KR20230173044A - Composition comprising extract Davallia mariesii root extract for preventing or treating neurodegenerative diseases - Google Patents

Abstract

Provided is a composition for preventing, improving, or treating neurodegenerative diseases, comprising as an active ingredient an extract of Neukline fern, a fraction thereof, or a compound isolated therefrom.

Description

Composition for preventing or treating neurodegenerative diseases comprising extract of the fern fern as an active ingredient {Composition comprising extract Davallia mariesii root extract for preventing or treating neurodegenerative diseases}

The present invention relates to a pharmaceutical composition for preventing, ameliorating, or treating degenerative neurological diseases containing an extract of Neorthyria fern as an active ingredient, and more specifically, to a pharmaceutical composition for treating degenerative neurological diseases containing as an active ingredient a compound isolated from an extract of Neorthyria fern or a fraction thereof. It relates to a composition for preventing, improving, or treating neurological diseases.

Degenerative neurological disease is a disease that occurs in the nervous system, including the brain, among degenerative diseases that occur with age, and its incidence is significantly increasing as the average lifespan of humans increases. For example, neurodegenerative diseases can be classified into Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, etc., considering the main symptoms and affected brain areas. Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and is characterized by gradual memory decline and cognitive loss. As the average lifespan of humans increases, its incidence rate is significantly increasing.

One of the pathological characteristics of Alzheimer's disease is senile plaques that accumulate on the outside of nerve cells. The causative agent of this is beta-amyloid (beta-amyloid or amyloid-β, β-amyloid, Aβ). I can hear it. Beta-amyloid (Aβ) is a protein fragment cut from a protein called amyloid precursor protein (APP). The most important enzyme involved in beta-amyloid production is beta-secretase, also known as beta-site APP-cleaving enzyme (BACE1). APP cut by beta-secretase (BACE1) has an N-terminal domain called beta-secretase derived secreted form of APP (sAPPβ) of about 90 kDa and beta-secretase It is divided into a cytoplasmic domain called β-secretase derived secreted C-terminal fragment of APP (CTFβ) (C99). The sAPPβ produced in this way is secreted out of the cell, and C99 is cut again by gamma secretase (γ-secretase) to generate beta-amyloid of 4 kDa. Therefore, substances that inhibit the activity of beta secretase (BACE1) are expected to have a therapeutic or preventive effect on neurodegenerative diseases, including Alzheimer's disease, by inhibiting the production of beta-amyloid.

However, most of the drugs currently on the market or in development are drugs that improve the quality of life of Alzheimer's patients by improving their symptoms. Examples of such drugs include tacrine, an acetylcholine degradation inhibitor developed based on the cholinergic nervous system hypothesis; Examples include acetylcholinesterase inhibitors such as donepezil, rivastigmine, and galantamine, and memantine, an NMDA receptor antagonist. However, most of these treatments have only a temporary effect of improving clinical symptoms in the early stages of the disease and also have side effects, so there are many problems with their actual use, and there is a need to develop medicines that can provide more fundamental treatment for neurodegenerative diseases.

The present inventors sought to develop a therapeutic agent with the goal of fundamental treatment based on the cause of neurodegenerative diseases, which develops when beta-amyloid is produced in large amounts due to abnormal metabolism of amyloid precursor protein (APP), causing toxicity to brain cells. The present invention was completed after efforts to develop a drug that can inhibit the production of beta-amyloid protein to treat neurodegenerative diseases.

Selkoe DJ. 1991. The molecular pathology of Alzheimer’s disease. Neuron 6:487-498

The object of the present invention is to provide a composition that prevents, improves, or treats neurodegenerative diseases, including Alzheimer's disease and dementia, using a fern extract that is highly effective in inhibiting beta-amyloid production.

One aspect is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a compound isolated from an extract or a fraction thereof, a pharmaceutically acceptable salt thereof, or a combination thereof as an active ingredient. will be.

Another aspect is to provide a food composition for preventing or ameliorating neurodegenerative disease, comprising as an active ingredient a compound isolated from the Neortium fern extract or a fraction thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

Another aspect is a health functional food for improving learning or memory, or improving cognitive function, comprising a compound isolated from the ethyl acetate fraction of the ethanol extract of the Neukline fern, a pharmaceutically acceptable salt thereof, or a combination thereof as an active ingredient. is to provide.

Another aspect is to provide a feed composition for preventing or improving degenerative neurological diseases using a compound isolated from a Neorline fern extract or a fraction thereof, a pharmaceutically acceptable salt thereof, or a combination thereof as an active ingredient.

Other objects and advantages of the present application will become clearer from the following detailed description together with the appended claims. Contents not described in this specification can be fully recognized and inferred by anyone with ordinary knowledge in the technical field of this application or a similar technical field, so description thereof is omitted. The contents of all publications incorporated by reference herein are hereby incorporated by reference in their entirety. All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods are also included in the scope of the present invention.

In one aspect, (-)-epiafzelechin-3-O-β-D-allopyranoside ((-)-epiafzelechin-3-O-β isolated from Davallia mariesii extract or fractions thereof -D-allopyranoside), (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-β-D-glucopyranoside ((2S)-5,7,3', 5'-tetrahydroxy-flavanone 7-O-β-D-glucopyranoside), (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperidoside ((2S)- 5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperidoside), 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopi Neurodegenerative disease containing lanoside (7-O-methyl-epiafzelechin-(4α→8)-epiafzelechin-3-O-β-glucopyranoside), a pharmaceutically acceptable salt thereof, or a combination thereof as an active ingredient A pharmaceutical composition for prevention or treatment is provided.

Davallia mariesii (also written as Davallia mariesii T. Moore ex Baker) used in this specification is a fern belonging to the genus Davallia of the family Davalliaceae. It grows attached to the surface of rocks or the trunks of trees. The rhizome is 3 to 5 mm in diameter, thick and extends long to the side, is brown or grey-brown, and has linear scales growing densely on the surface. Leaves grow sparsely. It is distinguished from other ferns in that the underground stem is exposed on the rock and crawls long to the side, and the densely attached scales are linear, lanceolate, or shield-shaped. It grows throughout the Korean Peninsula and is distributed in Japan, Taiwan, and China.

The above-mentioned fern can be purchased commercially or used as one collected or cultivated in nature.

The above-mentioned fern extract can be obtained from the roots and rhizome of the four-lined fern. Preferably, in one embodiment, the extract of the Neoline fern may be an extract of the root of the root of the fern, the rhizome of the root of the fern, or a combination thereof.

The term “extract” used in this specification refers to an extract, such as an extract obtained by extracting and processing a fern, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a mixture thereof. It includes extracts of all formulations that can be formed on their own and using extracts.

The method for extracting the four-lined fern is not particularly limited, and can be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include cold needle extraction, hot needle extraction, hot water extraction, ultrasonic extraction, filtration, and reflux extraction, which can be performed alone or by combining two or more methods.

The type of extraction solvent used to extract the four-lined fern is not particularly limited, and any solvent known in the art can be used. In one embodiment, the extraction solvent for the extract of the fern extract may be water, C ₁ to C ₄ alcohol, or a mixture thereof. In one specific example, the extraction solvent for the Neoline fern extract may be ethanol. In one embodiment, the alcohol may be 100% alcohol or may be provided by diluting the alcohol in water, such as 1 to 99% (v/v) of alcohol.

The ethanol is 10% (v/v) to 100% (v/v), 20% (v/v) to 100% (v/v), 30% (v/v) to 99% (v/v) , it may be at a concentration of 50% (v/v) to 95% (v/v), or 70% (v/v) to 90% (v/v). In one embodiment, the ethanol may have a concentration of 20% (v/v) to 100% (v/v).

The extraction temperature of the extract may be performed at 4°C to 120°C. Additionally, the extraction time may be 12 to 120 hours. If the extraction temperature is lower than 4°C, extraction may not be successful, and if it exceeds 120°C, the extract may be denatured. If the extraction time is less than 12 hours, some of the active ingredients may not be extracted, and if it exceeds 120 hours, impurities other than the active ingredients may be extracted together, which is not desirable. Alternatively, the extraction temperature may be room temperature.

If necessary, the extraction process can be repeated 1 to 10 times, for example, 2 to 5 times.

Additionally, the extract may be prepared and used in the form of a dry powder after extraction, and may be concentrated and used as needed, but is not limited thereto.

As used herein, the term “fraction” refers to the result obtained by performing fractionation to separate a specific component or specific group of components from a mixture containing various various components.

