KR20240042861A - Coumaroyl triterpene derivatives derived from Actinidia polygama and their use for preventing or treating diabetes - Google Patents

Abstract

The present invention was completed by confirming the diabetes prevention or treatment effect of coumaroyl triterpene derivatives isolated from Actinidia vulgaris, and relates to a composition for improving, preventing, or treating diabetes containing an extract of Actinidia vulgaris as an active ingredient. The present invention has the advantage that there are very few or no side effects due to use by using Actinidia perilla, a natural product that has been proven to be highly safe for a long time. Accordingly, the present invention is expected to be used in various fields, such as for medicinal purposes for improving, preventing, or treating diabetes, for external medicinal use, for cosmetic materials, for improving physical vitality, for functional bio materials, and for functional food materials.

Description

Coumaroyl triterpene derivatives derived from Actinidia polygama and their use for preventing or treating diabetes {Coumaroyl triterpene derivatives derived from Actinidia polygama and their use for preventing or treating diabetes}

The present invention was completed by confirming the diabetes prevention or treatment effect of coumaroyl triterpene derivatives isolated from Actinidia vulgaris, and relates to a composition for improving, preventing, or treating diabetes containing an extract of Actinidia vulgaris as an active ingredient.

Due to recent advances in medicine and improvements in eating habits, the elderly population is continuously increasing worldwide. Accordingly, the problems of various metabolic diseases are rapidly emerging due to aging, and diabetes in particular is a chronic disease that is increasing at the fastest rate. Diabetes is a metabolic disease in which blood sugar levels rise due to problems in the secretion or reception of insulin, which plays a role in lowering blood sugar levels by absorbing sugar in the blood into cells. Diabetes is known to cause diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, vascular disorders, neuropathy, and arteriosclerosis complications. For diabetic patients, the average life expectancy is reduced by 8 years compared to the general population due to complications, and the total mortality rate is higher. is known to be 2 to 3 times higher.

Diabetes is divided into 'type 1 diabetes', in which insulin is not secreted properly due to problems with the pancreas that secretes insulin, and 'type 2 diabetes', in which insulin is secreted properly but does not function properly due to various reasons. Normally, when blood sugar levels increase, pancreatic β-cells release insulin to induce insulin secretion. Insulin is activated by binding to receptors on the plasma membrane, causing autophosphorylation of tyrosine residues on the receptors, triggering signaling downstream. I order it.

Treatments for diabetes are largely divided into insulin secretion stimulants such as sulfonylureas (glibenclamide, gliclazide, etc.), insulin sensitizers such as thiazolidinediones (pioglitazone, rosiglitazone, etc.), and carbohydrate digestive enzyme inhibitors such as acarbose. It is divided into three categories. Among them, sulfonylurea is mainly used as a second-generation product with a short action time and strong blood sugar-lowering effect, and is used for adult diabetes patients who are not obese, and thiazolidinedione is a representative drug for improving insulin resistance in patients with type 2 diabetes. am. Acarbose is an α-glucosidase inhibitor and is used to improve postprandial blood sugar levels as an additional therapy in diabetic patients who cannot achieve sufficient blood sugar control despite diet and exercise therapy. However, these drugs have been reported to be associated with serious side effects. Among these, gliclazide has been reported to cause abdominal pain and dizziness, and the common side effects of acarbose are diarrhea and abdominal pain.

Currently, there is a need for a safe oral medication that has fast-acting effects, prevents postprandial blood sugar levels from rising, does not cause unnecessary hypoglycemia, and can simultaneously improve metabolic disorders accompanying diabetes. Treatment methods using traditional medicine or natural materials are generally known to have low toxicity and various therapeutic effects due to their diverse content, so they are known to have sufficient potential to simultaneously treat insulin-dependent and non-insulin-dependent diabetes.

Accordingly, the present inventors believed that in modern society where aging is accelerating, the demand for drugs and health functional foods that safely and effectively prevent and treat diabetes without side effects will continue to increase, and thus natural products that can prevent and treat diabetes are expected to increase. By searching, the present invention was completed by isolating and identifying coumaroyl-triterpene derivatives with anti-diabetic activity from Actinidia perilla leaf extract.

