KR20170057165A - A pharmaceutical composition comprising derhamnosylmasin or pharmaceutically acceptable salt thereof as an active ingredient for prevention and treatment of diabetes mellitus - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising derhamnosylmasin or a pharmaceutically acceptable salt thereof as an active ingredient for preventing and treating diabetes mellitus. As a result of examining the effect of derhamnosylmasin on muscle cells, it was confirmed that an intracellular glucose absorption factor is increased; peroxisome proliferator-activated receptor (PPAR) activity is increased; and an insulin signal transmission path is activated. Accordingly, derhamnosylmasin can be used for preventing and treating diabetes mellitus.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of diabetes mellitus comprising diramnosylmecycline or a pharmaceutically acceptable salt thereof as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing and treating diabetes comprising deramnosylmasin or a pharmaceutically acceptable salt thereof as an active ingredient.

In recent years, Korea has shown a very desirable phenomenon in terms of rapid economic growth, improvement of living standards, and westernization. On the other hand, adults suffer from stress due to excessive intake of high calorie foods, lack of exercise, It is increasingly becoming westernized mainly by adult diseases. Representative examples of adult diseases include liver disease, hypertension, diabetes, obesity, and hyperlipemia.

Diabetes mellitus is a non-communicable chronic disease that lacks insulin secreted from the pancreatic β-cells and does not function properly. Glucose can not be used as an energy source in the body and remains in high concentrations in the blood. It is an excreted disease. Diabetes mellitus can be categorized into insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM), depending on the cause and symptoms of the disease. In Korea, more than 95% of diabetic patients are NIDDM patients. Diabetes is not a large disease in itself, but rather a complication that arises from long-term illnesses such as diabetic neuropathy, retinopathy, cataract, and nephropathy. It is a societal problem because patients can not only lead a normal life but also cause catastrophic consequences.

Currently, commonly used diabetes drugs are broadly divided into oral hypoglycemic agents and insulin injections. In general, insulin-dependent diabetes mellitus patients who do not have insulin secretion in the body, gestational diabetes mellitus patients, and oral insulin-dependent diabetes mellitus patients whose insulin-dependent diabetes mellitus is poorly controlled due to oral hypoglycemia, Non-insulin dependent diabetes mellitus patients who do not control blood glucose levels are generally given oral hypoglycemic agents. Oral hypoglycemic agents can be classified into sulfonylurea drugs and acetaminophen drugs.

Sulfonylurea drugs such as glipizide, gliclazide, gliquidone, glibenclamide, and chlorpropamide show actions that promote insulin secretion in the pancreas However, it can not be used in patients with insulin-dependent diabetes without any insulin secretion in the pancreas. It can not be used in fertile women because of concerns about non-insulin dependent diabetes mellitus or aberrant birth, abortion, and stillbirth in which the insulin secretory ability is relatively decreased in the pancreas There are disadvantages. In addition, since most sulfonylurea drugs are metabolized in the liver and excreted in the kidney, they should be used with caution in patients with hepatic and renal dysfunction.

Metformin and other acetaminophen drugs have not been shown to have a mechanism of action, but they do not appear to increase insulin secretion in the pancreas. They have a lower blood glucose lowering effect than sulfonylurea drugs, low. However, the incidence of gastrointestinal adverse reactions is high, causing nausea, vomiting, diarrhea, and rash at the beginning of treatment, causing lactic acidosis and causing life-threatening side effects. have.

Therefore, there is a need to develop a safe hypoglycemic agent which can treat diabetes mellitus which is increasing in domestic and foreign countries with fewer side effects, because the currently used sulfonylurea or acetaminophen drugs have the disadvantages and side effects described above .

Thus, many serious side effects of currently chemically synthesized anti-diabetic drugs occur and drug effects due to the occurrence of drug resistance are lowered. Therefore, a lot of research is underway to find new substances having novel anti-diabetic activity.

As a prior art relating to the effect of diramnosylchemicin on diabetes, Korean Patent Laid-Open No. 10-2015-006115 discloses the effect of Mechin on the prevention of obesity, and Hyung Jun Park discloses a centipedegrass extract ( Eremochloa ophiuroides ) (BMC Complementary and Alternative Medicine 2012, 12: 230), the contents of the study on diramnosylmeycine to date, including the above-mentioned prior art, It is only to identify the ingredients, but the direct effect on it is unknown.

