KR102688683B1 - Composition for preventing or treating degenerative brain diseases comprising bellflower extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating degenerative brain diseases containing bellflower root extract as an active ingredient. The bellflower root extract of the present invention effectively inhibits the accumulation of Aβ in 5XFAD mice, an animal model of Alzheimer's disease, and prevents Aβ toxicity. It was confirmed that it effectively recovers damage to nerve cells, inhibits ROS production, and improves cognitive ability, and can be effectively used in the prevention and treatment of degenerative brain diseases.

Description

Composition for preventing or treating degenerative brain diseases comprising bellflower extract as an active ingredient}

The present invention relates to a composition for preventing or treating degenerative brain diseases containing bellflower root extract as an active ingredient.

Korea's average life expectancy is expected to reach 71.3 years in 1990, 74.3 years in 2000, and 76.95 years in 2020, and the elderly population aged 65 or older will also increase from 2,144,000 in 1990 to 3,168,000 in 2000 and 6,333,000 in 2020. It is predicted that it will account for about 12.5% of the total population, and as soon as Korea enters the 21st century, various degenerative brain diseases, which are prevalent in the elderly population, will emerge as a major medical and social problem. In addition, as family composition changes rapidly toward nuclear families and traditional values of filial piety change, it is also predictable that elderly people who cannot maintain a normal life due to degenerative diseases may end up wandering the streets without receiving care from their families. Moreover, due to the social belief that senile dementia or 'senility' is a 'natural' occurrence in the aging population, it is difficult for senile dementia patients to receive intensive treatment at medical institutions, and it is difficult to develop new treatments for patients with degenerative brain diseases. Therefore, research on degenerative brain diseases is urgently needed not only from a medical perspective but also from a socio-economic perspective, and is an area that should be given priority consideration in establishing health care policies to realize a 21st century welfare state.

Nerve cells constantly undergo apoptosis during the process of development and synapse reorganization, and cell death caused by stress and cytotoxic drugs is a major factor in degenerative brain diseases. Among these, oxidative stress is known to be closely related to the causes of degenerative neurological diseases such as dementia, Alzheimer's, forgetfulness, Parkinson's disease, stress, aging, stroke, and Huntington's disease, and recent studies have shown that chronic stress and Oxidative stress causes oxidative stress in the hypothalamus-pituitary-adrenocortical system, hippocampus, striatum, substantia nigra, and forebrain cortex, increasing cell death and reducing neurons and growth factors, leading to Alzheimer's disease, Parkinson's disease, stress, and aging. , is known to cause stroke and Huntington's disease.

Meanwhile, dementia is a type of degenerative brain disease that is a neurological symptom caused by damage to brain nerve cells. It refers to a pathological condition in which mental ability is reduced to the extent that it interferes with social life and makes it impossible to lead daily life without the help of others. Sexual dementia and cerebrovascular dementia are the most common, and other causes include Parkinson's disease and brain tumors. Degenerative neurological diseases, including dementia, have a variety of causes, and cell biology is particularly important due to reduced blood supply, damage to nerve cells, inflammatory substances secreted from glial cells (microglia), which are inflammatory cells in the brain, and nerve cell death resulting from this. Regulation of glial cell activity and neuronal protection are key targets for the development of prevention, control, and treatment of degenerative brain diseases, including stroke and dementia.

Many researchers both domestically and internationally have made efforts to prevent brain diseases by studying the mechanisms of neuronal death, but in the case of stroke, no clear treatment has currently been developed, and many researchers have so far studied excitatory neurotransmitters. Attempts were made to develop antagonists or antioxidants for phosphorus glutamate receptors as treatments for neurodegenerative diseases, but all failed due to minimal efficacy or toxicity of the drugs.

Meanwhile, herbal medicines have traditionally been used to treat human diseases and protect the body for a long time, and most of them are of plant origin and are mainly based on complex prescriptions of herbal medicine raw materials, and are based on prescriptions in herbal medicine books such as Donguibogam. These herbal medicines are used as preventive and therapeutic agents for various diseases, and interest is growing due to the chemical side effects of many synthetic drugs currently being developed from an Oriental medicine perspective due to their complex effects and low side effects. Republic of Korea Patent Application Nos. 2002-44507, 2002-30648, and 2002-30642 report that herbal medicines such as Angelica root, Trillium chinensis and Schisandra chinensis show protective activity for brain nerve cells, but the effects are not good enough to be applied clinically. It wasn't. Therefore, in order to prevent and improve degenerative brain diseases, there is a need for the development of new cognitive function improvement substances that delay the destruction and aging of brain cells, protect brain cells, restore cognitive function, and promote brain cell regeneration. .

