KR102137147B1 - Compositions for preventing or treating diabetes comprising Benzyl α-D-galactopyranoside or (-)-secoisolariciresinol-9'-O-β-D-glucopyranoside - Google Patents

Abstract

The present invention prevents diabetes comprising benzyl alpha-D-galactopyranoside or (-)-secoisolariciresinol-9'-O-beta-D-glucopyranoside derived from mallow as an active ingredient or It relates to a therapeutic pharmaceutical composition, a food composition and a method for preventing or treating diabetes.
Compounds derived from the extract or fractions of malt above the present invention exhibit excellent anti-diabetic effects such as restoring damaged pancreatic islets and increasing glucose absorption in pancreatic islet cells, and thus are included as an active ingredient in pharmaceutical compositions or health functional foods for diabetes It can be used for the development of therapeutic agents or improvers.

Description

Composition for preventing or treating diabetes including benzyl alpha-D-galactopyranoside or (-)-secoisolariciresinol-9'-O-beta-D-glucopyranoside comprising Benzyl α-D-galactopyranoside or (-)-secoisolariciresinol-9'-O-β-D-glucopyranoside}

The present invention is a pharmaceutical agent for preventing or treating diabetes comprising benzyl alpha-D-galactopyranoside or (-)-secoisolariciresinol-9'-O-beta-D-glucopyranoside as an active ingredient. A composition, a food composition, and a method for preventing or treating diabetes and a method for preparing the compound.

Diabetes mellitus is a disease in which insulin deficiency or insensitivity to insulin causes an abnormality in carbohydrate metabolism. Insulin, a polypeptide hormone, is made from β-cells of 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 abnormalities 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 glucose uptake in pancreatic islet cells can 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 susceptibility to insulin, such as pioglitazone and rosiglitazone, which enhance the response of insulin, and metformin, which improves insulin resistance. And sulfonyllurea and meglitinide sequences that stimulate insulin secretion in pancreatic β-cells. 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 resulted in benzyl alpha-D-galactopyranoside and (-)-secoisolaric acid derived from the extract of malt aboveground or a fraction thereof. Resinol-9'-O-beta-D-glucopyranoside was isolated and benzyl alpha-D-galactopyranoside or (-)-secoisolariciresinol-9'-O-beta-D- When the glucopyranoside was treated, the size of the damaged pancreatic islet was recovered, confirming that the anti-diabetic effect appeared, and the present invention was completed.

One object of the present invention is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof; Or to provide a pharmaceutical composition for preventing or treating diabetes comprising a compound represented by the formula (II) or a pharmaceutically acceptable salt thereof; as an active ingredient.

[Formula Ⅰ]

[Formula Ⅱ]

Another object of the present invention is to provide a method for preventing or treating diabetes, comprising administering the pharmaceutical composition to an individual.

Another object of the present invention is a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof; Or to provide a food composition for preventing or improving diabetes comprising the compound represented by the formula (II) or a pharmaceutically acceptable salt thereof; as an active ingredient.

Another object of the present invention comprises the steps of separating the compound represented by the formula (I) or the compound represented by the formula (II) from the mallow extract or a fraction thereof, a method for preparing the compound represented by the formula (I) or formula (II) Is to provide

In order to achieve the above object, one aspect of the present invention is a compound represented by Formula I (hereinafter,'Compound 1') or a pharmaceutically acceptable salt thereof; Or it provides a pharmaceutical composition for preventing or treating diabetes comprising a compound represented by the formula (II) (hereinafter,'Compound 2') or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula Ⅰ]

[Formula Ⅱ]

In the present invention, the term "compound 1" refers to a compound having the structure of formula (I): its name is benzyl alpha-D-galactopyranoside [benzyl α-D-galactopyranoside].

[Formula Ⅰ]

The term "compound 2" of the present invention means a compound having the structure of formula (II) below, whose name is (-)-Secoisolariciresinol-9'-O-beta-D-glucopyranoside [( -)-secoisolariciresinol-9'-O-β-glucopyranoside].

