KR20040064572A - Inhibition activity of cinnamon extract against sucrase, maltase and glucoamylase and compositions containing cinnamon extract for regulation of blood sugar - Google Patents

Abstract

PURPOSE: Provided is an inhibitor of an extract of Cinnamomi cortex which is mass-produced in a cost effective manner, and effectively inhibits only the absorption of carbohydrate without inhibiting the absorption of other necessary inorganic ions and proteins, thereby controlling the blood sugar and treating diabetes and adiposity. CONSTITUTION: A method of manufacturing the inhibitor comprises the steps of: making a Cinnamomi cortex extract with an activity of inhibiting sucrase, maltase and glucoamylase; and separating harmful materials from the extract. The proteins of sucrase, maltase and glucoamylase are derived from the small intestine of pigs, rats, mouses, Guinea pigs, rabbits, or persons.

Description

Inhibitory activity of broiler extracts against sucrase, maltase and glucoamylase and composition for controlling blood glucose using the same

The present invention relates to a method for producing broiler extracts having inhibitory activity against sucrase and maltase and glucoamylase, and a composition for controlling blood glucose using the same, and specifically, sucrose and maltase and glucose, which are carbohydrate degrading enzymes derived from swine intestine Isolating amylase; Searching for natural products having inhibitory activity against the sucrase and maltase and glucoamylase; And separating the active substance from the alcoholic extract of broiler with inhibitory activity, using a method for producing broiler extract with inhibitory activity against sucrose and maltase and glucoamylase and broiler extract produced by the method. The present invention relates to a glycemic control composition for inhibiting blood glucose increase in diabetic patients by inhibiting the activity of sucrose and maltase and glucoamylase, which are carbohydrate degrading enzymes in vivo.

In vivo digestion of ingested food usually results in the breakdown of some proteins in the stomach, including the initial breakdown of carbohydrates in the oral cavity. However, full-fledged carbohydrate and fat digestion and absorption occur mainly in the area from the duodenum to the ileum, and in general, it is known that resorption of water and salts and absorption of free fatty acids occur mainly in the large intestine. Therefore, the most important part of digestive absorption occurs in the small intestine, where proteolytic enzymes such as trypsinogen, chymotrypsinogen, and carboxy peptidase, lipolytic enzymes such as pancreatic lipase and sphyxin, pancreatic amylase, amylosin Carbohydrate degrading enzymes such as, maltase and various digestive enzymes such as various peptidase, nucleic acid degrading enzyme, alginase, phosphatase, and lactose degrading enzyme which are various glycolytic enzymes in the small intestine Absorbed by the nutrients absorbed through the dark ducts and blood vessels will be moved into the body.

The activity of sucrase, maltase and glucoamylase, important disaccharide degrading enzymes, which are indispensable for the transport of monosaccharides caused by the breakdown of carbohydrates, are very high in the chorionic membrane transporter. These enzymes increase the absorption of monosaccharides in the body. It is known. However, hydrolysis by disaccharide degrading enzymes in the intestine does not occur much during the upper part of the ileum, which means that the so-called chondrocytes of the chorionic cell membrane transporter are largely dependent on the membrane digestion. Indeed, it can be proved that the absorption of monosaccharides is relatively low compared to the total monosaccharide absorption until it reaches the ileal region where sucrase, maltase and glucoamylase are found to have much activity.

Diabetes is a chronic disease that is hard to cure yet. It is a disease that must be managed continuously by taking insulin injections or taking medicine for life. Diabetes is one of the 10 leading causes of death in Korea and is included as the only single disease.

In recent years, diabetes has been increasing rapidly. The government has decided to use diabetes as a special management disease and promote diabetes management at the national level. However, most of the functional foods for diabetics are low-calorie foods, and functional ingredients that help direct blood sugar control in Korea. Absent

Therefore, for diabetics who need blood sugar management, the differentiation suitable for diabetic patients who can inhibit the absorption of carbohydrates without inhibiting the absorption of essential ions and proteins other than the diet used for conventional diabetes without limiting nutrient intake. There is a need for the development of an established method.