The fractionation method for obtaining the above fraction is not particularly limited and may be performed according to methods commonly used in the art. Non-limiting examples of the fractionation method include solvent fractionation performed by treating various solvents, ultrafiltration fractionation performed by passing an ultrafiltration membrane with a certain molecular weight cut-off value, and various chromatography methods (size, charge, hydrophobicity). or chromatographic fractionation methods (designed for separation based on affinity), and combinations thereof. In one specific example, there is a method of obtaining a fraction from the extract by treating the extract obtained by extracting the fern with a predetermined solvent. In one embodiment, the extract of Neorjoule fern can be fractionated according to the polarity of the solvent.

The type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fractionating solvent include polar solvents such as water and C ₁ to C ₄ alcohols; Nonpolar solvents such as hexane (n-hexane, Hx), dichloromethane (DCM), and ethyl acetate (EA); Or a mixed solvent thereof, etc. may be mentioned. These may be used alone or in combination of one or more types, but are not limited thereto.

In one embodiment, after obtaining the extract of the Neoline fern, fractions can be obtained by fractionating according to the polarity of the solvent. In one embodiment, the fraction may be fractionated with hexane, dichloromethane, ethyl acetate, or water.

In one embodiment, the fraction may be fractionated with ethyl acetate. In one specific example, the fraction may be an ethyl acetate fraction of the ethanol extract of Neorline fern.

The organic solvent used in the fractionation solvent may be, for example, an aqueous solution of 0% (v/v) to 99% (v/v) or a solvent with a concentration of 100% (v/v). The fractionation temperature may be performed at 4°C to 120°C. Alternatively, the fractionation temperature may be room temperature. If necessary, the fractionation process can be repeated 1 to 10 times, for example, 2 to 5 times. If necessary, the extract may be suspended before the fractionation process. The fractionated extract obtained by performing the above fractionation process can be concentrated using a vacuum rotary concentrator or vacuum dryer and then stored at room temperature.

Additionally, the extract or fraction may be prepared and used in the form of a dry powder after extraction, and may be concentrated and used as needed, but is not limited thereto.

Amyloid precusor protein (APP) is divided by α,β,γ-secretase, N-terminal proteins are released into the supernatant, and C-terminal proteins remain within the cell. do. Beta-amyloid (Aβ) is a fragment cut from the APP protein, and the most important enzyme involved in its production is beta-secretase (BACE1).

In one embodiment, the pharmaceutical composition may include as an active ingredient a compound isolated from the ethyl acetate fraction of the ethanol extract of Neukline fern. In one specific example, as a result of measuring the ability to inhibit beta-amyloid production, the ethyl acetate fraction of the ethanol extract of Neukline fern showed the best beta-amyloid production inhibitory activity.

In one specific example, the roots and rhizome of the Four-lined Fern were extracted with 80% ethanol, fractionated according to solvent polarity, and each fraction was measured for inhibition of beta-amyloid production. As a result, the ethyl acetate layer (ethyl acetate fraction) most effectively inhibited beta-amyloid. Production was suppressed. The inhibitory effect of the ethyl acetate layer (ethyl acetate fraction) on beta-amyloid production was due to reducing the activity of beta-secretase, the most important enzyme in beta-amyloid production.

In addition, the extract, fractions, and compounds isolated therefrom according to one embodiment have the effect of inhibiting beta-amyloid aggregation and dismantling beta-amyloid aggregates, and can be effectively used in the prevention, improvement, and treatment of neurodegenerative diseases. .

In one embodiment, a compound that has an inhibitory effect on beta-amyloid production can be isolated from the Neukline fern extract or a fraction thereof using an activity-guided isolation method.

In one embodiment, the compound isolated from the extract or fraction thereof is (-)-epiaphzelechin-3-O-β-D-allopyranoside ( 1 ), (2S)-5,7,3',5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside ( 2 ), (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O- It may be hesperidoside ( 4 ), 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside ( 5 ), or mixtures thereof. there is. The compounds may be represented by the following formulas 1 to 2, 4, and 5.

[Chemical formula]

The above compounds are derived from the fern and significantly inhibit myloid production in a concentration-dependent manner, which is due to reducing beta-secretase activity.

In addition, the above compounds are derived from the four-lined fern and not only inhibit beta-amyloid aggregation, but also dismantle aggregated beta-amyloid to reduce the amount of aggregates and reduce neurotoxicity caused by beta-amyloid aggregates.

Accordingly, beta-amyloid production is reduced, which can prevent, improve, and treat degenerative neurological diseases, including Alzheimer's disease, or improve learning or memory or cognitive function, and can be a more fundamental treatment for dementia.

The above compounds may also include those derived from other plants, and may specifically be derived from the four-lined fern, but are not limited thereto.

In one specific example, the active ingredient may be obtained from the ethyl acetate fraction of the Neoline fern extract. In one specific example, the fraction may be an ethyl acetate fraction of the ethanol extract of Neorline fern.

The term "neurodegenerative disease" used herein is deeply related to aging, and unlike the normal aging process, the term "neurodegenerative disease" occurs rapidly in parts of the nervous system or the entire brain, causing death of abnormal nerve cells in the brain and spinal cord. It refers to a disease in which cognitive ability, walking-motor ability, etc. are reduced due to loss of function. For example, it refers to a disease that manifests as expressive dysfunction due to reasons such as degeneration or loss of brain and spinal cord cells of the central nervous system. In one embodiment, the neurodegenerative disease may be a degenerative brain disease.

The term “degenerative brain disease” used herein is one of the degenerative neurological diseases and refers to a disease that occurs in the brain as one ages. Due to causes that are not well known to date, certain groups of brain cells in the brain gradually lose their functions, death of brain nerve cells that are most important in transmitting information in the brain nervous system, problems with the shape or function of synapses that transmit information between brain nerve cells, or It is known to be caused by an abnormal increase or decrease in the electrical activity of cranial nerves.

The term “amyloid” used herein refers to a fibrous structure formed by specific interactions between proteins as water-soluble proteins exhibit water-insoluble properties in various biochemical processes, and is associated with the neurodegenerative diseases mentioned herein. Corresponds to common protein aggregates observed in .

In one embodiment, the neurodegenerative disease includes Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, dementia associated with beta-amyloid, and Parkinson's disease. Parkinson's disease, mild cognitive impairment, senile dementia, amyotrophic lateral sclerosis or Lou Gehrig disease, Huntington's disease, Niemann-Pick Disease, Dutch Consists of amyloidosis, amyloid stroke, Down's syndrome, senility, multiple sclerosis, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, and dementia. It may be any one selected from the group, but is not limited thereto.

In another embodiment, the neurodegenerative disease may be any one selected from the group consisting of Alzheimer's disease, cerebral amyloid angiopathy, systemic amyloidosis, beta-amyloid-related dementia, Parkinson's disease, mild cognitive impairment, and senile dementia.

The term “Alzheimer's disease” used herein is a degenerative neurological disease in which brain cells are gradually destroyed, leading to gradual loss of memory and cognitive ability and ultimately death. In the brain tissue of patients with Alzheimer's disease, senile plaques called beta-amyloid (Aβ) accumulate and neurofibrillary tangles (NFTs) appear in brain cells, resulting in widespread degenerative atrophy throughout the brain tissue. It happens. In this specification, “Alzheimer’s disease” may be used interchangeably with Alzheimer type dementia.

The term “cerebral amyloid angiopathy” used herein is a disease in which amyloid is deposited on the walls of cerebral blood vessels in the cortex and leptomeninges. Cerebral amyloid angiopathy causes lobar cerebral hemorrhage, dementia, transient ischemic attack, and epileptic seizures. Some studies have shown that the frequency of cerebral amyloid angiopathy in Alzheimer's disease reaches 80-90%, and it is known to be deeply related to Alzheimer's disease.

The term “systemic amyloidosis” used herein refers to a disease in which amyloid protein accumulates excessively in tissues or organs throughout the body, causing dysfunction of tissues or organs.

As used herein, the term “Dementia associated with beta-amyloid” refers to dementia caused by Alzheimer's disease, that is, dementia that develops when beta-amyloid is deposited in brain tissue, and is caused by beta-amyloid deposition. Includes dementia. Patients with this disease not only gradually lose their memory, but also lose the ability to accurately understand the situation they are in, their learning ability, language ability, and judgment ability decline, and they even lose the ability to take care of themselves. It leads to dementia. As the brain damage of patients with Alzheimer's disease gradually worsens, the functions of the central nervous system as well as the autonomic nervous system are violated, worsening the overall health condition.

The term "Parkinson's disease" used herein is caused by the gradual loss of dopamine neurons distributed in the substantia nigra of the brain and causes resting tremor, rigidity, bradykinesia (slow movement), and postural instability. It is a chronic, progressive degenerative disease of the nervous system that is characteristically manifest.

The term “mild cognitive impairment” used in this specification refers to a state in which the decline in memory or other cognitive functions is clearly deteriorated to the extent that it can be confirmed in an objective test, but the ability to perform daily life is preserved and is not yet dementia. .

