KR100506710B1 - Chitosan oligosaccharide ascorbic acid salt having anti-diabetic effect - Google Patents

Abstract

The present invention relates to chitosan oligosaccharide ascorbate which has an antidiabetic effect.

Chitosan oligosaccharide ascorbate of the present invention, the purified chitosan is added to distilled water in a ratio of 1 to 20% by weight to make a chitosan suspension, and ascorbic acid in the chitosan suspension 60 to 100% by weight of the chitosan After stirring for 2 to 3 hours to dissolve, the pH was adjusted to 4 to 6, and the chitosanase enzyme was decomposed for 12 to 13 hours at 30 to 50 ℃ by adding 1 to 5 units per 1 g of chitosan. After heat treatment for 10 to 20 minutes at 80 ~ 90 ℃, filtered, and then dried to prepare a chitosan oligosaccharide ascorbate.

The present invention provides chitosan oligosaccharide ascorbate which lowers blood sugar by increasing glucose tolerance and insulin secretion.

In addition, it reduces blood triglyceride (triglyceride) and cholesterol, and protects blood vessel damage, thus preventing the complications of diabetes and distribution. Chitosan oligosaccharide ascorbate is provided to increase stability during storage.

Description

CHITOSAN OLIGOSACCHARIDE ASCORBIC ACID SALT HAVING ANTI-DIABETIC EFFECT}

The present invention relates to chitosan oligosaccharide ascorbate which has an antidiabetic effect.

Chitosan was added to the chitin, a polymer polysaccharide in which the N-acetyl-D-glucosamine monomer was polymerized by β- (1,4), to give an alkali, etc., resulting in a proportion of D-glucosamine produced by the acetyl group falling from the N-acetyl-D-glucosamine. When it is 70% or more, it is called chitosan (Korea Food and Drug Administration, Food Additives Process) (see Fig. 1).

Chitosan is easily dissolved at room temperature in dilute hydrochloric acid, acetic acid, and lactic acid, which are monovalent ions, and sulfuric acid, phosphoric acid, malic acid, succinic acid, citric acid, and fumaric acid, which have divalent or higher ions, are easily dissolved when heat is applied. It is applied to various industrially.

Since chitosan is a cationic polyelectrolyte, research on its functionality has led to the development of applications in medicine, food, cosmetics, agriculture, and the development of low molecular weight chitosan oligosaccharides. Its application range, such as diabetes treatment, is becoming more and more fragmented.

Diabetes is a non-infectious chronic disease that occurs a lot in modern people. The lack of insulin, a hormone secreted by the body's pancreas, causes the body's metabolism to fail, resulting in increased blood sugar in the blood and sugar in the urine. It is caused by a combination of environmental and genetic factors such as obesity, stress, pregnancy, infection, drug abuse, and endocrine disorders.

Diabetes is largely divided into two types, one is insulin-dependent diabetes, type 1 diabetes, called pediatric diabetes mellitus, occurs frequently before childhood or 30 years of age, and the type of diabetes occurs because insulin is not secreted.

The other, insulin-independent diabetes, is called type 2 diabetes and adult diabetes, usually occurring after age 40, and 80-90% of all diabetics belong to it.

Diabetes suffers more because of its complications, including hypoglycemia, ketoacidosis, diabetic coma, diabetic asisosis, retinopathy, nephropathy, neurosis, myocardial infarction, arteriosclerosis, infections, and cerebrovascular disease.

Among these complications, atherosclerosis is a disease that can be improved only by controlling blood glucose levels.

However, most of the diabetic patients have an increase in blood glucose and fatty acid concentrations. Such hyperglycemic and hyperlipidemic conditions result in damage of vascular endothelial cells due to the increase of free radicals in blood vessels.

Therefore, chitosan, which has been reported to lower cholesterol and antioxidant activity, is likely to be a functional food for improving diabetes.

Regarding the liver function impairment effect of chitosan oligosaccharide, Korean Patent Laid-Open Publication No. 1999-026532 (an agent for preventing and improving liver function disorder caused by chitosan oligosaccharide), which has been filed by the inventor of the present application, is separated and purified from chitin into chitin and chitosan. From chitin and chitosan, chitosan oligosaccharides having a degree of polymerization of 2 or more D-glucosamine called N-acetyl-D-glucosamine were prepared as enzymatic or chemical decomposition products of chitosan and chitosan. It is disclosed that the powder, tablets, and liquid liver function disorder prevention and improvement agent containing the obtained specific chitosan oligosaccharide are improved.