Bellflower root ( Platycodon grandiflorum ) is also called Gilgyeong, Dorat, Gilgyeongchae, Baekyak, Plantain, and Sand bellflower. It grows in mountains and fields. The roots are thick and the stems grow straight. When cut, white juice comes out. The fruit is a capsule that is egg-shaped and ripens with a calyx piece. Propagation is done by seeds, and the roots are collected in spring and fall and eaten raw or as a vegetable. The main ingredient of bellflower root is known to be saponin, and herbal medicine Gilgyeong (桔梗) is made by peeling or drying the root, and in oriental medicine, it is used for dentition, lung fever, tonsillitis, and diarrhea. However, there is a lack of research using bellflower root extract for its effect on degenerative brain diseases and its protective effect against neuronal cell death.

Therefore, while researching compositions for preventing or improving degenerative brain diseases using bellflower root extract, the present inventors confirmed that bellflower root extract inhibits the accumulation of Aβ, a neurotoxic factor, and restores nerve cells. The invention was completed.

The purpose of the present invention is to provide a health functional food composition for preventing or improving degenerative brain diseases containing bellflower root extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases containing bellflower root extract as an active ingredient.

Another object of the present invention is to provide a method for increasing the content of platycoside E and platycodin D in bellflower extract.

Another object of the present invention is to provide a method for producing bellflower extract with increased platycoside E and platycodin D.

The present invention provides a health functional food composition for preventing or improving degenerative brain diseases containing bellflower root extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing bellflower root extract as an active ingredient.

Additionally, the present invention provides a method for increasing the content of platycoside E and platycodin D in bellflower extract.

Additionally, the present invention provides a method for producing bellflower extract with increased platycoside E and platycodin D.

It was confirmed that the bellflower root extract of the present invention effectively inhibits the accumulation of Aβ, effectively restores damage to nerve cells caused by Aβ toxicity, inhibits ROS production, and improves cognitive ability in 5XFAD mice, an animal model of Alzheimer's disease. , it can be used effectively in related industries by confirming that it can be effectively used in the prevention and treatment of degenerative brain diseases.

Figure 1 is a diagram showing the analysis of platycoside E and platycodin D of the bellflower extract of the present invention by HPLC-ELSD (A: confirmation of detection time, B: comparison of content with standard material )
Figure 2 is a diagram showing the results of confirming the cognitive ability improvement effect of the bellflower extract of the present invention in wild-type mice and Alzheimer's animal models through a Y-maze experiment (A: experiment schedule, B: quantification of change behavior, C: total branch change comparison).
Figure 3 is a diagram confirming changes in 4G8 positive cells in the brain tissue of 5 D: Quantification of plaque number).
Figure 4 shows immunofluorescence analysis performed on brain tissue of wild-type mice and 5 (A: SYN and NeuN positive cell immunofluorescence analysis results, B: SYN factor CA1 assay quantification, C: SYN factor CA3 assay quantification, D: SYN factor ML assay quantification, E: NeuN positive cell assay quantification).
Figure 5 shows the level of ionized calcium binding adapter molecule (Iba1) and glial fibrillary acidic protein (Glial fibrillary acidic protein), which are neuroinflammatory factors, in wild-type mice and 5XFAD mice according to administration of the bellflower extract of the present invention. This is a diagram showing the expression of GFAP) positive cells analyzed by immunochemical analysis (A: Expression analysis of Iba1 and GFAP positive cells, B: Iba1 area analysis, C: Quantification of the number of Iba1 positive cells, D: GFAP area analysis, E: GFAP Quantification of number of positive cells).
Figure 6 is a diagram showing analysis of Ki67 and DCX positive cells related to cognitive function in wild type mice and 5 , B: Ki67 assay quantification, C: DCX assay quantification).
Figure 7 is a diagram confirming the cell survival rate according to the treatment concentration of bellflower root extract in the HT22 cell line in which cell death was induced by Aβ of the present invention.
Figure 8 is a diagram confirming the inhibition of ROS production according to the treatment concentration of bellflower root extract in the HT22 cell line in which ROS production was induced by Aβ of the present invention.

The present invention provides a health functional food composition for preventing or improving degenerative brain diseases containing bellflower root extract as an active ingredient.

