KR20160068073A - Compositions comprising flavonoids for the antidiabetes - Google Patents

Abstract

The present invention relates to quercetin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating diabetes, which comprises rutin or a pharmaceutically acceptable salt thereof as an active ingredient; A food composition for preventing or improving diabetes; And a feed composition.
A mixture of quercetin and a rutin, or a mixture of isquercetin, quercetin and a rutin, can significantly increase the size of a damaged pancreas and the damaged pancreas in intracellular glucose levels synergistically, and thus can be usefully used for the prevention, treatment and improvement of diabetes .

Description

Compositions comprising flavonoids for the antidiabetes < RTI ID = 0.0 >

The present invention relates to quercetin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating diabetes, which comprises rutin or a pharmaceutically acceptable salt thereof as an active ingredient; A food composition for preventing or improving diabetes; And a feed composition.

Diabetes mellitus (diabetes mellitus) is lack of insulin or insulin sensitivity to the carbohydrate metabolism (carbohydrate metabolism) is a disease that is abnormal. Insulin, a polypeptide hormone, is produced in the beta cells of the Langerhans islet in the pancreas, and most cells in the body are required to use glucose, glucose. In the case of diabetic patients, the body's ability to use glucose normally becomes impaired, resulting in an increase in blood glucose levels and an accumulation of glucose in the blood, resulting in the excretion of excess sugar in the urine.

There are two types of diabetes. Type I is an insulin-dependent diabetes mellitus (IDDM), formerly called burning diabetes, requiring insulin injections. Because the insulin is not secreted from the pancreas, insulin should be supplied via injection. Type II is non-insulin-dependent diabetes mellitus (NIDDM), also known as adult type diabetes, and can be regulated by diet. This has been attributed to poor insulin secretion in the pancreas and tissue rejection of insulin. Type II diabetes has previously been classified as burning diabetes and adult type diabetes, but both can occur at any age. Non-insulin dependent diabetes mellitus, however, is much more common, accounting for 90% of all diabetic patients.

As described above, insulin injections are mostly used as a method of treating insulin-dependent diabetes, that is, type 1 (type I) diabetes.

In addition, conventional diabetes medicines show side effects due to each mechanism. For example, toxicity to the liver, kidney, muscle and heart as well as weight gain symptoms are typical side effects. Therefore, it is urgently required to develop a therapeutic agent for diabetes which can effectively treat diabetes or prevent diabetes at the same time without causing side effects and causing weight gain and the like.

Zebrafish ( Danio rerio ) is a small tropical freshwater fish from the Indian carp family. In vitro fertilization is performed and the embryo is transparent and fluorescently stained, so that the formation and differentiation process of specific cells constituting various organs can be easily observed under a general anatomical microscope. Currently, studies of insulin-dependent diabetes mellitus (type 1 diabetes) have been performed by administering alloxan and streptozotocin, known as diabetes-inducing drugs, to experimental animals. Alloates selectively accumulate rapidly in the beta cells of the pancreas, inhibiting beta-cell glucokinase and reducing insulin secretion.

Under these circumstances, the present inventors have made extensive efforts to find a substance having an antidiabetic effect derived from a natural product. As a result, it has been found that a quercetin and a mixture of rutin, or a mixture of isquercetin, quercetin and a mixture of rutin are more effective than pancreatic islets ) And the damaged pancreas can increase the intracellular glucose level synergistically. Thus, the present invention can be used for preventing, treating and improving diabetes.

Korean Patent Publication No. 10-2013-0134295 Korean Patent Publication No. 10-2014-0125594

One object of the present invention is the use of quercetin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating diabetes comprising, as an active ingredient, rutin or a pharmaceutically acceptable salt thereof

Another object of the present invention is to provide a composition comprising quercetin or a physiologically acceptable salt thereof; And a physiologically acceptable salt thereof as an active ingredient. The present invention also provides a food composition for preventing or improving diabetes mellitus.

Another object of the present invention is to provide a quercetin or a physiologically acceptable salt thereof; And a physiologically acceptable salt thereof as an active ingredient. The present invention also provides a composition for preventing or ameliorating diabetes mellitus.

In order to achieve the above object, one aspect of the present invention relates to quercetin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for the prevention or treatment of diabetes, which comprises, as an active ingredient, rutin or a pharmaceutically acceptable salt thereof.

In addition, the pharmaceutical composition may further comprise isoquercetin or a pharmaceutically acceptable salt thereof.

The quercetin, rutin or isoquercetin alone has an antidiabetic effect, but the quercetin and the rutin mixture or the isoquercetin, quercetin and the rutin mixture have a synergistic effect and have a much higher antidiabetic effect than those used individually Has never been reported. Specifically, the composition has an effect of restoring damaged pancreatic beta cells or increasing the intracellular glucose level and lowering the blood glucose level, so that the composition can be effectively used for preventing or treating diabetes.

Said quercetin, rutin or isoquercetin means a compound having the formula shown in Fig. The compositions of the present invention include both pharmaceutically acceptable salts thereof and possible solvates and hydrates thereof which may be prepared therefrom, and may include all possible stereoisomers. Also, the solvates, hydrates and stereoisomers of the above quercetin, rutin or isoquercetin can be prepared from the compounds shown in Fig. 1 using conventional methods.

