WO2018105929A1 - Composition for preventing or treating cognitive impairment including atemoya leaf extract or fraction thereof - Google Patents

Abstract

The present invention relates to: a composition which is for preventing or treating cognitive impairment, and includes an atemoya leaf extract or a fraction thereof; a method for preventing or treating cognitive impairment using the composition; and a health functional food composition which is for preventing or alleviating cognitive impairment, and includes an atemoya leaf extract or a fraction thereof.

Description

A composition for the prevention or treatment of cognitive dysfunction, including atemonia leaf extract or fractions thereof

The present invention relates to a composition for the prevention or treatment of cognitive dysfunction comprising an extract of Atenoya leaf (Annona atemoya leaf), specifically, for the prevention or treatment of cognitive dysfunction comprising the atetoya leaf extract or fractions thereof A composition, a method for preventing or treating cognitive dysfunction comprising administering the pharmaceutical composition, and a nutraceutical composition comprising the extract or a fraction thereof.

Cognitive impairment, which loses the ability of cognitive function, can be divided into dementia and mild cognitive impairment. Mild cognitive dysfunction complains of subjective memory impairment or abnormalities found in objective tests, but it does not interfere with daily life and mental function is maintained so that it is not dementia. Before the development of mild cognitive dysfunction, the process of killing nerve cells is already in progress in the brain, but there are no symptoms at this time and it is difficult to distinguish from memory impairment due to normal aging. Mild cognitive dysfunction is more common than dementia, and 15 to 30% of 60-year-olds may fall within this category and increase with age. The condition of mild cognitive dysfunction is the current cognitive impairment between normal and Alzheimer's disease, and about 15% annually develops symptoms of dementia. Dementia is expected to surpass 100 million people with dementia, 3.1 times larger than 2013, by 2050. Korea is expected to become the fastest growing country for dementia in the world due to the aging population of over 65%. As a significant social concern.

Dementia, not a specific disease name, refers to a specific state of cognitive decline, and there are about 100 kinds of causative diseases. Among them, Alzheimer's disease, which is a degenerative brain disease, accounts for over 50% of dementia, and the exact cause and treatment of symptoms such as irreversible behavior, personality changes, and decreased thinking ability caused by Alzheimer's disease are not known. Although, the amyloid-beta protein accumulation and oxidative stress due to the impaired function of the cholinergic nervous system, the neurotransmitter system, and inflammatory reactions have been reported (Lancet, 21, 1403, 1976; J. Biol. Chem., 273). , 29719, 1988; Science, 262, 689, 1993), has been found to be closely associated with impaired cognitive function. Accordingly, efforts to maintain the concentration of acetylcholine by inhibiting the activity of acetylcholinesterase (AChE), an acetylcholine degrading enzyme in the brain, have been actively carried out until recently to suppress amyloid beta aggregation in the early stage of dementia. It is becoming. In addition, the method of adjusting the electron transport system (DPPH, 1,1-diphenyl-2-picrylhydrazyl radical scavenging ability; ABTS, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid scavenging ability) to measure the antioxidant activity and They are looking for ways to show neuroprotective effects against free radicals (ROS), which can cause cell death.

However, to date, extensive and diverse studies on the causes and treatments of dementia have been conducted, but the cause is insufficient and the development of effective treatments is also insufficient. Currently used therapeutic agents are limited drugs such as delaying disease progression and alleviating symptoms under the premise of 'if found early', and the protective effect on neurons is that as the death of neurons progresses, The effect is diminished, and in the case of severe dementia the effect is even weaker.

Athemoya is a tropical fruit of the Anonaaceae, a hybrid of the American family of annona squamosa and annona cherimola, with a variety of varieties such as Hillary White and Pinks Marmose. Its shape resembles the head of a Buddha, and in China and Taiwan, it has no sour taste and is said to have the highest sugar content among fruits, and is grown in Jeju in Korea. Athemoya's immature fruits are dysentery, tumors, rashes, etc., petals are watery, antispasmodic, and the ethanol extract of seeds is used for folk remedies as insecticides and insect repellents, and roots are used for diarrhea and fever. And has been reported to inhibit tumors.

In addition, the antithrombotic effect of atemoya seed ethanol extract can be used as an anticancer drug (BMC Complement Altern Med. 2014 Sep 23; 14: 353), and antibacterial, antioxidant and Anti-inflammatory activity has been reported (Food Chem. 2016 Jul 1; 202: 176-83) and the treatment or prevention of degenerative encephalopathy with tryptamine, the active ingredient of atemeya seed extract, has been reported ( U.S. Patent No. 8614246), the effect on the cognitive dysfunction of the atmoya leaf extract has not been found.

