KR102239947B1 - Compositions for preventing, improving or treating cognition and stress-related disease comprising cephalotocin - Google Patents

Abstract

The present invention relates to a novel use of the octopus-derived peptide, and more particularly, to a composition for the prevention, improvement or treatment of cognitive dysfunction and stress-related diseases comprising the octopus-derived peptide as an active ingredient. It was confirmed that the octopus-derived peptide according to the present invention increases calcium in astrocytes, reduces the dwell time in the corner of the chamber in the open field test, and maintains the movement at a level similar to that of oxytocin in the tail hanging test. This means that separotocin has an effect of improving cognitive function and reducing stress, and can be used in various fields in pharmaceuticals and foods.

Description

Compositions for preventing, improving or treating cognition and stress-related disease comprising cephalotocin

The present invention relates to a novel use of the octopus-derived peptide, and more particularly, to a composition for the prevention, improvement or treatment of cognitive dysfunction and stress-related diseases comprising the octopus-derived peptide as an active ingredient.

One of the important causes of memory and cognitive decline in degenerative brain diseases such as Alzheimer's dementia is a lack of acetylcholine due to degeneration of cholinergic neurons. This phenomenon is further aggravated by an increase in the activity of acetylcholinesterase (AChE), an acetylcholine-degrading enzyme. Therefore, AChE inhibitors such as tacrine, donepezil, galantamine, and rivastigmine have been developed as treatments for Alzheimer's disease. However, since drugs that inhibit brain cell damage or directly regenerate brain cells have not been developed, it is urgent to develop an effective and safe memory and cognitive function improvement agent.

On the other hand, separotocin is a peptide secreted from Octopus vulgaris and belongs to the vasopressin/oxytocin superfamily. In previous studies, it has been reported that injecting separotocin to cuttlefish belonging to the cephalopod of invertebrates such as octopus increases long-term memory, and injecting separotocin to octopuses belonging to the same invertebrate octopus regulates the osmotic pressure of blood lymph. there was. However, the effect of administration of separotocin to vertebrates has not been previously studied, and it is known that vertebrates may have a different pharmacological reactivity than invertebrates from various cases including anesthetic studies. In the case of human vasopressin and oxytocin belonging to the same superfamily, two out of nine amino acid sequences are different and thus have different functions. In addition, felipressin obtained by modifying two amino acid sequences of vasopressin is used for dental treatment. From this, it can be seen that even peptides belonging to the same superfamily have different effects and uses depending on the amino acid sequence.

Accordingly, the present inventors completed the present invention by confirming the cognitive function improvement and anti-stress effect of separotocin, an octopus-derived peptide, in order to solve the problems of the prior art as described above.

Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating cognitive dysfunction comprising as an active ingredient separotosine represented by the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a composition for preventing, improving or treating stress-related diseases, comprising as an active ingredient separotocin represented by the amino acid sequence of SEQ ID NO: 1.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising separotocin represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating stress-related diseases comprising as an active ingredient separotocin represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a food composition for preventing or improving cognitive dysfunction comprising as an active ingredient separotosine represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a food composition for preventing or improving stress-related diseases comprising as an active ingredient separotocin represented by the amino acid sequence of SEQ ID NO: 1.

It was confirmed that the octopus-derived peptide according to the present invention increases calcium in astrocytes, reduces the residence time in the corner of the chamber in the open field test, and maintains the movement at a level similar to that of oxytocin in the tail hanging test. This means that separotocin has an effect of improving cognitive function and reducing stress, and can be used in various fields in pharmaceuticals and foods.

1 is a diagram showing the results of confirming the effect of improving cognitive function of separotocin through an open field test.
Figure 2 is a diagram showing the results of analyzing the calcium signal according to the treatment of separotocin at the cellular level (a: astrocytes, b: myelin sheath cells, c: neurons).
3 is a schematic diagram of a method for confirming the stress reduction effect of separotocin through a tail suspension test (TST).
4 is a diagram showing the results of confirming the stress reduction effect of separotocin through a tail hanging experiment.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides a composition for preventing, improving or treating cognitive dysfunction comprising as an active ingredient separotosine represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a composition for preventing, improving, or treating stress-related diseases comprising as an active ingredient separotocin represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, separotocin may be an octopus-derived peptide, and in particular, as a peptide secreted from Octopus vulgaris , it belongs to the vasopressin/oxytocin superfamily. The present inventors analyzed the full-length genome and transcript information of the previously published California two-spotted octopus ( Octopus bimaculoides ) and octopus ( Octopus minor ), and confirmed that they also have genes that are annotated as separotocin, and these It was predicted that the separotosine active peptide portion of was predicted to have the same amino acid sequence as that of the octopus. Since the octopus-derived cephalotosine dealt with in the present invention has two amino acids different from vasopressin and three different from oxytocin, it is apparent that the effectiveness and effect of cephalotosin in mammals can also be known only through actual experiments.

