KR102634246B1 - Composition for preventing or treating cognitive dysfunction or neuroinflammation comprising extracts of cnidium monnieri - Google Patents

Abstract

The present invention relates to bee dead extract ( Cnidium monnieri ) and its cognitive function enhancing effects. Specifically, bee casualty extract exhibits excellent cognitive function enhancement or neuroinflammation protection effects, and can be used to prevent or treat cognitive dysfunction or neuroinflammation.

Description

COMPOSITION FOR PREVENTING OR TREATING COGNITIVE DYSFUNCTION OR NEUROINFLAMMATION COMPRISING EXTRACTS OF CNIDIUM MONNIERI}

The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction or neuroinflammation containing an extract of bee killer ( Cnidium monnieri ) as an active ingredient. In addition, the present invention relates to a health functional food and feed composition for preventing or improving cognitive dysfunction or neuroinflammation containing the extract as an active ingredient.

As human lifespan increases in modern society, the elderly population is rapidly increasing, and the development of preventive or therapeutic agents for geriatric diseases is called for. In particular, diseases such as dementia are receiving great social attention because, if the disease progresses, it places a great burden on individuals, families, and society and has a significant negative impact on the quality of life.

Dementia can be classified into several types, including Alzheimer's disease, Parkinson's disease, and frontotemporal lobe degeneration, depending on the cause, and among these, Alzheimer's type dementia accounts for 50-60% of all dementia. Alzheimer's dementia causes symptoms of cognitive decline, such as memory loss, language ability, poor spatial and temporal understanding, and poor judgment, which interferes with basic daily life. However, currently no drug has been developed to fundamentally treat dementia, and the best way to relieve symptoms or delay the progression of the disease is through continuous management upon onset. Accordingly, there is a need for the development of new treatment methods that can ease the burden on patients and their families while also being effective.

In addition to dementia, cognitive dysfunction may occur due to diseases and symptoms that are common in modern life, such as high blood pressure, lack of exercise, and depression. In particular, the correlation is known to be highest in the case of depression, which has recently been rapidly increasing in the number of patients of all ages. Therefore, it is necessary to develop methods to improve cognitive dysfunction and protect the brain as a means to lead a healthy daily life.

In the case of diseases that require continuous management while taking drugs for a long period of time, such as cognitive dysfunction, reducing side effects is an important task. For example, donepezil, which is commonly used to treat dementia in Korea, can cause side effects such as muscle pain and loss of appetite, as well as hallucinations and abdominal disorders in severe cases, so the development of a new dementia treatment is urgently needed. Therefore, the development of natural medicines with fewer side effects than synthetic medicines in diseases related to cognitive dysfunction can be of great help to patients.

Therefore, treatments using natural substances are being developed to minimize side effects, for example, a composition for preventing or treating cognitive dysfunction containing fermented purple carrot extract as an active ingredient (Korean Patent No. 10-1495815), comb meat A composition for preventing or treating diseases related to cognitive impairment containing tree extracts (Korean Patent Registration No. 10-1877860) is known.

Meanwhile, it has been known that the extract of Cnidium monnieri exhibits an immune-enhancing effect and has an anti-cancer effect (Korean Patent No. 10-1484709). However, to date, there has been no disclosure that bee sting extract has an effect on cognitive dysfunction or neuroinflammation.

Against this background, the present inventors have conducted continuous research to develop a method to improve cognitive dysfunction and protect the brain. As a result, the use of bee dead extract has excellent cognitive function enhancement and brain protection effects, and furthermore, The present invention was completed by discovering that it is also effective in nerve inflammation.

Korean Patent No. 10-1495815 Korean Patent No. 10-1877860 Korean Patent No. 10-1484709

The present invention seeks to provide a pharmaceutical composition for the prevention or treatment of cognitive dysfunction or neuroinflammation containing an extract of Cnidium monnieri as an active ingredient.

The present invention also seeks to provide a health functional food for preventing or improving cognitive dysfunction or neuroinflammation containing the extract as an active ingredient.

