KR102586388B1 - Composition for ameliorating memory or cognitive ability comprising Trigonotis peduncularis extract as effective component - Google Patents

Abstract

The present invention relates to a composition for improving cognitive function or memory containing an extract of flower marigold as an active ingredient. The extract of flower marigold, which is an active ingredient of the present invention, increases the expression level of p-CREB in neurons of hippocampal tissue of fetal rats and reorganizes synapses. Not only does it increase the expression level of related proteins, it also increases long-term and short-term memory in animal models, and increases the expression level of synaptic reorganization factor and neuroplasticity factor proteins, making it a medicine, health functional food, or medicine to improve cognitive function or memory. It can be usefully used as a feed additive, etc.

Description

Composition for improving cognitive function or memory comprising Trigonotis peduncularis extract as an effective component {Composition for ameliorating memory or cognitive ability comprising Trigonotis peduncularis extract as effective component}

The present invention relates to a composition for improving cognitive function or memory containing flower marigold extract as an active ingredient.

Cognitive impairment refers to mental and behavioral disorders caused by brain disease, brain injury, addiction, aging, etc., and mental disorders caused by endocrine system abnormalities, metabolic and nutritional abnormalities, drugs, etc. Depending on the severity, it is a type of brain disease that occurs not only in amnesia, which is a temporary short-term memory disorder, but also in neurodegenerative diseases such as cognitive dysfunction and dementia that cause impaired judgment.

Mild cognitive impairment refers to a condition in which cognitive ability is reduced compared to people of the same age. Cognitive areas such as memory, language ability, and judgment are deteriorated. Mild cognitive impairment is a syndrome with various causes and symptoms, and is caused by various causes such as degeneration due to aging, psychiatric factors such as vascular depression or anxiety due to vascular causes, and other medical factors.

In addition, as we become an aging society, the number of elderly people has increased rapidly. In this social environment, senile dementia and Parkinson's disease are recognized as degenerative central nervous system diseases, and as the number of patients increases day by day, social issues increase and prevention efforts are needed accordingly. , the importance of improvement and treatment is emerging day by day.

Patients with Alzheimer's disease have abnormalities in almost all nerves, including cholinergic, adrenergic, GABAergic, and glutamatergic nerves, but damage and loss of cholinergic nerves has been studied to be the most serious. Meanwhile, senile dementia, a disease in which cognitive ability is gradually lost, is also related to the activity of cholinergic neurons in the central nervous system, and this is mainly caused by a significant decrease in the activity of acetylcholine and choline acetyltransferase in the brain. It is known. Accordingly, the development of treatments is being actively conducted in the direction of recovery and improvement to complement the ideal cholinergic nerve.

Meanwhile, Trigonotis peduncularis is a biennial plant of the dicotyledonous plant Lamiaceae family Boraginaceae and is also called pine radish. It grows in fields, field banks, or roadsides, has stems that grow up to 10-30cm, has short hairs all over, and splits into several pieces at the bottom. The leaves that emerge from the roots have long petioles, grow in clusters, and are egg-shaped or oval-shaped. The leaves coming out of the stem are alternate, have a long oval shape or a long egg shape, have smooth edges, and do not have petioles. Flowers bloom in light sky blue from April to July and form racemes at the end of the stem. The upper part of the inflorescence is curled, and as it unwinds like a spring, flowers bloom one after another from the bottom. The calyx is split into 5 pieces, and the split pieces are triangular and have hairs. The corolla is about 2mm in diameter and splits into 5 pieces. There are 5 surgeries. The fruit is a meristematic fruit divided into four divisions, has a short stalk, and is surrounded by a calyx. The segment is smooth and the top is pointed. Young shoots are used as vegetables. In oriental medicine, it is used as a medicine for muscle paralysis of the hands and feet, enuresis, colitis, dysentery, and boils. Distributed throughout Korea and in temperate and subtropical regions of Asia.

