KR20230140956A - Composition comprising extract Metasequoia glyptostroboides Hu & Cheng for preventing or treating neurodegenerative diseases - Google Patents

Abstract

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

Description

Composition for preventing or treating neurodegenerative diseases comprising extract of metasequoia as an active ingredient {Composition comprising extract Metasequoia glyptostroboides Hu & Cheng for preventing or treating neurodegenerative diseases}

The present invention relates to a pharmaceutical composition for preventing, improving or treating degenerative neurological diseases containing metasequoia extract as an active ingredient, and more specifically, to a pharmaceutical composition containing metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient. 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, showing symptoms of gradual memory decline and cognitive loss. It is characterized by a very gradual onset and gradual progression, and in the early stages, mainly memory for recent events. It is a disease that starts with problems and progresses to include abnormalities in other cognitive functions such as language function and judgment, and eventually leads to loss of all daily life functions.

The main neuropathological finding of Alzheimer's disease is the aggregation and deposition of beta-amyloid (Aβ) and hyperphosphorylated tau protein. Outside the brain neurons, beta-amyloid accumulates and becomes tangled, forming senile plaques, which causes neurotoxicity, and inside the neurons, neurofibrillary tangles made of hyperphosphorylated tau protein accumulate, causing Alzheimer's disease. It is known to cause disease.

Acetylcholinesterase enzymes such as tacrine, donepezil, rivastigmine, and galantamine relieve dementia symptoms by replenishing the lack of acetylcholine, a conventional neurotransmitter. ) inhibitors are known to be a treatment for Alzheimer's disease, and these acetylcholine hydrolase inhibitors are effective in improving symptoms by lowering the rate at which acetylcholine is removed from synapses. However, these drugs are not a fundamental treatment for degenerative neurological diseases, including Alzheimer's disease, which are mainly caused by beta-amyloid aggregates formed by overproducing beta-amyloid, and are known to have side effects such as hepatotoxicity, nausea, nausea, and diarrhea. .

Therefore, as a more fundamental treatment for degenerative diseases mainly caused by beta-amyloid aggregates, there is a need to develop medicines that can inhibit the formation of beta-amyloid aggregates and dismantle the aggregates.

Selkoe DJ. 1991. The molecular pathology of Alzheimer’s disease. Neuron 6:487-498 Selkoe DJ. 1996. Amyloid ß-protein and the genetics of Alzheimer’s disease. J Biol Chem. 271: 18295-18296. Coles, M.; Bicknell, W., Watson, A. A., Fairlie, D. P. Craik, D. J. 1998. Solution Structure of Amyloid β-Peptide (1-40) in a Water-Micelle Environment. Is the membrane spanning domain where we think it is? Biochemistry 37, 11064 Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K. 1985. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. Jun;82(12):4245-4249. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Iosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. 1998. Diffusible, nonfribrillar ligands derived from Aβ1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 95:6448-6453. Ward, R. V., Jennings, K. H., Jepras, R., Neville, W., Owen, D. E., Hawkins, J., Howlett, D. R. 2000. Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of beta-amyloid peptide. Biochemical Journal, 348(Pt 1), 137. Gong, Y., Chang, L., Viola, K. L., Lacor, P. N., Lambert, M. P., Finch, C. E., Krafft, G. A., and Klein, W. L. 2003. Alzheimer's disease-affected brain: presence of oligomeric Aβ ligands (ADDLs). suggests a molecular basis for reversible memory loss. Proceedings of the National Academy of Sciences, 100(18), 10417-10422. LeVine, III, H. 1993. Thioflavine T Interaction with Synthetic Alzheimer's Disease £] - Amyloid Peptides: Detection of Amyloid aggregation in Solution, Protein Science 2: 404-410. H. LeVine III. 1999. Quantification of β-sheet amyloid fibril structures with thioflavin T, Methods in Enzymology, vol. 309, pp. 274-284.

The goal of the present invention is to provide a composition that prevents, improves, or treats neurodegenerative diseases more fundamentally than existing treatments by inhibiting the formation of beta-amyloid aggregates, which are the root cause of neurodegenerative diseases such as dementia and Alzheimer's disease.