[Formula Ⅱ]

The present inventors conducted various studies to discover substances that exhibit excellent diabetes prevention or treatment effects from natural resources existing in nature, and as a result, it was confirmed that the mallow extract or its fraction shows the effect of restoring the size of the damaged pancreatic islet. Did. Thereafter, Compound 1 or Compound 2 was separated from the mallow extract or a fraction thereof, and it was confirmed that these compounds also exhibit anti-diabetic effects. The effect of preventing, treating or improving diabetes in these compounds is not known in the past, and can be said to be very significant in that it was first identified by the present inventors.

The term "mallow ( Malva verticillata )" in the present invention is an annual plant that grows to a size of about 35 to 150 cm. Native to East Asia, widely distributed in temperate regions of the northern hemisphere, edible leaves and young shoots, medicinal roots and fruits. Its seeds are used as a traditional Chinese medicine to treat various diseases. For the purposes of the present invention, mallow is not limited to the type, and can be used without limitation to its source, such as using cultivated or commercially available ones.

In the present invention, the term, "extract" means to extract the mallow, but is not limited thereto, an extract obtained by an extraction process, a diluent or concentrate of the extract, a dried product obtained by drying the extract, and their modulators , Extracts or mixtures thereof, and extracts of all formulations that can be formed using the extracts themselves and the extracts. In addition, it can be extracted from a variety of organs, hybrids, various plants of the mallow, for example, roots, roots, terrestrial parts, stems, leaves, fruits, etc., but may be specifically used mallow terrestrial parts, but is not limited thereto. .

The mallow extract of the present invention can be prepared using general extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but a method such as immersion extraction, hot water extraction, cold immersion extraction, reflux cooling extraction or ultrasonic extraction may be used.

The mallow extract may be extracted with water, alcohol or a mixed solvent thereof, but is not limited thereto. The extraction degree and loss degree of the active ingredient may vary depending on the organic solvent to be extracted, so select an appropriate organic solvent. Can be used. Specifically, methanol may be used, but is not limited thereto.

Further, the solvent extract may further include filtering the extract to remove floating solid particles. For example, the particles may be filtered using cotton, nylon, or the like, an ultrafiltration method, a freeze filtration method, or a centrifugal separation method, but is not limited thereto.

Concentration under reduced pressure and concentration of reverse osmosis may be used to concentrate the extract.

After the concentration and drying step, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, or infrared drying may be used. Optionally, a process of grinding the final dried extract may be further included.

In the present invention, the term "fraction" means a result obtained by performing a fraction to separate a specific component or a specific component group from a mixture comprising various various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited as long as it can obtain a fraction having a diabetes prevention or treatment effect, but may be performed according to a method commonly used in the art. For example, a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (separation according to size, charge, hydrophobicity or affinity) Chromatography), and combinations thereof.

In the present invention, the type of fractional solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractional solvent include polar solvents such as water, distilled water, and alcohol; And non-polar solvents such as hexane, ethyl acetate, chloroform and dichloromethane. These may be used alone or in combination of two or more. Specifically, ethyl acetate, n -butanol, or water may be used, but is not limited thereto.

In one embodiment of the present invention, after obtaining a methanol extract of mallow, water fraction thereof, ethyl acetate fraction, n -butanol fraction was obtained, and then the compound 1 and compound 2 of the present invention were separated from the fraction.

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 the relationship to the intake route Can be used in the present invention without.

The term "diabetes" of the present invention means a disease that occurs when the amount of insulin secretion is insufficient or the action and function of insulin 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 pancreatic islets present in the pancreas and secreted when the blood glucose level increases, and when it decreases, secretion is suppressed to regulate the proper activity of the energy source. 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, it can be useful for treating or preventing diabetes by recovering damaged pancreatic β-cells or increasing glucose uptake in pancreatic islet cells.

