KR20220137453A - Composition including triterpenoid as an active ingredient for treatment or prevention of neurodegenerative diseases - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or alleviating neurodegenerative diseases comprising a compound represented by chemical formulas 1 to 3 isolated from a Perilla frutescens extract, a derivative thereof, or a mixture thereof as an active component. Since an acid anhydride-based epoxy compound of the present invention directly participates in the curing reaction, phase separation does not occur when an external impact is applied, and the mechanical properties of a cured product as a whole can be improved.

Description

Composition including triterpenoid as an active ingredient for treatment or prevention of neurodegenerative diseases

It relates to a composition for treating or preventing neurodegenerative diseases comprising triterpenoid as an active ingredient, and more specifically, triterpe isolated from perilla leaves ( Perilla frutescens var. acuta ) for amyloid production and aggregation. Triterpe exhibits the anti-amyloidogenic effect of the nodal, more specifically, the effect of inhibiting the formation of beta-amyloid aggregates isolated from the perilla extract, as well as dissolving the produced beta-amyloid aggregates. It relates to the treatment, prevention, or improvement of neurodegenerative diseases based on nooid compounds.

Alzheimer's disease (AD), a typical degenerative neurological disease, is a disease showing symptoms of progressive memory loss and cognitive loss. One of the pathological features of Alzheimer's disease is senile plaques that accumulate on the outside of nerve cells. As an important constituent of senile plaque, beta-amyloid (beta-amyloid or amyloid-β, Aβ) protein is known.

Existing treatments for Alzheimer's include acetylcholinesterase inhibitors tacrine, donepezil, rivastigmine, and galantamine developed based on the cholinergic nervous system hypothesis. , and memantine, which is an antagonist of NMDA receptors on which glutamate acts, and the like. However, most of the drugs currently on the market or under development are drugs that improve the symptoms of Alzheimer's patients and improve the quality of life. In addition, existing therapeutic agents mainly only have an acetylcholinesterase inhibitory effect, and there are not many therapeutic agents for Alzheimer's dementia patients in which beta-amyloid has already been generated and aggregated.

The present inventors have tried to develop a therapeutic agent with the goal of a fundamental treatment based on the cause of the invention of Alzheimer's disease, and not only inhibit the generation of beta-amyloid aggregates, but also dismantle the generated beta-amyloid aggregates to solve the underlying cause of degenerative diseases. Research aimed at developing therapeutic agents for neurological diseases.

Korean Patent Registration No. 10-2053868

One aspect relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising as an active ingredient a compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from a perilla extract.

Another aspect relates to a food composition for preventing or improving neurodegenerative diseases, comprising as an active ingredient a compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from a perilla leaf extract.

(1) A pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising a compound represented by the following formulas 1 to 3, a derivative thereof, or a mixture thereof, as an active ingredient, isolated from a perilla leaf extract.

[Formula 1]

[Formula 2]

[Formula 3]

(2) The extraction solvent for the perilla leaf extract is water, a C ₁ to C ₄ alcohol, or a mixture thereof.

(3) The pharmaceutical composition wherein the extraction solvent for the perilla leaf extract is ethanol.

(4) The compound represented by Formulas 1 to 3 is a pharmaceutical composition that is separated from the dichloromethane fraction of the ethanol extract of perilla leaf.

(5) The compounds represented by Chemical Formulas 1 to 3 inhibit the production of beta-amyloid aggregates or the resulting beta-amyloid aggregates are dismantled; pharmaceutical compositions.

(6) The neurodegenerative disease is Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, Lou Gehrig disease (Amyotrophic lateral sclerosis or Lou Gehrig disease), Huntington's disease (Huntington's disease) ), Niemann-Pick Disease, Dutch Amyloidosis, Amyloid Stroke, Parkinson's Disease, Mild Cognitive Impairment, Senility, Dementia ), Lewy Body Dementia Multi-infarct dementia, Down's syndrome, Dementia associated with stroke, Dementia associated with Parkinson's disease, or beta - A pharmaceutical composition for preventing or treating amyloid-associated dementia (Dementia associated with beta-amyloid).

(7) The neurodegenerative disease is Alzheimer's disease, cerebral amyloid angiopathy, systemic amyloidosis, Lou Gehrig's disease, Huntington's disease, and a pharmaceutical composition that is selected from the group consisting of Niemann-Pick disease.

(8) The neurodegenerative disease is a degenerative brain disease pharmaceutical composition.

(9) The degenerative brain disease in the brain is beta- a pharmaceutical composition that is a disease mainly caused by amyloid accumulation.

(10) The neurodegenerative disease is Alzheimer's disease pharmaceutical composition.