Korean Laid-Open Patent Publication No. 2001-0044323 (polymeric water-soluble chitosan) discloses a glycemic-controlled diabetic improver that is effective in controlling blood sugar and preventing complications by administering a polymer-soluble chitosan having an average molecular weight of 10,000 to 1.5 million units for a period of time. .

US Pat. No. 5,981,510 (Method for treating and improving diabetes) discloses the use of therapeutically effective amounts of chitin oligosaccharides, chitosan oligosaccharides and pharmaceutically acceptable salts for the treatment of diabetes.

However, it uses salts prepared with acetic acid or hydrochloric acid, and the hypoglycemic effect did not lower blood sugar by increasing glucose tolerance and insulin secretion, but lowering blood sugar by dietary reduction, and ascorbic acid. The reduction of ascorbic acid could not cure complications such as atherosclerosis.

In addition, the conventional chitosan oligosaccharides are easily carbonized during storage to reduce the content, there was a problem in the stability of the quality that can occur during distribution.

In order to solve the above problems, an object of the present invention is to provide chitosan oligosaccharide ascorbate which lowers blood sugar by adding glucose ascorbic acid to the chitosan and reacting to increase glucose tolerance and insulin secretion.

In addition, it reduces blood triglyceride (triglyceride) and cholesterol, and protects blood vessel damage, thereby providing chitosan oligosaccharide ascorbate which can prevent the complications of diabetes.

Also, distributors. The purpose of the present invention is to provide chitosan oligosaccharides ascorbate which can improve stability during storage.

The present invention relates to chitosan oligosaccharide ascorbate which has an antidiabetic effect.

Chitosan oligosaccharide ascorbate of the present invention, the purified chitosan is added to distilled water in a ratio of 1 to 20% by weight to make a chitosan suspension, and ascorbic acid in the chitosan suspension 60 to 100% by weight of the chitosan After stirring for 2 to 3 hours to dissolve, the pH was adjusted to 4 to 6, and the chitosanase enzyme was decomposed for 12 to 13 hours at 30 to 50 ℃ by adding 1 to 5 units per 1 g of chitosan. After heat treatment for 10 to 20 minutes at 80 ~ 90 ℃, filtered and dried to produce chitosan oligosaccharide ascorbate.

The polymerization degree of chitosan oligosaccharide of this invention is 2-10.

Chitosan oligosaccharides ascorbate of the present invention, unlike chitosan oligosaccharides, which reduced blood sugar levels due to conventional dietary reduction, increases glucose tolerance and insulin secretion, lowers blood sugar, lowers cholesterol and triglycerides, and reduces vascular damage. This protects against the complications of diabetes.

In addition, when prepared by adding the existing hydrochloric acid, acetic acid, lactic acid to chitosan oligosaccharides, the carbonization occurred during distribution and lack stability, the chitosan oligosaccharides ascorbin prepared by adding the ascorbic acid of the present invention Acid salts have proven to be stable over time, rapid temperature changes, and heat.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples of the present invention, but this does not limit the content of the present invention.

Example 1 Preparation of Chitosan Oligosaccharide Ascorbate

Chitosan with 99% deacetylation was prepared.

55 g of the prepared chitosan was added to 500 ml of distilled water, and stirred for 10 minutes to prepare a 11% chitosan suspension.

35 g of ascorbic acid was added to the chitosan suspension and stirred for 2 hours to dissolve, and then the pH was adjusted to 5.0 with ascorbic acid.

Chitosanase enzyme (Sigma) 55 units were added and digested at 38 ° C. for 12 hours.

After the heat treatment for 10 minutes at 80 ℃ to inactivate the enzyme, and filtered to prepare a chitosan oligosaccharide composition.

The composition was lyophilized at -40 ° C to give 84 g of chitosan oligosaccharide ascorbate powder.

As a result of analyzing the obtained chitosan oligosaccharide ascorbate, the polymerization degree of chitosan oligosaccharide was 2-10, the oligoglucosamine content was 55%, and the glucosamine which was a monosaccharide was not detected.

Example 1 Chitosan oligosaccharide ascorbic acid salt of the present invention Chitioligo-C ^TM termed prepared, which was to use this effect experiments.