The present invention also seeks to provide a feed composition for preventing or improving cognitive dysfunction or neuroinflammation containing the extract as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction or neuroinflammation containing an extract of Cnidium monnieri as an active ingredient.

In the present invention, " Cnidium monnieri " is an annual herb of the Umbelliferae family, mainly distributed in China and Korea, and is widely used in the treatment of purulent dermatitis and female genital diseases (Yang, LL et al., 2003. Cytotoxic activity of coumarins from the fruits of cnidium monnieri on Leukemia cell lines. Planta Med. 69, 1091-109).

The present invention is characterized in that it contains a bee casualty extract as an active ingredient, and when the extract is used, it shows an excellent effect in preventing or treating cognitive dysfunction or neuroinflammation, even when compared to existing known substances. do.

As used herein, the term "extract" refers to a liquid component obtained by immersing the target material in various solvents and then extracting it at room temperature or at a heated state for a certain period of time, and a solid component obtained by removing the solvent from the liquid component. It can mean. In addition, in addition to the above results, it can be comprehensively interpreted to include all dilutions of the above results, concentrates thereof, crude purifications, purifications thereof, etc. The extract of the present invention can be prepared and used in the form of a dry powder after extraction.

The method for obtaining the extract is not particularly limited as long as it is possible to obtain an extract that has the effect of preventing or treating cognitive dysfunction or neuroinflammation based on the protection of brain neurons of the present invention. For example, a cold needle extraction method in which a mixture of dried or processed bee dead boxes is immersed in a solvent and extracted at a room temperature of 10 to 25 degrees Celsius, a heat extraction method in which the mixture is extracted by heating to 40 to 100 degrees Celsius, an ultrasonic extraction method in which the mixture is extracted by applying ultrasonic waves, and a reflux cooler. It can be extracted according to methods commonly used in the technical field, such as reflux extraction using . Non-limiting examples of the extraction solvent include water, alcohol, ethyl acetate, acetone, or a mixed solvent thereof. When alcohol is used as a solvent, C1 to C6 alcohol can be used as an example. Preferably, the extract may be a water and/or ethanol extract. In one embodiment, the water may be distilled water, and the ethanol may be a 10 to 80% ethanol aqueous solution, preferably a 20% ethanol aqueous solution or a 70% ethanol aqueous solution.

As used herein, the term "cognitive function" refers to various intellectual abilities such as attention, perception, memory, language ability, and executive ability. For the purpose of the present invention, the term "cognitive dysfunction" refers to a condition in which cognitive function as described above is deteriorated. It is a concept that can include the condition that occurs, the resulting disease, and all diseases that have symptoms, and can specifically mean a disorder that occurs due to damage to brain nerve cells. For example, it includes memory decline and memory impairment due to mental and behavioral disorders, endocrine abnormalities, metabolic and nutritional abnormalities, drugs, etc. due to brain disease, brain injury, addiction, etc. Specifically, cognitive dysfunction includes Alzheimer's disease, Huntington's disease, vascular dementia, Parkinson's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, dementia due to head injury, learning disability, mild cognitive impairment, Pick's disease, agnosia, amnesia, and aphasia. , apraxia, delirium, and combinations thereof, but is not limited thereto.

The term “neuroinflammation” used herein refers to inflammation occurring in the brain and is an important factor causing diseases related to cognitive dysfunction. It is known that when inflammatory cells in the brain become excessively activated, the secretion of pro-inflammatory cytokines increases, and this over-activation of the brain inflammatory response causes cognitive dysfunction due to damage to brain cells.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of cognitive dysfunction or neuroinflammation by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to any action that suppresses or delays the onset of cognitive dysfunction or neuroinflammation by administration of the pharmaceutical composition according to the present invention. It refers to any action that improves or beneficially changes the symptoms of a subject suspected of having cognitive dysfunction or neuroinflammation.

In one embodiment, the extract according to the present invention shows an effect of improving memory and cognitive function in behavioral experiments in animal models.

