KR102197295B1 - Composition for preventing or treating post traumatic stress disorder comprising tetramethylpyrazine - Google Patents

Abstract

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

Description

Composition for preventing or treating post-traumatic stress disorder comprising tetramethylpyrazine {Composition for preventing or treating post traumatic stress disorder comprising tetramethylpyrazine}

The present invention relates to a composition for preventing or treating post-traumatic stress disorder comprising tetramethylpyrazine (TMP), and specifically, the present invention comprises tetramethylpyrazine 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 preventing or treating post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder that shows symptoms such as arousal, escape, and manifest memory, and the memory of fear has a great influence on post-traumatic stress. The characteristics of this disorder include re-experience symptoms of shock, avoidance symptoms, and recollection of memories of fear, and post-traumatic recollections occur by re-experience of experiencing events similar to those of trauma. Re-experience symptoms are caused by memories of abominable traumas that appear day or night or suddenly, and 10% of the group with PTSD disorders show 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 studies have been actively conducted. For example, in International Publication No. WO 2015-134133, the treatment of patients suffering from post-traumatic stress disorder (PTSD) and/or hot flushes by administering a mixture prepared from a combination of a long-acting local anesthetic in combination with clonidine. A kit and a method are disclosed, 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 a post-traumatic stress disorder patient. However, since these treatment methods have side effects that cause damage to nerve tissues, they are not actually utilized, and therefore, development of treatment methods with less side effects is urgently required.

On the other hand, tetramethylpyrazine (TMP) is one of the important active ingredients present in the traditional Asian herbal medicine Ligusticum wallichii Franch . Since it is known that tetramethylpyrazine can pass through the blood-brain barrier, studies on the treatment of neurovascular and cardiovascular diseases are actively being conducted. For example, Alzheimer's disease (Zhu F. et al., BMC Neurosci. 2006;78-87), Parkinson's disease (Kwon IH. Et al., Neurosci Lett. 2010; 486:29-33), forebrain ischemia (Qin) -Wei Z. et al., Exp Ther Med. 2016;11:978-984), tetramethylpyrazine has been reported to have neuroprotective, antioxidant, anti-cancer and anti-inflammatory effects in diseases related to the central nervous system. However, as to whether the tetramethylpyrazine has a useful effect on post-traumatic stress disorder, research results have not been published.

Under this background, the present inventors did not show side effects, and as a result of intensive research efforts to develop a method for treating post-traumatic stress disorder, it was found that tetramethylpyrazine derived from a natural product harmless to the human body exhibits a therapeutic effect on post-traumatic stress disorder. And completed the present invention.

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

The term "tetramethylpyrazie" of the present invention means a compound having the structure of the following formula (I). Tetramethylpyrazine is known to have properties that relieve pain relief, anti-aging, anti-diabetes, anti-inflammatory, anti-obesity, antioxidant, and nerve protection, but its effects on preventing, treating or improving post-traumatic stress disorder have not been known. It was first identified by the inventors.

[Formula I]

Compositions containing tetramethylpyrazine may include derivatives of tetramethylpyrazine in a range predictable by some skilled in the art, and are included without limitation as long as they have the same effect in the present invention. Tetramethylpyrazine can be obtained from an extract containing it. Moreover, it can be synthesized and used by a known method.

The term of the present invention, “Post-traumatic stress disorder (PTSD)” refers to a disease in which abnormal psychosomatic symptoms continue to appear after experiencing a traumatic event. If the criteria set forth in the American Psychiatric Association's Manual of Diagnosis of Psychiatric Disorders (DSM-IV-TR) are satisfied, it is diagnosed as post-traumatic stress disorder. The onset of the disease varies from person to person, and may begin immediately after shock, or may appear days, weeks, months or years.

The main symptoms include cognitive problems such as dissociation, panic attacks, hallucinations, etc., aggressive tendencies, impulse control disorders, depression, substance abuse, and decreased concentration and memory.

