KR20220076751A - Anti-oxidant composition comprising the extract of flower apple skk14 as an effective component - Google Patents

Abstract

The present invention relates to an antioxidant composition comprising, as an active ingredient, an extract of a flower apple of the SKK14 variety ( Malus prunifolia Wild. Borkh), and more specifically, the extract of a flower apple of the SKK14 variety as an active ingredient It relates to a pharmaceutical composition having strong DPPH anion scavenging ability, ABTS cation scavenging ability, nitrite scavenging ability and reducing power, and an antioxidant composition for use as a health functional food or functional cosmetic composition. As an active ingredient of the antioxidant composition of the present invention, the flower apple extract of SKK14 cultivar exhibits very strong scavenging ability against active anions, active cations and nitrite, as demonstrated by the Examples of the present specification, and exhibits excellent reducing power at the same time. The active ingredient is processed into various forms such as extracts, powders, pills, and tablets, and is a very useful invention to be applied to pharmaceutical compositions, health functional foods, or functional cosmetic compositions.

Description

Antioxidant composition containing extracts of flowering apples of SK14 varieties as active ingredients {ANTI-OXIDANT COMPOSITION COMPRISING THE EXTRACT OF FLOWER APPLE SKK14 AS AN EFFECTIVE COMPONENT}

The present invention relates to an antioxidant composition comprising, as an active ingredient, an extract of a flower apple of the SKK14 variety ( Malus prunifolia Wild. Borkh), and more specifically, the extract of a flower apple of the SKK14 variety as an active ingredient It relates to a pharmaceutical composition having strong DPPH anion scavenging ability, ABTS cation scavenging ability, nitrite scavenging ability and reducing power, and an antioxidant composition for use as a health functional food or functional cosmetic composition.

The human body that breathes oxygen cannot avoid oxidative stress, and this oxidative stress is caused by the accumulation of reactive oxygen species. That is, in the case of conversion to water (H ₂ O) by the reaction of the final electron acceptor with oxygen during the metabolic process of organic matter, cytotoxicity does not appear, but hydrogen peroxide (H ₂ O ₂ ), superoxide (O2·) due to an abnormal reaction When , hydroxy radicals (OH·), etc. are generated, the activated oxygen species maintain a high concentration of radicals in the cell, causing protein or DNA damage, and various oxidative stresses, which eventually damage the cell structure and destroy the cells. Loss of function or deterioration (Choi et al. 2008. J. Korean Soc. Food Sci. Nutr. 37: 276-281).

In addition, recent research results show that oxidative stress is directly related to thrombus formation associated with cardiovascular disease (Bijak M et al., 2012, Thrombosis Res. 130, 123-128), and intravascular fibrinogen ( fibrinogen) is known to promote thrombus formation and inhibit blood flow to induce thrombosis (Martinez M et al., 2013. Free Radical Biol. Med. 65, 411-418). According to a recent report, it has been confirmed that oxidative stress in vivo is associated with carcinogenesis, aging, dementia, inflammation, and various cardiovascular diseases (Zhang et al., 2013. Chines J. Cancer biotheraphy, 20. 153). -158; Cho Min-hyun, 2014. J. Korean Soc. Pediatr. Nephrol. 18: 7-12: Shin Jeong-hee et al., 2012. Journal of the Korean Society of Industry-Academic Technology 13: 3561-3569). Therefore, it is judged that, while ensuring stability, natural ingredients capable of removing various active radicals can suppress oxidative stress in cells, and thus considerable help can be expected.

