KR20220053243A - Pharmaceutical composition comprising the extract of glehnia littoralis 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 comprising an extract of Glehnia littoralis as an active ingredient for preventing or treating diabetes and, more specifically, to a pharmaceutical composition and a health functional food for preventing or treating and/or ameliorating diabetes through strong β-amylase and α-glucosidase inhibitory activity, comprising as an active ingredient the extract of Glehnia littoralis and an active fraction thereof cultivated in the open field or greenhouse. The extract of Glehnia littoralis and the active fractions thereof as an active ingredient of an anti-diabetes composition of the present invention has an excellent effect of being used as a medicine and health functional food for preventing or ameliorating diabetes as demonstrated through the examples of the present specification. Furthermore, the extract of Glehnia littoralis and the active fractions thereof according to the present invention have excellent thermal stability and no loss of starch degrading enzyme inhibitory activity even under acidic conditions of pH 2 and plasma, and thus can be easily processed into various forms such as liquid, cream, powder, pill, tablet, etc., which is very useful in the pharmaceutical industry and food industry.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of diabetes containing extract of liberated peony as an active ingredient

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes mellitus and a functional food for the prevention or treatment of diabetes containing the extract of Glehnia littoralis as an active ingredient, and more particularly, to an extract of liberated peony grown in the open air or in a house, and its extract It relates to a pharmaceutical composition and health functional food for preventing or treating/improving diabetes through strong β-amylase and α-glucosidase inhibitory activity containing the active fraction as an active ingredient.

Diabetes mellitus, one of the most common diseases among modern people, is a type of metabolic disease such as insufficient insulin secretion or failure to function normally. .

Diabetes mellitus is divided into type 1 and type 2, type 1 diabetes is caused by the inability to produce insulin at all due to genetic influence, and type 2 diabetes is caused by poor insulin function and insulin resistance (insulin resistance) ) is known to be the cause. In particular, it is known that environmental factors such as high calorie, high fat, and high protein diet, lack of exercise, and stress play a major role in type 2 diabetes due to westernization of diet.

For the treatment of diabetes, insulin injection is essential in the case of type 1 diabetes, and lifestyle modification and drug treatment are required in the case of type 2 diabetes. nide) and a carbohydrate absorption delaying agent in the small intestine [Glucoby: ingredient name-acarbose, Basin: ingredient name-voglibose], etc. are being used.

On the other hand, Glehnia littoralis is a perennial grass of the Umbelliferae that grows wild as a halophyte in the sandy soil of the coast of Korea. In particular, the root is used as a substitute for windbreak, and is also called Gaetbangpung, Binbangpung, and Sea ginseng.

Commonly known as windbreak, it refers to the root of Ledebouriella seseloides or Saposhikovia divaricata , and for food use, it is Peucedanum japonicum . It refers to the root of Thunberg, and it is also different from the liberation wind of the present invention in terms of plant taxonomic, use and efficacy.

In the case of Korea, although some native areas are formed along the coastline, recently, due to the development of cultivation technology, the liberation wind has been mass-produced through outdoor cultivation or house cultivation at the coastline. (Woo et. al., 45th Annual Meeting & International Symposium, KMB 2018; Woo et. al., The 59th Annual Meeting and International Symposium of Korean Society of Life Science; Woo et. al., The 59th Annual Meeting and International Symposium of Korean Society of Life Science).

Most of the research on liberation wind is research on the ingredients and efficacy of the root, and the research on the above-ground part used for food is very insignificant. First, in the case of roots, components belonging to coumarin, polyacetylene, lignan, and monoterpenoid have been reported (Zhao Y et al., 2007, Yao Xue Xue Bao. 42: 1070-1073; Eom Young-ran et al., 2010, J. Korean Chemical Soc 54: 701-706), and recently, monoterpenoids, alkyl glycosides, aromatic glycosides, phenolic acids (chlorogenic acid, quercetin-3-glucoside) and pyranocoumarins have been identified in the fruits and outposts of Haebangpung (Hyeonjeong Kim, 2015. Mokpo University Master's thesis). .