In a specific embodiment of the present invention, as a result of administering tetramethylpyrazine to a rat in which PTSD was induced by applying a single continuous stress, the number of entry into the open area and the dwell time when performing an elevated cross maze test (EPM) This increased (FIGS. 2A to 2E), and the grooming time also decreased (FIG. 2F). Even in the open space test (OPM), it was confirmed that the number and time of crossing the center of the chamber decreased due to SPS increased when tetramethylpyrazine was administered, and changes in behavior such as standing on the hind legs occurred (FIGS. 3a to 3f). In the fear test by learning through continuous electric shock, it was confirmed that the increased stiffness time due to the acquired fear was alleviated when TMP was administered (FIG. 4). In addition, it was confirmed through ELISA that the expression of 5-HT reduced in the brain due to SPS stimulation was recovered by TMP (FIG. 5), and the 5-HT-related substances TRP, 5-HIAA, and VMAT-2 Expression was measured in the hippocampus through RT-PCR, and it was confirmed that the substances decreased due to PTSD induction were increased by TMP (Fig. 6), and the stress-related hormones CORT, CRH and ACTH were increased by SPS stimulation. The decrease due to TMP administration was confirmed through ELISA (FIG. 7).

This indicates that tetramethylpyrazine has an effect of restoring behavioral and neurochemical changes related to memory impairment by inhibiting the increase of stress-related substances in the PTSD rat model, so it can be usefully used for the prevention, treatment and improvement of PTSD. It suggests.

The term "pharmaceutically acceptable salt" of the present invention refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bound by electrostatic attraction, and generally, metal salts, organic bases and 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.), aluminum salt, and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine May be salts with the like; Salts with inorganic acids may be salts of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids may be salts of 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; Salts with acidic amino acids may be salts with aspartic acid and glutamic acid.

The term "prevention" of the present invention means any action of inhibiting or delaying the onset of PTSD by administration of the pharmaceutical composition according to the present invention.

The term "treatment" of the present invention refers to any action in which symptoms of a suspected and onset subject of PTSD are improved or beneficially altered by administration of the pharmaceutical composition.

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

More specifically, the carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, 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, etc. are mentioned.

Each of the pharmaceutical compositions may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method, The form of the carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

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

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

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.

The content of tetramethylpyrazine contained in the pharmaceutical composition of the present invention is not particularly limited thereto, but the content of 0.0001 to 80% by weight, 0.0001 to 50% by weight, more specifically 0.01 to 20% by weight based on the total weight of the final composition Can be included as

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 humans, which has or is likely to develop post-traumatic stress disorder.

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

Since the pharmaceutical composition of the present invention exhibits a prophylactic or therapeutic effect of post-traumatic stress disorder, the method of the present invention including administering the same to an individual may be usefully utilized for preventing or treating post-traumatic stress disorder.

The term "administering" of the present invention means introducing the composition to a subject in an appropriate manner.

The term "individual" of the present invention refers to all animals such as rats, mice, livestock, etc., including humans that have or may develop post-traumatic stress disorder, and 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. It means the amount, and the effective dose level is the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion of the treatment period. , Factors including drugs that are used in combination or co-use with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or may be administered in combination with a known pterygium treatment agent. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects.

The pharmaceutical composition of the present invention can be administered using various methods such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, and transdermal, and the dosage is according to the patient's age, sex, and weight. It may vary and can be readily determined by a person skilled in the art.

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

In addition, the dosage of the pharmaceutical composition may be adjusted such that tetramethylpyrazine is administered at 80 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 tetramethylpyrazine or a pharmaceutically acceptable salt thereof as an active ingredient.

At this time, the definitions of the terms "tetramethylpyrazine", "post-traumatic stress disorder", "prevention" and "pharmaceutically acceptable salts" are as described above.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter, such as a symptom, associated with a condition being treated with administration of a composition comprising tetramethylpyrazine.

Since tetramethylpyrazine 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, and since the food composition can be consumed on a daily basis, the prevention or improvement of PTSD High effects can be expected.

In the present invention, 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., including all foods in the usual sense, as long as it may contain the tetramethylpyrazine of the present invention, but is not limited thereto.

The term "health functional food" of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and'functionality' refers to the structure of the human body. And it means to obtain a useful effect for health use, such as controlling nutrients for function or 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 food refers to food for the purpose of health supplement. In some cases, the terms of health functional food, health food, and health supplement food Can be used interchangeably.

