KR20200038700A - Pharmaceutical composition comprising the extract of an unripe apple as an effective component for prevention or treatment of diabetes and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating diabetes, comprising an unripe apple extract as an effective component, and more specifically, to a pharmaceutical composition and a health functional food comprising a hot-water or ethanol extract of unripe apples, for preventing or treating/alleviating diabetes through potent β-amylase and α-glucosidase inhibition activity. The unripe apple extract of the present invention as the effective component of the pharmaceutical composition and the health functional food for preventing or treating diabetes, as demonstrated in embodiments in the present specification, may be used as a medication and a health functional food for preventing or alleviating diabetes. Moreover, the unripe apple extract of the present invention has excellent thermal stability and does not show a loss in amylase inhibition activity even in acidic conditions of pH 2 or in blood plasma, and thus may be easily processed in various forms such as liquid, cream, powder, pills, tablets and the like and may be extremely usefully utilized in the drug and food industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF AN UNRIPE APPLE AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF DIABETES AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of diabetes (diabetes) containing the green apple (unripe apple) extract as an active ingredient, more specifically, the green apple or hot water or ethanol extract as an active ingredient The present invention relates to a pharmaceutical composition for preventing or treating / improving diabetes through a potent β-amylase (β-amylase) and α-glucosidase (α-glucosidase) inhibitory activity and a dietary supplement.

Diabetes, one of the most common diseases in modern people, is a type of metabolic disease such as lack of insulin secretion or normal function, and is characterized by high glucose concentration in the blood and causes various symptoms and signs due to high blood sugar. .

Diabetes is divided into type 1 and type 2, and type 1 diabetes is caused by the inability to produce insulin at all due to genetic effects, and type 2 diabetes is insulin resistance due to poor insulin function. ) Is known as the cause. In particular, type 2 diabetes is known to have a large environmental effect such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of diet.

In order to treat diabetes, insulin injection is essential in the case of type 1 diabetes, lifestyle correction and drug treatment are required in the case of type 2 diabetes, and insulin secretagogues (e.g., repaglinide, mitigle) are typical. Age) and a carbohydrate absorption retarder in the small intestine (glucobai: component name-acarbose, basin: component name-voglibose) and the like are used.

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 particular, studies of apples related to diabetes have been reported to show anti-diabetic activity when apples and buttercups are mixed with onions having excellent hypoglycemic activity (Song Mi-kyung, 2012, Master's Thesis, Gyeonggi University). Research is insignificant. In addition, no anti-diabetic activity against green apples has been reported, and in the case of apples, apples contain a lot of organic acids, so they are called "gold apples in the morning and poisonous apples in the evening." It is known that it promotes bowel movement and gastric juice secretion, and it is known that when eaten in the evening, the stomach is sore, or the stomach is uncomfortable, which is not good for a good night's sleep (Kwak Jeong-eun, 2008, monthly diabetes, 220: 28), and eat apples for diabetes prevention and treatment There is a problem that can not.

On the other hand, as patent documents related to apples and diabetes, for example, Mistletoe, Ogapi, Sari, Birch, Cheongmirae, Barley, Onion, Beet, Apple and Persimmon in a specific mineral concentrate in Korean Patent Registration No. 10-1216032 Known as a "pharmaceutical composition for the prevention and treatment of diabetes-related diseases" containing a raw material selected from one or more of the group consisting of, Republic of Korea Patent Publication No. 10-2016-0009001, including apples, ginseng, red ginseng, etc. "Meju and miso using apples having anti-hyperlipidemic and anti-hyperlipidemic properties and a method of manufacturing the same" are disclosed, and Korean functional patent No. 10-2013-0104704 includes apple functionalized powder or ginseng thin functional powder, The manufacturing method "is disclosed, and in Korean Patent Publication No. 10-2013-0104703," Functional red pepper paste and its manufacturing method "are disclosed, but the anti-diabetes of the apple extract itself Performance-related patents are not known.

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" How to manufacture vinegar using green apple "and the like are disclosed, but until now, research on antidiabetic activity of green apple has not been known at all.

KR 10-1194767 B KR 10-2018-0075802 A KR 10-2018-0065352 A KR 10-1216032 B KR 10-2016-0009001 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 prevent or treat / improve pharmaceutical compositions and health functional foods for diabetes containing green apple extract as an active ingredient. Is to provide.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing green apple extract as an active ingredient.

The green apple extract is preferably green apple hot water extract or ethanol extract.