Until now, substances capable of scavenging various reactive oxygen species and radical substances have been developed as antioxidants, representatively vitamin C (vitamin C), vitamin E (vitamin E), BHT (butylated hydroxy toluene) and BHA (butylated hydroxy). anisole) are used as food additives, health functional foods, and pharmaceuticals. However, side effects of BHT and BHA, which are synthetic antioxidants showing strong antioxidant activity, have been reported (Ha, Hyeon-Ju et al., 2014. Korean J. Culinary Res. 20: 16-26; Choe and Yang, 1982. Korean J. Food Sci. Technol. 14 : 283-288), it is necessary to develop natural antioxidants that can replace synthetic antioxidants. In particular, there is an urgent need to develop natural antioxidants with excellent heat resistance that can replace very unstable substances that are weak to heat such as vitamin C ( Ahn et al., 2009. Korean J. Microbiol. Biotechnol. 37: 266-272; Kim et al., 2009. Korean J. Microbiol. Biotechnol. 37: 133-139).

On the other hand, flowering apple is a deciduous broad-leaved tree belonging to the genus Malus of the Rosaceae family, and its origin is Europe, Asia, and North America. Flowering apples grow very well even in barren land, and they are used as rootstock for apple trees that are difficult to self-fertilize because they bloom a large amount of flowers at once.

The fruits of flowering apples are much smaller than normal apples and are rich in fructose, glucose, tartaric acid, and vitamin C, which is said to be good for maintaining health, recovering from fatigue, and for constipation. They are not used, and most of them fall naturally and are discarded due to lack of verification power. In folklore, it was also used as a meat tenderizer to soften meat when cooking, and it was also used medicinally because it was effective in relieving fatigue, increasing appetite, nervousness, insomnia, and constipation when soaked in alcohol. In oriental medicine, dried fruits are used for dry stomach, indigestion, anorexia, gastric acid deficiency or hyperacidity, and are known to be used as medicines for diarrhea, menstrual pain, frostbite, dry stomach, back pain, and intestinal bleeding.

Flowering apples do not have any genetic barriers, so they are characterized by free interspecies or intraspecies hybridization, and over 700 varieties have been developed worldwide. Flower color is white, light pink, pink, dark pink, crimson, etc. It blooms in April for 8 to 12 days, and most of the fruits are known to be less than 2.5 cm in diameter (So Dong-young. 2011, Konkuk University Master's thesis) ). In addition, flower apples have strong cold resistance, so they have excellent environmental adaptability, and they grow well in poor soil. In the case of apple trees, the flowering time is different for each variety, and there are differences in diseases and pests and cultivation methods, so pollination is required for each cultivated variety. , Floret, SKK-14, and SKK-16.

Usually, in the folklore, the fruit of the hawthorn (Chinese hawthorn Crataegus cuneata Sieb, Western hawthorn Crataegus oxyacantha L.) is similar to a flowering apple and is often called confusedly, but this should be distinguished.

As a study related to flowering apples, selection of flowering apples suitable for pollination by apple variety (Kang In-gyu et al., 2002. Journal of Horticultural Science and Technology 20: 330-334; Son Kwang-min et al., 2013. Protected Horticulture and Plant Factory, 22: 116- 122), research on the reasons why it grows well in poor soil (Huang L. et al., 2018, Plant Physiol Biochem. 127:185-193; Tan Y et al., 2017, Plant Physiol Biochem. 115: 219-228) is mainly conducted It has been reported that the anthocyanin pigment of flowering apples has excellent light resistance and heat resistance, making it easy to use as a natural red pigment resource (Kim Yong-hwan, 1999, Korean J. Food Nutr. 12: 85-90). However, to date, studies on useful components and useful physiological activities of flowering apples are very limited.

Recently, it has been reported that the polyphenol and flavonoid components of flower apples are about twice that of regular apples, and it is rich in tartaric acid and vitamin C (So Dong-young. 2011, Konkuk University master's thesis), and it has more gallic acid and protocatechuic acid than regular apples. It has been reported that the content of phenolic acids such as acid, chlorogenic acid, p-coumaric acid, and ferulic acid is high, and the content of flavonoids such as quercetin and myricetin is also high, indicating superior antioxidant activity than apples (KM Maria John et al., 2014. Food Chemistry 163: 46-50). Also, recently, flowering apples Malus 'Red jade', Malus hupehensis (Pamp.) Rehd. And Malus prunifolia (Willd.) citric acid, p-coumaric acid, hyperoside, myricetin, naringenin, quercetin, kampferol, gentiopicroside, ursolic acid and 8-epilganic acid were isolated from three Borkh species, and their cholesterol-lowering and hyperlipidemic prevention effects has been reported (Chao Wen et al., 2018. J. Pharmaceutical Biomedical Analysis 150: 144-151). However, until now, the strong antioxidant activity of flower apples, particularly, flower apple extracts of SKK14 cultivar was not known.