Accordingly, the present inventors tried to find a new substance having an effect on diabetes. As a result of examining the effect of diramnosylmethacin on muscle cells, the present inventors have found that PPARγ (peroxisome proliferator-activated receptor γ ) Activity and activates the insulin signaling pathway, thus confirming that diramnosylchemycin can be effectively used for the prevention and treatment of diabetes. The present invention has been completed based on this finding.

It is an object of the present invention to provide a pharmaceutical composition for prevention and treatment of diabetes comprising deramnosylmasin or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating diabetes comprising as an active ingredient deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

The present invention also provides a health functional food for preventing or ameliorating diabetes comprising deramnosylmasin represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for reducing blood glucose content comprising diramnosylmecine represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for hypoglycemia comprising diramnosylmecin or its pharmaceutically acceptable salt represented by the above formula (1) as an active ingredient.

By confirming that the diramnosylmaysin of the present invention increases glucose uptake into muscle cells, increases PPARy (peroxisome proliferator-activated receptor gamma) activity and activates the insulin signaling pathway, And can be usefully used for prevention and treatment of diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a compositional analysis of a single substance fraction extracted from a centipedegrass.
Figure 2a shows glucose uptake rates of diramnosylmecin, orientin, rhamnosylorientin, meicin, irremotin, isoorientin, chlorogenic acid, and luteolin;
Control: You have not done anything.
FIG. 2B is a graph showing the glucose uptake rate according to the concentration of the centipede glass extract (50% CGE).
FIG. 3A is a graph showing the results of RT-PCR experiments for confirming the expression pattern of insulin signaling molecules of diramnosylmecine,
FIG. 3B is a graph showing the results of western blot experiments for confirming phosphorylation of the insulin signal transduction of diramnosylmecine.
IR: insulin receptor, IRS-1: insulin receptor substrate-1, GLUT4: glucose transporter 4, pIRS: phosphorylated insulin receptor substrate.
4A is a luciferase assay for confirming the transcriptional activity of PPARγ of diramnosylmecin, orientin, rhamnosylorientin, meicin, irremotin, isoorientin, chlorogenic acid, and luteolin ) Shows experimental results:
control: You have not done anything.
GW1929: PPARgamma agonist.
FIG. 4B is a graph showing the results of luciferase assay for confirming the transcriptional activity of PPARγ according to an increase in the concentration of diramnosylmecine.
FIG. 4c is a graph showing the results of luciferase assay for confirming the degree of transcriptional activity of PPARγ according to the concentration of the centipede glass extract (50% CGE).
FIG. 5 is a western blot diagram showing changes in the binding of PPARγ and RXRα with time by diramnosylmecine treatment. FIG.
FIG. 6 is a graph showing an experiment result of an assay kit ((LanthaScreen TR-FRET Peroxisome Proliferator Receptor delta Coactivator Assay kit; Invitrogen, CA) for binding to well-known sites of diramnosylmethionine PPARγ .
FIG. 7 is a diagram showing a region well known as a binding site with a substance necessary for regulating the activity of PPARy and an unknown region predicted to bind diramnosylmethacin.
8 is a view showing the binding site of PPARγ and diramnosylmecine.
FIG. 9 is a graph showing the results of luciferase assay for confirming the binding of diramnosylmethacin to PPARγ causing point mutation. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing and treating diabetes comprising as an active ingredient deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

The present invention also provides a pharmaceutical composition for reducing blood glucose content comprising diramnosylmecine represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Preferably, the composition increases glucose uptake into muscle cells, increases PPARy (peroxisome proliferator-activated receptor gamma) activity, and activates the insulin signaling pathway.

It is also preferred that the diabetes is type 1 diabetes (insulin-dependent diabetes mellitus) or type 2 diabetes (insulin-independent diabetes mellitus).