The term "pharmaceutically acceptable salt" of 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 usually metal salts and organic bases. It may be a salt of, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like. 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.), aluminum salt, or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine Salt with the back; Salts with inorganic acids can be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.; Salts with organic acids can be 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, p-toluenesulfonic acid, etc.; Salts with basic amino acids can be salts with arginine, lysine, ornithine and the like; Salts with acidic amino acids can be salts with aspartic acid, glutamic acid, and the like.

The term "prevention" of the present invention means any action that inhibits or delays the onset of diabetes by administration of a pharmaceutical composition according to the present invention.

The term "treatment" of the present invention means any action in which the symptoms of a suspected diabetes mellitus and an affected individual are improved or beneficially altered by administration of the pharmaceutical composition.

In the present invention, the compound 1 or the compound 2 may be to increase the size of the damaged pancreatic islet. In a specific embodiment of the present invention, as a result of treating 10 μM of Compound 1 or Compound 2 with zebrafish having a reduced size of pancreatic islet due to oxalic acid-induced, the size of the pancreatic islet increased by 60.8% and 57.0%, respectively. And it was confirmed that the glucose absorption is improved (Fig. 2 and Fig. 5). This suggests that Compound 1 or Compound 2 exhibits an anti-diabetic effect to restore the reduced size of pancreatic islets, and thus may be useful for diabetes prevention, treatment, and improvement.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent, which carrier may include a non-naturally occurring carrier.

Specifically, carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid ( Poly Lactic Acid, poly-L-lactic acid, Mineral oil and the like.

The pharmaceutical composition may be formulated and used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution, respectively, according to a conventional method. The carrier may include various types of amorphous carriers, microspheres, nanofibers, and the like.

When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, such as starch, calcium carbonate, in the extract and its fractions. It can be prepared by mixing sucrose or lactose, gelatin, and the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

Another aspect provides a method of preventing or treating diabetes, comprising administering the pharmaceutical composition to an individual.

At this time, the description of the "diabetes", "prevention" and "treatment" is as described above.

Since the pharmaceutical composition of the present invention exhibits the effect of preventing or treating diabetes, the method of the present invention including the step of administering it to an individual can be usefully used for preventing or treating diabetes.

The term "administration" of the present invention means introducing the pharmaceutical composition to a subject in an appropriate manner.

The term "individual" of the present invention means all animals such as rats, mice, livestock, including humans, who may or may have diabetes, and may be, but not limited to, mammals, including humans, as specific examples. .

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term, "pharmaceutically effective amount" is sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. By volume, effective dose level refers to the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the invention used, the route of administration and the rate of discharge , Factors including drugs used in combination or coincidental with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known pterygium therapeutic agent. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects.

The pharmaceutical composition of the present invention can be administered using various methods such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, transdermal, and the dosage may vary depending on the patient's age, sex, and weight. It can vary and can be readily determined by one skilled in the art.

In addition, the dosage of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of poisoning of the disease, the age, weight, sex, history of the patient, or the type of substance used as an active ingredient.

Another aspect is a compound represented by the formula (I) (hereinafter,'Compound 1') or a pharmaceutically acceptable salt thereof; Or it provides a food composition for preventing or improving diabetes comprising a compound represented by the following formula (II) (hereinafter,'Compound 2') or a pharmaceutically acceptable salt thereof; as an active ingredient.

[Formula Ⅰ]

[Formula Ⅱ]

At this time, the definitions of the terms "compound 1", "compound 2", "pharmaceutically acceptable salt", "diabetes" and "prevention" are as described above.

The term "improvement" of the present invention means any action that at least reduces the extent of symptoms associated with parameters related to the condition being treated with administration of a composition comprising Compound 1 or Compound 2.

Since the compound 1 or the compound 2 according to the present invention exhibits an excellent diabetes treatment effect, it may be included in a food composition for the purpose of preventing or improving diabetes, and the food composition can be taken on a daily basis to prevent or improve diabetes. High effects can be expected.

