KR20220063143A - Composition for preventing, treating or improving of diabetes or diabetic complications comprising poncirin - Google Patents

Abstract

The present invention relates to a composition for the prevention, treatment, or alleviation of diabetes or diabetic complications comprising poncirin as an active ingredient. The composition containing poncirin of the present invention inhibits α-glucosidase, HRAR, and RLAR, inhibits AGE formation, activates the insulin signal pathway, and has an effect of alleviating insulin sensitivity, thereby being useful as the composition for preventing, treating or alleviating diabetes or diabetic complications.

Description

Composition for preventing, treating or improving diabetes or diabetic complications comprising poncirin, comprising poncirin

The present invention relates to a composition for preventing, treating or improving diabetes or diabetic complications comprising ponsirin as an active ingredient.

Diabetes mellitus (DM) is a chronic metabolic disease that seriously threatens human health and quality of life. The number of people with diabetes has increased rapidly worldwide over the past two decades. Most of these cases are related to type 2 diabetes mellitus (T2DM), and lifestyle changes are leading to a younger age of onset. T2DM is characterized by hyperglycemia due to deficiency of insulin secretion, insulin action, or both. Currently, T2DM is a leading health problem due to its global prevalence and high morbidity and mortality associated with chronic diabetic complications. In addition, persistent hyperglycemia contributes to long-term diabetic complications such as neuropathy, cataracts, retinopathy, nephropathy, embryopathies, atherosclerosis and delayed wound healing.

Currently known treatments for diabetes include a) α-glucosidase inhibitors that delay the digestion and absorption of carbohydrates, and b) protein tyrosine phosphatase 1B (PTP1B) that regulates insulin receptor phosphorylation along with insulin therapy. ) inhibitors, and c) regulation of polyol and advanced glycation end-product (AGE) formation pathways to reduce hyperglycemia and maintain normoglycemia have been proposed.

Based on the current understanding of the pathophysiology of diabetes, insulin and its analogs, thiazolidinediones, sulfonylurea derivatives, dipeptidyl peptidase 4, DPP-4), biguanides, glucagon-like peptide 1 (GLP-1) agonists, sodium-glucose cotransporter 2 (SGL-2), PTP1B and α-glucosida enzyme inhibitors have been developed. Despite the identification of these potent synthetic and semisynthetic antidiabetic drugs, because they have not reached clinical trials, these molecules still have poor in vivo efficacy, poor oral bioavailability, poor membrane permeability, poor selectivity, and numerous side effects. . Therefore, there is a need for new inhibitors with better safety and efficacy that can be used to manage these metabolic disorders.

Aldose reductase is the first rate-determining enzyme in the polyol pathway and catalyzes the reduction of glucose to sorbitol followed by the conversion of NADPH to NADP ⁺ . Sorbitol is converted to fructose with reduction of NAD+ by sorbitol dehydrogenase. During hyperglycemia, the polyol metabolic pathway is activated and an increased flux of glucose through the polyol pathway leads to the accumulation of sorbitol, which occurs primarily in tissues exhibiting insulin-independent absorption of glucose, such as the lens, kidney, retina, and peripheral nerves. . Excess sorbitol causes the lenses to absorb water, leading to cataract formation with an osmotic imbalance, increased sodium and decreased potassium levels and decreased glutathione levels. Therefore, inhibition of aldose reductase would constitute an important strategy for controlling secondary complications in DM without risk of hypoglycemia.

Postprandial hyperglycemia plays an important role in the development of DM and its associated complications. The most effective way to prevent hyperglycemia and diabetes is to control the level of glucose in the blood. Sugar in the blood comes from the hydrolysis of carbohydrates and is catalyzed by digestive enzymes such as α-glucosidase. Mammalian α-glucosidase (EC 3.2.1.20) contains a family of hydrolases located on the brush interface membrane of cells of the small intestine. α-glucosidase inhibitors slow the digestive process and absorption of carbohydrates by competitively blocking glucosidase activity. As a result, the peak concentration of postprandial blood sugar decreases and blood sugar levels are controlled. Several α-glucosidase inhibitors obtained from natural sources such as acarbose and voglibose effectively control blood sugar levels after food intake and have been clinically used to treat DM. Although only a few α-glucosidase inhibitors are commercially available, it has been reported that they are clinically associated with serious gastrointestinal side effects. Therefore, there is a need to develop an alternative that exhibits α-glucosidase inhibitory activity without side effects.

PTP1B is a major transmembrane protein tyrosine phosphatase and is an important factor in non-insulin-dependent DM. PTP1B is located on the cytoplasmic side of the endoplasmic reticulum and is ubiquitously expressed, including in classical insulin target tissues such as skeletal muscle, liver, and fat. Evidence from biochemical, genetic and pharmacological studies supports a role for PTP1B as a negative regulator in both insulin and leptin signaling. PTP1B can bind and dephosphorylate activated insulin receptors or insulin receptor (IR) substrates. Mice with the PTP1B gene knockout have improved insulin sensitivity and reduced body weight even when fed a high-fat diet. Therefore, it is not surprising that overproduction of this enzyme is implicated in the pathogenesis of T2DM, and inhibitors of PTP1B may be useful for the prevention and treatment of T2DM, and may be promising insulin-sensitive drug targets.

Insulin resistance, a hallmark of T2DM, is due to an imbalance in autophosphorylation of the insulin receptor (IR) and tyrosine kinase activity. Insulin resistance in skeletal muscle has been shown to play an important role in the pathogenesis of T2DM. In the body, skeletal muscle is the primary target organ for uptake of glucose and completing about 80% of insulin-stimulated postprandial glucose uptake and consumption. As insulin targets, muscle cells are important sites for energy balance, consumption and storage. Therefore, improving insulin resistance in skeletal muscle is an effective strategy for diabetes drug development.

The phosphatidyl inositol-3-kinase/phosphorylated protein kinase B (PI3K/Akt) pathway is one of the most important signaling pathways involved in the metabolic effects of insulin. A signaling cascade is triggered when insulin associates with a membrane receptor on a target cell. Insulin receptor substrate 1 (IRS-1) phosphorylated by the activated insulin receptor induces PI3K and Akt activation. Akt can then phosphorylate GSK3β (glycogen synthasekinase-3β) to promote glucose transport. Furthermore, glucose uptake, a rate-limiting step in glucose metabolism, has been shown to be stimulated in skeletal muscle cells by the insulin signaling pathway. Insulin signaling is activated via the insulin IRS-1/PI3-kinase/Akt pathway and promotes glucose transporter type 4 (GLUT-4) translocation from the intracellular pool to the plasma membrane. GLUT4 is expressed at very high levels in skeletal muscle cells through the highest levels of insulin-stimulated glucose uptake. Thus, downstream of GLUT4 and PI3K are important proteins in regulating glucose uptake and glycogen metabolism. Moreover, the lack of IRS-1 activity leads to decreased PI3K phosphorylation and decreased GLUT4 expression in skeletal muscle cells, resulting in insulin resistance. During T2DM, not only the skeletal muscle's sensitivity to insulin is reduced, but also the expression of GLUT4 is reduced, resulting in reduced glucose uptake and utilization and elevated blood glucose levels.

AGEs are complex groups of molecules formed through non-enzymatic reactions between the reduction of sugar and amine residues on proteins, nucleic acids or lipids. This reaction initiates complex rearrangements, condensation, oxidative transformations and fragmentation, often resulting in incompletely characterized heterogeneous products, collectively referred to as AGEs. Accumulation of AGEs in various types of tissues alters proteins, resulting in changes in their properties, physicochemical and biochemical properties. AGE overproduction due to hyperglycemia plays a major role in the aging process as well as in the pathogenesis of age-related disorders including diabetic complications, Alzheimer's disease, connective tissue disease and end-stage renal disease. In addition, the interaction of AGE with the receptor of AGE (RAGE) causes oxidative stress, leading to vascular inflammation and thrombosis. Another study showed that reactive oxygen species (ROS) formed by AGE induce DNA damage and induction of apoptosis. Therefore, recently, many studies have been made on the use of anti-glycosylation to alleviate diabetic complications. Glucose and fructose, the most common reducing sugars found in the blood circulation, react spontaneously with the amino groups of proteins in AGEs. Glucose plays an important role in AGE formation, but fructose is known to undergo protein glycosylation much faster than glucose.

Nε-(carboxymethyl) lysine ((Nε-carboxymethyl) lysine, CML) is a non-fluorescent AGE that is an indicator of AGE formation resulting from the oxidative degradation of the Amodori product or that glyoxal, methylglyoxal and 3-de It is produced in the polyol pathway mediated by α-oxoaldehyde, such as oxyglucosone. Furthermore, CML is one of the most chemically characterized AGEs in humans, formed by oxidative degradation mediated by the hydroxyl radical of the Amadori product, and during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of proteins. CML was found along with other AGEs in the retinal vessels of type 2 diabetic patients. In addition, high levels of CML have been reported in peripheral nerves in diabetic patients, leading to microangiopathy and neuropathy. Thus, compounds capable of reducing CML production or interfering with damage pathways involving CML may have the potential to reduce the development or progression of both retinopathy and neuropathy.

In the early stage of glycosylation, a nucleophilic addition reaction between the free amino group of a protein and the carbonyl group of a reducing sugar produces a free reversible Schiff base, which is then converted to a more stable ketoamine Amadori product such as fructosamin. Fructosamine is clinically used as an indicator for short-term control of blood sugar in diabetic patients. In addition, fructose formation increases complications of diabetes such as nephropathy, neuropathy and retinopathy. Therefore, reduction of fructose is a therapeutic method to delay vascular complications.