One aspect is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases, which includes an extract of Metasequoia ( Metasequoia glyptostroboides Hu & Cheng), a fraction thereof, or a compound isolated therefrom as an active ingredient.

Another aspect is to provide a food composition for preventing or ameliorating neurodegenerative disease, which includes a metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient.

Another aspect is to provide a health functional food for improving learning or memory, or cognitive function, containing metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient.

Another aspect is to provide a feed composition for preventing or improving neurodegenerative diseases comprising metasequoia extract or fractions thereof, or compounds isolated therefrom 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.

One aspect provides a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising an extract of Metasequoia ( Metasequoia glyptostroboides Hu & Cheng), a fraction thereof, or a compound isolated therefrom as an active ingredient.

“ Metasequoia glyptostroboides Hu & Cheng” used in this specification is a deciduous gymnosperm belonging to the genus Metasequoia of the Cupressaceae family. As a tall tree, the branches usually face each other and spread out to the sides, forming an overall pyramid shape. The bark of the tree is reddish-brown, but older ones are grey-brown and are shallowly cracked vertically and peeled off. Young branches face each other and droop downward. The leaves are opposite each other, dioecious, and pollinated from February to April before the leaves emerge. The fruit is a cone, round shape, 1.4~2.5cm long, 1.6~2.3cm wide, ripens in October~November. The seeds are about 5 mm long and have wide wings. The shape of the tree is similar to that of the taxidermy, but the branches and leaves are opposite each other, and the fruits have long stalks, making them distinct. It grows naturally in the upper reaches of the Yangtze River in Hubei Province, southwestern China. It is planted throughout Korea and around the world.

The metasequoia can be purchased commercially, or collected or cultivated from nature.

The term “extract” used in this specification refers to the extract itself, such as an extract obtained by extracting metasequoia, 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, etc. and extracts of all formulations that can be formed using the extract.

The method for extracting the metasequoia is not particularly limited, and can be extracted according to methods 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 metasequoia is not particularly limited, and any solvent known in the art can be used. In one embodiment, the extraction solvent for the metasequoia extract may be water, C ₁ to C ₄ alcohol, or a mixture thereof. In one embodiment, the extraction solvent for the metasequoia 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.

In one embodiment, the ethanol is 10% (v/v) to 100% (v/v), 20% (v/v) to 99% (v/v), or 50% (v/v) to 90% (v/v). The concentration may be % (v/v).

The metasequoia extract can be obtained from the fruit or leaves of metasequoia. Preferably, in one embodiment, the metasequoia extract may be a metasequoia fruit extract.

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 metasequoia with a predetermined solvent. In one embodiment, the metasequoia extract 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, the fraction may be fractionated with hexane, dichloromethane, ethyl acetate, or water.

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.

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

In one embodiment, the compounds isolated from the extract or fractions thereof include taxoquinone ( 1 ), sugarol ( 2 ), suginal ( 3 ), sandaracopimarinol ( 4 ) and sandaracopimaradien-19-ol (sandaracopimaradien-19-ol) ( 5 ), or a mixture thereof. The compounds can be represented by the following formulas (1) to (5).

[Chemical formula]

The above compounds are derived from metasequoia 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 to prevent degenerative neurological diseases such as Alzheimer's disease. It can prevent, improve, treat, improve learning or memory, or improve 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 metasequoia, but are not limited thereto.

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.

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

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 .

The degenerative neurological diseases include Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, Lou Gehrig's disease (Amyotrophic lateral sclerosis or Lou Gehrig disease), Huntington's disease, and Niemann's disease. -Niemann-Pick Disease, Dutch amyloidosis, amyloid stroke, Parkinson's disease, Mild cognitive impairment, Down's syndrome, Senility , multiple sclerosis, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, and dementia, but is not limited thereto.

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

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 any one selected from the group consisting of Alzheimer's disease, cerebral amyloid angiopathy, systemic amyloidosis, and dementia such as beta-amyloid-related dementia.