Separotosine according to the present invention may be in a form in which the N-terminus or C-terminus is modified or protected by several organic groups in order to protect against protein cleavage enzymes in vivo and increase stability. That is, if the C-terminus of the separotosine is in a form that can be modified to increase stability, there is no particular limitation, but it may be preferably modified with a hydroxy group (-OH) or an amino group (-NH ₂ ). In addition, as long as the N-terminus of the separotosine is in a form that can be modified to increase stability, there is no particular limitation, but preferably an acetyl group, a fluorenyl methoxy carbonyl (Fmoc) group, or a formyl (Formyl) ) Group, palmitoyl group, myristyl group, stearyl group, and polyethylene glycol (PEG).

In an embodiment of the present invention, the concentration of the separotosine is preferably 0.1 to 10 mg/kg, more preferably 1 to 5 mg/kg, more preferably 2 mg/kg.

In the present invention, it was confirmed that separotocin increases calcium in astrocytes, reduces the time that the rat stays in the corner of the chamber in the open field test, and that the movement of the rat is maintained at a level similar to that of oxytocin in the tail hanging test. I did. This means that separotocin has an effect of improving cognitive function and reducing stress, and thus it can be used in various fields of pharmaceuticals and food.

In the present invention, cognitive dysfunction refers to a disease in which cognitive functions such as memory processing, perception, and problem solving are not exhibited.

In an embodiment of the present invention, the cognitive dysfunction is preferably a degenerative brain disease, more preferably stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob ) It is one or more selected from the group consisting of disease, ischemic brain disease, and stroke.

In another embodiment of the present invention, the cognitive function disorder may be a learning disorder or a memory disorder.

In the present invention, stress-related disease refers to a physical or mental disease caused by stress.

In an embodiment of the present invention, the stressful disease is stress anxiety, depression, fatigue syndrome, headache, neurodegenerative disease, eating disorder, sleep disorder, anorexia nervosa, gastritis, gastric ulcer, drug addiction, drug withdrawal syndrome and alcohol withdrawal. It is preferable that it is at least one selected from the group consisting of syndrome diseases, and more preferably, it may be depression.

In an embodiment of the present invention, the composition according to the present invention may further comprise one or more known ingredients having a preventive, ameliorating or therapeutic effect of cognitive dysfunction.

In an embodiment of the present invention, the composition according to the present invention may further include one or more known ingredients having an effect of preventing, improving or treating stress-related diseases.

In an embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition or a food composition.

When the composition is a pharmaceutical composition, it may further include a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of separotosine. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration may be used.

Suitable dosages of the pharmaceutical composition of the present invention include formulation method, mode of administration, age, weight, sex, degree of disease symptoms, food, administration time, route of administration, excretion rate and response sensitivity of the subject animal and patient. It varies by factors, and usually an experienced physician or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

Formulations for oral administration comprising the pharmaceutical composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. It can be formulated as zero. For formulation into formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate , Lubricating oil such as calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned substances.

Formulations for parenteral administration containing the pharmaceutical composition of the present invention as an active ingredient include injectable forms such as subcutaneous injection, intravenous injection, or intramuscular injection, suppository injection method, or aerosol that enables inhalation through the respiratory tract, etc. It can be formulated for spray. In order to formulate a formulation for injection, the composition of the present invention may be prepared as a solution or suspension by mixing in water together with a stabilizer or buffer, and formulated for unit administration of ampoules or vials. When formulated for spraying such as an aerosol, a propellant or the like may be blended together with an additive so that the water-dispersed concentrate or wet powder is dispersed.

When the composition is a food composition, it can be used as a health functional food, food additive or dietary supplement. When used as a food additive, the separotocin may be added as it is, or may be appropriately used according to a conventional method, such as being used by mixing with other foods or food ingredients.

In addition, the mixing of the additional active ingredients may be suitably changed depending on the purpose of use (prevention, health or therapeutic treatment), and it is preferable to be included in 0.001 to 99.9% by weight based on the total weight of the food composition, and more It is preferably contained in 1 to 80% by weight. If the content is less than 0.001% by weight, the efficiency of intake may decrease, and if it exceeds 99.9% by weight, it is difficult to formulate.