The pharmaceutically acceptable salt is not particularly limited as it is commonly used in the art, such as an acid addition salt. Preferred pharmaceutically acceptable acid addition salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, orthophosphoric acid or sulfuric acid; Or organic acids such as, for example, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, acetic acid, propionic acid, lactic acid, citric acid, fumaric acid, malic acid, succinic acid, salicylic acid, maleic acid, glycerophosphoric acid or acetylsalicylic acid.

In addition, a pharmaceutically acceptable metal salt can be obtained by a conventional method using a base. For example, a compound represented by the formula (1) is dissolved in an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, the non-soluble compound salt is filtered, and the filtrate is evaporated and dried to obtain a pharmaceutically acceptable metal salt. At this time, it is preferable to prepare metal salts, in particular, sodium salts, potassium salts or calcium salts, and these metal salts can be reacted with a suitable salt (for example, nitrate).

In the present invention, the quercetin, the rutin, or the isoquercetin may be synthesized by a known synthesis method, or may be isolated and purified from plants, or may be commercially available.

In one embodiment of the present invention, the ethanol extract of Audi was fractionated with ethyl acetate and the compounds 1 and 2 were separated and purified by column chromatography. In addition, the ethanol extract of the olive was sequentially fractionated with water and n-butanol, and the compounds 3 and 4 were separated and purified by column chromatography. Further, the compounds 1 to 4 were analyzed for their structures through NMR, MS, IR and the like, and were identified as quercetin, astragalin, isoquercetin and rutin, respectively (Examples 2 and 3).

Further, in the present invention, said quercetin or a pharmaceutically acceptable salt thereof; And rutin or a pharmaceutically acceptable salt thereof may be mixed in a weight ratio of 1 to 10: 1 to 10, particularly 1 to 5: 1 to 5, more specifically 1: 1, but are not limited thereto .

Further, in the present invention, said isoquercetin or a pharmaceutically acceptable salt thereof; Quercetin or a pharmaceutically acceptable salt thereof; And rutin or a pharmaceutically acceptable salt thereof are mixed in a weight ratio of 1 to 10: 1 to 10: 1 to 10, especially 1 to 5: 1 to 5: 1 to 5, more specifically 1: 1: 1 But is not limited thereto.

In one embodiment of the present invention, quercetin and rutin isolated and purified in Example 3 were mixed at a weight ratio of 1: 1, isoquercetin, quercetin and rutin in a weight ratio of 1: 1: 1 to prepare 10 μM, As a result of exposure to the alloxan-treated zebrafish larva for 1 hour, it was confirmed that the mixture could significantly increase the size of the damaged pancreatic islet and the damaged pancreatic intracellular glucose level synergistically, Prevention, treatment and improvement.

In the present invention, the term "diabetes" means a disease that occurs when insulin secretion is insufficient or insulin action and function are not sufficiently achieved. In case of this disease, excessive decomposition of glycogen, protein, Resulting in diabetes and ketoneuria, resulting in abnormal blood and electrolyte metabolism, and concomitant conditions such as circulatory disturbances and renal disorders, as well as blood concentration due to electrolyte loss. Insulin is secreted in the beta cells of the islets of the islets in the pancreas, secreted when the glucose level in the blood is increased, and secreted is suppressed when the blood glucose level is increased. The disease is divided into insulin - dependent diabetes mellitus (type I) and non - insulin dependent diabetes mellitus (type II). Diagnosis of diabetes is usually made by measuring blood glucose levels, which vary according to standards. In humans, diabetes is usually diagnosed when the blood glucose level is more than 200 mg / dl, and fasting is more than 140 mg / dl. Thus, diabetes can be treated or prevented by restoring damaged pancreatic beta cells or by increasing intracellular glucose levels and lowering blood glucose levels. The present inventors have found that a mixture of quercetin and a rutin or a mixture of isquercetin, quercetin and a rutin can significantly increase the size of damaged pancreas and the intracellular glucose level of the damaged pancreas more synergistically than using each compound alone The composition can be effectively used for preventing or treating insulin-dependent diabetes mellitus.

The term "prophylactic" in the present invention refers to any act which inhibits or delays the onset of diabetes by administration of the composition according to the invention.

As used herein, the term "treatment" refers to any action that improves or alters the symptoms of the disease upon administration of the composition of the present invention.

The composition may comprise a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not disturb the biological activity and properties of the compound being injected, without irritating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more.

The composition comprising a pharmaceutically acceptable carrier may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

In particular, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, quercetin, rutin, isoquercetin or a pharmaceutically acceptable salt thereof may be administered at a daily dose of 0.0001 to 1000 mg / kg, preferably 0.001 to 100 mg / kg.

Such administration means introducing the composition of the present invention to a patient in any appropriate manner, and the administration route of the composition can be administered through any conventional route as long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal administration.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or two or three times. When the two active ingredients are each monoglyceride, the number of administrations may be the same or different. In addition, the composition of the present invention can be used alone or in combination with other drug therapy for the prevention or treatment of diabetes mellitus. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The term " individual " means all animals such as mice, mice, livestock, etc., including humans that have developed or may develop diabetes. Preferably, it may be a mammal, including a human.