Experimental Example 1 Comparison of Normal and Insulin-Independent Diabetic Mice Before Chitioligo-C ^TM Treatment

Sprague-Dawley rats were prepared.

STZ (Streptozotocin) dissolved in 0.1 M citrate buffer (pH 5) was intraperitoneally injected at 3 days old SD Rats to induce insulin-independent diabetes (NIDDM).

In the case of the control group, only citrate buffer was administered in the same manner.

Body weight, plasma glucose, cholesterol, triglyceride, and glucose tolerance were measured in normal and insulin-independent diabetic rats.

Intraperitoneal glucose tolerance (IPGT) test was performed after growth for 4 and 8 weeks.

For the glucose tolerance test, glucose (2 g / kg) was intraperitoneally administered to rats fasted for 18 hours.

Blood samples were taken from the tail vein at 0, 1 and 2 hours after administration to separate plasma and stored in a deep freezer at -70 ℃ to measure the amount of glucose and insulin remaining in the blood.

Glucose concentration was measured by glucose analyzer 2 (Becman, USA), and plasma insulin was measured by rat insulin ELISA kits (Crystal Chem Inc, USA).

The cholesterol-E kit and TG kit (Yeongdong Pharmaceutical Corp, Korea) were used to measure the amount of cholesterol and triglyceride in the blood.

<Table 1> Comparative Experiment Results for Normal and Insulin-Independent Diabetic Rats

division Normal rat (n = 20) Insulin-Independent Diabetic Rats (n = 20) Body weight (g) 138.7 ± 15.1 139.0 ± 17.2 Plasma glucose (mg / dl)  87.5 ± 10.1  114.0 ± 18.6 * Cholesterol (mg / dl) 120.1 ± 20.1 142.5 ± 24.6 Triglyceride (mg / dl) 116.5 ± 20.3 104.5 ± 13.7 Glucose tolerance (mg / dl) 274.5 ± 41.3   583.0 ± 101.4 *

* Significance was given at confidence interval P <0.05.

As shown in Table 1, the weights of the normal and diabetic rats were not significantly different, but the plasma glucose levels of the diabetic rats were 87.5 ± 10.1 mg / dl and 114.0 ± 18.6 mg / dl, respectively. Diabetic rats had higher cholesterol levels (120.1 ± 20.1 mg / dl in diabetic rats, 142.5 ± 24.6 mg / dl in diabetic rats), 274.5 ± 41.3 mg / dl in diabetic rats, and 583.0 ± 101.4 mg / dl in diabetic rats. That figure was twice as high.

Experimental Example 2 Effect of Chitioligo-C ^TM on Normal and Insulin-Independent Diabetic Rats

Mice used in the experiment the normal rats (control group), one group (control group treated with Chitioligo-C ^TM), diabetic rats (NIDDM control group) administering an Chitioligo-C ^TM in normal mice, the Chitioligo-C ^TM in diabetic rats NIDDM treated with Chitioligo-C ^™ .

Chitioligo-C ^TM The experiment was conducted with 8 weeks of administration.

In the Chitioligo-C ^TM group, 0.3% Chitioligo-C ^TM was ingested freely, and the control group (non-administered group) was free intake of 0.1% vitamin C.

The results of experiments on the effects of chitosan oligosaccharides and ascorbate in normal and diabetic rats on fasted rats and fed rats are shown in Table 2 below.

<Table 2> Effect of Chitioligo-C ^TM on Normal and Insulin-Independent Diabetic Rats

division Normal rat Insulin-Independent Diabetic Rats Control Treatment group Control Treatment group In fasting rats plasma glucose (mg / dl) 90 ± 10 102 ± 14 111 ± 15 90 ± 16 Triglyceride (mg / dl) 81.3 ± 19.9 69.5 ± 30.4 78.9 ± 26.9 65.9 ± 22.4 Cholesterol (mg / dl) 145.0 ± 21.7 133.2 ± 34.8 150.0 ± 26.2 122.0 ± 33.2 In fed rats plasma glucose (mg / dl) 120 ± 11.5 125 ± 10 135 ± 18 133 ± 14 Triglyceride (mg / dl) 198.6 ± 134.9 124.0 ± 21.4 222.2 ± 80.5 114.0 ± 32.6 Cholesterol (mg / dl) 140.1 ± 12.4 124.0 ± 10.7 126.8 ± 18.4 111.9 ± 13.4