As the main pharmacological activity of haebangpung root, antioxidant, anti-inflammatory, peroxynitrite scavenging activity of haebangpung methanol extract (Heejeong Lee et al., 2004. Korean J. Biotechnol Bioeng. 19: 57-61), protective effect of liberation wind against hepatotoxicity caused by alpha-amanitine (Bohyun Kim et al., 2017, Journal of the Korean Society of Clinical Toxicology, 15: 107-115), cancer cell growth inhibitory activity (Kim Ji-hoon et al., 2008. Korean J. Medicinal Crop Science 16: 44-50) and platelet aggregation inhibitory activity according to thromboxane synthesis inhibition (Wang SR, 1993, Zhongguo Zhong Xi Yi Jie He Za Zhi, 13: 167-170), a substance isolated from outpost containing liberation wind roots (6-carboxyethyl-4,7-dimethoxy-5-hydroxybenzofuran, 5-O The anticoagulant activity of -β-D-glucopyranoside, chlorogenic acid, 3'-O-methylchlorogenic acid, quercetin 3-O-β-D-glucoside, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside) has been reported. Yes (Hyun-Jin Lim, Hyun-Jeong Kim et al., 2016. Journal of Herbal Medicine 47: 49-54; Hyun-Jung Kim, 2015. Mokpo National University Master’s thesis). Also, recently, research on callus induction and plant redifferentiation for artificial cultivation and liberation wind (Byeong-guk Lee et al., 2004. Korean J Plant Res 17: 153-160) is known.

In addition, it is reported that the ethyl acetate extract (GLEA: Glehnia littoralis leaf ethyl acetate) of Haebangpung leaf contributes to the improvement of cardiovascular diseases and lipid metabolism diseases through 15-PGDH (15-Hydroxyprostaglandin dehydrogenase) inhibition and HMG-CoA reductase inhibition (Kyung-Hun Seon) , 2013, Chosun University School of Medicine doctoral thesis), and the growth inhibitory effect of Haebangpung young leaf essential oil and its main components (α-pinene and β-pinene) on antibiotic-resistant Streptococcus pneumoniae strains (Shin Seung-won, 2005. Journal of Pharmacy 49: 312-316), antibacterial activity of hot water extract from the underground part of Haebangpung (Kim CM et al., 1991. Arch. Pharm, Res. 14, 87-92), root antibacterial (Matsuura H et al., 1996, Planta Med) 62: 256-259) and antifungal activity (McCutcheon AR et al., 1994. J Ethnopharmacol. 44: 157-169) and the like are known. However, until now, the antidiabetic activity of haebangpung according to strong inhibition of β-amylase and α-glucosidase has not been known.

In addition, as a patent related to Haebangpung, [Pharmaceutical composition for the prevention or treatment of cardiovascular diseases including Gaetbangpung extract] is known in Republic of Korea Patent No. 10-1787566, which contains a butanol fraction of a Haebangpung extract as an active ingredient, Registered Patent No. 10-1620153, [composition for preventing or treating arthritis containing Gaetbangpung extract as an active ingredient], and Registered Patent No. 10-1817242, [Composition for external application for skin containing Gaetbangpung extract] are known. In addition, Patent Registration No. 10-1164083 [a composition for relieving a hangover containing haebangpung extract as an active ingredient], and in No. 10-1207214 [a fraction prepared from haebangpung extract as an active ingredient, prevention and treatment of inflammatory diseases] Therapeutic composition], [Composition for the prevention and treatment of edema or dermatitis containing Haebangpung extract as an active ingredient] in No. 10-0919852, and [Use of Haebangpung extract to modulate PGE2 activity] in No. 10-1413363 is known In addition, recent patents for Haengbangpung for food include [Method for manufacturing Gaetbangpung extract for food additive] in Registered Patent No. 10-1867480, and [Method for manufacturing medicinal liquor and takju using Haebangpung] in No. 10-1812258. -1789183 [Method for producing noodles with Gaetbangpung added] and [Method for producing Dutch coffee with Gaetbangpung added] are known in No. 10-1610267. In addition, Patent Publication No. 10-2014-0077483 discloses [composition for preventing or improving ischemic cerebrovascular disease comprising Gaetbangpung extract] having an effect of inhibiting brain neuronal cell death, and in No. 10-2010-0037781, Pharmaceutical composition for prevention and treatment of cancer containing extract of Bangpung], [Method for manufacturing Gaetbangpung pickled radish] in No. 10-2018-0067861 and [Gaetbangpung leaf tea and manufacturing method thereof] in No. 10-2015-0064864 this is disclosed. However, to date, there is no known patent for the strong antidiabetic activity of Haebangpung extract.