The term “prevention” used in the present invention refers to all actions that suppress the symptoms or delay the progression of a specific disease by administering the composition of the present invention.

As used herein, the term “improvement” refers to any action that reduces at least the severity of a parameter, such as a symptom, related to the condition being treated.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, 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. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating degenerative brain diseases. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that is related to the structure and function of the human body. It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions of the food additive code and general test methods approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, the health functional food in the form of a tablet is made by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention and excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agents. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

According to one embodiment of the present invention, the bellflower extract may be extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohol, lower alcohol aqueous solution, hexane, chloroform, and acetate, preferably water, It is not limited to this.

According to one embodiment of the present invention, the bellflower extract may inhibit the formation of Aβ plaques.

According to one embodiment of the present invention, the bellflower extract may contain platycoside E and platycodin D.

According to one embodiment of the present invention, the bellflower extract may inhibit neuronal cell death induced by Aβ toxicity in the subiculum of the hippocampus.

According to one embodiment of the present invention, the bellflower extract may improve cognitive function.

According to one embodiment of the present invention, the bellflower extract may increase the number of cells positive for the nerve cell factors neuronal nuclei (NeuN) and synaptophysin (SYN).

According to one embodiment of the present invention, the bellflower extract reduces the number of ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) positive cells, which are indicators of neuroinflammation. It may be reducing it.

According to one embodiment of the present invention, the degenerative brain disease is dementia, and the brain disease is Alzheimer disease, Pick disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. disease), preferably dementia or Alzheimer's disease, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing bellflower root extract as an active ingredient.

The term “treatment” used in the present invention refers to any action that improves or beneficially changes the symptoms of a specific disease by administering the composition of the present invention.

The pharmaceutical composition of the present invention may further include adjuvants in addition to the active ingredients. The auxiliary agent may be any one known in the art without limitation, but the effect may be increased by further including, for example, Freund's complete auxiliary agent or incomplete auxiliary agent.

The pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Examples include calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods. .

When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition according to the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected taking into account the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the target, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

Additionally, the present invention provides a method for increasing the content of platycoside E and platycodin D in bellflower extract.

Additionally, the present invention provides a method for producing bellflower extract with increased platycoside E and platycodin D.

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

<Example 1> Bellflower extract and animal model

<1-1> Preparation of bellflower extract

To prepare the bellflower ( Platycodon grandiflorum ) extract (PEG) of the present invention, it was extracted using 3-year-old bellflower root harvested in 2018 in Boeun, North Chungcheong Province. The bellflower was placed in a cylindrical washing machine and foreign substances such as dirt and vinyl were completely removed. The washed bellflower root was dried in a hot air dryer at 55°C for 72 hours. The dried bellflower root was soaked in 10 times more water, extracted under reflux at 90°C for 6 hours, and the filtrates obtained by filtration were combined and freeze-dried to prepare bellflower root extract.

<1-2> Analysis of bellflower extract using HPLC-ELSD analysis

Among various bellflower saponins, platycoside E and platycodin D are known as major physiologically active molecules and indicator components. For component analysis of the bellflower extract of the present invention, platycoside E and platycodin D are known as the standard substances. The contents of side E and platycodin D were analyzed. Specifically, analytical grade platycoside E and platycodin D were purchased from Phytochemical Ltd. It was purchased and used from (Cheongdu, Sichuan, China). Specifically, bellflower extract was dissolved in 40 mL of distilled water and then degreased with diethyl ether in a separatory funnel. The separated aqueous layer was extracted three times with water-saturated n-butanol, the n-butanol layer was evaporated at 50°C, and the resulting residue was dissolved in methanol and analyzed. The retention times of Platicoside E and Platicodin D were confirmed by matching standard materials of the corresponding analytical grade, and the content was analyzed using a standard curve. Content analysis was performed using high-performance liquid chromatography (HPLC). ) and an evaporative light scattering detector (ELSD). The analysis was performed using a Waters Alliance 2695 HPLC system (2424 ELSD, Waters, Milford, MA, USA), and the column used was a C-18 column (Luna C-18). , Phenomenox, 250 × 4.6 mm, 5 μm, Torrance, CA, USA) was used. As the mobile phase, water was used as solvent A and acetonitrile as solvent B. Specific analysis conditions included 21-21% solvent B for 0-3 minutes relative to the volume ratio of solvent A; 21-23% solvent B for 3-23 minutes; 23-24% solvent B for 23-38 minutes; 24-100% solvent B for 38-70 min; Analysis was performed using a solvent gradient of 100-100% solvent B for 70-75 minutes, the flow rate was 1.0 mL/min, the sample injection volume was 30 μL, and the column temperature was maintained at 40°C. The ELSD conditions were analyzed by maintaining the sprayer temperature at 42°C, the drift tube temperature at 85°C, and the N ₂ gas pressure at 50 psi.

