KR102131350B1 - Composition for preventing or treating diabetes comprising (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmene - Google Patents

Abstract

The present invention comprises 3,5,6,9-tetrahydroxy-megastigman-7-ene (3,5,6,9-tetrahydroxy-megastigman-7-ene) derived from mallow as an active ingredient, It relates to a pharmaceutical composition for preventing or treating diabetes, a food composition for preventing or improving.
Since the pancreatic degree damaged by the compound derived from the mallow fraction in the present invention is recovered and the pancreas is also confirmed to increase the absorption of glucose in cells, the composition of the present invention can be widely used in the development of anti-diabetic compositions and health functional foods. Will be able to.

Description

(3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmen containing composition for preventing or treating diabetes (Composition for preventing or treating diabetes comprising (3S,5R) ,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmene}

The present invention is derived from mallow (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmen [(3S,5R,6R,7E,9R)-3 ,5,6,9-tetrahydroxy-7-megastigmene] as an active ingredient, diabetes prevention or treatment pharmaceutical composition; And a food composition for prevention or improvement.

Diabetes mellitus is a disease in which insulin deficiency or insulin sensitivity decreases and carbohydrate metabolism occurs. Insulin, a polypeptide hormone, is produced by β-cells in the pancreatic islets (PI) in the pancreas. When the blood glucose level increases, it is secreted and when it decreases, secretion is suppressed to regulate the proper activity of the energy source. In diabetic patients, the decrease in the number of β-cells and its dysfunction occurred.

The American Diabetes Association divides the medical classification of diabetes into two types: insulin-dependent diabetes (type 1) and insulin-independent diabetes (type 2). The etiology of insulin-independent diabetes (type 2), which accounts for more than 95% of diabetes, is known to have two causes: a complex disorder of insulin secretion and insulin resistance. Insulin secretion disorder refers to a situation in which an appropriate amount of insulin is not secreted from β-cells in the pancreas according to the blood glucose level, which includes both a quantitative decrease in β-cells secreting insulin or a functional secretion disorder. Insulin resistance refers to a situation in which secreted insulin rides the bloodstream and reaches a target organ, but insulin action and sensitivity are degraded in the target cell, which is generally considered to be a signaling impairment after cell membrane receptor binding, and its cause is a genetic predisposition , Obesity, physical inactivity and hyperglycemia or dyslipidemia. With insulin resistance, a greater amount of insulin must be secreted to overcome the resistance, and conversely, hyperglycemia itself caused by insufficient insulin secretion may deteriorate insulin resistance again. If left untreated, diabetes can lead to diabetic retinopathy, kidney disease, mammary disease, cardiovascular complications and microvascular complications. Therefore, it is possible to prevent death due to diabetes and reduce the mortality rate by controlling insulin preparations or hypoglycemic drugs.

Thus, restoring damaged pancreatic β-cells or increasing pancreatic islet glucose uptake may be a significant treatment for diabetes (Sunil C et al ., Effect of ethanolic extract of Pisonia alba Span. leaves on blood glucose levels and histological changes in tissues of alloxan-induced diabetic rats.Int J Appl Res Nat Prod 2009;2:4-11). Materials used to treat diabetes to date are drugs that increase insulin secretion or increase sensitivity to insulin, such as pioglitazone and rosiglitazone, which enhance the response of insulin, and metformin, which improves insulin resistance. There are a group of sulfonylurea and meglitinide that stimulate insulin secretion in β-cells of the pancreas. However, since these drugs have side effects such as weight gain symptoms as well as toxicity to the liver, kidneys, muscles, and heart, there are few side effects and are effective for treating or preventing diabetes, without causing weight gain, etc. The need for development is in desperate need.

Under the above background, the present inventors tried to find a substance having an anti-diabetic effect derived from natural products, and (3S,5R,6R,7E,9R)-3,5,6,9-tetra derived from the fraction above Hydroxy-7-megastigmen and L-tryptophan were separated and identified, and (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmen were treated. When the size of the damaged pancreas is restored, especially when L-tryptophan is used together (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmen It was confirmed that a higher anti-diabetic effect appeared compared to using alone, and the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Another object of the present invention is to provide a food composition for preventing or improving diabetes, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

As one aspect for achieving the above object, the present invention comprises a compound represented by Formula 1 (hereinafter,'Compound 1') or a pharmaceutically acceptable salt thereof as an active ingredient, diabetes prevention or treatment pharmacy Provides a composition.

[Formula 1]

The'compound 1'is (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmen [(3S,5R,6R,7E,9R)-3, 5,6,9-tetrahydroxy-7-megastigmene], the effect of treating compound 1 on diabetes is not known and was first confirmed in the present invention.