The green apple is preferably a variety of varieties selected from the group consisting of Miyama Fuji, crimson, summering, hongro, and alpine auto.

In addition, the present invention provides a health functional food for preventing or improving diabetes containing green apple extract as an active ingredient.

The green apple extract is preferably green apple hot water extract or ethanol extract.

The green apple is preferably a variety of varieties selected from the group consisting of Miyama Fuji, crimson, summering, hongro, and alpine auto.

The pharmaceutical composition for prevention or treatment / improvement of diabetes of the present invention and the green apple extract as an active ingredient of a health functional food, as proved through the examples of the present specification, as a drug for preventing or improving diabetes and a health functional food There is an excellent effect that can be used. In addition, the green apple extract of the present invention has excellent thermal stability and does not exhibit starch degrading enzyme inhibitory activity even in acidic conditions of pH 2 and plasma, and is easily processed into various forms such as liquid, cream, powder, pill, and tablet. It can be very useful in the pharmaceutical industry and food industry.

1 shows a green apple of the Miyama Fuji variety,
Figure 2 shows the green apple of the crimson varieties,
Figure 3 shows a green apple of the summering varieties,
Figure 4 shows the green apple varieties of green,
Figure 5 shows the green apple of the Alps Otome variety,
Figure 6 shows the degree of α-glucosidase (α-glucosidase) inhibition of ethanol extract by green apple varieties, 1: active control (acarbose, 0.5mg / ml), 2: Miyama Fuji extract (0.5mg / ml), 3: Crimson extract (0.5 mg / ml), 4: Summering extract (0.5 mg / ml) 5: Hongo extract (0.5 mg / ml), 6: Alpsotome extract (0.5 mg / ml), respectively.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, to test the anti-diabetic effect on the green apple, prepared the green apple as an extract through a certain method, evaluated the anti-diabetic activity of the extract, and confirmed the anti-diabetic activity of the green apple extract, and the green apple extract Silver does not show hemolytic activity against human red blood cells, and by confirming that it has excellent thermal and acid stability characteristics, the green apple extract exhibiting anti-diabetic activity is used as a pharmaceutical composition for preventing or treating / improving diabetes and as a health functional food. I wanted to utilize it.

Specifically, the present inventors use a green apple that contains a large amount of antioxidants such as polyphenols or anthocyanins compared to a general mature apple to develop a pharmaceutical composition for preventing or treating / improving diabetes and a health functional food, green apple As a result of preparing each of the hot water extract and the ethanol extract, and evaluating the anti-diabetic activity of these extracts against β-amylase and α-glucosidase α-glucosidase, green apple extract, in particular , In the ethanol extract of green apple, strong β-amylase (β-amylase) and α-glucosidase (α-glucosidase) inhibitory activity was confirmed, and these hot water extracts and ethanol extract did not show hemolytic activity against human red blood cells. It was confirmed that practical use is possible.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing green apple extract as an active ingredient.

The green apple extract is preferably a green apple hot water extract or an ethanol extract, and more preferably an ethanol extract.

The green apple is preferably a variety selected from the group consisting of Miyama Fuji, Crimson, Summer King, Red Road and Alps Automem, more preferably Miyama Fuji, Summer King or Alps Automembrane, and most preferably Alps Automeat variety. .

In addition, the present invention provides a health functional food for preventing or improving diabetes containing green apple extract as an active ingredient.

The green apple extract is preferably a green apple hot water extract or an ethanol extract, and more preferably an ethanol extract.

The green apple is preferably a variety selected from the group consisting of Miyama Fuji, Crimson, Summer King, Red Road and Alps Automem, more preferably Miyama Fuji, Summer King or Alps Automembrane, and most preferably Alps Automeat variety. .

 Hereinafter, a method of manufacturing the green apple extract of the present invention and an efficacy test will be described in more detail.

The present invention comprises the steps of preparing an extract from a green apple; Evaluation step of anti-diabetic 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. As a preferred embodiment, the green apple of the present invention is preferably a cultivar of Miyama Fuji, crimson, summering, hongro and Alpine Otome. 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 "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 "foot apple extract" contained in the composition of the present invention comprises the steps of crushing the green apple; Extracting the green apple crushed material with an organic solvent; Filtering the extract using a filter net 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 present invention may be prepared as a 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 they maintain activity even at a heat treatment of 100 ° C. and a pH of 2 in the human stomach.

The green apple extract of the present invention exhibits protease inhibitory activity against potent β-amylase (β-amylase) and α-glucosidase (α-glucosidase), type 1 diabetes, type 2 diabetes or diabetic retinopathy, It can be used as a pharmaceutical composition related to the prevention and treatment / improvement of diabetic complications such as diabetic nephropathy and health functional food.