In one embodiment of the present invention, the size change of the zebrafish pancreas shown in FIG. 9 was measured in order to confirm the anti-diabetic effect of quercetin, astragalin, isoquercetin, rutin and a mixture thereof.

As a result, in the group treated with the quercetin and the rutin mixture, the size of the pancreas of about 36.5%, which is much higher than that of the quercetin (22.3%) or the rutin (11.3%), was increased (FIG. 10). This was equivalent to gemepiride, a commercially available diabetes drug. In addition, even if isoquercetin and quercetin, or isoquercetin and rutin are mixed, it is confirmed that the effect of treating diabetes is rather inhibited because the effect of increasing size of pancreas is lower than that of each compound.

This confirms that the quercetin and rutin mixture has a synergistic effect on the increase in the size of the injured pancreas, and thus it can be effectively used for the prevention, treatment and improvement of diabetes.

In addition, a group treated with a mixture of isoquercetin, quercetin and a mixture of rutin, isoquercetin, quercetin or a mixture of rutin, a mixture of isoquercetin and quercetin, a mixture of isoquercetin and rutin, or a mixture of quercetin and rutin, 45.8% of the pancreas also showed increased size (Fig. 10).

As a result, it has been confirmed that a mixture of isoquercetin, quercetin and rutin, unlike the mixture of isoquercetin and quercetin, or the mixture of isoquercetin and rutin, has a remarkable synergistic effect on the increase of pancreas size and can be used for prevention, treatment and improvement of diabetes .

In one embodiment of the present invention, in order to confirm the antidiabetic effect of astragalin, rutin, isoquercetin, quercetin, and a mixture thereof, the compound is treated with zephyrate, Respectively.

As a result, the quercetin and the rutin mixture showed much higher fluorescence intensity than each of quercetin or rutin (Fig. 11). It has been found through this that the quercetin and rutin mixture has a synergistic and remarkable effect on the intracellular glucose level increase of the injured pancreatic duct, and thus it can be effectively used for the prevention, treatment and improvement of diabetes.

Also, mixtures of isoquercetin, quercetin and rutin each treated isoquercetin, quercetin or rutin respectively, or exhibited a much higher fluorescence intensity increase than mixtures of isoquercetin and quercetin, isoquercetin and rutin, or quercetin and rutin 11). It has been found that the mixture of isoquercetin, quercetin and rutin has a synergistic effect on the intracellular glucose level increase of injured pancreatic duct, which can be useful for prevention, treatment and improvement of diabetes.

Another aspect of the present invention provides a method of treating diabetes comprising administering the pharmaceutical composition to a subject in need thereof.

The administration, diabetes, and treatment are as described above.

Another aspect of the present invention relates to quercetin or a physiologically acceptable salt thereof; And a physiologically acceptable salt thereof as an active ingredient. The present invention also provides a food composition for preventing or improving diabetes mellitus.

Specifically, the quercetin or a physiologically acceptable salt thereof; And rutin or a physiologically acceptable salt thereof may be mixed with 1 to 10: 1 to 10, particularly 1 to 5: 1 to 5, more specifically 1: 1 by weight, but not limited thereto .

In addition, the food composition may further comprise isoquercetin or a physiologically acceptable salt thereof.

 Specifically, the isoquercetin or a physiologically acceptable salt thereof; Quercetin or a physiologically acceptable salt thereof; And rutin or a physiologically acceptable salt thereof are mixed in a weight ratio of 1 to 10: 1 to 10: 1 to 10, especially 1 to 5: 1 to 5: 1 to 5, more specifically 1: 1: 1 But is not limited thereto.

Since quercetin, rutin and isoquercetin have been used for a long time and proved to be stable, quercetin, rutin and isoquercetin can be prepared in the form of foods which are common but can prevent or ameliorate diabetes.

In one embodiment of the present invention, quercetin and rutin isolated and purified in Example 3 were mixed at a weight ratio of 1: 1, isoquercetin, quercetin and rutin in a weight ratio of 1: 1: 1 to prepare 10 μM, Exposure to alloxan-treated zebrafish larvae for 1 hour resulted in a synergistic significant effect on the increase in size of the pancreas with damaged quercetin and rutin mixtures or isoquercetin, quercetin and rutin mixtures (Fig. 10) (Fig. 11) synergistic effect on the intracellular glucose level increase (Fig. 11), it can be used for prevention, treatment and improvement of diabetes.

Such quercetin, rutin, isoquercetin, diabetes, and prevention are as described above.

The term "physiologically acceptable " of the present invention is physiologically acceptable and when administered to an organism, the compound to be administered, without causing an allergic reaction such as gastrointestinal disorder, dizziness, or the like, Quot; means < / RTI >

The term "improvement" of the present invention means all actions in which the diabetes disease is alleviated or beneficially modified by the administration of the composition of the present invention.

The term "food" of the present invention is intended to encompass all kinds of foods, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Vitamin complex, health functional food, and health food, all of which include foods in a conventional sense.

The term "functional food" as used herein is the same term as "food for special health use" (FoSHU). In addition to nutritional supplementation, functional foods are processed so as to efficiently show the biological control function, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the food can also be produced without restrictions as long as they are formulations recognized as food. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjunct to improve the effectiveness of arthritis prevention or improvement.

