KR20210033365A - Composition for preventing or treating cognitive dysfunction or neuroinflammation comprising extracts of centella asiatica, cnidium monnieri, and lycium barbarum linne - Google Patents

Abstract

The present invention relates to a mixed extract of Centella asiatica, Cnidium monnieri, and Lycium barbarum, and a cognitive function enhancing effect thereof. Specifically, since the present invention confirms that the mixed extract of Centella asiatica, Cnidium monnieri, and Lycium barbarum has an excellent cognitive function enhancing effect compared to a single extract, the mixed extract is possible to be used to alleviate diseases or disorders related to cognitive decline and to protect the brain.

Description

A composition for the prevention or treatment of cognitive dysfunction or neuroinflammation comprising extracts of centella asiatica, honeysuckle, and subterranean goji extract {COMPOSITION FOR PREVENTING OR TREATING COGNITIVE DYSFUNCTION OR NEUROINFLAMMATION COMPRISING EXTRACTS OF CENTELLA ASIATICA, CNIDIUM MONNIERI, AND LY LINCIUM BARBARBARUM}

The present invention relates to a pharmaceutical composition for the prevention or treatment of cognitive dysfunction or nerve inflammation comprising a mixed extract of Centella asiatica , Cnidium monnieri and Lycium barbarum Linne 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 comprising the extract as an active ingredient.

In modern society, as the lifespan of humans increases, the elderly population rapidly increases, and the development of prevention or treatment of senile diseases is being urged. In particular, diseases such as dementia are receiving great social attention because, when the progression is deepened, it places a lot of burden on individuals, families, and society and has a great adverse effect on the quality of life.

Depending on the cause of the disease, dementia can be divided into several categories, such as Alzheimer's disease, Parkinson's disease, and frontotemporal degeneration, of which Alzheimer-type dementia accounts for 50-60% of all dementia. Alzheimer's-type dementia causes symptoms of cognitive decline, such as decreased memory, decreased language ability, decreased ability to grasp spatio-temporality, and decreased judgment, which interferes with basic daily life. However, as of now, a drug that fundamentally treats dementia has not been developed, and it is best to continuously manage the disease to relieve symptoms or delay the progression of the disease at the onset. Accordingly, there is a need to develop a new treatment method that is effective while reducing the burden on patients and their families.

In addition to dementia, cognitive dysfunction may occur due to diseases and symptoms that are easy to find in modern life such as high blood pressure, lack of exercise, and depression. In particular, it is known that there is the highest correlation in the case of depression, which is rapidly increasing in patients at all ages. Therefore, there is a need to develop a method for improving cognitive dysfunction and protecting brain as a means for leading a healthy daily life.

Reducing side effects is one of the important tasks in the case of diseases that require continuous management while taking drugs for a long time, such as cognitive dysfunction. For example, donepezil, which is commonly used in the treatment of dementia in Korea, can cause side effects such as hallucinations and abdominal disorders in severe cases other than muscle pain and loss of appetite, and the development of a new dementia treatment is urgent. Therefore, the development of natural drugs with fewer side effects than synthetic drugs in cognitive dysfunction related diseases can be of great help to patients.

Therefore, in order to minimize side effects, therapeutic agents using natural substances are being developed, for example, compositions for the prevention or treatment of cognitive dysfunction containing fermented purple carrot extract as an active ingredient (Korean Patent No. 10-1495815), A composition for preventing or treating cognitive disorders-related diseases including tree extract (Korean Patent No. 10-1877860) is known.

On the other hand, Centella asiatica is a creeping perennial herb belonging to the umbel family, and it has been known that the asiatic acid derivatives contained therein can be used for the treatment of dementia and cognitive impairment (Korean Laid-Open Publication No. 10-2001-0038149). . However, the compound alone does not exhibit a sufficient effect in the treatment of cognitive disorders.

Under this background, the present inventors continued research to develop a method that can improve cognitive dysfunction and protect the brain. The present invention has been completed by discovering that it has a better cognitive function enhancement and brain protection effect than that, and is also effective in neuroinflammation.

Korean Patent Registration No. 10-1495815 Korean Patent Registration No. 10-1877860 Korean Publication No. 10-2001-0038149

The present invention is to provide a pharmaceutical composition for the prevention or treatment of cognitive dysfunction or nerve inflammation comprising a mixed extract of Centella asiatica , Cnidium monnieri and Lycium barbarum Linne as an active ingredient.

