KR20100104482A - Puple corn anthocyanins with excellent blood glucose control activity and efficient protective activity for diabetic loss of pancreatic beta cells - Google Patents

Abstract

PURPOSE: A composition containing purple corn anthocyanins for preventing and treating diabetes is provided to protect pancreatic beta cell activity and blood glucose control. CONSTITUTION: A purple corn anthocyanin has activities of protecting pancreatic beta cells and controlling blood glucose. The purple corn anthocyanins are isolated from Zea mays. An medicinal composition contains the purple corn anthocyanins. A health food composition contains the purple corn anthocyanins.

Description

Pucorn corn anthocyanins with excellent blood glucose control activity and efficient protective activity for diabetic loss of pancreatic beta cells}

The present invention relates to black corn anthocyanin which is excellent in preventing pancreatic beta cell loss due to diabetes and protecting pancreatic beta cells and glycemic control activity, and more specifically, pancreas in db / db mouse, a type 2 diabetes model animal. It relates to a black corn anthocyanin and such anthocyanin excellent in the activity of protecting beta cells and glycemic control, the composition can be applied to the manufacture of health functional foods or pharmaceuticals.

Insufficient insulin, produced by pancreatic beta cells to reduce the concentration of sugar in the blood, produces high blood sugar. Diabetes caused by this insulin deficiency is called type 1 diabetes and type 2 diabetes is caused by weak insulin action. It is called diabetes. The former is common in young people, the latter is more common after middle age, and the onset of genetic factors, such as obesity, overeating, stress, and the onset of the disease slowly. Most diabetes is type 2 diabetes and is one of the representative adult diseases. Diabetes medications currently in use are mostly hypoglycemic agents used for insulin injection and oral administration. The hypoglycemic agents used for oral administration are as follows: α- which prevents the rapid rise of blood sugar levels after meals by delaying the absorption of carbohydrates in the pioglitazone and rosiglitazone of the thiazolidinedione family that activates bigforme, metformin and PPARγ. Glucosidase inhibitors, and sulfonylureas and non-sulfonylureas that promote insulin secretion in the β-cells of the pancreas. Recently, it inhibits the selective inhibition of the "DPP-IV" enzyme, which directly breaks down the "incretin" enzyme, which promotes insulin secretion and lowers blood sugar, thereby keeping the "incretin" enzyme at high concentrations for a long time. DPP-IV inhibitors with important functions have been developed and marketed. To date, while metformin and sulfonylurea dominate the market for primary type 2 diabetes, Januvia and Byetta (exenatide) compete with PPAR-gamma agonists in the secondary market. Januvia is usually prescribed after tertiary therapy.

Indeed, none of the drugs developed to treat diabetes is a narcotic agent that treats pancreatic beta cells lost or worsened, but is only an insulin secretory or hypoglycemic agent that shows a temporary hypoglycemic effect upon drug treatment. To date, the development of a source therapy that protects beta cells and restores lost beta cells has not been reported, and the development of this source therapy is urgently needed. The technical problem to be achieved by the present invention is an anthocyanin of black corn and such anthocyanin having an activity to make a healthy and abundant beta cells by fundamentally solving the loss and malfunction of pancreatic beta cells due to various causes It is to provide a composition for preventing or treating diabetes.

The damage and loss of pancreatic beta cells is a fundamental problem in both type 1 and type 2 diabetes, but there is currently no underlying cure for it. The present invention provides a method for isolating anthocyanin from black corn as shown in the example, and using beta / db mouse, type 2 diabetes model animal to isolate beta cells from damage and loss of pancreatic beta cells By providing an excellent protective activity and glycemic control activity is to make an anthocyanin of black corn and a composition containing such anthocyanin to be used as a dietary supplement or medicine for the prevention or treatment of diabetes.

As described above, the present invention utilizes a composition containing black corn anthocyanin and anthocyanin, which is excellent in protecting pancreatic beta cells and glycemic control activity, and the material of a health functional food or drug for preventing or treating diabetes. Can develop and supply

All simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Hereinafter, a preferred embodiment of the present invention. However, the following examples are only presented to aid the understanding of the present invention, but the scope of the present invention is not limited to the examples. The embodiment of the method of extracting anthocyanin from black corn is not new and any other separation method is possible. The db / db mouse used as a type 2 diabetes model animal has been widely used for type 2 diabetes research, and its blood glucose is increased after about 10 weeks, and the beta cells are gradually lost. db / db mouse was used as an experimental animal to demonstrate pancreatic beta cell loss and glycemic control activity of black corn anthocyanin for 7 weeks.