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

The food product of the present invention can be prepared by a method commonly used in the art, and during the production, raw materials and ingredients commonly added in the art may be added to prepare it. Specifically, the food composition may further include a physiologically acceptable carrier, and the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used. In addition, the food composition may contain food additives such as preservatives, disinfectants, antioxidants, coloring agents, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, reinforcing agents, emulsifying agents, thickening agents, coating agents, gum base agents, foam inhibitors, solvents, and improving agents. Can 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 food composition of the present invention can be prepared in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term use of drugs using food as a raw material, and is excellent in portability. Food can be consumed as an adjuvant to enhance the effect of preventing or improving PTSD.

Tetramethylpyrazine of the present invention may be included in a variety of weight percent in a food composition if it can exhibit the effect of preventing or improving PTSD. Specifically, it may be included in 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto. When ingested for a long time for health and hygiene purposes, the content below the above range may be included, 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 tetramethylpyrazine or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the definitions of the terms "tetramethylpyrazine", "post-traumatic stress disorder", "prevention", "improvement" and "pharmaceutically acceptable salts" are as described above.

Since tetramethylpyrazine 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 on a daily basis by animals, so preventing or improving PTSD. High effects can be expected.

The term "feed" of the present invention means any natural or artificial diet, one meal meal, etc., or a component of the one meal meal, for animals to eat, ingest, and digest.

The kind of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals or grain by-products; Animal feeds such as proteins, inorganic logistics, oils and fats, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more.

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

1 is a schematic diagram of an experiment in chronological order to create a post-traumatic stress disorder (PTSD) model, and an anxiety-like symptom induction process through a single prolonged stress (SPS) Represents.
2A is a graph showing the effect of tetramethylpyrazine on anxiety-like symptoms through the percentage of time (open/total) that PTSD-induced rats stay in the open area.
FIG. 2B is a graph showing the effect of tetramethylpyrazine on anxiety-like symptoms through the percentage of time that the PTSD-induced rat stays in the non-open area (non-open/total).
2C is a graph showing the effect of tetramethylpyrazine on anxiety-like symptoms through the number of PTSD-induced rats entering the open area.
FIG. 2D is a graph showing the effect of tetramethylpyrazine on anxiety-like symptoms through the number of PTSD-induced rats entering the closed area.
2E is a graph showing the effect of tetramethylpyrazine on anxiety-like symptoms through anxiety index derived by calculating the number of attempts to enter the open and non-open areas of the rat in which PTSD was induced and the time spent.
2F is a graph showing the effect of tetramethylpyrazine on anxiety-like symptoms through PTSD-induced rat grooming behavior period.
3A is a graph showing the effect of tetramethylpyrazine on motor activity and exploration activity through the number of times the PTSD-induced rat crosses the central part.
3B is a graph showing the effect of tetramethylpyrazine on motor activity and exploration activity through the number of times the PTSD-induced rat crosses the periphery.
3C is a graph showing the effect of tetramethylpyrazine on motor activity and exploration activity through the time that PTSD-induced rats stay in the central part.
3D is a graph showing the effect of tetramethylpyrazine on motor activity and exploration activity through the time that PTSD-induced rats stay in the periphery.
3E is a graph showing the effect of tetramethylpyrazine on motor activity and exploration activity through the movement distance of the rat in which PTSD was induced.
3F is a graph showing the effect of tetramethylpyrazine on motor activity and exploration activity through the frequency of PTSD-induced rats standing on their hind legs.
FIG. 4 is a graph showing the effect of tetramethylpyrazine on spastic behavior induced from fear reactions in a post-war situation in PTSD-induced rats.
5 is a graph showing changes in serotonin (5-HT) concentration in the medial frontal lobe, striatum, hippocampus, and amygdala of the rat brain in which PTSD was induced and the effect of TMP thereon.
Figure 6 shows the effect of TMP administration on tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and vesomonoamine transporter-2 (VMAT-2) in PTSD-induced rats. It is a graph represented by.
Figure 7 shows the effect of TMP on plasma corticosterone (CORT), corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) levels in PTSD-induced rats through enzyme immunoassay (ELISA). It is a graph.

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

Example 1. Use of experimental animals and effects of TMP

220-250g male Sprague-Dawley (SD) rats (6 weeks old, Samtako AnimalCo., Seoul, Korea), were used for the treatment of post-traumatic stress disorder (PTSD). Rats were subjected to a 12-hour light/dark cycle (lit at 8:00am and off at 8:00pm) with a maximum of 5 rats per polycarbonate cage, a temperature of 23±5°C and a relative of 55±5%. Housed under humidity. All rats were allowed to acclimate to these conditions for 7 days after arrival. All of the above methods and procedures were approved by the Animal Management Committee of Kyunghee University [KHUASP(SE)-15-115], and all procedures were performed according to the guidelines for the management of experimental animals.