The present invention also aims to provide a health functional food for preventing or improving cognitive dysfunction or nerve inflammation comprising the mixed extract as an active ingredient.

The present invention also aims to provide a feed composition for preventing or improving cognitive dysfunction or nerve inflammation comprising the mixed extract as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention or treatment of cognitive dysfunction or nerve inflammation comprising a mixed extract of Centella asiatica , Cnidium monnieri and Lycium barbarum Linne as an active ingredient.

In the present invention, " Centella asiatica " is a creeping perennial herb belonging to the umbel family, originating from Madagascar Island in Africa, but in other coastal areas of the Indian Ocean, northern Australia, and a wide range of hot and humid places in parts of Central and South America, etc. It grows wildly, and in Korea, it grows wildly in the islands of Jeju Island and southern regions, which belong to the temperate zone.

In the present invention, " Cnidium monnieri " is an annual herb of Umbelliferae, 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).

In the present invention, " Lycium barbarum Linne " is a wolfberry tree distributed in China, and its fruits are mainly used for medicinal purposes. As for the ingredients of wolfberry, it contains a large amount of functional ingredients such as carotenoids, choline, meliscic acid, zeaxanthin, physalin (dipalmityzeaxanthin), betaine, β-sitosterol, vitamin B1, rutin and unsaturated fatty acids. In addition, various studies have been reported such as antioxidant, anti-aging effect, antibacterial effect, improvement of liver function, lowering blood pressure and anti-diabetes, immunity enhancement, lowering blood cholesterol, prevention of cardiovascular disease, and anti-cancer effect.

As used herein, the term "mixed extract of Centella asiatica, Beolsa box, and Nerceus Gugija" refers to a mixture of extracts obtained by extracting Centella asiatica, Beolsa box and Nerceus Gugija, respectively, and an extract obtained by extracting a mixture of Centella asiatica, Bulsa Box, and Nerceus Gugija. It all means.

The present invention is characterized in that it comprises a mixed extract of Centella asiatica, Beolsa box, and Minus Gugija as an active ingredient, and by using the mixed extract, Centella asiatica, Bulsa Box, and Minoria Gugija, respectively, exhibits superior effects than when used alone. It is characterized.

In addition, when the mixed extract is used in a specific blending ratio, the effect is further maximized. For example, centella asiatica, a deer box, and a zero-to-earth period reporter may be included in a weight ratio of 1 to 10: 1 to 5: 1 to 5. Preferably, it may be included in a weight ratio of 1:1:1, 2:1:1, or 3:1:1.

The term "extract" as used herein refers to a liquid component obtained by immersing a target substance in various solvents and then extracting it for a certain period of time at room temperature or warming state, and a resultant product such as a solid component obtained by removing the solvent from the liquid component. It can mean. In addition, in addition to the result, it can be comprehensively interpreted as including all of the resulting diluted solution, the concentrated solution thereof, the crude purified product, and the purified product. The extract of the present invention may be prepared and used in a dry powder form after extraction.

In the method for obtaining the extract, as long as it is possible to obtain an extract having an effect of preventing or treating cognitive dysfunction or neuroinflammation based on the protection of brain neurons of the present invention, it is not particularly limited. For example, a cold sediment extraction method in which a mixture of centella asiatica, dried or processed products of a beetle, and a dried or processed product of sub-zero or less is immersed in a solvent and extracted at room temperature of 10 to 25, a heat extraction method that extracts by heating to 40 to 100, and extracts by applying ultrasonic waves. It can be extracted according to methods commonly used in the art, such as an ultrasonic extraction method and a reflux extraction method using a reflux cooler. Non-limiting examples of the extraction solvent may include water, alcohol, hexane, or a mixed solvent thereof, and when alcohol is used as a solvent, C1 to C6 alcohol may be used as an example. Preferably, the extract may be a water and/or ethanol extract. In one embodiment, as the ethanol, a 20% EtOH aqueous solution may be used.