In the present invention, the term "food" is meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , Vitamin complexes, health functional foods, and the like, and includes all foods in the usual sense, and are not limited thereto as long as they can contain Compound 1 or Compound 2 of the present invention.

The compound 1 or the compound 2 of the present invention may be added as it is or used with other foods or food ingredients, and may be suitably used according to conventional methods.

The food of the present invention can be manufactured by a method commonly used in the art, and the above-described preparation can be made by adding raw materials and ingredients commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, and the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used. In addition, the food composition is a food additive such as preservatives, fungicides, antioxidants, colorants, colorants, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, reinforcing agents, emulsifiers, thickeners, coating agents, gum herbicides, foam inhibitors, solvents, improvers, etc. It can contain. The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food can be prepared in various forms of the formulation without limitation as long as it is a formulation recognized as food. Unlike general medicines, food has the advantage of not having side effects that can occur when taking medicines for a long time and has excellent portability, so the foods of the present invention are ingested as supplements to improve the effect of preventing or improving diabetes. It is possible.

Another aspect comprises the step of separating the compound represented by the following formula (I) (hereinafter,'Compound 1') or the compound represented by the following formula (II) (hereinafter,'Compound 2') from the mallow extract or a fraction thereof. , Provides a method for preparing a compound represented by Formula I or Formula II.

[Formula Ⅰ]

[Formula Ⅱ]

At this time, the definitions of the terms "mallow", "extract", "fraction", "compound 1" and "compound 2" are as described above. In addition, the effect of treating compound 1 or compound 2 on diabetes is as described above.

Compounds derived from the extract or fractions of malt above the present invention exhibit excellent anti-diabetic effects such as restoring damaged pancreatic islets and increasing glucose absorption in pancreatic islet cells, and thus are included as an active ingredient in pharmaceutical compositions or foods for diabetes. It can be used for the development of therapeutic agents or improvers.

1 is a graph showing the analysis of changes in fluorescence intensity caused by 2-NBDG in pancreatic islets treated with each fraction (10 μg/mL) with Focus Lite software (NOR: normal) Group, AX: alloxane-derived group, GLM: glimepiride treated group, Et: EtOAc fraction treated group, Bu: n -BuOH fraction treated group, Aq: water fraction treated group, ^### p <0.001; with normal group Comparison, ^** p <0.01, ^*** p <0.001; comparison with alloxane-derived group).
2 is a graph showing the analysis of changes in fluorescence intensity caused by 2-NBDG in pancreatic islets treated with compounds 1 to 4 (10 μM) with Focus Lite software (NOR: normal group) , AX: alloxane-derived group, GLM: glimepiride treated group, ^### p <0.001; comparison with normal group, ^** p <0.01, ^*** p <0.001; comparison with alloxane-derived group) .
FIG. 3 shows a differential interference contrast fluorescence microscopy image of pancreatic islets in wild-type zebrafish and after 5 days of fertilization.
Figure 4 shows a fluorescence microscopy image showing the fluorescence intensity induced by 2-NBDG in pancreatic islets treated with each fraction (10 μg/mL) (scale bar = 100 μm).
FIG. 5 shows fluorescence microscopy images showing the fluorescence intensity induced by 2-NBDG in pancreatic islets treated with compounds 1 to 4 (10 μM) (scale bar = 100 μm).

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited to them.

Example 1. Plant material

In April 2016, the ground part of Mook was purchased from a commercial farm in Namyangju, Korea, and the voucher specimen (KHU20160419) is stored in the Laboratory of Natural Products Chemistry at Kyunghee University (Yongin, Korea).

Example 2. Reagents and instruments

All analytical reagents and instruments used in this study are identical to those used in previous studies (Thi NN et al., Phenylpropanoids from Lilium Asiatic hybrid flowers and their anti-inflammatory activities.Applied biological Chemistry 2017;60:527-533). . Aloxane and sea salt were purchased from Sigma Chemical Co. (St. Louis, MO, USA). GLM was obtained from Cayman Chemical Co. (ann Arbor, MI, 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. Fluorescence microscopy was performed with an Olympus 1X70 microscope (Tokyo, Japan). For image analysis, Focus Lite (Focus Co, Daejeon, Korea) was used.

