KR102015221B1 - Compositions comprising tangeretin for preventing or treating post traumatic stress disorder - Google Patents

Abstract

The present invention relates to a composition for preventing or treating post-traumatic stress disorder containing tangeretin. Specifically, the present invention relates to: a pharmaceutical composition, a food composition, and a feed composition for preventing or treating post-traumatic stress disorder containing tangeretin or a pharmaceutically acceptable salt thereof as an active component; and a method for preventing or treating post-traumatic stress disorder which comprises a step of administering the pharmaceutical composition. Since tangeretin of the present invention exhibits an excellent PTSD therapeutic effect, tangeretin can be contained in the pharmaceutical composition or food as the active component, thereby being able to be used for developing a therapeutic agent or an improving agent of PTSD.

Description

Compositions comprising tangeretin for preventing or treating post traumatic stress disorder}

The present invention relates to a composition for the prevention or treatment of post-traumatic stress disorder comprising tangeretin, specifically, the prevention or post-traumatic stress disorder comprising tangeretine or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention relates to a therapeutic pharmaceutical composition, a food composition, a feed composition, and a method for preventing or treating post-traumatic stress disorder comprising administering the pharmaceutical composition.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that has been reclassified as a trauma and stress related disorder and can be caused in response to a life-threatening situation. It usually occurs after events that deviate from what you might experience in your daily life, such as natural disasters, fires, wars, physical assaults, torture, sexual assaults, hostages, abuses, and other major accidents. The timing of symptoms varies from person to person, and may begin immediately after a shock or may occur several days, weeks, months or years later.

The symptoms of PTSD are hyperalertness (hyperarousal), re-experience or intrusion, emotional avoidance or paralysis (emotional numbness). In addition, headaches, indigestion, vomiting, stomach pain, hydration, hormonal changes may occur such as allergies, cognitive decline, memory loss, parting anxiety, school fear, outsider fears. Unreal feelings can lead to abuse and addiction depending on alcohol and drugs, autonomic disorders, sometimes hallucinations, and dissociative disorders or panic attacks.

Treatment of PTSD includes methods such as cognitive therapy, behavioral therapy, hypnosis therapy, group therapy, drug therapy, and neuroprotection therapy, and drug therapy includes tricyclic antidepressant, monovalent amine oxidase inhibitor, or Antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRIs) are mainly used.

Representative examples of the SSRIs described above include Fluoxetine under the trade name Prozac. It has been known in the literature that fluoxetine can be used to treat PTSD (Journal of Traumatic Stress, 1991 Jul, 4 (3): 419-423 .; Neurosciences (Riyadh). 2011 Jul, 16 (3): 257-62.), There are a variety of new PTSD drugs, but fluoxetine is known to be the most widely prescribed drug in many countries. However, since SSRIs have side effects that limit their efficacy, including cognitive impairment, weight gain, sexual dysfunction, sedation, dependence and withdrawal symptoms, efforts have been made to use safer drugs for long-term treatment.

Recently, attention has been directed to the development of natural products or medicinal plants for the treatment of PTSD, depression, anxiety disorders, etc. Accordingly, the composition for the prevention or treatment of depression or cognitive disorders containing rice bran extract as an active ingredient (Korea Patent Registration) Korean Patent Publication No. 10-1620163), a composition for treating and preventing mental stress, depression or dementia, containing a vitamin tree extract as an active ingredient (Korean Patent Publication No. 10-1676828), containing a compound herbal extract as an active ingredient A pharmaceutical composition for preventing or treating depression (Korean Patent Publication No. 10-1470241) has been developed. However, the effects are still being questioned.

Tangerine (TAN) is a polymethoxylated flavone (PMF) enriched in citrus peels and has anti-cancer, anti-metastatic, anti-cell death, antioxidant, anti-diabetic, anti-carcinogenic, anti-inflammatory and neuroprotective activities. Indicates. Tangeretetin reduces neuronal damage caused by dopamine neurotoxins (Choi SY. Et al., Biol Pharm Bull. 2007; 30: 772-778) and has a neuroprotective effect in rat models of Parkinson's disease (Datla KP. Et al. , Neuroreport. 2001; 12: 3871-3875). However, research into the treatment or amelioration of psychotic symptoms such as PTSD of tangeretine has been insignificant.