The health food refers to a food having an active health promotion effect or a health promotion effect compared to a general food, and a health supplement food refers to a health supplement food. In some cases, the terms health functional foods, health foods, and health supplements are used.

Specifically, the health functional food is prepared by adding a quercetin and a rutin mixture, or a mixture of isquercetin, quercetin and a rutin to a food material such as a beverage, a tea, a spice, a gum and a confectionery, or a food made by encapsulation, , Which means that it has a certain health effect when consumed. However, unlike general medicine, there is an advantage that there is no side effect that may occur when a drug is taken for a long time by using food as a raw material.

Since the food composition of the present invention can be routinely ingested, it is very useful as it can be expected to prevent or improve diabetes.

The composition may further include a physiologically acceptable carrier. The carrier is not particularly limited and any carrier conventionally used in the art may be used.

In addition, the composition may contain additional ingredients which are commonly used in food compositions and which can improve odor, taste, vision and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the composition can be used in combination with a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), a disinfectant (such as bleaching powder and highly bleached white powder, sodium hypochlorite), an antioxidant (butylhydroxy anisole (BHA) (Sodium hypophosphate), bleach (sodium sulfite), seasoning (sodium MSG glutamate, etc.), sweeteners (hemicellulose, cyclamate, saccharin, A food additive such as a flavor (vanillin, lactones), a swelling agent (alum, D-tartrate, potassium hydrogen), an emulsifier, a thickening agent (glue), a covering agent, a gum base agent, a foam inhibitor, and food additives. The additives may be selected and used in appropriate amounts depending on the type of food.

When said quercetin and rutin mixture or a mixture of isoquercetin, quercetin and rutin is used as a food additive, a mixture of quercetin and rutin, or a mixture of isquercetin, quercetin and rutin can be added intact or used together with other food or food ingredients, And can be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). In general, the food composition of the present invention may be added in an amount of not more than 50 parts by weight, preferably not more than 20 parts by weight, based on the food or drink. However, in case of long-term ingestion for health and hygiene purposes, the active ingredient may be contained in an amount not exceeding the above range and there is no problem in terms of safety.

As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, various flavors or natural carbohydrates can be added as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer, Alcohols or carbonating agents, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks, or vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, 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.

The food composition of the present invention may be contained in various weight percentages as long as it can exhibit diabetes prevention or ameliorating effect. Specifically, quercetin and a mixture of rutin, or a mixture of isquercetin, quercetin and rutin, in an amount of 0.00001 to 100 wt% Or 0.01 to 80% by weight.

Another aspect of the present invention relates to quercetin or a physiologically acceptable salt thereof; And a physiologically acceptable salt thereof as an active ingredient.

Specifically, the quercetin or a physiologically acceptable salt thereof; And rutin or a physiologically acceptable salt thereof may be mixed with 1 to 10: 1 to 10, particularly 1 to 5: 1 to 5, more specifically 1: 1 by weight, but not limited thereto .

In addition, the feed composition may further comprise isoquercetin or a physiologically acceptable salt thereof.

Specifically, the isoquercetin or a physiologically acceptable salt thereof; Quercetin or a physiologically acceptable salt thereof; And rutin or a physiologically acceptable salt thereof are mixed in a weight ratio of 1 to 10: 1 to 10: 1 to 10, especially 1 to 5: 1 to 5: 1 to 5, more specifically 1: 1: 1 But is not limited thereto.

Since quercetin, rutin and isoquercetin have been used for a long period of time and proved to be stable, quercetin, rutin and isoquercetin can be prepared in the form of diets that can prevent common diabetes and improve diabetes.

In one embodiment of the present invention, quercetin and rutin isolated and purified in Example 3 were mixed at a weight ratio of 1: 1, isoquercetin, quercetin and rutin in a weight ratio of 1: 1: 1 to prepare 10 μM, Exposure to alloxan-treated zebrafish larvae for 1 hour resulted in a synergistic significant effect on the increase in size of the pancreas with damaged quercetin and rutin mixtures or isoquercetin, quercetin and rutin mixtures (Fig. 10) (Fig. 11) synergistic effect on the intracellular glucose level increase (Fig. 11), it can be used for prevention, treatment and improvement of diabetes.

Such quercetin, rutin, isoquercetin, physiologically acceptable, diabetes, prevention, and improvement are as described above.

The term "feed" of the present invention is intended to include any natural or artificial diet, single meal or the like, or a mixture of quercetin and rutin, or a mixture of isoquercetin, quercetin or quercetin as an ingredient for eating, And rutin mixtures as active ingredients can be made into various types of feeds known in the art, and preferably include concentrates, rations, and / or special diets.

The quercetin, the lutein, the isoquercetin, the isomer thereof, the derivative or the physiologically acceptable salt thereof, or the mixture thereof contained in the feed composition of the present invention may vary depending on the purpose of use and the use conditions of the feed, May be included in an amount of 0.01 to 100% by weight, more specifically 1 to 80% by weight, based on the total weight of the feed composition.

A mixture of quercetin and a rutin, or a mixture of isquercetin, quercetin and a rutin, can significantly increase the size of a damaged pancreas and the damaged pancreas in intracellular glucose levels synergistically, and thus can be usefully used for the prevention, treatment and improvement of diabetes .