(11) A pharmaceutical composition wherein the compound represented by Formulas 1 to 3 or a derivative thereof is provided as a pharmaceutically acceptable salt or solvate.

(12) A food composition for preventing or improving neurodegenerative diseases, comprising as an active ingredient a compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract.

(13) The compound represented by Formulas 1 to 3 is a food composition that is separated from the dichloromethane fraction of the ethanol extract of perilla leaf.

(14) The compound represented by Formulas 1 to 3 inhibits the production of beta-amyloid aggregates or decomposes the produced beta-amyloid aggregates. A food composition.

Compounds represented by Formulas 1 to 3, derivatives thereof, or mixtures thereof isolated from the perilla leaf extract according to an aspect exhibit an effect of inhibiting the production of beta-amyloid aggregates or dissolving the produced beta-amyloid aggregates. Therefore, it can be used as a pharmaceutical composition or food composition for the prevention, treatment, alleviation, improvement of neurodegenerative diseases, including degenerative brain disease, and a use thereof, and a disease associated with intracerebral beta-amyloid accumulation, including Alzheimer's disease, It can be used as a medicine, adjuvant, health food or functional food for patients who may be ill. For example, the composition may be used for brain tissue regeneration for the treatment of Alzheimer's dementia.

1 is a diagram confirming the beta-amyloid aggregation inhibitory effect of a perilla extract solvent fraction.
2 is a diagram showing the structure of triterpenoids isolated from perilla leaf extract dichloromethane (DCM) fraction.
3 is a diagram confirming the beta-amyloid aggregation inhibitory effect of compounds isolated from the DCM fraction of perilla extract.
4 is a diagram confirming the effect of dissolving beta-amyloid aggregates of the compounds isolated from the DCM fraction of perilla extract.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are hereby incorporated by reference in their entirety.

One aspect provides a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising as an active ingredient a compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from a perilla extract.

Perilla leaves ( Perilla frutescens var. acuta ) refers to the leaves of Perilla frutescens var. acuta KUDO., an annual herb belonging to the Lamiaceae family.

The extraction solvent of the perilla leaf extract may be water, an organic solvent, or a mixture thereof. The organic solvent may be, for example, a C ₁ to C ₄ alcohol, a polar solvent such as ethyl acetate or acetone, or a non-polar solvent such as ether, chloroform, benzene, hexane or dichloromethane, or a mixed solvent thereof.

In one embodiment, the extraction solvent for the perilla leaf extract may be water, C ₁ to C ₄ alcohol, or a mixture thereof.

In one embodiment, the extraction solvent for the perilla leaf extract may be ethanol.

The alcohol may be 100% alcohol. For example, the extraction solvent may be 100% ethanol.

Alternatively, the alcohol may be provided by diluting the alcohol with dilution water such as 1 to 99% (v/v) of the alcohol in water.

The extraction temperature of the extract may be carried out at 4 ℃ to 120 ℃. In addition, the extraction time may be 12 hours to 120 hours. If the extraction temperature is lower than 4 ℃, the extraction may be difficult, and if it exceeds 120 ℃, 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 preferable. Alternatively, the extraction temperature may be room temperature.

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

The perilla leaf extract can be extracted with an extraction solvent, the leaflet residue is removed, filtered with filter paper, and the filtrate is concentrated using a vacuum rotary concentrator or vacuum dryer, and then stored at room temperature.

The perilla leaf extract may be obtained as a fraction by further performing a fractionation process simultaneously or sequentially using one or more organic solvents or water selected from the group consisting of hexane, dichloromethane, ethyl acetate, chloroform, and butanol. The organic solvent may be, for example, a 10% (v/v) to 99% (v/v) aqueous solution or a solvent having a concentration of 100% (v/v).

The fractionation temperature may be carried out at 4 ℃ to 120 ℃. Alternatively, the fractionation temperature may be room temperature.

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

If necessary, a suspension process of the extract may be performed before the fractionation process.

The fractionated extract obtained by performing the fractionation process may be concentrated using a vacuum rotary concentrator or a vacuum dryer, and then stored at room temperature.

In one embodiment, the fraction of the perilla leaf extract may be obtained by successively fractionating with n -hexane, dichloromethane, and ethylacetate. In one embodiment, the compounds represented by Formulas 1 to 3 may be isolated from the dichloromethane fraction of the ethanol extract of perilla leaf.

The perilla leaf extract may be prepared according to a conventional method for producing a plant extract, and specifically may be a cold extraction method, a warm needle extraction method, a heat extraction method, an ultrasonic extraction method, etc. can

It is possible to obtain a triterpenoid having the effect of inhibiting aggregation of beta-amyloid produced from the perilla leaf extract and decomposing the already formed beta-amyloid aggregate. The triterpenoid includes compounds represented by Formulas 1 to 3.