Example 3. Extract and fraction of mallow

The dried top of the mallow (3.1 kg) was extracted with 80% MeOH (54 L x 5) at room temperature for 24 hours. The extract was filtered through filter paper and evaporated under reduced pressure at 43° C. to yield 794.0 g of extract. The obtained MeOH extract was suspended in water (2 L), and then extracted successively with ethyl acetate (EtOAc, 2 L × 4) and n -butanol (n-BuOH, 2 L × 4). Each layer was concentrated under reduced pressure to obtain EtOAc fraction (MVE, 80 g), n- BuOH fraction (MVB, 77 g), and water fraction (MVW, 637 g).

Example 4. Separation of compound from mallow fraction

EtOAc fractions (MVE, 80 g) were applied to a silica gel column (φ 10×15 cm) and eluent n -hexane-EtOAc (10:1→8:1→5:1→3:1, 23.3 L each) By separation, 14 fractions (MVE-1 to MVE-14) were obtained. Fraction MVE-8 [1.74 g, elution volume/total volume (V _e /V _t ) 0.130 to 0.313] was applied to SiO ₂ column chromatography and CHCl ₃ -EtOAc (35:1→8:1→2:1). , 1.8 L), each separated by eluent to obtain 15 fractions (MVE-8-1 to MVE-8-15). Fraction MVE-8-7 (209.0 mg, V _e /V _t 0.074 to 0.107) was separated on an ODS column (φ 2.5×6 cm) and acetone-H ₂ O (3:5, 1.8 L) eluent Separated with 15 fractions (MVE-8-7-1 to MVE-8-7-15) and purified compound 4 [ trans- ferulic acid methyl ester, MVE-8-7-4, 16.9 mg, V _e /V _t 0.051 to 0.079, TLC (silica gel 60 F ₂₅₄ ) R _f 0.52, CHCl ₃ -EtOAc (15:1), TLC (RP-18 F _254s ) Rf 0.90, acetone-H ₂ O (5:1) ].

n- BuOH fraction (MVB, 77 g) was applied to a SiO ₂ column (φ 10×15 cm) and CHCl ₃ -MeOH-H ₂ O (25:3:1→22:3:1→20:3: 1→18:3:1→15:3:1→12:3:1, each 18.8 L) separated by eluent to give 14 fractions (MVB-1 to MVB-14). Fraction MVB-9 (4.0 g, V _e /V _t 0.385 to 0.557) was separated in an ODS column (φ 4×6 cm) and MeOH-H ₂ O (3:2→2:1→2.5:1→3). :1→5:1, 3.6 L each), and 16 fractions (MVB-9-1 to MVB-9-16) were obtained. Fraction MVB-9-1 (1.7 g, V _e /V _t 0.000 to 0.003) was applied to SiO ₂ column chromatography (φ 3×15 cm) and CHCl ₃ -MeOH-H ₂ O (12:3:1). , 7.8 L each), and 19 fractions (MVB-9-1-1 to MVB-9-1-19) were obtained. Fraction MVB-9-1-11 (364.2 mg, V _e /V _t 0.231 to 0.254) was subjected to SiO ₂ column chromatography (φ 2.5×15 cm) and EtOAc-n-BuOH-H ₂ O (16: Separation with 3:1, 3.7 L) eluent yielded 12 fractions (MVB-9-1-11-1 to MVB-9-1-11-12). Fraction MVB-9-1-11-3 (41.4 mg, V _e /V _t 0.027 to 0.043) was applied to ODS column chromatography (φ 1.5×8 cm), MeOH-H ₂ O (1:2, respectively) 0.8 L) separated into eluent, 10 fractions (MVB-9-1-11-3-1 to MVB-9-1-11-3-10) and purified compound 2 [(-)-secoisolariciresinol-9' -O-β-D-glucopyranoside, MVB-9-1-11-3-8, 6.8 mg, V _e /V= 0.175 to 0.288, TLC (silica gel 60 F ₂₅₄ ) R _f 0.80, CHCl ₃ -MeOH- H ₂ O (65:35:10), TLC (RP-18 F _254s ) R _f 0.12, MeOH-H ₂ O(1:2)].