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the chemical formulas of quercetin (1), astragalin (2), isoquercetin (3), and rutin (4).
2 is a graph showing the results of ¹ H NMR analysis of quercetin.
3 is a graph showing the results of ¹³ C NMR analysis of quercetin.
4 is a graph showing the ¹ H NMR analysis results of astragalin.
5 is a graph showing the results of ¹ H NMR analysis of isoquercetin.
6 is a graph showing the results of ¹³ C NMR analysis of isoquercetin.
7 is a graph showing the results of ¹ H NMR analysis of the routine.
8 is a graph showing the results of ¹³ C NMR analysis of the routine.
FIG. 9 is a fluorescence microscope image showing the anti-diabetic effect of treating zephiricin pancreatic islet with quercetin, astragalin, isoquercetin, rutin, and mixtures thereof with alosoxane (red circle: pancreatic duct).
10 is a graph showing changes in the size of damaged pancreas as a result of treatment with zephyrate, astragalin, isoquercetin, rutin, and mixtures thereof with alooxane (* p <0.05, ** p < 0.01, *** p < 0.001).
FIG. 11 is a graph showing that the pancreas in which isoquercetin, quercetin and a mixture of rutin are damaged also synergistically significantly increases intracellular glucose levels.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Materials, equipment and reagents

1-1. material

The morus used in this embodiment alba L.) was received from the Korea Food Research Institute in 2011, and the sample (KHU-NPCL-20110826) is stored in Kyunghee University Natural Chemical Laboratory.

1-2. Instruments and reagents

Lichroprep RP-18 (40-63 μm, Merck) was used for column chromatography silica gel 60 (70-230 mesh, Merck) and octadecyl silica gel (ODS) ) Used Kiesegel 60 F254 and RP-18 F254s (Merck). Nuclear magnetic resonance (NMR) spectra were measured at 400 MHz, Inova AS 400, FT-NMR spectrometer (Varian) and electron ionization / mass spectrometer (EI / MS) using JMS-700 (JEOL).

Alloxan monohydrate and sea salt were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2- (N- (7-nitrobenz-2-oxazol-4-yl) , 3-diazol-4-yl) amino) -2-deoxyglucose, 2-NBDG) was purchased from Invitrogen (OR, USA). The fluorescence microscope was an Olympus 1X70 (Olympus, Japan). The software for image analysis was Focus Lite (Focus Co, Daejeon, Korea) and Image J (National Institutes of Health).

Example  2. Solvent Extraction and Fractionation of Oddi

To 15.4 kg of oats, 100% ethanol (EtOH) (35 Lx1) was added, and the mixture was extracted at room temperature for 25 hours. Then, the mixture was filtered through a filter paper, and the residue was further extracted twice with 70% ethanol (35 Lx2). The obtained filtrate was combined and concentrated under reduced pressure to obtain EtOH extract (2219 g). The resulting EtOH extract was partitioned with H ₂ O (1.7 L) and ethyl acetate (EtOAC, 1.7 L x 4) and the H ₂ O (1.7 L)

4). Each layer was concentrated under reduced pressure to obtain EtOAc fraction (MAE, 41 g), n-BuOH fraction (MAB, 247 g) and H ₂ O fraction (MAH, 1932 g).

Example  3. Audi From the fraction  Isolation and Identification of Compounds

3-1. Audi From the fraction  Separation of compounds

EtOAc fraction (40 g) of Example 2 was subjected to SiO ₂ column chromatography (Φ 9 × 14 cm, n-hexane-EtOAc = 10: 1 → 8: 1 → 6: 1 → 3: 1 → 1: 1 → CHCl 3 -MeOH 14 fractions (MAE-1 to MAE-14) were obtained by TLC analysis of each aliquot.

Among MAE-13 fraction [800 mg, elution volume / total volume (Ve / Vt) 0.821-0.874] was purified by silica gel (SiO ₂₎ was purified by column chromatography (Φ 4 × 15 cm, CHCl 3 -MeOH-H 2 O = 16 (MAE-13-1 to MAE-13-30) in the same manner as in Example 1, except that Compound 1 (3: 1 → 12: 3: [MAE-13-15, 24 mg, Ve / Vt 0.186 - 0.202, TLC (RP-18 F254s) Rf 0.68, MeOH-H 2 O (3: 1)] were separated.

Further, the above MAE-13-22 (42 mg, Ve / Vt 0.380-0.411) was subjected to octadecyl silica (ODS) column chromatography (Φ 2 × 5 cm, MeOH-H ₂ O = 1: 1) (MAE-13-22-1 to MAE-13-22-8), and Compound 2 [MAE-13-22-5, 2.7 mg, Ve / Vt 0.486 - 0.502, TLC (RP-18 F254s) Rf 0.52, MeOH-H ₂ O (1: 1)].