In one embodiment, the extract according to the invention exhibits neurogenic or neuroregenerative effects in the brain. In the present invention, the term “neurogenesis or nerve regeneration” refers to the process of generating or regenerating neurons from neural stem cells or progenitor cells. Brain proteins such as DCX (Doublecortin), phospho-CREB, or Synaptophysin are involved in such nerve regeneration, and the degree of nerve regeneration can be confirmed through the expression levels of these proteins. The extract according to the present invention can increase the expression of the brain protein related to neurogenesis.

Additionally, the extract according to the present invention can induce neurite outgrowth. Neurons are connected through extensions of cellular bodies called dendrites, and this biological phenomenon is referred to as neurite outgrowth. Therefore, the more neurite outgrowth observed, the more active neurogenesis is in the brain.

Additionally, the extract according to the present invention can increase the production of nerve growth factor (NGF).

In one embodiment, the extract according to the present invention may improve memory by increasing cell survival through protection of brain neurons.

In one embodiment, the extract according to the present invention inhibits the inflammatory response in microglial cells activated by lipopolysaccharide (LPS), such as BV-2 cells. Specifically, the extract according to the present invention can reduce the amount of Nitric Oxide produced in LPS-activated cells.

In one embodiment, the extract according to the invention exhibits a cytoprotective effect against neurotoxicity of Aβ _25-35 plaques.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions.

The present invention also provides a food composition for preventing or improving cognitive dysfunction or neuroinflammation containing an extract of Cnidium monnieri as an active ingredient. In one embodiment, the food composition can also be used to improve memory and learning ability.

Since the food composition of the present invention can be ingested on a daily basis, it is very useful as it can be expected to prevent or improve cognitive dysfunction and neuroinflammation.

As used herein, the term "improvement" refers to any action that results in at least a reduction in the severity of symptoms, for example, parameters associated with the condition being treated by administration of a composition comprising an extract of the present invention.

The food composition of the present invention includes the form of pills, powders, granules, precipitates, tablets, capsules, or liquids. Foods to which the composition of the present invention can be added include, for example, various foods, such as These include beverages, gum, tea, vitamin complexes, and health supplements.

When the composition of the present invention is a food composition, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

The food composition of the present invention includes health functional foods and health foods.

The above-mentioned health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, with high medical effects. It means food. Here, “function” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it is made from food as a raw material and has the advantage of not having side effects that may occur when taking the drug for a long period of time. It is also excellent in portability and can be used as a pharmaceutical composition according to the present invention. Food can be consumed as a supplement to enhance the effect of preventing or improving cognitive dysfunction or neuroinflammation.

The present invention also provides an animal feed composition for preventing or improving cognitive dysfunction or neuroinflammation, comprising an extract of Cnidium monnieri as an active ingredient.

The bee casualty extract according to the present invention exhibits excellent cognitive function enhancement and brain protection effects, and can be effectively used to prevent, improve, or treat cognitive dysfunction or neuroinflammation.

Figure 1 is a graph showing the amount of NGF production measured in C6 glioma cells according to Example 1.
Figure 2 is a graph showing the cell survival rate of C6 glioma cells measured according to Example 2.
Figure 3 is a graph showing the concentration of Nitrite produced from BV2 microglial cells treated with LPS according to Example 3.
Figure 4 is a graph showing the cell survival rate of BV2 microglial cells treated with LPS according to Example 4.
Figure 5 is a graph showing the degree of cytoprotective efficacy against Aβ _25-35 plaque neurotoxicity in HT22 cells measured according to Example 5.

Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present application may be implemented in various forms and is not limited to the embodiments and examples described herein.

[Production Example 1]

Preparation of extract

600g of bee casualties were extracted with 10 x 20% EtOH at room temperature for 24 hours. The extraction process was repeated two more times to obtain a total of 12.27 g of bee dead extract with a yield of 2.05%.

Additionally, an extract was prepared using distilled water, 70% EtOH, ethyl acetate, acetone, or hexane in the same manner as above.