As for technology related to Flower Marigold, Korean Patent No. 2260037 discloses a composition for the prevention or treatment of bone disease containing the Extract of the Flower Marigold as an active ingredient, and Korean Patent Publication No. 2023-0106452 discloses a composition for the prevention or treatment of Bone Disease extract as an active ingredient. Cosmetic compositions containing antioxidant, anti-inflammatory, skin whitening, or wrinkle prevention and improvement have been disclosed, but no composition for improving cognitive function or memory containing the flower of the flower extract of the present invention as an active ingredient has been disclosed yet.

The present invention was developed in response to the above-mentioned needs, and the present invention provides a composition for improving cognitive function or memory comprising a flower stalk extract as an active ingredient, and the active ingredient, the flower stalk extract of the present invention, is used in the hippocampal tissue nerves of fetal rats. It not only increases the expression level of p-CREB in cells and the expression level of proteins related to synaptic reorganization, but also increases long-term and short-term memory in animal models, and increases the expression level of synaptic reorganization factor and neuroplasticity factor proteins. By confirmation, the present invention was completed.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction containing Trigonotis peduncularis extract as an active ingredient.

In addition, the present invention provides a health functional food composition for improving cognitive function or memory containing Trigonotis peduncularis extract as an active ingredient.

In addition, a feed additive for improving cognitive function or memory containing flower marigold extract as an active ingredient is provided.

In addition, a veterinary composition for preventing or treating cognitive dysfunction containing flower marigold extract as an active ingredient is provided.

The present invention relates to a composition for improving cognitive function or memory containing an extract of flower marigold as an active ingredient. The extract of flower marigold, which is an active ingredient of the present invention, increases the expression level of p-CREB in neurons of hippocampal tissue of fetal rats and reorganizes synapses. It not only increases the expression level of related proteins, but also increases long-term and short-term memory in animal models, and increases the expression levels of synaptic reorganization factor and neuroplasticity factor proteins.

Figure 1 is a photograph showing the freeze-dried powder of the flower stalk extract, which is an active ingredient of the present invention.
Figure 2 shows the results of confirming the effect of the flower extract on the level of CREB phosphorylation, which affects neuronal plasticity, in brain cells obtained from primary culture of hippocampus tissue of fetal rats. *** indicates a statistically significant increase in the p-CREB expression level of the positive control group (AMPA) or the group treated with the flower extract of the present invention compared to the control group (CTL), p<0.001.
Figure 3 is a Western blot result confirming the effect of the hot water extract of Flower Marigold on the expression of neuronal NMDA receptors (Subunits: NR2A, NR2B) and PSD95 protein.
Figure 4 shows the results confirming the effect of the flower extract on long-term memory (A) and short-term memory (B) through the Morris water maze test in SD-rats. *, ** indicate a statistically significant increase in long-term or short-term memory in the group administered the flower marigold extract of the present invention compared to the control group, * is p < 0.05, and ** is p < 0.01.
Figure 5 shows the results of a probe test derived from the Morris water maze test, showing the swimming time (A) in the quadrant where the escape zone was and the distance traveled to find the escape zone (B). It is shown. *, **, *** indicate a statistically significant increase in swimming time in the quadrant where the escape zone was located in the flower flower extract administration group (TP50, TP100 and TP200) compared to the control group, * is p < 0.05, ** is p<0.01, and *** is p<0.05. In Figure 5B, arrow S indicates the starting point.
Figure 6 shows Western blot results confirming changes in protein expression levels of NMDA receptors (NR2A, NR2B), AMPA receptors (GluA1), and PSD95 in hippocampal tissue extracted from SD-rats following administration of the flower stalk extract of the present invention.
Figure 7 shows the expression levels of ERK and CaMKII proteins in hippocampal tissue extracted from SD-rats, according to administration of the flower stalk extract of the present invention; And the results of confirming changes in p-ERK and p-CaMKII protein expression levels (A), and the results of confirming BDNF protein expression levels (B).

The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction containing Trigonotis peduncularis extract as an active ingredient.

The cognitive dysfunction is any one selected from Alzheimer's disease, cerebrovascular dementia, Pick's disease, Creutzfeldt-Jakob disease, dementia caused by head injury, and Parkinson's disease. It is preferable, but is not limited to this.