Under these backgrounds, the present inventors have made efforts to find natural products that have a low side effect and are harmless to the human body while exhibiting the effect of preventing, improving or treating cognitive dysfunction. As a result, the excellent amyloid beta aggregation inhibitory, antioxidant effect, and cell of Atemeya leaf extract or fractions thereof The present invention was completed by confirming that there is a nontoxic and neuronal cell protective effect, and confirming that it can be applied to drugs or foods.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cognitive dysfunction, including an extract of Atenoya leaf (Annona atemoya leaf) or a fraction thereof.

Another object of the present invention is to provide a method for preventing or treating cognitive dysfunction comprising administering the composition to a subject.

Still another object of the present invention is to provide a health functional food composition for preventing or improving cognitive dysfunction, including an extract of Annona atemoya leaf or a fraction thereof.

Still another object of the present invention is to provide a feed composition for preventing or improving cognitive dysfunction, including an Atenaya leaf (Annona atemoya leaf) extract or a fraction thereof.

The composition comprising the atemonia leaf extract or fractions thereof of the present invention inhibits amyloid beta aggregation, and has a high antioxidant effect, nontoxic to neurons and protects the neurons from external stimuli, thereby preventing cognitive dysfunction, It can be applied to medicine or health food for treatment or improvement.

Figure 1 shows the manufacturing process of the fraction according to an embodiment of the present invention.

Figure 2 is a graph showing the effect of the leaves of Athemoya leaves, seeds and pulp extract on amyloid beta aggregation (Morin; positive control group, the same below).

Figure 3 is a graph showing the antioxidant efficacy of the leaves, seeds and pulp extract through DPPH radical scavenging ability (Vit. C; positive control group, the same below).

Figure 4 is a graph showing the antioxidant efficacy of the leaves, seeds and pulp extract through DPPH radical scavenging ability.

FIG. 5 is a graph showing cytotoxicity of atemonia leaf, seed and pulp extract against neuronal hippocampal cell line ( ^** P <0.01).

Figure 6 is a graph showing the protective effect of the Athemoya leaves, seeds and pulp extract using HT22 cells (Carvedilol: positive control, ^### P <0.001 vs. extract untreated, ^** P <0.01, ^*** P <0.001 vs. H ₂ O ₂ single treatment group)

FIG. 7 is a graph showing the effect of atemonia leaf extract fractions on amyloid beta aggregation.

Figure 8 is a graph showing the antioxidant efficacy of the Athemoya leaf extract fraction through ABTS radical scavenging ability.

Figure 9 is a graph showing the antioxidant efficacy of the Atemeia leaf extract fractions through DPPH radical scavenging ability.

Figure 10 is a graph showing the effect on the improvement of cognitive dysfunction of Atehya leaf extract through behavioral experiment in Alzheimer's dementia animal model (Morin; positive control group, ### P <0.001 vs. normal group, * P <0.05, *** P <0.001 vs. Amyloid beta group).

FIG. 11 is a graph showing the effects of atorectile dysfunction of the Athemoya leaf extract through a behavioral experiment in a short-term memory loss animal model (Tacrine; positive control group, # P <0.05, ### P <0.001 vs. normal group) , * P <0.05, ** P <0.01 vs. Scopolamine administered group).

One aspect of the present invention for achieving the above object, provides a pharmaceutical composition for preventing or treating cognitive dysfunction, including an extract (Anona atemoya leaf) or fractions thereof.

The present invention also provides a prophylactic or therapeutic use of atemoya leaf extract or fractions thereof.

As used herein, the term "Annona atemoya" is a tropical fruit of the Anpoaceae family, which is a hybrid of the sugar apple (annona squamosa) and annona cherimola native to the US tropical region. In the present invention, in order to examine the effects on cognitive dysfunction, the leaves were compared to the leaves, seeds and pulp extract, the remarkably excellent effect of the leaves of the leaves and cognitive dysfunction was first identified by the present inventors. The athemoya leaves, seeds and pulp of the present invention can be purchased and used commercially.

As used herein, the term “extract” refers to an extract obtained by extracting the atamoya leaves, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, a mixture thereof, and the like. , Extracts themselves and extracts of all formulations that can be formed using the extracts. The extract or fraction of the present invention may be prepared and used in the form of a dry powder after extraction.

In the atamoya leaf extract of the present invention, the method of extracting the atamoya leaf is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction solvent may include water, alcohol or a mixed solvent thereof, and, when using alcohol as a solvent, it is possible to use C1 to C4 alcohol as an example. As another example, the atemonia leaf extract of the present invention may be an ethanol extract.

