KR20200071492A - Pharmaceutical composition comprising the extract of acorn pollen 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 health-aid food for preventing or treating diabetes, including an extract of Quercus acutissima Carruthers pollen as an active ingredient, particularly to a pharmaceutical composition and health-aid food including a hexene fraction or ethyl acetate fraction of an ethanol extract of Quercus acutissima Carruthers pollen, or a mixture thereof, as an active ingredient for preventing or treating/alleviating diabetes through the activity of inhibiting α-glucosidase and β-amylase. The extract of Quercus acutissima Carruthers pollen as an active ingredient of the pharmaceutical composition and health-aid food shows a strong anti-diabetes activity through the activity of inhibiting α-glucosidase and β-amylase, has excellent heat stability, causes no loss of anti-diabetes activity even under an acidic condition of pH 2 and in the blood serum, and thus is expected to be used for preventing and treating diabetes, particularly Type 2 diabetes. The active ingredient can be processed into various forms, such as an extract, powder, pills, tablets, or the like, so that it may be administered at all times, and thus shows high industrial applicability in the medicine and food industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF ACORN POLLEN 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 for the prevention or treatment of diabetes (diabetes) containing an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient and a health functional food, and more specifically, a hexene of an acorn pollen ethanol extract Pharmaceutical composition and health for prevention or treatment/improvement of diabetes mellitus through inhibitory activity of α-glucosidase and β-amylase using as an active ingredient a fraction, an ethyl acetate fraction or a mixture thereof Functional foods.

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 blood glucose concentration 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 not producing 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 impact on environmental factors 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 case of type 1 diabetes, lifestyle correction and drug treatment are required in case of type 2 diabetes, and insulin secretagogues (e.g., repaglinide, mitigle) are typical. Age) and carbohydrate absorption retarders in the small intestine (glucobia: component name-acarbose, basin: component name-voglibose), and the like.

On the other hand, acorn (acorn) is a generic term for a tree fruit belonging to the oak family, and more specifically, oak ( Quercus acutissima ), oak ( Quercus variabilis ), stone oak ( Quercus mongolica ), oak ( Quercus aliena ), sol Refers to oak fruits such as oak ( Quercus serrata ), water oak, and oak ( Quercus dentata ), and the pots produced by bees from the flowers of these oak plants are called acorn pots. Acorn pollen, like other Chungmae pollen, is a mixture of pollen (pollen), honey, and enzyme by worker bees, and is agglomerated like a dumpling, and humans have long used it as a health food.

In addition, pollen is a reproductive organ of related plants that make seeds, and is a structure surrounded by a sturdy shell to protect male spouses, and is mainly carried by wind or insects to reach female spouses and fertilize to form seeds. do. The pollen is divided into insect-mediated pollen pollen and wind-mediated pollen pollen, and the imperial pollen is divided into single plant pots such as acorn pots, acorn pots, and sundries with various plant pots. Songhwabun.

Normally, pollen contains 20~25% crude protein, 7~15% crude lipid, 2~5% crude ash, 20~50% crude carbohydrate, and 10~15% moisture. It has been used as a nutritional supplement and traditional medicine. However, the pollen is composed of a thick layer of sporopollenin (extine) and an endothelial (intime), so it is not digested and decomposed by itself, so the bioavailability is low at 10-15%. There is (Pyeon HI, 2018, Journal of Life Science 28: 605 ~ 614), microbial contamination can be caused by a variety of plants, there is also a disadvantage of easy decay. In addition, toxic substances (1,2-dehydro pyrollizidine alkaloids) suspected of liver toxicity, pulmonary toxicity, and carcinogenicity have been identified in Western fern flowers (Boppre, M et al., J. Agric. Food Chem. 53, 594- 600).