In addition, the composition of the present invention may further include a compound represented by the following Chemical Formula 2 (hereinafter,'Compound 2') or a pharmaceutically acceptable salt thereof.

[Formula 2]

The'compound 2'is one of α-amino acids, L-tryptophan (L-tryptophan), which is an essential amino acid that is not synthesized in the body.

The present invention has the efficacy of treating diabetes mellitus isolated from the extract or its fraction has the effect of treating diabetes, and further, when the compound 1 and the compound 2 are used in combination, the antidiabetic effect is compared to the case where the compound 1 or the compound 2 is used alone. It is based on the technical idea of further improvement, which was first identified by the inventors.

In the present invention, the compound 1 and the compound 2 may be identified by separating from the mallow extract or a fraction thereof. At this time,'Mook ( Malva verticillata )'can be purchased commercially, or used in nature or grown. The mallow extract can be extracted from various organs of natural, hybrid, and varietal plants, for example, it can be produced by extracting from mallow tissue culture as well as above ground parts, roots, stems, leaves, and especially using mallow above-ground parts. However, it is not limited thereto.

In the present invention, the'Mook extract' is a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, butanol, and mixed solvents thereof, and more specifically, methanol as a solvent. It may be the result of extraction. In addition, the extraction temperature may be 20 to 100°C, 20 to 80°C, 30 to 60°C, 40 to 50°C, and the extraction period may be hot water extraction, cold immersion extraction, reflux cooling extraction or ultrasonic extraction for about 1 hour to 4 days. It may be extracted using an extraction method such as, but is not limited thereto. Any method for producing a mallow extract to isolate Compound 1 having a diabetes treatment effect can be used without limitation.

In the present invention,'fractionate' means a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components. Specifically, the present invention includes the result of fractionation of the mallow extract by various methods such as a solvent fractionation method, an ultrafiltration fractionation method, and a chromatographic fractionation method, and specifically, may be an n -butanol fraction of the mallow extract.

According to an embodiment of the present invention, after obtaining a methanol extract of mallow and obtaining an n -butanol fraction thereof, silica gel column chromatography was applied to the fraction to separate compound 1 and compound 2.

In addition, Compound 1 and Compound 2 of the present invention may be separated from other natural sources except mallow, and may be chemically synthesized or commercially available materials through methods known in the art, including relationships with the intake route Can be used in the present invention without.

'Pharmaceutically acceptable salt' in the present invention means a salt in a form that can be used pharmacologically among salts, which are materials in which cations and anions are bound by electrostatic attraction, and are relatively non-toxic and harmless to the patient. Any concentration of any organic or inorganic addition salt of the compound that does not degrade the beneficial effects of Compound 1 and Compound 2 as a side effect due to this salt as a concentration having Such salts include acid addition salts formed by pharmaceutically acceptable free acids or metal salts formed by bases.

Organic and inorganic acids can be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and maleic acid can be used as the organic acid ( maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, etc. can be used, It is not limited to this.

In addition, bases can be used to make metal salts that are pharmaceutically or food acceptable. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the inexpensive compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to manufacture sodium, potassium, or calcium salts for pharmaceutical or food use, but is not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of Compound 1 or Compound 2 of the present invention include salts of acidic or basic groups that may be present in Compound 1 or Compound 2, unless otherwise specified. For example, pharmacologically or foodly acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups are hydrobromide, sulfate, hydrogen sulfate, phosphate , Hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like. It can be prepared through a known method of salt preparation.

In the present invention,'diabetes disease' refers to a disease that occurs when insulin secretion is insufficient or insulin action and function are not sufficiently achieved. If you have diabetes, excessive breakdown of glycogen, protein, and fat causes an abnormal increase in glucose concentration in the liver or blood, leading to diabetes and ketoneuria, circulatory disorder with blood concentration due to loss of electrolyte due to abnormality in moisture and electrolyte metabolism, This will lead to pathological conditions such as kidney failure. Insulin is secreted from β-cells of the pancreas, which is present in the pancreas, and is secreted when the blood glucose level is increased. Diabetes is generally diagnosed by measuring blood glucose concentration. In humans, diabetes is usually diagnosed when blood glucose is above 200 mg/dl and 140 mg/dl on an empty stomach. Therefore, the damaged pancreatic β-cells may be restored or the pancreas may also be useful for treating or preventing diabetes by increasing glucose uptake in the cells.