Against this background, the present inventors have diligently researched to develop drugs for treating post-traumatic stress disorder. As a result, tangeretin enhances stress-induced cognitive / memory function in rat models with post-traumatic stress disorder, and dopamine levels. It was confirmed to alleviate the above symptoms by increasing the present invention to complete the present invention.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of post-traumatic stress disorder, including tangeretine 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 to a subject.

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

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

In order to achieve the above object, one embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of post-traumatic stress disorder comprising tangerine or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "tangeretin" means a compound having the structure of Formula I below. Tangeretine is known to have anti-cancer, anti-metastatic, anti-cell death, antioxidant, anti-diabetic, anti-carcinogenic, anti-inflammatory and neuroprotective properties, but its prevention, treatment or improvement of post-traumatic stress disorder is unknown. It was first identified by the inventor.

[Formula I]

Compositions comprising tangeretine may include derivatives in the range predicted by some skilled in the art in tangeretine, and are included without limitation so long as they have the same effect in the present invention. Tangeretetin can be obtained from an extract comprising the same. Moreover, it can synthesize | combine and use by a well-known method.

As used herein, the term "post-traumatic stress disorder" (PTSD) refers to a disease in which abnormal mental and physical symptoms continue to occur after experiencing a traumatic event. If you meet the criteria set forth in the American Psychiatric Association's Statistical Manual of Psychiatric Disorders (DSM-IV-TR), you will be diagnosed with post-traumatic stress disorder. The onset of the disease varies from person to person and may begin immediately after the shock or may occur several days, weeks, months or years later. Major symptoms include dissociation, panic attacks, cognitive problems such as hallucinations, aggressive tendencies, impulse control disorders, depression, drug abuse, cognitive problems such as decreased concentration and memory.

In the present invention, tangeretine is characterized by increasing dopamine and serotonin levels. By effectively increasing the mRNA expression of tyrosine hydrozylase (TH) associated with dopamine and tryptophan hydroxylase-1 (Tph1) associated with serotonin, it can be usefully used as a prophylactic or therapeutic agent for post-traumatic stress disorder.

As used herein, the term "dopamine (Dopamine; DA)" is a catecholamine-based neurotransmitter and is involved in the regulation of various neurological functions such as motor function, cognition, emotion, mood, reward, addiction, and motivation. Is a major cause of various mental or neurological disorders.

As used herein, the term "serotonin (5-HT)" is a neurotransmitter and is involved in various behaviors and reactions such as sleep, eating, schooling, and memory. It is a major cause of disease.

The term "tyrosine hydrozylase" (TH) is an enzyme involved in the synthesis of dopamine, and "Tph1 (tryptophan hydroxylase-1)" is an enzyme involved in the synthesis of serotonin.

In one specific embodiment of the present invention, as a result of administering tangerin to the PTSD-induced rats, dopamine (FIG. 4) and serotonin (FIG. 5) levels are increased in the hippocampus, striatum, frontal cortex and amygdala of the rat. MRNA levels of tyrosine hydrozylase (TH) and tryptophan hydroxylase-1 (Tph1), which are factors that regulate the dopamine and serotonin systems, are increased (FIG. 6).

In another specific embodiment of the present invention, as a result of administering tangerine to a PTSD-induced rat, the smell recognition time increases for a new object during the object recognition test (FIG. 2), and is hidden during the Morris underwater maze test. The time to reach the platform is reduced and the time and distance to stay in the target quadrant is increased (FIG. 2).

This suggests that tangeretine may be useful for the prevention, treatment and improvement of PTSD, as it has the effect of restoring behavioral and neurochemical changes associated with memory impairment by increasing dopamine and serotonin levels in the PTSD rat model. will be.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a pharmaceutically usable form among salts in which a cation and an anion are bound by an 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.), 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 and the like; Salts with inorganic acids can be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids can 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 can be salts with arginine, lysine, ornithine and the like; Salts with acidic amino acids can be salts with aspartic acid, glutamic acid and the like.

As used herein, the term "prevention" means any action that inhibits or delays the development of PTSD by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "treatment" means any action by which administration of the pharmaceutical composition improves or advantageously alters the symptoms of a suspected and developing subject of PTSD.

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

Specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, 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 compositions may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.

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

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

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

The content of tangeretine included in the pharmaceutical composition of the present invention is not particularly limited, but may be 0.0001 to 80% by weight, 0.0001 to 50% by weight, and more specifically 0.01 to 20% by weight, based on the total weight of the final composition. May be included.