[Example 1]

Nerve Growth Factor (NGF) secretion evaluation

C6 glioma cells were distributed in a ²⁴ well plate at 1 After treatment with 25 or 50 μg/ml of each substance and 20 μM of 6-shogaol (6-sho; positive control), the cells were cultured for 24 hours. Culture medium was collected and centrifuged. The amount of Nerve Growth Factor (NGF) secreted in C6 glioma culture supernatants was quantified using the Competitive Enzyme-Linked Immuno Assay (ELISA) kit (R&D systems, Minneapolis, MN, USA). The NGF production amount (%) in each treatment group was calculated by taking the NGF production amount of the untreated normal control group (Ctl) as 100%. The results are shown in Figure 1.

As can be seen from Figure 1, when treated with distilled water (DW), 20% ethanol (20%), or 70% ethanol (70%) extract of the present invention, the amount of NGF production increased, especially similar to the positive control group. Or, compared to this, cases where NGF production increased more significantly were also confirmed.

From the above results, it can be seen that the extract according to the present invention exhibits excellent brain neurogenesis effects.

[Example 2]

Cytotoxicity evaluation

C6 glioma cells were distributed at 1 x 10 ⁵ cell/well in a 24-well plate and stabilized for 24 hours. After stabilization, cells were incubated with 25 or 50 μg/ml of each extracted substance, 6-shogaol (6), using distilled water (DW), 20% ethanol (20%), 70% ethanol (70%), or hexane (H) as a solvent. -sho; positive control) was treated with 20 μM and cultured for 24 hours.

After removing the medium, 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was treated for 1 hour, and the reduced formazan was dissolved in 150 μl of DMSO and measured at 570 nm. Cell viability was measured with a microspectrophotometer at different wavelengths. The cell survival rate of the untreated normal control group (Ctl) was set as 100%, and the cell survival rate when treated with each substance was evaluated. The results are shown in Figure 2.

As can be seen from Figure 2, the cell survival rate in the distilled water (DW), 20% ethanol (20%), or 70% ethanol (70%) extract treatment group was the same or almost similar to the normal control group (Ctl). Confirmed. On the other hand, when treated with 50 μg/ml of hexane (H) extract, the cell viability decreased to about 75%, and in the case of 6-shogaol (6-sho; positive control), the cell viability decreased to 80%. .

From the above results, it was confirmed that the extract according to the present invention does not exhibit cytotoxicity and can be used safely.

[Example 3]

Nitric Oxide production amount evaluation

Toxins such as lipopolysaccharide (LPS) excessively activate microglial cells, increasing the secretion of neurotoxins, inflammatory mediators, and inflammatory cytokines. Additionally, Nitric Oxide (NO) is a major mediating factor in chronic inflammation of the nervous system, along with pro-inflammatory cytokines. Therefore, in this example, cells were activated with LPS to artificially secrete NO, and then the effect of reducing NO production according to the extract of the present invention was evaluated to determine whether the extract of the present invention suppresses inflammatory response in microglial cells. .

BV2 microglial cells were distributed at 4 x 10⁴ cells/well into a 96 well plate and incubated at 37°C overnight. The next day, cells were treated with 25 or 50 μg/ml of each substance extracted using 70% ethanol (70%) or hexane (H) as a solvent, and 5 or 20 μM of L-NMMA (positive control), followed by LPS (100 μg/ml). ng/ml) and incubated at 37°C for 24 hours. Afterwards, the culture medium was collected and the amount of NO production was evaluated using Griess reagent solution (1% sulfanilamide and 0.1% N-1-naphthyl ethylenediamine dihydrochloride in 5% phosphoric acid). Specifically, a total of 50 μL of supernatant was moved to a new 96 well plate, mixed with an equal volume of Griess reagent solution, and OD was measured at 570 nm. The results are shown in Figure 3.

As can be seen from Figure 3, when treated with LPS, the Nitrite concentration increased to more than 60 μM and the NO production greatly increased. However, when the 70% ethanol (70%) extract of the present invention was used, the Nitrite concentration was 30 μM or less. It was confirmed that the amount of NO production was greatly reduced. In particular, the amount of NO production when using the 70% ethanol (70%) extract of the present invention was lower than when using the positive control L-NMMA or hexane (H) extract.