Expression of insulin signaling molecules is regulated by PPARγ (peroxisome proliferator-activated receptor γ) and has been reported by many previous studies. PPAR (Peroxisome Proliferator Activated Receptor, Peroxisome Proliferator Activated Receptor) is involved in metabolism and energy production and is known to have the highest expression of PPARγ after ingestion. Activation of PPARγ lowers the concentration in the blood by putting fatty acids into fat cells, which results in a decrease in insulin resistance due to reduced use of fatty acids in muscle. Such abnormalities of PPAR may cause diabetes, hyperlipemia and obesity.

PPARs form heterodimers with retinoid X receptor α (RXRs), which, when coupled with ligands, cause a structural change in AF2 (activation function 2) The coactivator binds and the PPAR-RXR isoform promotes or inhibits the transcription of the target gene by binding to the peroxisome proliferator-response element (PPRE) in the DNA sequence, where the most affected molecules are insulin signaling molecules .

In a specific example of the present invention, diramnosylmecin was treated on muscle cells to examine the effect thereof. As a result, it was found that it increased the intracellular glucose uptake rate (see FIG. 2A), increased PPARγ (peroxisome proliferator-activated receptor γ) 4) to activate the insulin signaling pathway (see FIG. 3).

It was also confirmed that when diramnosylmethacin was treated, the binding of PPARγ and RXRα was rapidly induced (see FIGS. 5 and 4), and that diramnosylmecine binds to a site other than the ligand binding site of PPARγ 7), and it was confirmed that threonine, which is the 447th amino acid, plays the most important role (see FIGS. 8 and 9).

Therefore, diramnosylmecin can be usefully used as a pharmaceutical composition for prevention and treatment of diabetes or as a pharmaceutical composition for lowering blood glucose.

The present invention includes all of the compounds represented by formula (1) as well as pharmaceutically acceptable salts thereof, prodrugs thereof, and possible solvates, hydrates, racemates or stereoisomers thereof which can be prepared therefrom.

The present invention can be used in the form of a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from non-toxic organic acids, such as, for example, diesters, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinic acid, succinic acid, succinic acid, succinic acid, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and adding the salt to a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile To precipitate it. It is also possible to prepare the same by heating the compound represented by the general formula (1) and an acid aqueous solution or alcohol, followed by evaporating the mixture or drying the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The pharmaceutical composition of the present invention may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, Syrups, and other liquid preparations.

The pharmaceutical composition of the present invention can be used as a medicine for preventing or treating diabetes or diabetic complications.

Such diabetic complications include neuropathy, nephropathy, retinopathy, macroangiopathy, coronary artery disease, osteopenia, and also the pharmacological activity of the present invention The compositions can be used as medicaments for treating intraspinal gingival damage.

Specifically, the pharmaceutical compositions of the present invention may be formulated for oral administration, for example, tablets, troches, lozenges, aqueous or aqueous suspensions, prepared powders or granules, emulsions, hard or soft capsules, It is formulated into elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate, Lubricating oil such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection injection method for parenteral administration. In order to formulate the composition for parenteral administration, the mixed extract of the present invention is mixed with a stabilizer or a buffer in water to prepare a suspension, which is formulated into a unit dosage form of ampoule or vial, , Or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The dosage of the active ingredient according to the present invention is appropriately selected depending on the degree of absorption, inactivation rate and rate of absorption of the active ingredient in the body, age, sex and condition of the patient, and severity of the disease to be treated.

The present invention also provides a health functional food for prevention and improvement of diabetes comprising deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Chemical Formula 1]

In addition, the present invention provides a health functional food for hypoglycemia comprising diramnosylmecin or its pharmaceutically acceptable salt represented by the above formula (1) as an active ingredient.

The diabetes is preferably type 1 diabetes or type 2 diabetes.

As a result of confirming the effect of treatment of diramnosylmethacin on muscle cells, it was confirmed that it increased intracellular glucose uptake rate, increased PPARγ (peroxisome proliferator-activated receptor γ) activity and activated the insulin signal transduction pathway , Diramnosylmecin can be usefully used as a health functional food for prevention and improvement of diabetes or blood sugar lowering.

