KR20200038691A - Anti-oxidant composition comprising the extract of an unripe apple as an effective component - Google Patents

Abstract

The present invention relates to an antioxidant composition containing an unripe apple extract as an effective component, and more specifically, to an anti-oxidant composition which contains an extract of unripe Alps otome apples as an effective component and has potent DPPH anion scavenging activity, ABTS cation scavenging activity, nitrite scavenging activity, and reducing ability, wherein the antioxidant composition is for use in a pharmaceutical composition, a health functional food, or a functional cosmetic composition. The extract of unripe Alps otome apples as the effective component of the antioxidant composition of the present invention, as demonstrated in embodiments in the present specification, exhibits extremely potent scavenging activity with respect to active anions, active cations, and nitrite, and at the same time, exhibits excellent reducing ability. The effective component may be processed in various forms such as extract, powder, pills, tablets and the like and applied for use in pharmaceutical compositions, health functional food, or functional cosmetic compositions, and the like.

Description

Antioxidant composition containing green apple extract as an active ingredient {ANTI-OXIDANT COMPOSITION COMPRISING THE EXTRACT OF AN UNRIPE APPLE AS AN EFFECTIVE COMPONENT}

The present invention relates to an anti-oxidant composition containing an unripe apple extract as an active ingredient, and more specifically, a powerful DPPH containing an Alps otome variety of green apple extract as an active ingredient. Anionic scavenging ability, ABTS cation scavenging ability, Nitrite scavenging ability and reducing power, and relates to an antioxidant composition for use in a health functional food or functional cosmetic composition.

The oxygen breathing human body cannot avoid oxidative stress, and this oxidative stress is caused by the accumulation of active oxygen species. That is, in the metabolic process of organic matter, when the final electron acceptor and oxygen react and convert to water (H ₂ O), cytotoxicity does not appear, but hydrogen peroxide (H ₂ O ₂ ), superoxide (O2 ·) by abnormal reaction When hydroxy radicals (OH ·) are generated, activated oxygen species maintain a high radical concentration in the cells, causing protein or DNA damage, and inducing various oxidative stresses, resulting in cell structure damage and cell damage. Loss of function or alteration (Choi et al .. 2008. J. Korean Soc. Food Sci. Nutr. 37: 276-281).

In addition, recent studies have shown that oxidative stress is directly related to thrombus production associated with cardiovascular disease (Bijak M et al., 2012, Thrombosis Res. 130, 123-128), and fibrinogen in blood vessels caused by oxidative stress ( It is known that structural changes of fibrinogen can promote thrombosis and inhibit blood flow, leading to thrombosis (Martinez M et al., 2013. Free Radical Biol. Med. 65, 411-418). In fact, recent reports have 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 Hee Shin et al., 2012. Journal of the Korean Academy of Industrial Science and Technology 13: 3561-3569). Therefore, while stability is secured, it is considered that a natural product component capable of removing various active radicals can suppress oxidative stress of the cell, and thus, it is expected that considerable help can be expected.

To date, various active oxygen species and substances capable of scavenging radical substances have been developed as antioxidants, and are typically vitamin C (vitamin C), vitamin E (vitamin E), butylated hydroxy toluene (BHT) and butylated hydroxy (BHA). anisole) are used as food additives, dietary supplements and medicines. However, side effects of BHT and BHA, synthetic antioxidants showing strong antioxidant power, have been reported (Hyun-Joo Ha, 2014. Korean J. Culinary Res. 20: 16-26; Choe and Yang, 1982. Korean J. Food Sci. Technol. 14 : 283-288) As it became necessary to develop natural antioxidants that can replace synthetic antioxidants, in particular, the development of natural antioxidants with excellent heat resistance to replace very weak and very unstable substances such as vitamin C is in desperate condition ( 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, apples are representative fruits with high taste and flavor, and are the most popular crops in the entire fruit-growing area since Korea has a temperature suitable for growing apples and has many effective slopes. In particular, green apples have various physiological activities because they contain 10 times or more of polyphenols compared to mature apples. Specifically, they are rich in potassium and quercetin, which show a blood cholesterol reduction effect, and prevent aging and protect the lungs from harmful air. The effect is known, and it is known that it is rich in vitamins B and C, which not only helps skin health, but also relieves constipation and restores cancer and fatigue.