In the present invention,'prevention' refers to all actions of suppressing or delaying diabetes by administration of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, in the present invention,'treatment' refers to all actions in which symptoms of diabetes are improved or cured by administration of a composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Diabetes prevention or treatment in the present invention may be achieved by increasing the size of the damaged pancreas. When the compound 1 of the present invention is treated, the damaged pancreas is restored, the size of the pancreas is increased by 22.3%, and absorption of glucose is improved. In addition, when treating the composition of the compound 1 and compound 2 mixture, it was confirmed that the pancreas size increased by 48.6% and the absorption of glucose was improved. That is, the compound 1 can be used as an active ingredient of the composition for anti-diabetic by exhibiting the efficacy of treating diabetes, and in particular, by using a mixture of compound 1 and compound 2, it is possible to prepare a composition with improved anti-diabetic effect.

Furthermore, the mixing ratio of compound 1 and compound 2 in the present invention may affect the effect of treating diabetes. Specifically, in the present invention, the mixing ratio of Compound 1 and Compound 2 is 5:1 to 1:1, 4:1 to 1:1, 4:1 to 1.5:1, and 3.5:1 to 1.5:1, based on weight. 3: 1 to 1.5: 1, 2.5: 1 to 1.5: 1, 2: 1 to 1.5: 1, 2.2: 1 to 1.8: 1, or 1.96: 1, but is not limited thereto. The mixing ratio of the compound 1 and the compound 2 may be changed according to the treatment target, the severity of the disease, the treatment environment, and the like, and the appropriate mixing ratio can be determined and used by those skilled in the art as long as it can achieve the diabetes treatment effect.

In one embodiment of the present invention, the weight of Compound 1 and Compound 2 contained in the n -butanol fraction of the mallow extract was analyzed to confirm that Compound 1 and Compound 2 contained 5.58 ± 0.16% and 2.85 ± 0.13%, respectively. . That is, in the n -butanol fraction of the mallow extract, Compound 1 and Compound 2 are mixed in an amount of about 1.96:1, and in this case, a better diabetes treatment effect is seen than Compound 1 alone.

In the present invention, the'pharmaceutical composition' means that it is prepared for the purpose of preventing or treating a disease, and can be administered in various dosage forms, oral and parenteral, during actual clinical administration. It can be prepared using diluents or excipients such as extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.

In addition, pharmaceutically acceptable additives may be further included according to each formulation, and pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol and talc can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the mixed extract of the present invention, such as starch, calcium carbonate, water It can be prepared by mixing Sucrose, Lactose, or gelatin. In addition, lubricants such as magnesium stearate talc may be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, are included. Can.

Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The composition of the present invention may be administered orally or parenterally depending on the desired method, and when administered parenterally, it is used for external or intraperitoneal injection, intracutaneous injection, subcutaneous injection, intravenous injection, intramuscular injection, or intramuscular injection. You can choose The dosage may vary in range depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and disease severity.

The composition of the present invention can be administered in a pharmaceutically effective amount. The pharmaceutically effective amount is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and means an amount that does not cause side effects, and the effective dose level is the patient's health condition, type of disease, severity, The activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including the drug used in combination or co- and other factors well known in the medical field can be determined. In addition, the pharmaceutical composition of the present invention may be used alone or in combination with other pharmaceutically active compounds exhibiting an anti-diabetic, therapeutic, or improving effect or in an appropriate collection.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount capable of obtaining a maximum effect in a minimum amount without causing side effects, and can be easily determined by those skilled in the art.

As another aspect of the present invention, there is provided a food composition for preventing or improving diabetes, comprising the compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In addition, the composition of the present invention may further include a compound represented by the following Chemical Formula 2 or a pharmaceutically acceptable salt thereof.

[Formula 2]

Compound 1, compound 2, pharmaceutically acceptable salts, diabetes, the description of the prevention is as described above. Incidentally, the diabetes treatment effect of compound 1 and the treatment effect of diabetes when compound 1 and compound 2 are mixed are as described above.

In the present invention,'improvement' refers to all actions related to a condition related to a condition to be treated by ingestion of the food composition, for example, reducing the severity of symptoms.

In the present invention, the food composition may include a food additive that is food-pharmaceutically acceptable in addition to Compound 1 and/or Compound 2, which are active ingredients.

In the present invention,'food supplement additive' means a component that can be added to food supplementally, and is added to prepare a health functional food of each formulation and can be appropriately selected and used by those skilled in the art. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like, but the types of food supplements of the present invention are not limited by the examples.