The term “senile dementia” used in this specification refers to a condition in which a person who has been leading a normal life experiences damage to brain function due to various causes after the age of 65, resulting in a continuous and overall decline in cognitive function compared to before, causing significant disruption in daily life. It is a disease that gives.

The term "dementia" used in this specification is a disease in which a person's brain function is damaged due to various causes and the cognitive function is continuously and overall deteriorated compared to before in a person who has been leading a normal life, causing significant disruption in daily life. Alzheimer's dementia, Dementia associated with beta-amyloid, Senile dementia, Lewy Body Dementia, Multi-infarct dementia, Dementia associated with stroke), Dementia associated with Parkinson's disease, vascular dementia, and frontotemporal dementia.

In one embodiment, the neurodegenerative disease may be Alzheimer's disease.

In one embodiment, the neurodegenerative disease may be caused by beta-amyloid aggregation.

In one embodiment, the neurodegenerative disease may be a disease whose main cause is beta-amyloid accumulation in the brain.

In one embodiment, the pharmaceutical composition can inhibit beta-amyloid (β-amyloid) production.

In one embodiment, the pharmaceutical composition may reduce beta-secretase activity.

In one embodiment, the pharmaceutical composition can inhibit beta-amyloid aggregation and/or dismantle aggregated beta-amyloid.

In one embodiment, the pharmaceutical composition can increase acetylcholine content.

In one embodiment, the pharmaceutical composition may reduce acetylcholinesterase activity.

In one embodiment, the pharmaceutical composition can reduce beta-amyloid deposition.

In one embodiment, the pharmaceutical composition can increase the expression of BDNF and p-CREB, which are involved in synaptic plasticity that is important for memory and cognitive function.

Neurotrophic factor (brain-derived neurotrophic factor, BDNF) is known to have a cognitive and memory-enhancing effect and is closely related to long-term memory formation.

Cyclic AMP-response element binding protein (CREB) is known to play a role in brain neuronal plasticity and long-term memory formation.

Additionally, in one embodiment, the pharmaceutical composition can significantly improve the memory of a subject administered it.

According to one embodiment, a pharmaceutical composition comprising as an active ingredient an ethanol extract or fraction of the root or rhizome of the root or rhizome of the Four-lined Fern, or a compound of formulas 1 to 4 isolated therefrom, inhibits beta-amyloid production or is the most effective agent for beta-amyloid production. Degenerative neurological diseases can be treated, improved, and prevented by reducing the activity of beta-secretase, an important enzyme.

In addition, according to one embodiment, ethanol extracts, fractions, and compounds isolated therefrom from the roots or rhizome of Neukline fern significantly improve the memory of mice administered scopolamine.

As used herein, the term “included as an active ingredient” means included in an amount sufficient to achieve the desired efficacy or activity.

The content of the fern extract, fraction or compound derived therefrom in the composition according to the present invention can be appropriately adjusted depending on the type and age of the applied livestock, application form, desired effect, etc., for example, 1 to 99% by weight, preferably. Can be used in an amount of 10 to 90% by weight, more preferably 20 to 80% by weight, but is not limited thereto.

In the composition according to one embodiment, the extract or fraction thereof, or the compound of Formulas 1 to 5 isolated therefrom, is contained in an amount of 0.1 μg/mL to 1000 μg/mL, for example, 0.1, 0.5, 0,75, 1, 2. , 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 150, 200, 250, 300 , 500, 700, 900, 1000 ㎍/mL, or a range with any of the preceding values as the upper or lower limit, such as 1 ㎍/mL to 100 ㎍/mL, 5 ㎍/mL to 100 ㎍/mL, 10 ㎍/mL. to 100 μg/mL, 1 to 90 μg/mL, 1 μg/mL to 80 μg/mL, 1 μg/mL to 70 μg/mL, 1 μg/mL to 60 μg/mL, or 1 μg/mL to 50 μg/mL. It may be ㎍/mL.

According to one embodiment, the dosage of the composition containing the extract or fractions thereof of Neukline fern, or the compounds of formulas 1 to 5 isolated therefrom, is 0.1 mg/kg to 1000 mg/kg, for example, 0.1, 0.5, 0, 75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 150, 200, 250, 300, 500, 700, 900, 1000 mg/kg or a range with any of the preceding values as the upper or lower limit, such as 1 mg/kg to 500 mg/kg, 5 mg/kg to 400 mg/kg , 5 mg/kg to 200 mg/kg, 10 mg/kg to 200 mg/kg, 10 mg/kg to 100 mg/kg, 1 to 400 mg/kg, 10 mg/kg to 300 mg/kg, 10 mg /kg to 200 mg/kg, 20 mg/kg to 200 mg/kg, or 50 mg/kg to 200 mg/kg.

The term “treatment” used herein refers to any action in which a neurodegenerative disease is improved or beneficially altered by administration of the pharmaceutical composition.

As used herein, the term “prevention” refers to any action that inhibits or delays the progression of a neurodegenerative disease by administering the pharmaceutical composition of the present invention.

As used herein, “pharmaceutically acceptable” means exhibiting non-toxic properties to cells or humans exposed to the composition. In one embodiment, the active ingredient may be included in a pharmaceutically acceptable salt form.

As used herein, the term "pharmaceutically acceptable salt thereof" or "foodologically acceptable salt thereof" is generally safe, non-toxic, and is not biologically or otherwise unsuitable, so it can be used as a pharmaceutical and food composition. Indicates that it can be used in the manufacture of.

As used herein, “pharmaceutical composition” may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may be a non-naturally occurring carrier. carrier).

The type of the carrier is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in combination of two or more types. In addition, if necessary, other common additives such as antioxidants, buffers, and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to form solutions such as aqueous solutions, suspensions, emulsions, etc. It can be formulated and used in dosage form, pills, capsules, granules, or tablets.

The administration method of the pharmaceutical composition for preventing or treating neurodegenerative diseases according to the present invention is not particularly limited and may follow methods commonly used in the art. As a non-limiting example of the administration method, in one embodiment, the pharmaceutical composition may be for oral or parenteral administration. Additionally, the composition for preventing or treating planetary diseases of the present invention can be prepared in various formulations depending on the desired administration method. In one embodiment, the pharmaceutical composition may be formulated with a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspending agents, syrups, injections, and granules.

Another aspect is (-)-epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7,3' isolated from Davallia mariesii extract or fractions thereof. ,5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperido Seed, 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside, pharmaceutically acceptable salts thereof, or a combination thereof are effective. Provided is a food composition for preventing or improving neurodegenerative diseases, including the composition as an ingredient.

Another aspect provides a method for preventing or treating neurodegenerative disease, comprising administering to a subject a pharmaceutical composition containing an extract of Neukline fern, a fraction thereof, or a compound isolated therefrom as an active ingredient.

At this time, the definitions of the neurodegenerative disease, extracts, fractions, compounds isolated therefrom, prevention and treatment are as described above.

As used herein, the term “administration” means introducing a substance into a subject by an appropriate method.

As used herein, the term “individual” refers to mammals such as rats, mice, dogs, cats, hamsters, guinea pigs, birds such as parrots and canaries, including humans who have or may develop degenerative neurological diseases, and other livestock. This means all animals. As a specific example, it may be a mammal, including humans, or it may be a companion animal that lives with humans.

In one embodiment, the method for preventing or treating neurodegenerative diseases may include administering to a subject a pharmaceutical composition containing an extract of the fern or a fraction thereof, or a compound isolated therefrom as an active ingredient in a pharmaceutically effective amount. You can.

As used herein, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's gender. , factors including age, weight, health status, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors. It can be readily determined by a person skilled in the art according to factors well known in the medical field.

The pharmaceutical composition of the present invention can be administered at 0.0001 to 500 mg/kg of body weight per day, more specifically 0.01 to 500 mg/kg of body weight, based on solid content. The recommended dosage may be administered once a day, or it may be administered several times.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Additionally, it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

In the method for preventing or treating neurodegenerative diseases according to one embodiment, the administration route and method of administering the composition are not particularly limited, and any administration method may be used as long as the composition containing the composition can reach the target area. It may depend on the route and method of administration. Specifically, the composition may be administered through various oral or parenteral routes, and non-limiting examples of the administration route include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, and nasal. It may be administered intralaterally or through inhalation.

Another aspect is (-)-epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7,3' isolated from Davallia mariesii extract or fractions thereof. ,5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperido Seed, 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside, pharmaceutically acceptable salts thereof, or a combination thereof are effective. Provided is a food composition for preventing or improving neurodegenerative diseases, including the composition as an ingredient.

Another aspect is (-)-epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7,3' isolated from Davallia mariesii extract or fractions thereof. ,5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperido Seed, 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside, pharmaceutically acceptable salts thereof, or a combination thereof are effective. It provides health functional foods that contain ingredients for improving learning or memory, or improving cognitive function.

