KR101929682B1 - Compositions comprising oleuropein for preventing or treating post traumatic stress disorder - Google Patents

Abstract

The present invention relates to a composition comprising oleuropein for preventing or treating post traumatic stress disorders, and specifically, to a pharmaceutical composition and a health functional food composition comprising oleuropein or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating post traumatic stress disorders, and a method for preventing or treating post-traumatic stress disorders. Since the oleuropein of the present invention exhibits excellent PTSD therapeutic effect, the oleuropein is contained in the pharmaceutical composition or the health functional food to be used as an active ingredient to develop a therapeutic agent or an improving agent for PTSD.

Description

[0001] The present invention relates to a composition for preventing or treating post traumatic stress disorder,

The present invention relates to a composition for preventing or treating post-traumatic stress disorder comprising oleuropein, and more particularly to a composition for preventing or treating post-traumatic stress disorder comprising olerupine or a pharmaceutically acceptable salt thereof as an active ingredient. A health functional food composition, and a method for preventing or treating post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is an anxiety disorder reclassified as a trauma and stress-related disorder that can be caused by a response to life-threatening conditions. It usually occurs after events such as natural disasters, fire, war, physical assault, torture, rape, hostage, abuse, and other major accidents, The timing of the symptoms varies from person to person, and may start immediately after the impact, or even after several days, weeks, months, or years.

Symptoms of PTSD are hyperalertness and re-experience or intrusion, emotional avoidance or emotional numbness. Allergies such as headache, dyspepsia, vomiting, stomach cramps, hysterectomy, and hormonal changes can occur, and can be manifested in many forms such as cognitive decline, memory loss, separation anxiety, school fear, and outsider fear. Unrealistic emotions rely on alcohol and drugs to lead to abuse and poisoning, autonomic nervous system disorders, sometimes hallucinations, dissociative disorders or panic attacks.

In the treatment of PTSD, cognitive therapy, behavior therapy, hypnotherapy, group therapy, medication, and nerve block therapy are used. The tricyclic antidepressant, monoamine oxidase inhibitor (MAO inhibitor) Antidepressants such as selective serotonin reuptake inhibitors (SSRIs) are mainly used.

A representative example of the SSRIs described above is Fluoxetine having 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 remedies, but fluoxetine is among the most widely prescribed drugs in many countries. However, efforts have been made to use safer drugs in long-term treatment, as SSRIs present side effects that limit their efficacy, including cognitive impairment, weight gain, sexual dysfunction, sedation, dependence and withdrawal symptoms.

Recently, attention has been focused on the development of natural products and medicinal plants for the treatment of PTSD, depression, anxiety disorder and the like. Accordingly, a composition for prevention or treatment of depression or cognitive disorders comprising rice bran extract as an active ingredient 10-1620163), a composition for treating and preventing mental stress, depression or dementia containing vitamin-tree extract as an active ingredient (Korean Patent Laid-Open Publication No. 10-2016-0045256), a herbal medicine extract as an active ingredient And a pharmaceutical composition for preventing or treating depression (Korean Patent Registration No. 10-1470241) have been developed. However, the effect is still questionable.

On the other hand, oleruprin is a phenolic compound present in the leaves of olive tree ( Olea europaea L.) and has various pharmacological properties such as weight loss effect and neuroprotective effect. In particular, the leaves of olive trees have been used for a long time as private medicines in Mediterranean countries such as France, Greece, Italy, Morocco, Spain, Tunisia and Turkey. It is known that it contains various bioactive compounds which can exhibit characteristics such as antioxidant, antihypertensive agent, antithrombotic agent and anti-inflammatory agent. However, research as a therapeutic or ameliorating agent for psychotic symptoms such as the leaves of olive trees or PTSD of europallins has been limited.

Under these circumstances, the present inventors have made intensive efforts to develop a drug for treating post-traumatic stress disorder. As a result, olerupin has been shown to enhance the stress-induced cognitive function of a rat model with post-traumatic stress disorder, Confirming that the expression level of the factor is increased and that the symptoms are alleviated by reducing the expression of the inflammatory mediator, thereby completing the present invention.

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

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

It is still another object of the present invention to provide a health functional food composition for preventing or ameliorating post traumatic stress disorder, which comprises ollaur pin as an active ingredient.

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

The term " oleuropein " of the present invention means a compound having the structure of the following formula (1). It has been known that oleruprin has various pharmacological properties such as weight loss effect and neuroprotective effect. However, its preventive, therapeutic or ameliorative effect on post-traumatic stress disorder has not been known and it was first discovered by the present inventor.