Seon Kyung-hoon, 2013, Chosun University School of Medicine Ph.D. thesis

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 diabetes mellitus containing an extract of the above-ground or underground part of the liberated wind grown in the open air and in the house as an active ingredient. An object of the present invention is to provide a pharmaceutical composition for improvement and a health functional food.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing an extract of liberation pung ( Glehnia littoralis ) as an active ingredient.

It is preferable that the liberation wind extract is a liberated wind above ground extract grown in the open air or a liberated wind underground extract grown in a house.

The liberated wind extract is preferably a fraction selected from the group consisting of the following (1) to (5):

(1) the butanol fraction of the underground extract of liberated wind grown in the open field;

(2) the hexene fraction of the above-ground extract of liberated wind grown in the house;

(3) the hexene fraction of the underground extract of liberation wind grown in the house;

(4) the ethyl acetate fraction of the underground extract of Haebangpung grown in the house; and

(5) Butanol fraction of Haebangpung underground extract grown in a house.

In addition, the present invention provides a health functional food for the prevention or improvement of diabetes containing the extract of liberation pung ( Glehnia littoralis ) as an active ingredient.

It is preferable that the liberation wind extract is a liberated wind above ground extract grown in the open air or a liberated wind underground extract grown in a house.

The liberated wind extract is preferably a fraction selected from the group consisting of the following (1) to (5):

(1) the butanol fraction of the underground extract of liberated wind grown in the open field;

(2) the hexene fraction of the above-ground extract of liberated wind grown in the house;

(3) the hexene fraction of the underground extract of liberation wind grown in the house;

(4) the ethyl acetate fraction of the underground extract of Haebangpung grown in the house; and

(5) Butanol fraction of Haebangpung underground extract grown in a house.

As an active ingredient of the anti-diabetic composition of the present invention, the liberated peony extract and its active fractions have excellent effects that can be used as pharmaceuticals and health functional foods for the prevention or improvement of diabetes, as demonstrated through the examples of the present specification. In addition, the liberated wind extract of the present invention and its active fractions have excellent thermal stability, and no loss of starch degrading activity even in an acidic condition of pH 2 or in plasma, liquid, cream, powder, pill, tablet, etc. Since it can be easily processed into a shape, it can be very usefully used in the pharmaceutical industry and the food industry.

1 shows the above-ground part of the liberation wind cultivated in the open field,
Figure 2 shows the underground part of the liberation wind cultivated in the open field,
3 shows the above-ground part of the liberation wind cultivated in the house,
4 shows the underground part of the liberation wind cultivated in the house.

Hereinafter, the present invention will be described in detail.

In order to test the anti-diabetic efficacy of liberated pung, the inventors of the present invention prepared an extract in a certain method targeting open-field and house-cultivated liberated wind, and evaluated the activity to determine the liberated wind extract exhibiting antidiabetic activity, and then Each fraction as an antidiabetic active ingredient was recovered by preparing a hexene fraction, an ethyl acetate fraction, a butanol fraction, and an organic solvent fraction from the liberated wind extract, and the hexene fraction, the ethyl acetate fraction and the butanol fraction have thermal stability and acid stability. It was confirmed that it has these excellent characteristics.

Specifically, the present inventors prepared an ethanol extract of open-field and house-cultivated liberated wind, respectively, to develop an anti-diabetic composition using liberated wind, which is used for food and medicine in the private sector, and evaluated the antidiabetic activity, and cultivated in the open field. Antidiabetic activity was confirmed in the above-ground part and underground part of the house cultivation, and thereafter, the extracts of liberated wind were sequentially fractionated with hexene, ethyl acetate, and butanol with hexene, ethyl acetate, and butanol, respectively, and finally, a water residue was prepared after organic solvent fractionation. As a result of evaluating the inhibitory activity on β-amylase and α-glucosidase of each extract and fraction, it was confirmed that the hexene fraction, ethyl acetate fraction and butanol fraction of the house-cultivated Haebangpung underground extract showed excellent antidiabetic activity, and it was confirmed that house cultivation Excellent antidiabetic activity was also confirmed in the hexene fraction of the above-ground part of the liberation wind and the butanol fraction of the underground part of the open-air cultivation liberation wind.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing an extract of liberation pung ( Glehnia littoralis ) as an active ingredient.

It is preferable that the liberation wind extract is a liberated wind above ground extract grown in the open air or a liberated wind underground extract grown in a house.

