stress disorder (PTSD) and/or hot flashes by administering a mixture prepared in combination with a long-acting local anesthetic in combination with clonidine. A kit and a method are disclosed for this, and U.S. Patent Publication No. 2018-0340947 discloses a method of treating a post-traumatic stress disorder patient by administering a bufodienolide antagonist to the post-traumatic stress disorder patient. However, these treatment methods have a side effect of causing damage to the nervous tissue, and since they are not actually utilized, the development of a treatment method with few side effects is urgently required.

On the other hand, hesperidin (HSD) is a kind of polyphenol that is abundantly present in citrus fruits, and is also known as vitamin P along with rutin and quercetin as a vitamin-like substance. It helps collagen production, has anti-inflammatory action, is effective in diseases related to aging, and is known to affect joints and cartilage (Japanese Patent No. 4838772, Japanese Patent Application Laid-Open No. 2005-255527), and if the human body lacks vitamin It is known that decreased absorption of C, arteriosclerosis, and decreased immunity occur. However, the useful efficacy of hesperidin for post-traumatic stress disorder is not known.

Under this background, the present inventors made intensive research efforts to develop a method for treating post-traumatic stress disorder without showing side effects. As a result, it was confirmed that hesperidin derived from a natural product harmless to the human body exhibits a therapeutic effect on post-traumatic stress disorder. , the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating post-traumatic stress disorder comprising administering the pharmaceutical composition.

One embodiment of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term “hesperidin” refers to a compound having the structure of Formula 1 below. Hesperidin helps collagen production, has anti-inflammatory action, is effective against aging-related diseases, and is known to affect joints and cartilage. On the other hand, the prevention, treatment or improvement effect of the post-traumatic stress disorder was not known, and was first identified by the present inventors.

The composition containing hesperidin may include a range of derivatives of hesperidin expected by those skilled in the art, and is included without limitation as long as the same effect is obtained in the present invention. Hesperidin can be obtained from an extract comprising it. Moreover, it can be synthesize|combined and used by a well-known method.

As used herein, the term “post-traumatic stress disorder (PTSD)” refers to a disease in which abnormal psychosomatic symptoms continuously appear after experiencing a traumatic event. If the criteria presented in the Diagnostic Statistical Manual of Mental Disorders (DSM-IV-TR) of the American Psychiatric Association are satisfied, post-traumatic stress disorder is diagnosed. The onset of the disease varies from person to person, and may begin immediately after the shock or may appear days, weeks, months, or years later.

The main symptoms include dissociative phenomena, panic attacks, paresthesias such as auditory hallucinations, aggressive tendencies, impulse control disorders, depression, drug abuse, and cognitive problems such as poor concentration and memory.

In a specific embodiment of the present invention, as a result of administering hesperidin to PTSD-induced rats by giving a single continuous stress, SPS stimulation resulted in a change in the rat's body weight (Fig. 2) and a change in circus intake (Fig. 3). ), increased immobility time in the forced swimming experiment (FST) (Fig. 4A), a decrease in the number of traversing the center (Fig. 4D) and an increase in the contextual stiffness time (Fig. 4G) also in the open space test (OFT). etc., but it was confirmed that HSD administration had the effect of alleviating it. In addition, it was confirmed through ELISA experiments that corticosterone, serotonin, norepinephrine and dopamine levels in plasma and brain were changed due to SPS stimulation, and it was confirmed that HSD administration inhibited the changes caused by SPS (Figs. 5 to 6). ), in addition, changes in tryptophan, homovanillic acid, and 5-HIAA levels and their alleviation by HSD administration were confirmed ( FIG. 7 ). In addition, through RT-PCR, it was confirmed that the level of TPH1 was changed by SPS, and HSD administration alleviated it (FIG. 8), indicating that HSD has the effect of suppressing and alleviating hormonal changes similar to PTSD caused by SPS. Confirmed.

This suggests that hesperidin can be usefully used for the prevention, treatment and improvement of PTSD because it exhibits the effect of restoring behavioral and neurochemical changes related to memory impairment by inhibiting changes in stress-related substances in the PTSD rat model. will be.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts, which are substances in which cations and anions are bonded by electrostatic attraction. salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with, etc.; salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid, or the like.