As a specific example, when preparing food or beverage, the separotosine of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, it may be added in an amount below the above range, and since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range. have. There is no particular limitation on the type of the food, but examples of foods to which the separotocin of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream. Dairy products including, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the usual sense.

When the food composition of the present invention is prepared as a beverage, it may include additional ingredients such as various flavoring agents or natural carbohydrates, like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

The food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid. It may include a carbonation agent used in beverages, and may include flesh for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage, but is not limited thereto. These components may be used independently or in combination. The ratio of the additives is not largely limited, but is preferably contained within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the food composition of the present invention can be taken for a long time because there is no problem in terms of safety.

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

Example 1. Confirmation of the effect of improving cognitive function of Separotocin through an open field test

In order to confirm the effect of improving cognitive function, an open-field test was performed using an experimental animal administered with separotocin. The separotosine is an octopus-derived peptide and is represented by the amino acid sequence of SEQ ID NO: 1.

First, C57BL/6NCrlOri male rats (7 weeks) were purchased from Orient Bio Co., Ltd. (Seongnam, Korea). The purchased rats were allowed to acclimatize to the new breeding environment for about 8 days. Habitations were recorded to evaluate the amount of gait activity during the acclimatization period. All animals were reared at 4 to 5 per cage at a light/dark cycle of 12 hours, and were reared in an environment that allowed free access to food and water. Animal experiments in this example were conducted in accordance with the safety evaluation laboratory standard work instructions, test animal welfare method, and experimental animal management and use guide.

The prepared rats were divided into a physiological saline-administered group (VC), an ethanol-administered group (PC), and a separotocin-administered group (T1). Each experimental group was administered intraperitoneally with each substance at the doses disclosed in Table 1.

After administration of each test substance, an open field test (OFT) in dark conditions using each experimental animal according to the method described in Denenberg, et al. (1969) and Sturman O, et al. (2018). Was carried out. Specifically, the experimental animals administered with the test substance were 30 in a gait activity chamber (SAC-283422-NIR, 71.1x86.4x55.9cm) in a sound attenuating cubicle (Med associates, Allectown, PA, USA). Let it acclimate for a minute. After the adaptation was completed, data on the amount of gait activity was collected for 60 minutes through the infrared camera (NIR-100, Med associates) at the top of the gait activity level chamber. The collected gait activity data were analyzed using the EthoVisionXT program, and the results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that the time to stay in the corner of the chamber in the separotocin-administered group (T1) was increased compared to that of the physiological saline-administered group (VC). This example is an open field test measured in a dark condition, and the result means that separotocin improved the exploratory ability (ie, cognitive ability) of the experimental animal.

Example 2. Analysis of calcium signal according to treatment with separotocin in glial cells

Neurons used in this example were obtained from the brain of rat P2. Specifically, neurons were isolated by treating rat brain tissue with 2 ml of 0.05% trypsin enzyme. The isolated nerve cells were seeded at a concentration of ³ ×10 5 cells/ml on a cover glass coated with 0.1 mg/ml poly D-lysine. The seeded neurons were maintained for 14 days with neurobasal+1X B27 medium, and the medium was added or exchanged every 2 days. Astrocytes were obtained from the rat P2 brain in the same manner as the neurons, and were cultured in DMEM (10% FBS, 10% horse serum) medium. Myelin sheath cells were obtained in the same manner as the neurons and maintained in DMEM medium containing 10% FBS.

12 days after seeding, each cell was treated with separotocin. The cells were stirred at 500 rpm for 1 hour to obtain a supernatant of each cell, which was incubated for 10 minutes in a Petri dish. The cultured supernatant was centrifuged at 2000 rpm for 10 minutes and then seeded on a coverslip at a concentration of 2 ^{×10 5 cells/ml.} After 1 day, each cell was subjected to calcium imaging. Specifically, for the calcium imaging, 5uM Fura-2 was dissolved with 5ul of fluronic acid and then added to 1ml Hepes buffer to prepare a mixture. The prepared mixture was treated with cells seeded on coverslips, and reacted at room temperature for 40 minutes. After the reaction was completed, the supernatant was removed and exchanged with 1X Hepes buffer. After placing the coverslip on an inverted microscope, fura-2 was activated with wavelengths of 340 and 380 nm. The detected signal was expressed in a ratio of 340 nm/380 nm. A buffer was prepared in the chamber connected to the channel 1, and a solution containing separotosine was prepared in the chamber connected to the channel 2, and the calcium signal was analyzed by alternating the chamber. The calcium signal analysis results are shown in FIG. 2.