In addition, the extract may be concentrated or removed by performing a filtration process under reduced pressure, or further concentrated and / or lyophilized after performing the extraction or fractionation process, the obtained atemaya leaf extract is used until Can be stored in a quick freezer.

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

In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. As a non-limiting example of the fractionation method, there is a method of obtaining a fraction from the extract by treating the extract obtained by extracting the leaves of Athemoya with a predetermined solvent.

The kind of the fractionation solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water and alcohols; Non-polar solvents such as hexane, ethyl acetate, chloroform, dichloromethane and the like. These may be used alone or in combination of two or more thereof. In the case of using alcohol in the fractionation solvent, alcohols of C1 to C4 may be preferably used. As another example, the fractions of the atmoeya leaf extract of the present invention may be specifically hexane, ethyl acetate, saturated butanol and water fractions.

As used herein, the term "cognitive function" refers to various intellectual abilities such as attention, perception, memory, language, and executive ability. As a concept that can include the disease according to the disease, as a symptom thereof, it may mean a disorder caused by damage to brain neurons. For example, the cognitive dysfunction may mean a degenerative brain disease selected from the group consisting of forgetfulness, Alzheimer's disease, dementia, Parkinson's disease, Peak disease, and Huntington's disease.

Dementia is a syndrome in which the cognitive function of various areas such as memory, language, and judgment are reduced, and thus cannot perform daily life properly, and the brain function is damaged by various causes. As a result, the overall decline may result in significant disruption in daily life. The dementia can be divided into dozens of causative diseases, but among them, Alzheimer's disease is the most common cause and exceeds 50%. In addition, dementia can be caused by degenerative brain diseases such as Lewy dementia, frontal lobe degeneration, Parkinson's disease, and various causes such as normal pressure hydrocephalus, head trauma, brain tumors, metabolic diseases, deficiency diseases, addictive diseases, and infectious diseases. have.

In a specific embodiment of the present invention, it was confirmed that the amyloid beta aggregation inhibitory activity is significantly superior to the Athemoya seed and pulp extract of the Athemoya leaf extract of the present invention (Fig. 2); ABTS and DPPH radical scavenging ability was confirmed to have a high antioxidant activity (Fig. 3 and 4). In addition, the Atemonia leaf extract showed no toxicity to neurons (FIG. 5); It was confirmed that the survival rate of neurons was improved by using HT22 cells induced neuronal damage (FIG. 6). In addition, behavioral tests such as passive avoidance experiments and Y-maze experiments using mice induced with Alzheimer's dementia and short-term memory loss confirmed the effects of improving the cognitive function of the Athemoya leaf extract (FIGS. 10 and 11). ).

This suggests that the atmoeya leaf extract can be very useful for preventing, treating or improving cognitive dysfunction.

In addition, in a specific embodiment of the present invention, as a result of treating the fractions of the atemonia leaf extract of the present invention, the ethyl acetate fractions compared to the hexane, saturated butanol and water fractions were found to have significantly superior amyloid beta aggregation inhibitory activity (Fig. 7); High antioxidant activity was confirmed through ABTS and DPPH radical scavenging activity (FIGS. 8 and 9). This suggests that the fraction of the atamoya leaf extract, specifically, the ethyl acetate fraction can be very usefully used for the prevention, treatment or improvement of cognitive dysfunction.

As used herein, the term "prevention" refers to any action that inhibits or delays cognitive dysfunction by administration of a pharmaceutical composition comprising the atemonia leaf extract of the present invention or a fraction thereof.

As used herein, the term “treatment” refers to any behavior in which cognitive dysfunction is improved or beneficially altered by administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may include 0.01% to 100% by weight of the atemonia leaf extract or fractions thereof, based on the total weight of the composition, specifically, 1% to 80% by weight, but is not limited thereto.

The pharmaceutical composition for preventing or treating a cognitive function-related disease according to the present invention may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier to be provided as a food, medicine, feed additive, and drinking water additive. Can be.

As used herein, the term “pharmaceutically acceptable carrier” may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound to be injected without stimulating the organism. The kind of the carrier usable in the present invention is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In this case, the carrier may include a non-naturally occuring carrier. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. The carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but Without limitation, conventional carriers, excipients or diluents can be used. The ingredients may be added independently or in combination with the active ingredient atemeya leaf extract.

Solid form preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or It can be prepared by mixing lactose, gelatin and the like. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the pharmaceutical composition of the present invention is a group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories It can have any one formulation selected from.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. There is no particular restriction on the dosage, and it may be changed according to body absorption, weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of disease. The pharmaceutical compositions of the present invention may be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way may be formulated using a specialized dosage method according to the judgment of a professional who monitors or observes the administration of the drug as required and the needs of the individual. It can be used or administered several times at regular time intervals. Specifically, the pharmaceutical composition of the present invention may be administered at 0.0001 to 1000 mg / kg per day, more specifically at 0.01 to 100 mg / kg, based on the amount of atemonia leaf extract. It may be administered once or divided several times.