The formation of carbonyl proteins and loss of protein thiols during glycosylation are indicators of protein oxidation. Other reactive carbonyl compounds such as methylglyoxal, glyoxalaldehyde and 3-deoxyglucosone can react with amino groups of intracellular or extracellular proteins to form AGEs . It has been reported that the increase in carbonyl content and loss of free thiol groups by modification of cysteine residues directly reflect protein oxidation of BSA. ROS generated during glycosylation and glycosylation, such as hydrogen peroxide, can oxidize the side chains of amino acid residues in proteins to form carbonyl derivatives and reduce thiol groups, thereby reducing the oxidative defense of proteins, resulting in damage to cellular proteins. Accumulation of reactive carbonyl species causes oxidative stress, apoptosis and vascular damage in DM.

In general, protein glycosylation is an important important factor influencing the conformational changes of polypeptides, especially amyloid cross-β structure. Insoluble aggregates can form amyloid cross-β structures, changing protein structure and stability. Amyloid cross-β is an aggregated structure of proteins that accumulates as fibrils deposited in the brains of patients with glycosylation-related diseases such as Alzheimer's disease. In addition, pancreatic β-amyloid deposits have been shown to be a pathological lesion in pancreatic β-cells in patients with type 2 diabetes, and long-term accumulation of amyloid cross-β structures in tissues can lead to the progression of pancreatic islet amyloidosis and , which directly destroys β-cells and impairs insulin secretion.

On the other hand, poncirin, a 7-O-neohesperidoside of isosakuranetin, is a flavanone glycoside abundantly present in many citrus species. Poncerin was mainly found in high concentrations in edible citrus fruits of Poncirus trifoliata . Potsirin has potential gastric diseases, anti-inflammatory properties, anti-Alzheimer's disease, protective and protective effects against bacterial or viral infection and stress, promotion of osteoblast differentiation in mesenchymal stem cells, anti-cancer effects and anti-H. It has been proven through pharmacological studies and clinical trials that it has various activities such as pylori effect. Orally administered poncirin to mice or rats is metabolized to pnciretin by the intestinal microflora. Although many potential health benefits of ponsirin have been reported, to date there have been no studies on its antidiabetic potential.

Therefore, the present inventors PTP1B, α- glucosidase, rat lens aldose reductase (rat lens aldose reductase, RLAR), human recombinant aldose reductase (human recombinant aldose reductase, HRAR) and ponsirin through AGE inhibition assays was investigated, and the present invention was completed by confirming the glucose uptake stimulating effect of ponsirin and the activation effect of the insulin signaling pathway in insulin-resistant C2C12 cells.

Korean Patent Publication No. 10-2014-0025914

An object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes or diabetic complications comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition and health food composition for preventing or improving diabetes or diabetic complications comprising poncirin, an isomer, derivative, or a food pharmaceutically acceptable salt as an active ingredient. .

Another object of the present invention is to provide an insulin sensitivity enhancer through inhibition of PTP1B enzyme activity, comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating diabetes or diabetic complications comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition and health food composition for preventing or improving diabetes or diabetic complications, comprising poncirin, an isomer, derivative, or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides an insulin sensitivity enhancer through inhibition of PTP1B enzyme activity, comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

The ponsirin of the present invention has the effect of inhibiting α-glucosidase, HRAR and RLAR, inhibiting AGE formation, activating an insulin signaling pathway, and improving insulin sensitivity, thereby preventing, treating or improving diabetes or diabetic complications. It may be useful as a composition for

Specifically, the ponsirin of the present invention can stimulate glucose uptake and decrease PTP1B expression by binding to a specific PTP1B site, increase phosphorylation of IRS-1, PI3K, GSK3β and Akt, and increase the expression of GLUT4 in insulin-resistant C2C12 muscle cells. It has the effect of improving the insulin sensitivity by improving the expression level.

In addition, the ponsirin of the present invention can effectively inhibit the formation of fluorescent AGE and non-fluorescent AGE, for example, CML and fructosamine levels, and reduce the formation of protein carbonyl groups and inhibit the modification of protein thiol groups, thereby reducing the formation of glycosylated proteins. It can inhibit oxidation and inhibit the formation of amyloid cross-β structures in BSA.

Furthermore, ponsirin may be useful for controlling hyperglycemia through inhibition of α-glucosidase, activating the insulin signaling pathway through PTP1B inhibition, and ameliorating diabetes-related complications through inhibition of AR and AGE related pathways. .

1 shows an enzyme kinetic plot for inhibition of PTP1B, α-glucosidase, RLAR and HRAR by ponsirin; (a) Lineweaver-Burk plot for PTP1B inhibition by ponsirin: 0 μM (Δ), 2.0 μM (▼), 10 μM (○) and 50 μM (●)), (b) PTP1B inhibition by ponsirin Dixon plots for: pNPP 2.0 mM (●), 1.0 mM (○) and 0.5 mM (▼), (c) Lineweaver-Burk plots for α-glucosidase inhibition by ponsirin: 0 μM ponsirin (■), Dixon plots for α-glucosidase inhibition by 7.81 μM (Δ), 15.62 μM (▼), 31.25 μM (○) and 62.5 μM (●), (d) ponsirin: pNPG 2.5 mM (●), 1.25 Lineweaver-Burk plots for RLAR inhibition by ponsirin in mM (○) and 0.625 mM (▼), (e): ponsirin 0 μM (Δ), 5.0 μM (▼), 10 μM (○) and 20 μM (●), (f) Dixon plots for RLAR inhibition by ponsirin: DL-glyceraldehyde 25 mM (●), 50 mM (○) and 100 mM (▼), (g) Lineweaver-Burk plots for HRAR inhibition by ponsirin: Dixon plots for HRAR inhibition by ponsirin 0 μM (Δ), 0.2 μM (▼), 1.0 μM (○) and 5.0 μM (●), and (h) ponsirin: DL-glyceraldehyde 0.005 M (●) , 0.01 M (○) and 0.02 M (▼).
Figure 2 shows compound 23 (a) and ponsirin (b) in the active site of protein tyrosine phosphatase 1B (1NNY); and a 3D binding pose of co-crystalline ligands of α-D-glucose (c), acarbose (d) and ponsirin (e) within the active site of α-glucosidase (3A4A); and genarestat (f) and ponsirin (g) within the active site of human aldose reductase (1IEI); and 3D interaction poses of cognate ligands of sulindac (h), quercetin (i) and von sirin (j) in the active site of human aldose reductase (3RX2).
Figure 3 is (a) the chemical structure of ponsirin; (b) results of confirming cell viability through MTT assay to evaluate the effect of ponsirin on C2C12 cell viability; (c) results of measuring insulin-stimulated 2-NBDG uptake to evaluate the effect of ponsirin on glucose uptake in insulin-resistant C2C12 cells; (d) the result of measuring the relative density of PTP1B versus β-actin to evaluate the effect of ponsirin on PTP1B expression in insulin-resistant C2C12 cells; and (e) quantification of the PTP1B protein band intensity with respect to the β-actin level.
Figure 4 is the expression level of each protein was measured by Western blotting to evaluate the effect of ponsirin on the levels of total and phosphorylated IRS-1, Akt, PI3K, GSK-3 and GLUT-4 in insulin-resistant C2C12 cells. the results are shown.
5 is a result confirming the effect of ponsirin and aminoguanidine (AG) on the formation of fluorescent AGE in the BSA-glucose-fructose system.
6 is a view illustrating the effect of ponsirin and aminoguanidine (AG) on fructosamine formation in the BSA-glucose-fructose system.
7 is a result confirming the effect of ponsirin and aminoguanidine (AG) on (Nε-carboxymethyl) lysine (CML) formation in the BSA-glucose-fructose system.
FIG. 8 is a graph illustrating the effect of ponsirin and aminoguanidine (AG) on protein carbonyl content in the BSA-glucose-fructose system.
9 is a result confirming the effect of ponsirin and aminoguanidine (AG) on thiol formation in the BSA-glucose-fructose system.
10 is a result confirming the effect of ponsirin and aminoguanidine (AG) on the formation of amyloid cross-β structure in the BSA-glucose-fructose system.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for preventing or treating diabetes or diabetic complications

The present invention provides a pharmaceutical composition for preventing or treating diabetes or diabetic complications comprising poncirin or a pharmaceutically acceptable salt thereof as an active ingredient.

In the pharmaceutical composition according to the present invention, the diabetes may be insulin resistant diabetes.

In the pharmaceutical composition according to the present invention, the diabetes or diabetes complications are α-glucosidase, protein tyrosine phosphatase (PTP), rat lens aldose reductase (RLAR), and human recombinant aldose. It may be one or more enzyme-related pathway-dependent diabetes or complications of diabetes selected from the group consisting of reductase (HRAR).

The pharmaceutical composition according to the present invention may exhibit an α-glucosidase inhibitory effect.

The pharmaceutical composition according to the present invention may improve insulin resistance by activating the insulin signaling pathway through protein tyrosine phosphatase 1B (PTP1B) inhibitory activity.

The pharmaceutical composition according to the present invention may increase glucose absorption through protein tyrosine phosphatase 1B (PTP1B) inhibitory activity.

The pharmaceutical composition according to the present invention may exhibit an aldose reductase and advanced glycation end-product inhibitory effect.

The pharmaceutical composition according to the present invention may inhibit glycated protein oxidation.

According to one embodiment of the present invention, ponsirin of the present invention inhibits PTP1B, α-glucosidase, RLAR and HRAR, and has the activity of inhibiting AGE formation, and enzymatically, PTP1B and RLAR inhibit mixed type; α-glucosidase and HRAR may exhibit competitive inhibition (see Experimental Examples 1 and 2).

According to one embodiment of the present invention, ponsirin of the present invention is a competitive α-glucosidase inhibitor, and the α-glucosidase-ponsirin inhibitor complex has a negative binding free energy and a stable stereotactic pocket within the catalytic pocket of the enzyme. It can represent the structure (see Experimental Example 3). Through inhibition of α-glucosidase, it may have an effect of controlling hyperglycemia.

According to an embodiment of the present invention, the ponsirin of the present invention can exhibit a number of hydrogen bonds having negative binding free energy within the active pocket of PTP1B, and thus has an excellent effect of stabilizing and inhibiting it within the PTP1B protein. can be represented (see Experimental Example 3).