[Chemical Formula 1]

The term " post-traumatic stress disorder " (PTSD) of the present invention means a disease in which abnormal psychosomatic symptoms persist after experiencing a shock event. If the criteria presented in the American Psychiatric Association's DSM-IV-TR are met, it is diagnosed as post-traumatic stress disorder. The onset of the disease varies from person to person, and may begin immediately after the impact, or even after several days, weeks, months, or even years. Major symptoms include cognitive problems such as dissociation, panic attacks, perceptual anomalies such as hallucinations, aggressive behavior, impulse control disorder, depression, substance abuse, concentration and memory impairment.

In the present invention, the oleruprin increases the level of BDNF (Brain-derived neurotrophic factor) or CREB (cAMP response element-binding protein), or increases TNF-alpha (tumor necrosis factor-?) Or interleukin -1?).

"Brain-derived neurotrophic factor (BDNF)" and "cAMP response element-binding protein (CREB)" refer to proteins found in hippocampus. It plays an important role in the pathological state of the central nervous system (CNS), and it is known that the above factors are lacking in patients suffering from depression, obsessive compulsive disorder or the like, or those receiving psychiatric trauma.

In addition, the above-mentioned "tumor necrosis factor-α" and "IL-1β (interleukin-1β)" are inflammatory mediators and are known to cause an acute neuroinflammatory response in the brain. In general, it is known that nerve damage in the hippocampus causes deterioration of cognitive ability or memory, irreversible brain dysfunction, and thus aggravates depression and anxiety symptoms of PTSD patients.

Thus, increasing levels of BDNF or CREB and decreasing levels of TNF- [alpha] or IL-1 [beta] may reduce symptoms such as depression, anxiety and the like, thereby improving or treating PTSD.

In a specific embodiment of the present invention, administration of 100 mg / kg olfurane to rats induced with PTSD resulted in decreased serum corticosterone levels in the rats, increased cognitive ability and memory, , The number of cells expressing brain-derived nutrients BDBF and CREB and the mRNA level of the factor were increased and the protein and mRNA levels of TNF-α and IL-1β, inflammatory mediators, were decreased 2 to 6). This suggests that oleruprin improves the cognitive ability / memory reduction phenomenon due to anxiety / depressive behavior of the PTSD rat model, increases the level of brain-derived nutritional factors, and decreases the level of inflammatory mediators, And may be useful for prevention, treatment and improvement of PTSD.

The term " pharmaceutically acceptable salt " of the present invention means a salt that can be used pharmacologically in a salt in which a cation and an anion are bound by an electrostatic attraction, and usually a metal salt, an organic base, 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.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt and the like; Examples of salts with organic bases include salts with organic bases such as triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, And the like; The salt with inorganic acid may be a salt 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 and the like.

The term " prophylactic " of the present invention refers to any action that inhibits or delays the onset of PTSD by administration of a pharmaceutical composition according to the present invention.

The term " treatment " of the present invention means any suspicion of PTSD by the administration of the pharmaceutical composition and any action that alters or alleviates symptoms of the onset.

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

Examples of the carrier, excipient and diluent which can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium But are not limited to, silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, Lactic Acid, poly-L-lactic acid, Mineral oils and the like.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method. 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 fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

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

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

The content of oleruprin contained in the pharmaceutical composition of the present invention is not particularly limited, but may be in the range 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 .

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

Here, the above-described " post-traumatic stress disorder ", " prevention ", and " treatment "

Since the pharmaceutical composition of the present invention shows the effect of preventing or treating post-traumatic stress disorder, the method of the present invention including the step of administering it to a subject can be effectively utilized for preventing or treating post-traumatic stress disorder.

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

The term " individual " as used herein means all animals such as rats, mice, livestock and the like, including humans who have developed or may develop post-traumatic stress disorder, and specific examples thereof include mammals including humans, Do not.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term, " pharmaceutically effective amount " refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable for medical treatment or prevention And the effective dose level refers to the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, administration time of the composition of the present invention, route of administration, Factors including, but not limited to, the drugs used in combination with or co-used with the compositions of the present invention used, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered alone or in combination with a known therapeutic agent for pterygium. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

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

The dosage of the pharmaceutical composition may be determined by those skilled in the art in consideration of the intended use, the degree of addiction to the disease, the age, body weight, sex, history, or the kind of the substance used as the 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, more specifically 1 mg / kg to 100 mg / kg, more specifically 20 to 40 mg / kg per adult, The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses. The dose is not intended to limit the scope of the invention in any way.

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

Another embodiment provides a health functional food composition for preventing or ameliorating post traumatic stress disorder comprising oleruprin as an active ingredient.

Herein, the terms 'oleruprin' and 'post-traumatic stress disorder' and 'prevention' are as described above.

The term " improvement " of the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated by administration of a composition comprising oleruprin.

Because ollular pin according to the present invention exhibits excellent PTSD therapeutic effect, it can be included in a health functional food composition for the purpose of preventing or improving PTSD. Since the health functional food composition can be ingested routinely, A high effect can be expected on improvement or improvement.