The term "cognitive function" as used herein refers to various intellectual abilities such as attention, perception, memory, language ability, and executive ability, and for the purposes of the present invention, the term "cognitive function disorder" is a result of a decrease in cognitive function as described above. It is a concept that can include a state, a disease resulting therefrom, and all diseases having the same as a symptom, and specifically, may mean a disorder that occurs due to damage to brain neurons. For example, it includes mental and behavioral disorders and endocrine disorders due to brain diseases, brain injury, addiction, etc., metabolic and nutritional disorders, memory decline and memory impairment caused by drugs. Specifically, cognitive dysfunction is Alzheimer's disease, Huntington's disease, vascular dementia, Parkinson's disease, Lou Gehrig's disease, Creutzfeldt-Yakov's disease, dementia due to head injury, learning disability, mild cognitive impairment, Pick's disease, false recognition, forgetfulness, aphasia. , Fecundity, and delirium, but may be selected from the group consisting of, but is not limited thereto.

The term "neuroinflammation" as used herein refers to inflammation occurring in the brain, and is an important factor causing cognitive dysfunction related diseases. It is known that excessive activation of inflammatory cells in the brain leads to increased secretion of inflammatory cytokines, and cognitive dysfunction is caused by damage to brain cells by overactivation of this encephalopathic response.

The term "prevention" in the present invention 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, and "treatment" is recognized by administration of the pharmaceutical composition. It refers to any action in which symptoms of an individual suspected of having a dysfunction or neuroinflammation and symptoms are improved or beneficially changed.

In one embodiment, the mixed extract according to the present invention exhibits an effect of improving memory and cognitive function in a behavioral experiment of an animal model.

In one embodiment, the mixed extract according to the present invention exhibits an effect of neurogenesis or neural regeneration of the brain. In the present invention, the term "neurogenesis or neurogenesis" refers to a process in which neurons are generated or regenerated 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 level of these proteins. The mixed extract according to the present invention can increase the expression of the brain protein related to neurogenesis.

In addition, the mixed extract according to the present invention can induce neurite outgrowth. Neurons connect through the expansion of a cellular body called dendrite, a biological phenomenon called neurite outgrowth. Therefore, the more neurite outgrowth is observed, the more active neurogenesis in the brain is.

In addition, the mixed extract according to the present invention may increase the production amount of nerve growth factor (NGF).

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

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

In one embodiment, the mixed extract according to the present invention reduces the accumulation of Amyloid Beta (Aβ), thereby exhibiting the effect of Aβ destabilization.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

The present invention also provides a food composition for preventing or improving cognitive dysfunction or nerve inflammation comprising a mixed extract of Centella asiatica , Cnidium monnieri and Lycium barbarum Linne as an active ingredient. In one embodiment, the food composition may also be used to improve memory and learning ability.

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

The term "improvement" of the present invention means any action that at least reduces the severity of a parameter, such as a symptom, related to the condition being treated by administration of a composition comprising an extract of the present invention.

The food composition of the present invention includes forms such as pills, powders, granules, needles, tablets, capsules or liquids, and as foods to which the composition of the present invention can be added, for example, various foods, for example, Beverages, gum, tea, vitamin complexes, and dietary supplements are available.

When the composition of the present invention is a food composition, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

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

The health function (functional food) is the same term as food for special health use (FoSHU). In addition to supplying nutrition, functional food is a medicine that is processed so that the bioregulatory function is effectively displayed, and has high medical effects. Means food. Here, the term "function (sex)" means obtaining useful effects for health, such as controlling nutrients or physiological effects on the structure and function of the human body. The food product of the present invention can be prepared by a method commonly used in the art, and during the production, raw materials and ingredients commonly added in the art can be added to prepare it. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be prepared in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and is excellent in portability. Food can be consumed as an adjuvant for enhancing the effect of preventing or improving cognitive dysfunction or neuroinflammation.

The present invention also provides a feed composition for preventing or improving cognitive dysfunction or nerve inflammation comprising a mixed extract of Centella asiatica , Cnidium monnieri and Lycium barbarum Linne as an active ingredient.

The mixed extract of Centella asiatica, beolsa box and subterranean gugija according to the present invention has an excellent cognitive function enhancement and brain protection effect, which shows superior efficacy than a single extract of Centella asiatica, beolsa box and subterranean gugija. Accordingly, the mixed extract can be effectively used for the prevention, improvement, or treatment of cognitive dysfunction or neuroinflammation.