Another aspect of the invention provides a method of preventing or treating a post traumatic stress disorder, comprising administering the pharmaceutical composition to a subject.

At this time, 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 comprising administering the same to a subject may be useful for preventing or treating post-traumatic stress disorder.

As used herein, the term "administration" means introducing the composition to an individual in a suitable manner.

As used herein, the term "individual" means all animals including rats, mice, and livestock, including humans, who may develop or develop post-traumatic stress disorder, and may be, for example, mammals including humans, but are not limited thereto. Do not.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, wherein the term “pharmaceutically effective amount” is sufficient to treat or prevent the disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level refers to the severity of the disease, the activity of the drug, the age, body weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the invention used, the route of administration and the rate of release used. And factors well known in the art and other ingredients, including drugs used in combination or coincidental with the compositions of the invention used. The pharmaceutical compositions of the present invention may be administered alone or in combination with known pterygium therapeutics. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

The pharmaceutical composition of the present invention may be administered by various methods such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, transdermal, and the dosage is depending on the age, sex and weight of the patient. And may be readily determined by one skilled in the art.

In addition, the dosage of the pharmaceutical composition may 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 type of substance used as an active ingredient of the patient. For example, the pharmaceutical composition of the present invention can be administered at 1 mg / kg to 200 mg / kg, specifically 1 mg / kg to 100 mg / kg, more specifically 20 to 40 mg / kg per adult However, the frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or multiple times in divided doses. The dosage does not limit the scope of the invention in any aspect.

In addition, the dosage of the pharmaceutical composition may be adjusted to be administered to 80 to 120 mg / kg, more specifically 100 mg / kg, but is not limited thereto.

Yet another aspect of the present invention provides a food composition for preventing or ameliorating post-traumatic stress disorder comprising tangeretine or a pharmaceutically acceptable salt thereof as an active ingredient.

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

As used herein, the term " improvement " means any action that at least reduces the parameters associated with the condition being treated with the administration of a composition comprising tangeretine, eg, the degree of symptoms.

Tangeretine according to the present invention shows an excellent PTSD treatment effect, it can be included in the food composition for the purpose of prevention or improvement of PTSD, the food composition can be ingested on a daily basis, so it is high for prevention or improvement of PTSD You can expect the effect.

In the present invention, the term "food" is meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products, including ice cream, various soups, beverages, tea, drinks, alcoholic beverages It includes all foods in the usual sense, such as vitamin complexes, health functional foods, and so on, as long as it can include the tangeretine of the present invention.

As used herein, the term "health functional food" refers to a food prepared and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functionality" refers to the structure of the human body. And it means to obtain a useful effect for health use, such as regulating nutrients or physiological action for function. On the other hand, health food refers to foods that have active health maintenance or promotion effect compared to general foods, and health supplement food means foods for the purpose of health supplement, in some cases, the term functional health food, health food, health supplement food Can be used interchangeably.

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

The food of the present invention may be prepared by a method commonly used in the art, and the preparation may be performed by adding raw materials and ingredients commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of the carrier is not particularly limited and may be any carriers commonly used in the art. In addition, the food composition may contain food additives such as preservatives, fungicides, antioxidants, colorants, coloring agents, bleaches, seasonings, sweeteners, flavoring agents, swelling agents, reinforcing agents, emulsifiers, thickeners, coatings, gum herbicides, foam inhibitors, solvents, improvers, etc. It 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 the formulation is recognized as a food. Food composition of the present invention can be prepared in various forms of formulation, unlike the general medicine has a merit and excellent portability, such as side effects that can occur when taking long-term use of the drug as a raw material, the present invention May be taken as a supplement to enhance the effect of preventing or improving PTSD.

Tangeretine of the present invention can be included in various weight percent in the food composition if it can exhibit a prophylactic or ameliorating effect of PTSD. Specifically, it may be included as 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. In the case of long-term intake for health and hygiene purposes, it may include the content 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.

Yet another aspect of the present invention provides a feed composition for preventing or ameliorating post-traumatic stress disorder, comprising tangeretine or a pharmaceutically acceptable salt thereof as an active ingredient.

At this time, the definition of the term "tangeretine", "post-traumatic stress disorder", "prevention", "improvement" and "pharmaceutically acceptable salt" is as described above.