The term " health functional food " of the present invention means a food prepared and processed by using a raw material or ingredient having a useful function in the human body according to Law No. 6727 on Health Functional Food, ≪ / RTI > and to obtain beneficial effects in health applications such as controlling nutrients and physiological actions. On the other hand, the health food refers to food having an active health promotion effect or enhancement effect compared to the general food, and the health supplement food means food for health supplement. In some cases, the term of health functional food, health food, Can be mixed.

The oleruprin pin of the present invention may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to conventional methods.

The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. Specifically, the health functional food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier conventionally used in the art can be used. In addition, the health functional food composition can be used as a food such as a preservative, a bactericide, an antioxidant, a coloring agent, a coloring agent, a bleaching agent, a seasoning, a sweetener, a flavoring, a swelling agent, a strengthening agent, an emulsifier, a thickener, Additives. The additives may be selected and used in appropriate amounts depending on the type of food.

In addition, the formulation of the health functional food can be manufactured without restrictions as long as it is a food formulation. The composition for a health functional food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has advantages of being free from side effects that may occur when a food is used as a raw material for a long period of time, The food of the present invention can be ingested as an adjuvant to promote the effect of preventing or improving PTSD.

The oleruprin pin of the present invention may be contained at various weight percentages in the health functional food composition as long as it exhibits the preventive or ameliorative effect of PTSD. Specifically, it may include, but is not limited to, 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the health functional food composition. In case of long-term ingestion for health and hygiene purposes, it may contain 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 exceeding the above range.

Since ollaurin pin of the present invention exhibits excellent PTSD therapeutic effect, it can be used as an active ingredient in a pharmaceutical composition or a health functional food to be used for the development of a therapeutic agent or an improvement agent for PTSD.

FIG. 1 is a graph showing the post-traumatic stress disorder PTSD) model and shows the process of inducing anxiety-like symptoms through single prolonged stress (SPS). In this case, IMO is immobilized, FS is forced swim, Rest is rest, Ether is ether exposure, OFT is open field test, ORT is object recognition task, MWM refers to the Morris water maze test, and Sampling refers to the brain (hippocampal) tissue acquisition.
Figure 2 is a graph showing the effect of oleruprin on serum corticosterone levels in rats in which PTSD was induced by SPS. PTSD + OLE20 was exposed to SPS, and PTSD + OLE50 was treated with 10 mg / kg of oleruprin after exposure to SPS. PTSD + OLE50 was exposed to SPS, kg of PTSD + OLE1000, PTSD + OLE1000 was treated with 100 mg / kg of oleruprin after exposure to SPS, and PTSD + IBU40 was a positive control treated with 40 mg / kg of ibuprofen after exposure to SPS . At this time, * p <0.01 is compared with CON, and # p <0.05 represents statistical significance in comparison with PTSD.
FIG. 3 is a graph showing the effect of oleruprin on the cognitive function increasing effect in rats induced by PTSD by SPS. A is the sniffing time in seconds, B is the discrimination index, C is the time to reach the hidden platform, and D is the target quadrant. Time spent in the target quadrant, and E is the distance spent in the target quadrant. PTSD + OLE20 was exposed to SPS, and PTSD + OLE50 was treated with 10 mg / kg of oleruprin after exposure to SPS. PTSD + OLE50 was exposed to SPS, kg of PTSD + OLE1000, PTSD + OLE1000 was treated with 100 mg / kg of oleruprin after exposure to SPS, and PTSD + IBU40 was a positive control treated with 40 mg / kg of ibuprofen after exposure to SPS . * P <0.05, ** p <0.01, *** p <0.001 showed statistical significance when compared with CON, and #p <0.05 and ## p <0.01 compared with PTSD.
Figure 4 is a graph showing the effect of oleruprin on spontaneous motor activity and search activity in rats induced by PTSD by SPS and relates to the total distance traveled in space. PTSD + OLE20 was exposed to SPS, and PTSD + OLE50 was treated with 10 mg / kg of oleruprin after exposure to SPS. PTSD + OLE50 was exposed to SPS, kg of PTSD + OLE1000, PTSD + OLE1000 was treated with 100 mg / kg of oleruprin after exposure to SPS, and PTSD + IBU40 was a positive control treated with 40 mg / kg of ibuprofen after exposure to SPS . * P <0.05, ** p <0.01 indicates statistical significance when comparing with CON, and # p <0.05 and ## p <0.01 compared with PTSD.
Figure 5 is an image and graph showing the effect of oleruprin on the expression level of BDNF (Brain-derived neurotrophic factor) and CREB (cAMP response element-binding protein) in rats induced by PTSD by SPS. A is the number of cells showing a positive response to BDNF, B is the number of cells showing a positive response to CREB, D is the amount of mRNA expression of BDNF and CREB, and E is the expression of the mRNA And the result of quantifying the amount. PTSD + OLE20 was exposed to SPS, and PTSD + OLE50 was treated with 10 mg / kg of oleruprin after exposure to SPS. PTSD + OLE50 was exposed to SPS, kg of PTSD + OLE1000, PTSD + OLE1000 was treated with 100 mg / kg of oleruprin after exposure to SPS, and PTSD + IBU40 was a positive control treated with 40 mg / kg of ibuprofen after exposure to SPS . * P <0.05, ** p <0.01 indicates statistical significance when comparing with CON, and # p <0.05 and ## p <0.01 compared with PTSD.
Figure 6 is a graph showing the effect of IBU on changes in the expression levels of inflammatory mediators (TNF-a and IL-l [beta]) in rats in which PTSD was induced by SPS. A is the concentration of TNF-α protein present in serum, B is the concentration of IL-1β protein present in serum, C is the amount of mRNA expression of TNF-α and IL-1β and D is the amount of mRNA expression It is about the result. PTSD + OLE20 was exposed to SPS, and PTSD + OLE50 was treated with 10 mg / kg of oleruprin after exposure to SPS. PTSD + OLE50 was exposed to SPS, kg of PTSD + OLE1000, PTSD + OLE1000 was treated with 100 mg / kg of oleruprin after exposure to SPS, and PTSD + IBU40 was a positive control treated with 40 mg / kg of ibuprofen after exposure to SPS . * P <0.05, ** p <0.01 indicates statistical significance when comparing with CON, and # p <0.05 and ## p <0.01 compared with PTSD.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Experimental Example 1: Use of experimental animals and administration of oleruprin