According to one embodiment of the present invention, ponsirin of the present invention has the effect of remarkably enhancing insulin-stimulated 2-NBDG uptake by insulin-resistant C2C12 cells and reducing the expression of PTP1B, through which glucose uptake in muscle cells It may be involved in a signal transduction pathway (see Experimental Example 4).

According to one embodiment of the present invention, ponsirin of the present invention increases the level of p-IRS-1 expression (Tyr-895) in insulin-resistant C2C12 muscle cells and activates the downstream PI3K/Akt/GSK-3β signaling pathway. Increases phosphorylation of IRS-1, PI3K, GSK3β, and Akt, thereby stimulating glucose uptake and activating GLUT4 through activation of IRS-1/PI3K/Akt and GSK3β signaling pathways, thereby improving insulin sensitivity. (See Experimental Example 5).

According to one embodiment of the present invention, ponsirin of the present invention can inhibit AGE formation in glycated BSA. In addition, the ponsirin of the present invention may exhibit the effect of reducing diabetic vascular complications through fructosamine inhibitory activity in glycated BSA, and may exhibit the effect of inhibiting microangiopathy and neuropathy by inhibiting the formation of CML, By inhibiting protein carbonyl group formation and thiol group loss, it can prevent protein oxidative damage caused by hyperglycemia, inhibit apoptosis and vascular damage, and inhibit the development of debilitating degenerative diseases through inhibition of amyloid cross-β structure formation can be represented (see Experimental Example 6).

In the pharmaceutical composition according to the present invention, the diabetic complications are diabetic neuropathy, diabetic retinopathy, cataract, diabetic nephropathy, renal failure, sexual dysfunction, skin disease, high blood pressure, arteriosclerosis, stroke, cerebral infarction, heart disease, embryopathology , may be at least one selected from the group consisting of gangrene and delayed healing of wounds.

The term “isomer” refers to a compound in which the molecular formula is the same, but the connection method or spatial arrangement of constituent atoms in the bonsai is not the same. Isomers include, for example, structural isomers, and stereoisomers. The stereoisomer may be a diasteromer or an enantiomer. An enantiomer refers to an isomer that does not overlap its mirror image as in the relationship between the left hand and the right hand, and is also called an optical isomer. Enantiomers are divided into R (Rectus: clockwise) and S (sinister: counterclockwise) when 4 or more substituents differ from each other at the chiral central carbon. Diastereomers refer to stereoisomers that are not mirror images, and can be divided into cis-trans isomers due to the spatial arrangement of atoms.

The term "derivative" refers to a compound obtained by substituting a part of the structure of the compound with another atom or group of atoms.

The term "pharmaceutically acceptable salt" refers to any organic or inorganic addition of ponsirin in a concentration having an effective action that is relatively non-toxic and harmless to the patient, and the side effects resulting from the salt do not diminish the beneficial efficacy of ponsirin. means salt. For these salts, inorganic acids and organic acids can be used as free acids, and hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc. can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, and fumarin can be used as organic acids. acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid Phonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid may be used. Further, these salts include alkali metal salts (sodium salt, potassium salt, etc.) and alkaline earth metal salt (calcium salt, magnesium salt, etc.) and the like. For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, hebenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, maleate, malate ate, malonate, mesylate, methylsulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate Late, stearate, succinate, tartrate, tosylate, trifluoroacetate, aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salts and the like may be included.

The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving ponsirin in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and adding an organic or inorganic acid to filter and dry the resulting precipitate. It can be prepared by distilling the solvent and excess acid under reduced pressure and then drying or crystallization in an organic solvent.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg silver nitrate).

Furthermore, the present invention may include all possible solvates, hydrates, isomers, optical isomers, etc. that can be prepared therefrom, as well as the above ponsirin and pharmaceutically acceptable salts thereof.

In one embodiment of the present invention, the ponsirin may be derived from a branch (fruit of Poncirus trifoliata ), but is not limited thereto, and commercially purchased ones may be used. In addition, the ponsirin of the present invention may exhibit a significantly better prevention or treatment effect of diabetes or diabetic complications compared to the extract or fraction extracted from the fruit.

The pharmaceutical composition according to the present invention may be formulated for oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration, or topical administration.

The ponsirin, an isomer, derivative, or pharmaceutically acceptable salt thereof of the present invention may be administered in various oral and parenteral dosage forms during clinical administration, and in the case of formulation, commonly used fillers, extenders, binders, It is prepared using a diluent or excipient such as a wetting agent, a disintegrant, and a surfactant.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and such solid preparations include one or more compounds of the present invention and at least one excipient, for example, starch, calcium carbonate, water It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like can be used.

The pharmaceutical composition may further include a carrier, excipient or diluent. Carriers, excipients, or diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

In the pharmaceutical composition according to the present invention, the ponsirin, isomer, derivative, or pharmaceutically acceptable salt thereof may be included in an amount of 0.001 to 99.9% by weight based on the total weight of the composition.

In addition, the effective dose of the ponsirin, an isomer, derivative, or pharmaceutically acceptable salt thereof of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health status and disease degree, and generally to about 0.001 to 100 mg/kg/day, and preferably 0.01 to 35 mg/kg/day. Based on an adult patient weighing 70 kg, it is generally 0.07 to 7000 mg/day, preferably 0.7 to 2500 mg/day, and once a day at regular time intervals according to the judgment of a doctor or pharmacist It may be administered in several divided doses.

The pharmaceutical composition of the present invention may further include a known active ingredient having preventive or therapeutic activity for diabetes or diabetic complications as an active ingredient, and may be used in combination with other known treatments for the treatment of these diseases.

Health functional food or health food composition for preventing or improving diabetes or diabetic complications

The present invention provides a health functional food composition for preventing or improving diabetes or diabetic complications, comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, pills, liquids, powders and granules, etc. using raw materials or ingredients useful for the human body. Here, 'functionality' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method commonly used in the ordinary technical field, and at the time of the preparation, it can be prepared by adding raw materials and components commonly added in the conventional technical field. In addition, the dosage form of the health functional food may also be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The health functional food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, it uses food-derived ingredients as raw materials and has the advantage of not having side effects that may occur during long-term administration of the drug, and has excellent portability. , The health functional food of the present invention can be ingested as an adjuvant for enhancing the effect of a therapeutic agent for diabetes or diabetes complications.

In addition, the present invention provides a health food composition for preventing or improving diabetes or diabetic complications, comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

In the health functional food composition or health food composition according to the present invention, the diabetes may be insulin resistant diabetes.

In the health functional food composition or health food composition according to the present invention, the diabetes or diabetes complications are α-glucosidase, protein tyrosine phosphatase (PTP), and rat lens aldose reductase (RLAR). ) and one or more enzyme-related pathway-dependent diabetes or complications of diabetes selected from the group consisting of human recombinant aldose reductase (HRAR).

The health functional food composition or health food composition according to the present invention is for the purpose of preventing or improving related diseases through antidiabetic or antidiabetic complication activity. It can be added to health functional food or health food such as food and beverage.

There is no particular limitation on the type of the food. Examples of foods to which ponsirin, isomers, derivatives, or pharmaceutically acceptable salts thereof according to the present invention can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, Ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and the like, include all health food and health functional food in the ordinary sense.

The health food and health functional food composition according to the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the ponsirin, an isomer, a derivative thereof, or a pharmaceutically acceptable salt thereof according to the present invention may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, the amount of ponsirin, an isomer, derivative or a pharmaceutically acceptable salt thereof in health food and health functional food may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake for health maintenance or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health food and health functional food composition of the present invention is not particularly limited in other ingredients except for containing ponsirin according to the present invention, an isomer, a derivative or a food pharmaceutically acceptable salt thereof according to the present invention as an essential ingredient in the indicated ratio. It may contain various flavoring agents or natural carbohydrates as additional ingredients, such as beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the dietary supplement of the present invention.

In addition to the above, the health food and health functional food composition containing ponsirin, isomers, derivatives, or food pharmaceutically acceptable salts thereof according to the present invention are various nutrients, vitamins, minerals (electrolytes), synthetic flavorants and natural flavorants. Used in flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages It may contain a carbonation agent and the like. In addition, the health food and health functional food composition of the present invention may contain natural fruit juice, fruit juice, and fruit for the production of a vegetable drink.

These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the active substance of the present invention.

Insulin sensitivity enhancer through inhibition of PTP1B enzyme activity

The present invention provides an insulin sensitivity enhancer through inhibition of PTP1B enzyme activity, comprising poncirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient.

According to an embodiment of the present invention, since ponsirin of the present invention can function as a PTP1B inhibitor by inactivating the PTP1B enzyme active site, such ponsirin, an isomer, derivative, or pharmaceutically acceptable salt thereof as an active ingredient A composition comprising a can be usefully used as an insulin sensitivity enhancer.

That is, when a composition comprising ponsirin, an isomer, derivative, or a pharmaceutically acceptable salt thereof as an active ingredient inhibits PTP1B activity, the intracellular insulin signal is increased, and thus insulin resistance is improved to enhance insulin sensitivity. can do it Therefore, the composition can be usefully used in the treatment of diseases related to high blood glucose concentration, type 2 diabetes mellitus showing insulin resistance, and diabetes-related diseases.

In particular, when the insulin sensitivity enhancer of the present invention is used in combination with insulin, a higher effect can be expected.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. In addition, various modifications and changes within the scope of the technical spirit of the present invention based on these examples can be easily determined by those skilled in the art.