Tangeretine according to the present invention shows an excellent PTSD treatment effect, it can be included in the feed composition for the purpose of preventing or improving PTSD, the feed composition is to prevent or improve PTSD because the animal can be ingested daily A high effect can be expected.

As used herein, the term " feed " means any natural or artificial diet, one meal, or the like or any ingredient of the one meal for the animal to eat, ingest and digest.

The kind of the feed is not particularly limited, and may be used a feed commonly used in the art. Non-limiting examples of the feed include, but are not limited to, vegetable feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

Tangeretine of the present invention shows an excellent PTSD therapeutic effect, it can be included in the pharmaceutical composition or food as an active ingredient and used in the development of a therapeutic or improvement agent for PTSD.

1 is a post-traumatic stress disorder; PTSD) is a schematic of the experiments in chronological order for constructing the model, showing the induction process of anxiety-like symptoms through a single prolonged stress (SPS).
2 is a graph showing the effect of tangeretin on the cognitive memory increasing effect in PTSD induced rats. A is the time to smell the object in seconds, B is the differentiation index, C is the time it takes to find the hidden platform in seconds, D is the time to stay in the target quadrant, and E is the distance to stay in the target quadrant. It is about. SAL was normal control group treated with saline solution, PTSD was negative control group exposed to SPS, PTSD + TAN20 was experimental group treated with 20 mg / kg of tangerine after exposure to SPS, and PTSD + TAN50 was exposed to SPS. The experimental group treated with mg / kg, PTSD + TAN100 means experimental group treated with 100 mg / kg of tangeretine after exposure to SPS, and the PTSD + FLX means positive control group treated with 10 mg / kg of fluoxetine after exposure to SPS. do. At this time, * p <0.05, ** p <0.01, *** p <0.001 are statistically significant when compared with SAL, ^# p <0.05, ^## p <0.01 when compared with PTSD.
FIG. 3 is a graph showing the effect of tangeretin on motor activity and search activity in PTSD induced rats, relating to the total distance traveled in space and the number of hindlimbs. SAL was normal control group treated with saline solution, PTSD was negative control group exposed to SPS, PTSD + TAN20 was experimental group treated with 20 mg / kg of tangerine after exposure to SPS, and PTSD + TAN50 was exposed to SPS. The experimental group treated with mg / kg, PTSD + TAN100 means experimental group treated with 100 mg / kg of tangeretine after exposure to SPS, and the PTSD + FLX means positive control group treated with 10 mg / kg of fluoxetine after exposure to SPS. do. At this time, * p <0.05, ** p <0.01, *** p <0.001 are statistically significant when compared with SAL, ^# p <0.05, ^## p <0.01 when compared with PTSD.
4 is a graph showing the effect of tangeretin on dopamine concentration in PTSD induced rats. A relates to hippocampus, B to striatum, C to frontal cortex, and D to amygdala. SAL was normal control group treated with saline solution, PTSD was negative control group exposed to SPS, PTSD + TAN20 was experimental group treated with 20 mg / kg of tangerine after exposure to SPS, and PTSD + TAN50 was exposed to SPS. The experimental group treated with mg / kg, PTSD + TAN100 means experimental group treated with 100 mg / kg of tangeretine after exposure to SPS, and the PTSD + FLX means positive control group treated with 10 mg / kg of fluoxetine after exposure to SPS. do. At this time, * p <0.05, ** p <0.01, *** p <0.001 are statistically significant when compared with SAL, ^# p <0.05, ^## p <0.01 when compared with PTSD.
5 is a graph showing the effect of tangeretin on serotonin concentration in PTSD induced rats. A relates to the hippocampus, B to striatum, C to frontal cortex, and D to serotonin concentration (ng / g tissue) in the amygdala. SAL was normal control group treated with saline solution, PTSD was negative control group exposed to SPS, PTSD + TAN20 was experimental group treated with 20 mg / kg of tangerine after exposure to SPS, and PTSD + TAN50 was exposed to SPS. The experimental group treated with mg / kg, PTSD + TAN100 means experimental group treated with 100 mg / kg of tangeretine after exposure to SPS, and the PTSD + FLX means positive control group treated with 10 mg / kg of fluoxetine after exposure to SPS. do. At this time, * p <0.05, ** p <0.01, *** p <0.001 are statistically significant when compared with SAL, ^# p <0.05, ^## p <0.01 when compared with PTSD.
Figure 6 is a graph showing the effect of tangeretin on mRNA expression levels in PTSD induced rats. A relates to tyrosine hydrozylase (TH) and B relates to tryptophan hydroxylase-1 (Tph1). SAL was normal control group treated with saline solution, PTSD was negative control group exposed to SPS, PTSD + TAN20 was experimental group treated with 20 mg / kg of tangerine after exposure to SPS, and PTSD + TAN50 was exposed to SPS. The experimental group treated with mg / kg, PTSD + TAN100 means experimental group treated with 100 mg / kg of tangeretine after exposure to SPS, and the PTSD + FLX means positive control group treated with 10 mg / kg of fluoxetine after exposure to SPS. do. At this time, * p <0.05, ** p <0.01, *** p <0.001 are statistically significant when compared with SAL, ^# p <0.05, ^## p <0.01 when compared with PTSD.