Male Sprague-Dawley (SD) male rats (6 weeks old, Samtako Animal Company; Seoul, Korea), 200-220g, were used for animal experiments. All rats were housed in an access-restricted rodent facility with five animals per polycarbonate cage. The animal room was adjusted to maintain a temperature of 22 ± 2 ° C and a relative humidity of 55 ± 15%. The cage was adjusted to receive 12 hours of light, and the sterilized drinking water and standard meals were randomly supplied Respectively.

The treatment effect of PTSD was confirmed by treating the drug in 6-7 rats belonging to different groups. The standard dose and duration of Oleuropein (OLE) in rats was established based on previous studies. (1 mg / kg, IBU, Sigma-Aldrich Chemical Co.), which is a positive control drug, and olerupine (20, 50 or 100 mg / kg body weight, Sigma- Aldrich Chemical Co. St. Louise, Were administered intraperitoneally (ip) once daily for 21 days, and between 9:00 and 10:00 am.

Experimental Example 2: Single prolonged stress

Post-traumatic stress disorder; Single prolonged stress (SPS) model to induce PTSD was performed according to the known method (Serova L. et al., Neuroscience. 2013; 236: 298-312). First, the rats were immobilized (IMO) in a restraint tube (height 20 cm, diameter 7 cm) for 2 hours. The rats were then forced to swim (FS) for 20 minutes in a glass cylinder (50 cm in height, 20 cm in diameter) filled with 2/3 of fresh water at 24 ° C. The rats were dried and allowed to recover for 15 minutes and then exposed to ether vapors until unconscious. The SPS process was performed between 10:00 am and 4:00 pm. After giving the SPS stressors, one rat per cage was placed and was not disturbed for 14 days to show PTSD-like symptoms.

Parameters such as changes in serum corticosterone levels were measured to observe the effects of psychosomatic development by the SPS model of PTSD. A behavioral test was conducted to observe anxiety and other behaviors 24 hours after the end of the 14 day uninterrupted condition. The overall test schedule of SPS and behavioral testing is shown in FIG. Each test was performed separately or between each test to avoid carry-over from one test to another. After the behavioral test, all rats were sacrificed and brain tissue was immediately collected and stored at -70 ° C.

Experimental Example 3. Object Recognition Task (ORT)

When PTSD is triggered, the cognitive ability decreases due to depression and anxiety. Therefore, in order to evaluate the object recognition ability of the experimental animals, an object recognition test was performed to confirm whether a new object can be recognized in a familiar environment (Boultadakis A, 2010, Behav Brain Res 207: 208-214).

Specifically, the rats were exposed to two similar objects, and then to see if they could identify new objects placed at the same time as the familiar objects. Experiments were carried out on rectangular boxes made of wood painted in black with a size of 45 × 45 × 45 cm ³ and the boxes were not cleaned during the experiment to saturate the olfactory stimulus. Two transparent glasses (A1 and A2) were prepared as familiar objects, and white cement was placed in them so that the animals could not move freely. Thereafter, a metal container (B) containing white cement of the same size as a new object was prepared. The size of the object was assumed to be 2.5 times the size of the rat, so that the rats could smell the object. The rats were then placed in the middle of the box, allowing the box and object to be fully recognized.