As another aspect of the present invention, there is provided a method for treating cognitive dysfunction, comprising administering to the subject a pharmaceutical composition comprising the atemonia leaf extract or a fraction thereof as an active ingredient.

At this time, the atmoya, extracts, fractions, cognitive dysfunction, prevention and treatment are as described above.

As used herein, the term "administration" refers to the introduction of a predetermined substance to a subject in an appropriate manner and the route of administration of the composition can be administered via any general route as long as it can reach the desired tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

As used herein, the term "individual" means any animal, including humans, who may or may have a cognitive dysfunction. As a specific example, it may be a mammal including a human.

Another aspect of the invention provides a dietary supplement for the prevention or improvement of cognitive dysfunction, including the Athemoya leaf extract.

At this time, the atmoya, extracts, fractions, cognitive function, prevention and treatment are as described above.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with the condition, e.g., the degree of symptoms, treated with the administration of a composition comprising the atemonia leaf extract of the present invention or a fraction thereof.

When the composition of the present invention is used as a health functional food additive, the atamoya leaf extract or a fraction thereof may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), and the composition of the present invention is environmentally friendly and there is no problem in terms of stability, so there is no big limitation in the mixing amount.

Since the health functional food composition of the present invention can be expected to be ingested on a daily basis, the effect of improving cognitive dysfunction can be expected, and thus can be very useful for health promotion purposes.

The term "health functional food" of the present invention is the same term as a food for special health use (FOSHU), and foods having high medical effects and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. Means. Here, the term 'function' refers to obtaining a useful effect for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a food. The health functional food composition of the present invention can be prepared in various forms of formulations, unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material, and excellent portability In addition, the health functional food of the present invention can be ingested as an adjuvant for enhancing the cognitive dysfunction effect.

The health functional food means a food having an active health maintenance or promotion effect compared to the general food, and health supplement food (health supplement food) means a food for health supplement purposes. In some cases, the terms nutraceutical, health food, dietary supplement are used interchangeably.

Specifically, the health functional food is a food prepared by adding the compound of the present invention to food materials such as beverages, teas, spices, gums, confections, or the like, encapsulated, powdered, suspensions, etc. Means to bring effect, but unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material.

The health functional food composition may further include a physiologically acceptable carrier, and the type of carrier is not particularly limited and may be used as long as it is commonly used in the art.

In addition, the health functional food composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. Examples may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn) and copper (Cu); And amino acids such as lysine, tryptophan, cysteine, valine and the like.

In addition, the health functional food composition is a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), Butylhydroxytoluene (BHT), etc.), colorant (such as tar pigment), coloring agent (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (such as MSG glutamate), sweetener (ducin, cyclate , Saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen titanate, etc.), reinforcing agents, emulsifiers, thickeners (pigments), coatings, gum herbicides, foam inhibitors, solvents, modifiers, etc. May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

An example of the health functional food composition of the present invention can be used as a health beverage composition, in which case it may contain various flavors or natural carbohydrates as additional ingredients, such as conventional drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin, cyclodextrin; Sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumartin, stevia extract; Synthetic sweeteners such as saccharin and aspartame; The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohol or carbonation agent and the like. Others may contain fruit flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The present invention also provides a feed composition for the prevention or improvement of cognitive dysfunction, including the Athemoya leaf extract or a fraction thereof.

At this time, the definition of atemoya, extracts, fractions, cognitive impairment, prevention and improvement are as described above.

The content of atomeya leaf extract in the feed composition for preventing or improving cognitive dysfunction may be appropriately selected depending on the species, age, weight, and breeding conditions of the feed animals, and 0.01 to 95% by weight relative to the total weight of the feed composition, specifically As may be the ratio of 0.1 to 80% by weight.

The feed composition may include a feed additive. The feed additive of the present invention corresponds to a feed supplement in the Feed Control Act.

As used herein, the term "feed" may refer to any natural or artificial diet, one meal, or the like or a component of the one meal for the animal to eat, ingest, and digest.

The kind of the feed is not particularly limited, and may be used a feed commonly used in the art. Non-limiting examples of the feed may include plant feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

In addition, the feed additive may additionally contain a carrier that is acceptable to the unit animal. In the present invention, the feed additive may be added as it is, or a known carrier, stabilizer, or the like. If necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants, and other additives may be added. Powders, granules, pellets, suspensions and the like may be in a suitable state. In the case of supplying the feed additive of the present invention, the unit animal may be supplied alone or mixed with feed.