As used herein, the term “prevention” refers to any action that inhibits or delays the onset of PTSD by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which the symptoms of a subject suspected and affected of PTSD are improved or beneficially changed by administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, which may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid, mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The content of hesperidin included in the pharmaceutical composition of the present invention is not particularly limited thereto, but is included in an amount of 0.0001 to 80 wt%, 0.0001 to 50 wt%, and more specifically 0.01 to 20 wt%, based on the total weight of the final composition. can

Another embodiment of the present invention provides a method for preventing or treating post-traumatic stress disorder, comprising administering the pharmaceutical composition to an individual, other than a human, who has or is likely to develop post-traumatic stress disorder.

In this case, the description of "post-traumatic stress disorder", "prevention" and "treatment" is the same as described above.

Since the pharmaceutical composition of the present invention exhibits a preventive or therapeutic effect on post-traumatic stress disorder, the method of the present invention comprising administering it to an individual can be usefully utilized for the prevention or treatment of post-traumatic stress disorder.

As used herein, "administration" means introducing the composition to a subject by an appropriate method.

As used herein, the term "individual" refers to all animals, such as mice, mice, and livestock, including humans, that have or can develop post-traumatic stress disorder, and specifically, may be mammals including humans, but is not limited thereto does not

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term, "pharmaceutically effective amount" is sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. means the amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the rate of excretion Treatment period , factors including drugs used in combination with or concurrently with the composition of the present invention and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known therapeutic agent for pterygium. Taking all of the above factors into consideration, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects.

The pharmaceutical composition of the present invention may be administered using various methods such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, and transdermal, and the dosage may depend on the age, sex, and weight of the patient. may vary and can be readily determined by one of ordinary skill in the art.

In addition, the dosage of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at an amount of 1 mg/kg to 200 mg/kg, specifically 1 mg/kg to 100 mg/kg, and more specifically 20 to 40 mg/kg per adult. , The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage does not limit the scope of the present invention in any way.

In addition, the dosage of the pharmaceutical composition may be adjusted so that hesperidin is administered at 10 to 120 mg/kg, more specifically, 100 mg/kg, but is not limited thereto.

Another embodiment of the present invention provides a food composition for preventing or improving post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the definitions of the terms “hesperidin”, “post-traumatic stress disorder”, “prevention” and “pharmaceutically acceptable salt” are as described above.

As used herein, the term “improvement” refers to any action that at least reduces a parameter, eg, the severity of a symptom, associated with a condition to be treated by administration of a composition comprising hesperidin.

Since hesperidin according to the present invention exhibits an excellent PTSD treatment effect, it can be included in a food composition for the purpose of preventing or improving PTSD. can be expected

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, etc. are included, and as long as they can contain the hesperidin of the present invention, it is not limited thereto.

As used herein, the term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. and regulating nutrients for function or obtaining useful effects for health applications such as physiological action. On the other hand, health food refers to food that has an active health maintenance or promotion effect compared to general food, and health supplement refers to food for the purpose of health supplementation. can be mixed.

The hesperidin of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, fungicides, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, emulsifiers, thickeners, filming agents, gum base agents, foam inhibitors, solvents, and improving agents. may include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and is excellent in portability, so the present invention Foods of PTSD can be ingested as a supplement to enhance the effect of prevention or improvement of PTSD.

Hesperidin of the present invention may be included in various weight % in the food composition as long as it can exhibit an effect of preventing or improving PTSD. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. When ingested for a long period of time for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention provides a feed composition for preventing or improving post-traumatic stress disorder comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the definitions of the terms “hesperidin”, “post-traumatic stress disorder”, “prevention”, “improvement” and “pharmaceutically acceptable salt” are as described above.

Since hesperidin according to the present invention exhibits an excellent PTSD treatment effect, it can be included in a feed composition for the purpose of preventing or improving PTSD, and the feed composition can be consumed by animals on a daily basis. A high effect can be expected.

As used herein, the term "feed" means any natural or artificial diet, meal, etc., or a component of said meal, intended for or suitable for eating, ingestion, and digestion by an animal.

The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton or food. These may be used alone or in mixture of two or more.

Since hesperidin of the present invention exhibits excellent PTSD treatment effect, it can be included as an active ingredient in a pharmaceutical composition or food and used to develop a therapeutic agent or an ameliorating agent for PTSD.