Zheng, Y. et al., Nat. Rev. Endocrinol., 2018, 14, 88-98

The technical problem to be achieved by the present invention is to provide a composition derived from natural products for improving, preventing, or treating diabetes.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

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

In addition, the present invention provides a food composition for improving or preventing diabetes containing Actinidia ginseng extract as an active ingredient.

In addition, the present invention provides a method for preventing or treating diabetes comprising the step of administering an extract of Actinidia vulgaris to a subject, and the method may further include measuring the blood sugar level of the subject before and after administration of the Actinidia vulgaris extract. .

In addition, the present invention provides the use of the Actinidia ginseng extract for preventing or preparing a treatment for diabetes.

As an embodiment of the present invention, the Actinidia Actinidia extract may be an extract of Actinidia Actinidia leaf extract, and the extraction solvent may be one extracted using ethanol, preferably 70% ethanol.

As another embodiment of the present invention, the extract of Actinidia Actinidia may be an ethyl acetate fraction of the ethanol extract of Actinidia Actinidia.

As another embodiment of the present invention, the extract of Actinidia chinensis may promote insulin secretion from pancreatic beta cells and may inhibit the activity of alpha-glucosidase.

As another embodiment of the present invention, the extract of Actinidia chinensis may include Compound 1 represented by Formula 1 below and/or Compound 2 represented by Formula 2 below.

[Formula 1]

[Formula 2]

Additionally, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing Compound 1 and/or Compound 2 as an active ingredient.

Additionally, the present invention provides a food composition for improving or preventing diabetes comprising Compound 1 and/or Compound 2 as an active ingredient.

In addition, the present invention provides a method for preventing or treating diabetes comprising the step of administering Compound 1 and/or Compound 2 to an individual, the method comprising: An additional step of measuring blood sugar may be included.

Additionally, the present invention provides the use of Compound 1 and/or Compound 2 for preparing a drug for preventing or treating diabetes.

Additionally, the present invention provides a method for extracting Compound 1 and Compound 2 comprising the following steps:

(1) Extracting Actinidia Actinidia leaves with ethanol to obtain Actinidia Actinidia extract;

(2) fractionation by adding water to the Actinidia serrata extract and sequentially contacting it with hexane, dichloromethanol, ethyl acetate, and butanol; and

(3) Performing column chromatography with the ethyl acetate fraction.

The present invention uses a novel coumalo-triterpene derivative with insulin secretion promoting and alpha-glucosidase inhibitory activity from Actinidia parais, a natural product that has been proven to be highly safe for a long time, and has the advantage of having very little or no side effects due to use. Accordingly, the present invention is expected to be used in various fields, such as for medicinal purposes for improving, preventing, or treating diabetes, for external medicinal use, for cosmetic materials, for improving physical vitality, for functional bio materials, and for functional food materials.

Figure 1 is a diagram depicting the separation process of compounds derived from Ga. Actinidia.
Figure 2 is a chromatogram obtained by HPLC analysis of the ethanol extract of Actinidia chinensis.
Figure 3 shows the results of alpha-glucosidase inhibitory ability of compounds 1 to 2 and acarbose.
Figure 4 shows the cytotoxicity measurement results of compounds 1 to 2 and gliclazide in INS-1 cells.
Figure 5 shows the results confirming that compounds 1 to 2 and gliclazide promote insulin secretion by glucose stimulation in INS-1 cells.
Figure 6 shows the results confirming that compounds 1 to 2 and corosolic acid promote insulin secretion by glucose stimulation in INS-1 cells.
Figure 7 shows the results confirming that compounds 1 and 2 increase the expression of PPAR-γ, PDX-1, IRS-2 (Ser731), P-PI3K, PI3K, P-Akt (Ser473), and Akt in INS-1 cells. am.