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

Production Example  One : Athemoya  Preparation of Extract

Purified 5% of 70% (v / v) ethanol was added to 500.0 g of dried atemoya leaves, seeds and flesh, and ultrasonically extracted three times for 1 hour using an ultrasonic extractor. The ultrasonic extract was filtered using an Advantec No. 2 (110 mm) filter paper to remove insoluble matters, and then concentrated under reduced pressure at 40 ° C. with a condenser equipped with a cooling condenser. To completely remove the solvent of the concentrated extract under reduced pressure, 500 mL of purified water was added and suspended, and then concentrated using a lyophilizer. Table 1 shows the yields of extracts of each part of atemoya.

extract Yield (%) Athemoya Leaf 16.98 Atemoya Seeds 3.28 Atemoya pulp 23.30

Production Example  2 : Athemoya  Of leaf extract Fraction  Produce

200 mL of purified water was added and suspended in 10 g of the atemoya leaf extract prepared in Preparation Example 1, and 200 mL of n-hexane was repeatedly added three times, and then partitioned into a water layer and an organic layer using a separatory funnel. The organic layer fractions were concentrated under reduced pressure at 40 ° C. with a condenser equipped with a cooling condenser to obtain 413 mg of n-hexane fractions. Then, 200 mL of ethyl acetate was repeatedly added to the water layer three times, and the organic layer fractions were concentrated under reduced pressure at 40 ° C. with a condenser equipped with a cooling condenser using a separatory funnel and 424 mg of ethyl acetate. Fractions were obtained. 200 mL of saturated n-butanol was repeatedly added to the water layer three times, and the organic layer fraction and water layer obtained by fractionating the water layer and the organic layer using a separatory funnel were concentrated under reduced pressure at 40 ° C. using a condenser equipped with a cooling condenser at 1.373. g of saturated n-butanol fractions and 5.209 g of water fractions were obtained (FIG. 1). Table 2 shows the yield of the fractions of Atemoya leaf extract.

Fraction of Atemonia Leaf Extract Yield (%) n-hexane 4.13 Ethyl acetate 4.24 Saturated n-butanol 13.73 water 52.09

Example  One : Athemoya  Analysis of Physiological Indicators of Cognitive Dysfunction of Extracts by Parts

Example  1-1: Measurement of amyloid beta aggregation inhibitory efficacy

Amyloid beta aggregation inhibition was measured using a fluorescence assay (Thioflavin T assay). Amyloid beta (Aβ _{1 -42} ) peptide was stored at minus 80 ℃, the final concentration used for the measurement was 100 μg / mL. 2 mM thioflavin T as a fluorophore was used as a buffer solution for assays (assay buffer) (50 mM Tris / 150 mM NaCl (pH 7.2), 20 mM HEPES / 150 mM NaCl (pH 7.2), 10). prepared by dissolving in mM phosphate / 150 mM NaCl (pH 8.0). Samples were used to prepare a final concentration of 100 μg / mL of each of the leaves, seeds and pulp extract of Preparation Example 1 in the assay buffer. Aβ _{1 -42} in order to measure the degree of agglutination inhibition 96 well plates (black microplate) thio flavin T 10 μL with Aβ aggregation was put _1-42 in 85 μL, and mixed with 5 μL sample made with a certain concentration fluorescence spectroscopy Measured by an analyzer. The wavelength value of the fluorescence spectrometer used at this time was 440 nm / 484 nm (excitation / emission), and it measured in total for 2 hours at 37 degreeC in 20 minute intervals. Morin was used as a positive control for activity comparison.

As shown in FIG. 2, the amyloid beta aggregation inhibitory activity of the athemoya leaf extract at a concentration of 100 μg / mL of the sample showed a significant activity of 69.7% of the positive control activity in the leaf extract, and 25 to At 100 μg / mL, the concentration-dependent tendency of amyloid beta aggregation inhibitory activity increased with increasing concentration. In addition, the IC ₅₀ for the inhibition of beta aggregation of the atmoeya leaf extract was 87.19 μg / mL.

On the other hand, the extract of Athemoya showed a result of increasing amyloid beta aggregation rather, and it was confirmed that the atomeya pulp extract had no effect on amyloid beta aggregation.

From the above results, it can be seen that the Athemoya leaf extract has superior amyloid beta aggregation inhibitory effect compared to the seed and pulp extract, and can be effective in preventing cognitive dysfunction.

