KR101381590B1 - Inhibitory effects of the human intestinal maltase - Google Patents

Abstract

The present invention relates to inhibitors of the activity of human small intestine maltase, and more particularly, to the inhibitory activity of human small intestine maltase and its composition comprising epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient. Human small intestine maltase according to the present invention does not inhibit the activity of human pancreatic amylase and specifically inhibits maltase, an enzyme that directly increases blood sugar, thereby preventing post-prandial hyperglycemia and preventing glucose metabolism-related diseases. Or it may be usefully used for treatment.

Description

Inhibitory effects of the human intestinal maltase

The present invention relates to inhibitors of the activity of human small intestine maltase, and more particularly, to the inhibitory activity of human small intestine maltase and its composition comprising epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient.

Diabetes, a serious chronic disease, is not just limited to developed countries, but is emerging in most of Asia, where population growth, aging, and full-scale urbanization are experiencing. It is also estimated that by 2030, the prevalence of diabetes will increase to 4.4% (approximately 360 million people) of the world population.

Diabetes is a group of diseases that collectively refers to all diseases in which blood glucose levels are increased. Sugar, which is a lot of rice, flour, and potatoes that we eat as a stock, is digested in the intestines, converted into glucose, and then into the blood to become blood sugar. In this process, a hormone called insulin acts.

By the action of the insulin hormone, diabetes is divided into 'type 1' which is absolutely lacking in the production of insulin and 'type 2' where the relative insulin deficiency and function is reduced. In type 1 diabetes, insulin is necessary in the treatment of diabetes because the pancreas destroys insulin-secreting cells, resulting in an absolute lack of insulin.

Type 2 diabetes accounts for more than 90% of all diabetics, and the incidence increases with age and obesity, and about half of the patients are obese over normal weight. Thus, the treatment of type 2 diabetes is insufficient insulin, but the pancreatic insulin secretion ability remains, so dietary and exercise therapy can be controlled blood sugar, if the blood glucose control is not possible by such a method (oral blood glucose) Hypoglycemic) or insulin injections to control blood glucose. Eating hypoglycemics are largely divided into insulin secretagogues and insulin sensitivity improvers.

In the treatment of type 2 diabetes, development of inhibitory material of alpha-glucosidase is very important. The use of acarbose, boglibose, and miglitol, inhibitors of alpha-glucosidase, inhibits the breakdown of polysaccharides in the small intestine and delays the digestion and absorption of complex carbohydrates, resulting in postprandial hyperglycemia. It acts to suppress and reduce hyperinsulinemia. However, gastrointestinal side effects such as bloating, nausea, gas swelling and diarrhea are known, and more effective hypoglycemic agents are required while minimizing these side effects (Application 10-2009-0120314, Screening of Inhibitors of Human Intestinal Maltase) Method and maltase inhibitor using the same).

On the other hand, the main component that causes various physiological activities of green tea is catechin contained in the green tea leaves. The catechins contained in the green tea leaves are catechin, epicatechin, epigallocatechin, epicatechin gallate, catechin gallate, and epigallocatechin gallate. gallate). The interest in these green tea catechins is increasing because the catechin compounds have strong antioxidant properties and antibacterial effects, as well as various activities such as anti-ulcer, anticancer, and anti-allergy.

However, these catechin compounds do not dissolve well in water, and are easily decomposed and oxidized by light or heat to cause browning. Therefore, there are many limitations in using them as food additives, cosmetics, and medicines. On the other hand, Patent No. 10-0716797 biotransformation method, that is, the modified products synthesized by transferring these catechins through the enzymatic reaction sugar has not only increased the stability to light but also has a property of being soluble in water, It has been reported that catechins have a strong bitter and astringent taste.

In this patent, catechins, resveratrol, acubin, paranitrophenol, paranitrophenyl-O-α-D-glucopyranoside, tyrosol, alpha-acarbose, nodakenin using glycan sucrase or levansukraase. Only enzymatic synthesis of sugar derivatives, such as ferulic acid, quercetin and alcohols, is described, but it also shows the activity of human intestinal maltase (α-glucosidase), which directly affects blood sugar. There is no report of inhibitory activity.