Therefore, research on the existing pollen is mainly focused on the development of extraction conditions and pretreatment methods to improve digestion and absorption by decomposing the nutritional properties of the pollen and the solid outer shell. Recently, research on useful physiological activity has been conducted, and it is known that it is effective for various diseases such as vascular and circulatory system, digestive system, metabolic system, aging, and antibacterial and antioxidant efficacy, immune enhancing effect, prostatic hyperplasia and prostatitis treatment Effects have been reported (Choi et al., 2007, Korean J. Food preserv. 14, 87-93; Abouda et al., 2011, J. Microbiol. 6, 376-384; Li et al., 2009, Carbohydrate Polymers 78 , 80-88; Hong In-pyo et al., 2016, Journal of Apiculture 31, 219-225; Hong In-pyo, 2014, Journal of Apiculture 29, 137-142; Park Yong-ki et al., 2015. Journal of Apiculture 30, 299-306; Kim Seok-jung et al., 2005 , Korean journal of food science 37, 833-837).

On the other hand, studies on various components and efficacy studies of the fruit (acorn) and leaves of the oak plant have been extensively conducted, but studies related to acorn pollen have been almost absent. Studies related to acorn pollen include changes in ingredients by physical treatment of acorn pollen (pollen) (Hong In-pyo, 2013. Korean J. Apiculture 28, 217-221), antibacterial activity of acorn pollen germination solution (Choi Jun-hyuk, 2007, Korean food storage Distribution Journal, 14, 87-93) and antioxidant activity (Hong In-Pyo, 2014, Journal of Apiculture 29, 137-142) are known, and other activities related to acorn pollen are unknown.

In addition, as a patent related to acorn pollen, Korean Patent No. 10-1237215 [Pharmaceutical composition for preventing or treating enteritis] is known, and other patents related to physiological activity are not known.

To date, the anti-diabetic effect of pollen has been recognized in the private sector, but in some, contrary opinions exist because it is understood as a food ingredient that diabetics should avoid due to the large amount of sugar in the pollen. Among the by-products of beekeeping, anti-diabetic effects are generally recognized in royal jelly and propolis, but pollen is highly difficult to understand the activity related to diabetes so far, since its composition varies greatly depending on the type of wheat plant.

Anti-diabetic effects of pollen on scientific basis include polymer polysaccharides in willow pollen (Lei X et al., 2018. J Ethnopharmacol. 224:169-176) and polymer polysaccharides in glycosylated pollen (Li X et al. 2017, Molecules. 22(5) : E699) is known, but it is limited to diabetes complications and insulin resistance improvement effect by pollen polymer polysaccharide. In addition, it has been reported that the flavone component of the willow pollen improves insulin resistance (Feng, Xiao-Tao et al., 2014, Bioscience, Biotechnology, Biochemistry 78: 1738-1742), and the date palm pollen alleviates glycemia. Ham is known (Mohamed, Nema A. 2018, Biomedicine & Pharmacotherapy 97: 9-18).

However, until now, it is not known that a specific fat-soluble component of acorn pollen exhibits anti-diabetic activity through strong α-glucosidase and β-amylase inhibition.

WO 2016127463 A

Lei X et al., 2018. J Ethnopharmacol. 224: 169-176. Li X et al. 2017, Molecules. 22(5): E699. Feng, Xiao-Tao et al., 2014, Bioscience, Biotechnology, Biochemistry 78: 1738-1742. Mohamed, Nema A. 2018, Biomedicine & Pharmacotherapy 97: 9-18).

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 a pharmaceutical composition for preventing or treating diabetes and a health functional food containing acorn pollen extract 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 diabetes containing acorn pollen (Pollen of Quercus sp.) extract as an active ingredient.

As an active ingredient of the pharmaceutical composition, the acorn pollen extract is preferably a hexene fraction, an ethyl acetate fraction of acorn pollen ethanol extract or a mixture thereof.

In addition, the present invention provides a dietary supplement for preventing or improving diabetes, which contains an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient.

It is preferable that the acorn pollen extract as an active ingredient of the health functional food is a hexene fraction, an ethyl acetate fraction of acorn pollen ethanol extract or a mixture thereof.

Acorn pollen extract as an active ingredient in the pharmaceutical composition for preventing or treating diabetes of the present invention and a health functional food exhibits strong anti-diabetic activity through α-glucosidase and β-amylase inhibitory activity, and has thermal stability. It is excellent, and it is expected that it can be used for the prevention and treatment of diabetes, especially type 2 diabetes, because it does not exhibit anti-diabetic activity in pH 2 and acidic conditions and plasma, and the active ingredient is an extract, powder, pill, It is a very useful invention in the pharmaceutical industry and the food industry because it has an excellent effect that can be prepared in a form that can be taken at all times by being processed in various forms such as tablets.