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

Experimental Example 1. Use of experimental animals and administration method of tangeretine

For animal experiments, 200-220 g male SD (Sprague-Dawley) rats (7 weeks old, Samtako Animal Co., Seoul, Korea) were used. Rats were housed under a controlled temperature of 21 ± 2 ° C. and a relative humidity of 55 ± 10% under a 12 hour light / dark cycle (8:00 a.m. lit and 8:00 p.m. lit off). All the rats were allowed to adapt to these conditions for 7 days after arrival. All the methods and procedures were approved by Kyung Hee University Animal Care Committee [KHUASP (SE) -15-115], and all procedures were performed according to the guidelines for the management of experimental animals.

Six or seven rats from different groups were used for drug treatment and testing in randomized controlled animal experiments. Administration was done to all experimental animals, including control and drug-treated groups. Tangerine (20, 50, and 100 mg / kg, body weight; Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and positive drug fluoxetine (10 mg / kg, FLX, fluoxetine hydrochloride; Sigma) Intraperitoneally (ip) was injected after exposure to Single prolonged stress (SPS). Tangerete and fluoxetine were dissolved in 0.9% physiological saline before use, and the overall experimental schedule of all drug administration and behavioral tests is shown in FIG. 1.

Experimental Example 2. Single prolonged stress

Rats were exposed to a single prolonged stress (SPS) for 14 consecutive days with a slight modification of the method known by Patki's group (Patki G. et al., Physiol Behav. 2014; 130: 47-53). It was made. Rats were placed in a Plexiglas cylinder for 2 hours and then immediately forced to swim for 20 minutes (FS). Rats were dried and recovered for 15 minutes and then exposed to ether vapor until unconscious. After the SPS factor was given, one rat per cage was left undisturbed for 14 days to develop PTSD-like symptoms.

Experimental Example 3 object recognition task (ORT)

A new object recognition test (ORT) was used to assess cognitive abilities. A 45 × 45 × 45 cm hollow cube made of black painted wood was used. Two similar wooden toys (used objects, A1 and A2) were used as objects to distinguish. The wooden toy is heavy enough not to be carried by the rat, and the new wooden block (object) is different in color and shape compared to A1 and A2. The experiment was carried out in three steps; Habituation, training and testing. On the first day (habitation), rats were placed in the experimental box for 10 minutes. The next day (24 hours after habituation), the rats were placed in an experimental box with two similar objects (A1 and A2) and explored for 5 minutes (training). In the experimental phase, rats were exposed to one new object (B) and one familiar object for three minutes. The smelling time was then measured for new and familiar objects. The differential index was calculated as follows: (time to smell for a new object-time to smell for a familiar object) / (time to smell for a new object + time to smell for a familiar object).

Experimental Example 4. Morris water maze test (MWM)

After performing the ORT test according to Example 3, a Morris water maze test (MWM) was performed to measure the time and distance of swimming until reaching the locked platform. The MWM test includes a location navigation test and a space probe test. Location navigation tests were performed in a dark circular pool 2.0 m in diameter. The rats were trained three times a day for five consecutive days, during which time a hidden platform was discovered using visual cues presented around the experimental space. At this time, the water temperature was 22 ± 2 ° C. Each training was terminated after 180 seconds or when the rat found the platform. The space exploration test was then performed 6 days after the escape platform was removed; After each training for 60 seconds, the frequency of crossing the platform, swimming speed, swimming distance, and time spent in the target quadrant were measured. All training and navigation paths were recorded with a ceiling mounted video camera and the data analyzed with the help of a tracking program (S-MART; PanLab Co., Barcelona, Spain).