As used herein, the term " health functional food "refers to foods prepared and processed in the form of tablets, capsules, powders, granules, liquids, and circles by using raw materials and components having useful functions in the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional 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 that are conventionally added in the art. In addition, unlike general medicines, there is an advantage that there are no side effects that may occur when a medicine is taken for a long time by using a food as a raw material, and it is excellent in portability and the health functional food of the present invention is effective in improving the effect of sleep, It can be taken as an adjuvant for.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the compound of the formula (1) of the present invention is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight of the raw material composition in the production of food. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient like ordinary foods. The natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

≪ Example 1 > Determination of the content of active ingredients of sentiped grass

The centipedegrass harvested in autumn was extracted with 80% methanol for one week. The ratio of centipedegrass to 80% methanol was 1:20 (v / w). After the extraction, impurities (residue) were removed through centipede grasping and filtering. Methanol was then removed using a concentrator, and the water layer extract was recovered. The recovered extract was treated with hexane to remove lipid components and chlorophyll, and the hexane layer proceeded to a green state. Then, the active ingredients were extracted again by using ethyl acetate, and then concentrated to a powder state, which was refrigerated for use in the following experiments. The powdered Centipedgrass extract was dissolved in 50% and 70% methanol at a concentration of 1 mg / ml for analysis of the components, and the active ingredient was analyzed by HPLC analysis under the conditions shown in Table 1 below.

device Agilent 1260 liquid chromatography Measuring instrument UV (360 nm) column YMC-Pack ODS-A 150 X 4.6 mm. S-5 [mu] M, 12 nM Mobile phase Solventa; 0.1% Formic acid, Solvent B; 100% methanol Temperature 30 ℃ Flow rate 0.5 mL / min Density mode Time (minutes) Mobile phase A B 0.0 100 0 10.0 100 0 30.0 50 50 60.0 0 100 60.01 stop

As a result, as shown in Fig. 1, a single substance extracted and fractionated from centipedegrass was treated with a solution of diramnosylmaysin, orientin, rhamnosylisoorientin, maysin, , Isoorientin, and cholorogenic acid and eremonetin (FIG. 1). As a result of the examination of the content of each single substance, a total of 1 mg of Sentifedgrass extract ( 50% CGE) contained 57 μg (about 5%) of diramnosylchemicin (Table 2).

The chemical formulas of each single substance are shown in the following table.

< Example  2> Diramnosylmecine  Ready

For the separation of diramnosylmaysin, primary column chromatography was performed using Toyopearl HW-40 gel filler. First, a column having a diameter of 2.5 cm × 54 cm was set up and filled with gel. The column was then washed and activated with a column volume of methanol three times, then the column was replaced with 20% methanol, the starting solvent for column elution, and stabilized. The ethyl acetate layer was dissolved in a small amount of 20% methanol, adsorbed on packed gel, and then eluted with 20%, 40%, 60% and 100% of H ₂ O: MeOH using a solvent system of stepwise gradient mode. The column was eluted with methanol. Then, each of the eluting layers was concentrated under reduced pressure to remove the solvent, and then secured in powder form.

Next, a second column chromatograpy using an ODS AQ 120S filler was performed using a 40% methanol elution layer containing diramnosylmecine, which was presumed to contain diramnosylmecicin. The column was eluted with a small amount of H ₂ O, which was the starting solvent for column elution, and adsorbed on a packing material. Then, H ₂ O: The methanol content was increased by 5% using a solvent system of MeOH and step wise gradient mode. Each elution layer was fractionated in a test tube by 200 drops using a fraction device, and only a fraction of high purity was collected through HPLC monitoring to obtain a substance after concentration.

After the separation, the purity and molecular weight of the diramnosylmecine-enriched substance were confirmed by HPLC-MS. After confirming the purity, 1D NMR of 1H and 13C was measured and the structure of the compound was used for the following experiments.

< Experimental Example  1-1> Diramnosylmecine  Confirm glucose absorption effect

Glucose uptake experiments were carried out using C2C12 muscle cells (American Type Culture Collection, ATCC, USA) in the following manner to confirm the effect of controlling the glucose concentration of diramnosylchemicin.