Another aspect is (-)-epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7 isolated from the ethyl acetate fraction of the ethanol extract of Davallia mariesii. ,3',5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-Tetrahydroxy-flavanone 7-O- Hesperidoside, 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside, pharmaceutically acceptable salts thereof, or their We provide health functional foods for improving learning or memory or cognitive function that contain the combination as an active ingredient.

As used herein, the term “improvement of learning or memory” refers to any action in which symptoms of decreased learning function or memory loss are improved or beneficially changed by the extract, fraction, or compound derived therefrom according to the present invention. Also, , The term "cognitive function improvement" in the present invention refers to all actions in which symptoms of cognitive decline are improved or beneficially changed by the extract, fraction, or compound derived from the fern according to the present invention.

At this time, the definitions of the neurodegenerative disease, extracts, fractions, compounds isolated therefrom, and prevention are as described above.

As used herein, the term "improvement" means any action that results in at least a reduction in the severity of a parameter, such as a symptom, associated with the condition being treated by administration of a composition according to the invention.

The term “food” used in this specification refers to meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcohol. There are beverages, vitamin complexes, health functional foods, and health foods, and include all foods in the conventional sense.

In one embodiment, since other food compositions can be consumed on a daily basis, a high Parkinson's disease improvement effect can be expected, so they can be very useful for health promotion purposes.

The term “functional food” used in this specification is the same as food for special health use (FoSHU), which refers to medical and medical products processed to efficiently exhibit bioregulatory functions in addition to supplying nutrients. It means food with high effectiveness. Here, ‘function’ means controlling nutrients for 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 types of formulations, and unlike general drugs, it is made from natural products 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 highly portable, so it can be used as a raw material. The food of the invention can be consumed as a supplement to enhance the effect of improving degenerative neurological diseases.

Health food refers to food that has a more active health maintenance or promotion effect than regular food, and health supplement food refers to food for health supplement purposes. In some cases, the terms health functional food, health food, and health supplement are used interchangeably.

The health functional food is a food manufactured by adding the extract of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspending it, and has specific health effects when consumed. However, unlike regular drugs, it has the advantage of being made from food and eliminating the side effects that can occur when taking the drug for a long period of time.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art can be used.

In addition, the food composition may contain preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), if necessary. Colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), seasonings (MSG monosodium glutamate, etc.), sweeteners (dulcine, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents It may contain food additives such as (alum, D-potassium hydrogen tartrate, etc.), emulsifiers, thickeners, coating agents, anti-foam agents, solvents, and improvers. The additives can be selected depending on the type of food and used in an appropriate amount.

Another aspect is (-)-epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7,3' isolated from Davallia mariesii extract or fractions thereof. ,5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperido Seed, 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside, pharmaceutically acceptable salts thereof, or a combination thereof are effective. Provided is a composition for feed for preventing or improving neurodegenerative diseases, including the composition as an ingredient.

At this time, the definitions of the neurodegenerative disease, extracts, fractions, compounds isolated therefrom, and prevention or improvement are as described above.

The composition for feed can be used to prevent or treat the development of neurodegenerative diseases in subjects other than humans, such as livestock or companion animals, and can be used as a functional feed additive or composition for feed.

For administration, the composition for feed additionally contains organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid; Phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate; One or more types of natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, and tannic acid can be used in combination. Additionally, diluents, dispersants, surfactants, binders, or lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., capsules, granules, or tablets.

When using the composition for feed as a feed additive, the composition may be added as is or used together with other ingredients, and may be used appropriately according to conventional methods. The dosage form of the composition for feed can be prepared as an immediate-release or sustained-release formulation by combining it with a non-toxic pharmaceutically acceptable carrier. Such edible carriers may be corn starch, lactose, sucrose, or propylene glycol.

The composition for feed can be mixed with livestock feed in an amount of about 10 to 500 g, specifically 10 to 100 g per 1 kg on a dry weight basis, and supplied as a mash after complete mixing, or pounded through an additional processing process. It can be through latting, expansion, or extrusion processes.

As used herein, terms such as “have,” “may have,” “includes,” or “may include” indicate the presence of the corresponding feature (e.g., numerical value, or component such as an ingredient). indicates, does not rule out the presence of additional features.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the examples described above are illustrative in all respects and not restrictive. All numbers expressing the amounts of components used herein, properties such as molecular weight, reaction conditions, etc. are used in all cases, unless otherwise specified. is understood to be modified by the term “about”. Accordingly, the numbers set forth herein are approximations that may vary depending on the desired properties to be achieved by the present invention. For example, the term “about” used herein means within 5% of a predetermined value or range, preferably within 1% to 2%.

According to one embodiment, a pharmaceutical composition containing an extract of the fern extract, a fraction thereof, or an active ingredient isolated therefrom reduces the production of beta-amyloid, a causative agent of neurodegenerative diseases such as Alzheimer's disease, and inhibits the production of beta-amyloid. It can reduce the activity of beta secretase, the most important enzyme. In addition, according to one embodiment, a pharmaceutical composition containing an extract of Neukline fern, a fraction thereof, or an active ingredient isolated therefrom increases the acetylcholine content, reduces acetylcholinesterase activity, and reduces beta-amyloid deposition. and increases the expression of BDNF and p-CREB, which are involved in synaptic plasticity, which is important for memory and cognitive function. Accordingly, it provides a more fundamental treatment for neurodegenerative diseases that occur when beta-amyloid is produced in large amounts due to abnormal metabolism of amyloid precursor protein (APP) and causes toxicity to brain cells.

Accordingly, the composition according to one embodiment can be usefully used as a medicine, food, or health functional food for the prevention, improvement, treatment, or improvement of neurodegenerative diseases, including Alzheimer's disease. Therefore, it can be used as a medicine, supplement, health food, or functional food for patients who suffer from or may suffer from diseases related to beta-amyloid accumulation in the brain, including Alzheimer's disease, and can also be used for brain tissue regeneration for the treatment of Alzheimer's disease. can be used

Figure 1 is a graph showing the results of measuring the cytotoxicity of the hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) fractions of the Neoline fern extract (Y-axis: cell survival rate relative to the control group) (%)).
Figure 2 is a diagram showing the effect of the ethanol extract of Neukline fern on beta-amyloid production. Figure 2A shows the results of Western blot analysis in which sAPPβ and beta secretase proteins were separated through electrophoresis. Figures 2B and 2C show the contents of sAPPβ and betasecretase (Y-axis: expressed as % relative to the control group) measured by antibody binding method, respectively.
Figure 3 is a graph showing the results of measuring the effect of the hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) fractions of the Neorline fern extract on beta-amyloid production inhibition. Figure 3A shows the results of Western blot analysis in which sAPPβ and beta secretase proteins were separated through electrophoresis. Figures 3B and 3C show the contents of sAPPβ and betasecretase (Y-axis: expressed as % relative to the control group) measured by antibody binding method, respectively.
Figure 4 is a schematic diagram showing the separation process of the compounds of formulas 1 and 2 separated from the Neoline fern extract or fractions thereof.
Figure 5 is a schematic diagram showing the separation process of the compound of Chemical Formula 4 isolated from the Neoline fern extract or fractions thereof.
Figure 6 is a schematic diagram showing the separation process of the compound of Chemical Formula 5 isolated from the Neoline fern extract or fractions thereof.
Figure 7a shows ¹ H-NMR data measured for the compound of Formula 1. Figure 7b shows ¹³ C-NMR data measured for the compound of Formula 1.
Figure 8a shows the results of ¹ H-NMR analysis of the compound of Formula 2. Figure 8b shows the results of ¹³ C-NMR analysis of the compound of Formula 2.
Figure 9a shows the results of ¹ H-NMR analysis of the compound of Formula 4. Figure 9b shows the results of ¹³ C-NMR analysis of the compound of Formula 4. Figures 9c and 9d show 2D NMR data results for ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 4, respectively.
Figure 10a shows the results of ¹ H-NMR analysis of the compound of Formula 5. Figure 10b shows the results of ¹³ C-NMR analysis of the compound of Formula 5.
Figure 11 shows the structural formulas of compounds of formulas 1, 2, 4, and 5 isolated from the Neoline fern extract or fractions thereof.
Figure 12 is a graph showing the results of measuring the cytotoxicity of compounds of formulas 1, 2, 4, and 5 isolated from the Neoline fern extract or fractions thereof (Y-axis: cell survival rate (%) relative to the control group).
Figure 13 is a graph showing the results of measuring the effect of compounds of Formulas 1 and 2 on inhibiting beta-amyloid production.
Figure 14 is a graph showing the results of measuring the effect of the compound of Formula 4 on inhibiting beta-amyloid production.
Figure 15 is a graph showing the results of measuring the effect of the compound of Formula 5 on inhibiting beta-amyloid production.
Figure 16 is a graph showing the results of measuring the residence time in the dark room of scopolamine-induced mice (Y-axis: residence time (seconds)).
Figure 17 is a graph showing the results of measuring the escape latency of scopolamine-induced mice (Y-axis: escape delay time (seconds)).
Figure 18 shows the change in acetylcholine content due to the extract of Neukline fern (X-axis: normal group (N), scopolamine-treated group (C), donepezil 0.75 mg/kg + scopolamine-treated group, positive control group ( P), Four-lined fern 200 mg/kg+scopolamine-treated group (AL), Four-lined fern 500 mg/kg+scopolamine-treated group (AH), Y-axis: concentration (pg/mL)).
Figure 19 shows the change in acetylcholinesterase activity due to the extract of Neukline fern (X-axis: normal group (N), scopolamine-treated group (C), donepezil 0.75 mg/kg + scopolamine-treated group, Positive control group (P), Four-lined fern 200 mg/kg+scopolamine-treated group (AL), Four-lined fern 500 mg/kg+scopolamine-treated group (AH), Y-axis: Activity (mU/mL))
Figure 20 shows the effect of beta-amyloid reduction by the extract of Neorjoule fern (normal group (N), scopolamine-treated group (C), donepezil 0.75 mg/kg + scopolamine-treated group, positive control group (P), Neopjoule Bracken 200 mg/kg+scopolamine treated group (AL), Neorline fern 500 mg/kg+scopolamine treated group (AH)).
Figure 21 shows changes in the expression of cerebral BDNF, CREB, and p-CREB by extract of Neokline fern (normal group (N), scopolamine-treated group (C), donepezil 0.75 mg/kg + scopolamine-treated group , positive control group (P), four-lined fern 200 mg/kg+scopolamine-treated group (AL), four-lined fern 500 mg/kg+scopolamine-treated group (AH)).
Figure 22 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the Neoline fern extract or fractions thereof. From this, the effect of inhibiting beta-amyloid aggregation is confirmed.
Figure 23 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the Neoline fern extract or fractions thereof. From this, the effect of dismantling beta-amyloid aggregates is confirmed.
Figure 24 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the active ingredients isolated from the Neoline fern extract. From this, the effect of inhibiting beta-amyloid aggregation is confirmed.
Figure 25 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the active ingredients isolated from the Neoline fern extract. From this, the effect of dismantling beta-amyloid aggregates is confirmed.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Preparation Example 1. Cell culture of transfected cell line (using APP-CHO cells) overexpressing amyloid precursor protein (APP)