The n-butanol fraction (247 g) was further divided into eight fractions (MAB-1 to MAB-8) by Diaion HP-20 column chromatography (Φ 12 × 38 cm, H ₂ O → 50% MeOH → MeOH) . 5 fractions (MAB-5-1) were obtained by conducting ODS column chromatography (Φ 4.5 × 10 cm, MeOH-H ₂ O = 2: 3) on a small fraction MAB-5 (1.31 g, Ve / Vt 0.522-0.601) ~ MAB-5-12). Among them, MAB-5-7 fraction (271 mg, Ve / Vt 0.247-0.369) was subjected to SiO ₂ column chromatography (Φ 2.5 × 15 cm, CHCl 3 -MeOH-H ₂ O = 8: 3: 1) (MAB-5-7-1 to MAB-5-7-12). Compound 3 [MAB-5-7-6, 11.4 mg, Ve / Vt 0.104-0.185, TLC (RP-18 F254s) Rf 0.35, MeOH-H ₂ O = 3: 2] and compound 4 [MAB-5-7-9, 58 mg, Ve / Vt 0.210-0.275, TLC (RP-18 F254s) Rf 0.28, MeOH-H ₂ O = 3: 2].

3-2. Identification of isolated compounds

The structures of the four compounds (compounds 1 to 4) isolated and purified from the oder were identified by NMR, MS, IR and the like.

As a result of the analysis of the NMR data (FIG. 2 and FIG. 3) of the compound 1 as described below, it was confirmed that the compound 1 was quercetin, which is a flavonoid compound.

Quercetin: IR (KBr, cm ^-1 ): 3420, 1662, 1600, 1502; positive FAB / MS m / z 303 [M + H] &lt; ⁺ &gt;; ^{1 H-NMR (400 MHz,} pyridine, δ) 8.54 (1H, d, J = 2.4 Hz, H-2 '), 8.06 (1H, dd, J = 8.0, 2.4 Hz, H-6'), 7.30 ( (1H, d, J = 8.0 Hz, H-5 '), 6.70 (1H, d, J = 2.0 Hz, H-8), 6.64 (1H, d, J = 2.0 Hz, H-6); ^{13 C-NMR (100 MHz,} pyridine, δ) 177.2 (C-4), 165.4 (C-7), 162.3 (C-5), 157.4 (C-9), 147.6 (C-4 '), 147.0 ( C-3 '), 146.9 (C-2), 137.8 (C-3), 123.8 ), 104.3 (C-10), 99.1 (C-6), 94.1 (C-8).

Analysis of the NMR data (FIG. 4) of Compound 2 as described below revealed that Compound 2 was a flavonoid compound astragalin.

Astragalin: [?] _D +16 ( c = 1.0, MeOH); IR (KBr, cm ^-1 ): 3345, 2922, 1654, 1608; positive FAB / MS m / z 471 [M + H] &lt; ⁺ &gt;; ^{1 H-NMR (400 MHz,} CD 3 OD, δ) 8.01 (2H, d, J = 8.8 Hz, H-2 ', 6'), 6.88 (2H, d, J = 8.8 Hz, H-3 ', 5 '), 6.39 (1H, d, J = 1.6 Hz, H-8), 6.19 (1H, d, J = 1.6 Hz, H-6), 5.21 (1H, d, J = 8.0 Hz, H-1 ''), 3.68 (1H, dd, J = 12.0, 2.4 Hz, H-6''a), 3.53 (1H, dd, J = 12.0, 2.4 Hz, H-6''b), 3.44 ~ 3.37 ( 4H, overlapped, H-2 '', 3 '', 4 '', 5 ''); ^{13 C-NMR (100 MHz,} pyridine, δ) 178.6 (C-4), 166.0 (C-7), 162.7 (C-5), 161.6 (C-4 '), 157.5 (C-9), 157.2 ( (C-2), 136.0 (C-3), 131.9 (C-2 ', 6'), 121.9 (C-1 ''), 99.9 (C-6), 94.6 (C-8), 79.1 (C-4 ''), 62.6 (C-6 '').

As a result of analyzing NMR data (FIG. 5 and FIG. 6) of Compound 3 as described below, it was confirmed that Compound 3 was isoquercetin as a flavonoid compound.

Isoquercetin: [?] _D -12.5 ( c = 0.9, MeOH); IR (KBr, cm- ¹ ): 3382, 2942, 1656; positive FAB / MS m / z 487 [M + H] &lt; ⁺ &gt;; ^{1 H-NMR (400 MHz,} CD 3 OD, δ) 7.88 (1H, d, J = 2.0 Hz, H-2 '), 7.78 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 6.84 (1H, d, J = 8.4 Hz, H-5 '), 6.39 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H-6), 5.20 (1H, d, J = 7.6 Hz, H-1 ''), 3.70 (1H, dd, J = 12.0, 2.4 Hz, H-6''a), 3.57 (1H, dd, J = 12.0, 5.6 Hz , H-6'b), 3.34 to 3.19 (4H, overlapped, H-2 '', 3 '', 4 '', 5 ''); ^{13 C-NMR (100 MHz,} pyridine, δ) 179.4 (C-4), 166.0 (C-7), 163.0 (C-5), 159.0 (C-9), 158.4 (C-2), 149.8 (C (C-2 '), 145.9 (C-3'), 135.6 (C-3), 123.1 ), 105.6 (C-10), 104.3 (C-1 &quot;), 99.9 (C-6), 94.7 75.7 (C-2 ''), 71.2 (C-4 ''), 62.5 (C-6 '').