Example 1 Preparation of Black Corn Anthocyanin Extract

1-1. Securing Black Corn

Black corn is cultivated mainly in China and Peru. Its name is Zea mays, and black corn is one of the varieties. In China, Peru, Canada, and domestic, the composition of anthocyanin is slightly different, and the activity of protecting pancreatic beta cell loss and the hypoglycemic activity may be slightly different.

1-2. Preparation of Black Corn Anthocyanin Extract

100 g of dried black corn is added to 200 ml of water, crushed in a mixer, and the mixed solution is heated at 80 ° C. for 5 minutes. 300 ml of ethanol (alcohol) are added with 5 ml of acetic acid and anthocyanin is extracted for 2.5 hours. The mixture is centrifuged at 4 ° C. for 30 minutes and the extracts are collected separately. To the residue was added 60% ethanol containing 600 ml of 1% acetic acid and extracted in the same way. The extracts were combined and combined with the primary extract. The collected supernatant is removed from the solvent in a rotary evaporator at 40 ° C. The concentrate is dissolved in water containing 0.1% acetic acid, placed in a nonionic polymeric absorbent column (Amberlite, Diaion, etc.) and washed with water containing 0.1% acetic acid. After washing again with water, anthocyanins are eluted with ethanol containing 0.1% acetic acid. The eluate is dried in a vacuum rotary evaporator and the dried product is dissolved in water containing 0.1% acetic acid.

Example 2 Diabetes Improvement Efficacy Evaluation Using Beta Cells

  2-1. Determination of viability of cells in vitro

HIT-T15, a beta cell line of hamster pancreas, was treated with RPMI1640 medium containing 10% FBS (fatal bovine serum), 2 mM L-glutamine, 100 IU of penicillin-G and 100 μg / ml of streptomycin. Incubate using a cell incubator at CO2, 37 ° C. Inoculate the cells into a cell culture dish or multiwell plates at a density of 1.05.0 × 105 cells / ml and, when reaching 80%, add viable material to the cells and test for viability. Change the medium every 2 days and add and assess different concentrations of anthocyanin to 1 × 10 5 HIT-T15 cells in 24-well plates to assess the effects of anthocyanins on the viability of HIT-T15. Cell viability was analyzed by MTT reduction assay. As shown in FIG. 1, black corn anthocyanin was not toxic to cells up to 100 μg / ml, but rather promoted cell growth.

  2-2. Insulin Secretion and Insulin Analysis of Cells

HIT-T15 cell line was treated with glucose-free KRB buffer (Krebs-Ringer Bicarbonate Buffer, 115 mM NaCl, 4.7 mM KCl, 2.56 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 1.2 mM MgSO ₄ , 20 mM for 30 minutes at 37 ° C). Incubate in NaHCO ₃ , pH 7.4), change to KRB buffer containing 11.1 mM glucose, and incubate for 60 minutes. Centrifuge the cell culture at 3000g for 5 minutes and store the supernatant at -80 ° C for insulin secretion assay. Insulin secreted from the cell culture solution is measured using the ELISA insulin kit as rat insulin. Protein was analyzed using albumin as a standard using BCA protein assay reagent kit (PIERCE). As shown in FIG. 2, black corn anthocyanin promoted insulin secretion at 100 μg / ml.

Example 3 Diabetes Amelioration Efficacy of Type 2 Diabetes Mice

 3-1. Laboratory Animals and Diet

This experiment was carried out by purchasing Specific-pathogen-freen male C57BL / KsJ-db / db mice (Central Experimental Animal Co., Ltd.) under the constant temperature (25 ℃) and humidity (50 ± 5%). After one week of adaptation it was used for the experiment. Six mice were used as an experimental group (sample treated group), a control group (sample untreated group), and a positive control group (glimepiride) to evaluate diabetic improvement efficacy. Each group received oral gavage at 7 mg / kg body weight for 7 weeks. Changes in body weight were measured weekly and blood obtained by orbital blood collection with heparinized capillaries was centrifuged to obtain plasma.

 3-2.Weight change

Body weight was measured weekly using a scale and as shown in FIG. 3, the change in body weight remained almost constant regardless of the group.

3-3. Blood sugar change

Once weekly fasting blood glucose of the control and experimental groups were measured weekly using a portable glucose meter (OneTouch Ultra). As shown in FIG. 4, the blood sugar of the black corn anthocyanin (purple corn, PCP) -administered group showed a significant decrease compared to the control group or the positive control group (glimepiride-administered group). Black corn anthocyanin showed better hypoglycemic effect at the same dose than glimepiride, an insulin secretion drug.