Therefore, the present inventors directly inhibit the digestion and absorption of carbohydrates by using the broiler extract, which separates the enzymes sucrase and maltase and glucoamylase, which play important roles in the digestion and absorption of carbohydrates, from the small intestine of pigs. The present invention has been completed by revealing that it can be usefully used as a composition for improving and treating diseases.

The present invention can significantly improve the quality of life of obese and diabetic as a functional food ingredient that can alleviate the restriction of carbohydrates in the final body absorption by controlling the blood sugar and the restriction of food intake occurring in the diet of diabetic patients.

It is an object of the present invention to provide a method for isolating and producing extracts of broilers having inhibitory activity against carbohydrate degrading enzymes.

Another object of the present invention to provide a composition for controlling blood sugar comprising the broiler extract.

1 is a result of the separation of sucrase, maltase and glucoamylase from membrane proteins of the small intestine using anion exchange column chromatography according to the present invention,

Figure 2 shows the change in reaction rate with the temperature of the sucrase and maltase isolated in Figure 1 ,

Figure 3 is will showing a change of the reaction rate according to the number of pH Klein azepin maltase and remove it from the first.

Figure 4 shows the change in reaction rate according to the temperature and pH of the glucoamylase isolated in Figure 1 ,

Figure 5 shows the separation process of broiler extract,

Figure 6 shows that the broiler extract prepared according to the present invention directly inhibits the activity of swine intestine-derived maltase,

Figure 7 shows that the broiler extracts prepared according to the present invention directly inhibit the activity of swine small intestine-derived sucrase,

8 shows that the broiler extracts prepared according to the present invention directly inhibit glucoamylase activity of pig small intestine,

9 is a result of measuring the inhibition of the increase in the concentration of sugar in the body by the broiler extract of the present invention at the time of sucrose overload test using the animal model rat,

10 is a result of measuring the inhibition of the increase in the body sugar concentration by the broiler extract of the present invention during the maltose overload experiment using the animal model rat,

Figure 11 is the result of measuring the inhibition of the increase in the concentration of sugar in the body by the broiler extract of the present invention during the water-soluble starch overload test using an animal model rat.

In order to achieve the above object, the present invention provides a method for separating the extract of broiler inhibiting sucrase and maltase and glucoamylase.

In addition, the present invention provides a pharmaceutical composition for controlling blood sugar, which contains the broiler extract produced by the method as an active ingredient.

In addition, the present invention provides a food and beverage composition containing the broiler extract produced by the method as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for separating sucrase and extract of broiler that inhibits maltase and glucoamylase.

The separation method is

1) using broth and maltase and glucoamylase proteins as target enzymes to produce alcoholic extracts of broilers that inhibit them;

2) separating the spirit extract containing the inhibitor using silica gel column chromatography; And

3) separating the silica gel column chromatography active fractions using Alp-18 column chromatography.

The above steps will be described in more detail below.

In a preferred embodiment of the present invention, in step 1), membrane proteins are separated from the small intestine of pigs, and sucase, maltase and glucoamylase are purified using anion exchange column chromatography (see FIG. 1 ). In order to determine the enzymatic activity of purified sucrase, maltase and glucoamylase, the optimum reaction temperature and pH of each enzyme activity were examined. As a result, sucase was found at 40 ℃ and weakly acidic pH (pH 5-6). At 50 ° C. and near neutral pH (pH 6-7), glucoamylase showed the highest activity at 50 ° C. and weakly acidic pH (pH 5-6) (see FIGS. 2, 3 and 4 ).

The sucrase and maltase and glucoamylase used as target proteins in the present invention can be isolated from pig small intestine as well as from rat, mouse, guinea pig, rabbit or human and human-derived cell lines, and include genes encoding these enzyme proteins. Those expressed in recombinant microorganisms can be used separately, and chemically synthesized ones can also be used.

The broiler extract extracts the inhibitors of sucrase, maltase and glucoamylase from the above by using dinitrosalicylic acid (3,5-dinitrosalicylic acid). It was found that there is an inhibitory activity.