1 is a graph showing the change behavior measured in a Y-shaped maze experiment according to Example 2.
2 and 3 are results of analyzing the expression levels of DCX and phospho-CREB, which are brain proteins related to neurogenesis, in brain tissue by performing immunohistochemical analysis according to Example 3. 2 is a graph showing the number of DCX positive cells measured. 3 is a graph showing the number of phospho-CREB positive cells measured.
4 and 5 are graphs showing results of evaluation of the effect of the extract of the present invention on Neurite Outgrowth according to Example 4. 4 is a graph showing the measurement of neurite outgrowth in C6 glioma cells. 5 is a graph showing the measurement of neurite outgrowth in N2a cells.
Figure 6 shows the measurement of the amount of NGF produced in C6 glioma cells treated with 6-shogaol 10 μg/ml (positive control), bursa box (KYJ2), and mixed extract at 25 μg/ml according to Example 5. It is a graph.
7 is a case of treating BV2 cells activated with LPS according to Example 6 with 5 or 10 μg/ml of L-NMMA 5 or 20 μg/ml (positive control), KYJ2, and 5 or 10 μg/ml of mixed extracts, respectively. It is a graph showing by measuring the cell viability of.
8 is a LPS-activated BV2 cells according to Example 7 treated with 5 or 10 μg/ml of L-NMMA 5 or 20 μg/ml (positive control), KYJ2, and mixed extracts, respectively. It is a graph showing by measuring the concentration of Nitrite generated from cells.
Figure 9 is according to Example 8, 30 μg / ml (positive control), centella (KYJ1), bulsa box (KYJ2), zero-equivalent (GG1), and a mixed extract (mixture) in Aβ _1-42 monomer peptide, each 1 _{A graph showing the degree of accumulation of Aβ 1-42} after μg/ml treatment and a schematic diagram showing the experimental process thereof.

Hereinafter, embodiments and examples of the present disclosure will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present disclosure. 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

1) Preparation of Centella asiatica extract (KYJ1)

600 g of Centella asiatica was extracted with 10 x 20% EtOH for 24 hours at room temperature. The extraction process was repeated two more times to obtain a total of 66.13g centella asiatica extract in a yield of 11.02%.

2) Preparation of bee box extract (KYJ2)

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

3) Preparation of the extract (GG1)

600g of minuscule Giza was extracted with 10 x 20% EtOH for 24 hours at room temperature. The extraction process was repeated two more times to obtain a total of 112.34 (+α) g of the extract of sub-zero chinensis.

4) Preparation of mixed extract ("Mixture" or "Mix")

200 g of Centella asiatica, bee sac, and minus celeryus were prepared at a ratio of 1:1:1, and a total of 600 g of the mixture was extracted with 10 x 20% EtOH for 24 hours at room temperature. The extraction process was repeated two more times to obtain a total of 83.23 g of mixed extract in a yield of 13.87%.

[Example 1]

Preparation and administration of experimental animals

A 5-week-old male ICR mouse is distributed in an environment with a temperature of 23±1℃ and a humidity of 60±10% for 12 hours each night/day, and it is used in the experiment after acclimatizing in the animal room for 7 days while supplying enough water and general diet. I did. As shown in Table 1, the mixed extract 200 mg/kg/day oral administration group (n=11), centella extract 200 mg/kg/day oral administration group (n=11), the same amount of physiological saline ( vehicle) (normal control, n=11), piracetam 200 mg/kg/day intraperitoneal administration group (positive control, n=10) was set as a total of 4 groups, and administration was performed once a day for a total of 16 days.

Administration substance Population Mixture 200 mg/kg/day
(Oral administration) 11 Centella asiatica (KYJ1) extract 200 mg/kg/day
(Oral administration) 11 Saline solution 5 ml/kg/day
(Oral administration) 11 piracetam 200 mg/kg/day
(Intraperitoneal administration) 10

How to process statistics

The experimental score was expressed as mean ± standard error (Mean ± SEM), and the difference in the mean was tested by Student's t-test, and it was determined that there was a statistically significant difference when P <0.05.

[Example 2]

Confirming the effect of improving memory and cognitive function through behavioral experiment: Y-maze test

On the 13th day of administration, administration was performed 1 hour before the experiment, and then the experiment was performed. This is a behavioral experiment that evaluates learning and memory in space.In an experimental device with three arms at 120° intervals, each branch is 40cm long, 12cm high, and 3cm apart, place the experimental animal on one branch and move it to the other branch. The order of movement was recorded and observed for 8 minutes. The result was measured as change behavior (%) = (number of changes) / (total number of entries -2) x 100.