Experimental Example 5. Open field test (OFT)

Before the MWM test of Example 4 was completed, an open field test (OFT) was performed. The open space test was performed according to known methods (Lee B. et al., Biomil Ther (Seoul). 2014; 22: 213-222). Specifically, rats were individually placed in a dimly lit room container (60 × 60 × 30 cm). This is the best contrast between a video camera light placed above the center of the room and a white rat in a dim room. Motor activity was represented by travel speed and travel distance and monitored by a computerized video-tracking system using the S-MART program (PanLab Co., Barcelona, Spain). Later, the number of rerearing of each rat was also recorded to analyze motor activity in OFT.

Experimental Example 6 Measurement of dopamine (DA) and serotonin (5-HT)

After 21 days of induction of post-traumatic stress disorder, dopamine (DA) and serotonin (5-HT) concentrations in brain tissue were analyzed using known methods (Lee B. et al., Biomil Ther (Seoul). 2014; 22: 213-222). Four rats of each group were deeply anesthetized with isoflurane (1.2%) and sacrificed one day after the behavioral test. Subsequently, the medical prefrontal cortex, hippocampus, striatum and amygdale were dissected in the rat brain in random order. Dopamine and serotonin concentrations were determined using a competitive enzyme-linked immunoassay (ELISA) using mouse monoclonal DA and 5-HT antibodies (DA and 5-HT ELISA Kit; Abcam, Cambridge, MA, USA). Measured through.

Experimental Example 7 Total RNA Preparation and RT-PCR

Tyrosine hydroxylase (TH) and tryptophan hydrozylase-1 (Tph1) mRNA expression levels were measured by RT-PCR according to known methods (Yeom M et al., J Appl Microbiol. 2015; 119: 560-570). . Total RNA was isolated from the hippocampus of each rat using TRIzol reagent according to the manufacturer's instructions. cDNA was synthesized from 2 μg total RNA using reverse transcriptase (Takara Bio, Otsu, Japan), followed by Taq DNA polymerase (Takara, Kyoto, Japan) in a thermal cycler (MJ Research, Watertown, MA, USA). ) Was amplified by PCR (TH: 60 ° C, 30 cycles, Tph1: 58 ° C, 30 cycles). Data was normalized to glyceraldehydes 3-phosphate dehydrogenase (GADPH) expression in the corresponding samples.

Experimental Example 8. Statistical Analysis

The results are expressed as mean ± standard error. Differences in or between normally distributed data were analyzed using ANOVA and Tukey's post hoc tests using SPSS (version 13.0; SPSS, Inc., Chicago, IL, USA). P-values <0.05 are considered significant.

Example 1 Analysis of the Effect of TAN on Cognitive Memory

Example 1-1. Analysis of the impact of TAN on object cognitive memory

In order to confirm the therapeutic effect of Tangeretin (TAN) on PTSD, after giving SPS, rats' object cognition ability and memory of each group were analyzed according to the method of Experimental Example 3 above.

As a result, as can be seen in Figure 2A, compared to the SAL group, rats exposed to SPS was confirmed that there is no significant difference in smelling time for a familiar object.

On the other hand, it was confirmed that the smelling time for the new object was significantly reduced, which means that the cognitive ability to distinguish objects by SPS was decreased and memory was decreased. However, rats in the PTSD + TAN100 group compared with the PTSD group were found to recover significantly in the time of smelling new objects that were reduced by SPS.

In addition, as shown in B of Figure 2, compared to the SAL group, rats exposed to SPS was confirmed that the discrimination index is significantly reduced. A discrimination index of 0 means no preference for two different objects, and 1 means a preference only for new objects. A decrease in the discrimination index means that the two different objects cannot be distinguished. This means that the cognitive ability to classify is reduced and memory is reduced.

However, when compared with the PTSD group, the rats of the PTSD + TAN100 group were found to recover significantly from the discrimination index which was reduced by the SPS. In addition, the discrimination index in the PTSD + TAN100 group was almost similar to the discrimination index in the PTSD + FLX group.

Through the above results, it can be seen that administration of tangeretin can restore the object cognition ability and memory.

Example 1-2. Analysis of the impact of TAN on spatial cognitive memory

In order to confirm the therapeutic effect of Tangeretin (TAN) on PTSD, after giving SPS, the spatial cognitive ability and memory of rats belonging to each group were analyzed according to the Morris underwater maze test of Experimental Example 4.