Six or seven rats belonging to different groups were used for drug treatment and testing in randomized controlled animal experiments. Tetramethylpyrazine (TMP 10, 20, and 40 mg/kg, body weight; Sigma-Aldrich Chemical Co. St. Louise, MO, USA) dissolved in 0.9% physiological saline (SAL) and the positive drug fluoxetine (10 mg/kg, FLX, fluoxetine hydrochloride; Sigma) was injected into a single prolonged stress (SPS) for 14 days and then intraperitoneally (ip). After the rats were exposed to SPS, TMP and FLX were administered once a day for 14 days at a dose of 1 ml/kg. In addition, SAL was administered to rats not exposed to SPS for the control of the experiment (FIG. 1).

Example 2. Single prolonged stress

The rat prepared in Example 1 was placed in the holder for 2 hours, and then immediately forced swimming (FS) for 20 minutes. The rats were allowed to recover for 15 minutes and then subjected to a single prolonged stress (SPS) stimulation of exposure to isoprulane until unconscious. After SPS stimulation, two rats were placed per cage, and there was no disturbance for 14 days to develop PTSD-like symptoms.

Example 3. Analysis of the effect of TMP on SPS stimulation

Example 3-1. Analysis of the effects of TMP on behavioral changes in open areas caused by anxiety

To study the effect of TMP on behavioral changes in the open area due to anxiety in rats, an elevated plus maze (EPM) test was performed.

More specifically, for the Elevated plus maze (EPM) test, the rat was moved to a maze of a four-armed wooden platform in the shape of a cross. The maze device was painted with black enamel and was 50 cm high from the bottom, and all arms had a length of 50 cm and a width of 10 cm and were gathered in the center to form a 10 cm ² central platform. At this time, the two areas facing each other are closed while the other two areas are open. At the beginning of each experiment, the rats were allowed to move freely for 5 minutes, and the video footage of this session was scored. Anxiety index was measured by measuring the ratio of the attempted entry to the open area and the total time spent in the open sphere (FIGS.

As a result, as shown in Figs. 2A and 2C, the ratio of the number of times the rats enter the open area of the maze and the dwell time was significantly reduced in the PTSD group compared to the control group. However, rats in the PTSD + TMP40 group showed a significant recovery in the number of open zone entry compared to the PTSD group. On the other hand, as shown in FIGS. 2B and 2D, it was confirmed that the ratio of the time spent in the non-open area of the maze and the number of times of entering the non-open area did not differ significantly for each group. Compared with the PTSD group, the rats in the PTSD + TMP 40 mg/kg group recovered significantly the ratio of the time spent in the open area of the maze, which was reduced by SPS. In addition, as shown in FIG. 2A, it was confirmed that the ratio of the time spent in the open area of the maze in the PTSD + TMP40 group was almost similar to the ratio of the time in the open area of the maze in the PTSD + FLX group. In addition, as shown in Fig. 2e, it was confirmed that the anxiety index measured through the above times also increased in the PTSD group and was relieved when TMP was administered.

In addition, the grooming behavior may vary depending on the response to new things or other stress factors and intensity.As shown in Fig.2f, it was confirmed that the duration of the grooming behavior increased after SPS exposure was recovered when TMP 40mg/kg was administered. .

Through this, it was confirmed that TMP is related to suppressing symptoms such as PTSD by relieving anxiety symptoms caused by SPS stimulation with PTSD.

Example 3-2. Analysis of the effect of TMP on changes in motor and exploration activities caused by anxiety

To study the effect of TMP on changes in motor activity and exploration activity due to anxiety in rats, an open field test (OFT) was conducted.

More specifically, before the EPM test is completed, exercise activity in a rectangular open space area surrounded by dark opaque plexiglass (60×60cm) and walls (30cm in height) was evaluated in the S-MART program (PanLab Co., Barcelona, Spain) using a video-tracking system. The athletic activity was evaluated in terms of the total distance traveled in the container, and the painted white line divides the space into 16 spaces (15×15 cm each), allowing the rat to freely explore the arena for 5 minutes. Thereafter, exercise (cross-section of the center) and the time spent in the center and periphery were measured, and the number of times that the rat stood on its hind legs was also recorded in order to analyze the exercise activity in the OFT (Figs. 3A to 3F).