The pharmaceutical composition containing the flower marigold extract according to the present invention is preferably, but not limited to, any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. In addition to the above flower stalk extract, it may further contain a pharmaceutically acceptable carrier, excipient, or diluent. Carriers, excipients, or diluents that may be included in the pharmaceutical composition containing the flower stalk extract include lactose, dextrose, sucrose, and oligosaccharide. , sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl Examples include hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. The preferred dosage of the flower extract of the present invention varies depending on the patient's condition and weight, degree of disease, drug type, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the pharmaceutical composition containing the flower extract of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably at 0.001 to 10 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

In addition, the present invention relates to a health functional food composition for improving cognitive function or memory containing Trigonotis peduncularis extract as an active ingredient.

The flower extract may be prepared by a method including the following steps, but is not limited to this:

(1) extracting dried flowers by adding an extraction solvent;

(2) filtering the extract of step (1); and

(3) Preparing an extract by drying the filtered extract of step (2).

In step (1), the extraction solvent is preferably selected from water, C ₁ -C ₄ lower alcohols, or mixtures thereof, more preferably water or ethanol, and even more preferably water, but is not limited thereto. In the above manufacturing method, all conventional methods known in the art can be used as the extraction method, such as hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. It is preferable to extract the extraction solvent by adding 5 to 40 times the weight of the flower. The extraction temperature is preferably 20 to 100°C, but is not limited thereto. In addition, the extraction time is preferably 1 to 5 hours, and more preferably 2 to 4 hours, but is not limited thereto. In step (3), the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, and more preferably freeze drying, but is not limited thereto.

The extract of the cauliflower is preferably an extract of cauliflower leaves, flowers, stems, above-ground parts, underground parts, or whole plants, and more preferably is an extract of cauliflower leaves, but is not limited thereto.

The health functional food composition is preferably manufactured in a dosage form selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto. The health functional food composition of the present invention can be prepared by adding the flower extract as is or mixing it with other foods or food ingredients, and can be prepared appropriately according to conventional methods. Examples of foods to which the flower marigold extract can be added include caramel, meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. , it may be in any form selected from drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense. That is, there are no particular restrictions on the type of food. The health functional food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, it may contain pulp for the production of natural fruit juice and vegetable drinks. The above components can be used independently or in combination.

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, and the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, dextrin, and cyclodextrin. Polysaccharides such as xylitol, sorbitol, and erythritol are sugar alcohols. The ratio of the natural carbohydrate is not very important, but is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, per 100 g of the composition of the present invention, but is not limited thereto. As sweeteners, natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.

Additionally, the present invention relates to a feed additive for improving cognitive function or memory containing flower marigold extract as an active ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals. The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.

In addition, the present invention relates to a veterinary composition for the prevention or treatment of cognitive dysfunction containing flower marigold extract as an active ingredient.

The veterinary composition of the present invention may further include appropriate excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, ethanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, Examples include polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anti-aggregants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention provides rapid and sustained release of the active ingredient after administration to an animal. or may be formulated using methods well known in the art to provide sustained release, the dosage form being powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, It may be in the form of a suppository, sterile injectable solution, or sterile topical medication. The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. Severity of the disease or condition, sensitivity to the active ingredient of the present invention depending on the individual's age, weight, health condition or gender, administration route, administration period, factors including other compositions mixed or used simultaneously with the composition, and other physiological or It can be determined based on factors well known in the veterinary field.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Example 1. Preparation of flower mariage hot water extract

15 liters of distilled water was added to 500 g of dried flower stalk leaves and heated at 100°C for 3 hours to perform hot water extraction. The extracted Kkotmari hot water extract was concentrated under reduced pressure and freeze-dried to obtain 59.2 g of Kkotmari hot water freeze-dried material.