The liberated wind extract is preferably a fraction selected from the group consisting of the following (1) to (5):

(1) the butanol fraction of the underground extract of liberated wind grown in the open field;

(2) the hexene fraction of the above-ground extract of liberated wind grown in the house;

(3) the hexene fraction of the underground extract of liberation wind grown in the house;

(4) the ethyl acetate fraction of the underground extract of Haebangpung grown in the house; and

(5) Butanol fraction of Haebangpung underground extract grown in a house.

In addition, the present invention provides a health functional food for the prevention or improvement of diabetes containing the extract of liberation pung ( Glehnia littoralis ) as an active ingredient.

It is preferable that the liberation wind extract is a liberated wind above ground extract grown in the open air or a liberated wind underground extract grown in a house.

The liberated wind extract is preferably a fraction selected from the group consisting of the following (1) to (5):

(1) the butanol fraction of the underground extract of liberated wind grown in the open field;

(2) the hexene fraction of the above-ground extract of liberated wind grown in the house;

(3) the hexene fraction of the underground extract of liberation wind grown in the house;

(4) the ethyl acetate fraction of the underground extract of Haebangpung grown in the house; and

(5) Butanol fraction of Haebangpung underground extract grown in a house.

Hereinafter, the preparation method and efficacy experiments of the liberated pung extract and the antidiabetic active fraction of the present invention will be described in more detail.

The present invention prepares the basement and the basement of the open air and house cultivation liberation wind; preparing each extract; Antidiabetic activity evaluation step of Haebangpung extract; a step of preparing sequential organic solvent fractions of hexene, ethyl acetate, and butanol from the liberated wind extract and preparing the water residue obtained thereafter; Antidiabetic activity evaluation step of the fraction; and a step of examining the stability of the antidiabetic active fraction.

The "liberation wind extract" contained in the composition of the present invention includes the steps of collecting mature liberated wind and washing it; classifying the liberation wind into an underground part and an underground part; It can be obtained by extracting with an organic solvent for each part of the liberation wind, filtering the extract using a filtration network of 0.06 mm or less, and concentrating it under reduced pressure.

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

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

The liberated wind extract of the present invention and its active fractions can be prepared into powder through a conventional powdering process such as drying under reduced pressure and freeze-drying, or spray-drying. They are not degraded by various degrading enzymes in plasma, and they maintain activity even at 100°C heat treatment and at pH 2 in the human stomach.

Haebangpung extract and its active fractions of the present invention exhibit strong β-amylase and α-glucosidase inhibitory activity against diabetic complications such as type 1 diabetes, type 2 diabetes or diabetic retinopathy, diabetic nephropathy, etc. It can be used as a material for pharmaceutical compositions and health functional foods related to prevention and treatment/improvement.

The pharmaceutical composition comprising the active ingredient of the present invention can be prepared according to a conventional method according to the purpose of use. Oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injection solutions It can be formulated and used in various forms, such as oral administration, or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, etc.1

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

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

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

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

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

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

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

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

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

In addition, the health functional food of the present invention can be used in various ways, such as food and beverage effective for preventing or improving diabetes. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, tea, vitamin complexes, health supplements, and the like, and may be used in powder, granule, tablet, capsule or beverage form. .

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

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

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

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

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

[Example]

Example 1: Preparation of Haebangpung extract and analysis of components of the extract

In 2017, Haebangpung grown in the open air at the coast of Yeongdeok, Gyeongsangbuk-do and Haebangpung grown in a house in the interior of Yeongdeok were purchased, cleaned, and separated into aboveground and underground parts. In the case of the open-air culture liberation wind, the leaves were smaller and relatively harder than the house cultivation, and the underground root was smaller and black than the house cultivation ( FIGS. 1 to 4 ). After that, an ethanol extract was prepared for each plantation site and each part. Specifically, 10 times of ethanol was added to the liberated wind sample, extracted three times at room temperature, the extract was collected, filtered, and concentrated under reduced pressure to make a powder did. The results of each extraction efficiency and component analysis of the extract are shown in Table 1. The content of total polyphenols, total flavonoids, total sugars and reducing sugars was determined by component analysis. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample solution, and the absorbance was measured at 725 nm after standing at room temperature for 1 hour. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1 N NaOH was added again, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] Analysis of extraction efficiency and useful components of ethanol extracts above and below the open air and house cultivation liberation winds