As used herein, the term “Alzheimer's disease” is a 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" as used herein is caused by the gradual loss of dopamine neurons distributed in the substantia nigra of the brain and is characterized by resting tremor, rigidity, bradykinesia (slow movement), and postural instability. It is a chronic progressive degenerative disease of the nervous system.

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.

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 the neurodegenerative disease, beta-amyloid aggregates may be the main cause of the disease.

In one embodiment, the pharmaceutical composition can inhibit beta-amyloid aggregation.

In one embodiment, the pharmaceutical composition can disintegrate aggregated beta-amyloid.

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

According to one embodiment, a pharmaceutical composition containing as an active ingredient an ethanol extract or fraction of metasequoia fruit, or a compound of formula 1 to 5 isolated therefrom, inhibits beta-amyloid aggregation or dismantles aggregated beta-amyloid (aggregates). , Degenerative neurological diseases can be treated, improved, and prevented by reducing aggregates.

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 metasequoia 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 1 to 99% by weight. It may 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 metasequoia extract or fractions thereof, or the compounds of formulas 1 to 5 isolated therefrom, are contained in an amount of 0.1 to 1000 μg/mL, for example, 0.1, 0.5, 1, 2, 3, 4, 5, 6. , 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500, 700, 900, 1000 μg/mL or any of the preceding values. It may be a range with either the upper or lower limit, such as 1 to 100, 4 to 100, or 10 to 100 μg/mL.

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) may be included.

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 provides a method for preventing or treating neurodegenerative disease, comprising administering to a subject a pharmaceutical composition containing a metasequoia extract or 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 an individual a pharmaceutical composition containing a metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient in a pharmaceutically effective amount. there is.

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 provides a food composition for preventing or improving neurodegenerative disease, comprising a metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient.

One specific example is the prevention of neurodegenerative disease comprising taxoquinone, sugarol, resinal, sandaracopimarinol, sandaracopimaradien-19-ol, or mixtures thereof isolated from metasequoia extract or fractions thereof as active ingredients, or A composition for improving food is provided.

Another aspect provides a health functional food for improving learning or memory, or cognitive function, containing metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient.

One specific example includes taxoquinone, sugarol, resinal, sandaracopimarinol, sandaracopimaradien-19-ol, or mixtures thereof isolated from metasequoia extract or fractions thereof as an active ingredient, improving learning or memory, Alternatively, health functional foods for improving cognitive function are provided.

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 metasequoia extract, fraction, or compound derived therefrom according to the present invention. In addition, As used herein, the term “cognitive function improvement” refers to all actions in which symptoms of cognitive decline are improved or beneficially altered by the extract, fraction, or compound derived from metasequoia 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 improve the effect of improving neurodegenerative 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 provides a feed composition for preventing or improving neurodegenerative disease, comprising a metasequoia extract or a fraction thereof, or a compound isolated therefrom as an active ingredient.

One specific example is the prevention of neurodegenerative disease comprising taxoquinone, sugarol, resinal, sandaracopimarinol, sandaracopimaradien-19-ol, or mixtures thereof isolated from metasequoia extract or fractions thereof as active ingredients, or A composition for improving feed is provided.

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 a metasequoia extract, a fraction thereof, or a compound of formulas 1 to 5, which are active ingredients isolated therefrom, not only inhibits beta-amyloid aggregation, which is the causative agent of neurodegenerative diseases such as Alzheimer's disease. It can also dismantle aggregated beta-amyloid. In addition, as the formation of beta-amyloid aggregates is suppressed and the amount of already formed aggregates is also reduced, nerve cells can be protected from neurotoxins caused by beta-amyloid aggregates. Therefore, it can be usefully used as a medicine, food, or health functional food to prevent, improve, and treat degenerative neurological diseases, including Alzheimer's disease, or to improve popular functions. 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 Thioflavin T fluorescence analysis of metasequoia extract or fractions thereof.
Figure 2 is a schematic diagram showing the process of producing metasequoia extract or fractions thereof.
Figure 3 is a schematic diagram showing the separation process of compounds of formulas 1 to 3.
Figure 4 is a schematic diagram showing the separation process of compounds of formulas 4 and 5.
Figure 5 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 1.
Figure 6 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 2.
Figure 7 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 3.
Figure 8 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 4.
Figure 9 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 5.
Figure 10 shows the structural formulas of compounds of formulas 1 to 5, which are active ingredients isolated from metasequoia extract.
Figure 11 is a graph showing the results (%) of Thioflavin T fluorescence analysis of compounds of Formulas 1 to 5. From this, the effect of inhibiting beta-amyloid aggregation is confirmed.
Figure 12 is a graph showing the results (%) of Thioflavin T fluorescence analysis of compounds of Formulas 1 to 5. From this, the effect of dismantling beta-amyloid aggregates is confirmed.
Figure 13 is a graph showing the cell survival rate (%) of compounds of formulas 1 to 5. From this, the effect of protecting nerve cells through inhibition of beta-amyloid aggregation is confirmed.
Figure 14 is a graph showing the DPPH scavenging activity (%) of compounds of Formulas 1 to 5. From this, the DPPH radical scavenging ability 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.