Example 2. Primary culture of fetal rat hippocampus neurons

The cells used in Examples 3 and 4 of the present invention were primary cultured Hippocampal neurons, which were isolated from SD rat fetuses on days 18 and 19 of pregnancy. The separated hippocampal nerve tissue was placed in HBSS (Hank's Balanced Salt Solution) containing 0.25% trypsin and reacted at 37°C for 10 minutes. After washing several times with HBSS, the cells were carefully separated into single cells using a 1 ml pipette. Afterwards, the separated cells were loaded onto a culture dish previously coated with poly-L-lysine (0.5mg/ml), and 0.5mM L-glutamine, 25μM glutamate, and 25μM 2-mergaptoethanol (2 -mercaptoethanol), 100U/ml penicillin, and 100㎍/ml streptomycin were filled with Neuro basal/B27 medium and cultured at 37°C and 5% CO ₂ conditions. The medium was changed on days 4 and 11 of cell culture, and cells from the 14th day of culture were used in the experiment.

Example 3. Confirmation of CREB (cAMP-response element binding proein) phosphorylation level in hippocampal neurons of fetal rats

1 hour before treatment with the flower stalk extract, in Example 2, the medium of primary cultured hippocampal neurons cultured for 14 days was exchanged with medium without Neuro basal/B27. As a positive control group, 100 μM AMPA, an agonist of the AMPA receptor (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor), was used, and 3, 10, and 30 μg/ml of water extract of the flower were used for 10 minutes each. After treatment, the cells were lysed after washing with ice-cold PBS. The dissolved cell fluid was quantified according to the BCA protein quantification method, and the same amount was measured using the phospho-CREB (Ser133) sandwich ELISA Kit (Cell signaling) according to the manufacturer's instructions.

As a result, as shown in Figure 2, the level of CREB phosphorylation in the experimental group and positive control group treated with 3, 10, and 30㎍/㎖ of flower stalk extract compared to the control group was statistically significantly increased.

Example 4. Confirmation of expression level of factors involved in synapse remodeling in hippocampal neurons of fetal rats

In Example 2, primary cultured hippocampal neurons cultured for 14 days were treated with 3, 10, and 30 ㎍/㎖ flower stalk extract each for 1 minute, washed with ice-cold PBS, and then washed with Syn-PER ^TM synaptic protein. Using an extraction reagent, cells were lysed and subjected to Western blotting.

As a result, as shown in Figure 3, compared to the control group that was not treated with anything, the NMDA (N-methyl-D-aspartate) receptors (NR2A and NR2B) of the experimental group treated with 3, 10, and 30㎍/㎖ of the flower stalk extract of the present invention ) and PSD95 (postsynaptic density protein 95) protein expression increased.

Example 5. Morris water maze test

(1) Animal model

The 6-week-old male SD-rat used as an animal model was purchased from SAMTACO, Korea. After purchase, it was kept in an animal breeding room under certain conditions (temperature: 22 ± 2°C, humidity: 505%, light and dark: 12 hours). After acclimating to the light/dark cycle for a week, it was used in the experiment.

(2) Escape latency analysis

For behavioral evaluation of experimental animals, male SD-rats were orally administered the Komori extract (50, 100, and 200 mg/kg B.W.) of the present invention once a day for 21 days, and no Komalia extract was administered as a control group. An untreated group was used.

In the Morris water maze experiment, a circular pool (diameter: 180 cm, height: 75 cm) was filled with 25 ± 1 cm of water, and an escape platform (24 cm) was hidden 1 cm below the water surface. Spatial clues were installed on all four sides of the wall to confirm the location, and skim milk powder was dissolved in water to make the escape zone invisible to the naked eye. One day before the experiment, all experimental groups were allowed to swim freely for 1 minute in a swimming pool without an escape platform to acclimate them to swimming.

On the first day of this experiment, animals were started in a random order from two of the three quadrants excluding the quadrant where the escape zone was located and were asked to find the escape zone for 60 seconds. If the animal found an escape platform, it was allowed to stay on it for 20 seconds. If it did not find an escape platform within 45 seconds, the experimenter guided the animal to the escape platform by hand and made it stay there for 20 seconds. After 20 seconds of location learning, the animal was immediately started from a random quadrant and the time and distance traveled to the escape area were measured. After finding an escape platform and climbing up, 20 seconds were given and this was done continuously, a total of 3 times a day, for 3 days. The first trial in each experimental day is an indicator of long-term memory, and the last trial, the third swim in each experimental day, is an indicator of short-term memory.