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 a variety of foods and beverages effective in preventing or improving diabetes. 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.

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

To the green apple harvested in Example 1, distilled water of twice the weight 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.

[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. 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 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. 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. Table 4 shows the results of each extraction efficiency and component analysis of the extract.

Total polyphenols, total flavonoids, total sugars, and reducing sugars were 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 extracted 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. 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 1N NaOH again, reacting at 37 ° C. for 1 hour, and measuring absorbance at 420 nm. . 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: Evaluation of antidiabetic activity of green apple extract

The anti-diabetic activity of the green apple extract prepared in Example 3 was evaluated for β-amylase inhibitory activity and α-glucosidase inhibitory activity, and the results are shown in Table 5 and FIG. 6.

β-amylase (4-alpha-D-glucan maltohydrolase) inhibitory activity was mixed with 2.5 μL of sample and 25 μL of α-amylase (0.25 U / mL) diluted with 50 mM phosphate buffer (pH 6.8) for 1 min at 37 ° C for 10 min. After the reaction, 25 μL of 0.5% soluble starch (Samchun Chemicals Co., Korea) was added and the second reaction was performed at 37 ° C. for 10 minutes, followed by heating at 100 ° C. for 5 minutes to stop the reaction, and 150 μL of DNS (3) , 5-dinitrosalicylic acid, Sigma Co., st. Louis, USA) was added and heated at 100 ° C. for 5 minutes to develop color and then cooled to room temperature. The chromophore was measured for absorbance at 540 nm to calculate the inhibition rate.

Inhibition rate (%) = [1- (Enzyme activity in sample added / enzyme activity in control added)] x 100

On the other hand, α-glucosidase inhibitory activity was evaluated using pNPG (p-nitrophenol glucoside; Sigma Co., USA), α-glucosidase (0.25U) diluted with 2.5 μl of ginger extract sample and 50 mM Sodium acetate buffer (pH 5.6). / ml) 25 μl was mixed and reacted first at 37 ° C. for 10 minutes, and 25 μl of 1 mM pNPG solution was added to react at 60 ° C. for 10 minutes. Then, 25 μl of 1M NaOH was added to stop the reaction, and the absorbance was measured at 405 nm to calculate the inhibition rate.

Inhibition rate (%) = [1- (Enzyme activity in sample added / enzyme activity in control added)] x 100

[Table 5] Antidiabetic activity of green apple extract

As a result, as shown in Table 5, acarbose, an anti-diabetic agent used in clinical trials, inhibited β-amylase and α-glucosidase 60.5% and 70.2%, respectively, at a concentration of 0.5 mg / ml, confirming the basis for clinical use. In the case of green apple extract, 0.5mg / ml showed better β-amylase inhibitory activity in all varieties, but α-glucosidase disaster activity was found only in the Miyama Fuji variety.

On the other hand, the green apple ethanol extract showed excellent inhibition for both β-amylase and α-glucosidase, and the strongest β-amylase inhibition was confirmed in the Miyama Fuji extract (14.5% inhibition at 0.5 mg / ml concentration), and strong α- The glucosidasee inhibition showed 22.7% and 10.5% inhibition, respectively, in the Alpine autosomal and summering extracts, indicating stronger inhibition than other green apple extracts. Therefore, among the green apples, it was confirmed that the hot water extract of Miyama Fuji, the Alpine Otome, and the summering ethanol extract had particularly excellent anti-diabetic activity.

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, in order to evaluate the acute toxicity of the green apple extract, human red blood cell hemolytic activity was evaluated, and the results are shown in Table 6.

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 6] Human red blood cell hemolytic activity of green apple extract

As a result, as shown in Table 6, 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

Plasma stability, thermal stability and acid stability for anti-diabetic activity were confirmed for the green apple extract obtained in Example 3. The samples showed high stability by not showing a significant decrease in anti-diabetic 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 anti-diabetic activity during digestion and absorption and food manufacturing.

Claims (4)

A pharmaceutical composition for the prevention or treatment of diabetes containing green apple extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the green apple extract is a green apple hot water extract or an ethanol extract. The method of claim 1, wherein the green apple is a pharmaceutical composition, characterized in that the varieties selected from the group consisting of Miyama Fuji, crimson, summering, hongro and Alpine Otome. A health functional food for preventing or improving diabetes, comprising the active ingredient according to any one of claims 1 to 3.

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