Specifically, C2C12 cells in an undifferentiated state were added to each well of a 6-well plate at 1 × 10 ⁵ , and DME medium (Dulbecco's modified Eagle's medium containing 10% FBS (Fetal bovine serum) Gibco, Life technologies, USA) in a 5% CO ₂ incubator maintained at 37 ° C for 24 hours. Then, the culture medium was discarded and a medium for assisting differentiation into muscle cells was added to induce differentiation into muscle cells for two days. The composition of the culture medium for supporting the differentiation of muscle cells is DMEM medium containing 2% FBS. Thereafter, a single substance (derhamnosylmaysin, orientin, rhamnosylorientin, or the like) extracted and fractionated in a centipedegrass by the method of Examples 1 and 2 during differentiation into muscle cells rhamnosylisoorientin, maysin, eremonetin (luteoin-6-C-boivinopyranoside), isoorientin, cholorogenic acid, luteolin) And cultured for 4-6 days until mature muscle cells were obtained. Thereafter, the medium for inducing differentiation was changed every two days, and the single substances were treated to a concentration of 10 μg / ml. When grown to mature muscle cells, the cells were cultured in serum-free media for 12 hours, and then 2-NBDG (2- (N- (7-Nitrobenz-2-oxa-1,3- diazol- 4-yl) Amino) -2-Deoxyglucose), and the intracellular glucose was observed through a fluorescence microscope and a fluorescence analyzer (Infinite M200 Tecan, Austria). 2-NBDG is a structure in which the deoxy-form of glucose is bound to a fluorescent structure. It enters the cell like glucose but does not decompose, so it can accumulate fluorescence. Fluorescence was confirmed by naked eyes and compared with photographs, and the relative amounts of 2-NBDG in the cells were numerically compared using a fluorescence analyzer.

As a result, it was confirmed that the glucose absorption rate of diramnosylmecin was significantly higher than that of the other components as shown in FIG. 2A (FIG. 2A).

< Experimental Example  1-2> Sentiped Grass  Identification of glucose absorption effect of extract

In the same manner as in EXPERIMENTAL EXAMPLE 1-1, 6.25, 12.5, 25, 50, and 100 μg / mL of the centrifuged granular extract isolated in Example 1 were dissolved in 50% methanol (50% Glucose uptake experiments were performed. As a result, it was confirmed that the intracellular glucose uptake increased as the concentration of the centipedgras extract increased as shown in FIG. 2B (FIG. 2B). However, in comparison with the results in Fig. 2a, it was confirmed that the treatment of a single substance of diramnosylmecine increased the glucose uptake rate at a similar concentration as compared with the treatment with the centipedragus extract.

< Experimental Example  2> Regulation of insulin signaling molecule expression

Insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) are key insulin signaling molecules that are activated by insulin and induce glucose to enter the cells when blood glucose levels are high . In order to investigate the effect of derhamnosylmaysin on the expression of insulin signaling molecules, experiments were conducted to compare the mRNA expression and protein activity of the above three substances. Specifically, the cells were cultured in a culture medium in the same manner as in the method of differentiating C2C12 cells in undifferentiated state in <Experimental Example 1-1>. From the starting point of differentiation, diramnosylmecin was treated at a concentration of 10 μg / ml for 4 days, and then total RNA and proteins were isolated by collecting the cells from the treatment and control groups.

First, Trizol reagent (MRC, Cincinnati, OH, USA) was used to isolate total RNA, and then the expression levels of IR, IRS-1 and GLUT4 mRNA were compared through RT-PCR experiments (FIG. The total RNA isolation was performed according to the manufacturer's instructions. RIPA lysis buffer was used for the separation of proteins, and proteins were separated by SDS-PAGE to analyze the dissolved proteins.

Then, the amount and activity of the target proteins IRS-1 and Akt among the proteins separated through Western blotting were confirmed (FIG. 3B). The degree of activation can be compared by confirming the degree of phosphorylation of each protein. Western blotting was performed by a conventional method known in the art, and all of the antibodies used therein were purchased from cell signaling technology (USA).