Actinidia polygama is a deciduous vine of the Actinidiaceae family and is native to Korea, Japan, and Siberia. It grows at altitudes of 100 to 1,500 m above sea level in Korea and grows in well-drained areas. It is known that Gaisella is traditionally used to treat syphilis, urinary stones, sea fever, abdominal pain, arthritis, and stroke. In the private sector, Actillae has been used to relieve pain, diuresis, high blood pressure, and bronchitis. In modern medicine, it is known to be effective in pain relief, gout, anti-inflammatory, asthma, and arteriosclerosis. Actinidia ginseng contains flavonoid compounds, iridoids, and various types of terpenoids, but little research has been done using these components.

The present inventors separated 3-O-coumaroyl-maslinic acid and 3-O-coumaroyl-hydroxyursolic acid from the extract of Actinidia Actinidia. and identified, and confirmed the insulin secretion promoting and α-glucosidase inhibitory activities of the above two coumaroyl triterpene derivatives.

Therefore, the purpose of the present invention is to provide a composition for improving, preventing, or treating diabetes containing Actinidia ginseng extract as an active ingredient. The composition of the present invention can be used as a pharmaceutical composition for preventing or treating diabetes, and can be used as a food composition for improving or preventing diabetes.

In the present invention, the Actinidia Actinidia extract can be extracted with any conventionally used solvent, preferably water, lower alcohols with 1 to 4 carbon atoms, polyhydric alcohols with 3 to 6 carbon atoms, glycerin, propylene glycol, butylene. It may be extracted with a solvent selected from the group consisting of glycol and mixed solvents thereof, preferably ethanol, more preferably 70% ethanol.

The extraction method using the extraction solvent may be a conventional extraction method in the art, for example, ultrasonic extraction, subcritical extraction, high temperature extraction, high pressure extraction, filtration, or reflux extraction. Preferably, A reflux extraction method may be used, but is not limited thereto.

In addition, the above-mentioned Actinidia chinensis extract can be additionally fractionated using a conventional systematic fractionation method. Specifically, the Actinidia Actinidia fraction is obtained by suspending the above-mentioned Actinidia Actinidia extract in distilled water, sequentially fractionating it using hexane, dichloromethanol, ethyl acetate, and butanol, and obtaining the remaining solubles in distilled water to obtain hexane fraction, dichloromethane fraction, ethyl acetate fraction, A butanol fraction and a water fraction can be obtained.

In the present invention, the Actinidia chinensis extract can be used as is, but it can also be used in the form of a concentrated extract after filtration or in the form of a freeze-dried product by freeze-drying.

The present inventors performed a reflux extraction method using 70% ethanol to obtain an extract of Actinidia vulgaris, fractionated the extract of Actinidia vulgaris with hexane, dichloromethane, ethyl acetate, and n-butanol, and obtained the following formula 1 in the ethyl acetate fraction: Compound 1 (3-O-coumaroyl-maslinic acid) represented by and Compound 2 (3-O-coumaroyl-hydroxyursolic acid) represented by the following formula 2 were separated and identified.

[Formula 1]

[Formula 2]

The coumaroyl triterpene derivatives of Compounds 1 and 2 were obtained by eluting the ethyl acetate fraction of Ga. Actinidia using silica normal phase vacuum chromatography with appropriate solvents ranging from chloroform/methanol 20:1 to chloroform/methanol 0:1 and dividing them into six sub-fractions. When classified, it was separated from the first subfraction.

Compounds 1 and 2, isolated from the ethyl acetate fraction of the ethanol extract of Actinidia chinensis, had alpha-glucosidase inhibitory activity, were not toxic to pancreatic cells, promoted insulin secretion from the pancreas, and inhibited PPAR-γ, PDX-1, It was confirmed that the expression of IRS-2, PI3K, and Akt increased and the phosphorylation of IRS-2, PI3K, and Akt was promoted.

Accordingly, another object of the present invention is to provide a composition for improving, preventing, or treating diabetes containing Compound 1 and/or Compound 2 obtained from the above-mentioned Actinidia chinensis extract as an active ingredient. As described above, the composition can be used as a pharmaceutical composition for preventing or treating diabetes, and can be used as a food composition for improving or preventing diabetes.

In the present invention, Compound 1 and Compound 2 may be used in the form of pharmaceutically acceptable salts, and may be provided as stereoisomers, hydrates, or solvates thereof.