As a result, as shown in FIGS. 3C and 3D, the rats exposed to SPS had a longer staying time in the peripheral portion than in the central portion compared to the SAL group. However, as shown in FIGS. 3A and 3B, when exposed to SPS stimulation, the number of crossovers in the central region was significantly reduced. Through this, it was found that the rat's anxiety behavior and exercise and exploration activities due to SPS stimulation were closely related. Rats treated with TMP 40mg/kg showed a significant increase in the number of cross-sections in the central region compared to the PTSD group, and the behavior of the PTSD+TMP 40mg/kg group was similar to that of the PTSD+FLX group.

In addition, as shown in Figs. 3e and 3f, a difference related to PTSD was found in the motor activity (motor function) and the number of total hindlimbs (search activity) in OFT through the analysis of variance (one-way ANOVA). Rats exposed to SPS significantly reduced the distance traveled and the frequency of standing on the hind legs in the open area compared to the SAL group. However, when TMP was administered, the travel distance slightly increased compared to the PTSD group, and the frequency of the hind limbs recovered.

Through this, it was confirmed that SPS stimulation reduced motor activity and exploration activity. In addition, it was thought that TMP has an effect of recovering the decreased activity change similar to that of FLX administration.

Example 4. Stiffness behavior caused by fear learned through SPS and analysis of the effect of TMP on it

The degree of stiffness was measured to measure the stiffness behavior caused by fear acquisition and the relaxation effect through TMP.

More specifically, after exposing SPS to each group of rats that did not receive the EPM test, experiments were conducted for fear response and disappearance. The experiment was carried out over 3 days. The rat was placed in a chamber (26×30×22cm) to which an electric shock was applied, allowed to roam freely for 120 seconds, and observed through a camera. After 120 seconds, a total of 10 electric shocks (0.75 mA, 2 s duration and 74 s interval) were applied through the chamber floor and held for 300 seconds without any shock. On Days 1-2 of the test, rats were placed in the same chamber for 8 minutes to determine the extent of acquisition and extinction of the fear response. This promotes previous behavioral sensitization and can lead to re-experience of hateful events. The percentage of freezing responses to a specific stimulus was calculated by dividing the freezing time by the total time. Freezing behavior was defined as a complete immobility state excluding minor behaviors required for breathing (Fig. 4).

As a result, as shown in FIG. 4, the stiff response to fear that was not initially trained was the same in all groups, but after the acquisition of fear memory, stiffness time significantly increased. However, in the group to which TMP 40mg/kg was administered, it was confirmed that it was significantly relieved with repeated administration.

Through this, it was confirmed that the persistent fear response was associated with trauma, and it was found that TMP administration alleviated such trauma-related rigid behavior.

Example 5. Analysis of the effect of TMP on the change of 5-HT level in the brain by SPS stimulation

The effect of TMP on PTSD was analyzed by measuring the level of serotonin (5-HT), a neurochemical that is closely related to PTSD.

More specifically, 5-HT levels of the PTSD group in the medial prefrontal cortex (mPFC), striatum, hippocampus, and amygdala were measured through post-mortem tests. Four rats of each group were anesthetized and sacrificed by inhaling with isoflurane (1.2%) one day after the behavioral test. Special care was taken to avoid stress before sacrifice. Thereafter, brains were collected, digested by region, and 5-HT levels were measured through competitive enzyme-linked immunoassay (ELISA) using 5-HT antibodies (FIG. 5).

As a result, as shown in FIG. 5, it was confirmed that the level of 5-HT decreased significantly in the SD group, especially in the hippocampus, and when TMP was administered daily, the reduced 5-HT level in the striatum increased by 211.94%, and decreased in the amygdala. It was confirmed that the 5-HT increased by 214.12%. It was confirmed that the results of administering TMP 40mg/kg were similar to those of FLX 10mg/kg.

Through this, it was confirmed that PTSD and TMP are closely related, and it was confirmed that TMP can alleviate anxiety symptoms such as PTSD by controlling the 5-HT level.