The food composition of the present invention may include a health functional food. In the present invention, the term'health functional food' means the same food as a specific health food, a functional food, and a health food, and refers to a food with high medical and medical effects processed to efficiently exhibit bio-regulatory functions in addition to nutrition. Silver may be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills to obtain useful effects in preventing or improving hair loss-related diseases. Unlike general medicines, it has the advantage that there is no side effect that can occur when taking medicines for a long time using food as a raw material, and it has excellent portability, and the health functional food of the present invention can be ingested as an adjuvant for preventing or improving diabetes. .

There is no restriction on the form that the health functional food of the present invention can take, and may include all foods in a conventional meaning, and can be mixed with terms known in the art, such as functional food. Specifically, the food is a food prepared by adding the food composition of the present invention to food materials such as beverages, teas, spices, gums, confectionery, or by encapsulation, powdering, suspension, etc., for example, various foods, beverages , Gum, tea, vitamin complex, health functional food, etc. can be used. It may be prepared by mixing a known additive with other suitable auxiliary ingredients that may be included in the food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gums, ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and There are vitamin complexes, etc., and can be prepared by adding the compound represented by Chemical Formula 1 according to the present invention as a main component to juice, tea, jelly, and juice. It also includes foods used as feed for animals.

In another aspect of the present invention, the compound represented by Formula 1, comprising the step of separating the compound represented by Formula 1, a compound represented by Formula 2 or a mixture thereof from the mallow extract or a fraction thereof, Provided are methods for preparing the compounds or mixtures thereof.

[Formula 1]

[Formula 2]

Mallow, extract, fraction, compound 1, the description of compound 2 is as described above. Incidentally, the diabetes treatment effect of compound 1 and the treatment effect of diabetes when compound 1 and compound 2 are mixed are as described above.

Furthermore, the present invention provides a method of treating diabetes, comprising administering the pharmaceutical composition to a suspected diabetes subject.

Description of the pharmaceutical composition, diabetes is as described above.

In the present invention, the subject may refer to all animals, including humans, who may or may have diabetes. The animals may be mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc., which require treatment of similar symptoms as well as humans, but are not limited thereto. Diabetes can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to an individual. In addition, the pharmaceutical composition of the present invention may exhibit a synergistic effect by administration in parallel with an existing therapeutic agent.

Since the pancreatic degree damaged by the compound derived from the mallow fraction in the present invention is recovered and the pancreas is also confirmed to increase the absorption of glucose in cells, the composition of the present invention can be widely used in the development of anti-diabetic compositions and health functional foods. Will be able to.

Figure 1 shows the HPLC chromatogram (280 nm) of the n -BuOH fraction (1000 ppm) at the far end (Compound 1, RT: 6.87; Compound 2, RT: 8.04).
Figure 2 is a diagram showing the pancreas area and fluorescence intensity image, (A) pancreatic area ( ^### p <0.001; compared with the normal group), ( ^* p <0.05, ^*** p <0.001; alloxane Compared to the induction group), (B) DIC image and fluorescence image of the pancreas diagram, and (C) fluorescence image of the pancreas diagram.
FIG. 3 shows the recovery effect of Compounds 1, 2, and mixtures thereof on zebrafish cheers treated with alloxan (AX) and diazooxide (Dazoxide; DZ), (A) pancreatic area ( ^{# ##} p <0.001; compared to normal group), ( ^* p <0.05, ^*** p <0.001; compared to alloxane-derived group), ( ⁺ p <0.05, ⁺⁺ p <0.01), (B) pancreas It shows the fluorescent image of the figure.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

< Example 1> Materials, instruments, and reagents

1-1. Plant material

Mallow used in this example ( Malva The verticillata ) surface was collected in April 2016 at a commercial farm in Namyangju-si, Korea, and a sample sample (KHU20160419) is stored in the Natural Product Chemistry laboratory at Kyunghee University.

1-2. device

Compounds 1 and 2 were detected with UV lamp Spectroline Model ENF-240 C/F (Spectronics Corporation, USA) and 10% H ₂ SO ₄ solvent. The optical rotation was investigated on a JASCO P-1010 digital polarimeter (Jasco, Japan).

Infrared (IR) spectra were performed with a Perkin Elmer Spectrum One FT-IR spectrometer (England). Melting points were determined on Fisher-John's Melting Point Apparatus (Fisher Scientific, USA) and uncorr. EI-MS was performed with JEOL JMSAX-700 (Japan). ¹ H NMR spectrum (400 MHz) and ¹³ C NMR spectrum (100 MHz) and 2D NMR spectrum were performed with a Varian Unity Inova AS-400 FT-NMR spectrometer (USA) (Park JH et al ., Inhibition of NO production in LPS-stimulated RAW264. 7 macrophage cells with curcuminoids and xanthorrhizol from the rhizome of Curcuma xanthorrhiza Roxb. and quantitative analysis using HPLC.Appl Biol Chem 2014;57:407-412).