Example  1-2: Antioxidant efficacy measurement

Example  1-2-1: ABTS  Radical Scavenging power  Measure

Antioxidant activity using ABTS (3-ethyl-benzothiazoline-6-sulfonic acid) radical was measured by ABTS + · cation decolorization analysis. 7 mM ABTS and 2.45 mM potassium persulfate were uniformly mixed and left for 24 hours in the dark at room temperature to form ABTS cations (ABTS +). It was then diluted with PBS to have an absorbance value of 0.7 at 743 nm. After mixing 100 μl of each sample of Preparation Example 1 with ABTS + solution in a 96 well plate and reacting at room temperature for 30 minutes, the absorbance was measured at 743 nm using an Epoch microplate spectrophotometer. The radical scavenging activity of each sample was expressed as a percentage of the radical scavenging ability with respect to the control group using PBS as a control group. Vitamin C was used as a positive control for comparison of activity.

As shown in Figure 3, as the concentration of the leaves leaves, seeds and pulp extract showed a concentration-dependent tendency to increase the radical scavenging activity of ABTS. The seed and pulp extracts treated with 100 μg / mL concentration showed 46.74% and 76.74% radical scavenging activity, respectively, and the leaf extract treated with 100% ABTS radical scavenging activity. As well as the pulp extract, it was confirmed that the inhibitory activity was much higher than the positive control group having an ABTS radical scavenging ability of 77.05%.

Example  1-2-2: DPPH  Radical Scavenging power  Measure

Antioxidant activity evaluation using DPPH (1-1-diphenyl-2-picrylhydrazyl) radical was carried out using a 96 well plate. 0.15 mM DPPH solution and the sample of Preparation Example 1 were uniformly mixed in the same amount in a 96 well plate and reacted at room temperature for 30 minutes, and then absorbance was measured at 517 nm. The antioxidant activity of each site extract was expressed as a percentage of radical scavenging ability with respect to the control group using the solvent DMSO as a control. Vitamin C was used as a positive control for the activity comparison.

As shown in FIG. 4, the Athemoya leaf extract showed a more pronounced DPPH radical scavenging activity than the seeds and the flesh extract, and the concentration of the Athemoya leaf extract showed a concentration-dependent tendency to increase the DPPH radical scavenging activity.

Incorporating the results of the above Examples 1-2-1 and 1-2-2, it can be seen that the antioxidant activity of the leaf extract, particularly in the extract of each part of the leaves, seeds and pulp of the atmoya. In addition, the ABTS scavenging ability (100%) at the same concentration is higher than the DPPH scavenging ability (79.86%) of 100 μg / mL of the Athemoya leaf extract, while DPPH scavenging free radicals, while ABTS is a cationic radical It is reported that there is a difference in elimination of radicals and the radical scavenging ability is different because the degree of binding to the reactants reacting with the two substrates is different (J Korean Soc Food Sci Nutr 36, 250-254).

Example  2: Efficacy of Neuronal Cell Protection by HT22 Cells

Example  2-1: HT22 Cell Culture

HT22 cells were cultured in a 37% 5% CO ₂ incubator using DMEM medium added with 10% FBS.

Example  2- 1: Cell  Toxicity Measurement

In order to determine the cytotoxicity of each sample of Preparation Example 1, Cell Counting Kit-8 (CCK-8) was used according to the manufacturer's instructions. Samples were treated by concentration in HT22 cell lines (5 × 10 ³ cells / well) dispensed in 96 well plates and incubated for 24 hours. After incubating for 4 hours with 10 μL of CCK-8 solution, the absorbance was measured at 450 nm and compared with each control (each extract-free (0 μg / mL) group) to determine the relative percentage of cell viability. Calculation was made (FIG. 3).

As shown in Figure 5, when compared to the respective extracts and controls, the cell viability was rapidly dropped to 56.20% when treated with 100 μg / mL of Atemeya seed extract, the highest cytotoxicity, the flesh extract When treated, the cell viability of 86.37% and some cytotoxicity. However, it was confirmed that the cell viability was almost 100% when 100 μg / mL of the Atmoya leaf extract.

From the above results, the atamoya leaf extract showed no cytotoxicity with almost 100% cell viability at all concentrations of 12.5 to 100 μg / mL, thus confirming the harmless safety to the human body compared to the atomeya seed and pulp extract.

Example  3: measurement of neuronal protective activity

To ahtte Moya to determine the neuroprotective efficacy of the respective cuts of the extract, was treated to 250 μM of hydrogen peroxide (H ₂ O ₂₎ to the HT22 cells to induce a damage of nerve cells 6 hours, H ₂ O ₂ only treatment group and The cell protection effect was determined by comparing the change in cell viability of the sample and the H ₂ O ₂ co-treatment group. Carvedilol was used as a positive control.