As patents related to flowering apples, Korean Patent Registration No. 10-1081059, [Flower mixed extract having antioxidant and whitening activity, extraction method thereof, and cosmetic composition containing the flower mixed extract] and Japanese Patent JP-0137455 [ Production of nursery stock of pear tree] has been disclosed, but there are no patents or research results for active extracts with strong antioxidant activity and non-toxic activity of flower apple extract of SKK14 cultivar.

K. M. Maria John et al., 2014. Food Chemistry 163: 46-50. Metabolic variation and antioxidant potential of Malus prunifolia (wild apple) compared with high flavon-3-ol containing fruits (apple, grapes) and beverage (black tea)

The present invention has been devised to solve the problems of the prior art as described above, and an object to be solved in the present invention is to provide an antioxidant composition containing a flower apple extract of SKK14 variety as an active ingredient.

In order to solve the above problems, the present invention contains the SKK14 variety of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient for preventing or preventing a disease selected from the group consisting of cancer, dementia, inflammation and cardiovascular disease. A therapeutic pharmaceutical composition is provided.

The extract is preferably a hot water extract.

In addition, the present invention provides a health functional food for antioxidants containing SKK14 cultivar of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The extract is preferably a hot water extract.

In addition, the present invention provides a cosmetic composition for antioxidants containing SKK14 cultivar of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The extract is preferably a hot water extract.

As an active ingredient of the antioxidant composition of the present invention, the flower apple extract of SKK14 cultivar exhibits very strong scavenging ability against active anions, active cations and nitrite, as demonstrated by the Examples of the present specification, and exhibits excellent reducing power at the same time. The active ingredient is processed into various forms such as extracts, powders, pills, and tablets, and is a very useful invention to be applied to pharmaceutical compositions, health functional foods, or functional cosmetic compositions.

1 shows flowering apples of immature SKK14 variety at 70 days after flowering.
2 shows flowering apples of the mature SKK14 variety at 130 days after flowering.

Hereinafter, the present invention will be described in detail.

In order to test the antioxidant efficacy of flower apples, the inventors of the present invention harvested flower apples of various varieties to remove foreign substances, prepared them as extracts by a certain method, and evaluated the antioxidant activity of the extracts. As a result, very strong DPPH anion scavenging ability, ABTS cation scavenging ability, nitrite scavenging ability and reducing power were confirmed in the flower apple extract of SKK14 variety. It was attempted to utilize the flower apple extract of SKK14 cultivar as an antioxidant composition by confirming that it has

Specifically, the present inventors harvest immature and mature flowering apples of the SKK14 variety cultivated in Korea in order to develop an antioxidant composition using flowering apples used for various purposes in the private sector, and then prepare an extract for them and , As a result of evaluating the scavenging ability of these extracts against various active radicals, it was confirmed that the immature and mature flower apple extracts of SKK14 cultivar showed strong antioxidant activity. More specifically, in the case of immature flowering apples of SKK14, the RC ₅₀ of DPPH anion scavenging activity, ABTS cation scavenging activity, and nitrite scavenging activity were 106.5, 38.5, and 20.4 μg/ml, respectively, and in the case of mature flowering apples, 117.4, It was found to be 54, 6, and 93.9 μg/ml, confirming that it has about 5 to 10 times stronger antioxidant power compared to the Fuji variety, which is an ordinary apple. In addition, in the reducing power evaluation, at the concentration of 0.5 mg/ml, it was 0.965 for immature flower apples and 0.663 for mature flower apples, indicating stronger reducing power than general apples and flower apples of other varieties. In addition, it was confirmed that the flower apple extract of SKK14 cultivar did not show hemolytic activity against human red blood cells and did not cause acute toxicity, confirming that it has a very high practical use as an antioxidant in clinical practice.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating a disease selected from the group consisting of cancer, dementia, inflammation, and cardiovascular diseases, containing the extract of flower apples of SKK14 variety ( Malus prunifolia Wild. Borkh) as an active ingredient do.