1 is a schematic diagram of an experiment according to a time sequence for producing a post-traumatic stress disorder (PTSD) model, and the induction process of anxiety-like symptoms through a single prolonged stress (SPS) indicates
2 is a graph showing the change in body weight according to time of rats for each group.
Figure 3 is a graph showing the change in circus intake of rats by group.
Figure 4A is a graph showing the immobility time during the FST experiment of rats by group.
Figure 4B is a graph showing the climbing (climbing) time during the FST experiment of rats by group.
Figure 4C is a graph showing the swimming time during the FST experiment of rats by group.
Figure 4D is a graph showing the number of crossing the center during the OFT experiment of rats by group.
Figure 4E is a graph showing the number of crossing the outskirts during the OFT experiment of rats by group.
Figure 4F is a graph showing the number of grooming (grooming) during the OFT experiment of rats by group.
4G is a graph showing changes in the freezing pattern during the OFT experiment of rats by group.
5 is a graph showing changes in the plasma CORT concentration of rats for each group.
6A is a graph showing 5-HT levels in the hippocampus of rats by group.
6B is a graph showing the level of 5-HT in the medial frontal lobe of rats by group.
6C is a graph showing 5-HT levels in the amygdala of rats by group.
6D is a graph showing the level of 5-HT in the striatum of rats by group.
6E is a graph showing the level of NE in the hippocampus of rats by group.
6F is a graph showing the DA level in the hippocampus of rats by group.
7A is a graph showing the tryptophan level of rats by group.
7B is a graph showing the HVA level of rats by group.
7C is a graph showing 5-HTAA levels of rats by group.
Figure 7D is a graph showing the 5-HTAA / 5-HT ratio of rats by group
8 is a graph comparing the relative TPH-1 mRNA levels of rats by group.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1. Use of laboratory animals and effects of HSD

In an animal experiment for post-traumatic stress disorder (PTSD) treatment, 200-220 g SD (Sprague-Dawley) rats (6 weeks old, Samtako AnimalCo., Seoul, Korea) were used. Cages were maintained at a light/dark cycle of 12 h (lights on at 9:00am, lights off at 9:00pm), a temperature of 21±2°C, and a relative humidity of 55±10%. All of the above methods and procedures were approved by the Animal Care Committee of Kyunghee University [KHUASP(SE)-15-115], and all procedures were performed according to the guidelines for the management of laboratory animals.

After exposure to SPS, HSD (20, 50, and 100 mg/kg, body weight) dissolved in 0.9% saline and fluoxetine hydrochloride were administered intraperitoneally once a day for 14 days. The same dose of saline was intraperitoneally administered to the group and the SPS-induced rat group to serve as a control group.

In addition, rats were randomly assigned to 7 or 8 rats per group, saline-treated control group, saline group after SPS-induction (negative control group), 20 mg/kg HSD administration group after SPS-induction, 50 mg after SPS-induction. /kg HSD administration group, 100 mg/kg HSD administration group after SPS-induction, and 10 mg/kg FLX administration group after SPS-induction were divided into a total of 6 groups.

Changes in body weight of the rats were observed for 14 days, and the drug administration period and behavioral experiments are shown in FIG. 1 .

Example 2. Single prolonged stress

The rats prepared in Example 1 were put in a holder for 2 hours, and then immediately after being allowed to swim for 20 minutes (forced swimming; FS). The rats were allowed to recover for 15 min and then subjected to a single prolonged stress (SPS) stimulus exposing them to isoprulan until they became unconscious. At this time, the control group was separated into another space for a while while SPS stimulation was applied. After SPS stimulation, the rats were placed in a cage so that they were unable to engage in social interactions, so that PTSD-like symptoms were not disturbed for 7 days.

Example 3. Weight change

In order to confirm the weight change of rats by SPS, the weight of rats was measured.

Specifically, 14 days after induction of SPS, a one-way ANOVA repeated experiment was performed to measure the change in body weight of rats.

As a result, as can be seen in FIG. 2 , after exposure to SPS, the rats did not change their body weight on the 1st day, but gradually increased their weight from the 2nd day, but a continuous weight loss was confirmed compared to the SAL group. .

On the other hand, although not at a statistically significant level, it was found that weight loss was alleviated when 20, 50 and 100 mg/kg of HSD were administered.

Through this, it was found that the SPS stage causes extreme stress such as PTSD symptoms, and it was confirmed that the HSD administration partially suppressed the weight loss.