Example 1. Preparation of metasequoia extract and fractions

After crushing 4 kg of Metasequoia ( Metasequoia glyptostroboides Hu & Cheng) fruit, extracting it three times with 80% ethanol (three times at 5-6 L each, room temperature), the filtered filtrate was concentrated under reduced pressure to obtain 310.0 g of Metasequoia ethanol extract. did.

The obtained metasequoia ethanol extract was fractionated according to the polarity of the solvent, and fractions were obtained from each fraction layer. Specifically, the obtained extract was dissolved in a small amount of methanol (100-200 mL), suspended in water (5 L) , and mixed with n -hexane, dichloromethane, and ethyl acetate. The mixture was continuously fractionated (3 times, 5L each, at room temperature) to obtain a hexane fraction (115.2 g), a dichloromethane fraction (46 g), an ethyl acetate fraction (30.9 g), and a water fraction (115.8 g).

Figure 1 is a schematic diagram showing the process of producing metasequoia extract or fractions thereof.

Experimental Example 1. Thioflavin T fluorescence analysis of metasequoia extracts and fractions - measurement of beta-amyloid aggregation inhibitory activity

The beta-amyloid aggregation inhibitory activity of the metasequoia fruit ethanol extract prepared in Example 1 and each fraction was measured using a 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 thioflavin T fluorescence analysis to evaluate the degree to which self-aggregation into fibrils was inhibited by the metasequoia ethanol extract and each fraction obtained therefrom.

Beta-amyloid was dispensed into a 96 well plate to a final concentration of 20 μM, and then the metasequoia ethanol extract prepared in Example 1 at concentrations of 10, 20, and 50 μg/mL (final concentration), and hexane at a concentration of 50 μg/mL each. Fraction (Hx), dichloromethane 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 a group treated with only DMSO without treatment with metasequoia ethanol extract or fractions (CTR) and a group with only DMSO treated with beta-amyloid were used as the control group.

The Y-axis is the fluorescence intensity percentage relative to the group treated only with beta-amyloid, and the results are shown in Figure 2.

Figure 2 is a graph showing the results (%) of Thioflavin T fluorescence analysis of metasequoia extract or fractions thereof.

As shown in Figure 2, metasequoia extract inhibited the aggregation of beta-amyloid in a concentration-dependent manner.

In addition, the hexane fraction, dichloromethane fraction, ethyl acetate fraction, and water fraction obtained from the metasequoia extract showed an inhibitory effect on beta-amyloid aggregation, with the ethyl acetate fraction showing the best activity.

Example 2. Separation of active ingredients and identification of compounds of metasequoia extract

The active ingredient was isolated from the ethyl acetate fraction (fraction of the solvent-fractionated ethyl acetate layer) of the metasequoia extract based on Thioflavin T fluorescence analysis and activity tracking separation method.

Figure 3 is a schematic diagram showing the separation process of compounds of formulas 1 to 3.

Figure 4 is a schematic diagram showing the separation process of compounds of formulas 4 and 5.

Five diterpenoids obtained from this were isolated and referred to as compounds 1 to 5.