The term “pharmaceutically acceptable salt” as used herein refers to a salt that can be used pharmaceutically among salts that are substances in which cations and anions are combined by electrostatic attraction, and are usually metal salts and organic bases. It may be a salt with a salt, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, etc. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt, etc.; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine. It may be a salt with, etc.; Salts with inorganic acids may include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.; Salts with organic acids may include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; Salts with basic amino acids may include salts with arginine, lysine, ornithine, etc.; Salts with acidic amino acids can be salts with aspartic acid, glutamic acid, etc. Particularly preferred salts include, when the compound has an acidic functional group therein, inorganic salts such as alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and organic salts such as ammonium salts, and when the compound has a basic functional group therein, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc., acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, There are salts with organic acids such as succinic acid, methanesulfonic acid, and p-toluenesulfonic acid.

The composition of the present invention may further contain one or more known active ingredients that have a preventive or therapeutic effect on diabetes along with the Actinidia vulgaris extract or the compounds 1 and/or 2.

The composition of the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Additionally, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods.

Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, and methyl cellulose. , microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. In addition, non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

In addition, the present invention also provides a method for preventing or treating diabetes, comprising administering the composition to an individual, and a use of the composition for preventing or treating diabetes.

In the present invention, “individual” refers to a subject in a high-risk group for diabetes who has developed diabetes or is likely to develop diabetes, and more specifically, refers to a human or non-human primate, mouse, rat, dog, cat, or horse. and mammals such as cattle.

As used herein, the term “administration” means providing a given composition of the invention to an individual by any suitable method.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, and can be appropriately selected by a person skilled in the art. For a desirable effect, the amount of the Actinidia perilla extract of the present invention, its fraction, or the compounds 1 and/or 2 derived therefrom is 0.1 mg/kg to 1000 mg/kg per day, preferably 0.001 to 200 mg/kg. It can be administered in quantity, once a day or in divided doses.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, local injection.

The composition of the present invention can be added to food for the purpose of improving or preventing diabetes, and the food may be a health functional food. In the present invention, a health functional food refers to disease prevention or improvement, biological defense, immunity, It refers to foods that have bioregulatory functions such as recovery from illness and inhibition of aging, and must be harmless to the human body when taken over a long period of time.

When the composition of the present invention is used as a food additive, the Actinidia chinensis extract or Compound 1 and/or Compound 2 can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when producing food or beverages, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in amounts above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, etc. These include alcoholic beverages and vitamin complexes, and include all health foods in the conventional sense.

The health drink composition of the present invention may include various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may include carbonating agents used in carbonated drinks. Additionally, the composition of the present invention may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, various changes can be made to the embodiments, so the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

In addition, when describing with reference to the accompanying drawings, identical components will be assigned the same reference numerals regardless of the reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiments, if it is determined that detailed descriptions of related known technologies may unnecessarily obscure the gist of the embodiments, the detailed descriptions are omitted.

[Example]

Example 1: Preparation of Actinidia chinensis extract and its fractions

Actinidia polygama leaves were obtained from Wangsan-myeon, Gangneung-si, Gangwon-do in September 2016 and dried in warm air to obtain a dry weight of 300 g. The dried leaves of Actinidia were cut into pieces with a crusher, and then refluxed and extracted with an organic solvent of 70% ethanol/water (3L) for 10 hours. The extract was filtered through filter paper, transferred to a round bottom flask (capacity 20 L), and concentrated using a vacuum concentrator. Through this process, 110 g of crude extract of Actinidia perilla leaves was obtained, showing an extraction efficiency of about 37%.

The obtained crude extract was dissolved in water, brought into contact with hexane, the hexane layer was taken, and distilled under reduced pressure to obtain 6.4 g of hexane fraction (yield 5.8%). After removing the hexane layer, dichloromethane was brought into contact with the remaining water layer, and the dichloromethane layer was taken and distilled under reduced pressure to obtain 9.3 g of dichloromethane fraction (yield 8.5%). In addition, the water layer remaining in the process of preparing the dichloromethane fraction was brought into contact with ethyl acetate, the ethyl acetate layer was taken, and distilled under reduced pressure to obtain 9.8 g of ethyl acetate fraction (yield 8.9%). In addition, butanol was brought into contact with the water remaining in the process of preparing the ethyl acetate fraction, the butanol layer was taken, and distilled under reduced pressure to obtain 21.2 g of butanol fraction (yield 19.3%). Finally, the remaining water layer was distilled under reduced pressure to obtain a water fraction, which was freeze-dried and stored.