Example 4. Changes in circus intake

Anesthesia is one of the symptoms of depression and can be quantified through a circus intake experiment. In order to confirm the depressive symptoms caused by SPS-induction and to confirm the improvement effect thereof by HSD, the intake change of 1% circus solution after SPS-induction was confirmed.

More specifically, in order to acclimatize the rats before measuring the change in the intake of the circus solution, the circus solution was administered 24 hours before the experiment, water and food were removed 6 hours before the experiment, and the change in the circus intake for 3 hours on the day of the experiment To confirm the change in the weight of the water bottle was measured. The measurement was performed three times in total, 1 day before stress induction, 7 days after stress induction, and 14 days after stress induction.

As a result, as can be seen in FIG. 3 , compared to the SAL group, it was confirmed that the SPS-induced rat group significantly reduced circus intake. However, it was confirmed that the circus intake of rats administered with HSD 100 mg/kg was improved compared to the group not administered.

Through this, it was confirmed that SPS-induction induces depressive symptoms such as PTSD, and it was confirmed that HSD administration has the effect of alleviating/improving it in a concentration-dependent manner.

Example 5. Exercise ability and behavior change experiment

The following experiments were performed and compared to confirm the change in motor ability and behavior of rats by SPS-induction.

Example 5-1. Forced swimming test (FST)

One of the symptoms of depression is not responding to stress. When stress such as FST was applied, the degree of immobility was measured, and the relief effect by HSD was measured.

More specifically, a transparent plexiglass cage (20 cm diameter x 50 cm depth) was filled with water at 25 ° C. 30 cm, and rats were placed. The total experiment time was 6 minutes, and the initial 2 minutes were not measured as adaptation time. In addition, the immobility state that does not move was defined as the state of sticking the head out of the water and making minimal movements as the immobility state.

As a result, as can be seen in Figure 4, it was confirmed that the administration of HSD significantly reduced the immobility time in a concentration-dependent manner, and it was confirmed that FLX, a positive control, also significantly reduced the immobility time of rats during FST (Fig. 4A). ). However, there was no change in swimming behavior due to SPS in any group (Fig. 4C).

In addition, although not statistically significant, it was confirmed that the time of climbing during FST significantly increased ( FIG. 4B ).

Through this, it was confirmed that HSD has the effect of relieving the immobility time caused by stress such as PTSD.

Example 5-2. Behavioral change in the open field (OFT)

In order to study the effects of SPS stimulation and HSD-induced changes in motor activity and exploratory activity, an open field test (OFT) was performed.

More specifically, a rectangular container (60 × 60 cm × 30 cm) made of wood with a checkerboard floor in which the floor is drawn horizontally and vertically at intervals of 15 cm was prepared and divided into a central portion and a peripheral portion. Motor activity was measured through a video-tracking system using the S-MART program (PanLab Co., Barcelona, Spain), and motor activity was measured by the total distance traveled in the container, the speed, and the number of times and movements (central part) that crossed the line in 5 minutes. traverse) and the time spent in the central and peripheral regions were measured, and the number of grooming of the rats was also recorded to analyze motor activity in the OFT.

As a result, as can be seen in FIG. 4 , the time spent in the central region of rats exposed to SPS was reduced in comparison with the SAL group ( FIG. 5A ), and the tendency to pass through the central region was also reduced ( FIG. 4D ). On the other hand, there was no difference in the tendency to pass to the outer peripheral area between the groups (FIG. 4E). In particular, it was confirmed that the time spent in the central region was significantly increased in rats administered with HSD 100 mg/kg. This result was similar to the group administered with the positive control FLX.

In addition, as can be seen in FIG. 4F , although it was not a statistically significant value in rats administered with HSD 100 mg/kg, it was confirmed that grooming behavior increased.

Through this, it was confirmed that SPS stimulation reduced the motor activity, and it was confirmed that HSD had the effect of improving the changed motor ability similarly to the positive control FLX.

Example 5-3. Contextual rigid behavior experiment through SPS stimulation

In order to measure the spastic behavior of fear acquisition and the alleviation effect of HSD, the spastic behavior of rats after exposure to SPS was investigated.

SPS stimulation is the same as the method described in Example 2.

As a result, as can be seen in FIG. 4H , after exposure to SPS, the stiffness time was significantly increased in the SPS group compared to the SAL group. However, when HSD 100 mg/kg was administered, it was confirmed that the stiffness time was significantly reduced compared to the group not administered.