From this, it was confirmed that the extract of the present invention can suppress the inflammatory response in microglial cells.

[Example 4]

Cytotoxic recovery assessment

In this experiment, the degree of cytotoxicity recovery was evaluated when the extract of the present invention was used on cells that had induced cytotoxicity by treatment with LPS.

BV2 microglial cells were treated and incubated with the extract of the present invention and LPS in the same manner as in Example 3, and the extracts specifically included distilled water (DW), 20% ethanol (20%), and 70% ethanol (70%). Alternatively, hexane (H) extract was used.

After removing the culture medium, the culture medium was treated with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution for 1 hour, and the reaction mixture was carefully removed. 200 μL of dimethyl sulfoxide solution was added to the plate. OD was measured using a plate reader at a wavelength of 570 nm. Surviving cells reduced yellow MTT to purple formazan, and cell survival rate was measured by observing the degree of reduction. The cell survival rate of the control group treated with only LPS was set as 100%, and the cell survival rate when treated with each substance was evaluated. The results are shown in Figure 4.

As can be seen from Figure 4, the cell survival rate (100%) when treated with LPS was reduced compared to the cell survival rate of about 140% in the normal control group that was not treated with anything, especially at 50 μg/ml of hexane (H) extract. When treated with , cell viability decreased to about 70%. On the other hand, when the distilled water (DW) extract, 20% ethanol extract, or 70% ethanol extract of the present invention was used, the cell survival rate was greatly increased, showing a cell survival rate similar to or higher than that of the normal control or positive control.

From the above results, it was confirmed that the extract of the present invention has the effect of restoring cytotoxicity and increasing survival rate even when cytotoxicity appears.

[Example 5]

Neuroprotective efficacy evaluation

In this example, the cytoprotective efficacy of bee dead extract against Aβ _25-35 plaque neurotoxicity in HT22 cells was evaluated by MTT assay.

HT22 cells were dispensed at 4 x 10⁴ cells/ml and then incubated at 37°C for 18 hours. Afterwards, the cells were treated with 50 μg/ml of each substance extracted using distilled water (DW) or 20% ethanol (20%) as a solvent, or 50 μg/ml of hexane extract (H1, H2) at 37°C. Incubated for 3 hours.

Afterwards, it was treated with Aβ _25-35 (3 μM) and incubated at 37°C for 23 hours. Then, the cells were treated with MTT solution and incubated at 37°C for 6 hours to measure cell viability. The cell survival rate of the untreated normal control group (Ctl) was set as 100%, and the cell survival rate when treated with each substance was evaluated. The results are shown in Figure 5.

As can be seen from Figure 5, it was confirmed that the cell survival rate in the group treated with distilled water (DW) or 20% ethanol (20%) was the same or almost similar to the normal control group (Ctl) that was not treated with anything. On the other hand, when treated with hexane extract, cell survival rate was much reduced compared to the negative control group treated with only Aβ _25-35 .

From the above results, it was confirmed that the extract of the present invention exhibits neuroprotective efficacy.

Claims (12)

A pharmaceutical composition for preventing or treating neuroinflammation comprising an extract of Cnidium monnieri as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, C1 to C6 alcohol, ethyl acetate, acetone, and mixed solvents thereof.
The pharmaceutical composition according to claim 2, wherein the solvent is distilled water or a 10 to 90% ethanol aqueous solution.
The pharmaceutical composition according to claim 3, wherein the solvent is a 20% aqueous ethanol solution or a 70% aqueous ethanol solution.
delete delete The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition generates cranial nerves.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition increases the survival rate of brain nerve cells.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits the production of nitric oxide (NO).
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition exhibits a neuroprotective effect.
A food composition for preventing or improving nerve inflammation using bee dead extract (Cnidium monnieri) as an active ingredient.
A feed composition for preventing or improving nerve inflammation using bee dead extract (Cnidium monnieri) as an active ingredient.