As a result, platycoside E was detected at 14.2 minutes and platycodin D at 44.3 minutes (Figure 1A), with 1597.14 ± 21.71 μg/g for platycoside E and 942.32 ± 97.97 μg/g for platycodin D. It was confirmed that this was included (Figure 1B).

<1-3> Alzheimer's animal model

To confirm the effect of the bellflower root extract of the present invention, 6-month-old female 5XFAD mice expressing five mutations genetically associated with early onset of AD were used. The five mutations include the human PSEN1 mutations M146L and L286V and the APP mutations Swedish (K607N and M671L), Florida (I716V) and London (V717I), while control wild-type mice were female B6SJL/ It was obtained by crossing F1 mice and male 5XFAD mice. In order to specifically classify mice, DNA was isolated from the mouse's tail, genotype was analyzed using the primers listed in Table 1 below, and classified into wild type mice (WT) and Alzheimer's mice (5XFAD), and further classified into 4 Divided into groups.

Gene Sequence(5’ - 3’) APP F: AGG ACT GAC CAC TCG ACC AG R: CGG GGG TCT AGT TCT GCA T PSEN1 F: AAT AGA GAA CGG CAG GAG CA R: GCC ATG AGG GCA CTA ATC AT

Specifically, 1) a group in which saline was administered to wild-type mice (WT+vehicle); 2) A group administered bellflower root extract to wild-type mice (WT+PEG); 3) A group administered saline solution to Alzheimer's mice (5XFAD+vehicle); and 4) the group in which bellflower root extract was administered to Alzheimer's mice (5

<Example 2> Behavioral experiment (Y-maze experiment)

The control group and the Alzheimer's mouse animal model of Example 1-2 were adapted to the experimental environment for a week before the Y-maze experiment after 3 weeks, and after the last PEG administration, they were placed in the Y-maze (8 cm in width, 30 cm in length, and height, respectively). 15 cm) into branches A, B, and C, and it was measured whether the mouse freely entered each branch. The detailed experimental plan is shown in Figure 2A. When the mouse enters each new branch, 1 point is given (actual change), and the percentage of crossing behavior was calculated using Equation 1 below.

<Equation 1>

As a result, as shown in Figure 2B, compared to WT mice (WT+vehicle), it was confirmed that the crossing behavior of 5XFAD mice (5XFAD+vehicle) was significantly reduced. In addition, 5XFAD mice (5XFAD+PEG) treated with bellflower root extract showed a significant increase in crossing behavior compared to the vehicle treated group, and there was no significant difference in total branching changes between each group (Figure 2C), in conclusion, bellflower root extract It was confirmed that the administration of improved memory without affecting motor activity.

<Example 3> Immunohistochemical analysis

<3-1> Organization preparation

The mice of Example 2 were anesthetized after the behavioral experiment was completed, and their hearts were perfused and fixed with a mixture of 0.05 M PBS solution, 0.1 M PB, and 4% formaldehyde (PFA). Afterwards, the brain was removed, immersed in 4% PFA and fixed at 4°C for 20 hours, and then immersed in a solution of 30% sucrose in 0.05M PBS to prevent freezing. The immersed brain tissue was cut into 30 μm thick coronal sections using a cryostat at -25°C. The cut tissue was stored in 0.05M PBS buffer containing 25% ethylene glycol and glycerol at 4°C until immunohistochemical analysis.