<Preparation example> Sample and compound preparation and statistical analysis

p-nitrophenyl phosphate (pNPP), p-nitrophenyl α-D-glucopyranoside (p-nitrophenyl α-D-glucopyranoside, pNPG), yeast α-glucosidase (yeast α-glucosidase) ), acarbose, ursolic acid (>90% purity), poncirin (>98% purity), quercetin (>95% purity), rosiglitazone (>98% purity) ), insulin from bovine pancreas, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), 3-( 4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT), β- Nicotinamide adenine dinucleotide phosphate (β-Nicotinamide adenine dinucleotide phosphate, NADPH), guanidine hydrochloride, 2,4-dinitrophenylhydrazine (DNPH), nitroblue tetrazolium , NBT), 5,5'-dithiobis (2-nitrobenzoic acid) (5,5'-dithiobis (2-nitrobenzoic acid), DTNB), thioflavin T (thioflavin T), 1-deoxy-1- Demorpholino-D-fructose (1-deoxy-1-demorpholino-D-fructose, DMF), L-cysteine (L-cysteine), DL-glyceraldehyde dimer (DL-glyceraldehyde dimer) ), D-(-)-fructose (D-(-)-fructose), D-(+)-glucose (D-(+)-glucose) and aminoguanidine hydrochloride from Sigma-Aldrich Co. (St Louis, MO, USA). PTP1B (human recombinant) was purchased from Biomol® International LP (Plymouth Meeting, PA, USA). Dulbecco's modified Eagle medium (DMEM), penicillin-streptomycin, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), fetal bovine serum , FBS), sodium pyruvate and non-essential amino acids were purchased from Gibco-BRL Life Technologies (Grand Island, NY, USA). OxiSelect™ Nε-(carboxymethyl)lysine (CML) ELISA kit was purchased from Cell Biolabs (San Diego, CA, USA). Dithiothreitol (DTT) was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Fluorescent D-glucose analog and glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy -D-glucose (2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose, 2-NBDG) was obtained from Life Technologies (Carlsbad, CA, USA). Phospho-Akt (Phospho-Akt, Ser473), (D9E) rabbit monoclonal antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). PTP1B, IRS, p-IRS-1 (Tyr 895), Akt, PI3-kinase, p-PI3-kinase, anti-GSK-3β, anti-GLUT4 and anti-phospho-ser9-GSK-3β, β-actin and All secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Sodium azide is manufactured by Junsei Chemical Co., Ltd. (Tokyo, Japan). Human recombinant AR (0.4 units) was purchased from Wako Chemicals (Osaka, Japan). Other than that, all other chemicals and solvents used in the present invention were purchased from E. Merck, Fluka or Sigma-Aldrich.

All experiments were expressed as the mean±SEM of three experiments. Statistically significant values were analyzed using analysis of variance (ANOVA) and Duncan's test (Systat Inc., Evanston, IL, USA). A P -value of <0.05 was considered statistically significant.

<Experimental Example 1> PTP1B, α-glucosidase, RLAR, HRAR and ponsirin inhibitory activity against AGE

1-1. Analysis of PTP1B inhibitory activity of ponsirin

P-nitrophenyl phosphate (pNPP) analysis method (Ali, MY, et al., Chem. Biol. Interact . 2019, 305 , 180–194). In each well of a 96-well plate, 40 μL of PTP1B enzyme [0.5 units diluted in PTP1B reaction buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA and 1 mM DTT] and/or 10% sample It was dissolved in DMSO and added. Plates were pre-incubated at 37 °C for 10 min, and then 50 µL of PTP1B reaction buffer containing 2 mM pNPP was added. After incubation at 37° C. for 20 minutes, the reaction was terminated by addition of 10 M NaOH. The amount of p-nitrophenyl produced after enzymatic dephosphorylation of pNPP was analyzed by measuring absorbance at 405 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Ursolic acid was used as a positive control.

1-2. Analysis of α-glucosidase inhibitory activity of ponsirin

The α-glucosidase inhibitory activity of ponsirin was reported using a procedure reported using p-nitrophenyl α-D-glucopyranoside (pNPG) (Ali, MY, et al., Chem. Biol. Interact . 2019, 305 , 180-194). A total of 60 µL of reaction mixture containing 20 µL of 100 µM phosphate buffer (pH 6.8), 20 µL of 2.5 mM pNPG and 20 µL of sample dissolved in 10% DMSO is added to each well followed by 20 µL of α-glucosidase Aze [0.2 U/mL (pH 6.8) in 10 mM phosphate buffer] was added. The plate was incubated at 37° C. for 15 min, then the reaction was stopped by adding 80 μL of 0.2 M sodium carbonate solution. The absorbance was then recorded at 405 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Acarbose was used as a positive control.

1-3. Analysis of RLAR Inhibitory Activity of Ponsirin

To evaluate the rat lens aldose reductase (RLAR) inhibitory activity of ponsirin, rat lens homogenates were subjected to a previously known method with slight modifications (Ali, MY, et al., Chem. Biol. Interact . 2019, 305 , 180-194; Hayman, S. & Kinoshita, JH, J. Biol. Chem. 1965, 240 , 877-882). prepared by adding sodium phosphate dibasic (Na ₂ HPO ₄ .H ₂ O, 0.66 g) and sodium phosphate monobasic (NaH ₂ PO ₄ .H ₂ O, 1.27 g) to 100 mL of double distilled H ₂ O The lenses were homogenized in sodium phosphate buffer (pH 6.2). The homogenate was purified by centrifugation at 10,000 rpm at 4° C. for 20 minutes, and the resulting supernatant was frozen until use. A crude AR homogenate with a specific activity of 6.5 U/mg was used for all enzyme inhibition evaluations. The reaction solution consisted of 620 μL of 100 mM sodium phosphate buffer (pH 6.2), 90 μL of AR homogenate, 90 μL of 1.6 mM NADPH and 9 μL of sample. The substrate contained 90 μL of 50 mM DL-glyceraldehyde. AR activity was determined by measuring the decrease in NADPH absorption for 4 min at 340 nm using an Ultrospec®2100pro UV/Visible spectrophotometer. SWIFT II Applications software (Amersham Biosciences) was used for all data analysis. A well-known AR inhibitor (ARI), quercetin, was used as a control.

1-4. HRAR inhibitory activity assay of ponsirin

The human recombinant aldose reductase (HRAR) inhibitory activity of ponsirin was investigated by the same method as the RLAR (Ali, MY, et al., Chem. Biol. Interact . 2019, 305 , 180-194). became AR activity was measured by measuring the decrease in NADPH absorption at 340 nm for 1 min using an Ultrospec®2100pro UV/Visible spectrophotometer. SWIFT II Applications software (Amersham Biosciences, NJ, USA) was used for all data analysis. Quercetin, a well-known ARI, was used as a positive control.

1-5. Analysis of AGE inhibitory activity of ponsirin

The inhibitory activity of advanced glycation end-product (AGE) production was carried out by slightly modifying the known method. 10 mg/ml bovine serum albumin was dissolved in 0.2 M phosphate buffer (pH 7.4), and sodium azide 0.02% and 0.2 M fructose and 0.2 M bacterium inhibitor were used. M glucose was added and used. The sample was dissolved in 10% DMSO and mixed with the reaction mixture (950 μL) to obtain various concentrations between 0.5 and 500 μM. Incubated at 37° C. and fluorescence intensity (Ex: 350 nm, Em: 450 nm) was measured using a spectrofluorometric detector (FLx800 microplate fluorescence reader, Bio-Tek Instrument, Inc., Winooski, USA).

1-6. Analysis of PTP1B, α-glucosidase, RLAR, HRAR and AGE inhibitory activity of ponsirin

Table 1 shows the results of measuring the inhibitory activity of ponsirin against PTP1B, α-glucosidase, RLAR, HRAR and AGE analyzed by the methods of Experimental Examples 1-1 to 1-5 as IC ₅₀ values.

test sample IC ₅₀ (μM) ^a α-Glucosidase PTP1B RLAR HRAR AGE Poncirin 21.31±1.26 7.76±0.21 11.91±0.21 3.56±0.33 3.23±0.09 Acarbose ^b 122.34±1.56 Ursolic acid ^c 6.69±0.62 Quercetin ^d 5.47±0.63 4.93±0.23 Zenarestat ^e 0.79±0.14 Aminoguanidine ^f 471.32±2.58 ^a The 50% inhibitory concentration (μM) was calculated as a log-dose inhibition curve and expressed as the mean±SEM over three experiments. ^bf positive control

To evaluate the antidiabetic activity of ponsirin, the inhibition potential of PTP1B and α-glucosidase was evaluated using pNPP and pNPG as substrates. As shown in Table 1, inhibition of PTP1B and α-glucosidase Compared with ursolic acid (IC ₅₀ value 6.69 ± 0.62 μM) and acarbose (IC ₅₀ value 122.34 ± 1.56 μM) used as positive controls in each assay for activity, ponsirin was 7.76 ± 0.21 and 21.31 ± respectively. It showed strong inhibitory activity against PTP1B and α-glucosidase with an IC ₅₀ value of 1.26 μM.

In addition, ponsirin was a potent RLAR with IC ₅₀ values of 11.91±0.21 and 3.56±0.33 μM, respectively, compared to the positive control quercetin, which had an IC ₅₀ value of 4.91±0.23 μM for HRAR and 5.47±0.63 μM for RLAR. and HRAR inhibitory activity (Table 1). Ponsirin showed a stronger inhibitory activity on HRAR than RLAR, and from these results, it was confirmed that it can be used as an effective therapeutic agent for treating diabetic complications in humans.

As shown in Table 1, ponsirin exhibited strong AGE inhibitory activity with an IC ₅₀ value of 3.23±0.09 μM, and the positive control (aminoguanidine, aminoguanidine) had an IC ₅₀ value of 471.32±2.58 μM. These results show that ponsirin has potent AGE formation inhibitory activity. In particular, ponsirin showed 145-fold stronger inhibition of AGE formation than aminoguanidine, a well-known AGE formation inhibitor.

<Experimental Example 2> Enzyme kinetics of ponsirin on inhibition of PTP1B, α-glucosidase, RLAR and HRAR

2-1. Enzyme kinetic analysis of ponsirin in inhibition of PTP1B, α-glucosidase, HRAR and RLAR

Lineweaver-Burk and Dixon plots were generated for enzyme kinetic analysis of PTP1B, α-glucosidase, RLAR and HRAR inhibition by ponsirin. Substrates and concentrations in each assay are as follows.