Research related to apples mainly relates to food development targeting mature apples and various bioactive substances contained in apples, and in relation to green apples, it is limited to some studies to use green apples as food ingredients, for example, Quality characteristics of bread with added green apple powder (Bong-Hyun Park, Master's Thesis, Graduate School of Medicine and Food, Chung-Ang University, 2017) and fermentation studies using green apples from Mungyeong (Kwon Sun-Koo et al., Korea Institute of Industrial Convergence, 2016). In addition, as a patent document related to green apple, for example, Republic of Korea Patent Registration No. 10-1194767 "Cosmetic composition for inhibiting pore contraction and sebum secretion containing green apple extract", Republic of Korea Patent Publication No. 10-2018-0075802 Green apple powder manufacturing method ", Republic of Korea Patent Publication No. 10-2018-0065352" green vinegar using green apple "and the like are disclosed.

KR 10-1194767 B KR 10-2018-0075802 A KR 10-2018-0065352 A

 Bong-Hyeon Park, Quality Characteristics of Bread with Green Apple Powder, Master's Thesis, Graduate School of Food and Drug Administration, Chung-Ang University, 2017  Kwon, Soon-gu et al., Fermentation research using Mungyeong green apple, Proceedings of the Korean Society for Convergence of Industry, 2016

The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to provide an antioxidant composition containing an green apple extract, and more specifically, an green apple extract of the Alpine automedium as an active ingredient. Is what you want.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases selected from the group consisting of cancer, dementia, inflammation, and cardiovascular disease, which contains the green apple extract of the Alpine Otto variety as an active ingredient. do.

In addition, the present invention provides an antioxidant health functional food containing the green apple extract of the Alpine Otome variety as an active ingredient.

In addition, the present invention provides an antioxidant cosmetic composition containing the green apple extract of the Alpine Otome variety as an active ingredient.

As an active ingredient of the antioxidant composition of the present invention, the green apple extract of the Alpine automedium variety exhibits a very strong scavenging ability against active anions, active cations and nitrite, and proves excellent reducing power at the same time, as demonstrated through the examples of the present specification. The active ingredients are processed in various forms such as extracts, powders, pills, tablets, etc., and are very useful inventions to be applied to pharmaceutical compositions, health functional foods, or functional cosmetic compositions.

Figure 1 shows a green apple of the Alps Otome variety,
Figure 2 shows a green apple of the Miyama Fuji variety,
Figure 3 shows the green apple of the crimson varieties,
Figure 4 shows a green apple of the summering varieties,
Figure 5 shows the green apple varieties of green apples.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, in order to test the anti-oxidation efficacy against green apples, harvested green apples of various varieties to remove foreign substances, prepared them as extracts in a certain way, and evaluated the antioxidant activity of the extracts. As a result, Alpine Ottome varieties of green apple extract very strong DPPH anion scavenging ability, ABTS cation scavenging ability, Nitrite scavenging ability and reducing power was confirmed, and the extract does not show hemolytic activity against human red blood cells, but has excellent thermal stability and acid stability. By confirming that it has a try to use the green apple extract of the Alpine Otome variety as an antioxidant composition.