As a result, as shown in FIG. 3A, it can be seen that the degree of expression of insulin signal transduction factors regulated by insulin increases with the treatment of diramnosylchemicin. In addition, as shown in FIG. 3B, IRS-1, which is an increased insulin signal transduction factor, exhibits a relatively increased pattern of its activity (phosphorylation) as the amount thereof is increased. Activation (phosphorylation). This means that diramnosylmeicin is a substance capable of regulating blood sugar (FIG. 3).

< Experimental Example  4> PPARγ  Active regulation

Expression of insulin signaling molecules is regulated by PPARγ (peroxisome proliferator-activated receptor γ) and has been reported by many previous studies. The present inventors performed a luciferase assay to confirm the effect of diramnosylmeicin, which increases the expression of insulin signaling molecules, on the transcriptional activity of PPARγ. Luminescent enzyme analysis is a method for measuring the transcriptional activity of a specific gene. A fusion gene in which a luminescent enzyme gene is linked to a specific gene is introduced into cultured cells, and transcription activity is measured from the amount of luminescence.

Specifically, a plasmid capable of expressing PPARγ and a binding partner RXRα (retinoid X receptor α) sufficiently existed in 293T cells (ATCC, American Type Culture Collection, USA) was transformed and overexpressed in cells. Also, the binding sites exist so that these transcription factors can bind and express transcriptional activity, and as a result, plasmids expressing the luminescent enzyme are also transfected. The transformation was carried out using Lipofectamine LTX (Thermofisher, USA) according to the manufacturer's instructions. Subsequently, the transformed 293T cells were treated with the single substances extracted from centipedegrass used in <Experimental Example 1-1> at a concentration of 10 μg / ml for 24 hours, and then the cells were disrupted to produce luminescence enzyme Luciferase assay was performed. Luciferase Assay System (Promega, Madison, Wis., USA) was used for the luminescence analysis and the experiment was conducted as instructed by the manufacturer. In the case of diramnosylmecin, various experiments were conducted at a concentration of 0 to 20 μg / ml.

As a result, as shown in FIG. 4, it was confirmed that diramnosylmecin alone significantly increased the activity of PPARγ among the single substances extracted and fractionated in centipedegrass (FIG. 4A) (Fig. 4B). &Lt; tb &gt; &lt; TABLE &gt;

(Luciferase assay) (FIG. 4c) was performed by treating the centipede glass extract (50% CGE) used in Experimental Example 1-2, and when a single substance of diramnosylmecine was treated, It was confirmed that the activity of PPARγ was increased more at a similar concentration than when treated with a glass extract (50% CGE).

< Experimental Example  5> Diramnosylmecine PPARγ and Of RXRα  Effect on binding force

Immunoprecipitation was performed to confirm the effect of diramnosylmecin on the binding strength of PPARγ and RXRα. Immunoprecipitation is an experiment in which the binding force between two molecules is examined. After attaching a marker to one molecule and binding reaction with the other molecules, the substance capable of recognizing and separating the marker is treated, . At this time, if the molecule having the marker and another molecule are binding, the principle is separated. This way you can see how many molecules are binding, or how fast they are binding.

Specifically, a plasmid capable of expressing PPARγ by attaching a marker to PPARγ was transfected into 293T cells (ATCC; American Type Culture Collection, USA), overexpressed in cells, and disrupted and used as a sample. The transformation was carried out using Lipofectamine LTX (Thermofisher, USA) according to the manufacturer's instructions. Then, plasmids expressing RXRa were transfected into other 293T cells to prepare samples capable of reacting together. Subsequently, the immunoprecipitation experiment was carried out using the prepared two molecular samples. The sample was treated with diramnoxyline to determine its activity. A sample in which the PPARgamma protein was over-expressed was prepared so that the activity becomes more apparent by pretreatment of diramnosylchemicin. Thereafter, a sample in which RXRα protein was overexpressed and diramnosylmecin-pretreated PPARγ-overexpressed sample were mixed at a ratio of 1: 1 to induce the reaction. At this time, the reaction time was different, and the reaction time was checked. After completion of the reaction, the reaction solution was settled to remove the supernatant, and the reaction was not allowed to proceed through the eluent with the surfactant. Thereafter, binding of PPARy with RXR [alpha] was confirmed by Western blotting. Western blotting was performed by a conventional method known in the art, and all of the antibodies used therein were purchased from cell signaling technology (USA).