The structures of the isolated compounds were determined by comparing the obtained NMR data with reference literature: taxoquinone ( 1 ), sugarol ( 2 ), suginal ( 3 ), and sandaracopimarinol. ( 4 ) and sandaracopimaradien-19-ol ( 5 ).

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

Figure 6 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 2.

Figure 7 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 3.

Figure 8 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 4.

Figure 9 shows the results of ¹ H-NMR and ¹³ C-NMR analysis of the compound of Formula 5.

Figure 10 shows the structural formulas of compounds of formulas 1 to 5, which are active ingredients isolated from metasequoia extract.

Experimental Example 2. Thioflavin T fluorescence analysis of compounds of formulas 1 to 5 isolated from metasequoia extract - measurement of beta-amyloid aggregation inhibitory activity

The beta-amyloid aggregation inhibitory activity of the compounds of formulas 1 to 5 isolated from the metasequoia extract prepared in Example 2 was measured by thioflavin T fluorescence analysis as in Experiment 1.

Beta-amyloid was dispensed into a 96 well plate to a final concentration of 20 μM, and then reacted with compounds of formulas 1 to 5 prepared in Example 2 at concentrations of 4, 20, and 100 μg/mL, respectively, at 37°C for 24 hours. Afterwards, add thioflavin T test solution (thioflavin with 100 mM glycine, pH 8.5) to reach a final concentration of 3 μM, react for 20 minutes, and measure the amount of fluorescence using a fluorometer that measures 442 excitation and 485 emission. was measured. The same method was performed, but the group not treated with the compounds of formulas 1 to 5 (DMSO treated) was used as the control group.

The Y-axis is the fluorescence intensity percentage relative to the group treated only with beta-amyloid, and the results are shown in Figure 11.

Figure 11 is a graph showing the results (%) of Thioflavin T fluorescence analysis of compounds of formulas 1 to 5 isolated from metasequoia extract.

As shown in Figure 11, the compounds of Formulas 1 to 5 significantly inhibited the aggregation of beta-amyloid (Aβ) in a concentration-dependent manner.

Experimental Example 3. Thioflavin T fluorescence analysis of compounds of formulas 1 to 5 isolated from metasequoia extract - measurement of beta-amyloid aggregate disassembly activity

The activity of compounds of formulas 1 to 5 isolated from metasequoia extracts to dismantle already aggregated beta-amyloid aggregates 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, compounds of Formulas 1 to 5 were added at concentrations of 100, 20, and 4 μg/mL (final concentration), and 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 not treated with the compounds of formulas 1 to 5 (DMSO treated) was used as the control group.

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

Figure 12 is a graph showing the results (%) of Thioflavin T fluorescence analysis of compounds of Formulas 1 to 5.

As shown in Figure 12, the compounds of Formulas 1 to 5 exhibited activity in dismantling already aggregated beta-amyloid aggregates in a concentration-dependent manner.

In addition, compounds of formulas 1 to 5 obtained from metasequoia extracts also showed activity in dismantling beta-amyloid aggregates.

Experimental Example 4. Measurement of the neuronal protective effect of compounds of formulas 1 to 5 isolated from metasequoia extract

The neuronal protective effect of the active ingredient isolated from metasequoia extract was measured using P12 cells (PC12 cells).

After dispensing beta-amyloid to a final concentration of 20 μM in a 96 well plate, compounds of formulas 1 to 5 were added at concentrations of 4, 20, and 100 μg/mL, respectively, and reacted at 37°C for 24 hours (beta-amyloid and mixtures of compounds of formulas 1 to 5). Meanwhile, PC12 cells were stabilized by seeding ^4x10 cells in a 96 well plate. After 3 hours, PC12 cells in a 96 well plate were treated with a mixture of pre-reacted beta-amyloid and compounds of formulas 1 to 5, and each was reacted at 37°C for an additional 24 hours. At this time, to determine changes in cell viability, formazan formed 3 hours after treatment with 5 mM MTT solution was dissolved by adding 100 uL of DMSO for 30 minutes, and then the absorbance change was measured at 570 nm.

Figure 13 is a graph showing cell survival rate (%) when treated with compounds of Formulas 1 to 5.