Specifically, the present inventors prepared a hot water extract and an ethanol extract for 5 types of green apple varieties grown in Korea, respectively, in order to develop an antioxidant composition using green apples of various varieties, and various activities were targeted to these extracts As a result of evaluating the scavenging ability of the radicals, it was confirmed that it exhibits strong antioxidant activity in the green apple extract of the Alpine automedium variety. More specifically, the hot-water extract from the putsagwa of Alps Automation varieties, after evaluating the antioxidant activity at various concentrations, as a result of calculating the RC ₅₀ represents the 50% scavenging activity, DPPH anion scavenging activity, ABTS cation scavenging, the nitrite scavenging RC ₅₀ These were 235.7, 84.6, and 44.8 μg / ml, respectively, and the ethanol extracts were 246.2, 105.7, and 79.9 μg / ml, respectively, and were confirmed to have about 2 to 4 times stronger antioxidant power than other types of green apples. . In addition, even in the evaluation of reducing power, at a concentration of 0.5 mg / ml, the hot water extract was 0.639 and the ethanol extract was 0.677, indicating a stronger reducing power than other types of green apples. In addition, it was confirmed that the green apple extract of the above Alpine automedium does not exhibit hemolytic activity against human red blood cells and does not cause acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases selected from the group consisting of cancer, dementia, inflammation and cardiovascular disease, which contains the green apple extract of the Alpine Autome variety as an active ingredient.

In addition, the present invention provides an antioxidant health functional food containing the green apple extract of the Alpine Otome variety as an active ingredient.

In addition, the present invention provides an antioxidant cosmetic composition containing the green apple extract of the Alpine Otome variety as an active ingredient.

Hereinafter, the production method and efficacy test of the green apple extract of the Alpine Otome variety of the present invention will be described in more detail.

The present invention harvesting various types of green apples, removing foreign matter; Shredding green apples and preparing an extract; Evaluation of antioxidant activity of green apple extract; And a stability investigation step of the active extract.

"Foot apple" of the present invention means an immature apple in which seeds and seeds are not made within 70 days after flowering. In the present invention, Alpine Otome varieties, as well as Miyama Fuji, crimson, summering, and Hongro varieties of green apples were used. The "Alps Autome" varieties are varieties registered as a breed name registration number 03-0001-151 on October 15, 2014, and filed with the National Species Resource of Korea. The "Miyama Fuji" varieties are varieties registered as a breed name registration number 03-0001-80 on March 16, 2005, when the breed name registration was filed with the National Species Resource of Korea. The "crimson" varieties are varieties registered as a breed name registration number 03-0001-122 as of July 20, 2010, and filed with the National Species Resource of the Republic of Korea. The "Summer King" varieties are varieties registered as a breed name registration number 03-0001-124 on March 23, 2011. The "Hongro" varieties are varieties registered as a breed name registration number 03-0001-60 as of December 31, 1997, which was filed with the National Species Resource of the Republic of Korea.

The step of crushing the green apple of the Alps automembrane is included in the composition of the present invention. Extracting the green apple crushed material with an organic solvent; Filtering the extract using a filter 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 alcohols 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 green apple extract of the Alpine automedium variety of the present invention can be prepared as a powder through a conventional powdering process such as vacuum drying, freeze drying or spray drying. They are not degraded by various degrading enzymes in plasma, and they maintain activity even at a heat treatment of 100 ° C and a pH of 2 in the human stomach.

The active ingredient of the present invention can be used as a pharmaceutical composition for the prevention or treatment of diseases associated with oxidative stress. The diseases may be, for example, cancer, dementia, inflammation, and cardiovascular disease, and the cardiovascular disease may be myocardial infarction, chest pain, difficulty breathing, loss of consciousness, stroke, angina, hypertension, headache, dyskinesia, sensation Abnormality, poor vision, and pain in the persimmon.

The pharmaceutical composition containing the active ingredient of the present invention is an oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, injection of sterile injectable solution according to a conventional method according to each purpose of use. It can be formulated and used in various forms, such as oral administration or intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

The pharmaceutical composition may further include a carrier, excipient or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, 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-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

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

As a preferred embodiment, a liquid preparation for oral use may be exemplified by suspensions, solvents, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives, and the like may be included.

As a preferred embodiment, the formulation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. 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 can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal 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 patient's age, sex, and weight, and generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg daily Or it can be administered every other day or divided into 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable way, and the route of administration 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 target cells.

“Subject” in the present invention includes, but is not particularly limited to, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits, or guinea pigs, for example. 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 the prevention or improvement of diseases associated with oxidative stress. The diseases may be, for example, cancer, dementia, inflammation, and cardiovascular disease, and the cardiovascular disease may include myocardial infarction, chest pain, difficulty breathing, loss of consciousness, stroke, angina pectoris, hypertension, headache, dyskinesia, sensation Abnormalities, poor vision, chest pain, and the like.

Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, and dietary supplements, and may be used in the form of powders, granules, tablets, capsules, or beverages. .

The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

The dietary supplement of the present invention may contain, as an essential ingredient in the indicated proportions, the above compound as an essential ingredient, and may additionally contain, as an additional ingredient, food additives, such as natural carbohydrates and various flavoring additives.

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

As the flavoring agent, natural flavoring agents such as stevia such as taumatin, rebaudioside A or glycyrrhizine, and synthetic flavoring agents such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used in about 1 to 20 g, preferably about 5 to 12 g per 100 ml of 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, synthetic flavoring agents, flavoring agents such as natural flavoring agents, coloring agents and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloids It may contain a thickening agent, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is generally selected from 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. The effects according to the antioxidant activity include, for example, improved pigmentation, whitening effect, wrinkle improvement, skin elasticity improvement, and skin aging prevention.

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

In addition to the active ingredients of the present invention, the composition of the present invention can be used by further including a known natural product extract or other ingredients for the effect of antioxidant, anti-aging, and skin regeneration in a range that does not inhibit the effective activity of the present invention. have.

In addition, the composition is a component 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 nonionic emulsifiers, fillers, metal ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives or It may be molded into a specific formulation, including adjuvants or carriers 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, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, packs, soaps, surfactants- It may be formulated as containing cleansing, oil and spray, but is not limited thereto. More specifically, flexible lotion, nutrition lotion, nutrition 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 rinse.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as a carrier component. You 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, additionally 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, Fatty acid esters of 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can 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 following examples.

[Example]

Example 1: Green apple varieties and characteristics at harvest

The green apple used in this example was obtained by apple varieties listed in Table 1 below on the 70th day after flowering, grown in the test packing of the Cheongsong County Agricultural Technology Center in Cheongsong-gun, Gyeongsangbuk-do, 2018.

[Table 1] Green apple varieties and characteristics

Table 2 below shows the weight and size of the obtained green apples.

[Table 2] Weight and size of each harvested green apple

Example 2: Comparison of physicochemical properties of green apple extract

Distilled water of twice the weight of the green apple harvested in Example 1 was added, heated at 100 ° C. for 1 hour, and filtered to prepare a green apple extract. The physicochemical properties and raw tea analysis of the prepared green apple extract are shown in Table 3 below. In the case of the faucet of the Miyama Fuji variety, the hot water extract of Example 3 below was evaluated.

[Table 3] Physicochemical properties and color difference analysis of green apple extract harvested on the 70th day of flowering

As a result, as shown in Table 3, on the 70th day of flowering, the green apple showed pH 3.4, brix 3.8-4, and acidity 0.17-0.58, and the crimson variety with a sugar / acid ratio of 6.5 had the strongest sour taste. The peak of the Miyama Fuji variety was pH 4.6, brix 1.8, and acidity 0.08. On the other hand, as a result of color difference analysis, the brightness was highest in the Miyama Fuji and Alpine Ome varieties, the redness was the highest in the crimson varieties, and the yellowness was the highest in the Hongro varieties. At this time, the color difference analysis was measured using a Hunter Color Difference meter (Super color SP-80 Colormeter, Tokyo Denshoku Co., Japan), brightness (white 100 ~ 0 black), redness (red 100 ~ -80 green), The yellowness (yellow 70-80 black) was measured. As for the chromaticity of the standard white board, the L value was set to 92.44, the a value was -0.06, and the b value was 1.35, and the average value was obtained by measuring three times per sample, and the color difference (△ E) was calculated using the following equation. Did.

Example 3: Extraction efficiency and component analysis of green apple extract

The green apples harvested on the 70th day after flowering of Example 1 were removed by type, and put into a blender to prepare green apple crushed products, and hot water extracts and ethanol extracts were prepared as targets. For the hot water extract, 10 times of distilled water was added to the green apple crushed product, and the extract was collected and filtered three times for 1 hour at 100 ° C, filtered and concentrated under reduced pressure to prepare a powder. For the ethanol extract, 10 times of ethanol was added to the green apple crushed product, and the extract was collected and filtered three times at room temperature, filtered, and concentrated under reduced pressure to prepare a powder.