Example 2: Isolation and structural identification of Compound 1 and Chemical 2 from Actinidia chinensis extract

2-1) Separation of compounds 1 and 2

The ethyl acetate fraction was divided into a total of six subfractions by eluting with an appropriate solvent ranging from chloroform/methanol 20:1 to chloroform/methanol 0:1 using silica normal-phase vacuum flash chromatography. The first subfraction (292.4 mg) was subjected to preparative column chromatography under the conditions shown in Table 1 below to isolate 1.0 mg of Compound 1 having the structure of Formula 1 at a retention time of about 32.7 minutes, and the above was isolated at a retention time of about 37.5 minutes. 1.5 mg of Compound 2 having the structure of Formula 2 was isolated (Figures 1 and 2).

The separation conditions for compounds 1 and 2 are shown in Table 1 below.

Equipment used Gilson 321 HPLC column Phenomenex Luna C-18 RP HPLC column (21.2x250 ㎜, 20 ㎛) menstruum (a) Acetonitrile with 0.02% TFA (b) Water with 0.02% TFA Mobile phase composition Elution begins at 0 minutes with (a) solvent:(b) solvent volume ratio of 80:20. From 0 minutes to 60 minutes, (a) increases the solvent ratio from 80% to 100%. Mobile phase flow rate 8mL/min detector UV 280 nm

2-2) Identification of Compound 1

The molecular weight of Compound 1 isolated in Example 2-1 was determined to be 618 through MS measurement using an Agilent 1100 high-performance fluid chromatography-mass spectrometer (HPLC-ESI-MS), and was determined to be 618 using a nuclear magnetic resonance device (Varian 500 MHz, 125 MHz). Through 1H and 13C NMR spectrum analysis using MHz NMR), the structure was estimated to be 3-O-coumaroyl-maslinic acid having the structure of the following formula (1). In addition, the structure was identified by comparing the NMR data with data from existing literature (Lee et al. Biol. Pharm. Bull, 2004, 11, 1883-1886).

[Formula 1]

Molecular formula C39H54O6; ESI-MS: m/z 619 [M+H]+;

1H NMR (CDCl3, 400 MHz): δH 7.63 (1H, d, J = 15.5 Hz, H-7′), 7.47 (2H, d, J = 8.5 Hz, H-2′, H-6′), 6.81 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.40 (1H, d, J = 15.5 Hz, H-8′), 5.24 (1H, m, H-12), 4.64 (1H, d, J = 10.0 Hz, H-3), 3.62 (1H, m, H-2), 2.20 (1H, d, J = 11.5 Hz, H-18), 1.15 (3H, s, H3- 27), 1.05 (3H, s, H3-25), 0.96 (3H, s, H3-30), 0.96 (3H, s, H3-24), 0.91 (3H, s, H3-29), 0.90 (3H) , s, H3-23), 0.83 (3H, s, H3-26)

2-3) Identification of Compound 2

The molecular weight of Compound 2 isolated in Example 2-1 was determined to be 618 through MS measurement using an Agilent 1100 high-performance fluid chromatography-mass spectrometer (HPLC-ESI-MS), and was determined to be 618 using a nuclear magnetic resonance device (Varian 500 MHz, 125 MHz). Through 1H and 13C NMR spectrum analysis using MHz NMR), the structure was estimated to be jacoumaric acid having the structure of Formula 2 below. In addition, the structure was identified by comparing the NMR data with data from existing literature (Numata et al. Chem. Pharm. Bull, 1989, 37, 648-651).