Step 2) extracts broiler broiler for 72 hours using alcohol and extracted the inhibitor twice more in the same way and confirmed the activity. In addition, the broiler alcohol extract was concentrated under reduced pressure to reduce the volume of the sample, and then adsorbed onto silica gel resin to perform silica gel column chromatography. The silica gel column chromography activity fractions whose inhibitory activity was confirmed were concentrated and dissolved again in distilled water, followed by Alp-18 column chromatography to confirm the active fractions.

In order to use the extract of broiler as a raw material for glycemic control, it is economical to use the broiler's alcohol extract as it is, but silica gel column chromatography and Alp-18 column chromatography were used to confirm the properties of inhibitors.

The separated and purified broiler extract effectively inhibits the enzymatic activity of maltase, sukrase and glucoamylase (see FIGS. 6 , 7 and 8 ) and absorbs carbohydrates through inhibition of the enzyme activity in vivo. It was confirmed that the blood glucose is reduced by effectively inhibiting (see FIGS . 9 and 10 ).

The present invention also provides a pharmaceutical composition for inhibiting carbohydrate absorption containing the broiler extract produced by the method as an active ingredient.

The broiler extracts for sucralase, maltase and glucoamylase produced according to the present invention directly inhibit the breakdown of carbohydrates in vivo to reduce blood sugar levels, so that the pharmaceutical composition comprising the broiler extract as an active ingredient can be used without restriction of food intake. It can be usefully used for the prevention and treatment of diseases caused by excessive carbohydrate absorption, such as obesity, diabetes, hypertension and the like.

The pharmaceutical formulations can be prepared according to conventional methods using the compositions of the present invention. The formulation may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly Vinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. For humans, a typical daily dosage of purified broiler extract may range from 5 to 30 mg / kg body weight, preferably 10 to 20 mg / kg body weight, and may be administered once or in several doses.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the route of administration chosen, the age, sex, weight of the patient and the severity of the patient's symptoms, and thus the dosage may be The method does not limit the scope of the present invention.

In addition, the present invention provides a food and beverage composition containing the broiler extract produced by the method as an active ingredient.

Since the food and beverage composition of the present invention inhibits the breakdown of carbohydrates in the body, it is possible to maintain and improve health through blood sugar control, obesity control, and the like.

The broiler extract of the present invention is also effective for diseases caused by the absorption of excessive carbohydrates for the purpose of exerting a prophylactic and therapeutic effect such as diabetes, or as a dietary ingredient for improving obesity, e. It may be incorporated as a food supplement. As the food or beverage, meat; Vegetable juices (eg, carrot juice and tomato juice) and fruit juices (eg, orange juice, grape juice, pineapple juice, apple juice and banana juice); Chocolate; Snacks; confectionery; Pizza; Food products made of grain powder such as bread, cakes, crackers, cookies, biscuits, noodle, etc .; Gums; Dairy products such as milk, cheese, yogurt and ice cream; soup; Juicy; Pastes, ketchups and sauces; car; Alcoholic beverages; Carbonated drinks, vitamin complexes; And various health foods. In this case, the content of the broiler extract in the food or beverage may be in the range of 0.5 to 5% by weight, preferably 1 to 2% by weight.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Sucrase, Maltase and Glucoamylase Inhibitors

(Step 1) Isolation of Sucrase, Maltase and Glucoamylase from Porcine Small Intestine

To isolate sucrase, maltase and glucoamylase involved in digestive enzymes, fresh pig intestine was washed with phosphate saline buffer containing physiological salts (abbreviated as 'PBS') and scraped off mucosal layers. Then, 20 mM Tris-HCl buffer containing 200 mM mannitol (pH 6) was mixed at a ratio of 1:10. The mixture was homogenized, left to stand by addition of 50 mM calcium chloride and centrifuged to recover only the supernatant. The recovered supernatant was ultracentrifuged at 35,000 xg to separate the precipitated membrane protein. The isolated membrane protein was adsorbed onto a DEAE-Sepharose column (Amersham Pharmacia Biotech Co., Ltd.) equilibrated with 50 mM Tris-HCl buffer (pH 8) and the same containing 0-1.0 M sodium chloride. Proteins were selectively fractionated with buffer to separate sucrase from maltase and glucoamylase ( FIG. 1 ).