1-3. reagent

Distilled water was used in all experiments. HPLC-gradient water, acetonitrile, and methanol are J.T. It was purchased from Baker (Phillipsburg, NJ, USA). Alloxan monohydrate and sea salt are used by Sigma Chemical Co. (St. Louis, MO, USA).

2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) is purchased from Invitrogen (Life Technologies, Grand Island, NY, USA) Did. Glimepiride (GLM) is manufactured by Cayman Chemical Co. (Ann Arbor, MI, USA). Diazoxide (DZ) is a Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

An HPLC instrument was used for HPLC (Shimadzu, Japan), and an fluorescence microscope was used for Olympus 1X70 microscope (Olympus, Japan). Focus Lite (Focus Co, Daejeon, 85 Korea) and Image J software (National Institutes of Health) were used for image analysis (Nam YH et al ., Synergistic potentials of coffee on injured pancreatic islets and insulin action via K _ATP channel blocking in zebrafish. J Agric Food Chem 2015; 63:5612-5621).

< Example 2> Extract and fraction of mallow

The upper part of the dry mallow (3.1 kg) was extracted with 80% methanol (MeOH, 54.0 L×5) at room temperature for 24 hours. The extract was filtered through filter paper and evaporated under reduced pressure at 43° C. to give 794 g of extract. The obtained methanol extract was suspended in water (2 L), and extracted successively with ethyl acetate (EtOAc, 2 L×4) and n -butanol ( n- BuOH, 2 L×4). Each layer was concentrated to give ethyl acetate fraction (MVE, 80 g), n -butanol fraction (MVB, 77 g) and water fraction (MVW, 637 g).

< Example 3> mallow n - BuOH From fractions Isolation and identification of compounds

3-1. Mallow n - BuOH From fractions Separation of compounds

The n -butanol fraction of Example 2 (MVB, 77 g) was applied to silica gel column chromatography (φ 10×15 cm) and chloroform (CHCl ₃ )-methanol (MeOH)-water (H ₂ O) (25:3: As a result of separating 1 → 22: 3: 1 → 20: 3: 1 → 18: 3: 1 → 15: 3: 1 → 12: 3: 1, each 18.8L) as eluent, 14 fractions (MVB- 1 to MVB-14).

Fraction MVB-12 (11.0 g, elution volume/total volume (V _e /V _t ) 0.756-0.898) was applied to reverse phase chromatography (φ 6.5 × 5 cm) and methanol-water (1: 1 → 2: 1 → 3 : 1 → 5: 1, each eluted with 3.8 L) to obtain 18 fractions (MVB-12-1 to MVB-12-18).

Of these, fraction MVB-12-1 (450.3 mg, V _e /V _t 0.001-0.006) was subjected to silica gel column chromatography (φ 3.0×18 cm) and ethyl acetate- n -butanol-water (10:3: Compound 1 [MVB-12-1-6, 84.4 mg, V _e /, purified with 11 fractions (MVB-12-1-1 to MVB-12-1-11), eluting with 1, 4.6 L) V _t 0.139-0.200, TLC (SiO ₂ ) R _f 0.61, ethyl acetate- n -butanol-water (2: 3: 1), TLC (ODS) R _f 0.90, methanol-water (5: 10)] and compound 2 [ MVB-12-1-9, 64.3 mg, V _e /V _t 0.296-0.517, TLC (SiO ₂ ) R _f 0.45, ethyl acetate- n -butanol-water (2: 3: 1), TLC (ODS) R _f 0.75, methanol-water (5:10)] was obtained.

3-2. Identification of isolated compounds

NMR, IR and FAB-MS analysis were performed on the compounds 1 and 2 obtained in Example 3-1 to identify the structure of each compound.

The results of analyzing Compound 1 are as follows.