As shown in FIG. 6, in the case of atamoya seeds and pulp extract, cell viability was reduced compared to the cell viability in the H ₂ O ₂ treatment group compared to the control group without any treatment at all concentrations of 25 to 100 μg / mL. According to the cell protective effect by the extract could not be confirmed.

On the other hand, the cell survival rate increased gradually as the concentration increased in the group treated with Atemoya leaf extract and H ₂ O ₂ than in the H ₂ O ₂ alone group compared to the control group without any treatment. At 50 and 100 μg / mL concentrations, the survival rate of 80% similar to that of the positive control group was about 40% higher than that of the H ₂ O ₂ treated group.

From the above results, it can be seen that the leaves extract leaves the nerve cells to protect against external stimulation, thereby improving the survival rate of the nerve cells.

Example  4 : Athemoya  Leaf extract Fraction  Cognitive dysfunction

Example  4-1: Amyloid beta aggregation inhibitory efficacy measurement

Amyloid beta aggregation inhibition measurement of the atmoya leaf extract fraction of Preparation Example 2 was used for fluorescence analysis (Thioflavin T assay), the specific experimental method was carried out in the same manner as in Example 1-1.

As a result, as shown in Figure 7, the amyloid beta aggregation inhibitory activity of each fraction of 100 μg / mL of the sample of Atehoya leaf extract showed an excellent activity than the positive control in the ethyl acetate fraction.

From the above results, it can be seen that the Athemoya leaf extract fraction, specifically ethyl acetate fraction has an excellent amyloid beta aggregation inhibitory effect, through which it can be effective in preventing cognitive dysfunction.

Example  4-2: Antioxidant efficacy measurement

Example  4-2-1: ABTS  Radical Scavenging power  Measure

Antioxidant activity was measured using ABTS radicals of the Athemoya leaf extract fractions in accordance with the ABTS + cation decol orization assay using a 96-well plate, and the specific experimental method was the same as in Example 1-2-1. Was performed.

As a result, as shown in FIG. 8, all fractions of the atmoeya leaf extract exhibited radical scavenging ability of ABTS, and in particular, the ethyl acetate fraction and then the saturated saturated butanol fraction showed superior radical scavenging ability of ABTS.

Example  4-2-2: DPPH  Radical Scavenging power  Measure

Antioxidant activity measurement using DPPH (1-1-diphenyl-2-picrylhydrazyl) radicals of Atemeya leaf extract fractions was performed using a 96 well plate.

As a result, as shown in Figure 9, the fractions of Atehoya leaf extract showed excellent DPPH radical scavenging activity, in particular, the ethyl DP acetate and then the excellent DPPH radical scavenging ability of the saturated butanol fraction was confirmed.

Through this, it was confirmed the antioxidant activity of the fractions of Atemoya leaf extract.

Example  5: Athemoya  Of leaf extract Invivo ( in vivo ) Experiment

Example  5-1: Experimental animals  Preparations

23-28 g male mice (ICR, central laboratory animals) at 7 weeks of age had a temperature of 23 ± 3 ° C, a relative humidity of 55 ± 15%, a ventilation frequency of 10-20 times / hour, an illumination time of 12 hours 8 pm off) and roughness 150-300 Lux were kept in the animal room under conditions. All experimental procedures were conducted in accordance with the NIH Guidelines for the Management and Use of Laboratory Animals. Animal handling was conducted in accordance with the National Animal Welfare Law of Korea.

Example  5-2: Induction of Alzheimer's Dementia Animals and Athemoya  Leaf extract dosage

Mice were randomly divided into 5 groups after 1 week of accrual (n = 8), weighed amyloid beta, dissolved in sterile 0.1 M phosphate-buffered saline (pH 7.4) to a concentration of 1 μg / uL. Alzheimer's dementia was induced by cryopreservation for at least 3 weeks before administration to induce aggregation and administration to the third ventricle of the brain (intracerebroventricular injection, icv).

Atemeya leaf extract, a test substance, was dissolved in PBS, and was administered orally for a total of 23 days until the end of the experiment starting the administration of amyloid beta 5 days before administration. Morin, an amyloid aggregation inhibitor, was used as a positive control for the test substance (FIG. 10A).

Example  5-3: Induction of short-term memory loss and Athemoya  Leaf extract dosage

Mice were randomly divided into 5 groups after 1 week of accrual (n = 8). The test substance dissolved in PBS was administered orally for 2 weeks, and tacrine was used as a positive control group. At this time, scopolamine, a memory loss substance, was intraperitoneally administered 30 minutes before the behavioral experiment (FIG. 11A).