These small intestine-derived membrane proteins are glycoproteins bound to sugars and are composed of subunits having sucrase activity and maltase and glucoamylase activity, and have molecular weights of about 14 to 150,000. It also appears as one long polypeptide chain with molecular weight of 260,000 depending on the intestinal site (Siostrom, H. et al., J Biolog. Chem. 255 (23), 1980). As a result of SDS-PAGE electrophoresis analysis of the isolated sucralase, maltase and glucoamylase protein fractions, it was confirmed that protein bands were detected at positions of at least 200,000 and about 150,000 molecular weights.

(Step 2) Determination of Enzyme Activity of Isolated Sucrase, Maltase and Glucoamylase

In order to investigate the activity of the enzyme isolated in step 1, sucrose, maltose, and water-soluble starch were added as substrates, and an enzyme reaction was performed. The reaction solution contains a substrate 1%, 50 mM (phosphate buffer), enzyme (0.2 unit), distilled water and the reaction volume was reacted for 20 minutes at 37 ℃ with 0.1 ml. At this time, the temperature was reacted in the temperature range of 30 ℃ to 70 ℃ for the investigation of the optimum reaction temperature and pH of the enzyme and the pH was reacted in the pH range of 3 to 9. After the reaction, the enzyme activity was stopped at 100 ° C, and the resulting glucose concentration was quantified using GEL-zyme kit (Shinyang Chemical Co., Ltd.), a GOD-POD colorimetric method using glucose oxidase.

As a result, sucrase showed the highest enzymatic activity at 40 ℃ and weak acid (pH 5-6), maltase showed the highest activity at 50 ℃ and neutral (pH 6-7) and glucoamylase at 50 ℃ and pH The highest activity was shown at 5 ( FIGS. 2, 3, and 4 ).

(Step 3) Search for Enzyme Inhibitor

Purified sucrase, maltase and glucoamylase isolated from porcine intestine were used as target enzymes to search for natural extracts capable of inhibiting the activity of the enzymes. Natural products used natural herbal medicines listed in the Food Code to be used as food, and added grains and legumes such as broilers. Each 500 g of the natural product raw material was heated after cooling for 2 hours with 1 liter of cold water, and extracted using alcohol at 95-110 ° C. for 2-4 hours. After alcohol extraction, 100 ml of the supernatant of the extract was taken and used as an inhibitory sample.

Confirmation of inhibitory activity of the inhibitory samples was performed using a method of 3,5-dinitrosalicylic acid (hereinafter DNS) (hereinafter referred to as DNS method). In the preparation of DNS color development reagent, 1 g of DNS reagent is dissolved in 20 ml of 2M sodium hydroxide solution, and 30 g of sodium potassium tartrate tetrahydrate is slowly dissolved to adjust the final volume to 100 ml using distilled water. . Sucrose, maltose and water-soluble starch were used as a substrate of the enzyme in a concentration of 1% in 0.02M phosphate buffer at pH 6.9 containing 50 mM physiological salt. To prepare a calibration curve, a glucose solution was used. In order to quantify the activity of the enzyme inhibitor, the positive control group included 1% of substrate, 50 mM (phosphate buffer), enzyme (0.2 unit), and distilled water. The test of the enzyme inhibitor included 1% of substrate, 50 mM (phosphate buffer), enzyme (0.2 unit), natural extract, distilled water, and the reaction volume was 2 ml for 20 minutes at 37 ° C. After the reaction was completed, the reaction was terminated at 100 ° C. For the color reaction, 1 ml of the DNS reagent prepared above was added thereto, followed by cooling for 5 minutes at 100 ° C., followed by cooling to 10 ml of distilled water, and absorbance at 540 nm was measured. Absorbance measurements were measured for absorbance of each well with an ELISA reader in a 96 well microplate.