The APP-CHO cell line used in the experiment was cultured in an incubator at 37°C and 5% CO ₂ using RPMI medium containing 10% FBS, and the medium was changed every 3 days.

Example 1. Preparation of Neorline fern extract and fractions

Purchase 3 kg of roots and rhizomes of Davallia mariesii , grind them finely with a grinder, extract them 3 times with 80% ethanol (20 L of EtOH, 3 times), and then concentrate the filtered filtrate under reduced pressure to obtain 235.5 g of ethanol from the fern. The extract was obtained.

Fractions were obtained from each fraction layer obtained by fractionating the obtained ethanol extract of Neukline fern according to the polarity of the solvent. Specifically, the obtained extract was dissolved in a small amount of methanol (10-20 mL), suspended in water (4-5 L), and mixed with n -hexane, dichloromethane, and ethyl acetate ( ethylacetate) to obtain a hexane fraction (26.3 g), a dichloromethane fraction (1.6 g), an ethyl acetate fraction (29.1 g), and a water fraction (170.3 g).

Experimental Example 1. Cytotoxicity evaluation of extracts and fractions of Neukline fern (MTT assay)

MTT-based cytotoxicity assay was performed to test the cytotoxicity of the root and rhizome extracts of Neukline fern on APP-CHO cells. After culturing the drug-treated cells for 24 hours, MTT solution at a concentration of 5 mg/ml was added and cultured for 3 hours. As the treatment drug, the extract of Neorline fern obtained in Example 1 and fractions of hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) were used. Each fraction was treated at concentrations of 12.5, 25, 50, and 100 μg/mL.

The medium was removed, DMSO was added to dissolve for 30 minutes, and the absorbance was measured at 540 nm using an Emax precision microplate reader (Molecular Devices, CA, USA). The same method was performed, but the group that was not treated with the root extract of Neoline fern (DMSO treated) was used as the control group.

The treatment of APP-CHO cells with the ethanol extract of Neoline fern showed no toxicity. The extract of Neoline fern did not show cytotoxicity up to 100 ㎍/mL, the highest concentration treated in the experiment.

Figure 1 is a graph showing the results of measuring the cytotoxicity of the hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) fractions of the Neoline fern extract (Y-axis: cell survival rate relative to the control group) (%)).

As shown in Figure 1, fractions excluding the water layer showed no cytotoxicity up to 50 μg/mL.

Experimental Example 2. Evaluation of the inhibitory effect of Beta-amyloid production of Neukline fern extracts and fractions: Measurement of changes in sAPPβ and beta-secretase levels using Western blot analysis

The inhibitory effect of the extracts and fractions of Neorline fern on beta-amyloid production was measured, and whether this effect was due to inhibition of beta-secretase was evaluated. For this purpose, changes in the levels of beta-secretase and the resulting fragment, sAPPβ, were measured using western blot analysis. As a treatment drug, the extracts of the Neoline fern obtained in Examples 1 and 2 and their respective fractions of hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) were treated. As a positive control group (PC), a Chinese root extract (50 ㎍/mL) was used.

As the treatment drug, the Neorline fern extract obtained in Example 1 and its fractions of hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) were used. The Neoline fern extract was treated at concentrations of 12.5, 25, 50, and 100 ㎍/mL. Each fraction was treated at a concentration of 50 μg/mL, which was set as the highest concentration at which no cytotoxicity occurred.

The N-terminal proteins of APP split by α, β, and γ-secretase are released into the supernatant, and the C-terminal proteins remain within the cell. The APP-CHO cells prepared in Preparation Example 1 cultured in a 60 mm dish were treated with the extract or fractions of the Neukline fern at various concentrations for 24 hours, and the supernatant was centrifuged (1000 x g, 10 minutes) and stored separately, and the remaining The cells were washed with PBS, incubated with trypsin for 3 minutes, then removed and transferred to an EP tube. After centrifuging at 1000 x g to create a cell pellet, the supernatant was removed and 50 μl of lysis buffer was added. Proteins were extracted by dissolving at -20°C for 30 minutes and then quantified using the SMART™ BCA kit (Intron, Korea). Sample loading buffer was added to the extracted protein and supernatant, boiled in boiling water at 100°C for 10 minutes, and stored at -20°C until use. Proteins were separated through electrophoresis using 7.5% gel or 15% Acrylamide gel and then transferred to PVDF membrane. To block non-specific binding of antibodies to the PVDF membrane, react in blocking buffer (5% skin milk in PBS) for 1 hour, then wash three times for 15 minutes each with a PBS solution containing Tween 20 (0.1% PBST). Primary antibody against the verification protein was added and reacted at 4°C for 12 hours.

The primary antibodies used were β-secretase antibody (1:1000, Abcam), APP c-terminal antibody (1:1000, Sigma A8717), sAPPβ antibody (1:1000, IBL 18957), beta-actin antibody (1:1000) , BD biosciences, CA, USA).

The cells were washed three times for 15 minutes with PBST solution and then reacted with secondary antibody (5% skin milk in PBS) for 1 hour. After reaction with ECL-spray (Advanta, CA, USA), the protein was identified on G:Box iChemiXT Imager (Syngene, Cambridge, UK). The same method was performed, but the group that was not treated with the root extract or fractions of the root of the fern (DMSO treated) was used as a negative control group, and the positive control group was used as the group treated with the root extract of L. fern.

Figure 2 is a diagram showing the effect of the ethanol extract of Neukline fern on beta-amyloid production.

Figure 2A shows the results of Western blot analysis in which sAPPβ and beta secretase proteins were separated through electrophoresis. Figures 2B and 2C show the contents of sAPPβ and betasecretase (Y-axis: expressed as % relative to the control group) measured by antibody binding method, respectively.

As shown in FIG. 2, the Neorline fern extract inhibited the production of sAPPβ in a concentration-dependent manner at concentrations ranging from 12.5 ㎍/mL to 100 ㎍/mL, and also significantly reduced the expression level of beta secretase.

When treated with the Neoline fern extract, sAPPβ released into the supernatant was reduced in a concentration-dependent manner compared to the negative control group (Figure 2B).