Therefore, the present inventors found that epigallocatechin gallate-4'-O-alpha-di-glucopyranoside, which is a sugar transition compound derivative of epigallocatechin gallate, the main component of green tea catechin, breaks down maltose into glucose and lowers blood glucose. The present invention has been found to effectively inhibit the activity of human small intestine maltase (α-glucosidase), which has a direct effect.

It is an object of the present invention to provide an inhibitor of the activity of human small intestine maltase comprising epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient.

Still another object of the present invention is to provide a composition for the treatment or prevention of glucose metabolism-related diseases using epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient.

In order to achieve the above object, the present invention provides an inhibitor of the activity of human small intestine maltase using epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient.

In another aspect, the present invention provides a composition for the treatment or prevention of diseases associated with glucose metabolism comprising epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient.

Inhibitor of the human small intestine maltase of the present invention comprises epigallocatechin gallate or epigallocatechin gallate-glucoside as an active ingredient, and the active ingredient does not inhibit the activity of human pancreatic amylase and directly lowers blood glucose. It is characterized by the specific inhibition of only increasing maltase.

More specifically, the epigallocatechin gallate-glucoside is epigallocatechin gallate as the receptor, and epigallocatechin gallate is obtained by successively transferring glucose of sucrose to the receptor using dextran sucrose. Glucoside is synthesized.

Epigallocatechin gallate, epigallocatechin gallate-glucoside or mixtures thereof of the present invention does not inhibit the activity of human pancreatic amylase but specifically inhibits the human small intestine maltase. It is characterized in that it can provide a therapeutic or prophylactic composition.

Hereinafter, the present invention will be described in detail.

The present invention provides an inhibitor of the activity of human small intestine maltase using the compound of Formula 1 or the compound of Formula 2 as an active ingredient.

In the present invention, the compound of Formula 1 is characterized in that obtained by the reaction after the addition of the sugar transfer enzyme to a mixture of sucrose and epigallocatechin gallate (epigallocatechin gallate).

More specifically, the compound of Chemical Formula 1 may include the steps of: 1) generating epigallocatechin gallate-glucoside by transferring a glucosyl group of sucrose to epigallocatechin gallate as a receptor; 2) separating and purifying the receptor reactant; And the like.

In the present invention, the compound of Formula 1 is added to the solution containing the sucrose 0.01 to 1.0 M concentration of sucrose so that the reactor final active concentration is 0.1 to 100 U / ㎖, it is 15 to 70 ℃ Epigallocatechin gallate is added to the final concentration in the reactor to 0.01 to 10% (w / v), characterized in that it is obtained by reacting.

In the present invention, the sugar transfer enzyme is characterized in that the dextran sucrase derived from Leuconostoc mesenteroides species strain.

More specifically, to the solution containing 80 mM sucrose, dextran sucrase derived from Leuconostoc mesenteroides species strain was added so that the reactor final active concentration was 1 U / mL, Epigallocatechin gallate-glucoside was prepared by adding epigallocatechin gallate as a receptor to a final concentration of 0.2% (w / v) in a reactor at 28 ° C.

In the present invention, the inhibitor does not inhibit the activity of human pancreatic amylase, it is characterized in that it specifically inhibits only maltase that directly increases blood glucose.

More specifically, the epigallocatechin gallate-glucoside was confirmed to inhibit the activity of recombinant human small intestine maltase and human pancreatic amylase. As a result, epigallocatechin gallate and epigallocatechin gallate-glucoside were identified as human pancreatic maltase. It can be seen that it effectively inhibits and does not inhibit human pancreatic amylase at all. See FIGS. 2 and 3.

The human small intestinal maltase (HMA) used in the present invention expresses and purifies the amino terminal catalytic domain of the membrane protein present in epithelial cells of the small intestine called maltase and glucoamylase (maltase-glucoamylase). will be

The present invention is a composition for the treatment or prevention of any one or more sugar metabolic related diseases selected from obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia using the compound of Formula 1, the compound of Formula 2 or a mixture thereof as an active ingredient To provide.

In the present invention, the glucose-related metabolic disease is more preferably diabetes.

The compound of Formula 2 or a mixture of Compounds 1 and 2 according to the present invention may be used alone or in combination with a pharmaceutically acceptable carrier to be used as a hypoglycemic agent.