(Step 4) Separation of Broiler Extract

Inhibitors were isolated from the extracts of broiler broilers with high inhibitory activity from natural extracts with inhibitory activity against sucrase, maltase and glucoamylase. Separation of the inhibitor was performed by cold extraction for 72 hours by adding 2 liters of broiler 1kg broiler and repeated twice. After confirming the inhibitory activity in the alcohol extract, the broiler's alcohol extract was concentrated under reduced pressure at 56 ℃. The concentrated inhibitor was dissolved in chloroform and separated using silica gel column chromatography. 1 kg of silica gel was suspended in chloroform, degassed under reduced pressure, and filled in a glass column. Chromatography was performed by adding broiler extract dissolved in a small amount of chloroform on top of silica gel filled in a glass column. Chromatographic elution conditions were eluted with a stepwise mixture of chloroform and methanol. Chloroform 100%, Chloroform 99: MeOH 1, Chloroform 95: MeOH 5, Chloroform 90: MeOH 10, Chloroform 80: MeOH 20, Chloroform 50: MeOH 50, MeOH 100%. Silica gel column chromatography was carried out to confirm the inhibitory activity with the eluted fractions and the inhibitory activity was confirmed under solvent conditions containing 50-100% MeOH. The active fraction separated through silica gel column chromatography was concentrated under reduced pressure at 56 ° C., dissolved in a small amount of distilled water, and separated through Alp-18 column chromatography. 500 g of Alp-18 resin was suspended in distilled water, and the gas was removed under reduced pressure and filled in a glass column. Chromatography was performed by adding the concentrated silica gel column chromatography active fraction to the top of Alpi-18 packed in a glass column. Chromatographic elution conditions were eluted with a mixed solution of distilled water and methanol. Distilled water 100%, distilled water 90: MeOH 10, distilled water 80: MeOH 20, distilled water 70: MeOH 30, distilled water 60: MeOH 40, distilled water 50: MeOH 50, MeOH eluted under the solvent conditions. As a result of confirming the inhibitory activity of the eluted fraction by performing Alp-18 column chromatography, the inhibitory activity was confirmed under solvent conditions containing 0-10% MeOH. Thus, the broiler extract inhibitory active material was prepared through silica gel column chromatography and Alp-18 column chromatography.

<Example 2> Confirmation of inhibition of enzyme activity of broiler extract

The same method as in Step 2 of Example 1 to determine whether the broiler extract isolated and purified from Example 1 can actually inhibit the activity of sucrase and maltase and glucoamylase of the chorion cell membrane transporter. The enzymatic activities of sucrase, maltase and glucoamylase were measured, respectively. In the enzyme reaction, broiler extract was added at a concentration of 1 mg / ml to measure the degree of activity deactivation of the enzyme. In order to compare the inhibitory activity of broiler extracts against sucrase, maltase and glucoamylase, the control group without enzymes, no inhibitors and broiler extracts were used.

As a result, as shown in Figures 6, 7 and 8 , the sucrose and maltase by the broiler extract prepared in accordance with the present invention confirmed that about 60% glucoamylase enzyme activity of about 40% is inhibited It was.

<Example 3> Inhibition of carbohydrate absorption in vivo

Whether or not the broiler extract prepared according to the present invention exhibits absorption inhibitory activity of carbohydrates in vivo was investigated using SD rats. 10-week-old SD rats (Korean experimental animals) weighing 400 g were divided into 4 groups of 10 rats in each group and fasted for 12 hours, and then the control group was administered only saline, and the experimental group weighed broiler extract prepared according to the present invention. Administration at 1 mg or 5 mg per kg. When the broiler extract was dispersed in phosphate buffer containing physiological salts, the injection solution was orally administered using a horn. After 10 minutes after the broiler extract administration, 200 mg / kg sucrose and maltose were orally administered, respectively, and blood was collected by time to quantify blood glucose levels. Blood glucose levels were quantified using GEL-zyme kit (Shinyang Chemical), a GOD-POD colorimetric method using glucose oxidase.