The compounds represented by Formulas 1 to 3 isolated from the perilla leaf extract may be referred to as ursolic acid, tomentic acid, and corosolic acid, respectively.

The triterpenoids, ursolic acid, tomentic acid, and corosolic acid isolated from the perilla leaf extract have the effect of inhibiting the aggregation of the produced beta-amyloid and decomposing the already formed beta-amyloid aggregate, thus degenerative brain disease. It can be used for the treatment and prevention of neurodegenerative diseases, including. In addition, the therapeutic effect can be expected even for patients with brain tissue damage due to diseases such as Alzheimer's dementia already invented. In one embodiment, the compound may be used for brain tissue regeneration for the treatment of Alzheimer's dementia.

In one embodiment, the compound isolated from the perilla leaf extract can reduce the amount of beta-amyloid aggregates by inhibiting beta-amyloid aggregation and/or dismantling or decomposing the produced beta-amyloid aggregates at the same time. It can be used for the treatment and prevention of neurodegenerative diseases, including.

In one embodiment, the pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising the compound isolated from the perilla leaf extract may particularly include corosolic acid as an active ingredient.

As used herein, the term "neurodegenerative disease" is closely related to aging, and, unlike the normal process of aging, rapid death of abnormal nerve cells occurs in a part of the nervous system or the entire brain, resulting in 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 in which the brain and spinal cord cells of the central nervous system show expressive dysfunction due to degeneration or loss.

As used herein, the term "degenerative brain disease (degenerative brain disease)" is one of the neurodegenerative diseases, refers to a disease that occurs in the brain as we age. Due to unknown causes, a specific group of brain cells gradually loses their function in the brain, and the death of cranial nerve cells, which is the most important for information transmission in the cranial nervous system, problems with the shape or function of synapses that transmit information between cranial nerve cells and cranial nerve cells, or It is known to be caused by an ideal increase or decrease in electrical activity of cranial nerves.

In one embodiment, the neurodegenerative disease is Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, Lou Gehrig disease (Amyotrophic lateral sclerosis or Lou Gehrig disease), Huntington's disease ( Huntington's disease, Niemann-Pick disease, Dutch amyloidosis, amyloid stroke, Parkinson's disease, mild cognitive impairment, senility, dementia Dementia, Lewy Body Dementia Multi-infarct dementia, Down's syndrome, Dementia associated with stroke, Dementia associated with Parkinson's disease or beta-amyloid-associated dementia (Dementia associated with beta-amyloid), but is not limited thereto.

In one embodiment, the neurodegenerative disease may be selected from the group consisting of Alzheimer's disease, cerebral amyloid angiopathy, Lou Gehrig's disease, Huntington's disease, and Niemann-Pick's disease.

In one embodiment, the neurodegenerative disease may be a degenerative brain disease.

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

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

Alzheimer's disease is a disease in which brain cells are gradually destroyed, leading to a gradual loss of memory and cognitive ability, and eventually death. Patients with this disease not only gradually lose their memory, but also lose the ability to accurately understand their current situation, their learning ability, language ability and judgment ability, and even the ability to take care of themselves. leads to dementia. As the brain of patients with Alzheimer's disease is gradually damaged, the functions of the central nervous system as well as the autonomic nervous system are infringed and the overall health condition deteriorates. In the brain tissues of patients with Alzheimer's disease, senile plaques called beta-amyloid (Aβ) accumulate and neurofibrillary tangles (NFTs) appear in brain cells, resulting in extensive degenerative atrophy throughout the brain tissue. this happens For example, the Alzheimer's disease includes dementia caused by Alzheimer's disease, that is, dementia caused by deposition of beta-amyloid in brain tissue, and dementia caused by deposition of beta-amyloid is included in the scope of the present invention. .

Cerebral amyloid angiopathy (Cerebral amyloid angiopathy) is a disease in which amyloid deposits on the walls of blood vessels in the brain in the cortex and leptomeningeal membrane. Cerebral amyloid angiopathy causes lobar cerebral hemorrhage, dementia, transient ischemic attacks, and epileptic seizures. There are also studies that the frequency of brain amyloid angiopathy in Alzheimer's disease reaches 80-90%, and it is thought to be deeply related to Alzheimer's disease.

Systemic amyloidosis is a generic term for diseases in which amyloid protein builds up excessively in tissues or organs throughout the body, resulting in tissue or organ dysfunction.

As used herein, the term “amyloid” refers to a fibrous structure formed by specific interactions between proteins by showing water-soluble proteins exhibiting water-insoluble properties in various biochemical vessels. It corresponds to a common protein aggregate observed in the various neurodegenerative diseases mentioned above.

In one embodiment, the composition may inhibit beta-amyloid production.