In addition, the results of measuring the protein amount of β-secretase also showed a significant decrease compared to the positive control group in a concentration-dependent manner (Figure 2C).

Figure 3 is a graph showing the results of measuring the effect of the hexane (Hx), dichloromethane (DCM), ethyl acetate (EA), and water (DW) fractions of the Neorline fern extract on beta-amyloid production inhibition. Each of the above fractions was treated at a concentration of 50 μg/mL, which was set as the highest concentration at which no cytotoxicity occurred.

Figure 3A shows the results of Western blot analysis in which sAPPβ and beta secretase proteins were separated through electrophoresis. Figures 3B and 3C show the contents of sAPPβ and betasecretase (Y-axis: expressed as % relative to the control group) measured by antibody binding method, respectively.

As shown in Figure 3, among the fractions of the Neorline fern extract, the ethyl acetate (EA) fraction most significantly inhibited the production of sAPPβ and also significantly reduced the expression level of beta secretase.

Example 2. Isolation and identification of compounds of formulas 1, 2, 4, and 5 from Neorline fern extract

The active ingredients contained in the Neoline fern extract were isolated using activity-guided isolation, and as a result, four active substances were separated and their structures were identified.

Column chromatography was performed on the ethyl acetate layer (ethyl acetate fraction) obtained in Example 1. Column chromatography was performed using silica gel as the stationary phase and chloroform:MeOH = 100:1 as the mobile phase, and 13 subfractions (DME1 to DME13) were obtained. From this, compounds of formulas 1, 2, 4, and 5 were isolated by the following method.

(1) Compounds of formulas 1 and 2

Among the subfractions obtained above, DME8 was subjected to column chromatography using silica gel as a stationary phase and chloroform:MeOH = 100:5 as a mobile phase, and seven subfractions (DME8-1 to DME8-7) were obtained. Among these, DME8-5-3 was separated by column chromatography using Sephadex LH-20 (10% MeOH), resulting in DME8-5-3-1 (compound 1 , 32.0 mg) and DME8-5-3-3 (compound). 2 , 11.2 mg) was secured as a pure substance.

Figure 4 is a schematic diagram showing the separation process of the compounds of formulas 1 and 2 separated from the Neoline fern extract or fractions thereof.

(2) Compound of formula 4

As a result of separating DME11 from the subfractions obtained above using silica gel column chromatography (mobile phase chloroform: MeOH = 100:1), 9 subfractions (DME11-1 to DME11-9) were obtained, of which DME11-4 was separated using Sephadex LH- As a result of separation using column chromatography using 20 (10% MeOH), DME11-4-5 (compound 4 , 107.34 mg) was secured as a pure substance.

Figure 5 is a schematic diagram showing the separation process of the compound of Chemical Formula 4 isolated from the Neoline fern extract or fractions thereof.

(3) Compound of formula 5

Among the small fractions obtained above, DME11-6 was separated by column chromatography using Sephadex LH-20 (10% MeOH), and DME11-6-6 (compound 5 , 15.36 mg) was obtained as a pure substance.

Figure 6 is a schematic diagram showing the separation process of the compound of Chemical Formula 5 isolated from the Neoline fern extract or fractions thereof.

(4) Identification of compounds of formulas 1, 2, 4 and 5

Activity-guided isolation was performed to isolate the active ingredient that inhibits beta-amyloid production contained in the four-lined fern, and as a result, five substances were isolated. Among these, structural analysis was completed for four active compounds (compounds 1 , 2 , 4 , and 5 ). The structure of the compound was analyzed by comparing NMR results with reference literature. The analysis results are as follows.

Compound 1 : (-)-Epiafzelechin-3- O -β-D-allopyranoside

Compound 2 : (2S)-5,7,3',5'-tetradroxy-flavanone 7- O -β-D-glucopyranoside

Compound 4 : (2S)-5,7,3',5'-tetradroxy-flavanone 7- O -hesperidoside

Compound 5 : 7- O -methyl-epiafzelechin-(4α→8)-epiafzelechin-3- O -β-glucopyranoside

Figure 7a shows ¹ H-NMR data measured for the compound of Formula 1. Figure 7b shows ¹³ C-NMR data measured for the compound of Formula 1.

Figure 8a shows the results of ¹ H-NMR analysis of the compound of Formula 2. Figure 8b shows the results of ¹³ C-NMR analysis of the compound of Formula 2.

Figure 9a shows the results of ¹ H-NMR analysis of the compound of Formula 4. Figure 9b shows the results of ¹³ C-NMR analysis of the compound of Formula 4. Figures 9c and 9d show 2D NMR data results for ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 4, respectively.

Figure 10a shows the results of ¹ H-NMR analysis of the compound of Formula 5. Figure 10b shows the results of ¹³ C-NMR analysis of the compound of Formula 5.

Figure 11 shows the structural formulas of compounds of formulas 1, 2, 4, and 5, which are the four active ingredients isolated from the Neoline fern extract or fractions thereof.

Experimental Example 3. Cytotoxicity evaluation of compounds isolated from Neukline fern extract

The cytotoxicity of the compounds of formulas 1, 2, 4, and 5 isolated from the Neoline fern extract obtained in Example 2 was evaluated. The same method as Experimental Example 1 was performed, except that 10 and 50 μg/mL of compounds of Chemical Formulas 1, 2, 4, and 5 were used as treatment drugs.

Figure 12 is a graph showing the results of measuring the cytotoxicity of compounds of formulas 1, 2, 4, and 5 isolated from the Neoline fern extract or fractions thereof (Y-axis: cell survival rate (%) relative to the control group).

As shown in Figure 12, compounds of formulas 1, 2, 4, and 5 all showed no cytotoxicity at concentrations of 10 and 50 μg/mL.

Experimental Example 4. Evaluation of the inhibitory effect of beta-amyloid production of a compound isolated from a fern extract: Measurement of changes in sAPPβ and beta-secretase levels using Western blot analysis

The inhibitory effect of the compounds of formulas 1, 2, 4, and 5 isolated from the fern extract obtained in Example 2 on beta-amyloid production was measured, and whether this effect was due to inhibition of beta secretase was evaluated. The same method as Experiment 2 was performed, except that 10 and 50 μg/mL of compounds of Chemical Formulas 1, 2, 4, and 5 were used as treatment drugs.

Figure 13 is a graph showing the results of measuring the effect of compounds of Formulas 1 and 2 on inhibiting beta-amyloid production.

Figure 14 is a graph showing the results of measuring the effect of the compound of Formula 4 on inhibiting beta-amyloid production.

Figure 15 is a graph showing the results of measuring the effect of the compound of Formula 5 on inhibiting beta-amyloid production.

As shown in Figures 13 to 15, compounds of formulas 1, 2, 4, and 5 significantly inhibited beta-amyloid production in a concentration-dependent manner at concentrations of 10 and 50 ㎍/mL, and this effect was due to a decrease in beta-secretase expression and It matched.

It was confirmed that the four active substances in the extract of Neorline fern, compounds of formulas 1, 2, 4, and 5, inhibited the production of beta-amyloid in a concentration-dependent manner, and at the same time, the protein amount of beta-secretase was also significantly reduced.

Experimental Example 5. Evaluation of the cognitive function improvement effect of Nekline fern extracts and fractions

The cognitive function improvement effect of the Neukline fern extract was tested using a mouse model (ICR mouse, 6 weeks old, male) with memory loss induced by scopolamine.

Each experimental group was a normal group (N) (administered with vehicle only), negative control group (C) (administered with vehicle and then treated with scopolamine), positive control group (P) (administered with 0.75 mg/kg of donepezil and then treated with scopolamine), and four lines. Divided into a low-dose bracken fern extract treatment group (AL) (administered with 200 mg/kg of bracken fern extract and then treated with scopolamine), and a high-dose bracken fern extract treated group (AH) (treated with scopolamine after administered with 500 mg/kg of bracken fern extract). There were 9 animals in each group.

As test substances, vehicle, donepezil, or fern extract were administered for 28 days. After completion of administration, each group except the normal group was treated with 5 mg/kg of scopolamine intraperitoneally (i.p.).

(1) Behavioral test

(1.1) Passive avoidance test

To evaluate experimental animals with induced memory impairment, a passive avoidance experiment was conducted using avoidance system (GEMINI, USA) equipment. There are two rooms (dark, bright) measuring 25 cm × 15 cm, and a partition door (connecting guillotine door: 10 × 10 cm) is located between the rooms. When an experimental animal is placed in a bright room equipped with a light bulb and the partition is opened to enter a dark room without lighting, an electrical stimulus of 0.5 Ma flows through the grid floor for 3 seconds and the time the experimental animal stays in the dark box out of 60 seconds is measured. did.