(A) Lineweaver-Burk plots for PTP1B inhibition by ponsirin: ponsirin 0 μM (Δ), 2.0 μM (▼), 10 μM (○) and 50 μM (●));

(b) Dixon plots for PTP1B inhibition by ponsyrin: pNPP 2.0 mM (-), 1.0 mM (○) and 0.5 mM (▼);

(c) Lineweaver-Burk plot for α-glucosidase inhibition by ponsirin: ponsirin 0 μM (■), 7.81 μM (Δ), 15.62 μM (▼), 31.25 μM (○) and 62.5 μM (●) ;

(d) Dixon plots for α-glucosidase inhibition by ponsirin: pNPG 2.5 mM (●), 1.25 mM (○) and 0.625 mM (▼);

(e) Lineweaver-Burk plots for RLAR inhibition by ponsirin: ponsirin 0 μM (Δ), 5.0 μM (▼), 10 μM (○) and 20 μM (●);

(f) Dixon plots for RLAR inhibition by ponsyrin: DL-glyceraldehyde 25 mM (-), 50 mM (○) and 100 mM (▼);

(g) Lineweaver-Burk plots for HRAR inhibition by ponsirin: ponsirin 0 μM (Δ), 0.2 μM (▼), 1.0 μM (○) and 5.0 μM (●); and

(h) Dixon plots for HRAR inhibition by ponsirin: DL-glyceraldehyde 0.005 M (●), 0.01 M (○) and 0.02 M (▼).

Types of enzyme inhibition were plotted on a Lineweaver-Burk double reciprocating plot [1/enzyme rate (1/V) vs. 1/substrate concentration(1/[S])] was determined by analysis, and the inhibition constant (K _i ) was determined by interpretation of Dixon plots, where the values on the x-axis represent K _i .

2-2. Results of enzyme kinetic analysis of ponsyrin for inhibition of PTP1B, α-glucosidase, RLAR and HRAR

To elucidate the enzymatic inhibition mode of ponsirin, the results of kinetic analysis at different concentrations of the corresponding substrates (pNPP for PTP1B and pNPG for α-glucosidase) and various inhibitor concentrations are presented in Table 2 and below. 1 is shown.

test sample kinetic parameters α-Glucosidase PTP1B RLAR HRAR AGE K _i ^b 23.26 7.35 10.47 3.03 - Inhibition type ^c Competitive Mixed Mixed Competitive - ^a The inhibition constant (K _i ) was determined using the Dixon plot. ^b Dixon plots and Lineweaver-Burk plots were interpreted to determine the type of inhibition.

As shown in Fig. 1a-b, as a result of Lineweaver-Burk and Dixon plot analysis, ponsirin inhibits mixed PTP1B with a K _i value of 7.35 μM that can bind to the allosteric site of the free enzyme or the enzyme-substrate complex. was shown (Table 2). On the other hand, ponsirin showed a different mode of inhibition for α-glucosidase, ie, competitive inhibition with a K _i value of 52.33 μM ( FIGS. 1c-d and Table 2).

In addition, to determine how ponsyrin inhibits RLAR and HRAR, different concentrations of substrate (25, 50 and 100 mM for RLAR and 0.005, 0.01 and 0.02 M for HRAR) and inhibitor were used to determine how ponsirin inhibited RLAR and HRAR, Figure 1e- As shown in f, ponsirin exhibited mixed RLAR inhibition with a K _i value of 10.47 μM (Table 2) and competitive HRAR inhibition with a K _i value of 3.03 μM (Fig. 1g–h and Table 2). ).

<Experimental Example 3> Molecular docking analysis of ponsirin and target protein

3-1. Selection of protein structures and preparation of ligands

For docking studies of ponsirin with target proteins, target proteins PTP1B (PDB ID: 1NNY), α-glucosidase (PDB ID: 3A4A), HRAR (PDB ID: 1IEI) and The X-ray structure of RLAR (PDB ID: 3RX2) was extracted. The interaction of ponsirin with the protein's cognate ligand was investigated within the active site and the results presented in the form of 3D poses. The active pocket of each receptor was found using the site finder program within the Molecular Operating Environment (MOE) (http://www.chemcomp.com/MOE Molecular_Operating_Environment.htm). After positing the structure of the target protein using the AMBER99 force field, the energy was minimized in the RMSD gradient of 0.05 kcal/mol. MOE builder default parameters were used for structural preparation of ponsirin, acarbose and quercetin. Energy minimization for these structures was performed by applying an mMFF94x force field at the RMSD of 0.01 kcal/mol Å in the MOE. On the other hand, the cognate ligand was downloaded from the protein data bank.

3-2. Docking analysis method

Molecular modeling was performed with the help of LeadIT software (BioSolveIT GmbH, Germany) and default settings were maintained during the docking study. Prior to the docking assay, a confirmation step was performed by docking the cognate ligand within each protein, after which ponsirin was docked within the active site of the target protein. The top 50 binding poses were analyzed for HYDE visual affinity. The HYDE evaluation was used to select the final pose with favorable and unfavorable interactions in mind for all ligands. The postures with the lowest combined score and the highest preference were selected and visualized with the Discovery Studio Visualizer. The results are shown in Table 3 below.

Target Enzymes PDB ID Ligands H-bonds interacting residues π-π interacting residues Other interacting residues PTP1B 1NNY Poncirin Gly183, Arg221, Gly220, Gln266, Cys215, Asp48, Gln262 - π-alkyl linkage with Ala217, alkyl interactions with Val49 and Ile219 Compound 23 Gly259, Asp48, Gln266, Gln262, Gly220, Gly218, Ala217, Tyr20, Arg221 Tyr46 Salt bridge interaction with Arg254, charge to charge and pi-cation interactions with Arg24 and charge to charge interaction with Arg221, carbon hydrogen bond with Gln266, π-sigma and π-alkyl with Ala217 and pi-alkyl interactions with Arg24 α-Glucosidase) 3A4A Poncirin Tyr158, Pro312, Glu277, Ser157, Asp69, Asp215 Tyr72, Phe303 π-alkyl interactions with Val216, alkyl linkage with Lys156, pi-cation with Arg442 and pi-anion with Glu277 Acarbose Tyr158, Ser240, Glu277, Arg213, Asp352, Asp242 - Charge to charge salt bridge with Glu277 and charge-charge with Asp352 and pi-sigma with Phe303 Alpha-D-Glucose Arg213, Arg442, Asp352, Glu277, Asp69, Asp215 - pi-donar bond with Tyr72 and pi-sigma with Phe178 HRAR 1IEI Poncirin Gln49, Val47, Tyr48, Gln183, His110 Trp20 Pi-alkyl with Cys298 and pi-donar with Tyr48 Zenarestat Typ111, Tyr48, Cys298 and Tyr309 Trp111, Trp20 Charge to charge interactions with Lys77, pi-alkyl with Tyr48, Trp111, Val47, Trp20 and Tyr309, alkyl linkage with Val47 and Cys303, pi-sigma with Leu300 RLAR 3RX2 Poncirin Trp111, Arg296, His110, Val297, Val47 Trp20 Pi-sigma with Val47, pi-lone pair with Trp20 and pi-alkyl linkage with Trp219 and Cys298 Quercetin Thr19, Tyr48, Trp111, Ser210, Ile260, Asp43, Gly18 His110 and Tyr209 Pi-sigma bond with Ile260, pi-sulfur with Cys298 Sulandic Ser302, Trp111 and Tyr48 Trp20 and Phe122 Pi-alkyl linkage with Trp219, Trp111 and Val47, alkyl with Cys298 and Val47, charge to charge interactions with Lys77

3-3. Analysis of docking interactions of ponsirin and PTP1B cognate ligands

cognate ligand of protein tyrosine phosphatase 1B (PTP1B), 3-({5-[(N-acetyl-3-{4-[(carboxycarbonyl)(2-carboxyphenyl) )amino]-1-naphthyl}-L-alanyl)amino]pentyl}oxy)-2-naphthoic acid (3-({5-[(N-acetyl-3-{4-[(carboxycarbonyl)( Molecular modeling of 2-carboxyphenyl)amino]-1-naphthyl}-L-alanyl)amino]pentyl}oxy)-2-naphthoic acid, compound 23) revealed an interaction network including hydrogen bonds and pi-pi interactions. showed

As shown in Figure 2a, amino acid residues found to be involved in hydrogen bonding are Tyr20 (2.14 Å), Ser216 (2.98 Å), Ala217 (2.90 Å), Gly218 (3.25 Å), Ile219 (2.83 Å), Gly220 (2.62 Å). Å), Gln262 (3.19 Å), Gln266 (3.18 Å), Asp48 (1.89 Å and 1.73 Å), Arg221 (2.76 Å) and (2.53 Å), the carbon H bonds are Gln266 (3.76 Å), Gly259 (3.46 Å) ) and Asp48 (2.77 Å). In addition, pi-pi stacking interactions were observed between compound 23 and Tyr46 (5.47 Å) and (4.87 Å). Some amino acids showed pi-cation interactions, such as Arg254 (4.06 Å and 4.63 Å) and Arg24 (4.49 Å), while others were pi-alkyl such as Ala217 (4.62 Å) and Arg24 (4.68 Å and 4.94 Å) showed interaction. A pi-sigma interaction was observed by Arg254 (3.07 Å) between the salt bridges with Ala217 (3.60 Å) and Tyr46 (2.63 Å).

The compound ponsirin of the present invention exhibited a number of hydrogen bonds within the PTP1B activity pocket, and for this reason, it seems that stabilization within the protein and an excellent inhibitor role of the protein are possible. Figure 2b shows that ponsirin was well accommodated inside the PTP1B activity pocket, and the presence of ponsirin in the catalytic cavity allowed a conformational change of the protein. Important interactions include hydrogen with Gly183 (2.53 Å), Gly220 (2.79 Å), Arg221 (2.78 Å), Gln266 (3.10 Å), Cys215 (2.94 Å), Asp48 (2.12 Å and 1.65 Å), Gln262 (2.50 Å) bonds were included, and in addition to pi-alkyl bonds with Ala217 (4.57 Å), alkyl bonds with Val49 (3.92 Å) and Ile219 (4.67 Å) were identified.