Through this, it was confirmed that the response by SPS was similar to the initial trauma symptoms, and it was confirmed that HSD administration could alleviate it.

Example 6. Changes in plasma corticosterone by SPS stimulation

By measuring the concentration of stress-related hormones, PTSD symptoms were induced through SPS stimulation, and the hormonal changes and the effects of HSD were analyzed.

More specifically, after allowing PTSD to be induced for 14 days, plasma corticosterone (CORT) concentrations were measured. Four rats of each group were sacrificed by anesthesia by spraying isoflurene one day after the behavioral experiment. Isoflurene was sprayed in a closed cage at room temperature with 4% oxygen at 6 psi atmospheric pressure for 30 seconds according to Institutional Animal Care and Use Committee (IACUC) guidelines.

Thereafter, plasma was rapidly collected through the abdominal aorta. The concentration of CORT, a stress-related hormone, was measured from the collected plasma to induce PTSD symptoms through SPS stimulation, and the hormonal changes and the efficacy of HSD were analyzed.

As a result, as can be seen in FIG. 5 , it was confirmed that the CORT level in plasma of the SPS group was significantly increased compared to the SAL group.

On the other hand, it was confirmed that 100 mg/kg of HSD administration significantly suppressed the increased CORT level due to this SPS-stimulation.

Through this, it was found that SPS stimulation causes extreme stress such as PTSD symptoms, and it was confirmed that HSD administration significantly inhibited the increase in plasma CORT levels caused by SPS.

Example 7. Changes in Serotonin, Tryptophan, Norepinephrine, and Dopamine in the Brain

By measuring the concentration of stress-related hormones, PTSD symptoms were induced through SPS stimulation, and the hormonal changes and the effects of HSD were analyzed.

Specifically, after allowing PTSD to be induced for 14 days, the concentrations of serotonin (5-HT) in brain tissue, tryptophan, norepinephrine, dopamine, and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus were measured. The experimental method was performed in the same manner as in Example 6. After rapidly collecting plasma through the abdominal aorta, the brain was collected and the hippocampus, medial frontal lobe, striatum, and amygdala were isolated. Then, in order to measure changes in stress-related hormones serotonin (5-HT), tryptophan (TRP) norepinephrine (NB), and dopamine (DA) through a competitive ELISA experiment using an antibody, 5-HT, serotonin metabolites The concentrations of phosphorus 5-hydroxyindoleacetic acid (5-HIAA), TRP, NE, DA, and homovanillyric acid (HVA), which are metabolites of dopamine, were measured, and the alleviation efficacy of HSD PTSD symptoms was confirmed.

As a result, as can be seen in FIG. 8 , it was confirmed that 5-HT expression levels were different for each group in the brain through one-way ANOVA and post-hoc tests.

1) Serotonin (5-HT)

Compared to the SAL group, it was confirmed that the 5-HT level in the hippocampus of the SPS-induced group was lowered ( FIG. 8A ). However, it was confirmed that the decreased 5-HT was recovered in the hippocampus of the group administered with 100 mg/kg of HSD.

In addition, it was confirmed that the 5-HT level in the medial frontal lobe of the SPS-induced group was lowered compared to the SAL group ( FIG. 8B ). However, it was confirmed that the decreased 5-HT was recovered in the medial frontal lobe of the group administered with 100 mg/kg of HSD.

In addition, it was confirmed that 5-HT expression level was reduced even in the amygdala of the SPS-induced group compared to the SAL group ( FIG. 8C ). However, it was confirmed that the reduced 5-HT was recovered in the amygdala of the group administered with 100 mg/kg of HSD (391.45%).

In addition, it was confirmed that 5-HT expression level was reduced even in the striatum of the SPS-induced group compared to the SAL group ( FIG. 8D ). However, it was confirmed that the reduced 5-HT was recovered in the amygdala of the group administered with 100 mg/kg of HSD.

In addition, it was confirmed that the reduced 5-HT was recovered even when 10 mg/kg of FLX was administered.

2) Norepinephrine (NE)

It was confirmed that the level of NE in the hippocampus of the SPS-induced group was significantly increased compared to the SAL group (422.92%). On the other hand, it was confirmed that the increase in NE level was suppressed in the hippocampus of the group administered with HSD ( FIG. 6E ).