The results of each extraction efficiency and component analysis of the extracts are shown in Table 4. Total polyphenols, total flavonoids, total sugars, and reducing sugars were determined by component analysis. The total polyphenol content was 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ saturated solution in 400 μl of the extracted sample solution, allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. The total flavonoid content was extracted by stirring each sample with methanol for 18 hours, adding 4 ml of 90% diethylene glycol to 400 μl of the filtered extraction sample, adding 40 μl of 1 N NaOH again, and reacting at 37 ° C. for 1 hour to 420 nm. Absorbance was measured at. Rutin was used as a standard reagent. The reducing sugar was quantified using the DNS method and the total sugar was phenol-sulfuric acid method.

[Table 4] Analysis of extraction efficiency and useful components of green apple extract and ethanol extract

As shown in Table 4, the extraction efficiency of hot water was higher than that of ethanol, and the total flavonoid, total sugar, and reducing sugar contents of the extract were similar. In particular, the total polyphenol content and total flavonoid content were high in both the hot water extract and the ethanol extract in the crimson, hongro, and alpine automation varieties.

Example 4: Antioxidant activity of green apple extract

The antioxidant activity of the green apple extract of Example 3 was evaluated, and the extract was dissolved in dimethylsulfoxide (DMSO), diluted to a suitable concentration, and DPPH (1,1-diphenyl-2-picryl hydrazyl) anion radical scavenging ability, ABTS [2,2 -azobis (3-ethylbenzo thiazoline-6-sulfonate)] It was used to measure cationic radical scavenging capacity, nitrite scavenging capacity and reducing power. First, in the case of DPPH scavenging ability, 380 μl of a 2x 10 ^-4 M DPPH solution dissolved in 99.5% ethanol was added to 20 μl of samples diluted with various concentrations, mixed and reacted at 37 ° C. for 30 minutes, followed by microplate reader (Asys Hitech at 516 nm) , Expert96, Asys Co., Austria). DPPH radical scavenging activity was expressed as a percentage of sample-added and non-added. In the case of ABTS scavenging ability, 5 ml of 7 mM ABTS (Sigma Co., USA) and 88 ml of 140 mM potassium persulfate were mixed, and the light was blocked for 16 hours at room temperature to form ABTS cations, and the absorbance value was then 1.5 at 414 nm. It was diluted with ethanol. After mixing the prepared dilution solution 190ml and 10ml of the sample prepared at various concentrations and reacting for 6 minutes at room temperature, the absorbance at 734nm was measured, and the ABTS radical scavenging ability was calculated by the following equation.

On the other hand, in the case of nitrite scavenging capacity measurement, a sample solution was added to a nitrite solution (1 mM), and 0.1N HCl was added thereto to adjust the pH to 1.2, followed by reaction at 37 ° C for 1 hour, followed by Griess reagent (Sigma Co., USA) Was added and mixed. Then, after standing at room temperature for 15 minutes, the absorbance was measured at 520 nm to measure the amount of residual nitrite. The nitrite scavenging capacity (%) was calculated by the following equation.

Each activity evaluation was expressed as the mean and the deviation of the experiment repeated 3 times each.

On the other hand, in the case of evaluating the reducing power, it was measured by modifying the method of Oyaizu et al. To 2.5 ml of the sample dissolved in ethanol, 2.5 ml of 0.2M sodium phosphate buffer (pH 6.6) and 2.5 ml of 10% potassium ferricyanide were added, reacted at 50 ° C for 20 minutes, and then 2.5 ml of 10% trichloroacetic acid was added to terminate the reaction. And the supernatant was recovered by centrifugation at 4000 rpm for 10 minutes. The recovered supernatant was diluted twice with distilled water, mixed with a freshly prepared 0.1% ferric chloride solution in a 5: 1 (v / v) ratio, and evaluated by measuring absorbance at 700 nm. Table 5 shows the results of each antioxidant measurement.