When the compound is used as a post-prandial hyperglycemia inhibitor, it is preferable to administer it at a daily dose of 1 μg / kg body weight to 50 mg / kg body weight, but this may include the age, sex, diet, health condition, severity of the subject, The dosage may be increased or decreased depending on the administration method, administration time, drug mixture, and the like.

According to the present invention there is provided a pharmaceutical composition for the prevention and treatment of a disease containing the compound as an active ingredient and associated with the role of human small intestine maltase and requiring selective inhibition of human small intestine maltase, in particular the disease is postprandial hyperglycemia , Diabetes and obesity. The composition comprises at least one compound of the compounds described herein as an active ingredient, that is, a composition for treating or preventing a glucose metabolism related disease according to the present invention may include the compound of the present invention in any combination.

The composition may be administered by oral, rectal, nasal, lung, topical, transdermal, intraracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) routes. And may be specifically formulated for administration by any suitable route, such as by oral route. The preferred route will depend upon the general condition and age of the subject being treated, the nature of the condition being treated, and the active ingredient selected.

The composition prepared according to the invention may be in any suitable route, for example in tablets, capsules, powders, granules, pellets, oral tablets, dragees, pills or tablets, solutions in aqueous or non-aqueous liquids. Or parenterally in the form of oral or injectable solutions in the form of suspensions, or oil-in-water or water-in-oil liquid emulsions, elixirs, syrups and the like. Other compositions for parenteral administration include sterile powders composed of sterile injectable solutions or dispersions prior to use as well as dispersions, suspensions or emulsions. Depot injection formulations are also considered to be within the scope of the present invention. Other suitable dosage forms include suppositories, sprays, ointments, creams, gels, inhalants, skin patches, and the like. To prepare the composition, methods known in the art may be used, and any pharmaceutically acceptable carrier diluents, excipients or other additives commonly used in the art may be used.

Such carriers are those conventionally used at the time of formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, water, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may further contain an antiseptic, a stability improving substance, a viscosity improving or regulating substance, a solubility improving substance, a sweetener, a dye, a taste improving compound, a salt for varying osmotic pressure, a buffer solution,

In addition, the compositions of the present invention may include one or more other therapeutically useful substances, such as sulfonate agents (glymephride, glyclazide, glybenclamide, etc.), meglitinides (lepaglinide, nateglinide, mitigli) Amide, etc.), biguanide-based (metformin), chiazolidinedione-based (loziglitazone, pioglitazone, etc.), alphaglucosidase inhibitors (acarbose, bogglibose, miglitol) and the like. .

Human small intestine maltase according to the present invention does not inhibit the activity of human pancreatic amylase, and specifically inhibits glucose metabolism-related diseases as an inhibitor of postprandial hyperglycemia and diabetes treatment due to the advantage of specifically inhibiting maltase, an enzyme that directly increases blood glucose. It can be usefully used for prevention or treatment.

Figure 1 shows an individual map of the recombinant vector (pPICZαA-HMA) comprising the human small intestine maltase gene of the present invention,
2 is a result confirming the activity inhibitory of maltose to the human small intestine maltase of epigallocatechin gallate and epigallocatechin gallate-glucoside according to the present invention,
(M, maltose; G, glucose; 1, 20 mM maltose + 0.3% (w / v) epigallocatechin gallate; 2, 0.3% (w / v) epigallocatechin gallate; 3, 20 mM Maltose + 0.3% (w / v) epigallocatechin gallate + human small intestine maltase; 4, 0.3% (w / v) epigallocatechin gallate-glucoside; 5, 20 mM maltose + 0.3% (w epigallocatechingallate-glucoside; 6, 20 mM maltose + 0.3% (w / v) epigallocatechingallate-glucoside + human small intestine maltase; 7, 20 mM maltose; 8, 20 mM maltose + Small intestine maltase)
Figure 3 is a result confirming the inhibitory ability of starch degradation on human pancreatic amylase of epigallocatechin gallate and epigallocatechin gallate-glucoside according to the present invention.
(S, maltooligosaccharides; G, glucose; 1, 0.3% (w / v) epigallocatechin gallate; 2, 0.5% soluble starch + 0.3% epigallocatechin gallate; 3, 0.5% soluble starch + 0 .3% epigallocatechin gallate + recombinant human pancreatic amylase; 4, 0.3% (w / v) epigallocatechin gallate-glucoside; 5, 0.5% (w / v) soluble starch + 0.3% (w epigallocatechingallate-glucoside; 6, 0.5% (w / v) soluble starch + 0.3% (w / v) epigallocatechin gallate-glucoside + recombinant human pancreatic amylase; 7, 0.5% (w / v) soluble starch; 8, 0.5% (w / v) soluble starch + recombinant human pancreatic amylase)