As a result, as shown in Figure 1, the mixed extract (mixture) administration group (experimental group) showed a similar change behavior to the positive control piracetam administration group. In addition, the mixed extract (mixture) administration group showed excellent alteration behavior compared to centella (KYJ1) extract. From this, it was confirmed that the mixed extract is effective in improving memory and cognitive function.

[Example 3]

Immunohistochemical analysis: analysis of DCX and phospho-CREB protein expression in brain tissue

In this example, in order to confirm the neurogenesis effect according to the treatment with the mixed extract of the present invention, the expression levels of DCX and phospho-CREB, which are brain proteins related to neurogenesis, were analyzed.

Immunohistochemical staining method

After obtaining brain tissue by sacrificing the experimental animals to which each experimental group and control group were administered according to Example 1, the brain tissue was dehydrated in a 30% sucrose solution. After that, the brain tissue was subjected to coronal section in a frozen state to obtain a brain tissue section and stored in a stock solution.

After washing the brain tissue sections with 0.6% hydrogen peroxide and phosphate buffered saline, 10% normal serum and primary antibody (goat anti-DCX 1:100 dilution, rabbit anti-phosphor-CREB 1:100 dilution, mouse anti- synaptophysin 1:1000 dilution) and reacted overnight at 4°C. The next day, a secondary antibody (biotinylated anti-rabbit IgG or anti-mouse IgG 1:200 dilution) and avidin-biotin complex mixture were reacted in turn at room temperature for 1 hour, followed by 3'-diaminobenzidine color development process, and observed under a microscope. Between each process, washing was performed 3 times with PBS for 5 minutes each.

Example 3-1: DCX analysis

The hippocampal dentate gyrus tissue was collected from the experimental animals and stained with DCX by the above method. The number of DCX positive cells was measured in the tissue.

As shown in FIG. 2, the number of DCX-positive cells was increased in Piracetam (positive control) and mixed extract, compared to the normal control, and in particular, the mixed extract administered group was higher than Piracetam (positive control). Showed an increase. In addition, the number of DCX-positive cells in the mixed extract showed a higher increase compared to the extract of Centella asiatica (KYJ1).

Example 3-2: phospho-CREB analysis

The hippocampal dentate gyrus tissue was collected from the experimental animals and stained with CREB by the above method. The number of phospho-CREB positive cells was measured in the tissue.

As a result, as shown in FIG. 3, the number of phospho-CREB positive cells was increased in Piracetam (positive control) and mixed extract, compared to the normal control, and in particular, in the mixed extract administered group, Piracetam (positive Control) showed almost similar increase. In addition, the number of positive cells in the mixed extract showed a higher increase compared to the extract of Centella asiatica (KYJ1).

From the results of Examples 3-1 and 3-2 above, it was confirmed that the expression of DCX and phospho-CREB, which are brain proteins related to neurogenesis, was increased when the mixed extract of the present invention was treated. It showed a higher increase compared to, and it was confirmed that it exhibited a neurogenic effect equal to or higher than that of Piracetam (positive control).

[Example 4]

Neurite Outgrowth Assessment

Example 4-1: Evaluation in C6 glioma cells

C6 glioma cells ^{were dispensed into a 24 well plate at 1 x 10 5} cells/well, and the cells were treated with each material according to Table 2, and the presence or absence of neurite outgrowth was evaluated through InCucyte, a real-time cell observer. Each cell was measured as a photograph every 2 hours, and the number of neurites was measured and calculated through Incucyte software.

As a result, as shown in FIG. 4, when treated with the mixed extract, Neurite Outgrowth increased more than that of the extract of KYJ-2, and even more increased than that of the positive control (Cerebrolycin).

Processing material Concentration (μg/ml) Beehive Box Extract
(KYJ2) 25 Mixed extract
(Mixture) 25 Cerebrolycin
(Positive control) 10

Example 4-2: Evaluation in N2a cells

N2a cells were seeded in a 6-well plate at a density of 15 to 30 x 10 ^{3 cells/well and allowed to attach.} Subsequently, N2a cells were treated with each material according to Table 3 below, and neurite outgrowth/Neuritogenesis was evaluated through InCucyte, a real-time cell observer. Each well was monitored with a 20x objective lens in InCucyte Zoom, and the length of the neurite on the cell body was measured every 2 hours and expressed as mm/mm ² . In addition, a phase contrast image was captured every 2 hours for 24 hours. The number of neurites was measured and calculated through Incucyte software.