[Formula 2]

Molecular formula C39H54O6; ESI-MS: m/z 619 [M+H]+;

1H NMR (CDCl3, 400 MHz): δH 7.63 (1H, d, J = 16.0 Hz, H-7′), 7.46 (2H, d, J = 8.5 Hz, H-2′, H-6′), 6.80 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.34 (1H, d, J = 16.0 Hz, H-8′), 5.22 (1H, m, H-12), 4.10 (1H, d, J = 9.5 Hz, H-3), 3.62 (1H, m, H-2), 2.22 (1H, d, J = 11.5 Hz, H-18), 1.17 (3H, s, H3- 27), 1.09 (3H, s, H3-25), 0.96 (3H, d, J = 6.5 Hz, H3-30), 0.95 (3H, s, H3-24), 0.90 (3H, s, H3-23) ), 0.88 (3H, d, J = 6.5 Hz, H3-29), 0.88 (3H, s, H3-26)

Example 3: Confirmation of physiological activity of compounds 1 to 2

3-1) Measurement of alpha-glucosidase inhibitory activity

Alpha-glucosidase activity was measured using an assay kit (MAK 123, Sigma-Aldrich). The isolated compound was mixed with alpha-glucosidase enzyme solution (200 μL), kept at 37°C for 20 minutes, and measured using a microplate reader at 405 nm. Acarbose was used as a positive control.

The half-maximum inhibitory concentration (IC50) of compound 1 was 21.79 ± 0.81 μM, and the IC50 of compound 2 was 29.93 ± 3.68 μM, which was similar to the IC50 of the positive control acarbose, 29.67 ± 1.12 μM (Figure 3).

3-2) Measurement of toxicity to INS-1 cells

INS-1 cell line (Biohermes) is a rat insulinoma cell line and has the ability to secrete insulin.

Cultured INS-1 cells were supplemented with 10% fetal bovine serum (FBS), 1% phenesirin/streptomycin, 11mM D-glucose, 2mM L-glutamine, 10mM HEPES, 0.05mM The cells were cultured in RPMI-1640 (Cellgro) medium containing 2-mercaptoethanol and 1 mM sodium pyruvate in a 95% air/5% carbon dioxide incubator maintained at 37°C. Among the ingredients mentioned above, reagents without manufacturer mention were purchased from Gibco BRL, Life Technologies.

Cultured INS-1 cells were dispensed at ^1x104 per well into a 96-well plate and cultured to allow the cells to stabilize for 24 hours. Afterwards, Compound 1 and Compound 2 were treated at concentrations of 2.5 μM, 5 μM, and 10 μM and measured using an Ez-Cytox kit (Daeil Lab Service Co), and the measurement wavelength was 405 nm (Figure 4).

Both Compound 1 and Compound 2 showed no toxicity at the corresponding concentrations.

3-3) Measurement of effect on insulin secretion of INS-1 cells

Cultured INS-1 cells were distributed in 4x10 ⁵ cells in a 12-well plate and cultured to allow the cells to stabilize for 24 hours. Then, they were added with 4.4 mM potassium chloride (KCl), 129 mM sodium chloride (NaCl), and 1.2 mM potassium phosphate ( KH2PO4), 1.2mM magnesium sulfate (MgSO4), 2.5mM calcium chloride (CaCl2), 10mM Hepes, 5mM sodium bicarbonate (NaHCO3), and 0.1% bovine serum albumin (BSA) were mixed and the hydrogen ion concentration was adjusted to pH. Washed twice with Krebs-Ringer buffer adjusted to 7.4.

Afterwards, the Krebs-Ringer buffer was added to the washed cells and incubated for 2 hours, then treated with 2.5 μM, 5 μM, and 10 μM of Compound 1 and Compound 2 and incubated for 2 hours. The wells were treated with 2.8mM and 16.7mM of glucose for 1 hour, and the amount of insulin was measured using a rat insulin ELISA kit (Gentaur). Stimulation index (SI) is the value measured at high glucose concentration, i.e., the amount of insulin secreted by stimulation of 16.7mM glucose concentration, compared to the value measured at low glucose concentration, i.e., the amount secreted by stimulation of 3.3mM glucose concentration. It was calculated by dividing by the amount of insulin administered. Gliclazide was used as a positive control.