The extract is preferably a hot water extract.

In addition, the present invention provides a health functional food for antioxidants containing SKK14 cultivar of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The extract is preferably a hot water extract.

In addition, the present invention provides a cosmetic composition for antioxidants containing SKK14 cultivar of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The extract is preferably a hot water extract.

Hereinafter, the manufacturing method and efficacy experiment of the flower apple extract of the SKK14 variety of the present invention will be described in more detail.

The present invention comprises the steps of harvesting immature and mature flowering apples of SKK14 variety, and removing foreign substances; Chopping the flower apples and preparing an extract; evaluation step of antioxidant activity of flower apple extract; and a step of examining the stability of the active extract.

The "flowered apple of cultivar SKK14" of the present invention may be an immature fruit in which ovaries are not produced for less than 70 days after flowering, and mature fruits in which ovaries are formed after 130 days or more after flowering. The "SKK14" variety is a variety registered as a variety name registration number 03-0001-151 as of October 15, 2014 by applying for a variety name registration to the National Seed Resources of the Republic of Korea.

The "flower apple extract of SKK14 variety" contained in the composition of the present invention comprises the steps of crushing immature or mature flower apples of SKK14 variety; extracting the pulverized flower apple with an organic solvent; Filtering the extract using a filtration network of 0.06 mm or less; and concentrating it under reduced pressure.

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, ethyl acetate, etc.), a mixed solvent of the lower alcohol and water etc. can be used, and hot water or 95% ethanol extraction is most preferred.

The flower apple extract of SKK14 cultivar of the present invention may be prepared into powder through a conventional powdering process such as drying under reduced pressure, freeze drying, or spray drying. They are not degraded by various degrading enzymes in plasma, and remain active even at 100°C heat treatment and pH 2 in the human stomach.

The active ingredient of the present invention can be used as a pharmaceutical composition for preventing or treating diseases related to oxidative stress. The diseases may be, for example, cancer, dementia, inflammation and cardiovascular disease, wherein the cardiovascular disease is myocardial infarction, chest pain, shortness of breath, loss of consciousness, stroke, angina, hypertension, headache, dyskinesia, sensory Abnormality, decreased visual acuity, numbness pain, etc. are mentioned.

The pharmaceutical composition comprising the active ingredient of the present invention can be prepared according to a conventional method according to the purpose of use. Oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injection solutions It can be formulated and used in various forms, such as oral administration, or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

The pharmaceutical composition may further include a carrier, excipient or diluent, and the like, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. and the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may be used.

As a preferred embodiment, the oral liquid formulation may include suspensions, solutions, emulsions, syrups, etc., and various excipients, for example, wetting agents, sweetening agents, in addition to water and liquid paraffin, which are commonly used simple diluents, Perfumes, preservatives, and the like may be included.

As a preferred embodiment, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents, suppositories, etc. can be exemplified as formulations for parenteral administration. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight daily Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject through various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

In the present invention, the "subject" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. and preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways, such as antioxidant functional food and beverage. The active ingredient of the present invention can be used for prevention or improvement of diseases related to oxidative stress. The diseases may be, for example, cancer, dementia, inflammation and cardiovascular disease, wherein the cardiovascular disease is myocardial infarction, chest pain, shortness of breath, loss of consciousness, stroke, angina, hypertension, headache, dyskinesia, sensory Abnormalities, blurred vision, chest pain, etc. are mentioned.

Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, tea, vitamin complexes, health supplements, and the like, and may be used in powder, granule, tablet, capsule or beverage form. .

In general, the active ingredient of the present invention may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition may be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

The health functional food of the present invention may contain, as an additional ingredient, in addition to containing the above compound as an essential ingredient in the indicated ratio, a food pharmaceutically acceptable food supplement additive, such as natural carbohydrate and various flavoring agents.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and common sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

As the flavoring agent, a natural flavoring agent such as stevia, such as taumatine, rebaudioside A, or glycyrrhizin, and a synthetic flavoring agent such as saccharin and aspartame may be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice, and fruit for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

The active ingredient of the present invention can be used as a functional cosmetic composition for antioxidant use. Effects according to the antioxidant activity, for example, may include pigmentation improvement, whitening effect, wrinkle improvement, skin elasticity improvement, skin aging prevention, and the like.

The active ingredient of the cosmetic composition of the present invention may be included in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, and more preferably 1 to 10% by weight based on the total weight of the composition. In this content range, appropriate formulation stability can be ensured, and a desired antioxidant effect can be expected.

In addition to the active ingredient of the present invention, the composition of the present invention can be used by additionally including known natural extracts or other ingredients for the effects of antioxidant, skin aging prevention and skin regeneration promotion within the range that does not inhibit the effective activity of the present invention. have.

In addition, the composition may contain ingredients commonly added to cosmetic compositions, for example, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetics, such as fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives; It may be molded into a specific formulation including an adjuvant or carrier commonly used in the field of dermatology.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, pack, soap, surfactant- It may be formulated as containing cleansing, oil and spray, but is not limited thereto. More specifically, flexible lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, pact, lip gloss It can be prepared in formulations such as rose, lipstick, shadow, shampoo and conditioner.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used.

Hereinafter, the present invention will be described in more detail through examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example 1: Preparation of flower apples and common apple extracts and analysis of components of the extracts

In 2017, flower apples of SKK14 cultivar and general apples of Miami Fuji cultivar cultivated and harvested at the Agricultural Technology Center in Cheongsong-gun, Gyeongsangbuk-do were collected 70 days after flowering and 130 days after flowering to remove foreign substances, shredded, and extracted with hot water. was prepared. Information on flower apples and general apples used in the experiment is shown in Table 1 and FIG. 1 . SKK14 is used as pollination water for late-flowering apple cultivars (Fuji, Hwahong). The immature flower apple SKK14 used was 0.7 cm in size, whereas the normal green apple was 3.5 cm, which was more than 5 times the size, and the mature flower apple SKK14 was On the other hand, the size of a mature normal apple is 6.1 cm, about 8 times the size of 0.8 cm.

[Table 1] Size and characteristics of flowering apples and regular apples

On the other hand, the water content and physicochemical properties of the flower apples and ordinary apples used and the color difference of the extract obtained by double hydrolysis are shown in Table 2.

[Table 2] Physicochemical properties and color difference of flowering apples and ordinary apples

As shown in Table 2, in the case of flowering apples, as the moisture content decreased from 67.2% (70 days after flowering) to 60.6% (130 days after flowering) as they mature, the increase in brix and acidity was insignificant. , as the moisture content increased from 80.4% (70 days after flowering) to 87.1% (130 days after flowering) as it matured, brix and acidity increased significantly. In addition, as a result of color difference analysis of the hot water extract, both flower apples and regular apples showed increases in brightness and yellowness and decreases in redness as they mature.