As a result, as shown in Figure 2 C, compared to the SAL group, the rats exposed to SPS was found to significantly increase the time it takes to find the hidden platform.

However, compared to the PTSD group, rats of the PTSD + TAN100 group were found to significantly reduce the time taken to find the hidden platform that was increased by the SPS. In addition, it was confirmed that the time taken to find the hidden platform in the PTSD + TAN100 group was almost the same as the time taken to find the hidden platform in the PTSD + FLX group.

In addition, as shown in D of Figure 2, compared with the SAL group, the rats exposed to SPS was confirmed that the retention time in the target quadrant is significantly reduced.

However, compared with the PTSD group, the rats of the PTSD + TAN100 group were found to recover significantly in the target quadrant, which was reduced by SPS. In addition, it was confirmed that the time to stay in the target quadrant in the PTSD + TAN100 group was almost similar to the time to stay in the target quadrant in the PTSD + FLX group.

In addition, as shown in E of FIG. 2, compared with the SAL group, the rats exposed to the SPS showed that the distance to stay in the target quadrant was significantly reduced.

However, compared with the PTSD group, the rats of the PTSD + TAN100 group recovered from the target quadrant, which was reduced by SPS, although not statistically significant.

Through the above results, it can be seen that administration of tangeretin can restore the object cognition ability and memory.

Example 2 Analysis of the Effect of TAN on Voluntary Motor Activity and Search Activity

In order to confirm the depressive / anxiety improvement effect of tangeretine on PTSD, after the SPS, the exercise activity and the exploratory activity of rats in each group were analyzed according to the method of Experimental Example 5.

As a result, as shown in Fig. 3, the saline-treated SAL group, the SPSD-exposed PTSD group, the fluoxetine-administered PTSD + FLX group, and the tangeretine-administered PTSD + TAN group, It was confirmed that there is no big difference.

Through the above results, it was found that cognitive or memory impairment caused by SPS is not caused by a decrease in motor activity but by negative emotions such as depression or anxiety.

Example 3 Analysis of the Effect of TAN on Dopamine Concentration

In order to confirm the therapeutic effect of Tangeretin (TAN) on PTSD, dopamine concentrations in the frontal cortex, striatum, hippocampus and amygdala of rats in each group after SPS were administered according to the method of Experimental Example 7 above. Analyzed.

As a result, as shown in Figure 4A, it was confirmed that the dopamine concentration in the hippocampus was significantly reduced in rats exposed to SPS compared to the SAL group. However, rats of the PTSD + TAN100 group showed significantly increased dopamine concentrations in the hippocampus, which were reduced by SPS, compared to the PTSD group. In addition, it was confirmed that the dopamine concentration in the hippocampus in the PTSD + TAN100 group was almost similar to the dopamine concentration in the hippocampus in the PTSD + FLX group.

In addition, as shown in B of Figure 4, compared with the SAL group, rats exposed to SPS was found to decrease the dopamine concentration in the striatum, although not statistically significant. However, rats of the PTSD + TAN100 group compared with the PTSD group confirmed that the dopamine concentration in the striatum, which was decreased by SPS, was not statistically significant but increased.

In addition, as can be seen in Figure 4C, compared to the SAL group, rats exposed to SPS was confirmed that the dopamine concentration in the frontal cortex is not statistically significant, but decreased. However, the rats of the PTSD + TAN100 group compared with the PTSD group showed that the dopamine concentration in the frontal cortex, which was decreased by SPS, was not statistically significant but increased.

In addition, as shown in D of Figure 4, compared with the SAL group, rats exposed to SPS was confirmed that the dopamine concentration in the amygdala is significantly reduced. However, rats of the PTSD + TAN100 group showed significantly increased dopamine concentrations in the amygdala, which were reduced by SPS, compared to the PTSD group. In addition, it was confirmed that the dopamine concentration in the amygdala in the PTSD + TAN100 group was almost similar to the dopamine concentration in the amygdala in the PTSD + FLX group.

The results indicate that administration of TAN restores cognitive impairment, memory impairment and neurochemical changes by regulating the brain dopamine system of rats with PTSD.

Example 4 Analysis of the Effect of TAN on Serotonin Concentration

In order to confirm the therapeutic effect of Tangeretin (TAN) on PTSD, serotonin concentrations in the frontal cortex, striatum, hippocampus and amygdala of rats in each group after SPS were administered according to the method of Experimental Example 7 above. Analyzed.