Thereafter, the experiment was carried out in three stages: habituation, training, and testing. First, for habituation, the test box was pre-exposed for 10 minutes a day. Second, for training, the rats were placed in a box on the day following the pre-exposure (24 hours later) to allow recognition of two similar objects (A1 and A2) for 5 minutes. After 5 minutes, the rats were removed from the box and familiar objects (A1 and A2) were removed and left for 20 minutes. Finally, for the experiment, the rats were again placed in a box to allow the familiar object (A1) and the new object (B) to be recognized for 3 minutes and the time taken to smell the objects was measured. At this time, when the nose of the rat was positioned at about 2 cm from the object, the object was defined as being exploded.

The total time to search for the object was used to calculate the discrimination index for evaluating cognitive ability, and the equation 1 used in the calculation is shown below.

[Equation 1]

Differential index = (time to send to new object - time to familiar object) / (time to send to new object + time to familiar object)

In this case, the discriminant index is expressed in the range of -1 to 1, -1 indicates the preference only for familiar objects, 0 indicates no preference for two different objects, and 1 indicates preference for new objects it means. On the other hand, the difference in search for two objects is not a deficit of memory, and the differential index indicates that the search activity distinguishes a familiar object from a new object.

Experimental Example 4. Morris water maze test (MWM)

When PTSD is induced, spatial cognitive ability is reduced by depression and anxiety. Therefore, in order to evaluate the spatial cognitive ability of the experimental animals, the Morris water maze test was performed after performing the object recognition test according to the third embodiment.

Specifically, it was carried out in a small circular pool (diameter 2.0 m, depth 0.35 m) made of polypropylene and its interior was painted white. The pool was 30 cm deep, filled with water, and 1 kg of skim milk powder mixed to make the water opaque. The temperature of the water was maintained at 22 ± 2 ° C. The pool was then divided into equivalent quadrants and an escape platform (15 cm in diameter) was placed in one of the four zones, 1.5 cm below the surface of the water and about 50 cm from the wall. To record the behavior of the rats, a digital camera was installed on the ceiling directly above the center of the pool and equipped with a program (S-MART, PanLab Co., Barcelona, Spain) to automatically track the route of movement of the rat to the camera The recording system was connected.

MWM was initiated 16 days after administration of oleruprin and three times daily 30-40 minutes apart. The experiment lasted for a maximum of 180 seconds and the escape latency was expressed as the swimming time to find a submerged platform. At this time, the finding of the escape platform was defined to be located on the escape platform for at least 4 seconds before the end of 180 seconds of the experiment time. On the other hand, when the rat failed to find the escape platform in a limited time on the first attempt, he specified a waiting time of 180 seconds after placing the rat on the platform for 20 seconds.

EXPERIMENTAL EXAMPLE 5. Open field test (OFT)

Before the ORT and MWM of Examples 3 and 4 were completed, an open space test was used to evaluate emotions based on conflict situations. The open space consisted of a square stadium (60 × 60cm) made of opaque plexiglass surrounded by walls (30cm high) and provided illumination through a central light source (60W) on the ceiling. After 5 minutes of habituation, the total distance traveled by the rats in the stadium was measured in cm. All movement activities (laboratory animal movements) were measured with a video camera mounted on the center of the labyrinth and the video tracking system using a computerized S-MART program (PanLab Co., Barcelona, Spain) Were measured. Meanwhile, for another test, the bottom surface of each chamber was thoroughly washed with 70% ethanol.

Experimental Example 6. Measurement of corticosterone (CORT) and inflammatory mediator

The levels of corticosterone and inflammatory mediators in serum were measured. First, the neck of an unspecified rat was cut and blood was rapidly collected through the abdominal aorta. The rats were divided into 6 groups of 4 animals. Blood samples were centrifuged at 4000g for 10 minutes and serum was collected.

Concentrations of corticosterone, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-l [beta]) were measured using a competitive enzyme-linked immunosorbent assay immunosorbent assay (ELISA). A human TNF-α antibody (Abcam, Cambridge, MA, USA), a rabbit polyclonal CORT antibody (Abcam Corticosterone ELISA kit; Cambridge, MA, USA) And IL-1? Antibody (human IL-1b ELISA kit; Abcam).

A sample (or a standard reagent) was added to each well and the plate was incubated for 1 hour at room temperature without blocking. Each well was rinsed several times with buffer and then developed with the appropriate color and the optical density was measured at 450 nm using an ELISA reader (MultiRead 400; Authos Co., Vienna, Austria).