In one embodiment, the composition may inhibit the aggregation of the produced beta-amyloid.

In one embodiment, the composition may disintegrate or decompose the produced beta-amyloid aggregates.

In one embodiment, the compounds represented by Formulas 1 to 3 may inhibit the formation of beta-amyloid aggregates or disintegrate the produced beta-amyloid aggregates.

As used herein, the term "derivative" refers to a compound obtained by substituting a part of the structure of the compound with another atom or group of atoms. The "substitution" refers to introduced instead of a hydrogen atom when a derivative is formed by substituting one or more hydrogen atoms in an organic compound with another atomic group, and "substituent" refers to an introduced atomic group. The substituent is, for example, a hydroxyl group, a halogen atom (eg, fluorine (F), chlorine (Cl), bromine (Br), and iodine (I)), a C ₂ to C ₁₀ alkenyl group, a C ₂ to C ₁₀ alkynyl group. , C ₁ to C ₁₀ heteroalkyl group, C ₆ to C ₁₀ aryl group, C ₆ to C ₁₀ heteroaryl group, C ₁ to C ₁₀ alkoxy, nitro group, cyano group, amino group, carboxyl group or a salt thereof, sulfonyl group, It may be a sulfamoyl group, a sulfonic acid group or a salt thereof, phosphoric acid or a salt thereof, or a combination thereof.

The compounds represented by Formulas 1 to 3 or derivatives thereof include isomers thereof. The isomers may be structural isomers or stereoisomers.

As used herein, the term “solvate” refers to a compound solvated in an organic or inorganic solvent. The solvate is, for example, a hydrate.

As used herein, the term “salt” refers to an addition salt of an inorganic, organic, or metal salt of a compound. The inorganic acid salt may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salt is formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl salt, trichloroacetic acid, trifluoroacetate , benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartate It may be an acid. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt. The salt may be a pharmaceutically or food acceptable salt.

As used herein, the term "pharmaceutically acceptable salt thereof" or "food acceptable salt thereof" is generally safe, non-toxic, and is not biologically or otherwise unsuitable for pharmaceutical and food pharmaceutical compositions. indicates that it can be used in the manufacture of

Another aspect provides a food composition for preventing or improving neurodegenerative diseases, comprising as an active ingredient a compound represented by the following Chemical Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from a perilla leaf extract.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include various dairy products such as milk and yogurt, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and snacks such as bread, chocolate, candy, and confectionery. It includes all foods in the sense of phosphorus.

The food composition may be provided as a supplement or a health functional food.

In the composition, the compounds represented by Formulas 1 to 3, derivatives thereof, or mixtures thereof may be included in an effective amount pharmaceutically or foodologically. For example, the compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof may be present in a concentration of 0.001 to 1000 nM in the composition. For example, in the composition, 5 nM, 10 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 75 nM, 100 nM, or these values may be present in a range having an upper limit or a lower limit. For example, it may be present in a concentration of 1 to 10 M in the composition.

Or in the composition, the compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof is 0.001 to 100 μg/mL, for example, 0.5, 1, 2, 4, 5, 7, 8, 10, 15, 20 , 30 μg/mL or a range having the upper or lower limits thereof, such as 0.5 to 30 μg/mL, 1 to 20 μg/mL, 4 to 15 μg/mL, and 7 to 10 μg/mL.

For example, in the composition, the compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof is all, 1/2, or 1/ of the daily dose required for the prevention, treatment, or improvement of neurodegenerative diseases. 3 may be included.

The composition may further include a pharmaceutically or food-acceptable additive or carrier. Such additives or carriers may be appropriately selected, for example, in the range of 0.001 to 90 parts by weight per 100 parts by weight of the composition.

The composition may be prepared in a unit dose form by formulating using a pharmaceutically or food acceptable additive or carrier, or may be prepared by internalizing in a multi-dose container.

The additive that may be included in the pharmaceutical or food composition may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be exemplified by microcrystalline cellulose, lactose, or low-substituted hydroxycellulose, but is not limited thereto. The disintegrant may be exemplified by sodium starch glycolate, or anhydrous calcium monohydrogen phosphate, but is not limited thereto. The binder may be exemplified by polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, or hydroxypropyl cellulose, but is not limited thereto. The lubricant may be exemplified by magnesium stearate, silicon dioxide, or talc, but is not limited thereto.

The composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection or an external preparation for skin. The external preparation for skin may be exemplified as a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, or lotion, but is not limited thereto.

Administration of the composition may be administered by a method known in the art. For example, it can be administered directly to a subject by any means, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. . The administration may be systemically or locally.