As a result, as shown in Figure 5A, it was confirmed that the dopamine concentration in the hippocampus was significantly reduced in rats exposed to SPS compared to the SAL group. However, rats of the PTSD + TAN100 group showed significantly increased dopamine concentrations in the hippocampus, which were reduced by SPS, compared to the PTSD group. In addition, it was confirmed that the dopamine concentration in the hippocampus in the PTSD + TAN100 group was almost similar to the dopamine concentration in the hippocampus in the PTSD + FLX group.

In addition, as shown in B of Figure 5, compared to the SAL group, rats exposed to SPS was confirmed that the dopamine concentration in the striatum significantly decreased. However, rats of the PTSD + TAN100 group compared with the PTSD group confirmed that the dopamine concentration in the striatum, which was decreased by SPS, was not statistically significant but increased.

In addition, as shown in FIG. 5C, the rats exposed to SPS compared with the SAL group showed a significant decrease in the dopamine concentration in the frontal cortex. However, the rats of the PTSD + TAN100 group compared with the PTSD group showed that the dopamine concentration in the frontal cortex, which was decreased by SPS, was not statistically significant but increased.

In addition, as shown in D of Figure 5, compared with the SAL group, rats exposed to SPS was confirmed that the dopamine concentration in the amygdala is significantly reduced. However, rats of the PTSD + TAN100 group showed significantly increased dopamine concentrations in the amygdala, which were reduced by SPS, compared to the PTSD group. In addition, it was confirmed that the dopamine concentration in the amygdala in the PTSD + TAN100 group was almost similar to the dopamine concentration in the amygdala in the PTSD + FLX group.

The results indicate that administration of TAN restores behavioral and neurochemical changes associated with memory impairment by regulating the brain serotonin system of rats with PTSD.

Example 5 Analysis of the Effect of Tangeretine on TH and Tph1 mRNA Expression

To confirm the therapeutic effect of Tangeretin (TAN) on PTSD, mRNA levels of tyrosine hydrozylase (TH) and tryptophan hydroxylase-1 (Tph1) in rats belonging to each group after SPS were given The analysis was carried out according to the method of 7.

As a result, as shown in Figure 6A, compared with the SAL group, rats exposed to SPS was confirmed that the TH mRNA expression level is significantly reduced. However, compared with the PTSD group, rats of the PTSD + TAN100 group were found to significantly increase the level of TH mRNA expression that was reduced by SPS. In addition, it was confirmed that TH mRNA expression level in PTSD + TAN100 group was almost similar to TH mRNA expression level in PTSD + FLX group.

In addition, as shown in B of Figure 6, compared with the SAL group, rats exposed to SPS was confirmed that the Tph1 mRNA expression level is significantly reduced. However, compared to the PTSD group, rats of the PTSD + TAN100 group were found to significantly increase the Tph1 mRNA expression level which was reduced by SPS. In addition, it was confirmed that Tph1 mRNA expression level in PTSD + TAN100 group was almost similar to Tph1 mRNA expression level in PTSD + FLX group.

These results indicate that administration of TAN restores behavioral and neurochemical changes associated with memory impairment by modulating the brain dopamine and serotonin systems in rats with PTSD.

Taken together, it can be seen that tangeretine can be used for the prevention or treatment of post-traumatic stress disorder.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (8)

Pharmaceutical composition for the prevention or treatment of post-traumatic stress disorder comprising tangeretin (tangeretin) or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 1, wherein the tangeretine is represented by the following formula (I), pharmaceutical composition.
[Formula I]

The pharmaceutical composition of claim 1, wherein the tangeretine is contained in an amount of 0.01 to 80 wt%.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
A method of preventing or treating post-traumatic stress disorder, comprising administering the pharmaceutical composition of any one of claims 1 to 4 to a subject other than a human.
The method for preventing or treating post-traumatic stress disorder according to claim 5, wherein the administration is to administer tangeretine at 80 to 120 mg / kg.
Tangerine (tangeretin) or a pharmaceutically acceptable salt thereof as a active ingredient comprising a food composition for the prevention or improvement of post-traumatic stress disorder.
Feed composition for the prevention or amelioration of post-traumatic stress disorder comprising tangeretin (tangeretin) or a pharmaceutically acceptable salt thereof as an active ingredient.

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