The SI of compound 1 ranged from 1.29 to 4.29 between 2.5 μM and 10 μM, and the SI of compound 2 ranged from 2.11 to 3.93 between 2.5 μM and 10 μM. Gliclazide showed 3.11 to 11.37 at the same concentration. Although the SI of Compound 1 and Compound 2 was not superior to that of gliclazide, it showed a significant result, showing a nearly 4-fold increase compared to the untreated group (Figure 5).

3-4) Check the effect on protein expression

Cultured INS-1 cells were distributed in 8x10 ⁵ cells in a 6-well plate and cultured to allow the cells to stabilize for 24 hours, and then treated with Compound 1 and Compound 2 at a concentration of 10 μM. PPARγ, PDX-1, Phospho-IRS2 (P-IRS2) (Ser731), IRS-2, Phospho-PI3K (P-PI3K), PI3K, Phospho-Akt (P-Akt) (Ser473), and Akt were identified by Western blot. It was measured by . All antibodies were purchased from Cell Signaling. Cells were lysed with RIPA buffer containing protease inhibitors for 20 min. Samples containing a protein concentration of 20 μg were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The above membranes were cultured overnight at 4°C with PPARγ, PDX-1, P-IRS2, IRS-2, P-PI3K, PI3K, P-Akt, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and then incubated with The cells were incubated with dish peroxidase (HRP)-conjugated anti-rabbit secondary antibody at room temperature for 1 hour. Protein expression was visualized using enhanced chemiluminescence reagents and a chemiluminescence system.

The expression of PPAR-γ, PDX-1, IRS-2, PI3K, and Akt was increased by compound 1 and compound 2, and the phosphorylation of IRS-2, PI3K, and Akt was also shown to be promoted (Figure 7).

Example 4. Comparison with corosolic acid

The effect of Compound 1 and Compound 2 on insulin secretion in INS-1 cells was compared with that of corosolic acid by the method described in Example 3-2 above.

Compound 1 showed an insulin secretion ability of at least 140 ng/ml per hour at 5 μM and 10 μM, and compound 2 showed an insulin secretion ability of at least 150 ng/ml per hour at 5 μM and 10 μM. At the same concentration, corosolic acid showed an insulin secretion ability of 120 ng/ml per hour, so it was confirmed that Compound 1 and Compound 2 were superior to corosolic acid in promoting insulin secretion (FIG. 6).

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (10)

A pharmaceutical composition for preventing or treating diabetes containing an extract of Actinidia vulgaris as an active ingredient. According to paragraph 1,
A pharmaceutical composition, characterized in that the extract is an Actinidia vulgaris leaf extract. According to paragraph 1,
A pharmaceutical composition, characterized in that the extract is extracted with 70% ethanol as a solvent. According to paragraph 3,
A pharmaceutical composition, characterized in that the extract is an ethyl acetate fraction of an ethanol extract of Actinidia vulgaris. According to paragraph 1,
A pharmaceutical composition, wherein the extract of Actinidia vulgaris contains Compound 1 represented by the following Chemical Formula 1 and/or Compound 2 represented by the following Chemical Formula 2.
[Formula 1]

[Formula 2]
A pharmaceutical composition for preventing or treating diabetes, comprising Compound 1 represented by Formula 1 below and/or Compound 2 represented by Formula 2 below as an active ingredient.
[Formula 1]

[Formula 2]
According to clause 5,
A pharmaceutical composition, characterized in that Compound 1 and Compound 2 are derived from Actillae. A method for extracting compound 1 represented by formula 1 and compound 2 represented by formula 2, comprising the following steps:
(1) Extracting Actinidia Actinidia leaves with ethanol to obtain Actinidia Actinidia extract;
(2) fractionation by adding water to the Actinidia serrata extract and sequentially contacting it with hexane, dichloromethanol, ethyl acetate, and butanol; and
(3) Performing column chromatography with the ethyl acetate fraction.
[Formula 1]

[Formula 2]
A food composition for improving or preventing diabetes containing an extract of Actinidia vulgaris as an active ingredient. A food composition for improving or preventing diabetes, comprising Compound 1 represented by Formula 1 below and/or Compound 2 represented by Formula 2 below as an active ingredient.
[Formula 1]

[Formula 2]