A suitable dosage of the composition varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, and usually skilled in the art. A physician or the like can readily determine and prescribe an effective dosage for the desired treatment, prevention or improvement. According to one embodiment, the daily dose of the composition may be 0.001 to 10 g / kg.

For example, the dosage of the pharmaceutical composition for brain tissue regeneration for the treatment of degenerative neurological diseases such as Alzheimer's dementia can be determined in consideration of the absorption of the active ingredient, the inactivation rate, and the drug used in combination, based on the daily active ingredient 0.1 mg/kg (body weight) to 500 mg/kg (body weight), 0.1 mg/kg (body weight) to 400 mg/kg (body weight), or 1 mg/kg (body weight) to 300 mg/kg (body weight) may be administered, and may be administered once or dividedly into several doses, but is not limited thereto.

Another aspect is a degenerative neurological disease of an individual comprising administering to the individual a composition comprising a compound represented by the following Chemical Formulas 1 to 3, a derivative thereof, or a mixture thereof, as an active ingredient, isolated from a perilla leaf extract. Methods for preventing, ameliorating or treating are provided.

As used herein, the term "prevention" refers to inhibiting the development of a disease or disorder in an individual who has not been diagnosed with the disease or disease, but is prone to the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease or disorder in a subject, (b) alleviating the disease or disorder, and (c) eliminating the disease or disorder.

As used herein, the term “improvement” refers to any action in which a disease or symptom of a disease is improved in an individual.

As used herein, the term "individual" refers to monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, chimpanzees, etc. means a mammal of

As used herein, the term “contained as an active ingredient” refers to an amount sufficient to achieve the efficacy or activity of a compound represented by the following Chemical Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract. it means.

For example, the content of the active ingredient in the pharmaceutical composition for brain tissue regeneration for the treatment of degenerative neurological diseases such as Alzheimer's dementia is 0.001 wt% to 99.9 wt%, 0.1 wt% to 99 wt%, or 1 wt% to 50 wt% may be, but is not limited thereto, and may be appropriately adjusted to a desired content according to the usage aspect and method of use of the composition.

In one embodiment, the compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract may be included in an amount of 1 to 80% by weight based on the total weight of the pharmaceutical composition, and a solvate or It may also be provided in the form of a pharmaceutically acceptable salt.

In one embodiment, the compound represented by Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract may be included in an amount of 1 to 80% by weight based on the total weight of the food composition, and the solvate or It may also be provided in the form of a food-acceptable salt.

In addition, in the composition, in addition to the active ingredient, a therapeutic agent for degenerative neurological disease known in the art to have a known therapeutic effect on dementia, for example, tacrine, donepezil, rivastigmine, galantamine ( galantamine) and the like may be additionally included.

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

Example 1. Preparation of perilla ethanol extract

After grinding perilla leaf (6 kg) purchased from Samheung Construction Materials (Seoul, 2019), extracting it three times with 100% ethanol (10 L each, 3 times at room temperature), and then concentrating the filtered filtrate under reduced pressure to 650 g of ethanol The extract was obtained.

Example 2. Preparation of fractions of ethanol extract from perilla leaf

After adding and dissolving a small amount of methanol (100 mL) to the ethanol extract obtained in Example 1, it was suspended in water (5 L ) and successively with n -hexane, dichloromethane, and ethylacetate. was fractionated with (5 L each, 3 times, room temperature) to obtain n -hexane (116.1 g), dichloromethane (30 g), ethyl acetate (263.3 g), and an aqueous layer (226.7 g).

As a result, n -hexane (Hx) fraction, dichloromethane (DCM) fraction, ethyl acetate (EA) fraction, and water fraction (DW) were obtained.

Test Example 1. Evaluation of inhibition of beta-amyloid aggregation of ethanol extract of perilla leaf or each fraction (ThT assay)

The beta-amyloid aggregation inhibitory effect of the ethanol extract of perilla and each fraction prepared in Examples 1 and 2 was tested using ThT assay.

Aggregation of beta-amyloid (Aβ) from monomers to oligomers and fibrils is known to induce neurotoxicity and induce neuronal cell death. Therefore, the amount of beta-amyloid monomer (Aβ monomer) was performed through Thioflavin T assays (ThT assays) to evaluate whether the ethanol extract of perilla leaf and each fraction could inhibit self-aggregation into fibrils. was measured.

After aliquoting beta-amyloid in a 96-well plate to a final concentration of 20 μM, the ethanol extract of 4, 20, and 100 μg/mL (final concentration) prepared in Example 1 and each fraction, i.e., 4, 20, n -hexane (Hx) fraction, dichloromethane (DCM) fraction, and ethyl acetate (EA) fraction at a concentration of 100 μg/mL (final concentration), and a water fraction (DW) at a concentration of 4 and 20 μg/mL (DW) and 37 respectively The reaction was carried out at ℃ for 24 hours. Then, thioflavin T test solution (ThT solution) (thioflavin with 100 mM glycine, pH 8.5) was added so that the final concentration was 3 μM, and after 20 minutes of reaction, the amount of fluorescence was measured 442 excitation and 485 emission. Fluorescence values were measured with a measuring instrument. In the same manner, the group not treated with compounds 1 to 3 (THF treatment) was used as a control group.

