KR20040110962A - Inhibition activity of glycine max extract against sucrase, maltase and glucoamylase and compositions containing glycine max extract for regulation of blood sugar - Google Patents

Abstract

PURPOSE: Provided are the glycine max extract having inhibition activity against sucrase, maltase and glucoamylase as carbohydrases derived from porcine small intestine, an isolation and production method of an inhibitor against sucrase, maltase and glucoamylase and a composition containing the same for regulation of blood sugar level. The glycine max extract directly inhibits the decomposition of carbohydrate in the small intestine and is thus useful for the prevention and treatment of diabetes by regulating blood sugar level. CONSTITUTION: The isolation and purification method of an inhibitor against sucrase, maltase and glucoamylase comprises the steps of: preparing the glycine max extract having inhibition activity against sucrase, maltase and glucoamylase by using an organic solvent; and isolating and purifying an inhibitor from the extract. The pharmaceutical composition containing the glycine max extract as an active ingredient is used for inhibition of carbohydrate absorption, as well as prevention and treatment of diseases associated with obesity or diabetes.

Description

Inhibitory activity of black soybean extract against sucrase, maltase and glucoamylase and composition for glycemic control using the same

The present invention relates to a method for producing black soybean extract having inhibitory activity against sucrase, 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 the small intestine of pig Isolating amylase; Searching for natural products having inhibitory activity against the sucrase and maltase and glucoamylase; And separating the active substance from the hydrothermal extract of black soybeans with inhibitory activity, the method of producing black soybean extracts with inhibitory activity against sucrose and maltase and glucoamylase and black soybean produced by the above 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 the proteolytic enzymes such as trypsinogen, chymotrypsinogen, and carboxypeptidase, lipolytic enzymes such as pancreatic lipase, and stepin, 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.

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 extract of black soybean, which separates the enzymes sucrase and maltase and glucoamylase, which play an important role 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 diabetes 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 extracts of black soybeans with inhibitory activity against carbohydrate degrading enzymes and producing them.

Another object of the present invention to provide a blood sugar control composition comprising the black soybean 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 with temperature and pH of the glucoamylase isolated in Figure 1 ,

5 shows the separation process of black soybean extract,

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

Figure 7 shows that the black soybean extract prepared according to the present invention directly inhibits the activity of pig small intestine-derived sucrase,

8 shows that the black soybean extract prepared according to the present invention directly inhibits glucoamylase activity from pig small intestine,

9 is a result of measuring the inhibition of the increase in the concentration of sugar in the body by the black soybean extract of the present invention during the sucrose overload experiment using a rat animal model,

Figure 10 is the result of measuring the inhibition of the increase in the body sugar concentration by the black soybean extract of the present invention during the maltose overload experiment using the rat animal model.

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

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

In addition, the present invention provides a food and beverage composition containing the black bean 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 black soybeans that inhibits maltase and glucoamylase.

The separation method is

1) producing hydrothermal extracts of black soybeans using the sucrase and maltase and glucoamylase proteins as target enzymes to inhibit them;

2) mixing the hot water extract of the black soybean with the chloroform solution and stirring to convert the inhibitory active material into the chloroform solution; And

3) separating the chloroform solution containing the inhibitor using silica gel 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 ).

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 black soybean extract was searched for a natural product by using a 3,5-dinirosalicylic acid as an inhibitor that inhibits the isolated sucrase, maltase and glucoamylase. It was found that there is an activity that inhibits the activity of the enzyme.

Step 2) after 2 hours cold soaked black soybeans were extracted for 2-8 hours using hot water of 95-110 ℃ to extract the inhibitory substances and confirmed the activity. In addition, the hot water extract of black soybean was stirred with the chloroform solution to confirm that the inhibitory substance moved to the chloroform layer, and the inhibitory activity was confirmed in the chloroform layer. As a step of separating the inhibitor transferred to the chloroform layer, silica gel column chromatography was used, and the active fraction was identified.

In order to use black soybean extract as a functional material for blood sugar control, it is economical to use black soybean hot water extract as it is, but chloroform extract and silica gel column chromatography were used to confirm the properties of inhibitors.

The black bean extract isolated and purified as described above 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 ).

In another aspect, the present invention provides a pharmaceutical composition for inhibiting carbohydrate absorption containing black soybean extract produced by the above method as an active ingredient.

The black soybean extract for sucrase and maltase and glucoamylase produced in accordance with the present invention directly inhibits the breakdown of carbohydrates in vivo, thereby reducing blood sugar levels. Without limitation, 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.

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. A typical daily dosage of purified black soybean extract for humans 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 black bean 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.

Black soybean extract of the present invention is also effective for diseases caused by the absorption of excessive carbohydrates for the purpose of showing the prophylactic and therapeutic effect of diabetes, or as a dietary ingredient for improving obesity, for example, additives in food or beverage Or as a food supplement. In this case, the content of the black bean 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 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 x g 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 weights of 260,000 depending on the intestinal site (Siostrom, H. et el ., 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

Using a sucrase, maltase and glucoamylase isolated and purified from porcine intestine, as a target enzyme, natural extracts having the ability to inhibit the activity of the enzyme were searched. Natural products used natural herbal medicines listed in the Food Code to be used as food, and added black beans and legumes. 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 hot water at 95-110 ° C. for 2-4 hours. After hot water 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). Preparation of DNS color development reagent dissolve 1 g of DNS reagent in 20 ml of 2 M sodium hydroxide solution, slowly add 30 g of sodium potassium tartrate tetrahydrate, and then distilled water to obtain a final volume of 100. Adjust to ml. Sucrose, maltose and water-soluble starch were used as the substrate of the enzyme by dissolving to a concentration of 1% in 0.02 M 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), distilled water, and the reaction volume was reacted at 37 ° C. for 20 minutes at 2 ml. 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 reacted at 37 ° C. for 20 minutes at 2 ml. After the reaction was completed, the reaction was terminated at 100 ° C. For the color reaction, 1 ml of the above prepared DNS reagent was added, followed by cooling at 100 ° C. for 5 minutes, and then cooled to 10 ml of distilled water, and absorbance was measured at 540 nm. Absorbance measurements were measured for absorbance of each well with an ELISA reader in a 96 well microplate.