Figure 16 is a graph showing the results of measuring the residence time in the dark room of scopolamine-induced mice (X axis: normal group (N), negative control group (C), positive control group (P), low-dose fern extract treatment group (AL), high-dose treatment group of Neukline fern extract (AH), Y-axis: retention time (seconds)).

As shown in Figure 16, the results of the passive avoidance experiment showed that the time taken to make a passive avoidance response tended to increase due to scopolamine administration, and the normal group (N) compared to the negative control group (scopolamine treated group) (C) decreased to a significant level (p<0.005).

In addition, in the case of the mouse group administered with the root extract of the fern root (AL: 200 mg/kg administration group, AH: 500 mg/kg administration group), the passive avoidance reaction time tended to decrease compared to the negative control group (C) treated only with scopolamine. indicated.

(1.2) Water maze test

This is an experiment that can measure whether it helps in recovering the spatial perception ability and short-term and long-term memory of experimental animals. Applying the Morris method, a 100 cm (W) This made the escape platform less visible. The time it took for the experimental animal to find an escape platform and climb up was defined as the escape latency time, and the maximum swimming time per individual was set at 120 seconds.

Figure 17 is a graph showing the results of measuring the escape waiting time of scopolamine-induced mice (X-axis: normal group (N), negative control group (C), positive control group (P), low-dose fern extract treatment group (AL ), High-dose treatment group of Neukline fern extract (AH), Y-axis: Escape delay time (seconds)).

As shown in Figure 17, the results of the water maze experiment showed that the scopolamine-treated group (C) significantly increased compared to the normal group (N), and the amount of scopolamine-treated group (N) was significantly increased compared to the normal group (N). Administration group) The administration group showed a tendency to decrease compared to the negative control group (C) treated only with scopolamine.

In Figures 16 and 17, 1) Values are expressed as mean ± SE for 7 experimental groups.

2) *p<0.05 vs. C, #p<0.005 vs. C

As a result of behavioral experiments in a mouse model in which cognitive impairment was induced by scopolamine administration, it was confirmed that the group administered with Neorjul fern extract showed an effect of improving memory decline in a concentration-dependent manner and showed a tendency to improve cognitive function.

(2) ELISA

(2.1) Measurement of change in acetylcholine (ACh) content

Brain tissue extracted during mouse model autopsy was homogenized in cold 0.1M PBS. The supernatant was separated by centrifugation at 5,000 rpm for 10 minutes (4°C), and the acetylcholine content was measured using the Mouse Acetylcholine, Ach ELISA Kit (Cat.no#MBS160373, Mybiosource, USA). To measure acetylcholine content, 40 μL of brain tissue enzyme was dispensed into a 96-well plate and 10 μL of anti-Ach antibody was added. Additionally, 50 μL of streptavidin-HRP reagent was dispensed, mixed well, sealed, and incubated at 37°C. After washing the plate, 50 μL each of substrate solutions A and B were added, incubated for about 10 minutes at 37°C, blocked from light, and then 50 μL of stop solution was dispensed and the absorbance was measured at 450 nm.

Figure 18 shows the change in acetylcholine content due to the extract of Neopline fern (X-axis: normal group (N), negative control group (C), positive control group (P), low-dose treatment group of Neorline fern extract (AL), Extract high dose treatment group (AH), Y axis: concentration (pg/mL)).

As shown in Figure 18, compared to the negative control group (C), the content of acetylcholine increased in the low-dose treatment group (AL) and the high-dose treatment group (AH) of the Neoline fern extract.

(2.2) Measurement of changes in acetylcholinesterase (AChE) activity

Brain tissue removed at autopsy was homogenized in cold 0.1M PBS. The supernatant was separated by centrifugation at 5,000 rpm for 10 minutes (4°C), and acetylcholinesterase activity was measured using Mouse Acetylcholinesterase ELISA Kit (Cat.no#MBS721845, Mybiosource, USA). To measure acetylcholinesterase activity, 100 μL of brain tissue enzyme source was dispensed into a 96-well plate and 10 μL of balance solution was added. Then, 50 μL of conjugate reagent was added, mixed well, sealed, and incubated at 37°C. After washing the plate, 50 μL each of substrate A and B reagents were added and cultured at 37°C for about 15-20 minutes, shielded from light. After dispensing 50 μL of stop solution, the absorbance was measured at 450 nm.

Figure 19 shows the change in acetylcholinesterase activity due to the extract of the fern N. fern (X-axis: normal group (N), negative control group (C), positive control group (P), low-dose treatment group of the N. fern extract (AL) , High-dose treatment group of Neorline fern extract (AH), Y-axis: Activity (mU/mL))

As shown in Figure 19, compared to the negative control group (C), acetylcholinesterase activity decreased in the low-dose treatment group (AL) and the high-dose treatment group (AH) with the Neoline fern extract.

In Figures 18 and 19, 1) Values are expressed as mean ± SE for 7 experimental groups.

2) *p<0.05 vs. C, #p<0.005 vs. C

The results shown in Figures 18 and 19 show that when the extract of the fern of the line is administered, the activity of acetylcholinesterase (AChE) increased by scopolamine is reduced, thereby increasing the amount of ACh, a neurotransmitter, in the synapse, thereby improving cognitive function. suggests that it was helpful.

(3) Immunohistochemistry

Immunohistochemical staining was performed to measure the expression patterns of p-CREB, CREB, BDNF, and β-amyloid. Brain tissue extracted during autopsy was fixed in 10% formalin for 24 hours, then sequentially exchanged for 10% and 20% sucrose solutions for 12 hours each, and then stored in 30% sucrose solution for 3 days. Sections were prepared and attached to a slide, and then reacted with a primary antibody containing 1% bovine serum albumin and a biotinylated secondary antibody, respectively. The primary antibodies used were: -amyloid antibody (MOAB-2, 1:100, Novus Biologicals, Centennial, CO, USA), BDNF antibody (1:200, Novus Biologicals), CREB antibody (1:500) and p-CREB antibody (p-Ser133, 1:100, Novus Biologicals).

After washing, it reacted with avidin-biotin-peroxidase complex, developed color using a coloring reagent (0.05% 3,3-diaminobenzide and 0.02% H ₂ O ₂ ), and then used a general optical microscope (Nikon, E600, Japan) at 100x magnification. It was filmed with

p-CREB: activated CREB (phosphorylated upstream transcription factor).

CREB (cAMP response element-binding protein): A transcriptional regulator that is activated by mitogens, neurotrophins, and other nerve growth factors (CREB is in its pre-activated form).

BDNF (Brain-derived neurotrophic factor): Brain-derived neurotrophic factor (a protein that promotes the survival and growth of neurons (nerve cells), and also performs the function of protecting cognitive functions such as memory and thinking ability).

The expression of BDNF and p-CREB must increase to induce cognitive function improvement effects.

(3.1) Measurement of beta-amyloid deposition (Aβ deposition)

Figure 20 shows the effect of reducing beta-amyloid deposition by the Neorline fern extract (normal group (N), negative control group (C), positive control group (P), low-dose treatment group (AL), high-dose treatment with Neorline fern extract. County (AH)).

In Figure 20, the red arrow indicates the area where beta-amyloid is deposited.

As shown in Figure 20, beta-amyloid deposition was reduced in the low-dose treatment group (AL) and the high-dose treatment group (AH) of the Neoline fern extract compared to the negative control group (C).

(3.2) Measurement of expression changes of BDNF, CREB, and p-CREB

Figure 21 shows changes in the expression of cerebral BDNF, CREB, and p-CREB by extract of Neokline fern (normal group (N), negative control group (C), positive control group (P), and low-dose treatment group of Neokline fern extract (AL ), group treated with high dose of Neorline fern extract (AH)).

As shown in Figure 21, compared to the negative control group (C), the expression of BDNF and p-CREB increased in the low-dose treatment group (AL) and the high-dose treatment group (AH) of the Neoline fern extract.

It can be seen that fern extract helps improve cognitive function through BDNF/CREB signaling, which is involved in synaptic plasticity, which is important for memory and cognitive function.

Experimental Example 6. Evaluation of inhibition of beta-amyloid aggregation in extracts and fractions of Neukline fern: Thioflavin T assay (Thioflavin T fluorescence analysis)

The inhibitory effect of beta-amyloid aggregation of extracts and fractions of Neorline fern was measured using Thioflavin T fluorescence assay (ThT assay).

It is known that the aggregation of beta-amyloid (Aβ) from monomers to oligomers and fibrils induces neurotoxicity and causes neuronal cell death. The amount of beta-amyloid aggregates (Aβ aggregates) was measured using a thioflavin T fluorescence assay, and the extent to which self-aggregation into fibrils was inhibited by the ethanol extract of the Nessium fern and each fraction obtained therefrom was evaluated. .