[Table 5] Evaluation of antioxidant activity of green apple extract

As shown in Table 5, as a result of evaluating antioxidant activity by adjusting the green apple extract for each cultivar to a concentration of 0.5 mg / ml, the Alpine Autome cultivars in both hot water extract and ethanol extract exhibited the best active radical scavenging activity and reducing power. In the case of reducing power, in the order of hot water extract, Alpine Otome = Crimson>Hongro> Miyama Fuji> Summering was superior, and in the case of ethanol extract, Alpine Otome> Crimson = Miyama Fuji = Hongro> Summering was excellent. On the other hand, in the case of active radical scavenging activity, all exhibit strong antioxidant capacity, and for the determination of the antioxidant power, the concentration required to eliminate the active radical by 50% by evaluating the antioxidant power of each extract at various concentrations (RC ₅₀ ) It was determined, and the results are shown in Table 6.

[Table 6] Antioxidant activity evaluation of green apple extract (concentration evaluation showing 50% active radical scavenging activity)

As a result, in the case of DPPH scavenging activity, an RC ₅₀ value of 235.7 μg / ml in the hot water extract of Alpine Otome variety green apple, and 246.2 μg / ml in the ethanol extract, and in the case of ABTS scavenging activity, 84.6 μg / ml in the hot water extract, ethanol The extract showed an RC ₅₀ value of 105.7 μg / ml, and in the case of the Nitrite scavenging ability, the RC ₅₀ value of 44.8 μg / ml in a hot water extract and 79.9 μg / ml in an ethanol extract, the Alpine automembrane extract is a green apple extract of other varieties. Compared with it was confirmed that it shows a remarkably excellent antioxidant power. Considering that RC ₅₀ values for DPPH anion scavenging ability, ABTS cation scavenging ability, and Nitrite scavenging ability of pure purified vitamin C are 11.5, 5.4, and 18.9 μg / ml, respectively, the green apple varieties of green apple extract can be used as antioxidants for various purposes. Is judged.

Example 5: Human red blood cell hemolytic activity of green apple extract

Apple is a food ingredient that has been widely edible for a long time and has no safety. In the present invention, to evaluate the acute toxicity of the green apple extract, human erythrocyte hemolytic activity was evaluated, and the results are shown in Table 7. At this time, the hemolytic activity was evaluated according to an existing report (Kim Mi-sun et al., 2014. J. Life Sci. 24: 515-521). Simply, 100 μl of human red blood cells washed three times with PBS were added to a 96-well microplate and various After adding 100 μl of the sample solution of the concentration and reacting for 30 minutes at 37 ° C., the reaction solution was centrifuged (1,500 rpm) for 10 minutes to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin outflow due to hemolysis was measured at 414 nm. Did. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg / ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 7] Human red blood cell hemolytic activity of green apple extract

As a result, as shown in Table 7, first, DMSO used as a control did not show red blood cell hemolytic activity, and triton X-100 was confirmed to hemolytically red blood cells 100% at a concentration of 1 mg / ml. On the other hand, the green apple extract had no hemolytic activity in hot water extracts and ethanol extracts of all varieties. Therefore, it is judged that the green apple extract will not exhibit the problem of causing acute toxicity.

Example 6: Evaluation of plasma, acid and thermal stability of green apple extract

For the green apple extract obtained in Example 3, plasma stability, thermal stability, and acid stability for antioxidant activity were confirmed. The samples exhibited high stability by not showing a significant decrease in antioxidant 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. Therefore, the green apple extract is expected to maintain excellent antioxidant activity during the digestion and absorption process and the food manufacturing process.

Claims (3)

A pharmaceutical composition for the prevention or treatment of diseases selected from the group consisting of cancer, dementia, inflammation and cardiovascular disease, which contains the green apple extract of the Alpine Ome variety as an active ingredient. Antioxidant health functional food containing Alpine Otome variety green apple extract as an active ingredient. Antioxidant cosmetic composition containing Alpine Otome variety green apple extract as an active ingredient.

Images