<3-2> Immunofluorescence labeling

Accumulation of Aβ (amyloid-β, 4G8) is known to have a direct impact on the onset and progression of cognitive decline, so we sought to confirm Aβ plaque formation. In addition, to observe neuronal death, neuron biomarkers neuronal nuclei (NeuN) and synaptic endoplasmic reticulum protein synaptophysin (SYN) were analyzed, and ionized calcium-binding adapter molecule 1 (ionized calcium-binding adapter molecule 1), an indicator of neuroinflammation, was analyzed. Immunofluorescence analysis was performed to analyze positive cells for calcium binding adapter molecule (Iba1) and glial fibrillary acidic protein (GFAP), respectively. Specifically, the prepared brain tissue of Example 3-1 was separated at intervals of 150-180 μm (-1.46 and -2.06 mm with respect to bregma) and 3- at intervals of 270-350 μm (-2.70 and -3.80 mm with respect to bregma). Four sections were selected and stained. The prepared sections were briefly washed with PBS, and the primary antibody was diluted with PBS containing BSA and 0.3% Triton X-100 at a concentration of 0.5 mg/ml and reacted for 18 hours. The primary antibodies used were mouse anti-4G8 antibody (1:2,000), mouse anti-neuronuclear (NeuN) antibody (1:500), mouse anti-synaptophysin (SYN 1:500), and goat anti-ionization. Ionized calcium binding adapter molecule 1 (Iba1, 1:1,000) antibody and rat anti-glial fibrillary acidic protein (GFAP, 1:500) antibody were used. Brain tissue was treated with 70% formic acid for 20 minutes for antigen detection before reaction with mouse anti-4G8 antibody. Secondary antibodies were donkey Alexa 488-conjugated anti-mouse IgG (1:200) and donkey Alexa 594-conjugated anti-mouse IgG (1:200) diluted in PBS containing 0.3% Triton It was reacted with secondary antibody for minutes. Immunohistochemically stained sections were mounted on slides using Fluoroshield ^™ containing 4,6-diamidino-2-phenylindole (DAPI) and overlaid with a cover slip.

As a result, as shown in Figure 3, it was confirmed that the 4G8 positive area, average plaque size, and number of plaques were reduced when 5 Suppression was confirmed.

In addition, the density of SYN (CA1, CA3, and ML) was confirmed to be significantly reduced in the hippocampus of 5XFAD+vehicle mice compared to wild-type mice. When bellflower root extract was administered to 5XFAD mice, the density of SYN was significantly increased. (Figures 4A to 4D). Compared to wild-type mice, NeuN-positive cells were decreased in mice in the 5XFAD+vehicle group. However, when bellflower extract was administered to 5XFAD mice, NeuN-positive cells significantly increased. This was confirmed (Figure 4E).

In addition, the Iba1-positive area and GFAP-positive area, which are neuroinflammatory factors, were confirmed to be significantly increased in 5XFAD+vehicle mice compared to wild-type mice. When 5XFAD mice were treated with bellflower root extract, the Iba1-positive area and GFAP-positive area were significantly increased. It was confirmed that it decreased significantly (Figure 5).

<3-2> Immunoperoxidase labeling

To confirm the neuroprotective effect of bellflower extract on neurogenesis in the adult hippocampus, immunoperoxidase analysis was performed using the adult neurogenesis factors DCX and Ki-67. Specifically, 3-4 sections of the brain tissue prepared in Example 3-1 were obtained at intervals between 120 and 180 μm (-1.70 and 2.18 mm for bregma). The obtained sections were washed with PBS and treated with PBS containing 1% H ₂ O ₂ at room temperature for 15 minutes. The tissue was then treated with the primary antibodies, goat anti-doublecortin (DCX 1:1,000) antibody and rabbit anti-Ki67 antibody (1:2,000) at 4°C. The sections treated with primary antibody were reacted with biotinylated secondary antibody (1:200) for 1 hour at room temperature. After completion of the reaction, avidin-biotin-peroxidase complex (ABC) solution and DAB (3,3-diaminobenzidine) were used for color development. The visualized sections were placed on slides, dehydrated with alcohol and xylene, and then overlaid with a coverslip using mounting medium (Thermo Fisher Scientific, Waltham, MA, USA).

As a result, as shown in Figure 6, Ki67 and DCX positive cells were confirmed to be significantly reduced in 5XFAD+vehicle mice compared to wild type mice, but when bellflower extract was administered to 5XFAD mice, Ki67 and DCX positive cells were significantly reduced There was no increase.

<Example 4> Confirmation of cell viability

HT22 cells, a rat hippocampal neuronal cell line, were cultured in Dulbeccos Modified Eagles Media (DMEM), specifically in a medium supplemented with 10% fetal calf serum and 100 U/ml penicillin-streptomycin, 5% CO2, 37°C. It was cultured under these conditions. The cultured cells were inoculated into a 96 well plate at 1 x 10^4 cells. 24 hours after inoculation, bellflower extract (PEG) was pretreated at concentrations of 50, 100, and 200 μg/ml for 1 hour, and Aβ25-35 was treated. After 48 hours of treatment Aβ25-35 was treated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4 sulfophenyl)-2H according to the manufacturer's guide. Cell viability was quantified using the -tetrazolium (MTS) assay, and absorbance was measured at 490 nm using a multimode microplate reader (Biotek Inc., Winooski, VT, USA). All experiments were performed three times.