(Step 4) Isolation of Black Bean Extract

Inhibitors were isolated from the extracts of black soybeans, which were found to have high inhibitory activity, from the natural extracts from which the inhibitory activity against sucrase, maltase and glucoamylase was confirmed. Separation of inhibitors was carried out by adding 1 liter of black soybeans to 10 liters of distilled water for 2 hours, followed by 2-8 hours of hydrothermal extraction at a temperature of 95-110 ° C. The mixture was stirred for 8 hours using a magnetic stirrer with a 1/4 portion of chloroform solution to move the inhibitory substance to the chloroform layer. It was confirmed that the inhibitory activity moved to the chloroform layer. As a step of separating the inhibitor moved to the chloroform layer, the chloroform solution containing the inhibitor component was separated from the aqueous layer using separation filtration, and the separated chloroform solution was concentrated under reduced pressure at 56 ° C, and then dissolved in a small amount of chloroform solution. . Silica gel column chromatography was used to separate the components contained in the chloroform solution. 1 kg of silica gel was suspended in chloroform, degassed under reduced pressure, and filled in a glass column. Chromatography was performed by adding the inhibitory ingredients of the black soybean extract dissolved in a small amount of chloroform on top of the 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% eluted under solvent conditions. As a result of performing silica gel column chromatography, the inhibitory activity was confirmed by the eluted fraction, and the inhibitory activity was confirmed under solvent conditions containing 1-50% MeOH. Thus, black bean extract inhibitory active material was prepared through silica gel column chromatography (FIG. 5).

<Example 2> Confirmation of inhibition of enzyme activity of black soybean extract

In order to confirm whether the black bean extract isolated and purified from Example 1 can actually inhibit the activity of sucrase, maltase and glucoamylase of the chorion cell membrane transporter, the same procedure as in Step 2 of Example 1 By the method, the enzyme activities of sucralase, maltase and glucoamylase were measured, respectively. During the enzymatic reaction, black soybean extract was added at a concentration of 1 mg / ml to determine the degree of activity inhibition. In order to compare the inhibitory activity of black soybean extract against sucralase, maltase and glucoamylase, a control group without enzyme, no addition of inhibitor and black soybean extract addition group were used.

As a result, as shown in Figures 6, 7 and 8 , the sucrose and maltase by the black soybean extract prepared in accordance with the present invention inhibits about 57% glucoamylase enzyme activity of about 38% Confirmed.

<Example 3> Inhibition of carbohydrate absorption in vivo

Whether or not the black soybean extract prepared according to the present invention exhibits the 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 control group was administered only saline, and the experimental group was treated with black soybean extract prepared according to the present invention. 1 mg / kg body weight was administered at 5 mg. At the time of administration, the injection solution in which black bean extract was dispersed in phosphate buffer containing physiological salts was orally administered using a horn. After 10 minutes after the administration of the black soybean extract, 200 mg / kg of 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 not administered the black soybean extract, and increased 14 mg / dL at 30 minutes and 27.5 mg / dL at 4 hours. In the experimental group administered 1 mg or 5 mg of black soybean extract per 1 kg of body weight, blood glucose levels increased by 8 mg / dL and 5 mg / dL at 30 minutes, respectively. At 4 hours, blood glucose increases of 20.4 mg / dL and 12.6 mg / dL were shown, indicating an inhibitory effect of 26% and 54% compared to the control group ( FIG. 9 ).

In the case of maltose, in the control group, the blood glucose level increased to 12 mg / dL at 30 minutes and 24.9 mg / dL at 4 hours, whereas in the experimental group in which the black soybean extract was administered at 1 mg or 5 mg concentration per kg body weight, respectively The blood glucose levels were increased by 10.3 mg / dL and 8.4 mg / dL at 30 minutes, respectively, showing a 14% and 30% inhibition of blood glucose increase compared to the control group. At 4 hours, blood glucose increase was 20 mg / dL and 11.7 mg / dL, showing 19% and 53% inhibitory effect compared to the control group ( FIG. 10 ).

From this, it was confirmed that the black soybean extract prepared according to the present invention effectively inhibits the absorption of glucose in vivo in a concentration-dependent manner.

As described above, black soybean extracts for sucrase, maltase and glucoamylase according to the present invention can be produced safely and in large quantities using hot water, which is very economical and impairs absorption of other essential inorganic ions and proteins. By effectively inhibiting only the absorption of carbohydrates can be usefully used as a composition for improving and treating diabetes diseases and obesity through blood sugar control.

Claims (7)

1) producing a black soybean 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 black soybean 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, characterized in that they are expressed or chemically synthesized by recombinant microorganisms. How to. The method of claim 1, wherein the inhibitory substance is extracted from the black soybeans using an organic solvent directly. The method according to claim 1, wherein the black soybean-derived inhibitory compound is chemically synthesized. A pharmaceutical composition for inhibiting carbohydrate absorption, comprising the black soybean extract produced by the method of any one of claims 1 to 3, as an active ingredient, used for the prevention and treatment of obesity or diabetes-related diseases Composition. Food and beverage composition comprising the black soybean extract produced by the method of any one of claims 1 to 3 as an active ingredient. 7. A food and beverage composition according to claim 6, which is used for blood sugar control, obesity prevention or improvement for diabetic patients.