As a result, as shown in Fig. 5, when treated with the mixed extract, Neurite Outgrowth increased more than each single extract (i.e., KYJ1, KYJ2, GG-1), and even more than the positive control (Cerebrolycin). Increased.

Processing material Concentration (μg/ml) Centella asiatica extract
(KYJ1) 20 Beehive Box Extract
(KYJ2) 20 Minus Gugija Extract
(GG1) 20 Mixed extract
(Mixture) 20 Cerebrolycin
(Positive control) 10

From the results of Examples 4-1 and 4-2 above, it was confirmed that the mixed extract of the present invention exhibited a very excellent effect of increasing Neurite Outgrowth compared to each of the single extracts, and in particular, increased Neurite Outgrowth, which was excellent even compared to the positive control (Cerebrolycin). It was confirmed that the effect was shown.

[Example 5]

Evaluation of Nerve Growth Factor (NGF) secretion

C6 glioma cells ^{were dispensed into a 24 well plate at 1 x 10 5} cells/well, and the cells were treated with each material according to Table 4, and then cultured for 24 hours. After transferring the culture medium, the amount of secretion of Nerve Growth Factor (NGF) was quantified through the Enzyme-linked immunosorbent assay (ELISA) kit, which is an enzyme immunoassay method. The amount of NGF production (%) in each treatment group was calculated as 100% of the amount of NGF production in the normal control group (Ctl) that was not treated with anything. The results are shown in FIG. 6.

When treated with the mixed extract of the present invention, the amount of NGF produced was increased compared to the extract of bee sacks alone (KYJ2), and in particular, when treated with the mixed extract, the amount of NGF produced was increased more than that of the positive control group.

Processing material Concentration (μg/ml) Beehive Box Extract
(KYJ2) 25 Mixed extract
(Mixture) 25 6-shogaol
(Positive control) 10

From the results of Examples 3 to 5, it can be seen that the mixed extract according to the present invention exhibits superior brain neurogenic effects compared to a single extract.

[Example 6]

Cytotoxicity assessment

In this experiment, the degree of recovery of cytotoxicity was evaluated when the mixed extract of the present invention was used for cells that were treated with LPS to induce cytotoxicity.

BV2 cells (mouse, C57BL/6, brain, microglial cells) were treated with LPS to induce cytotoxicity, then ^{dispensed into a 24-well plate at 1 x 10 5} cells/well and stabilized for 24 hours. After stabilization, the cells were treated with an extract or a control material as shown in Table 5 and cultured in a _{CO 2 incubator for 24 hours.}

Processing material Concentration (μg/ml) Beehive Box Extract
(KYJ2) 5 or 10 Mixed extract
(Mix) 5 or 10 L-NMMA
(Positive control) 5 or 20 LPS
(Negative control) 100

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 to 570 nm. Cell viability was measured with a microspectrophotometer at wavelength. The cell viability of the normal control without any treatment was set to 100%, and the cell viability was evaluated when each substance was treated. The results are shown in FIG. 7.

As can be seen from FIG. 7, the cell viability when treated with LPS was reduced to about 50% compared to 100% of the cell viability of the normal control without treatment, but when the mixed extract of the present invention was used, the cells The survival rate was greatly increased. In particular, when the mixed extract of the present invention was used at a concentration of 10 μg/ml, the cell viability was nearly 100% similar to that of the positive control or the normal control.

From the above results, it was confirmed that the mixed extract of the present invention exhibits an effect of increasing the survival rate by recovering cytotoxicity even when toxicity to the cells is shown.

[Example 7]

Nitric Oxide production evaluation

Toxins such as Lipopolysaccharide (LPS) over-activate microglial cells, increasing the secretion of neurotoxins, inflammatory mediators, and inflammatory cytokines. In addition, Nitric Oxide (NO) is a major mediator of chronic inflammation of the nervous system, along with proinflammatory cytokines. Therefore, in this example, after activating cells with LPS to artificially secrete NO, by evaluating the effect of reducing the amount of NO production according to the extract according to the present invention, it is determined whether the mixed extract of the present invention inhibits the inflammatory reaction in microglia. Investigated.