As a result, it was confirmed that the cell survival rate of HT22 cells treated with Aβ decreased by 50% compared to the control group, and that the cell survival rate increased in HT22 cells treated with bellflower root extract (Figure 7).

<Example 5> Confirmation of intracellular ROS production

Intracellular ROS production was analyzed using a modified dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. HT22 cells were inoculated into a 96 well plate at a cell count of 1x10^4 and cultured for 24 hours. Afterwards, the bellflower extract was treated at the concentration described in Example 4 and further cultured for 24 hours. Cultured cells were treated with serum-free medium (SFM) containing 10 μM Aβ25-35 for 20 minutes, followed by SFM containing DCF-DA for 30 minutes. After 30 minutes, the cells were washed, and 100 μL of Dulbecco’s phosphate-buffer was added to each well. Afterwards, each fluorescence was measured at 485 nm/535 nm.

As a result, it was confirmed that when HT22 cells were treated with Aβ, the production of ROS increased by about 1.7 times compared to the control group. However, in cells treated with bellflower root extract, ROS production induced by Aβ was significantly reduced, confirming that bellflower root extract has neuroprotective and antioxidant effects (Figure 8).

<Example 6> Image analysis

Brains stained with antibodies using Zeiss LSM 700 (Carl Zeiss AG, Oberkochen, Germany) and Olympus CX23 (Olympus Corporation, Tokyo, Japan) microscopes to quantify the immune response in Examples 3-2 to 3-3. Images were obtained from the sections and analyzed using ImageJ software. Analyzes were performed on the ipsilateral and contralateral hippocampi of brain slices from each animal. To measure 4G8 immunoreactivity, the area fraction (%), average plaque size per area (mm ² ), and average plaque number for immunopositive signals in the dorsal hippocampal junction were quantified. Additionally, immunoreactivity of Iba1 and GFAP was quantified as the area fraction of immunopositive signal in the same dorsolateral hippocampal transition. SYN immunoreactivity was quantified by the fluorescence intensity (optical density) of the hippocampus, and DCX and Ki67 immunoreactivity was measured by positive cells per length (mm) of the subgranular zone in the hippocampal dentate gyrus (DG). It was quantified by the number of. Histological quantification, statistical analysis, and image acquisition were performed in a blinded manner for the four groups.

<Example 7> Statistical analysis

All examples of the present invention were performed blindly and randomly in four groups. All statistical analyzes were performed using GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA). All data were expressed as mean ± standard error of the mean (SEM), and the significance of differences between the two groups was determined independently. A t-test was used. Statistical analysis between the four groups was performed using one-way analysis of variance followed by Fisher's LSD and Tukey's post hoc test. If the p value was p<0.05, it was considered statistically significant.

Therefore, the bellflower extract of the present invention effectively inhibits the accumulation of Aβ in 5 It was confirmed that it can be effectively used for the prevention and treatment of degenerative brain diseases.

Claims (23)