1 is a diagram confirming the beta-amyloid aggregation inhibitory effect of a perilla extract solvent fraction.

FIG. 1 shows the results of measuring beta-amyloid aggregation inhibitory effects of perilla leaf ethanol extract, n -hexane fraction, dichloromethane fraction, ethyl acetate fraction, and water fraction using ThT assay. The horizontal axis represents the concentration (μg/mL) of the perilla leaf ethanol extract or each fraction, and the vertical axis represents the ThT fluorescence measurement result (expressed as a percentage of the control group).

Example 3. Identification of compounds of formulas 1 to 3

Compounds of Formulas 1 to 3 (hereinafter referred to as Compounds 1 to 3) were obtained by the following method. First, the dichloromethane layer (DCM fraction) obtained in Example 2 was applied to open column chromatography using silica gel as a stationary phase (chloroform: methanol = 100: 1 to 5: 5 to 0: 1) to secure 7 small fractions did. Among them, PFD 2 was fractionated by open column chromatography ( n -hexane: acetone = 8: 2) using silica gel as a stationary phase and purified (chloroform: methanol = 100: 1 to 50: 1) to obtain compound 1 (120 mg). got it

The small fraction PFD 4 was subjected to open column chromatography using silica gel as a stationary phase (chloroform : methanol = 100 : 1 ~ 20 : 1 ~ 0 : 1) to purify two of the small fractions obtained ((chloroform : methanol = 20 : 1) or (chloroform : acetone = 4.5 : 1 ~ 1 : 1)) to obtain compound 2 (41 mg) and compound 3 (11.7 mg).

The separated compounds 1 , 2 and 3 were identified as ursolic acid, tomentic acid, and corosolic acid by comparing the NMR and MS results with the literature, respectively.

2 is a diagram showing the structure of triterpenoids isolated from perilla leaf extract dichloromethane (DCM) fraction.

○ Compound 1 - White powder; C ₃₀ H ₄₈ O ₃ MS m/z 457 [M+H] ⁺ ; ¹ H NMR (500 MHz, Pyridine- d5 ): δ 5.47 (1H, brs, H-12), 3,44 (1H, dd, J =10.0, 5.5 Hz, H-3), 2.62 (1H, d, J =11.0 Hz, H-18), 2.31 (1H, td, J =13.0, 3.5 Hz, H-15), 2.10 (1H, td, J =13.5, 4.0 Hz, H-16), 1.23 (3H, s, Me-23), 1.21 (3H, s, Me-27), 1.03 (3H, s, Me-26), 1.01 (3H, s, Me-24), 0.98 (3H, d, J =6.0 Hz , Me-29), 0.95 (3H, d, J =6.0 Hz, Me-30), 0.87 (3H, s, Me-25); ¹³ C NMR (125 MHz, Pyridine- d5 ): δ 181.2 (C-28), 140.5 (C-13), 126.9 (C-12), 79.4 (C-3), 57.1 (C-5), 54.8 ( C-18), 49.3 (C-9, C-17), 43.7 (C-14), 41.2 (C-8), 40.7 (C-19), 40.6 (C-4, C-20), 40.3 ( C-1), 38.7 (C-22), 38.5 (C-10), 34.8 (C-7), 32.3 (C-21), 30.1 (C-23), 29.9 (C-15), 29.4 (C) -2), 26.2 (C-16), 25.2 (C-27), 24.9 (C-11), 22.3 (C-30), 19.9 (C-6), 18.6 (C-29), 18.5 (C- 26), 17.5 (C-24), 16.4 (C-25).