On the other hand, distilled water 20 times the weight of flower apples and ordinary apples was added, extracted once at 100 ° C. for 1 hour, the extracts were collected, filtered, and concentrated under reduced pressure to prepare a powder to prepare hot water extracts, respectively. The extraction efficiency and the results of component analysis of the extract are shown in Table 3. The content of total polyphenols, total flavonoids, total sugars and reducing sugars was determined by component analysis. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample solution, and the absorbance was measured at 725 nm after standing at room temperature for 1 hour. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample solution, 40 μl of 1 N NaOH was added again, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. . As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 3] Extraction efficiency and useful component analysis of hot water extracts of flower apples and general apples

As shown in Table 3, the extraction efficiency of flowering apples was lower than that of Fuji apples, and in particular, in the case of ordinary apples, the extraction efficiency increased as they mature, whereas in the case of flowering apples SKK14, the extraction efficiency of mature fruits decreased compared to immature fruits. was shown. Therefore, in the case of SKK14, it is presumed that many flowers bloom and bear fruit at the beginning, and then mature into a hard form without major changes and finally fall naturally.

On the other hand, in the case of immature flower apple SKK14 extract, the polyphenol content of 150.0 mg/g was shown, which was 2.3 to 11.7 times higher than that of mature flower apples, green apples, and mature apples. In addition, the flavonoid content of SKK14 extract from immature flower apples was also very high, 2.7 to 9.1 times higher than those of mature flower apples, green apples, and mature apples. However, the total sugar and reducing sugar contents were relatively low, and the acidity was about twice that of apples. Therefore, it was confirmed that immature and mature flower apple SKK14 has low functionality, making it difficult to use for food, and it is desirable to develop it as a functional material.

Example 2: Antioxidant activity of flower apple and common apple extract

The antioxidant activity of the flower apple and common apple extracts of Example 1 was evaluated, and the extract was dissolved in DMSO (dimethylsulfoxide) and diluted to an appropriate concentration to obtain DPPH (1,1-diphenyl-2-picryl hydrazyl) anion radical scavenging ability, ABTS [ 2,2-azobis (3-ethylbenzo thiazoline-6-sulfonate)] was used to measure cation radical scavenging ability, nitrite scavenging ability and reducing power. First, in the case of DPPH scavenging ability, 380 μl of a 2x10 ^-4 M DPPH solution dissolved in 99.5% ethanol was added to 20 μl of a sample diluted to various concentrations, mixed and reacted at 37° C. for 30 minutes, followed by a microplate reader (Asys Hitech, The absorbance was measured using Expert96, Asys Co., Austria). The DPPH radical scavenging ability was expressed as a percentage of the sample-added group and non-additive group. In the case of ABTS scavenging activity, 5ml of 7mM ABTS (Sigma Co., USA) and 88ml of 140mM potassium persulfate were mixed and light was blocked at room temperature for 16 hours to form ABTS cations. It was diluted with ethanol. 190 ml of the prepared diluted solution and 10 ml of samples prepared at various concentrations were mixed, reacted at room temperature for 6 minutes, absorbance was measured at 734 nm, and the ABTS radical scavenging ability was calculated by the following equation.

On the other hand, in the case of measuring nitrite scavenging ability, the sample solution was added to a nitrite solution (1 mM), and 0.1N HCl was added thereto to adjust the pH to 1.2, and after reacting at 37°C for 1 hour, Griess reagent (Sigma Co., USA) was added and mixed. After leaving at room temperature for 15 minutes, absorbance was measured at 520 nm to measure the amount of remaining nitrite. The nitrite scavenging ability (%) was calculated by the following formula.

In the antioxidant experiment, vitamin C (Sigma Co., USA) was used as a control, and DMSO was used as a solvent control. Each activity evaluation was expressed as the mean and deviation of each repeated experiment three times.

On the other hand, in the case of reducing power evaluation, it was measured by modifying the method of Oyaizu et al. (Sunmi Ahn et al., 2011. J. Life Sci. 21: 576-583). 2.5ml of 0.2M sodium phosphate buffer (pH 6.6) and 2.5ml of 10% potassium ferricyanide were added to 2.5ml of sample dissolved in ethanol, and after reacting at 50℃ for 20 minutes, 2.5ml of 10% trichloroacetic acid was added to terminate the reaction and centrifuged at 4000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was diluted two-fold with distilled water, mixed with a freshly prepared 0.1% ferric chloride solution at a ratio of 5:1 (v/v), and absorbance was measured at 700 nm to evaluate. The measurement results of each antioxidant power are shown in Table 4.