3-4. Docking interactions of ponsirin, acarbose and α-glucosidase cognate ligands

As shown in Figure 2c and Table 3, alpha-D-glucose, which is the cognate ligand of the α-glucosidase enzyme, is bound to conventional hydrogen bonds (Arg213 (2.98 Å), Arg442 (2.90 Å), Asp352 (1.87 Å and 2.59 Å) ), Glu277 (2.30 Å) and Asp69 (1.54 Å)) and carbon hydrogen bonds (Asp352 (2.78 Å) Glu277 (2.58 Å), Asp69 (2.80 Å and 2.35 Å) and Asp215 (2.27 Å and 1.97 Å) form) In addition, pi-donor interaction with Tyr72 (2.17 Å) and pi-sigma linkage with Phe178 (2.55 Å) played an important role in the α-glucosidase inhibitory activity of ponsirin. The binding pose of alpha-D-glucose was located deep inside the catalytic cavity of glucosidase.

The reasonable binding poses of acarbose suggested that the amino acids involved in hydrogen bonding were Arg213 (2.98 Å), Tyr158 (1.94 Å and 1.78 Å), Ser240 (2.96 Å), Asp352 (1.51 Å) and Glu277 (1.55 Å). , Asp242 (2.20 Å and 2.05 Å) and Glu277 (3.02 Å) were found to exhibit carbon hydrogen bonding (Fig. 2d).

In addition, when acarbose was docked in α-glucosidase, a charge charging pi-sigma interaction by Phe303 (2.31 Å) and interactions with Glu277 (2.04 Å salt bridge) and Asp352 (3.29 Å) was observed. became

As shown in FIG. 2E and Table 3, ponsirin exhibits conventional hydrogen bonding with Tyr158 (2.05 Å) and Pro312 (3.00 Å) of α-glucosidase and Glu277 (2.60 Å), Ser157 (2.46 Å), Asp69 ( 2.49 Å and 3.00 Å) and carbon hydrogen bonds with Asp215 (3.06 Å). Other interactions were also observed, such as a pi-cation with Arg442 (4.31 Å) and a pi-anion with Glu277 (3.50 Å). Ponsirin made pi-pi T-shaped interactions with Tyr72 (5.86 Å) and Phe303 (4.79 Å) and pi-alkyl bonds with Val216 (5.10 Å).

3-5. Docking interaction of ponsirin and human aldose reductase cognate ligand

A plausible mode of zenarestat, its cognate ligand, has been shown to exhibit important and key interactions within the active site of human aldose reductase, which interactions include hydrogen bonding Tyr48 (2.67 Å), Trp111 (2.95 Å). ), Tyr309 (3.09 Å) and Cys298 (2.84 Å) carbon-hydrogen bonds.

As shown in Figure 2f and Table 3, the amino acids exhibiting pi-pi stacking interactions are Trp20 (4.16 Å, 4.82 Å, 4.59 Å, and 4.24 Å) and Trp111 (4.27 Å and 3.70 Å), and the pi-alkyl bond is amino acids Tyr48 (4.52 Å and 4.52 Å), Trp111 (4.14 Å and 4.00 Å), Trp20 (4.25 Å), Tyr309 (5.07 Å) and Val47 (4.79 Å). Alkyl bonds were confirmed by Val47 (2.99 Å and 3.82 Å) and Cys303 (4.36 Å). In addition, genarestat showed a pi-sigma interaction with Leu300 (3.70 Å) and a charge-charge interaction with Lys77 (4.46A). The 7-chloro-2,4-dioxoquinazolin-1-yl moiety of genarestat was further positioned towards the cofactor, NAP350. On the other hand, the 4-bromo-2-fluorophenyl moiety of genarestat was shown to align near distinct sites of the hydrophilic pocket.

As shown in Figure 2g and Table 3, the main interaction of ponsirin within the binding site of human aldose reductase was the pi-pi stacking interaction with Trp20 (5.35 Å) near NAP350, Tyr48 (3.89A). pi-donor interaction with and pi-alkyl interaction with Cys298 (5.02 A). In addition, hydrogen bonding was seen in Gln49 (1.99 Å, 2.06 Å and 1.54 Å) and Val47 (2.10 Å) located near the hydrophobic pocket of the enzyme, Tyr48 (2.15 Å), Gln183 (1.73 Å) and His110 (3.08 Å) Carbon hydrogen bonding was observed within the hydrophilic cavity of the

3-6. Docking interactions of ponsirin and RLAR cognate ligands

The docking result of sulindac in human aldose reductase (Fig. 2h) shows that the amino acids involved in the pi-pi stacking interaction are nicotinamide adenine dinucleotide phosphate (NAP) in addition to Trp20 (4.95Å, 4.78 A, 5.36 A and 4.66 A) and Phe122 (5.98 A). Hydrogen bonds of Tyr48 (3.24 Å), Ser302 (3.01 Å) and Trp111 (2.91 Å) were found between sulindac and amino acids forming a substrate binding cavity, and Lys77 (5.24 Å) was in charge-charge interaction ( Table 3). Residues Trp111 (5.23 Å), Val47 (4.87 Å) and Trp219 (5.32 Å) were in pi-alkyl bonds with sulindac, with alkyl bonds represented by Cys298 (3.21 Å) and Val47 (2.81 Å).

A reasonable mode of binding for quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) within the activity pocket of the enzyme is that multiple hydrogen bonds are The hydrophilic head and the inlet cavity of the hydrophobic cavity were found to be responsible for the stabilization of quercetin (Fig. 2i). As shown in Table 3, the amino acids that form hydrogen bonds are Thr19 (2.81A), Tyr48 (2.15 Å), Trp111 (2.65 Å), Ser210 (2.74 Å, 2.62 Å and 2.27 Å), Asp43 (2.32 Å) and Ile260 (2.09 Å) and formed a carbon H bond with Gly18 (2.94 Å). Likewise, the amino acids responsible for the pi-pi T-form interaction were His110 (4.87 Å) and Tyr209 (5.38 Å). In addition, quercetin exhibited a pi-sigma interaction with Ile260 (3.98 Å) and a pi-sulfur interaction with Cys298 (5.78 Å and 4.36 Å).

Examination of the docking pose of ponsyrin revealed that the major amino acid residues Trp111 (2.91 Å), His110 (2.31 Å), Val297 (2.71 Å), Val47 (2.97 Å) and Arg296 (2.29 Å) are hydrogen within the active pocket of the enzyme. was shown to form a binding network (FIG. 2J and Table 3). Ponsirin exhibited pi-sigma binding with Val47 (3.44 Å and 3.68 Å) and pi-lone pair interaction with Trp20 (2.98 Å and 2.54 Å). In addition, pi-pi T-type interactions with Trp20 (5.14 Å) and pi-alkyl bonds with Trp219 (5.30 Å) and Cys298 (3.61 Å) are factors contributing to the binding of ponsirin towards the inlet cavity of the enzyme. confirmed to be

3-7. HYDE assessment of compounds for all targets

HYDE visual affinity for binding of selective poses was analyzed to investigate the efficiency of ligands within the active pockets of α-glucosidase (3A4A), RLAR (3RX2), PTP1B (1NNY) and HRAR (1IEI). HYDE is derived from the Gibbs-Helmholtz equation and is directly related to binding affinity, giving the results of visual inspection of poses and the highest pose with the highest affinity for the protein.

Table 4 shows the binding free energies and FlexX docking scores for ponsirin, cognate ligands and positive controls in all target proteins.

Ligands FlexX score of the top-ranking pose Binding free energy
ΔG (kJ mol ^-1 ) α-glucosidase (3A4A) Porcirin -26.57 -14 Acarbose -17.28 -18 Alpha-D-Glucose -31.43 -31 RLAR (3RX2) Porcirin -25.74 -29 Quercetin -31.05 -22 Sulandic -28.92 -24 PTP1B (1NNY) Porcirin -19.21 -14 Compound 23 -67.05 -20 HRAR (1IEI) Porcirin -24.87 -28 Zenarestat -27.52 -41

As shown in Table 4, as a result of confirming the inhibitory ability of ponsyrin on α-glucosidase, it was found that the inhibitory activity was 5.7 times stronger than that of acarbose, which is an α-glucosidase inhibitor currently used. In addition, as a competitive α-glucosidase inhibitor, ponsirin binds to the active site of the α-glucosidase enzyme and prevents the formation of the enzyme-substrate complex, and molecular docking assays show that the α-glucosidase-ponsirin inhibitor complex showed a stable conformation within the catalytic pocket of the enzyme with a binding free energy of -14.00 kJ mol ^-1 .

Molecular docking of ponsirin revealed a number of hydrogen bonds with negative binding free energy (-17.98 kJ mol ⁻¹ ) within the active pocket of PTP1B, which stabilized it inside the PTP1B protein and could show an excellent protein inhibitory effect. means there is

<Experimental Example 4> Effects of ponsirin on glucose uptake and PTP1B expression levels in insulin-resistant C2C12 skeletal muscle cells

The chemical structure of ponsirin is shown in FIG. 3A.

4-1. Cell culture and cell viability assays

Prior to examining the ability of ponsirin to increase glucose uptake, the cytotoxicity of ponsirin in C2C12 cells was measured by MTT assay.