1 is a photograph of an acorn pollen,
2 is a microscope (200 times) observation photograph of the acorn pollen.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared acorn pollen extracts in a certain way to test the anti-diabetic efficacy against acorn pollen, and recovered the hexene fraction and ethyl acetate fraction thereof as anti-diabetic active ingredients, the hexene fraction and ethyl acetate By confirming that the fraction has excellent characteristics of thermal stability and acid stability, the acorn pollen extract, its hexene fraction, and ethyl acetate fraction were intended to be used as a pharmaceutical composition for preventing and treating or improving diabetes and a health functional food.

Specifically, the present inventors prepared an ethanol extract for an acorn pollen using an acorn pollen known to have anti-inflammatory, hepatoprotective, and antihypertensive effects. After preparing a solvent fraction, the inhibitory activity of α-alpha glucosidase and β-amylase was evaluated, and the strong enzyme inhibitory activity was confirmed in the hexene fraction and ethyl acetate fraction of the acorn pollen extract.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing acorn pollen (Pollen of Quercus sp.) extract as an active ingredient.

As an active ingredient of the pharmaceutical composition, the acorn pollen extract is preferably a hexene fraction, an ethyl acetate fraction of acorn pollen ethanol extract or a mixture thereof.

In addition, the present invention provides a dietary supplement for preventing or improving diabetes, which contains an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient.

It is preferable that the acorn pollen extract as an active ingredient of the health functional food is a hexene fraction, an ethyl acetate fraction of acorn pollen ethanol extract or a mixture thereof.

Hereinafter, a method and an efficacy test of the acorn pollen extract of the present invention and each active fraction will be described in more detail.

The present invention comprises the steps of preparing an extract from acorn pollen; Sequential organic solvent fraction preparation of hexene, ethyl acetate, butanol from acorn pollen ethanol extract and preparation of water residues subsequently obtained; Evaluating antithrombotic activity of the extract and fraction; And a stability investigation step of extracts and fractions exhibiting activity.

The "acorn pollen extract" contained in the composition of the present invention can be obtained by extracting the acorn pollen with an organic solvent and 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, butanol, etc.), mixed solvents of the lower alcohol and water, etc. Can be used, hot water, or 95% ethanol extraction is most preferred.

As a preferred embodiment, the acorn pollen extract of the present invention can be used by extracting the acorn pollen with hot water or ethanol. Here, the hot water or ethanol extract may be sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate and butanol to additionally obtain a hexene fraction, ethyl acetate fraction and butanol fraction and water residue.

The acorn pollen extract of the present invention and its active fraction materials may be prepared as a powder through a conventional powdering process such as vacuum drying and lyophilization, 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 in the human stomach of pH 2.

The active ingredient of the present invention can be used for the prevention or treatment of diabetes and diabetes complications. The diabetes may be type 1 diabetes or type 2 diabetes, and the diabetes complications may be diabetic retinopathy or diabetic nephropathy.

The pharmaceutical composition comprising 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 carrier, excipient, or diluent 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 suspending agents, solvent solutions, 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, etc. may be included.

As a preferred embodiment, the preparation for parenteral administration may include sterilized 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 may 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 drugs, 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, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., so that the 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 the active ingredient to target cells.

“Subject” in the present invention is not particularly limited, and includes, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit or guinea pig. And, preferably, a mammal, more preferably a human.

In addition, the health functional foods of the present invention are various in foods and beverages effective in preventing or improving diabetes, for example, type 1 diabetes or type 2 diabetes and diabetes complications, for example, diabetic retinopathy or diabetic nephropathy. Can be used.