As a result of analyzing NMR data (FIG. 7 and FIG. 8) of Compound 4 as described below, it was confirmed that Compound 4 was a routine as a flavonoid compound.

Routine: [?] _D -8.2 ( c = 1.2, MeOH); IR (KBr, cm- ¹ ): 3382, 2932, 1656; positive FAB / MS m / z 633 [M + H] &lt; ⁺ &gt;; ^{1 H-NMR (400 MHz,} CD 3 OD, δ) 7.68 (1H, d, J = 2.0 Hz, H-2 '), 7.65 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 6.87 (1H, d, J = 8.4 Hz, H-5 '), 6.40 (1H, d, J = 2.0 Hz, H-8), 6.23 (1H, d, J = 2.0 Hz, H-6), 5.10 (1H, d, J = 7.6 Hz, H-1 ''), 4.46 (1H, d, J = 1.2 Hz, H-1 '''), 1.12 (3H, d, J = 6.4 Hz, H-6 '''); ¹³ C-NMR (100 MHz, pyridine,?) 179.9 (C-4), 154.3 (C-7), 161.9 (C-5), 160.2 ), 146.8 (C-3 '), 137.3 (C-3), 121.9 (C-1'), 114.3 , 118.7 (C-2 '), 103.5 (C-1''), 102.4 (C-1'''), 99.9 (C-6), 96.8 , 77.2 (C-3 ''), 72.7 (C-2 ''), 73.9 (C-4 ''), 69.7 (C-5 '''), 68.5 (C-6''), 17.9 (C-6''').

Example  4. Zebra fish  Pancreas degree ( pancreatic islet ) Change measurement

4-1. Zebrafish ) breed

Zebrafish fishes were maintained in zebrafish system S Type (1500 (W) x 400 (D) x 2050 (H) mm) (Genomic Design, Daejeon, Korea). Zebrafish embryos were collected after 12 hours in a 1: 1 ratio of female to male in a water tank equipped with a net for separating eggs and adult fish. After collecting, the cells were washed three times with 0.03% sea salt solution to remove foreign matters and then cultivated in an incubator set at 28.5 ° C light period (14 light: 10 dark).

4-2. Zebra fish  How to Measure Changes in Pancreas Degree

Zebrafish larvae of 5 dpf (day post fertilization) were placed in 96 wells and stained with 25 μM 2-NBDG for 12 hours. Then, the zebrafish larva was exposed to 100 μM of alloxan, a diabetic inducer that selectively destroyed only the pancreatic beta cells, for 15 minutes, and was exposed to the 0.03% sea salt solution for 5 hours. Fluorescence microscopy (Olympus 1X70, Olympus, Japan) was used to directly observe the pancreatic duct, and 25 μM 2-NBDG was re-stained for 1 h before fluorescence microscopy.

Zebrafish larvae exposed to the above allylate were subjected to a primary microscope and the compounds (quercetin, astragalin, isoquercetin, and rutin) isolated and purified in Example 3 were prepared at 10 μM and exposed for 1 hour. The above compounds were mixed at a weight ratio of 1: 1 or 1: 1: 1 and made to 10 μM, and exposed to the above-mentioned alloxane-exposed zebrafish larva for 1 hour. Gimepiride, an antidiabetic agent, was used as a positive control.

To observe changes in pancreatic tissue due to the compound, a second microscopic photograph was taken and 25 μM 2-NBDG was re-stained for 1 h before fluorescence microscopy as in the first microscopic photograph. The fluorescence intensity of the zebrafish (FIG. 10) and the fluorescence intensity of the pancreas (FIG. 11) were analyzed using the Focus Lite and Image J software images obtained through fluorescence microscopy (FIG. 9). Measurement of the 2-NBDG staining intensity in the image of FIG. 9 enables the evaluation of intracellular glucose uptake ability, and thus the staining intensity is used as an index for evaluating anti-diabetic activity.

4-3. Statistical analysis

Experimental results were analyzed by one-way ANOVA using Graphpad Prism (version 5). Post-analysis for comparison between groups was verified by Tukey and statistically significant only at P <0.05. The experimental results are shown as mean ± standard deviation.

Example  5. Zebra fish  Pancreas size change measurement result

In order to confirm the anti-diabetic effect of quercetin, astragalin, isoquercetin, rutin and a mixture thereof, the size change of the zebrafish pancreas shown in FIG. 9 was measured and shown in FIG. 10, Increase and decrease.

Alloxacin treatment group Pancreas size reduction%

= (Pancreas size of alloxan treatment group - pancreas size of normal group) / normal group pancreas size X 100

Drug treatment group Pancreas size increase%

= (Drug treated group, the pancreas also size-alloxan treated groups pancreas in size) / alloxan treatment group pancreatic the size X 100

First, zebrafish exposed to alloxan was reduced in size by 40.9% compared to zebrafish without alloxan, and the pancreatic duct was damaged. The damaged pancreas was exposed to gemepiride As a result, gemepiride was found to increase the size of the reduced pancreas by 25.3%.

In contrast, in the group treated with the quercetin and rutin mixture, the size of the pancreas of about 36.5%, which is much higher than that of the quercetin (22.3%) or the rutin (11.3%), increased. This was equivalent to gemepiride, a commercially available diabetes drug.