-Dark brown powder

-[α] _D +35.1° ( c 0.10, MeOH)

-Melting point 98-100 ℃

-UV λ _max = 232, 280 nm

-positive FAB-MS m/z 245 [M+H] ⁺

-IR (KBr, ν) 3383, 1623 cm ^-1

^-1 H-NMR (CD ₃ OD, 400 MHz, δ _H , coupling pattern, J in Hz): 0.97 (3H, s, H-13), 1.15 (3H, s, H-12), 1.18 (3H, s, H-11), 1.21 (3H, d, 6.8, H-10), 1.41 (1H, dd, 9.6, 13.0, H-2b), 1.76 (1H, dd, 2.0, 13.0, H-2a), 1.89 (1H, dd, 8.0, 14.6, H-4b), 2.45 (1H, dd, 5.6, 14.6, H-4a), 4.48 (1H, m, H-3), 4.28 (1H, m, H-9 ), 5.67 (1H, dd, 6.0, 15.4, H-8), 5.89 (1H, d, 15.4, H-7);

^-13 C-NMR (100 MHz, CD ₃ OD, δ _C ): 20.2 (C-13), 23.8 (C-10), 25.4 (C-12), 29.4 (C-11), 35.8 (C-1 ), 38.9 (C-4), 44.2 (C-2), 67.3 (C-5), 68.6 (C-9), 71.1 (C-6), 73.3 (C-3), 125.5 (C-7) , 139.2 (C-8).

As a result of the analysis, Compound 1 was confirmed to be (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmen (D'Abrosca B et al ., Structure elucidation and phytotoxicity of C ₁₃ nor-isoprenoids from Cestrum parqui.Phytochem 2004;65:497-505; Greca MD et al ., Stratioside II-a C ₁₃ Norterpene Glucoside from Pistia stratiotes . Nat Prod Lett 1996; 8:83-86).

The results of analyzing Compound 2 are as follows.

-Dark yellow powder

-Melting point 280-285 ℃

-[α] _D -33.0° ( c 0.20, H ₂ O)

-UV λ _max = 280 nm

-positive FAB-MS m/z 205 [M+H] ⁺

-IR (KBr, ν) 3404, 3254, 1730, 1631, 1588 cm ^-1

^-1 H-NMR (CD ₃ OD and D ₂ O, 400 MHz, δ _H , coupling pattern, J in Hz): 3.20 (1H, dd, 9.7, 15.0, H-8b), 3.46 (1H, dd, 4.5 , 15.0, H-8a), 3.91 (1H, dd, 4.5, 8.7, H-9), 7.09 (1H, dd, 7.8, 7.8, H-5), 7.17 (1H, dd, 7.8, 7.8, H- 6), 7.25 (1H, s, H-2), 7.42 (1H, d, 7.8, H-4), 7.70 (1H, d, 7.8, H-7)

^-13 C-NMR (CD ₃ OD and D ₂ O, 100 MHz, δ _C ): 27.9 (C-8), 57.9 (C-9), 108.8 (C-3), 111.8 (C-7), 118.8 (C-4), 119.7 (C-5), 122.1 (C-6), 124.3 (C-2), 127.7 (C-3a), 138.0 (C-7a), 173.6 (C-10).

As a result of the analysis, it was confirmed that compound 2 is L-tryptophan (Wang X et al ., Isolation, purification and identification of antioxidants in an aqueous aged garlic extract.Food Chem 2015;187:37-43).

< Example 4> Compounds 1 and 2 HPLC analysis

The relative amounts of Compound 1 and Compound 2 in the n- BuOH fraction of Mallow were calculated through HPLC analysis.

Each compound (1.0 mg) was dissolved in 1 mL of a 6:4 mixture of methanol and water, and compounds 1, 62.5, 31.3, 15.6, 7.8, and 3.9 μg diluted to sequential concentrations of 125, 62.5, 31.3, and 15.6 μg/mL. A calibration curve was created by injecting Compound 2 standard solution diluted to a sequential concentration of /mL. The average area (n=3) resulting from the standard solution was plotted as a concentration versus peak area to establish a calibration equation for Compound 1 and Compound 2.

For quantitative analysis of each compound, 1 g of the upper part of the dried mallow was extracted with 80% methanol (100 mL × 3) at room temperature for 24 hours. The extract was filtered through filter paper and evaporated under reduced pressure at 43° C. to give 234 mg of extract. The obtained methanol extract was suspended in 100 mL of water, and extracted successively with ethyl acetate (EtOAc, 100 mL × 3) and n -butanol ( n -BuOH, 100 mL × 3). Each layer was concentrated to give ethyl acetate fraction (MVE, 58.2 mg), n -butanol fraction (MVB, 41.3 mg) and water fraction (MVW, 134.5 mg).