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 contain, as an additional ingredient, food additives, such as natural carbohydrates and various flavoring additives.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and sugars such as xylitol, sorbitol, and erythritol, and common sugars 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 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, 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: Physicochemical properties of acorn pollen

In 2018, acorn pots harvested from Korea were purchased and used for experiments.Acorn pots used in the experiment were dark yellow granules with a brightness of 40.18, a redness of 3.58, and a yellowness of 20.29. Shown. In addition, the acorn pollen exhibited pH 4.6, brix 52.0, and acidity 0.74, which showed higher sugar content and acidity than that of Darae pollen pH 5.6, brix 40.0, and acidity 0.68. The moisture content was 11.5% in a very dry state, and was allowed to absorb moisture again when left at room temperature for a long time (FIG. 1, Table 1). As a result of observation under a microscope (200 times), the acorn pollen was isolated into a prolate, the polar surface was circular, and the surface was confirmed to be granular or granular (Fig. 2).

[Table 1] Physicochemical properties and color difference analysis of acorn pollen

As a result of nutritional component analysis, crude protein 25.1%, crude fat, 12.5%, crude carbohydrate showed 48.0%, which was excellent at 404.9 kcal/100g, and the ash content also showed 1.9%, which contained various minerals in large quantities (Table 2). It can be seen that it contains more crude fat when compared to 35.6%, crude fat, 8.1%, and 48.3% crude carbohydrate content of Darae pollen.

[Table 2] Analysis of Nutritional Components of Acorn Pollen

Example 2: Acorn pollen extract, fraction preparation and analysis of useful components thereof

In 2018, acorn pollen harvested in Korea was purchased and used to prepare hot water and ethanol extracts. Specifically, 10 times ethanol was added to the acorn pollen sample, the extract was collected twice, filtered at room temperature, filtered, concentrated under reduced pressure to produce an ethanol extract, and in the case of acorn pollen hot water extract, acorn 10 times sterile distilled water was added to the pollen sample, repeated extraction at 100° C. for 1 hour twice, followed by filtration and concentration as described above. Among the prepared acorn pollen extracts, ethanol extracts were subjected to sequential organic solvent fractionation with hexene, ethyl acetate, and butanol, and finally water residues were recovered. Table 3 shows the results of the fractional efficiency and component analysis.

Total polyphenols, total flavonoids, total sugars and reducing sugars were determined by analyzing the components of the acorn pollen extract and fractions. 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, and allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol stirring for 18 hours, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1 N NaOH was added again, and the absorbance at 420 nm was measured after reaction at 37° C. for 1 hour. 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 3] Analysis of extraction efficiency and useful components of acorn pollen extract and fractions

As shown in Table 3, the extraction efficiency of the acorn pollen ethanol extract was 46.4%, which was lower than that of the hot water extract (56.5%), but the total polyphenol content of the ethanol extract was 1.5 times higher than the hot water extract, and the total sugar And the reducing sugar content of the hot water extract was 1.3 times and 1.36 times higher than the ethanol extract. On the other hand, the total flavonoid content was similar in hot water extract and ethanol extract.

On the other hand, the largest portion of the acorn pollen extract was transferred to the butanol fraction (64.8%), and water residues accounted for 25.1% of the extract. The ethyl acetate fraction showing the smallest yield showed 4.9% of the acorn pollen extract, and 2.27 g per 100 g of acorn pollen could be recovered. On the other hand, as a result of analyzing the total polyphenol and total flavonoid content, the highest content of polyphenol content (225.0 mg/g) and flavonoid content (222.1 mg/g) in the ethyl acetate fraction was shown. This was 2,250 times higher polyphenol content and 158.6 times higher flavonoid content compared to the lowest water residue. Therefore, the ethyl acetate fraction of the acorn pollen extract was expected to exhibit various physiological activities, and as a result of actual analysis, the fraction exhibited strong antioxidant activity and nitrite scavenging activity. Therefore, the ethyl acetate fraction of acorn pollen is expected to contribute to blood circulation improvement and anti-diabetic activity by showing excellent antioxidant activity and carcinogenesis inhibitory activity.

Example 3: Evaluation of anti-diabetic activity of acorn pollen extract and fractions

To evaluate the anti-diabetic activity of the acorn pollen extract prepared in Example 2 and its fraction, in-vitro β-amylase inhibitory activity and α-glucosidase inhibitory activity were measured, respectively. First, 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) and mixed at 10° C. for 10 min. After the first 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 in the reaction solution (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. In the color developing solution, the absorbance was measured at 540 nm to calculate the inhibition rate.