As shown in FIG. 2A, when the astrocytes were stimulated with separotocin (013V), the concentration of calcium increased, which was similar to the increase in calcium caused by ATP and oxytocin.

In addition, as shown in Figure 2b, myelin cells (oligodendrocyte precursor cell, OPC) did not respond to separotosine (013V), but it was confirmed that there is a response to acetylcholine (acetylcholine, Ach).

In addition, as shown in Figure 3c, it was confirmed that the nerve cells do not have the capacity to accept separotocin (013V).

The above results indicate that separotocin specifically acts on the oxytocin receptor of astrocytes.

Example 3. Confirmation of the stress reduction effect of Separotocin through tail hanging test

A tail suspension test (TST) to confirm the stress reduction effect of separotocin was performed as shown in FIG. 3. The tail hanging test is known as a test to evaluate the stress, and if the stress persists, the movement of the rat decreases.

Specifically, C57BL/6NCrlOri male rats (7 weeks) were purchased from Orient Bio Co., Ltd. (Seongnam, Korea). The purchased rats were allowed to acclimatize to the new breeding environment for about 5 days. Rats were reared at 4 to 5 per cage in a 12-hour light/dark cycle, and were reared in an environment where free access to food and water was possible. Animal experiments in this example were conducted in accordance with the safety evaluation laboratory standard work instructions, test animal welfare method, and experimental animal management and use guide.

A pre-test of the tail hanging test was performed using rats that were purified for 5 days after acquisition. In addition, on the 7th day after acquisition, stereotaxic surgery for mounting a guide cannula was performed. Specifically, the surgery was performed using an intracranial positioning device, and the needle portion of the guide cannula was positioned at the CA1 coordination coordinates of the rat hippocampus (AP -4mm, ML 2mm, DV 3mm). The cement was mixed to fill the periphery of the guide cannula and then dried. After the cement was dried, the holder of the guide cannula was removed. The desired solution can be administered to the hippocampus through an installed guide cannula. After 6 days of surgery (13 days after receipt), 5 ml of 0.1 mM separotocin was administered, and a tail hanging test was performed. In the tail hanging test, 5ml of saline was administered to the negative control group instead of separotocin, and oxytocin was administered to the positive control group. The results of the tail hanging test are shown in FIG. 4.

As shown in FIG. 4, it was confirmed that the negative control group to which saline was administered had very little movement due to stress. On the other hand, even though the separotocin-administered group performed the tail hanging test, it was confirmed that movement was statistically significant (*1 P=0.0354, *2 P=0.0434). It was confirmed that the effect of increasing movement of the separotocin-administered group was similar to that of the positive control group. The above results indicate that separotocin reduces the stress caused by the tail hanging test, that is, has an anti-stress effect.

Overall, the present inventors found that separotocin increases calcium in astrocytes, reduces the time the rat stays in the corner of the chamber in the open field test, and that the rat's movement is maintained at a level similar to that of oxytocin in the tail hanging test. Confirmed. This means that separotocin has an effect of improving cognitive function and reducing stress, and can be used in various fields in pharmaceuticals and foods.

Hereinafter, the present invention will be described in more detail through formulation examples. Formulation examples are for illustrative purposes only, and are not to be construed as limiting the scope of the present invention.

Formulation Example 1. Preparation of pharmaceutical composition

1-1. Preparation of powder

Separotocin 20 mg

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

Separotocin 10 mg

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

1-3. Preparation of capsules

Separotocin 10 mg

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

1-4. Preparation of injections

Separotocin 10 mg

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO ₄ 2H ₂ O 26 mg

It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.

1-5. Preparation of liquid

Separotocin 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of the liquid formulation, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill in a brown bottle. It is sterilized to prepare a liquid formulation.

Above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY National Marine Biodiversity Institute of Korea <120> Compositions for preventing, improving or treating cognition and stress-related disease comprising cephalotocin <130> 1.8P-1 <150> KR 1020190048492 <151> 2019-04-25 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Octopus vulgaris <400> 1 Cys Tyr Phe Arg Asn Cys Pro Ile Gly 1 5

Claims (10)

delete delete delete delete delete delete Containing separotosine represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
A pharmaceutical composition for preventing or treating depression. delete delete Containing separotosine represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
Food composition for preventing or improving depression.

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