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Preparation Example 1 Preparation of Dextran Sucrase Enzyme Liquid

Can dextran used in the present invention Klein azepin are flow Pocono stock methoxy Centennial Lloyds B-512FMCM (Leuconostoc mesenteroides B- 512FMCM) and flow Pocono stock methoxy Centennial Lloyds B-1299CB4 BF563 (Leuconostoc mesenteroides B -1299CB4 BF563) enzyme obtained from the strain Was used. Dextran sucrose is Leuconostoc mesenteroides ( Leuconostoc mesenteroides) B-512FMCM) and Leuconostoc mesenteroides B-1299CB4 BF563 (Luconostoc mesenteroides B-1299CB4 BF563) were cultured using LM medium containing 2% (w / v) glucose as a carbon source and the growth temperature was maintained at 28 ° C. It was.

Example 1 Epigallocatechin gallate-glucoside synthesis, purification and structural analysis

(1) Enzymatic Reaction for Epigallocatechingallate-glucoside Synthesis

Epigallocatechingallate-glucoside synthesis reaction conditions were 80 mM sucrose, 1 unit of dextran sucrase per ml of reaction solution, and the final concentration 0.2% (w / v) epigallocatechingal represented by the following compound Receptor reaction solution prepared in the rate (epigallocatechin gallate) was reacted at 28 ℃ until all the sucrose of the reactor was consumed (about 6.5 hours), and the reaction mixture was boiled for 5 minutes to stop the enzyme reaction. One unit of enzyme used is expressed as the amount of enzyme liberating 1 μmol fructose per minute.

Production of the receptor reaction product and confirmation of complete reaction of sucrose were carried out on a TLC plate ((Merck K6F, Darmstadt, Germany) by taking 1 μl of the reaction solution, followed by ethyl acetate: acetic acid: water = 3: 1: 1 (v / v / v) was developed using a solvent prepared as a component of the carbohydrate, and the separated carbohydrate was prepared by thin layer chromatography (TLC) plates containing 0.5% (w / v) α-naphthol and 5% (v / v) sulfuric acid. Analysis was performed using a color reagent (Mukerjea R. et al. Simplified and improved methylation analysis of saccharides using a modified procedure and thin-layer chromatography, Carbohydrate Research, 1996, 292, 11-20).

(2) Epigallocatechin gallate - Glucoside  refine

Epigallocatechin gallate-glucoside produced in 1) can be isolated and purified by conventional methods. The present invention administers a sample to a column filled with Sephadex LH-20 (GE Healthcare Biosciences AB), a gel filtration chromatography (Gel filtration Chromatograph), fractional elution by ethanol (0 to 100%) concentration gradient. The degree of sugar separation was confirmed using TLC. Purified synthetic products were collected by size, lyophilized and prepared in powder form.

(3) refined Epigallocatechin gallate  Receptor Reaction products  Structure determination

After purification by size using Sephadex LH-20 column chromatography, the molecular weight of each material was analyzed using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF). To this end, the purified reaction product (1mg / 1mL) is diluted with distilled water and mixed with 2,5-dihydroxybenzoic acid (dehyeroxy-benzoic acid, 1mg / 1mL) in a volume of 1: 1. 1 μl of the mixed solution was added to a stainless steel matrix and then slowly dried at room temperature.

The mass spectrum was analyzed using MALDI-TOF-MS (Voyager DE-STR; Appled Bio Systems, USA), at which time 65 kV acceleration voltage was used in positive mode. From the MALDI-TOF mass spectrometry, it was confirmed that the purified reaction product increased the molecular weight by the molecular weight of glucose from which water molecules were separated by one molecule of glucose into epigallocatechin gallate.