As a result, in the case of sucrose, the blood glucose level increased with time in the control group that was not administered broiler extract, and 12 mg / dl at 30 minutes and 30 mg / dl at 4 hours. In the experimental group administered 1 mg or 5 mg of broiler extract per kg of body weight, blood glucose levels increased by 4.8 mg / dL and 2.9 mg / dL at 30 minutes, respectively. At 4 hours, blood glucose increase of 19 mg / dL and 14 mg / dL was shown, showing 37% and 53% inhibitory effect compared to the control group. ( FIG. 9 ).

In the case of maltose, the blood glucose level in the control group increased to 10.5 mg / dL at 30 minutes and 24 mg / dL at 4 hours, whereas the blood glucose level was increased in the experimental group in which the broiler extract was administered at 1 mg or 5 mg / kg body weight, respectively. At 30 minutes, 9.5 mg / dL and 7.5 mg / dL increased, respectively, and showed an inhibitory effect on blood glucose increase of 10% and 29% compared to the control group. At 4 hours, the blood glucose increase was 19 mg / dL and 12 mg / dL, showing an inhibitory effect of 20% and 50% compared to the control group ( FIG. 10 ).

It was confirmed that the broiler extract prepared according to the present invention effectively inhibits the absorption of glucose in vivo in a concentration-dependent manner.

<Preparation Example 1> Preparation of a pharmaceutical formulation

Capsules were prepared by mixing the following ingredients to fill hard gelatine capsules:

<Preparation Example 2> Preparation of functional food containing broiler extract

Functional foods containing the broiler extract of the present invention were prepared as follows.

(1) Manufacture of dairy products

Broiler extract was added to the milk in an amount of 5% by weight and milk was used to prepare various dairy products such as butter and ice cream.

However, broiler extract was added to the coagulated milk protein in cheese production and to the coagulated milk protein obtained after fermentation in yogurt production.

(2) Preparation of Mixed Drinks

1 g of broiler extract was added to 1,000 ml of fruit juice such as tomato, carrot, apple, or grape juice to prepare a mixed beverage for health promotion.

(3) Manufacture of dietary supplements

The broiler extract was added at a concentration of 5% by weight to prepare dietary supplements and dietary supplements for diabetic patients, including Cheongung and Sukji.

As described above, the broiler extracts for sucrase, maltase and glucoamylase according to the present invention can be produced safely in large quantities using alcohol, which is very economical and does not inhibit absorption of other essential inorganic ions and proteins. By effectively inhibiting the absorption of carbohydrate can be usefully used as a composition for improving and treating diabetes diseases and obesity through blood sugar control.

Claims (10)

1) producing a broiler extract having inhibitory activity against sucrase and maltase and glucoamylase; And 2) a method for producing an inhibitor against sucrase and maltase and glucoamylase comprising the step of separating the inhibitor from the broiler extract. The method of claim 1, wherein the sucrase and maltase and glucoamylase proteins are isolated from porcine small intestine, rat, mouse, guinea pig, rabbit or human and human derived cell lines. The method of claim 1, wherein the sucrase and maltase and glucoamylase proteins are expressed or chemically synthesized by the recombinant microorganism. The method of claim 1, wherein the inhibitory active substance is extracted from the broiler using an organic solvent directly. The method according to claim 1, wherein the broiler-derived inhibitory active substance is chemically synthesized. A pharmaceutical composition for inhibiting carbohydrate absorption, comprising a broiler extract produced by the method of any one of claims 1 to 4 as an active ingredient. 6. A pharmaceutical composition according to claim 5 for use in the prevention and treatment of obesity or diabetes related diseases. Food and beverage composition comprising the broiler extract produced by the method of any one of claims 1 to 4. 9. A food and beverage composition according to claim 8, which is used for glycemic control for diabetic patients. The food and beverage composition according to claim 8, which is used for preventing or improving obesity.