As shown in Figure 13, the compounds of Formulas 1 to 5 showed the effect of inhibiting beta-amyloid (Aβ) aggregation and protecting nerve cells from neurotoxicity caused by beta-amyloid.

From the results of Experimental Examples 2, 3, and 4, the compounds of formulas 1 to 5 isolated from metasequoia extract not only inhibit the aggregation of beta-amyloid, but can also effectively decompose and dismantle already formed aggregates, and prevent the aggregation caused by beta-amyloid. It was confirmed that it had a protective effect on nerve cells by significantly suppressing neurotoxicity.

Experimental Example 5. Antioxidant effect of active ingredients isolated from metasequoia extract (DPPH radical scavenging ability)

The antioxidant activity of compounds of formulas 1 to 5 isolated from metasequoia extract was measured using the DPPH method.

First, dissolve DPPH in ethanol to make a 0.2 mM solution. Add 190 μL of DPPH and 10 μL of compounds of formulas 1 to 5 into a 96 well plate and mix well. At this time, the final concentrations of compounds of formulas 1 to 5 are 20, 50, and 150 μg/mL. Leave it at room temperature for 30 minutes, shielded from light, and then measure the absorbance at 517 nm.

Figure 14 displays the DPPH scavenging activity (%) of compounds of Formulas 1 to 5. From this, the DPPH radical scavenging ability can be seen.

As shown in Figure 14, the compounds of Formulas 1 to 5 showed high DPPH scavenging activity in a concentration-dependent manner at concentrations of 20, 50, and 150 μg/mL.

It was confirmed that compounds 1 to 5 isolated from the ethyl acetate fraction layer of the metasequoia extract had excellent antioxidant effects.

This research project was supported by the National Research Foundation of Korea mid-career researcher support project (2019R1A2C100621412).

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.

Claims (16)

A pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an extract of Metasequoia ( Metasequoia glyptostroboides Hu & Cheng), a fraction thereof, or a compound isolated therefrom as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extraction solvent of the metasequoia extract is water, C ₁ to C ₄ alcohol, or a mixture thereof. The pharmaceutical composition according to claim 1, wherein the fraction is fractionated with hexane (n-hexane), dichloromethane (DCM), ethyl acetate (EA), or water. The pharmaceutical composition according to claim 1, comprising as an active ingredient a compound isolated from the ethyl acetate fraction of the ethanol extract of Metasequoia. The method according to claim 1, wherein the compounds isolated from the extract or fractions thereof include taxoquinone, sugarol, suginal, sandaracopimarinol, and sandaracopimaradien-19-ol ( sandaracopimaradien-19-ol), or a mixture thereof, a pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the metasequoia extract is a metasequoia fruit extract. 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 method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease (AD), cerebral amyloid angiopathy, systemic amyloidosis, and dementia associated with beta-amyloid. A pharmaceutical composition selected from the group consisting of: 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 caused by beta-amyloid (β-amyloid) aggregation. The pharmaceutical composition according to claim 1, which inhibits beta-amyloid aggregation and dismantles aggregated beta-amyloid. The pharmaceutical composition for preventing or treating neurodegenerative diseases 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, suspensions, syrups, injections, and granules. enemy composition. The pharmaceutical composition for preventing or treating neurodegenerative diseases according to claim 1, which is for oral or parenteral administration. Food for preventing or improving degenerative neurological diseases containing taxoquinone, sugarol, resinal, sandaracopimarinol, sandaracopimaradien-19-ol, or mixtures thereof isolated from metasequoia extract or fractions thereof as active ingredients Composition. Improving learning or memory or improving cognitive function containing taxoquinone, sugarol, resinal, sandaracopimarinol, sandaracopimaradien-19-ol, or mixtures thereof isolated from metasequoia extract or fractions thereof as active ingredients. Health functional food. A feed composition for preventing or improving degenerative neurological diseases containing taxoquinone, sugarol, resinal, sandaracopimarinol, sandaracopimaradien-19-ol, or mixtures thereof isolated from metasequoia extract or fractions thereof as active ingredients. .