The C2C12 cell (skeletal muscle cell) line was purchased from the American Type Culture Collection (Manassas, VA, USA). C2C12 cells were cultured at 37° C. in DMEM supplemented with 10% FBS and streptomycin-penicillin (Hyclone, Mordialloc, VIC, Australia) in a humidified atmosphere of 5% CO ₂ . The viability of C2C12 cells was determined using the tetrazolium dye colorimetric test (MTT). Initially, C2C12 cells were cultured in 96-well plates (2×10 ⁵ cells/well) for 24 hours. After the cell density reached 90%, it was treated with various concentrations of ponsirin. After incubation for 24 hours, MTT reagent was added to each well, and the plate was incubated at 37° C. for 2 hours. The medium was removed and the wells washed twice with PBS (pH 7.4). To determine the percentage of viable cells, the medium was replaced with 100 μL DMSO (100%). The resulting absorbance values were measured at 570 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

C2C12 cells were incubated for 24 hours and then pre-treated with ponsirin at a concentration of 20 μM or less, as shown in FIG. 3b , as ponsirin did not show cytotoxicity at 15 μM or less, so subsequent glucose uptake at a concentration of 15 μM or less Analysis was performed.

4-2. Analysis of muscle cell differentiation and glucose uptake

To investigate the ability of ponsirin to increase glucose uptake, an insulin-stimulated 2-NBDG uptake assay was performed with insulin-resistant C2C12 cells.

C2C12 cells were cultured in 96-well plates (2×10 ⁵ cells/well) with DMEM containing 10% FBS and 1% P/S at 37° C. and exposed to a 5% CO ₂ atmosphere. When the cells reached the desired confluency, they were differentiated for 5 days in DMEM supplemented with 2% horse serum. Cells were then starved for 24 h in hypoglycemic serum-free DMEM, followed by a 2-NBDG assay to assess glucose uptake. Briefly, insulin resistance was induced by treatment with insulin (100 nM) for 12 h, then cells were treated with various concentrations of ponsirin or 5.0 µM rosiglitazone for 24 h, followed by 20 µM 2-NBDG for 24 h. treated for hours. To stop the reaction, cells were washed three times with ice-cold PBS and in a microplate reader (Bio-Tek Instruments Inc., FL×800, Winooski, USA) at 490 nm excitation and 535 nm emission wavelengths. 2-NBDG fluorescence intensity was measured and quantified. Five replicate wells were established, and each experiment was repeated three times.

As a result, the positive control rosiglitazone significantly increased insulin-stimulated glucose uptake in insulin-resistant C2C12 cells. Ponsirin significantly enhanced insulin-stimulated 2-NBDG uptake by insulin-resistant C2C12 cells at concentrations of 2.5, 10 and 15 μM compared to the control group (Fig. 3c). These results suggest that ponsirin is involved in the glucose uptake signaling pathway in muscle cells.

4-3. PTP1B expression analysis

To determine the effect of ponsirin on the expression level of protein tyrosine phosphatase 1B (PTP1B) in insulin-resistant C2C12 cells, insulin-resistant C2C12 cells were incubated with selected concentrations of ponsirin for 24 hours. The relative densities of PTP1B versus β-actin were measured and shown in FIG. 3D, and quantitative values obtained by normalizing the PTP1B protein band intensity to β-actin levels through density measurement were shown in FIG. 3E.

As a result, as shown in FIGS. 3D and 3E , treatment of insulin resistant C2C12 cells with 10 and 15 μM ponsirin reduced the expression level of PTP1B.

<Experimental Example 5> Effects of ponsirin on GLUT4 activation through IRS-1/PI3K/Akt and GSK3β signaling pathways

To determine the molecular mechanism affecting insulin signaling of ponsirin, we investigated the expression levels of various proteins involved in the insulin-signaling pathway by Western blotting. Rosiglitazone was used as a positive control.

5-1. Preparation of Protein Lysates and Western Blot Analysis

Specifically, insulin-resistant C2C12 cells (2×10 ⁵ cells/well) in 6-well plates were treated with different concentrations of ponsyrin for 24 hours. After stimulation with 100 nM insulin for 30 min at 37 °C, cells were washed three times with ice-cold PBS and collected, followed by sample buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 0.5% NP-40, 1 mM PMSF; 1 mM DTT, 0.2% aprotinin, 0.5% leupeptin, 20 mM NaF and 1 mM Na ₃ VO ₄ ) on ice. After incubation for 30 minutes, insoluble materials were removed by centrifugation at 25,000×g for 20 minutes.

Insulin-resistant C2C12 cells total and phosphorylated insulin receptor substrate 1 (IRS-1), phosphorylated protein kinase (Akt), phosphatidyl inositol-3-kinase (Phosphatidyl inositol-3- Western blotting to evaluate the effect on the level of kinase, PI3K), glycogen synthasekinase-3 (GSK-3), and glucose transporter type 4 (GLUT-4) The expression level was measured by Each protein concentration was determined by a modified Bradford protein assay kit. Total protein (50 μg) was electrophoresed on dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in blocking buffer (5% skim milk in Tris buffered saline (TBST) containing 0.1% Tween 20) and incubated overnight with primary antibody at 4°C followed by incubation with appropriate secondary antibody for 4 h at room temperature. did. The membrane was washed 3 times with TBST for 40 min. Bands were detected using Super signal West Pico chemiluminescence substrate (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Immuno-reactive protein bands were detected using an Odyssey scanning system (LI-COR, Lincoln, NE, USA). Density measurement analysis of the data was performed using a cooled CCD camera system EZ-Capture II and CS analyzer version 3.00 software (ATTO Co., Tokyo, Japan).

5-2. Analysis of the effect of ponsirin on GLUT4 activation through IRS-1/PI3K/Akt and GSK3β signaling pathways

As shown in FIG. 4 , it was confirmed that ponsirin activated IRS-1 by enhancing phosphorylation in tyrosine 895 (Tyr 895) to the level observed in control insulin-sensitive cells. Ponsirin also significantly increased the relative expression levels of p-Akt, p-PI3K and p-GSK3β without changing the expression levels of total Akt, PI3K or GSK-3β ( FIG. 4 ).

These results suggest that ponsirin increases p-IRS-1 expression (Tyr-895) levels and activates the downstream PI3K/Akt/GSK-3β signaling pathway, thus stimulating glucose uptake in insulin-resistant C2C12 muscle cells. It shows that it can improve insulin sensitivity.

In skeletal muscle cells, GLUT4 plays a pivotal role in insulin-dependent and/or uptake of glucose in the basal state. Cellular expression of GLUT4 was significantly enhanced when ponsirin was treated at concentrations of 10 and 15 μM ( FIG. 4 ).

Several previous studies have reported that PTP1B inhibitors increase tyrosine phosphorylation of the insulin receptor (IR) and activate molecules downstream of insulin signaling such as IRS-1, Akt, PI3K and GSK-3β. Therefore, inhibitors of PTP1B are potential antidiabetic agents because they enhance insulin sensitivity in T2DM. From the above results, it was found that ponsirin decreased the expression of PTP1B, which increased phosphorylation of IRS-1, PI3K, GSK3β, Akt and GLUT4, in insulin-resistant C2C12 skeletal muscle cells. As a potential modulator of PTP1B, it was confirmed that it could exert an antidiabetic effect by improving insulin resistance in skeletal muscle by regulating the PTP1B-IRS1-Akt-GLUT4 signaling pathway.

<Experimental Example 6> Effect of ponsirin on glycation process

6-1. In vitro glycosylation of BSA

The formation of glycosylated bovine serum albumin (BSA) was investigated according to a modified method (Islam, MN, et al., Food Chem. Toxicol . 2014, 69 , 55-62). To prepare an advanced glycation end-product (AGE) reaction solution, 10 mg/mL bovine serum albumin (BSA) in 50 mM sodium phosphate buffer (pH 7.4) was added to 0.2 M fructose and 0.2 M glucose, and bacteria 0.02% sodium azide was added to prevent growth. The reaction mixture (3.8 mL) was then mixed with 200 μL of 10% DMSO in which various concentrations of ponsyrin and aminoguanidine hydrochloride were dissolved. Controls were prepared using BSA and sugar, and blanks were prepared using only BSA in the same buffer. The reaction mixture was incubated at 37° C. for 4 weeks. An aliquot of the reaction mixture was analyzed for determination of AGE formation inhibitory activity.

6-2. Inhibition assay of AGE formation

Inhibitory activity on the formation of final glycation products was evaluated according to a previously reported method (Islam, M.N et al., Food Chem. Toxicol. 2014, 69, 55-62) with slight modifications. To prepare the AGE reaction solution, add 10 mg/mL bovine serum albumin (BSA) in 50 mM sodium phosphate buffer (pH 7.4) with 0.02% sodium azide to 0.2 M fructose and 0.2 M glucose to prevent bacterial growth. did The reaction mixture (950 μl) was then combined with samples of various concentrations (50 μl, test concentration range 0.5-500 μM) dissolved in 10% DMSO. After incubation at 37°C, formation of AGEs in aliquots using a spectroscopic fluorescence detector (FLx800 microplate fluorescence reader, Bio-Tek Instrument, Inc., Winooski, USA) with excitation and emission wavelengths at 350 nm and 450 nm, respectively. was measured at weekly intervals for 4 weeks. Aminoguanidine, a nucleophilic hydrazine compound, was used as a control in the AGE analysis.

6-3. Effect of ponsirin on the formation of fluorescent AGEs

As a result of monitoring the formation of AGEs weekly by measuring the fluorescence intensity of the BSA-glucose-fructose solution, as shown in FIG. 5 , as the incubation time increased, the fluorescence increased, confirming that BSA glycosylation was significantly increased. When ponsirin was added to the reaction medium containing the BSA-glucose-fructose system, a significant decrease in fluorescence intensity was observed over the duration of the experiment. At 4 weeks of incubation, the percentage inhibition of AGE formation by ponsirin was found to be 19.42%, 31.65%, 60.90% and 81.68% at concentrations of 0.5, 2.0, 10 and 50 μM, respectively, while the positive control aminoguanidine had a concentration of 500 μM. concentration inhibited AGE formation by 47.50%. In particular, ponsirin at a concentration of 50 μM showed stronger inhibition on glycosylated BSA formation compared to aminoguanidine (500 μM).