Experimental Example 7. Immunohistochemistry for BDNF and CREB

To perform immunohistochemical tests on BDNF (Brain-derived neurotrophic factor) and CREB (cAMP response element-binding protein), three rats in each group were treated with sodium pentobarbital (80 mg / kg , And saline (0.9%) followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) to perfuse the ascending aorta. The brains were removed in a random order, fixed overnight and then cryopreserved at 4 ° C in 20% sucrose in 0.1 M PBS. 30 [mu] m thick coronal sections were successively sectioned through the hippocampus using a cryostat (Leica CM1850; Leica Microsystems Ltd., Nussloch, Germany) and Paxinos and Watson's rat brain walk (Paxinos G, Watson C. New York : Academic Press; 1986. p. 54-85.) (Hippocampus, bregma -2.6 and -3.6).

BDNF and CREB expression of the fragments was then immunostained using the avidin-biotin-peroxidase complex (ABC) method. Sections were incubated with primary rabbit anti-BDNF antibody (1: 200 dilution, Cell Signaling, Boston, Mass., USA) and rabbit anti-CRB antibody (1: 200 dilution, Cell Signaling) at 4 &lt; 0 &gt; C for 48 hours. The sections were incubated with secondary antibodies for 120 minutes at room temperature. The secondary antibody was obtained from Vector Laboratories Co. (Burlingame, Calif., USA) and diluted 1: 200 in PBST containing 2% normal serum. The sections were incubated in ABC reagent (Vectastain Elite ABC kit; Vector Labs Co., Burlingame, Calif., USA) for 90 minutes, DAB (3,3'- diaminobenzidine, Sigma) and 0.01% And incubated for 1 minute with a solution containing H ₂ O ₂ .

Images were imaged using the AxioVision 3.0 imaging system (Carl Zeiss, Inc., Oberkochen, Germany) and processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA, USA). The sections were observed at 200x magnification. The number of immunopositive cells in at least three different hippocampal sections per rat brain was counted and calculated only when the density reached a darkness above background level. The brown points for BDNF and CREB were observed only in the cytoplasm of the hippocampal cytoplasm, and they were placed anatomically according to the stereotactic rat brain walk of Paxinos and Watson. Brightness and contrast between images were not adjusted to exclude the subjective choice of immune response cells.

Experimental example 8. Total RNA preparation and RT-PCR

Expression levels of TNF-α, IL-1β, CREB and BDNF mRNA were confirmed by reverse transcription polymerase chain reaction (RT-PCR). Brain hippocampal tissue isolated from three rats per group was used. Total RNA was isolated from the above tissues using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA), and complementary DNA was synthesized from the total RNA using reverse transcriptase (Takara Co., Shiga, Japan). PCR was performed using a PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, Mass., USA). All primers were designed using the known mRNA sequence of cytokines and primer design software (Primer 3) provided by Whitehead Biomedical (Cambridge, MA, USA, http://primer3.wi.mit.edu). The PCR products were separated on a 1.2% agarose gel and stained with ethidium bromide. The density of each band was quantified using an image analysis system (i-Max ^TM , CoreBio System Co., Seoul, Korea). Relative density of target bands such as TNF-α, IL-1β, CREB, and BDNF were calculated for GAPDH (glyceraldehyde 3-phosphate dehydrogenase)

Experimental Example 9: Statistical Analysis

All measurements were made through independent investigators who did not know the experimental conditions. Results were expressed as mean ± standard error (SE). Differences within or between normally distributed data were analyzed using ANOVA with SPSS (version 13.0, SPSS, Inc., Chicago, IL, USA) and Tukey's post hoc test was performed. Statistical significance was set at p <0.05.

Example 1. Analysis of Olla-Europa Effect on Changes in Serum CORT Levels

In order to confirm the therapeutic effect of Oleuropein (OLE) on PTSD, single prolonged stress (SPS) was given, and then, according to the method of Experimental Example 6, The concentration of the corticosterone (CORT) was measured for 7 days.

As a result, as shown in FIG. 2, the negative control group (PTSD) exposed to SPS significantly increased the serum CORT concentration, and the amount thereof was found to be 206.54% higher than that of the normal control (CON). However, it was confirmed that serum level of CORT induced by SPS was decreased in the positive control (PTSD + IBU) in which ibuprofen was administered. Especially, in the experimental group (PTSD + OLE100) in which 100 mg / kg of oleruprin was administered, the level of serum CORT induced by SPS was significantly decreased and the level was similar to that of the normal control group.

From the above results, it was confirmed that the SPS process induces an anxiety-like symptom in rats. Therefore, it was confirmed that a rat model in which PTSD was induced by SPS can be produced, and furthermore, the europtera has a corticosterone level in the SPS- (PTSD), respectively.