As a result, as shown in Fig. 5, it was confirmed that diramnosylmecin induced binding of PPARy with RXRa more rapidly. There was no significant difference in the binding of the two proteins for 5 minutes. When the binding reaction was performed for 10 minutes, the difference between the treated and untreated diramnosylchemic groups was remarkable, And the difference was more apparent in the group to which the reaction was performed (Fig. 5).

< Experimental Example  6> Diramnosylmeysin PPARγ and Of RXRα  Surface to identify the effect on binding Plasmon  Resonance experiment

Surface plasmon resonance experiments were conducted to examine in more detail the effect of diramnosylmethacin on binding of PPARγ and RXRα. In the surface plasmon resonance experiment, the degree of binding between molecules can be confirmed in real time, and the optimal affinity for binding between two molecules can be compared and analyzed to reveal the binding affinity between two molecules.

Specifically, a stationary phase and a mobile phase are designated to perform surface plasmon resonance experiments. The immobilized phase is coded on the gold chip, and when the mobile phase flows at a constant rate, it binds to the target molecule present in the mobile phase. At this time, the concentration of molecules in the mobile phase plays an important role in showing the binding affinity. The PPARγ protein was coated on a gold chip in a stationary phase, and the RXRα protein was prepared as a mobile phase by dissolving the protein in PBS buffer. The PPARγ and RXRα proteins used in this experiment were purchased from CUSABIO. The gold chip coated with the PPAR gamma protein is mounted on the Reichert SR7500DC system to prepare the experiment. Before dripping the mobile phase of the RXRα protein, diramnosylcysine to be used as a comparator or to be used as an experimental group is flowed into the pretreatment. Diramnosylmethacin or roglitazone is flowed through the pretreatment for 5 minutes, and then the mobile phase containing the RXRα protein is poured to compare the binding affinities. Scrubber2 software was used to analyze the binding affinity. .

As a result, as shown in Table 4, the treatment of diramnosylmecin was found to increase the binding of PPARγ and RXRα by about 3 times, which is higher than that of rosiglitazone, which is already known as a therapeutic agent for diabetes.

EXPERIMENTAL EXAMPLE 7-1 Confirmation of binding site of diramnosylmecine in PPARy protein

The activity of PPARγ, which regulates the expression of insulin signaling factors, is enhanced by the binding of certain substances to well-known ligand binding sites. It is reported that the 473rd amino acid sequence tyrosine residue deeply participates in the ligand binding site. Therefore, in order to confirm whether diramnosylmethionine binds to a well-known site of PPARγ to increase the activity of the protein, an assay kit ((LanthaScreen) in which only a ligand binding site of PPARγ exists and a specific activity is required to bind directly to the ligand binding site TR-FRET Peroxisome Proliferator Receptor delta Coactivator Assay kit (Invitrogen, CA).

Specifically, a single substance extracted from centipedegrass (derhamnosylmaysin, orientin, and the like) was analyzed by the method of Example 1 to analyze whether it binds to the ligand binding site of PPARγ. Isomers of rhamnosylisoorientin, maysin, eremonetin (luteolin-6-C-boivinopyranoside), isoorientin, cholorogenic acid, luteolin, ) Is dissolved in methanol and prepared. At this time, the concentration of the single substances makes the final treatment concentration 10 μg / ml. GW1929, which binds to the ligand binding site of PPARγ and increases its activity, and GW9662, which binds to the ligand binding site and inhibits its activity, were prepared as comparative groups. The experimental method was carried out according to the method provided by the analysis kit.

As a result, as shown in FIG. 6, when diramnosylmecin was treated, the activity of PPARγ was not increased, and it was confirmed that diramnosylmecin did not bind to the well-known ligand binding site of PPARγ.

<Experimental Example 7-2> Confirmation of diramnosylmecine binding site in PPARγ protein

Since diramnosylmethacin has been found not to bind to known sites in increasing the activity of PPARy, the present inventors have searched through computer modeling which part of diramnosylmeicin binds to PPARy.