Water fraction (MVW, 637 g) was applied to a Dianion HP-20 column (φ 9×30 cm) and H ₂ O-MeOH (H ₂ O→5:1→3:2→2:3→1:4) , 7.2 L), and separated into eluents to obtain 9 fractions (MVW-1 to MVW-9). Fraction MVW-5 (18.3 g, V _e /V _t 0.478 to 0.778) was subjected to ODS CC (φ 5×15 cm) and separated with MeOH-H ₂ O (1:4, 2.6 L) eluent, Nine fractions (MVW-5-1 to MVW-5-9) were obtained. Fraction MVW-5-4 (1.4 g, V _e /V _t 0.167 to 0.189) was targeted for SiO ₂ CC (φ 4.5×15 cm), and CHCl ₃ -MeOH-H ₂ O (9:3:1→ 6:4:1, both 4.4 L) separated by eluent, 17 fractions (MVW-5-4-1 to MVW-5-4-17) and two purified compounds 1 [Benzyl α-D-galactopyranoside , MVW-5-4-14, 46.9 mg, V _e /V= 0.628 to 0.690, TLC (silica gel 60 F ₂₅₄ ) R _f 0.52, CHCl ₃ -MeOH-H ₂ O (6:4:1), TLC (RP-18 F _254s ) R _f 0.82, acetone-H ₂ O(2:3)] and compound 3 [ trans -ferulic acid, MVW-5-4-15, 33.5 mg, V _e /V= 0.691 to 0.756 , TLC(silica gel 60 F ₂₅₄ ) R _f 0.52, CHCl ₃ -MeOH-H ₂ O(6:4:1), TLC(RP-18 F _254s ) R _f 0.82, acetone-H ₂ O(2:3 )].

Example 5 Evaluation of recovery efficacy against alloxan-induced pancreatic islet damage in zebrafish pom

5-1. Manage and maintain zebrafish

The adult zebrafish was maintained in a zebrafish system S-type (1500 [W] x 400 [D] x 2050 [H] mm) (Daejeon, Seoul) and 14 hours light/10 hours dark cycle at 28.5°C. Two pairs of adult zebrafish were kept in spawning boxes overnight to obtain zebrafish cheer. The zebrafish spawned for a 30-minute light period. For culture, zebrafish embryos were collected after 3 hours of fertilization and kept in an incubator of 0.03% sea salt solution, 14:10 light-dark photoperiod at 28.5°C. The zebrafish was managed according to the standard zebrafish protocol, approved by the Animal Care and Use Commottee (KHUASP[SE]-15-10).

5-2. Statistical analysis

Statistical analysis was performed using GraphPad Prism (version 5, GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as the standard error of the mean (SEM) for 3 replicates. Statistical significance was determined using repeated one-way ANOVA followed by Tukey's post-hoc test. P <0.05 was considered statistically significant.

5-3. Zebrafish pancreatic islet size change measurement

The zebrafish cheer was divided into the following groups: normal group (NOR), alloxane-derived group (AX, control), glimepiride treated group (GLM, positive control), fractions treated with fractions (Et, Bu, Aq) Acid-derived group, alloxane-derived group treated with compounds 1 to 4 (1, 2, 3, 4, respectively).

Wild-type zebrafish cheers 5 days after fertilization were placed in 24-well plates. Pancreas islet injury was induced by exposing the pom to 600 μM alloxane for 3 hours. To measure the recovery efficacy of the fractions and compounds, the alloxane-derived pom was treated with 10 μg/mL fractions or 10 μM compounds for 12 hours.