BV2 cells were dispensed into a 96 well plate at 4 x 10⁴ cells/well, and then stabilized for 24 hours. BV2 cells were treated with LPS (100 μg/ml) for 30 minutes. After LPS pretreatment, the medium was removed, and the cells were treated with each material according to Table 6, and then cultured for 24 hours. After culturing, the culture medium was collected and reacted with a Griess reagent solution (1% sulfanilamide and 0.1% N-1-naphthyl ethylenediamine dihydrochloride in 5% phosphoric acid) to evaluate the amount of NO production. The reaction solution was analyzed with a microspectrophotometer at a wavelength of 540 nm. The results are shown in FIG. 8.

Processing material Concentration (μg/ml) Beehive Box Extract
(KYJ2) 5 or 10 Mixed extract
(Mix) 5 or 10 L-NMMA
(Positive control) 5 or 20 LPS
(Negative control) 100

As can be seen from FIG. 8, when treated with LPS, the concentration of Nitrite increased to about 90 uM, resulting in a very increase in the amount of NO production, but when the mixed extract of the present invention was used, the concentration of Nitrite decreased to about 60 uM, resulting in the amount of NO produced. It was confirmed that this was greatly reduced. Particularly, when the mixed extract of the present invention was used at a concentration of 5 ug/ml, the amount of NO produced was lower than when the positive control L-NMMA was used at a concentration of 5 ug/ml.

From this, it was confirmed that the mixed extract of the present invention can inhibit the inflammatory response in microglia.

[Example 8]

Amyloid Beta (Aβ) destabilization evaluation

In this example, the Aβ destabilization ability of the mixed extract of the present invention was evaluated.

_{Aβ 1-42} monomer peptide was incubated at 37° C. for 72 hours to generate aggregates, and then each drug was treated. After 3 hours, 150 μl of Thioflavin T (Th T) reagent was added and reacted for 30 minutes, and then absorbance (excitation: 470 nm, emission: 520 nm) using a FLUOstar Omega multimode microplate reader (BMG LABTECH GmbH, Ortenberg, Germany). Was measured. The results are shown in FIG. 9.

Processing material Concentration (μg/ml) Centella asiatica extract
(KYJ1) One Beehive Box Extract
(KYJ2) One Minus Gugija Extract
(GG1) One Mixed extract
(Mixture) One Jogudeung extract
(Positive control) 30

There was no significant change in Aβ accumulation in the group treated with the extracts of Centella asiatica (KYJ1) and Bursa (KYJ2). Aβ accumulation was decreased in the group treated with Gigija (GG1) and mixed extract, especially in the group treated with mixed extract (mixture). From this, it was confirmed that the Aβ destabilization effect of the mixed extract of the present invention is superior to that of the single extract (FIG. 9).

Claims (11)

A pharmaceutical composition for the prevention or treatment of cognitive dysfunction or neuroinflammation comprising a mixed extract of centella asiatica, beolsa box, and subterranean gugija as an active ingredient.
The pharmaceutical composition of claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, C1 to C6 alcohol, and a mixed solvent thereof.
The pharmaceutical composition of claim 2, wherein the solvent is a 20% aqueous ethanol solution.
The method of claim 1, wherein the cognitive dysfunction is Alzheimer's disease, Huntington's disease, vascular dementia, Parkinson's disease, Lou Gehrig's disease, Creutzfeldt-Yakov's disease, dementia due to head injury, learning disability, mild cognitive impairment, Pick's disease, false recognition, Forgetfulness, aphasia, aphasia, and delirium, characterized in that selected from the group consisting of, pharmaceutical composition.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition improves cognitive ability or memory.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition generates a cranial nerve.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition increases the survival rate of brain neurons.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits the production of nitrogen monoxide (NO).
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition reduces the accumulation of amyloid beta.
A food composition for preventing or improving cognitive dysfunction or neuroinflammation using a mixed extract of centella asiatica, beesa box, and quasi-gugija as an active ingredient.
A feed composition for the prevention or improvement of cognitive dysfunction or neuroinflammation as an active ingredient using a mixed extract of centella asiatica, bee sacrum, and subterranean goji.

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