Specifically, experiments were conducted based on the X-ray crystal structure (PDB ID: 4JAZ) of PPARγ reported in the literature for computer modeling. Numerical calculations of diramnosylcysine and PPARγ for computer modeling were performed using the GOLD Suite 5.2.2 (The Cambridge Crystallographic Data Center, UK) program. Using the Clean Protein protocol of the Discovery Studio (DS) 3.5 program, diramnosylmethacin was analyzed for binding sites to PPARγ. All binding sites within 15 Å of the amino acid residues of PPARγ and the diramnosylmecin molecule were first selected, and then the two sites most likely to bind were selected using the Minimization protocol.

As a result, as shown in Fig. 7, another site not yet known was selected as a site where diramnosylmeicin is expected to bind PPARy. The amino acid sequences that are believed to be involved in binding of diramnosylchemicin in unknown new sites are represented by Tyr320, His323, Glu324, Arg443, Gln444, Thr447, Ile472 residues. Among them, Glu324, Arg443 and Thr447, which have strong binding strength, are considered to be important (FIG. 8).

<Experimental Example 7-3> Confirmation of diramnosylmecine binding site in PPARγ protein

Glu324, Arg443, and Thr447 were selected as the amino acids expected to play an important role in binding among the amino acid sequences of Tyr320, His323, Glu324, Arg443, Gln444, Thr 447, and Ile472, which are expected to bind PPARγ with diramnosylcysine (Fig. 8).

The present inventors have confirmed through the luciferase activity that diramnosylmethacin is directly involved in regulating the activity of PPARγ through point mutation of the above amino acid and that this phenomenon will be linked to an unknown site.

Specifically, in order to generate a point mutation of PPARγ, a single base sequence was substituted for the DNA sequence of each site, and QuikChange IIXL Site Directed Mutagenesis Kit (Agilent, USA) was used for this experiment. The method was carried out according to the manual provided by the manufacturer. Each amino acid sequence of PPARγ that caused point mutation was replaced with Ala. The plasmid expressing the point mutated PPARγ was transformed into 293T cells and luciferase assay was carried out. At this time, the experimental procedure was the same as that used in Experimental Example 4, except that the plasmid only overexpressing PPARγ was replaced with a point mutation. The transformed 293T cells were treated with diramnosylchemicin at concentrations of 10 μg / ml and 20 μg / ml for 24 hours, and then cells were disrupted and used for luciferase assay.

As a result, as shown in FIG. 9, it can be confirmed that the activity of PPARγ having a normal amino acid sequence is increased by diramnosylmethacin. It was also confirmed that the activity of PPARγ was further increased when alanine (glutamate), which is the 443rd amino acid, was substituted with alanine.

However, PPARγ in which threonine, which is the 447th amino acid, was replaced with alanine showed no increase in the activity of PPARγ due to diramnosylmecin. This suggests that diramnosylmethacin can not bind to PPARγ and thus does not increase its activity.

Thus, diramnosylchemicin modulates the activity of transcription factors that regulate the expression of insulin signaling factors, thereby regulating abnormal glucose levels by introducing intracellular glucose, which does not bind to already well known regions of the transcription factor, (Threonine), which is the 447th amino acid, plays an important role.

Claims (9)

A pharmaceutical composition for prevention and treatment of diabetes comprising derhamnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

The pharmaceutical composition for preventing and treating diabetes according to claim 1, wherein the composition increases glucose uptake into muscle cells.
The pharmaceutical composition for the prevention and treatment of diabetes according to claim 1, wherein the composition increases PPARy (peroxisome proliferator-activated receptor gamma) activity.
The pharmaceutical composition for the prevention and treatment of diabetes according to claim 1, wherein the composition activates an insulin signaling pathway.
The pharmaceutical composition for the prevention and treatment of diabetes according to claim 1, wherein the diabetes is type 1 diabetes or type 2 diabetes.
A dietary supplement for preventing and improving diabetes comprising deramnosylmasin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

7. The dietary supplement according to claim 6, wherein the diabetes is type 1 diabetes or type 2 diabetes.
A pharmaceutical composition for lowering blood glucose comprising diramnosylmecine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

A health functional food for hypoglycemia comprising diramnosylmecine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

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