Example 2. Effect of OLE on cognitive function

Example 2-1. The impact of OLE on object recognition

In order to confirm the therapeutic effect of Oleuropein (OLE) on PTSD, the object recognition ability and memory of the rats belonging to each group were analyzed according to the object recognition test of Experimental Example 3 after the SPS.

As a result, as shown in FIG. 3A, the negative control group (PTSD) exposed to SPS as compared with the normal control group (CON) significantly decreased the sniffing time for a new object by about 7 seconds Respectively. This means that cognitive ability to distinguish objects by SPS decreases and memory capacity decreases.

However, the positive control group (PTSD + IBU) to which ibuprofen was administered after SPS exposure significantly increased the time for smelling new objects which had been reduced by SPS as compared with the PTSD group, and was remarkably increased by about 15 seconds. In particular, (PTSD + OLE100), which was administered in mg / kg, was markedly increased by about 14 seconds for a new object which was reduced by SPS.

As can be seen from FIG. 3B, the negative control group (PTSD) exposed to SPS in comparison with the normal control group (CON) showed a significant reduction of the discrimination index for familiar objects and new objects by about 0.1. A differential index of 0 means that there is no preference for two different objects and a value of 1 indicates preference only for a new object. The reduction of the differential index means that two different objects can not be distinguished. And the cognitive ability to distinguish between the cognitive ability is decreased, and the memory is decreased.

However, the positive control group (PTSD + IBU) to which ibuprofen was administered compared with the PTSD group showed an increase of the discrimination index which was decreased by SPS to about 0.6, and in particular, the test group (PTSD + OLE100 ) Was also found to be significantly higher than that of the normal control group because the discrimination index for the new object which was decreased by SPS was about 0.5.

The above results of FIGS. 3A and 3B show that oleruprin can restore the object recognition ability and memory ability in the PTSD induced rats by SPS, thereby improving the depression and anxiety. Thus, PTSD Can be used as a therapeutic agent.

Example 2-2. Analysis of the effect of OLE on spatial cognition

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

As a result, as shown in FIG. 3C, the negative control group (PTSD) exposed to the SPS in comparison with the normal control group (CON) during 5 days during the experiment showed that the time required for searching the hidden platform was significantly increased Respectively.

However, in comparison with the PTSD group, the time required to visit the platform in which the ibuprofen was administered after the SPS exposure (PTSD + IBU) was reduced by the SPS decreased, and in particular, PTSD + OLE100) was found to be similar to that of the normal control group.

In addition, as shown in FIG. 3 D, the negative control group (PTSD) exposed to SPS in the quadrant where the hidden platform is located is reduced to about 15 seconds in comparison with the normal control group (CON).

However, when compared with the PTSD group, the time taken for the positive control (PTSD + IBU) in which ibuprofen was administered after SPS exposure was reduced to about 28 seconds by SPS, and in particular, the test group administered with 100 mg / (PTSD + OLE100) was also found to be about 28 seconds, which was similar to that of the positive control group and the normal control group.

In addition, as shown in FIG. 3E, the distance between the negative control group (PTSD) exposed to the SPS and the quadrant where the hidden platform is located is reduced to about 15 as compared with the normal control group (CON).

However, the positive control group (PTSD + IBU) to which ibuprofen was administered after SPS exposure increased the distance reduced by SPS to about 25 in comparison with the PTSD group, and in particular, the test group (100 mg / kg) PTSD + OLE100) was also increased by about 26 in the distance reduced by SPS, and was found to be higher than that of the positive control group.

As described above, the results of C to E in FIG. 3 indicate that oleruprin can restore the object recognition ability and memory ability reduced by depression and anxiety in the PTSD induced rats by SPS, And thus it can be used as a therapeutic agent for PTSD.

Example 3: Analysis of the influence of OLE on voluntary athletic activity and search activity

In order to confirm the effect of ollopular pinning on olfactory pins in PTSD, voluntary athletic activities and search activities of rats belonging to each group after SPS were analyzed according to the open space test of Example 5.

As a result, as shown in FIG. 4, the control group (CON), the negative control group exposed to SPS (PTSD), the positive control group (PTSD + IBU) administered with ibuprofen, ) Showed no significant difference in exercise or seeking activity.

These results indicate that cognitive ability or memory impairment due to SPS is not caused by a decrease in athletic activity but by negative emotions such as depression or anxiety. In addition, oleruprin was shown to be effective as a therapeutic agent for PTSD since it exhibits an effect of increasing exercise and searching activity.

Example 4. Effect of OLE on changes in BDNF and CREB expression levels

In order to confirm the therapeutic effect of olerupine on PTSD, the level of expression of BDNF (Brain-derived neurotrophic factor) and CREB (cAMP response element-binding protein) in rats belonging to each group after SPS was measured in Experimental Example 7 .