3-4. Glycated hemoglobin (HbA1c)

Glycosylated hemoglobin was measured by a method of knowing the average state of blood glucose for a long time. In this experiment, type 2 diabetes model db / db mice were treated with untreated (control), Glimepiride (positive control), black corn anthocyanin (treated group, PCP) and HbA1c was measured at 1 and 7 weeks. As shown in FIG. 5, the glycated hemoglobin (Hb1AC) was slightly lower than the black corn anthocyanin treated group (PCP) than the positive control group, Glimiperide, which proved that black corn anthocyanin has excellent long-term glycemic control ability.

2-5. Oral Glucose Tolerance Test

To test the ability of glycemic control for a short period of time, the mice were fasted after 7 weeks of treatment, orally administered glucose (0.2g / kg), and blood samples were measured in the orbital vein for several hours (0-120 min) to measure blood glucose. Controls were compared. As shown in FIG. 6, the black corn anthocyanin treated group (PCP) showed better blood glucose reduction than the positive control group, Glimepiride, and the rats treated with black corn anthocyanin normalized short-term blood glucose control ability. .

2-6. Immunohistochemical Analysis of Beta Cells (Immunohistochemistry)

Mice are completely anesthetized with phenobarbital sodium (10 mg / 100 g body weight i.p.) and perfused through the heart with 0.9% saline and 4% paraformaldehyde in 0.1 mol / L PBS (pH 7.4). The pancreas is cut out and fixed in 4% paraformaldehyde for 6 hours and embedded in paraffin. Cut into 5-μm thick slices and incubate for 14 hours at 4 ° C with primary antibody (guinea pig anti-insulin antibody) diluted in PBS containing 1% bovine serum albumin (BSA). Next, the cells were incubated with a secondary antibody (biotinylated goat anti-guinea pig IgG antibody 1: 1000) for 30 minutes at room temperature, and treated with horseradish peroxidase-conjugated streptavidin to develop color, observed under a microscope, and photographed. As shown in FIG. 7, glycated hemoglobin (Hb1AC) was slightly lower than Glim, a positive control group, in black corn anthocyanin treated group (PCP), demonstrating that black corn anthocyanin has excellent long-term glycemic control ability. The number of insulin secreting cells (beta cells) of pancreatic islet was significantly decreased in the islet in the control group of the type 2 diabetes model db / db mouse (non-treated group), but slightly increased in the glimepiride treated group, which was positive control, and treated with black corn anthocyanin. A marked increase was observed in the group (PCP), demonstrating that black corn anthocyanin protects beta cells secreting insulin.

2-7. Basic Safety Assessment in Type 2 Diabetic Mice

After 6 weeks of oral administration of black corn anthocyanin at 10 mg / kg / day to db / db mouse, a type 2 diabetic model animal, the behavioral test showed no difference with the untreated group. As a result, no difference was found with the untreated group. In conclusion, it was proved that oral administration of db / db mice was not toxic to black corn anthocyanin.

1 is a result of the black corn anthocyanin at various concentrations in the cell culture of HIT-T15, a beta cell line of hamster pancreas, and whether the growth of the cells is inhibited by MTT analysis, and FIG. 2 is the addition of black corn anthocyanin. These results show the insulin secretion of beta cell line HIT-T15. Figures 3 and 4 analyze the changes in body weight (Figure 3) and blood sugar (Figure 4) while ingesting control animals glimepiride (insulin secretion accelerator) and black corn anthocyanin for 7 weeks in db / db mouse, a type 2 diabetes animal model One result. Figure 5 shows the results of the analysis of glycated hemoglobin (Hb1Ac) showing a long-term average blood glucose state at 1 week and 7 weeks, Figure 6 is an oral glucose tolerance test (OGTT) showing a short-term glycolysis Show results. FIG. 7 shows the results of immunohistochemical staining of black corn anthocyanin and positive control group glimepiride in db / db mouse after 7 weeks of inlet analysis of beta cells in pancreatic islet.

Claims (4)

Black corn anthocyanin with excellent activity to protect pancreatic beta cells and glycemic control The black corn anthocyanin according to claim 1, which is separated from black corn ( Zea mays ). External pharmaceutical composition containing black corn anthocyanin according to claim 1 Health functional food composition comprising black corn anthocyanin according to claim 1

Images