A 10-μL aliquot of n -butanol fraction solution (500 μg/mL) was injected into the HPLC system. The analysis was performed using Shimadzu LC-20 (Kyoto, Japan) and Shimadzu SPD-20AV UV detector (280 nm). As a column, Shim-pack GIS (250 mm×4.6 mm, particle size 5 μm) was used. As the mobile phase, 0.01% TFA (solvent A) and acetonitrile (solvent B) in water were used, and B: 15% (0.01 min) at a flow rate of 1.0 mL/min → B: 15% (10 min) → B: 90% (40 min) → B: eluted with a gradient elution of 15% (50 min). Quantitative analysis was repeated 3 times.

The HPLC chromatogram is shown in FIG. 1. Table 1 shows the regression equations and correlation coefficients (r ² , 0.998-1.000) for Compound 1 and Compound 2. It was confirmed that the analysis was reliable through a high r ² value.

compound Residence time (minutes) Regression equation r ² density(%) One 6.87 y = 824.98x + 32719 r ² =0.998 5.58 ± 0.16 2 8.04 y = 10956x + 9986.3 r ² =1.000 2.85 ± 0.13

The relative concentrations of Compound 1 and Compound 2 were determined to be 5.58 ± 0.16% and 2.85 ± 0.13%, respectively, using the peak areas in the chromatogram and the regression equations in Table 1.

< Example 5> From zebrafish Pancreatic islet Recoverability Measure

5-1. Zebrafish ( Zebrafish , ZF ) Management and maintenance

The adult zebrafish was maintained in an S-type ZF system (1500[W] × 400[D] × 2050[H] mm) (Daejeon, Korea), and repeated 14 hours light and 10 hours dark photoperiod at 28.5°C. . To obtain a zebrafish larva, two pairs of adult zebrafish were placed in the spawning box overnight. The zebrafish lay eggs for 30 minutes while giving light. For incubation, zebrafish embryos were harvested after 3 hours of fertilization and maintained in 0.03% seawater solution at 28.5° C. for 14 hours light and 10 hours dark photoperiod. The zebrafish was processed according to the standard zebrafish protocol, and all experiments were approved by the Animal Care and Use Committee of Kyunghee University (KHUASP [SE] -15-10).

5-2. Statistical analysis

Statistical analysis was performed using Graphpad Prism (version 5). Data were expressed as the mean of the standard error of the mean (SEM) at 3 replicates. The effectiveness was confirmed using repeated one-way ANOVA and Tukey test. p <0.05 was considered statistically significant.

5-3. Zebrafish Pancreas size change measurement

remind n-According to the mixing ratio of Compound 1 and Compound 2 contained in the BuOH fraction (about 1.96: 1), a composition in which Compound 1 and Compound 2 were mixed in a ratio of 1.96: 1 (hereinafter referred to as'MX') was prepared.

The zebrafish pom was treated with normal (NOR), alloxane-derived group (AXI), allo-induced n- BuOH fraction treatment group (Bu), compound 1 treatment group (1), compound 2 treatment group (2), and MX treatment. Group (MX), Glymepiride treatment group (GLM) was divided into a total of 7 groups.

Wild-type zebrafish cheers, 5 days after fertilization, were placed in 96-well plates. The zebrafish cheer was exposed to 600 μM alloxane for 3 hours to induce pancreatic damage.

Then, to confirm the recovery efficacy of the n -BuOH fraction and compound, n -BuOH fraction (10 μg/mL), compound 1 (1 μg/mL), compound 2 (1 μg/mL), MX (1 μg/ mL) was treated for 12 hours according to the 7 group classification.

As a result of treatment with alloxane on zebrafish pom, the size of pancreas was reduced by 48.9% ( p <0.0001) compared to the normal group. When the n- BuOH fraction was treated, the size of the damaged pancreas was increased by 70.4% ( p = 0.0008) compared to the alloxane-induced group. In the group treated with Compound 1, the size of the damaged pancreas was increased by 22.3% ( p = 0.0114) compared to the alloxane-induced group, and increased by 18.9% ( p = 0.1522) in the compound 2 treated group.

In addition, as a result of the MX treatment, it was confirmed that the size of the pancreas was increased by 48.6% ( p = 0.0004) compared to the alloxane-induced group.

5-4. Zebrafish The pancreas is also intracellular Glucose Absorption capacity analysis

In order to analyze the intracellular glucose uptake capacity, fluorescence staining intensity was measured to analyze changes in fluorescence staining intensity. The fluorescent substance is 2-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-[ N -(7-nitrobenz-2- oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose, 2-NBDG) was used. The zebrafish cheer was stained with 40 μM of 2-NBDG for 30 minutes and rinsed with a 0.03% seawater solution for 20 minutes.