Example 6. Changes in TRP, 5-HIAA, and VMAT-2 levels in the hippocampus by SPS stimulation and analysis of the effects of TMP on it

Existing research results have shown that tryptophan (TRP) is an important marker for synthesizing 5-HT and observing the response of fear or anxiety along with traumatic stress, and neurons such as vesicular monoamine transporter-2 (VMAT-2) It was also found to be closely related to the transporter and the sertonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA). By measuring the expression levels of these substances, the effects of TMP on relieving fear and anxiety such as PTSD were analyzed.

More specifically, mRNA expression levels of TRP, 5-HIAA and VMAT-2 were measured through reverse transcription poly chain reaction (RT-PCR) of rats. Total RNA was isolated from the hippocampus of each rat using Trizol reagent. cDNA was synthesized from 2 μg total RNA using reverse transcriptase (Takara Bio, Otsu, Japan), and then Taq DNA polymerase (Takara, Kyoto, Japan) in a thermal cycler (MJ Research, Watertown, MA, USA) Using PCR (TRC: 60 ℃, 28 cycles, 5-HIAA: 60 ℃, 30 cycles, VMAT-2: 56 ℃, 35 cycles). The data obtained through the above procedure were normalized to the expression of glyceraldehydes 3-phosphate dehydrogenase (GADPH) (FIG. 6).

As a result, as shown in FIG. 6, it was confirmed that the mRNA levels of TRP, 5-HIAA, and VMAT-2 were lowered in the rats of the PTSD group. On the other hand, when TMP 40mg/kg was administered, the lowered mRNA levels of TRP and 5-HIAA recovered to levels similar to those of the SAL group. In addition, the level of mRNA of VMAT-2 recovered to a level similar to that of the group administered with 10 mg/kg of FLX.

Through this, it was confirmed that PTSD reduced the mRNA expression of TRP, 5-HIAA and VMAT-2, and TMP was thought to alleviate the expression of the mRNA reduced by PTSD.

Example 7. Changes in levels of CORT, CRH, and ACTH by SPS stimulation and TMP effect therefor

Hormonal changes due to PTSD and the effect of TMP by measuring the concentration of stress-related hormones corticosterone (CORT), adrenocorticotropic hormone (ACTH), and corticotrophin releasing hormone (CRH). Was analyzed.

More specifically, four rats of each group were anesthetized and sacrificed by inhaling with isoflurane (1.2%) one day after the behavioral test. Special care was taken to avoid stress before sacrifice. Plasma was rapidly collected through the abdominal aorta. Plasma CORT, CRH, and ACTH concentrations were measured for expression changes through an enzyme-linked immunoassay (ELISA) using antibodies corresponding to each substance (FIG. 7).

As a result, as shown in FIG. 7, it was confirmed through ELISA analysis that the levels of CORT, CRH, and ATCH in plasma increased by 317.42%, 246.87% and 282.63% compared to rats of the SAL group when SPS stimulation was given. On the other hand, it was confirmed that the level of the hormone, which had been increased when TMP was administered, decreased.

Through this, it was confirmed that TMP is associated with suppressing symptoms such as PTSD by reducing the level of hypothalamic-pituitary-adrenal stress hormone.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

Claims (8)

As a pharmaceutical composition for preventing or treating post-traumatic stress disorder, comprising tetramethylpyrazine or a pharmaceutically acceptable salt thereof as an active ingredient,
The tetramethylpyrazine is represented by the following formula (I), a pharmaceutical composition.

[Formula I]

delete The pharmaceutical composition of claim 1, wherein the tetramethylpyrazine is contained in an amount of 0.01 to 98% by weight.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
The post-traumatic stress disorder comprising the step of administering the pharmaceutical composition of any one of claims 1, 3, and 4 to an individual other than humans, which has or is likely to develop post-traumatic stress disorder. Prevention or treatment method.
The method of claim 5, wherein the administration is 80 to 120 mg/kg of tetramethylpyrazine.
As a food composition for preventing or improving post-traumatic stress disorder comprising tetramethylpyrazine or a pharmaceutically acceptable salt thereof as an active ingredient,
The tetramethylpyrazine is represented by the following formula (I), food composition.

[Formula I]

As a feed composition for preventing or improving post-traumatic stress disorder comprising tetramethylpyrazine or a pharmaceutically acceptable salt thereof as an active ingredient,
The tetramethylpyrazine is represented by the following formula I, feed composition.

[Formula I]

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