○ Compound 2 - White powder; C ₃₀ H ₄₈ O ₅ MS m/z 489 [M+H] ⁺ ; ¹ H NMR (500 MHz, Pyridine- d5 ): δ 5.52 (1H, brs, H-12), 4.04 (1H, ddd, J =10.5, 9.5, 4.5 Hz, H-2), 3.32 (1H, d, J =9.5 Hz, H-3), 3.06 (1H, ddd, J =13.5, 12.5, 4.5 Hz, H-16), 2.98 (1H, s, H-18), 1.65 (3H, s, Me-27) ), 1.37 (3H, s, Me-29), 1.20 (3H, s, Me-23), 1.05 (3H, d, J =6.0 Hz, Me-30), 1.04 (3H, s, Me-26) , 1.01 (3H, s, Me-24), 0.93 (3H, s, Me-25); ¹³ C NMR (125 MHz, Pyridine- d5 ): δ 180.4 (C-28), 144.6 (C-13), 127.7 (C-12), 83.6 (C-3), 72.4 (C-19), 68.3 ( C-2), 55.7 (C-5), 54.3 (C-18), 48.5 (C-17), 48.1 (C-9), 48.0 (C-1), 42.6 (C-20), 42.1 (C) -14), 40.1 (C-8), 39.8 (C-4), 38.5 (C-10, C-22), 33.2 (C-7), 29.2 (C-23), 29.1 (C-15), 26.8 (C-29), 26.7 (C-21), 26.1 (C-16), 24.6 (C-27), 24.5 (C-11), 18.9 (C-6), 17.5 (C-24), 17.1 (C-26), 16.7 (C-25), 16.4 (C-30).

○ Compound 3 - White powder; C ₃₀ H ₄₈ O ₅ MS m/z 489 [M+H] ⁺ ; ¹ H NMR (500 MHz, Pyridine- d5 ): δ 5.38 (1H, brs, H-12), 4.18 (1H, d, J =11.0, 9.0, 4.5 Hz, H-2), 3.48 (1H, d, J =6.0 Hz, H-19), 3.18 (1H, d, J =9.0 Hz, H-18), 3.19 (1H, d, J =9.0 Hz, H-3), 1.62 (3H, s, Me- 27), 1.25 (3H, s, Me-23), 1.17 (3H, s, Me-29), 1.09 (3H, s, Me-30), 1.06 (3H, s, Me-24), 1.05 (3H) , s, Me-25), 1.00 (3H, s, Me-26); ¹³ C NMR (125 MHz, Pyridine- d5 ): δ 179.1 (C-28), 139.0 (C-13), 125.3 (C-12), 83.5 (C-3), 68.3 (C-2), 55.6 ( C-5), 53.2 (C-18), 47.8 (C-9, C-17), 47.7 (C-1), 42.3 (C-14), 39.7 (C-8), 39.6 (C-4) , 39.2 (C-20), 39.1 (C-19), 38.2 (C-10), 37.2 (C-22), 33.2 (C-7), 30.8 (C-21), 29.1 (C-23), 28.4 (C-15), 24.6 (C-16), 23.6 (C-27), 23.4 (C-11), 21.1 (C-29), 18.6 (C-6), 17.4 (C-24), 17.2 (C-26, C-30), 16.7 (C-25).

Test Example 2. Beta-amyloid aggregation inhibition evaluation of compounds 1 to 3 (ThT assay)

The isolated beta-amyloid aggregation inhibitory effect of Compound 1-3 was tested using a ThT assay.

Aggregation of beta-amyloid (Aβ) from monomers to oligomers and fibrils is known to induce neurotoxicity and induce neuronal cell death. Therefore, in order to evaluate whether compounds 1 to 3 can inhibit the self-aggregation of Aβ to fibrils, the amount of beta-amyloid monomer (Aβ monomer) was measured through thioflavin T assays (ThT assays). measured.

After dispensing beta-amyloid in a 96-well plate to a final concentration of 20 μM, compounds 1 to 3 at a concentration of 0.5, 1, 2, 4, and 8 μg/mL (final concentration) obtained in Example 3 and each at 37° C. for 24 hours reacted. Then, thioflavin T test solution (ThT solution) (thioflavin with 100 mM glycine, pH 8.5) was added so that the final concentration was 3 μM, and after 20 minutes of reaction, the amount of fluorescence was measured 442 excitation and 485 emission. Fluorescence values were measured with a measuring instrument. In the same manner, the group not treated with compounds 1 to 3 (THF treatment) was used as a control group.

3 is a diagram confirming the beta-amyloid aggregation inhibitory effect of compounds isolated from the DCM fraction of perilla extract.

In FIG. 3 , the horizontal axis indicates the concentration (μg/mL) of Compounds 1 to 3 , and the vertical axis indicates the ThT fluorescence measurement result (expressed as a percentage of the control group).

As shown in FIG. 3 , compounds 1-3 exhibited a similar beta-amyloid aggregation effect, and inhibited aggregation in a concentration-dependent manner. In particular, at a concentration of 8 μg/mL, compounds 1 to 3 inhibited beta-amyloid aggregation by 50% or more.

Test Example 3. Beta-amyloid aggregate degradation (disintegration) evaluation of compounds 1 to 3 (ThT assay)

The effect of decomposing the isolated beta-amyloid aggregates of Compound 1-3 was tested using a Th T assay.