Structural analysis of the reaction product using NMR was performed by dissolving 2 to 3 mg of purified reaction product in 250 μl of DMSO-d6, and then placing it in an NMR (3 mm diameter) tube using a VNMRS spectrometer (Varian, Palo Alto, CA). , 1H NMR with 600 MHz equipment and 13C NMR with 150 MHz equipment, respectively.

Epigallocatechin gallate and the linkage site between sugars are homoclear correlation spectroscopy (COSY) that connects hydrogen to each other and heteronuclear single quantum coherence (HSQC) that connects hydrogen and carbon, and hydrogen to carbon. The results of analysis using heteronuclear multiple bond correlation (HMBC) connected by bonds are shown in Table 1 below.

As a result of NMR analysis, epigallocatechin gallate-glucoside represented by the following compound was isolated and confirmed to be epigallocatechin gallate-4'-O-alpha-di-glucopyranoside.

[ Example  2] With epigallocatechin gallate Epigallocatechin gallate - Glucoside  Inhibition of Human Small Intestine Maltase Activity

(1) Activity test for recombinant human small intestine maltase

To confirm the activity of recombinant human small intestine maltase, maltose, a substrate, was dissolved in 50 mM potassium phosphate buffer (pH 6.5) at a concentration of 10 mM.

The recombinant human small intestine maltase is a yeast bacterium Pichia using the pPICZαA-HMA of FIG. 1 prepared by the inventor of the present invention filed 10-2009-0120314 (name of the invention: activity inhibitor screening method of human small intestine maltase and maltase inhibitor using the same) The recombinant human small intestine maltase obtained from the culture of the transforming strain by transformation into pastoris X-33 (Invitrogen, USA) was used.

The recombinant human small intestine maltase (0.04 U / ml) was mixed with epigallocatechin gallate or epigallocatechin gallate-glucoside (final concentration 0 μM to 150 μM) for 20 minutes at 37 ° C., followed by 2M Tris -Cl (pH 8.0) is added to stop the reaction. The reaction product glucose was measured for glucose activity after quantitative analysis using a glucose measuring reagent (Asan Pharmaceutical Co., Ltd.). One unit of enzyme was determined by the amount of enzyme that produced 1 μmole of glucose per minute from the substrate.

When the final concentration of the inhibitor 100 μM, epigallocatechin gallate and epigallocatechin gallate-glucoside inhibition of human small intestine maltase activity was as shown in Table 2.

The comparative group displays the activity of the enzyme solution without epigallocatechin gallate and its glycoside as 100% activity.

As can be seen from the results of Table 2 and Figure 2, it was confirmed that epigallocatechin gallate and epigallocatechin gallate-glucoside effectively inhibited human small intestine maltase.

(2) Investigation of starch resolution for human pancreatic amylase

In order to investigate the inhibitory activity on starch resolution against human pancreatic amylase, the substrate starch was dissolved in 50 mM sodium phosphate buffer (pH 7.0) at a final concentration of 0.5% (w / v). After mixing 0.3% (w / v) epigallocatechin gallate or epigallocatechin gallate-glucoside with recombinant human pancreatic amylase (0.04 U / ml), starch and buffer for 1 hour at 37 ° C, Remaining activity and reaction products of human pancreatic α-amylase were confirmed using TLC.

As a result, as shown in FIG. 3, when epigallocatechin gallate is not added or epigallocatechin gallate-glucoside is added, starch, which is a substrate in human pancreatic amylase enzyme reaction, is converted into malto oligosaccharide. Decomposition was performed, but it was confirmed that substrate starch was not degraded at all in the reaction with epigallocatechin gallate.

Indirectly confirming that epigallocatechingallate-glucoside of the present invention effectively inhibits human small intestine maltase but does not inhibit human pancreatic amylase, thereby indirectly reducing the side effects of bloating. Could.

Claims (9)

delete delete delete delete delete delete delete A method for inhibiting human small intestine maltase activity in vitro by treatment with a compound of Formula 1 or a compound of Formula 2.

10. The method of claim 8, wherein the activity of human pancreatic amylase is not inhibited but only maltase is specifically inhibited.

Images