6-4. Effects of ponsirin on fructosamine formation

The level of the Amadori product fructosamine was nitroblue-tetrazolium ( NBT) dye was used to measure the AGE in the same reaction mixture. Briefly, 10 μL of glycated BSA was added to 100 μL of 0.5 mM NBT in 0.1 M carbonate buffer (pH 10.4) and incubated at 37° C. for 15 min. Absorbance was measured at 530 nm using a VERSA max microplate reader. The concentration of fructosamine was calculated and compared with DMF as standard.

The effect of ponsirin on the level of fructosamine is shown in FIG. 6 .

Fructosamine levels of glucose-fructose-glycosylated BSA were significantly increased during the 4 weeks of the experiment. At the end of the experimental period, ponsirin at concentrations of 0.5, 2.0, 10 and 50 μM inhibited fructosamine formation in the BSA-glucose-fructose system, compared to 15.4% inhibition of fructosamine formation by the positive control, 500 μM, aminoguanidine. 9.8%, 17.9%, 30.3% and 31.5% inhibition, respectively. Therefore, it was confirmed that ponsirin can reduce diabetic vascular complications.

6-5. Effects of ponsirin on CML formation

After 1, 2, 3 and 4 weeks of culture, Nε-(carboxymethyl)lysine (CML), a major antigenic AGE structure, was determined in the same reaction mixture in which the AGE was measured using an ELISA kit according to the manufacturer's protocol. The absorbance of the sample was measured at 450 nm and compared to the absorbance of the CML-BSA standard provided in the assay kit.

As a result of measuring the level of Nε-CML, a biomarker for non-fluorescent AGE formation, at 4 weeks of incubation, glucose-fructose-induced glycated BSA showed a 9.6-fold increase in CML formation compared to non-glycosylated BSA at 4 weeks ( FIG. 7 ). On the other hand, CML formation was inhibited in a concentration-dependent manner upon treatment with ponsirin, and treatment with ponsirin at concentrations of 0.5, 2.0, 10 and 50 μM at week 4 significantly reduced the formation of CML by 6.64%, 20.49%, 38.10% and 46.21%, respectively. appeared to be suppressed. Aminoguanidine reduced CML levels by about 41.9% at a concentration of 500 μM.

6-6. Effect of ponsirin on protein carbonyl group formation

After culturing for 1, 2, 3, and 4 weeks, the carbonyl group of glycated BSA, an indicator of protein oxidative damage, was identified by the previous method of Levine and colleagues (Adisakwattana, S., et al., Int. J. Mol. Sci . 2012, 13 , 1778-1789) and a slightly modified method. Briefly, 400 μL of 10 mM DNPH dissolved in 2.5 M HCl was added to 100 μL of glycosylated sample. After incubation in the dark for 60 min, 0.50 mL of 20% (w/v) TCA was used for protein precipitation (5 min on ice), followed by centrifugation at 10,000 g for 10 min at 4°C. The protein pellet was washed 3 times with 500 μl of an ethanol/ethyl acetate (1:1) mixture and resuspended in 250 μl of 6.0 M guanidine hydrochloride. Absorbance was measured at 370 nm. The carbonyl content of each sample was calculated based on the extinction coefficient of DNPH (ε= 22,000 M ⁻¹ cm ⁻¹ ). Results are expressed in nM carbonyl/mg protein.

As a result of evaluating the level of carbonyl content as an indicator of protein oxidation during glycosylation, as shown in FIG. 8 , the exposure of BSA to high glucose-fructose increased as the exposure time increased, compared to non-glycosylated BSA. The carbonyl content was significantly increased, and the carbonyl content at week 4 in the BSA-glucose-fructose system was increased by more than 7.8 times compared with the non-glycosylated BSA. At 4 weeks of incubation, ponsyrin in the BSA-glucose-fructose system significantly reduced protein carbonyl levels by 34.0%, 42.9%, 49.8% and 55.29% at concentrations of 0.5, 2.0, 10 and 50 μM, respectively. Aminoguanidine was shown to reduce the protein carbonyl content by 50.12% at a concentration of 500 μM, and it was found that ponsirin at 10 μM or higher was more effective than aminoguanidine (500 μM). Therefore, ponsirin may be effective in preventing oxidative damage to proteins due to hyperglycemia.

6-7. Effect of ponsirin on thiol group formation

Protein thiol groups were determined according to previously published methods with slight modifications (Islam, MN, et al., Food Chem. Toxicol . 2014, 69 , 55-62). Briefly, 70 μL of glycated samples were incubated with 5 mM DTNB in 0.1 M PBS, pH 7.4 at 25 °C for 15 min. The absorbance of the sample was measured at 410 nm. The concentration of free thiol was calculated from the L-cysteine standard and expressed in μM.

As a result of analyzing the formation of thiol groups to examine protein oxidation mediated by the glycosylation process, as shown in FIG. 9, glucose-fructose-mediated saccharification of BSA is a gradual change of thiol groups in BSA with increasing incubation time. decreased, and at week 4, there was a 51.2% thiol loss in BSA. On the other hand, the loss of thiol groups was significantly inhibited in a concentration-dependent manner upon treatment with ponsirin, and at 4 weeks, the loss of thiol groups was 62.59%, 67.95%, and 69.59% at the concentrations of 0.5, 2.0, 10 and 50 μM of ponsirin, respectively. and 74.69% inhibition. In addition, the level of thiol was significantly improved after the addition of aminoguanidine, and 67.5% of thiol loss was prevented at a concentration of 500 μM.

6-8. Effects of ponsirin on amyloid cross-β structure

The amyloid cross-β structure, a common marker for protein aggregation, uses a thioflavin T assay according to a previous method with slight modifications (Islam, MN, et al., Food Chem. Toxicol . 2014, 69 , 55-62). Thus, the AGE was measured in the same reaction mixture. Briefly, 100 µL of 64 µM Thioflavin T in 100 µL of 0.1 M PBS at pH 7.4 was added to 10 µL of the glycated sample and incubated at 25 °C for 1 h. Fluorescence intensity was measured at an excitation wavelength of 435 nm and an emission wavelength of 485 nm.

As shown in FIG. 10 , compared to unglycosylated BSA, glycosylated BSA had an increased amyloid cross-β conformation over the 4 weeks of the experiment. At week 4, glycosylated BSA exhibited a 2.9-fold increase in amyloid cross-β conformation compared to unglycosylated BSA. On the other hand, ponsirin treatment reduced the level of amyloid cross-β structures by 19.3%, 33.2%, 66.1% and 68.8%, respectively, at concentrations of 0.5, 2.0, 10 and 50 μM. Similarly, aminoguanidine reduced the level of amyloid cross-β structures by 30.1% at a concentration of 500 μM. Consequently, treatment with ponsirin at concentrations of 10 and 50 μM reduced the level of amyloid cross-β structures to normal levels, and ponsirin was found to be more effective than aminoguanidine (500 μM). These beneficial effects of ponsirin may help reduce the risk of developing debilitating degenerative diseases in diabetic patients.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (11)

Containing poncirin, an isomer thereof, or a pharmaceutically acceptable salt as an active ingredient,
Pharmaceutical for preventing or treating insulin-resistant diabetes mellitus or diabetic complications induced by an increase in protein tyrosine phosphatase 1B (PTP1B) and a decrease in glucose transporter type 4 (GLUT-4) composition. According to claim 1,
The insulin-resistant diabetes mellitus or complications of diabetes are α-glucosidase, protein tyrosine phosphatase (PTP), rat lens aldose reductase (RLAR) and human recombinant aldose reductase (HRAR)-related pathway dependence. Diabetes mellitus or a complication of diabetes, the pharmaceutical composition. According to claim 1,
The pharmaceutical composition is characterized in that it exhibits the effect of activating the glucose uptake signal transduction pathway in muscle cells, the pharmaceutical composition. According to claim 1,
The pharmaceutical composition 1) improves insulin resistance and increases glucose absorption by activating the insulin signaling pathway through PTP1B inhibitory activity, 2) aldose reductase (AR) and advanced glycation end -product, AGE) that exhibits an inhibitory effect, a pharmaceutical composition. According to claim 1,
The pharmaceutical composition is characterized in that inhibiting glycosylated protein oxidation, pharmaceutical composition. According to claim 1,
The diabetic complications are diabetic neuropathy, diabetic retinopathy, cataract, diabetic nephropathy, renal failure, sexual dysfunction, skin disease, high blood pressure, arteriosclerosis, stroke, cerebral infarction, heart disease, embryopathy, gangrene and delayed wound healing. One or more diseases selected from, the pharmaceutical composition. Containing poncirin, an isomer thereof, or a food pharmaceutically acceptable salt as an active ingredient,
A dietary supplement composition for preventing or improving insulin resistance diabetes or diabetes complications induced by an increase in protein tyrosine phosphatase 1B (PTP1B) and a decrease in glucose transporter type 4 (GLUT-4). 8. The method of claim 7,
The insulin-resistant diabetes mellitus or complications of diabetes are α-glucosidase, protein tyrosine phosphatase (PTP), rat lens aldose reductase (RLAR) and human recombinant aldose reductase (HRAR)-related pathway dependence. Diabetes mellitus or complications of diabetes, health functional food composition. 8. The method of claim 7,
The health functional food composition 1) improves insulin resistance and increases glucose absorption by activating the insulin signaling pathway through PTP1B inhibitory activity, and 2) inhibits aldose reductase (AR) and final glycation product (AGE). It represents, a health functional food composition. 8. The method of claim 7,
The health functional food is a food in the form of a tablet, capsule, pill or liquid, health functional food composition. Containing poncirin, an isomer thereof, or a pharmaceutically acceptable salt as an active ingredient,
Insulin-resistant diabetes mellitus or diabetes complications induced by an increase in protein tyrosine phosphatase 1B (PTP1B) and a decrease in glucose transporter type 4 (GLUT-4) through inhibition of PTP1B enzyme activity by inactivating the PTP1B enzyme active site A composition for enhancing insulin sensitivity.

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