After dispensing beta-amyloid to a final concentration of 20 μM in a 96-well plate, leave it at 37 °C to allow beta-amyloid to aggregate. After 24 hours, 0.0625, 0.125, 0.25, 0.5, 1, and 2 μg/mL of compounds 1 to 3 obtained in Example 3 were added and reacted at 37° C. for 24 hours, respectively. Then, thioflavin T test solution (ThT solution) (thioflavin with 100 mM glycine, pH 8.5) was added so that the final concentration was 3 μM, and after 20 minutes of reaction, the amount of fluorescence was measured 442 excitation and 485 emission. Fluorescence values were measured with a measuring instrument. In the same manner, the group not treated with compounds 1 to 3 (THF treatment) was used as a control group.

4 is a diagram confirming the effect of dissolving beta-amyloid aggregates of the compounds isolated from the DCM fraction of perilla extract.

In FIG. 4 , the horizontal axis represents the concentration (μg/mL) of Compounds 1 to 3 , and the vertical axis represents the ThT fluorescence measurement result (expressed as a percentage of the control group).

As shown in FIG. 4 , compounds 1 to 3 effectively decomposed beta-amyloid aggregates, and the effect was concentration-dependent.

Test Example 4. Measurement of antioxidant activity of compounds 1 to 3 (DPPH method)

The antioxidant activity of the isolated compound 1-3 was measured using the DPPH method. All of the compounds of Compound 1-3 showed very low DPPH scavenging activity of 5% or less compared to the control group at a concentration of 64 mg/mL.

Substances showing an effect on Alzheimer's disease, etc. have antioxidant activity, but Compound 1-3 did not show antioxidant activity, and as shown in the results of Test Example 1 or Test Example 2, which are not antioxidant activity, beta-amyloid aggregation inhibition or aggregates It can be seen that the decomposition will show an effect on degenerative neurological diseases.

statistical analysis

All data and figures are expressed as mean ± standard deviation.

This patent was researched with support from the Ministry of Agriculture, Food and Rural Affairs, a research support project, with the support of the Agricultural Food Technology Planning and Evaluation Institute's customized innovative food and natural safe material technology development project (119012-3).

Those of ordinary skill 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 are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (14)

A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, comprising as an active ingredient a compound represented by the following Chemical Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract.
[Formula 1]

[Formula 2]

[Formula 3]

The pharmaceutical composition according to claim 1, wherein the extraction solvent for the perilla leaf extract is water, C ₁ to C ₄ alcohol, or a mixture thereof.
The pharmaceutical composition according to claim 1, wherein the extraction solvent for the perilla leaf extract is ethanol.
The pharmaceutical composition according to claim 1, wherein the compounds represented by Formulas 1 to 3 are separated from the dichloromethane fraction of the ethanol extract of perilla leaf.
The pharmaceutical composition according to claim 1, wherein the compounds represented by Chemical Formulas 1 to 3 inhibit the production of beta-amyloid aggregates or disintegrate the produced beta-amyloid aggregates.
The method according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, Lou Gehrig disease (Amyotrophic lateral sclerosis or Lou Gehrig disease), Huntington's disease ( Huntington's disease, Niemann-Pick disease, Dutch amyloidosis, amyloid stroke, Parkinson's disease, mild cognitive impairment, senility, dementia Dementia, Lewy Body Dementia Multi-infarct dementia, Down's syndrome, Dementia associated with stroke, Dementia associated with Parkinson's disease Or beta- amyloid-related dementia (Dementia associated with beta-amyloid) for the prevention or treatment of a pharmaceutical composition.
The pharmaceutical composition of claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, cerebral amyloid angiopathy, systemic amyloidosis, Lou Gehrig's disease, Huntington's disease, and Niemann-Pick's disease.
The pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is a degenerative brain disease.
The pharmaceutical composition according to claim 1, wherein the degenerative brain disease is a disease mainly caused by accumulation of beta-amyloid in the brain.
The pharmaceutical composition of claim 1, wherein the neurodegenerative disease is Alzheimer's disease.
The pharmaceutical composition according to claim 1, wherein the compound represented by Formulas 1 to 3 or a derivative thereof is provided as a pharmaceutically acceptable salt or solvate.
A food composition for preventing or improving neurodegenerative diseases, comprising as an active ingredient a compound represented by the following Chemical Formulas 1 to 3, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract.
[Formula 1]

[Formula 2]

[Formula 3]

The food composition according to claim 12, wherein the compounds represented by Chemical Formulas 1 to 3 are separated from the dichloromethane fraction of the ethanol extract of perilla leaf.
The food composition according to claim 12, wherein the compounds represented by Chemical Formulas 1 to 3 inhibit the production of beta-amyloid aggregates or disintegrate the produced beta-amyloid aggregates.

Images