[Table 4] Antioxidant activity of hot water extracts of flower apples and ordinary apples

As shown in Table 4, the antioxidant activity was evaluated by using the flower apple extract of the SKK14 variety and the Fuji apple extract at a concentration of 0.5 mg/ml. As a result, the flower apple extract of the SKK14 variety showed excellent activity, especially the immature flower apple. The extract was the strongest. This phenomenon was also found in the ethanol extract of flower apples of SKK14 varieties, indicating that flower apples SKK14 varieties contain a large amount of antioxidants.

Therefore, the antioxidant activity was evaluated by varying the concentrations of each flower apple and Fuji apple extract, and the results are shown in Table 5.

[Table 5] Antioxidant activity of flower apple extract and general apple extract (concentration evaluation showing 50% active radical scavenging ability)

As shown in Table 5, the flower apple extract of SKK14 cultivar has 4 to 12 times better DPPH anion scavenging ability, 4.5 to 8 times better ABTS cation scavenging ability, 5 to 20 times stronger nitrite scavenging ability, and 2 to 10 times better reducing power than Fuji apple. , and the immature flower apple extract showed very strong ABTS cation scavenging activity, nitrite scavenging activity, and priming power compared to the mature flower apple extract. The above results suggest that it is possible to use the flower apple extract of SKK14 variety, particularly the immature flower apple extract, as a commercial antioxidant composition.

Example 3: Human hemolytic activity of flower apple and common apple extract

Flowered apples have been processed and eaten for a long time into jam, vinegar, and alcoholic beverages. In addition, apples are widely used for food as raw fruits and processed foods. In the present invention, human erythrocyte hemolytic activity was evaluated to evaluate the acute toxicity of immature flower apple extract of SKK14 cultivar, and the results are shown in Table 6. At this time, hemolytic activity was evaluated according to an existing report (Miseon Kim et al., 2014. J. Life Sci. 24: 515-521), and 100 μl of human red blood cells washed three times with PBS was simply added to a 96-well microplate and various After adding 100 μl of the sample solution of the concentration, the reaction solution was reacted at 37° C. for 30 minutes. Then, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. did DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 6] Human erythrocyte hemolytic activity of flower apple and common apple extract

As a result, as shown in Table 6, first, DMSO and water used as controls had no hemolytic activity, and it was confirmed that triton X-100 hemolyzed red blood cells 100% at a concentration of 1 mg/ml. On the other hand, both flower apple and common apple extract had no hemolytic activity regardless of maturity. Therefore, it is judged that the flower apple extract of SKK14 cultivar will not show a separate problem of causing acute toxicity.

Example 4: Evaluation of Plasma, Acid and Thermal Stability of Flower Apple Extract of SKK14 Varieties

Plasma stability, thermal stability, and acid stability with respect to antioxidant activity were confirmed for the flower apple extract of SKK14 cultivar obtained in Example 1 above. The samples showed a significant decrease in antioxidant activity of DPPH anion scavenging activity, ABTS cation scavenging activity, and nitrite scavenging activity even after 1 hour heat treatment at 100° C., 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. It showed high stability. Therefore, the above extract is expected to maintain excellent antioxidant activity during digestion and absorption process and food manufacturing process.

Claims (4)

A pharmaceutical composition for the prevention or treatment of diseases selected from the group consisting of cancer, dementia, inflammation and cardiovascular diseases, containing as an active ingredient a flower apple extract of SKK14 variety ( Malus prunifolia Wild. Borkh). The pharmaceutical composition according to claim 1, wherein the extract is a hot water extract. A health functional food for antioxidants containing the active ingredient according to claim 1 or 2. A cosmetic composition for antioxidants containing the active ingredient according to claim 1 or 2.

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