The stained pancreas was observed with a fluorescence microscope and analyzed using Focus Lite software. To investigate the fluorescence intensity of the pancreas, a histogram of Image J software was used for the pixel intensity (green) of fluorescence indicated from 0 to 255.

The fluorescence intensity of pancreatic β-cells was decreased in the alloxane-induced group compared to the normal group. On the other hand, in the case of treatment with glimepiride, compound 1, and compound 2 after induction of alloxane, it was confirmed that the pancreas size increased compared to before treatment. In addition, when treated with n- BuOH fraction and MX, a more significant increase in pancreas was observed (FIG. 2 ).

< Example 6> From zebrafish Of diazoxide (DZ) Action

By using diazo oxide to act on the K _ATP channel (ATP dependent K ⁺ channel) to suppress insulin secretion due to K _ATP channel stimulation and restore pancreatic β-cells, the mechanism of the pancreatic degree recovery effect was also identified.

The zebrafish cheer was divided into 14 groups. Specifically, the normal group (NOR), the normal group treated with diazo oxide (NOR + DZ), the alloxane-derived group (AX), the diazooxide-treated group after induction of alloxane (AX + DZ), the glimepiride treated group ( GLM), glimepiride and diazooxide treatment group (GLM + DZ), n -BuOH fraction treatment group (Bu), n -BuOH fraction and diazooxide treatment group (Bu + DZ), compound 1 treatment group (1), Compound 1 and DZ treatment group (1 + DZ), compound 2 treatment group (2), compound 2 and diazooxide treatment group (2 + DZ), MX treatment group (MX), MX and diazooxide treatment group (MX + DZ).

The wild-type zebrafish cheer, which was 5 days after fertilization, was added to 10 zebrafish cheers per well in a 96-well plate. The zebrafish pom was exposed to 600 μM oxalic acid for 3 hours to induce damage to the pancreas and rinsed with 0.03% seawater. After 3 hours, n- BuOH fraction (10 μg/ml), compound 1 (1 μg/ml), compound 2 (1 μg/ml), MX (1 μg/ml), diazooxide (25 μM) Using, treatment was performed for 12 hours according to the classification of the 14 groups.

After completion of treatment, zebrafish pom was stained with 40 μM 2-NBDG for 30 minutes and rinsed with 0.03% seawater solution for 20 minutes to obtain pancreatic images. Pancreatic images were captured using a fluorescence microscope and analyzed using Focus Lite software.

Compared to the normal group without diazooxide treatment, it was confirmed that the size of the pancreas was reduced by 45.7% when treated with diazooxide (45.7%, p = 0.0001).

Glimepiride a crude diamond oxide, n -BuOH fraction, or if treated with MX, glimepiride, n -BuOH fraction, or by the MX compared with the single treatment with, respectively 31.4% (p <0.0001), 30.2% (p = 0.0004), 28.3% ( p <0.0001) The size of the pancreas was reduced (FIG. 3 ).

That is, when the n- BuOH fraction or MX was treated with diazo oxide, the result of the pancreatic degree recovery effect was slightly lowered compared to the single treatment. Through this, it was found that the n- BuOH fraction and MX stimulated insulin secretion by blocking the K _ATP channel in β-cells of the pancreas, thereby showing the effect of treating diabetes.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the claims, which will be described later, rather than the above detailed description, and all modified or modified forms derived from the equivalent concepts thereof.

Claims (9)

A pharmaceutical composition composition for preventing or treating diabetes comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, and a mixture of a compound represented by Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient:
Mixing (weight) ratio of the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof and the compound represented by the formula (2) or a pharmaceutically acceptable salt thereof is 5: 1 to 1: 1, diabetes prevention or Therapeutic pharmaceutical composition.
[Formula 1]

[Formula 2]

The composition of claim 1, wherein the compound represented by Formula 1 is derived from an mallow extract or a fraction thereof.
delete The method according to claim 1, wherein the mixing (weight) ratio of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof and the compound represented by Formula 2 or a pharmaceutically acceptable salt thereof is 2.0 to 1.9:1. , Composition.
The composition of claim 1, 2, or 4, wherein the composition increases the size of the damaged pancreas.
As a food composition for preventing or improving diabetes comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, and a mixture of a compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient,
Mixing (weight) ratio of the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof and the compound represented by the formula (2) or a pharmaceutically acceptable salt thereof is 5: 1 to 1: 1, diabetes prevention or Food composition for improvement.
[Formula 1]

[Formula 2]

delete The compound (weight) ratio of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof and the compound represented by Formula 2 or a pharmaceutically acceptable salt thereof according to claim 6 is 2.0 to 1.9:1. , Composition.
delete

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