BDNF and CREB, on the other hand, are proteins found in the hippocampus and play an important role in the pathological state of the central nervous system (CNS). (Shafia S, 2017, Neurobiol Learn Mem. 139: 165-178, Takei S, 2011, J Psychiatr Res 45: 223-241). In addition, it has been reported that patients with depression, obsessive compulsive disorder, 460-468).

As a result, as shown in FIGS. 5A to 5E, the negative control group (PTSD) exposed to SPS as compared to the normal control group (CON) showed that cells that immunoreacted with BDNF and CREB in both hippocampal CA1 and CA3 Respectively, to 64.15 and 61.00%, respectively.

However, when compared to the PTSD group, the number of immunoreactive cells that were reduced by SPS was significantly increased in the experimental group (PTSD + OLE100) in which 100 mg / kg of oleruprin was administered after SPS exposure, In the experimental group (PTSD + OLE100) administered with 100 mg / kg, the number of cells was increased by SPS to a similar level to that of the positive control group and the normal control group.

In addition, for each group of rats, mRNA expression levels of BDNF and CREB in the hippocampus were analyzed according to the method of Experimental Example 8.

As a result, as shown in D and E of FIG. 5, in the test group (PTSD + OLE100) in which 100 mg / kg of oleruprin was administered after the SPS exposure, the amount of mRNA expression of BDNF and CREB decreased by SPS was increased Levels were similar to the normal control (CON) and positive control (PTSD + IBU).

From the above results of FIGS. 5A to 5E, oleruprin can be used as a therapeutic agent for PTSD since it can restore the levels of reduced BDNF and CREB in hippocampus of rats induced by PTSD by SPS Could know.

Example 5. Effect of OLE on changes in the expression level of inflammatory mediators

In order to confirm the therapeutic effect of oleruprin on PTSD, protein and mRNA levels of inflammatory mediators (TNF-α and IL-1) in rats belonging to each group after the administration of SPS were measured by the method of Experimental Examples 6 and 8 Respectively.

First, after conducting behavioral experiments, the amount of TNF-α and IL-1β protein present in the serum of rats was analyzed.

As a result, as shown in FIGS. 6A and 6B, the negative control group (PTSD) exposed to SPS in comparison with the normal control group (CON) showed protein concentrations of TNF-α and IL-1β increased by 299.69 and 242.33% Respectively.

However, in comparison with the PTSD group, the protein concentration of TNF-α and IL-1β, which were increased by SPS, was significantly reduced in the experimental group (PTSD + OLE100) in which 100 mg / kg of oleruprin was administered after SPS exposure , And the experimental group (PTSD + OLE100) in which oleruprin 100 mg / kg was administered also showed that the protein concentration increased by SPS was similar to that of the positive control group and the normal control group.

Then, the amount of mRNA of TNF-α and IL-1β present in the rat hippocampus was analyzed.

As a result, as shown in FIGS. 6C and 6D, in the experimental group (PTSD + OLE100) in which 100 mg / kg of oleruprin was administered after the SPS exposure, the amount of TNF-α and IL-1β mRNA expression , And the level was similar to that of the normal control (CON) and the positive control (PTSD + IBU).

6A to 6D, oleruprin is able to reduce the levels of inflammatory mediators (TNF-α and IL-1β) increased in the PTSD-induced rats by SPS, Can be used as a therapeutic agent.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (10)

A pharmaceutical composition for preventing or treating cognitive dysfunction, which comprises oleuropein or a pharmaceutically acceptable salt thereof as an active ingredient, which is administered to a subject suffering from a decrease in BDNF in the hippocampus by post-traumatic stress disorder. 2. The pharmaceutical composition according to claim 1, wherein the ollauron pin is represented by the following formula (1).
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the oleruprin fins increase the level of BDNF (Brain-derived neurotrophic factor) or CREB (cAMP response element-binding protein) in the hippocampus. 2. The pharmaceutical composition according to claim 1, wherein the oleruprin fins reduce the level of TNF-alpha and IL-1 beta (interleukin-1 beta). The pharmaceutical composition according to claim 1, wherein the oleruprin pin is included in the composition in an amount of 0.0001 to 80% by weight. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier. A health functional food composition for prevention or amelioration of hypo-cognitive impairment by administering oleuropein as an active ingredient to an individual whose BDNF has been reduced in the hippocampus by post-traumatic stress disorder. 8. The health functional food composition according to claim 7, wherein the ollaur pin is contained in the composition in an amount of 0.01 to 80% by weight. A method for preventing or treating hypo-cognitive dysfunction caused by post-traumatic stress disorder, comprising administering the pharmaceutical composition of any one of claims 1 to 6 to a subject other than a human. 10. The method according to claim 9, wherein the administration is administering oleruprin at 80 to 120 mg / kg.

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