3) Dopamine (DA)

Compared with the SAL group, it was confirmed that the DA level in the hippocampus of the SPS-induced group was significantly reduced (21.13%). On the other hand, it was confirmed that the decrease in DA level was suppressed in the hippocampus of the group administered with HSD ( FIG. 6F ).

4) Tryptophan (TRP)

Compared to the SAL group, it was confirmed that the expression of TRP in the hippocampus of the SPS-induced group was significantly reduced (44.95%). In addition, it was confirmed that 5-HIAA levels in the hippocampus of rats exposed to SPS for 14 days were increased compared to the SAL group (256.03%) ( FIG. 7A ).

5) Homovanillic acid (HVA)

Compared with the SAL group, it was confirmed that the HVA level in the hippocampus of the SPS-induced group was significantly reduced (27.22%). On the other hand, it was confirmed that the decrease in HVA level was suppressed in the hippocampus of the group administered with HSD ( FIG. 7B ).

6) 5-hydroxyindoleacetic acid (5-HIAA)

It was confirmed that 5-HIAA expression in the hippocampus of the SPS-induced group was increased compared to the SAL group. On the other hand, it was confirmed that the SPS-induced increase in 5-HIAA expression was suppressed in the hippocampus of the group administered with 100 mg/kg of HSD ( FIG. 7C ). In addition, it was confirmed that continuous HSD administration (100 mg/kg) decreased the 5-HIAA/5-HT ratio in the hippocampus ( FIG. 7D ).

Through this, it was found that SPS stimulation causes extreme stress, such as PTSD symptoms, and it was confirmed that HSD administration significantly inhibited SPS-induced changes in the concentration of 5-HIAA, TRP, NE, and DA in the brain.

Example 8. Changes in tryptophan hydroxylase and tyrosine hydroxylase mRNA in the hippocampus

Through the results of previous studies, it was confirmed that tryptophan (TRP) is an important marker for the response of fear or anxiety along with traumatic stress, and by measuring the expression level of tryptophan hydroxylase (TPH-1), The alleviation efficacy of fear and anxiety was analyzed.

More specifically, to measure the efficacy of HSD on the expression of TPH-1 in the hippocampus of SPS-induced rats, the mRNA expression of TPH-1 was analyzed by RT-PCR. Total RNA was extracted from the hippocampus of each rat using TRIzol reagent. To quantify this, the RNA was diluted 50-fold, and absorbance was measured through a UV/vis spectrophotometer at 260 nm. RT-PCR premix (Bioneer, Korea), sense primer, antisense primer (Bioneer), RNA, and DEPC-treated sterile water were added to make the final volume 50 μl, and then RT-PCR was performed. Thereafter, cDNA was amplified by PCR at 60° C., 28 times to produce TPH-1, and then using Taq DNA polymerase (Takara) at 58° C., 28 times in a thermal cycler (MJ Research, Watertown, MA). , USA) to produce cDNA from 2 μg of total RNA. The PCR product was electrophoresed by flowing a 50v current on a 1.2% agarose gel containing 0.5 μg/ml ethidium bromide (EtBr; Sigma) for 40 minutes, and then confirmed by UV. The data were normalized through GADPH expression of the same sample.

As a result, as shown in FIG. 8 , the expression of TPH-1 mRNA in the hippocampus of the SPS group was significantly lower than that of the SAL group, whereas the TPH-1 mRNA expression in the hippocampus of the group administered with 100 mg/kg of HSD was higher than that of the SAL group. It was confirmed that the recovery was similar to the group.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (8)

A pharmaceutical composition for preventing or treating post-traumatic stress disorder, comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the hesperidin is represented by the following formula (1):
[Formula 1]

.
The pharmaceutical composition according to claim 1, wherein the hesperidin is included in an amount of 0.01 to 98 wt%.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
A method for preventing or treating post-traumatic stress disorder, comprising administering the pharmaceutical composition of any one of claims 1 to 4 to an individual other than a human, which has or is likely to develop post-traumatic stress disorder.
The method of claim 5, wherein the administration is 10 to 120 mg/kg of hesperidin.
A food composition for preventing or improving post-traumatic stress disorder, comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.
A feed composition for preventing or improving post-traumatic stress disorder, comprising hesperidin or a pharmaceutically acceptable salt thereof as an active ingredient.

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