However, on the 2nd and 3rd days, the initial learning leading to the escape zone was omitted in order to evaluate the long-term memory acquired the previous day. All data were captured by a camera installed above the swimming pool and analyzed using a video tracking system (Panlab, Barcelona, Spain).

As a result, as shown in Figure 4A, the escape latency of the flower root extract administration group (TP50mg/kg, TP100mg/kg, and TP200mg/kg) compared to the control group (CTL) in the first attempt, which is a long-term memory indicator, was statistically significantly lower. There was a significant decrease, and as shown in Figure 4B, even in the last trials, which were the third swim every day, the escape delay time of the flower root extract administration group (TP50mg/kg, TP100mg/kg, and TP200mg/kg) compared to the control group (CTL) ( escape latency) decreased statistically significantly.

(3) Probe test

A probe test was conducted on the 4th day, 24 hours after completing the 3-day escape delay time test. Free swimming was conducted for 90 seconds without an escape platform, and a test was conducted to determine whether the participant had memory of the previous location of the escape platform (swimming time in the quadrant where the escape platform was located among the four quadrants).

As a result, as shown in Figure 5, the swimming time (A) and the distance traveled to find the escape zone (B) in the quadrant where the escape zone was located in the flower stalk extract-administered group compared to the control group increased.

Example 6. Confirmation of expression levels of synapse remodeling factor and neuroplasticity factor protein in hippocampal tissue extracted from SD-rats

On the day after all behavioral evaluations were completed, hippocampal tissue was extracted from SD-rats (n=3 in each group) to determine changes in expression levels of synaptic reorganization factors AMPA receptor (GluA1), NMDA receptor (NR2A and NR2B), and PSD95 protein and neuronal activity. Changes in the expression levels of plasticity factors ERK, CaMKII, and BDNF proteins were confirmed using Western blot technique.

As a result, as shown in Figure 6, the expression level of proteins of NMDA receptors (NR2A and NR2B), AMPA receptors (GluA1), and PSD95, which are synaptic reorganization factors expressed in the hippocampal tissue of the group administered the flower marigold extract of the present invention compared to the control group, significantly increased. did.

In addition, as shown in Figure 7, the expression levels of phosphorylated ERK and CaMKII, which are active forms of the signaling pathways ERK and CaMKII involved in regulating synaptic plasticity, were significantly increased in the group administered with the flower extract of the present invention compared to the control group (Figure 7A), and synaptic The expression level of BDNF (brain-derived neurotrophic factor) protein, a brain-derived nerve growth factor involved in the formation of nerve cells, increased (Figure 7B).

Claims (9)

A pharmaceutical composition for the prevention or treatment of cognitive dysfunction comprising a thermal water extract of Trigonotis peduncularis as an active ingredient. The method of claim 1, wherein the cognitive dysfunction is Alzheimer's disease, cerebrovascular dementia, Pick's disease, Creutzfeldt-Jakob disease, dementia due to head injury, and Parkinson's disease. A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that it is any one selected from disease). The pharmaceutical composition for preventing or treating cognitive dysfunction according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the water extract of the flower. A health functional food composition for improving cognitive function or memory containing Trigonotis peduncularis thermal water extract as an active ingredient. delete The health functional food composition for improving cognitive function or memory according to claim 4, wherein the hydrothermal extract of the flower plant is a hot water extract of the leaf, flower, stem, aerial part, underground part, or whole plant of the flower plant. The health functional food composition for improving cognitive function or memory according to claim 4, wherein the composition is manufactured in a dosage form selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage. A feed additive for improving cognitive function or memory that contains the thermal water extract of Flower Marigold as an active ingredient. A veterinary composition for the prevention or treatment of cognitive dysfunction containing the thermal water extract of flower marigold as an active ingredient.