(1) with or without Chitioligo-C ^TM treatment Change in plasma glucose concentration

Fasting plasma glucose levels in normal rats were not significantly different between the control group and the control group for 4 weeks after the start of the experiment. However, blood glucose levels were decreased in the diabetic rats treated with 0.3% Chitioligo-C ^TM for 4 weeks (Table 2). , Figure 2)

There was no change in the normal rats, the administration of the postprandial blood glucose trend is 0.3% Chitioligo-C ^TM, blood glucose was reduced when the diabetic rats to 0.3% Chitioligo-C ^TM 8 weeks (Table 2, Fig. 3)

(2) with or without Chitioligo-C ^TM treatment change in glucose tolerance

To investigate the change in glucose tolerance in diabetic rats treated with chitoligo-C ^TM , the blood glucose AUC (area under curve (AUC0-2)) of diabetic rats was measured for 2 hours after intraperitoneal administration with glucose 2g / kg concentration. One result showed a significant decrease in blood glucose.

This phenomenon was more significant when Chitoligo-C ^TM was repeatedly administered for 4 weeks, which indicates that repeated intake of Chitoligo-C ^{TM has} an effect of increasing glucose tolerance (FIG. 4).

When 0.1% vitamin C (ascorbic acid) was administered as a control, there was no change in glucose tolerance, indicating that vitamin C had no direct effect.

In the case of normal mice, no change was found through this experiment.

(3) According to Chitioligo-C ^TM or without treatment Changes in insulin levels

In order to investigate the blood insulin concentration of Chitoligo-C ^TM in diabetic rats, the blood insulin AUC (area under curve (AUC0-2)) of diabetic rats showed a significant increase.

In particular, the blood insulin concentrations of the chitoligo-C ^TM treated group (NIDDM treated group) and normal mice were found to be similar (FIG. 5).

These results suggest that the postprandial glucose reduction mechanism of Chitoligo-C ^TM may be due to increased insulin secretion.

(4) with or without Chitioligo-C ^TM treatment changes in triglyceride levels

When 0.3% Chitoligo-C ^TM was administered, the concentration of triglyceride in blood was significantly decreased in both normal and diabetic rats (FIG. 6).

The blood triglyceride concentration increased from 100 mg / dl to about 200 mg / dl for 8 weeks in the control group (Normal and diabetic rats not administered Chitoligo-C ^TM ) but for 8 weeks in the 0.3% Chitoligo-C ^TM group. No increase was seen.

These results suggest that Chitoligo-C ^TM can be used as a functional food with lipid-lowering effect and glycemic control ability that can effectively inhibit various complications caused by increased blood lipid concentration in most diabetic patients.

<Experimental Example 3> damage protection experiments on cardiac muscle cells according to Chitioligo-C ^TM or without treatment

STZ (Streptozotocin) dissolved in 0.1 M citrate buffer (pH 5) was intraperitoneally injected at 3 days old SD Rats to induce insulin-independent diabetes (NIDDM).

Chitoligo-C ^™ prepared in Example 1 of the present invention was prepared at 0.3%.

A 0.3% Chitoligo-C ^TM to prepare diabetic rats After 8 weeks of treatment, the cardiac tissues were observed under an electron microscope and compared with the untreated group.

Diabetic rats treated with Chitoligo-C ^{™ for} 8 weeks were able to prevent the myocardial structure from changing, whereas untreated diabetic rats showed changes in myocardial tissues in typical diabetes (FIG. 7).

This shows that the heart muscle cells are destroyed by myocardial infarction due to blood vessel damage caused by diabetes, and the experimental group treated with Chitoligo-C ^™ of the present invention prevents blood vessel damage, thereby not destroying the heart muscle cells. It can be said that it did not show.

<Experiment 4> stability testing for Chitoligo-C ^TM

Chitosan oligosaccharide ascorbate of Example 1 of the present invention was prepared.

Conventional chitosan oligosaccharide acetate and chitosan oligosaccharide hydrochloride were prepared.

(1) Experimental Changes of Salt Content of Chitosan Oligosaccharides with Time

After storage for 2 weeks the content of chitosan oligosaccharides was measured.