Example  5-4: Manual Evasion Experiment

Passive avoidance experiments were performed at the same time for three consecutive days at 24 hour intervals. On the first day, the dementia-induced mice were kept in the shaded area for 2 minutes, and then placed in the illuminated area and immediately moved to the shaded area and immediately taken out for adaptive training. On the second day (after 24 hours), two training sessions were held at two-minute intervals: In the first training, the mouse was placed in the passive avoidance test instrument chamber for 60 seconds to adapt to the instrument. In this case, the guillotine door was opened without lighting, and the mouse was freely moved in and out. After 60 seconds of adaptation, the lamp was re-lit and allowed to freely enter and exit for 120 seconds. After moving to the shading area, the guillotine door was closed and 0.20 mA of scrambled shock was received for 2 seconds. The mice that did not move at this time were excluded from the experiment. On the last day, the time to move to the shaded area was measured by measuring the time as well as opening the guillotine door in the illuminated area.

As a result, as shown in Figure 10B, when the test substance was administered from the day after the administration of amyloid beta, when administered 100 mg / kg of the Atmoya leaf extract, the moving air increased three times more than the positive control (morin) Showed time.

In addition, as shown in Figure 11B, even when administered the test substance for two weeks and 30 minutes before the experiment to examine the passive avoidance reaction, 100 mg / kg of Atehoya leaf extract, even when administered the memory loss substance scopolamine When administered, the movement latency was increased more than two times than the positive control (tacrine).

Example  5-5: Y-Maze Experiment

The Y-maze is made of black acrylic in the shape of a Y-shaped blocky maze, and three branches are defined as A, B, and C, respectively, and the mouse is carefully placed on one branch and allowed to move freely for 8 minutes. The branches containing the mice were recorded in order. The case was entered completely to the tail, and the case was entered again to the branch which once entered. One point was awarded for three different branches. Change behavior was calculated by the following equation.

Spontaneous alternation (%) = [Actual Changes / Total Entry-2] x 100

As a result, as shown in Figure 10C, when the test substance was administered from the day following the administration of amyloid beta, when administered 100 mg / kg of Atmoeya leaf extract, showed a better alteration behavior than the positive control (morin).

In addition, as shown in Figure 11C, even when administered the test substance for two weeks and 30 minutes before the experiment to examine the passive avoidance reaction, when the scopolamine, a memory loss substance, 50 and 100 mg / kg of atmoya Each dose of leaf extract showed altered behavior corresponding to the positive control (tacrine).

From the behavioral test results of the Alzheimer's dementia animals, it can be seen that the Athemoya leaf extract may be effective in preventing cognitive dysfunction.

Example  5-6: Statistical Analysis

Statistical analysis was performed using GraphPad Prism Ver. 6 was used and tested for significance with the control. Test substance administration group was performed to test the ANOVA (One-way ANOVA) members and, to test the significance Duncan (Duncan) or Dunnett t-test. In addition, the normal group and the control group were tested for significance using Student's t-test . P <0.05 was determined to be statistically significant.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (12)

Annona atemoya leaf Pharmaceutical composition for the prevention or treatment of cognitive dysfunction comprising an extract or a fraction thereof as an active ingredient. The pharmaceutical composition of claim 1, wherein the cognitive dysfunction is a degenerative brain disease selected from the group consisting of forgetfulness, Alzheimer's disease, dementia, Parkinson's disease, peak disease, and Huntington's disease. The pharmaceutical composition of claim 1, wherein the atamoya leaf extract is obtained by extracting the atamoya leaf with water, C1 to C4 alcohol, or a mixed solvent thereof. The pharmaceutical composition of claim 1, wherein the atamoya leaf extract is obtained by extraction with ethanol. The pharmaceutical composition of claim 1, wherein the fraction of the atamoya leaf extract is one or more fractions selected from the group consisting of hexane, ethyl acetate, butanol and water fractions. The pharmaceutical composition of claim 1, wherein the fraction is an ethyl acetate fraction. The pharmaceutical composition of claim 1, wherein the fraction is a saturated butanol fraction. The pharmaceutical composition of claim 1, wherein the atamoya leaf extract or fractions thereof have acetylcholine degrading enzyme inhibition, amyloid beta aggregation inhibition, antioxidant and neuronal cell protective effects. A method of preventing or treating cognitive dysfunction comprising administering to a subject a composition of any one of claims 1 to 8. Health functional food composition for preventing or improving cognitive dysfunction comprising atemeya leaf extract or fractions thereof as an active ingredient. The pharmaceutical composition of claim 10, wherein the cognitive dysfunction is a degenerative brain disease selected from the group consisting of forgetfulness, Alzheimer's disease, dementia, Parkinson's disease, peak disease, and Huntington's disease. Feed composition for the prevention or improvement of cognitive dysfunction, including atemonia leaf extract or fractions thereof.

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