The alloxane-derived group (AX) significantly reduced the pancreatic islet size to 44.3% ( p <0.0001) compared to the normal group (FIGS. 1 and 2 ). Glymepiride-treated group significantly increased pancreatic islet size compared to the alloxane-derived group (FIGS. 1 and 2 ).

The EtOAc fraction-treated group, n- BuOH fraction-treated group, and water fraction-treated group had 62.4% ( p = 0.0072) and 68.6% ( p = 0.0012) of the size of the damaged pancreatic islet, respectively, compared to the alloxane-derived group. And 90.2% ( p =0.0001) significantly (FIG. 1 ).

In addition, as a result of treating compounds 1 to 3 in the alloxane-derived group, the size of the damaged pancreatic islet was significantly 60.8% (p=0.0051), 57.0% (p=0.0059) and 83.8% (p=0.0007), respectively. It was confirmed to increase. As a result of treatment with Compound 4, it was confirmed that the size of the damaged pancreatic islet was not statistically significant but increased by 33.4% (p=0.0072).

That is, it was confirmed that the compounds 1 and 2 of the present invention are substances that increase the size of the damaged pancreatic islets by 60.8% and 57.0%, thereby realizing the diabetes treatment effect.

5-4. Analysis of glucose absorption capacity in zebrafish pancreatic islet cells

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) is a fluorescent dye used to monitor glucose uptake. When observed under a fluorescence microscope, an increase in the amount of 2-NBDG absorbed into the zebrafish indicates an increase in fluorescence intensity emitted from the absorbed 2-NBDG. At this time, an increase in fluorescence intensity indicates that the test substance has a diabetes treatment effect.

Zebrafish pom was stained for 30 minutes using 40 μM of 2-NBDG as a fluorescent dye and rinsed with 0.03% sea salt solution for 20 minutes. Pancreatic islets were observed under a fluorescence microscope and analyzed using Focus software (FIG. 3).

The oxalic acid-derived group had a reduced fluorescence intensity compared to the normal group (FIGS. 4 and 5 ). The fluorescence intensity was increased in the glimepiride-treated group compared to the alloxane-induced group (FIGS. 4 and 5 ).

The EtOAc fraction treatment group, the n- BuOH fraction treatment group, and the water fraction treatment group had an increased fluorescence intensity compared to the alloxane-derived group (FIG. 4). In addition, when the compounds 1 to 4 were treated, it was confirmed that the fluorescence intensity increased compared to the alloxane-derived group (FIG. 5 ).

Through the above results, it was found that when the compound 1 or the compound 2 of the present invention was treated, the ability to absorb glucose was improved in the pancreatic islet. This suggests that, as in the results of Example 5-3, treatment of Compound 1 or Compound 2 of the present invention on the damaged pancreatic islet showed the effect of treating diabetes.

From the above results, it was found that fractions and compounds (1 to 4) derived from mallow can be used for diabetes prevention or treatment.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as 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 are described later, rather than the above detailed description, and all modified or modified forms derived from equivalent concepts thereof.

Claims (7)

A pharmaceutical composition for preventing or treating diabetes, comprising the compound represented by the following formula II or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula Ⅱ]

The composition according to claim 1, wherein the compound represented by the formula (II) is derived from an mallow extract or a fraction thereof.
The composition of claim 1, wherein the compound represented by Formula II increases the size of the damaged pancreatic islet.
A method for preventing or treating diabetes, comprising administering the pharmaceutical composition of any one of claims 1 to 3 to an individual other than a human.
A compound represented by the following formula (II) or a salt acceptable as a food thereof; a food composition for preventing or improving diabetes comprising as an active ingredient.
[Formula Ⅱ]

The composition according to claim 5, wherein the compound represented by Formula II is derived from a mallow extract or a fraction thereof.
Method for producing a compound represented by the formula (II) comprising the step of separating the compound represented by the following formula (II) from the mallow extract or a fraction thereof.
[Formula Ⅱ]

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