A health functional food composition for preventing or improving Alzheimer's disease containing water extract of bellflower root ( Platycodon grandiflorum ) as an active ingredient,
The bellflower water extract is extracted by mixing dried bellflower root and water at a weight ratio of 1:10,
The dried bellflower root is bellflower root dried at 55°C for 72 hours,
The bellflower water extract contains 1596 to 1598 μg/g of platycoside E and 941 to 943 μg/g of platycodin D,
The bellflower water extract inhibits the accumulation of amyloid-β (4G8) and increases the number of neuronal nuclei (NeuN) and synaptophysin (SYN) positive cells in the hippocampus,
The composition is administered to a group of Alzheimer's disease patients with increased expression of human PSEN1 mutations M146L and L286V and APP mutations Sweden (K607N and M671L), Florida (I716V) and London (V717I),
The bellflower extract inhibits neuronal cell death caused by Aβ toxicity in the subiculum of the hippocampus, and inhibits ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, which are indicators of neuroinflammation. A composition that reduces the number of positive cells (protein, GFAP). delete delete delete delete According to clause 1,
The bellflower extract is a health functional food composition for preventing or improving Alzheimer's disease, characterized in that it inhibits Aβ-induced ROS production. According to clause 1,
The bellflower extract is a health functional food composition for preventing or improving Alzheimer's disease, characterized in that it improves cognitive function. delete delete delete A pharmaceutical composition for preventing or treating Alzheimer's disease containing a water extract of bellflower root ( Platycodon grandiflorum ) as an active ingredient,
The bellflower water extract is extracted by mixing dried bellflower root and water at a weight ratio of 1:10,
The dried bellflower root is bellflower root dried at 55°C for 72 hours,
The bellflower water extract contains 1596 to 1598 μg/g of platycoside E and 941 to 943 μg/g of platycodin D,
The bellflower water extract inhibits the accumulation of amyloid-β (4G8) and increases the number of neuronal nuclei (NeuN) and synaptophysin (SYN) positive cells in the hippocampus,
The composition is administered to a group of Alzheimer's disease patients with increased expression of human PSEN1 mutations M146L and L286V and APP mutations Sweden (K607N and M671L), Florida (I716V) and London (V717I),
The bellflower water extract inhibits neuronal cell death induced by Aβ toxicity in the subiculum of the hippocampus, and inhibits ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, which are indicators of neuroinflammation. A composition that reduces the number of acidic protein (GFAP) positive cells. delete delete delete delete According to claim 11,
A pharmaceutical composition for preventing or treating Alzheimer's disease, wherein the bellflower extract inhibits Aβ-induced ROS production. According to claim 11,
The bellflower extract is a pharmaceutical composition for preventing or treating Alzheimer's disease, characterized in that it improves cognitive function. delete delete delete As a method of increasing the content of platycoside E and platycodin D in bellflower water extract,
The bellflower water extract is extracted by mixing dried bellflower root and water at a weight ratio of 1:10,
The dried bellflower root is bellflower root dried at 55°C for 72 hours,
The bellflower water extract contains 1596 to 1598 μg/g of platycoside E and 941 to 943 μg/g of platycodin D,
The bellflower water extract inhibits the accumulation of amyloid-β (4G8) and increases the number of neuronal nuclei (NeuN) and synaptophysin (SYN) positive cells in the hippocampus,
The bellflower water extract is administered to Alzheimer's disease cells with increased expression of human PSEN1 mutations M146L and L286V and APP mutations Sweden (K607N and M671L), Florida (I716V) and London (V717I),
The bellflower water extract inhibits neuronal cell death induced by Aβ toxicity in the subiculum of the hippocampus, and inhibits ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, which are indicators of neuroinflammation. A method of reducing the number of acidic protein (GFAP) positive cells. A method for producing bellflower water extract with increased platycoside E and platycodin D,
The bellflower water extract is extracted by mixing dried bellflower root and water at a weight ratio of 1:10,
The dried bellflower root is bellflower root dried at 55°C for 72 hours,
The bellflower water extract contains 1596 to 1598 μg/g of platycoside E and 941 to 943 μg/g of platycodin D,
The bellflower water extract inhibits the accumulation of amyloid-β (4G8) and increases the number of neuronal nuclei (NeuN) and synaptophysin (SYN) positive cells in the hippocampus,
The bellflower water extract is administered to Alzheimer's disease cells with increased expression of human PSEN1 mutations M146L and L286V and APP mutations Sweden (K607N and M671L), Florida (I716V) and London (V717I),
The bellflower water extract inhibits neuronal cell death induced by Aβ toxicity in the subiculum of the hippocampus, and inhibits ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, which are indicators of neuroinflammation. A method of reducing the number of acidic protein (GFAP) positive cells. isolated from an Alzheimer's disease animal model with increased expression of human PSEN1 mutations M146L and L286V and APP mutations Sweden (K607N and M671L), Florida (I716V), and London (V717I) in vitro. Processing bellflower root water extract in hippocampal cells; inhibiting the accumulation of Aβ (amyloid-β, 4G8) containing and increasing the number of neuronal nuclei (NeuN) and synaptophysin (SYN) positive cells. As a method,
The bellflower water extract is extracted by mixing dried bellflower root and water at a weight ratio of 1:10,
The dried bellflower root is bellflower root dried at 55°C for 72 hours,
The method wherein the bellflower water extract contains 1596 to 1598 μg/g of platycoside E and 941 to 943 μg/g of platycodin D.