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

α-glucosidase inhibitory activity was evaluated using pNPG (p-nitrophenol glucoside; Sigma Co., USA), α-glucosidase (0.25 U/ml) diluted with 2.5 μl of ginger extract sample and 50 mM Sodium acetate buffer (pH 5.6) ) 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 for 2 min 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 addition/enzyme activity in control)] x 100

[Table 4] Anti-diabetic activity of acorn pollen extract and its fraction

As a result, as shown in Table 4, acarbose used as a therapeutic agent for diabetes in the clinic showed potently dependent β-amylase inhibitory activity and α-glucosidase inhibitory activity. On the other hand, in the case of acorn pollen extract, both β-amylase and α-glucosidase inhibitory activity was not observed in both hot water and ethanol extracts, but very strong β-amylase inhibition at 0.5 mg/ml concentration in the hexene fraction and ethyl acetate fraction of ethanol extract In particular, in the ethyl acetate fraction, 57.1% β-amylase inhibition and 13.0% α-glucosidase inhibition were simultaneously observed at a concentration of 0.5 mg/ml. The anti-diabetic activity of the ethyl acetate fraction of the acorn pollen ethanol extract was more potent during high concentration treatment and showed concentration-dependent inhibitory activity. Therefore, it was confirmed that the hexene fraction and the ethyl acetate fraction of the acorn pollen extract can be used for the prevention and treatment of diabetes, especially type 2 diabetes.

Example 4: Human red blood cell hemolytic activity of acorn pollen extract and fractions thereof

Acorn pollen is a food that has been secured by being registered as a food ingredient. To evaluate the acute toxicity potential of acorn pollen ethanol extract and its fractions, human erythrocyte hemolysis activity was evaluated, and the results are shown in Table 5. At this time, the hemolytic activity was evaluated according to an existing report (Hohn Son Son, 2014 ㆍKorean J. Microbiol. Biotechnol. 42: 285-292), and simply 100 μl of human red blood cells washed three times with PBS in a 96-well microplate. After adding 100 μl of sample solution of various concentrations 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 414 nm. It was measured at. 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 5: Human red blood cell hemolytic activity of acorn pollen extract and its fractions

First, DMSO and water used as controls had no hemolytic activity, and triton X-100 was confirmed to hemolytically erythrocyte 100% at a concentration of 1 mg/ml. In addition, in the case of amphotericin B, which is used as an anti-cancer agent and anti-fungal agent, it was confirmed that hemolysis of 50% of red blood cells was carried out at a concentration of 0.00625 mg/ml. The acorn pollen ethanol extract of the present invention was found to have no acute toxicity and erythrocyte hemolytic activity since no erythrocyte hemolysis occurred in fractions except the hexene fraction up to a concentration of 1 mg/ml. The hexene fraction exhibited red blood cell hemolytic activity of 89.9% at a concentration of 1 mg/ml, 53.5% at 0.5 mg/ml, and 7.5% at 0.25 mg/ml, and thus was expected to have acute toxicity potential.

Example 5: Plasma, acid and thermal stability evaluation of acorn pollen extract and its active fractions

Plasma stability, heat stability and acid stability for anti-diabetic activity were confirmed for the acorn pollen ethanol extract obtained in Example 2 and the hexene fraction and ethyl acetate fraction thereof. Acorn pollen ethanol extract and its active fractions have almost no reduction of β-amylase and α-glucosidase inhibitory 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. Did not appear. Therefore, it was confirmed that the acorn pollen extract and its active fraction contained anti-diabetic active substances having acid resistance and heat resistance.

Claims (4)

Acorn pollen (Pollen of Quercus sp.) A pharmaceutical composition for the prevention or treatment of diabetes containing an extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the acorn pollen extract is a hexene fraction, an ethyl acetate fraction, or a mixture thereof of ethanol extract of acorn pollen. A dietary supplement for the prevention or improvement of diabetes, which contains the extract of acorn pollen (Pollen of Quercus sp.) as an active ingredient. 4. The dietary supplement according to claim 3, wherein the acorn pollen extract is a hexene fraction, an ethyl acetate fraction of acorn pollen ethanol extract or a mixture thereof.

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