As shown in Table 1, the extraction efficiency of the underground part of the liberation wind was about 1.5 times higher than that of the above-ground part, and the extraction efficiency of the above-ground part and underground part of open-air cultivation was slightly higher than that of house cultivation. As a result of the analysis of the total polyphenol content, the above-ground part was about 4.4 to 6.7 times higher than that of the underground part, and it was higher in the open-air cultivation than in the house cultivation. In the case of total flavonoid content, the above-ground part was also 7.3~15.1 times higher than that of the underground part. As a result of evaluating the total sugar and reducing sugar content, the total sugar and reducing sugar content were found to be very low in the above-ground part grown in the open air, whereas the content in the underground part was higher than that of the house-cultivated liberation wind. Therefore, it was confirmed that the liberation wind showed many component differences depending on the cultivation method.

Example 2: Evaluation of antidiabetic activity of Haebangpung extract

The β-amylase inhibitory activity and α-glucosidase inhibitory activity were evaluated for the antidiabetic activity of the liberated peony extract prepared in Example 1, and the results are shown in Table 2. The inhibitory activity of β-amylase (4-alpha-D-glucan maltohydrolase) was obtained by mixing 2.5 μL of sample and 25 μL of α-amylase (0.25 U/mL) diluted with 50 mM phosphate buffer (pH 6.8) at 37°C for 10 minutes. After the reaction, 25 μL of 0.5% soluble starch (Samchun Chemicals Co., Korea) was added, followed by a secondary reaction 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) solution was added, heated at 100° C. for 5 minutes to develop color, and then allowed to cool at room temperature. The inhibition rate was calculated by measuring the absorbance at 540 nm of the color developer.

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

Meanwhile, α-glucosidase inhibitory activity was evaluated using pNPG (p-nitrophenol glucoside; Sigma Co., USA), and α-glucosidase (0.25U) diluted with 2.5 μl of a ginger extract sample and 50 mM sodium acetate buffer (pH 5.6). /ml) 25 μl of the mixture was first reacted at 37° C. for 10 minutes, and 25 μl of 1 mM pNPG solution was added, followed by a second reaction 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 the sample addition group/enzyme activity in the control group)] x 100

[Table 2] Antidiabetic activity of Haebangpung extract

As shown in Table 2, acarbose, which is clinically used as a therapeutic agent for diabetes, showed strong β-amylase inhibitory activity and α-glucosidase inhibitory activity in a concentration-dependent manner. Among the extracts of Haebangpung, antidiabetic activity was shown in the above-ground extracts grown in open-air cultivation and the extracts of the underground parts of house cultivation, and in particular, good α-glucosidase inhibitory activity was confirmed in the extracts of the underground part cultivated in house.

Example 3: Preparation of Sequential Organic Solvent Fractions of Haebangpung Extract by Cultivation Method and Site and Analysis of Their Components

The various liberated wind extracts of Example 1 were subjected to sequential organic solvent fractionation with hexene, ethyl acetate, and butanol, and finally the water residue was recovered. The yield of fractions and the results of component analysis of fractions are shown in Table 3.

[Table 3] Comparison of yield and component analysis of sequential fractions of Haebangpung extract by cultivation method and region

As shown in Table 3, in the above-ground extracts of liberation wind, the hexene fraction was high as 20.56~36.82% of the extract in both open field and house cultivation, whereas the underground extract showed 6.2% and 3.8% in open field and house cultivation, so that the above-ground extract was fat-soluble. It was found to contain a large amount of ingredients. On the other hand, the underground extract contained a large amount of water-soluble components, and in the case of house-cultivated liberation wind, water residues accounted for 64% of the extract, suggesting that most of them were water-soluble components. However, in the case of the underground part cultivated in the open field, the butanol extract and the water residue accounted for 47.0% and 44.9%, indicating a significantly different pattern from that of the house-cultivated underground part. These results mean that the components of the Haebangpung extract are very different depending on the cultivation method and each part.

The fraction with the highest total polyphenol and total flavonoid content was identified as the ethyl acetate fraction of each extract, and the above-ground part showed a significantly higher content than the underground part. These results mean that the fraction of the above-ground part extract, which has been mainly used for food so far, can be used as medicinal and health functional food. Also in the present invention, it was expected that various physiological activities of the extract of Haebangpung would appear in the ethyl acetate fraction. In this example, 0.088, 0.096, 0.078 and 0.099 g of ethyl acetate fractions could be recovered from the above-ground and underground parts of 100 g liberation wind grown in the open air, and 0.088, 0.096, 0.078 and 0.099 g of ethyl acetate fractions, respectively, from the above-ground and underground parts of 100 g liberation wind of house cultivation.