On the other hand, the pancreatic size of about 20.1% (FIG. 12) in the group treated with the isoquercetin and quercetin mixture and about 16.1% (FIG. 13) in the group treated with the isoquercetin and the rutin mixture showed an increase in the amount of isoquercetin %) Or quercetin (22.3%), respectively.

These results suggest that even if isoquercetin and quercetin, or isoquercetin and rutin are mixed, the pancreas is less effective in increasing the size of each compound than that of each compound, and the effect of treating diabetes is rather hindered. On the other hand, the quercetin and rutin mixture showed a synergistic effect on the increase of the pancreas size, indicating that it can be useful for the prevention, treatment and improvement of diabetes.

In addition, a group treated with a mixture of isoquercetin, quercetin and a mixture of rutin, isoquercetin, quercetin or a mixture of rutin, a mixture of isoquercetin and quercetin, a mixture of isoquercetin and rutin, or a mixture of quercetin and rutin, 45.8% of the pancreas also showed increased size (Fig. 10).

As a result, it has been confirmed that a mixture of isoquercetin, quercetin and rutin, unlike the mixture of isoquercetin and quercetin, or the mixture of isoquercetin and rutin, has a remarkable synergistic effect on the increase of pancreas size and can be used for prevention, treatment and improvement of diabetes .

Example  6. Zebra fish  The pancreas is also intracellular Glucose  Numerical analysis result

In order to analyze the intracellular glucose level in Example 4, the fluorescence intensity was measured and the fluorescence intensity change analyzed by the following equation is shown in FIG.

Alloxan treated group Fluorescence staining intensity change%

= (Fluorescein value of pancreas in alooxa treated group - fluorescence value in normal group pancreas) / normal group Pancreas degree fluorescence value X 100

Fluorescent staining intensity change in drug treatment group%

= (Drug treatment group pancreas fluorescence - alooxa treated group pancreas fluorescence value) / alloxan treatment group pancreas fluorescence value X 100

As a result, it was confirmed that the fluorescence intensity of zebrafish exposed to alloxan was lower than that of unoxidized zebrafish (normal group), and in the alooxan positive control group, The fluorescence intensity increased by 49.4%.

On the other hand, in the group treated with the quercetin and the rutin mixture, the fluorescence intensity of the quercetin and the rutin was much higher than that of the quercetin or the rutin, respectively.

As a result, it was found that the quercetin and the rutin mixture had a synergistic effect on the increase of fluorescence intensity. Therefore, it has been confirmed that the quercetin and the rutin mixture has a synergistic and remarkable effect on the intracellular glucose level increase of the injured pancreatic duct, and thus can be effectively used for the prevention, treatment and improvement of diabetes.

In addition, in the group treated with a mixture of isoquercetin, quercetin and rutin, isoquercetin, quercetin or rutin were treated respectively, or a mixture of isoquercetin and quercetin, isoquercetin and rutin, or quercetin and rutin, Indicating an increase in staining intensity (Fig. 11).

As a result, it was found that the mixture of isoquercetin, quercetin and rutin had a synergistic effect on the increase of fluorescence intensity. Therefore, it has been confirmed that a mixture of isoquercetin, quercetin and rutin has a synergistic effect on the intracellular glucose level increase of injured pancreatic duct, and thus can be effectively used for prevention, treatment and improvement of diabetes.

In comparison, 33.5% of the quercetin-treated group, 32.6% of the isoquercetin-treated group and 22.4% of the rutin-treated group showed an increase in fluorescence intensity. The fluorescence intensity of the astragalin treatment group was not increased by 4.22%.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (12)

Quercetin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating diabetes mellitus comprising rutin or a pharmaceutically acceptable salt thereof as an active ingredient.
2. The method of claim 1, wherein the quercetin or a pharmaceutically acceptable salt thereof; And lutin, or a pharmaceutically acceptable salt thereof, are mixed in a weight ratio of 1 to 10: 1 to 10.
The pharmaceutical composition of claim 1, wherein the composition further comprises isoquercetin or a pharmaceutically acceptable salt thereof.
4. The method of claim 3, wherein the isoquercetin or a pharmaceutically acceptable salt thereof; Quercetin or a pharmaceutically acceptable salt thereof; And lutin, or a pharmaceutically acceptable salt thereof, are mixed in a weight ratio of 1 to 10: 1 to 10: 1 to 10.
The pharmaceutical composition of claim 1, wherein said composition restores damaged pancreatic beta cells or increases intracellular glucose levels.
2. The pharmaceutical composition according to claim 1, wherein the diabetes is insulin dependent diabetes.
The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
Quercetin or a physiologically acceptable salt thereof; And a rutin or a physiologically acceptable salt thereof as an active ingredient.
9. The food composition of claim 8, wherein the composition further comprises isoquercetin or a physiologically acceptable salt thereof.
9. The food composition of claim 8, wherein the composition further comprises a physiologically acceptable carrier.
Quercetin or a physiologically acceptable salt thereof; And a diuretic or a physiologically acceptable salt thereof as an active ingredient.
12. The feed composition according to claim 11, wherein the composition further comprises isoquercetin or a physiologically acceptable salt thereof.

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