<Table 3> Experimental Results of Chitosan Oligosaccharide Content Changes over Time

division Chitosan oligosaccharide acetate Chitosan oligosaccharide hydrochloride Chitosan oligosaccharide ascorbate 0 days 68.2% 57.5% 57.5% 7 days 6.9% 14.1% 56.1% 14 days 3.7% 12% 55.6%

As shown in Table 3, the acetate or hydrochloride was found to be easily carbonized during storage, so that the content of chitosan oligosaccharide was easily lowered.

(2) Experimental Changes of Salts of Chitosan Oligosaccharides Under Rapid Temperature Changes

After storage for 7 days under the condition of rapid temperature change (distribution process) from 20 ° C. to 50 ° C., each sample was made into a 5% solution and filtered, and the filter paper was observed by drying at room temperature.

As shown in FIG. 8, chitosan oligosaccharides treated with hydrochloride, lactate, and acetate were severely carbonized, and thus, many impurities were generated.

(3) Thermal Stability Test of Salts Treated with Chitosan Oligosaccharides

Chitosan oligosaccharide ascorbate was most stabilized when preservation experiment of each chitosan oligosaccharide salt product at 50 ℃, and the content of hydrochloride, acetate and lactate rapidly decreased from the beginning of preservation, so that chitosan oligosaccharide was almost after 6 weeks. It was not detected.

However, in the case of desalted high purity chitosan oligosaccharides, no change in content was observed during the storage period, and only slight color changes were observed (FIG. 9).

According to the present invention, there is provided a chitosan oligosaccharide ascorbate having an antidiabetic effect of lowering blood sugar by increasing glucose tolerance and insulin secretion.

In addition, it reduces blood triglycerides (triglycerides) and cholesterol, and protects blood vessel damage, thus preventing the complications of diabetes and circulation. Chitosan oligosaccharide ascorbate is provided to improve quality stability during storage.

BRIEF DESCRIPTION OF THE DRAWINGS The structure of D-glucosamine and N-acetyl-D-glucosamine, and the structure of chitosan using these as a polymer unit.

Figure 2 is according to the treatment with or without Chitioligo-C ^TM chitosan oligosaccharide ascorbate of the present invention On an empty stomach Graph showing the change in blood glucose concentration.

Figure 3 according to the presence or absence of the treatment of Chitioligo-C ^TM After a meal Graph showing the change in blood glucose concentration.

4 is according to the treatment of Chitioligo-C ^TM Graph showing changes in glucose tolerance.

5 is treated with or without Chitioligo-C ^TM Graph showing changes in insulin levels.

6 shows whether Chitioligo-C ^TM is treated or not. Graph showing changes in triglyceride levels.

Figure 7 is an electron micrograph showing the damage protection effect on cardiomyocytes with and without Chitioligo-C ^TM treatment.

A: Electron micrograph of heart tissue of Chitioligo-C ^TM treated diabetic rat.

B: Electron micrograph of cardiac tissue of untreated diabetic rats treated with Chitioligo-C ^TM

Figure 8 is a photograph of the change of salt of chitosan oligosaccharides with a rapid temperature change.

    A: chitosan oligosaccharide ascorbate B: chitosan oligosaccharide hydrochloride

    C: chitosan oligosaccharide lactate D: chitosan oligosaccharide acetate

9 is a graph of the thermal stability test of salts treated with chitosan oligosaccharides.

    1: high purity chitosan oligosaccharide 2: chitosan oligosaccharide ascorbate

    3: chitosan oligosaccharide lactate 4: chitosan oligosaccharide acetate

    5: chitosan oligosaccharide hydrochloride

Claims (3)

The purified chitosan was added to distilled water at a ratio of 1 to 20% by weight, followed by stirring to form a chitosan suspension. After adjusting the pH to 4 to 6, the chitosanase enzyme was added to decompose for 12 to 13 hours at 30 to 50 ° C, heat treated at 80 to 90 ° C for 10 to 20 minutes, and then filtered and dried. Containing chitosan oligosaccharide ascorbate having a degree of polymerization of 2 to 10, A composition for preventing and treating diabetes that has an antidiabetic effect. delete The method of claim 1, Increases insulin secretion, reduces blood cholesterol, reduces blood triglycerides (triglycerides), and protects blood vessel damage, A composition for preventing and treating diabetes that has an antidiabetic effect.