Example 4: Evaluation of antidiabetic activity of sequential organic solvent fractions of Haebangpung extract

The antidiabetic activity of the fractions of the liberation pung extract obtained in Example 3 was evaluated in the same manner as in Example 2, and the results are shown in Table 4.

[Table 4] Antidiabetic activity of sequential fractions of various Haebangpung extracts

As shown in Table 4, most of the fractions of the above-ground and underground extracts of open-field and house-cultivated liberated wind did not show β-amylase and α-glucosidase inhibition. However, the hexene fraction of the house-cultivated above-ground extract inhibited α-glucosidase by 24.1% at a concentration of 0.5 mg/ml, and the butanol fraction of the underground extract grown in the open air inhibited β-amylase by 21.0% at a concentration of 0.5 mg/ml, thereby inhibiting antidiabetic activity was expected to represent However, strong simultaneous inhibition of β-amylase and α-glucosidase was confirmed in the hexene fraction, ethyl acetate fraction and butanol fraction of the house-cultivated underground extract.

Example 5: Human Hemolytic Activity of Sequential Organic Solvent Fractions of Haebangpung Extract

The underground part of liberation wind has been used for medicinal purposes for a long time, and the above-ground part has been eaten as a vegetable and spice plant. In the present invention, in order to evaluate the acute toxicity of the Haebangpung extract and its fractions by cultivation method and site, the hemolytic activity of human red blood cells was evaluated for the Haebangpung fractions prepared in Example 3, and the results are shown in Table 5. At this time, hemolytic activity was evaluated according to an existing report (Miseon Kim et al., 2014. J. Life Sci. 24: 515-521), and 100 μl of human red blood cells washed three times with PBS was simply added to a 96-well microplate and various After adding 100 μl of the sample solution of the concentration, the reaction solution was reacted at 37° C. for 30 minutes. Then, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. did DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 5] Human erythrocyte hemolytic activity of Haebangpung extract and fractions

As a result, as shown in Table 5, first, DMSO and water used as controls had no hemolytic activity, and it was confirmed that triton X-100 hemolyzed red blood cells 100% at a concentration of 1 mg/ml. On the other hand, the liberation wind extract showed more than 50% hemolytic activity in the ethyl acetate fraction of the above-ground part of open-air cultivation, the ethanol extract of the underground part of the open-field cultivation, the hexene fraction and the ethyl acetate fraction of the underground part of the open-air cultivation and house cultivation. Considering that, it is expected that the fractions do not present a problem of causing serious acute toxicity. On the other hand, the hexene fraction of the above-ground extract of house-cultivated liberation wind, the butanol fraction of the open-air underground part, and the butanol fraction of the underground part cultivated in the house did not show any hemolytic activity at all.

Example 6: Evaluation of Plasma, Acid and Thermal Stability of Haebangpung Extract and its Active Fractions

Plasma stability, thermal stability, and acid stability with respect to antidiabetic activity were confirmed for the liberated wind extract and active fractions obtained in Examples 1 and 3 above. The samples exhibited high stability because no significant decrease in antidiabetic activity was observed 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, it is expected that the liberated wind extract and its active fractions will maintain excellent antidiabetic activity during the digestion and absorption process and food manufacturing process.

Claims (4)

A pharmaceutical composition for preventing or treating diabetes containing an extract of liberation pung ( Glehnia littoralis ) as an active ingredient. The pharmaceutical composition according to claim 1, wherein the Haebangpung extract is an extract of the above-ground part of the liberation wind grown in the open air or the extract of the basement part of the liberation wind grown in a house. The pharmaceutical composition according to claim 1, wherein the liberated wind extract is a fraction selected from the group consisting of the following (1) to (5):
(1) the butanol fraction of the underground extract of liberated wind grown in the open field;
(2) the hexene fraction of the above-ground extract of liberated wind grown in the house;
(3) the hexene fraction of the underground extract of liberation wind grown in the house;
(4) the ethyl acetate fraction of the underground extract of Haebangpung grown in the house; and
(5) Butanol fraction of Haebangpung underground extract grown in a house. The health functional food for the prevention or improvement of diabetes containing the active ingredient in any one of Claims 1-3.

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