Beta-amyloid was dispensed into a 96 well plate at a final concentration of 20 μM, then the ethanol extract of Neukline fern at concentrations of 100, 20, and 4 μg/mL (final concentration) prepared in Example 1, the hexane fraction (Hx), and dichloromethane extract. Methane fraction (DCM), ethyl acetate fraction (EA), and water fraction (DW) were added and each reacted at 37°C for 24 hours. Afterwards, thioflavin T test solution (ThT solution) (thioflavin with 100 mM glycine, pH 8.5) was added to reach a final concentration of 3 μM, reacted for 20 minutes, and the amount of fluorescence was measured at 442 excitation and 485 emission. The fluorescence value was measured with a measuring instrument. The same method was performed, but the group that was not treated with the ethanol extract or fraction of Neorthyra fern (DMSO treated) was used as the control group.

The Y-axis is the fluorescence intensity % for the group treated with only beta-amyloid, and the results are shown in Figure 18.

Figure 22 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the Neoline fern extract or fractions thereof. From this, the effect of inhibiting beta-amyloid aggregation is confirmed.

As shown in Figure 22, the extract of Neorline fern inhibited the aggregation of beta-amyloid in a concentration-dependent manner.

In addition, the hexane fraction, dichloromethane fraction, and ethyl acetate fraction obtained from the Four-lined Fern extract also showed an inhibitory effect on beta-amyloid aggregation, and the dichloromethane and ethyl acetate fractions showed the best activity.

Experimental Example 7. Evaluation of beta-amyloid aggregate disassembly activity of extracts and fractions of Neukline fern: Thioflavin T assay (Thioflavin T fluorescence assay)

The activity of disassembling already aggregated beta-amyloid aggregates of the Neorline fern extract and fractions was measured using a Thioflavin T fluorescence assay (ThT assay).

Beta-amyloid was dispensed into a 96 well plate to a final concentration of 20 μM and then reacted at 37°C for 24 hours to form beta-amyloid aggregates. Afterwards, ethanol extract of Neorthyra fern at concentrations of 100, 20, and 4 ㎍/mL (final concentration), and hexane fraction (Hx), dichloromethane fraction (DCM), ethyl acetate fraction (EA), and water fraction (DW) were added. Each was reacted at 37°C for an additional 24 hours. Afterwards, thioflavin T test solution (ThT solution) (thioflavin with 100 mM glycine, pH 8.5) was added to reach a final concentration of 3 μM, reacted for 20 minutes, and the amount of fluorescence was measured at 442 excitation and 485 emission. The fluorescence value was measured with a measuring instrument. The same method was performed, but the group that was not treated with the ethanol extract or fraction of Neorthyra fern (DMSO treated) was used as the control group.

The Y-axis is the fluorescence intensity % for the group treated with only beta-amyloid, and the results are shown in Figure 19.

Figure 23 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the Neoline fern extract or fractions thereof. From this, the effect of dismantling beta-amyloid aggregates is confirmed.

As shown in Figure 23, the extract of Neorjoule fern showed activity in dismantling already aggregated beta-amyloid aggregates in a concentration-dependent manner.

In addition, the hexane fraction, dichloromethane fraction, and ethyl acetate fraction obtained from the Neorline fern extract also showed activity in dismantling beta-amyloid aggregates, with the ethyl acetate fraction showing the best activity.

Experimental Example 8. Evaluation of the activity of inhibiting beta-amyloid aggregation and disassembling aggregates of active ingredients isolated from fern extract: Thioflavin T assay (Thioflavin T fluorescence analysis)

As in Experimental Examples 6 and 7, the inhibitory effect of beta-amyloid aggregation and the activity of dismantling already aggregated beta-amyloid aggregates of the five active ingredients isolated from the extract of Neorline fern were tested using a Thioflavin T fluorescence assay (ThT assay). ) was measured.

Figure 24 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the active ingredients isolated from the Neoline fern extract.

As shown in Figure 24, the active ingredients isolated from the fern extract inhibited the aggregation of beta-amyloid in a concentration-dependent manner. From this, the effect of inhibiting beta-amyloid aggregation is confirmed.

Figure 25 is a graph showing the results (%) of Thioflavin T fluorescence analysis of the active ingredients isolated from the Neoline fern extract. From this, the effect of dismantling beta-amyloid aggregates is confirmed.

As shown in Figure 25, the active ingredients isolated from the fern extract showed activity in dismantling already aggregated beta-amyloid aggregates in a concentration-dependent manner.

As shown in the results above, all of the active ingredients of Formulas 1 to 5 not only inhibited the aggregation of beta-amyloid but also showed the effect of dismantling already aggregated beta-amyloid aggregates.

Among these ingredients, the compound of formula 2 was the best in both effects.

Judging from the above experimental results, it can be seen that the fern extract, ethyl acetate fraction layer, and active ingredients have the effect of inhibiting the production of beta-amyloid, and the mechanism is related to the inhibition of expression of beta-secretase. I was able to. Therefore, the active ingredients of the Neoline fern extract, the ethyl acetate fraction layer, and the compounds of formulas 1, 2, 4, and 5 separated therefrom can be developed as preventive and therapeutic agents for degenerative neurological diseases such as Alzheimer's dementia, and as health functional foods. .

A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

This research project was conducted under the National Research Foundation of Korea's mid-career researcher support project (2019R1A2C100621412).

Claims (16)

(-)-Epiafzelechin-3-O-β-D-allopyranoside ((-)-epiafzelechin-3-O-β-D- isolated from Davallia mariesii extract or fractions thereof allopyranoside), (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-β-D-glucopyranoside ((2S)-5,7,3',5'- tetrahydroxy-flavanone 7-O-β-D-glucopyranoside), (2S)-5,7,3',5'-tetrahydroxy-flavanone 7-O-hesperidoside ((2S)-5,7 ,3',5'-tetrahydroxy-flavanone 7-O-hesperidoside), 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside ( Prevention or treatment of neurodegenerative diseases containing 7-O-methyl-epiafzelechin-(4α→8)-epiafzelechin-3-O-β-glucopyranoside), their pharmaceutically acceptable salts, or a combination thereof as an active ingredient. Pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the extract of the Neorline fern is an extract of the root, the rhizome of the Neorline fern, or a combination thereof. The pharmaceutical composition according to claim 1, wherein the extraction solvent of the Neoline fern extract is water, C ₁ to C ₄ alcohol, or a mixture thereof. The pharmaceutical composition according to claim 1, wherein the extraction solvent of the Neorjoule fern extract is ethanol. The pharmaceutical composition according to claim 4, wherein the ethanol has a concentration of 20% (v/v) to 100% (v/v). The pharmaceutical composition according to claim 1, wherein the fraction is an ethyl acetate fraction of an ethanol extract of Neorjoule fern. The method of claim 1, wherein the neurodegenerative disease includes Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, dementia associated with beta-amyloid, and Parkinson's disease. A pharmaceutical composition selected from the group consisting of Parkinson's disease, mild cognitive impairment, and senile dementia. The pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease. The pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is a disease whose main cause is beta-amyloid accumulation in the brain. The pharmaceutical composition according to claim 1, wherein the composition inhibits the production of beta-amyloid (β-amyloid). The pharmaceutical composition according to claim 1, wherein the composition reduces beta-secretase activity. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is for oral or parenteral administration. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is formulated with a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspending agents, syrups, injections, and granules. (-)-Epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7,3',5'- isolated from Davallia mariesii extract or fractions thereof Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-Tetrahydroxy-flavanone 7-O-hesperidoside, 7- O-methyl-epiafzelechin-(4α→8)-epiafzelechin-3-O-β-glucopyranoside, a pharmaceutically acceptable salt thereof, or a combination thereof as an active ingredient Food composition for preventing or improving neurodegenerative diseases. Ethyl acetate fraction of ethanol extract of Davallia mariesii extract, or (-)-epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7, isolated from the fraction. 3',5'-Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-Tetrahydroxy-flavanone 7-O-Hess Peridoside, 7-O-methyl-epiaphzelechin-(4α→8)-epiaphzelechin-3-O-β-glucopyranoside, pharmaceutically acceptable salts thereof, or a combination thereof A health functional food for improving learning or memory or cognitive function containing as an active ingredient. (-)-Epiafzelechin-3-O-β-D-allopyranoside, (2S)-5,7,3',5'- isolated from Davallia mariesii extract or fractions thereof Tetrahydroxy-flavanone 7-O-β-D-glucopyranoside, (2S)-5,7,3',5'-Tetrahydroxy-flavanone 7-O-hesperidoside, 7- O-methyl-epiafzelechin-(4α→8)-epiafzelechin-3-O-β-glucopyranoside, a pharmaceutically acceptable salt thereof, or a combination thereof as an active ingredient Composition for feed for preventing or improving degenerative neurological diseases.

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