KR101651908B1 - Composition for Prevention or Treatment diabetes comprising Tenebrio molitor larva or extract suspension of Tenebrio molitor larva - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetes comprising a larva of Tenebrio molitor or an extract thereof as an active ingredient.
The pharmaceutical composition for the prevention and treatment of diabetes or the composition for prevention and improvement of diabetes according to the present invention is characterized by comprising a larva of Tenebrio molitor or an extract thereof as an active ingredient.
According to the present invention, there is provided a method for preventing or treating diabetes mellitus comprising an effective amount of Tenebrio molitor larvae or an extract thereof, which exhibits an effect of reducing oxidative stress, increasing the survival rate of pancreatic beta cells and increasing the secretion of insulin, Therapeutic compositions are provided.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating diabetes comprising an effective amount of a larval larva or an extract thereof,

The present invention relates to a composition for preventing or treating diabetes comprising an effective amount of a larva or extract thereof. More particularly, the present invention relates to a composition for protecting pancreatic cells against damage, suppressing damage from oxidative stress, and increasing insulin secretion ability And a composition for preventing or treating diabetes.

 In Korea, about 4.2 million people are diabetic, accounting for about 10% of the total population, and about 10% of the world are known to have diabetes. Although diabetes has been studied for a number of reasons, it has not been able to find the underlying cause of diabetes because of the variety of causes.

Type 1 diabetes (insulin dependent) is a disease in which insulin secretion does not occur due to the destruction of the pancreatic beta cells by immunological mechanisms, and type 2 diabetes (insulin independent type) ) Produces insulin in the body, but increases insulin resistance, accounting for 90% to 95% of diabetic patients.

As a representative cause of type 2 diabetes, insulin secretion ability is expected to be lowered due to a decrease in pancreatic beta cell function. To date, several factors have been known to decrease the function of pancreatic beta cells. However, it is known that glucose toxicity due to hyperglycemia, lipid toxicity due to fatty acids, and oxidative stress due to the enhancement of reactive oxygen species (ROS).

In particular, oxidative stress is an important cause of damage, because pancreatic beta cells are much less susceptible to oxidative stress than antioxidant enzymes. In addition, since oxidative stress is involved in diabetic complications, there have been reports that suppression of oxidative stress, elimination and improvement of lipid metabolism have been suggested as preventive measures for diabetic complications.

What is dangerous when you have diabetes is high blood sugar levels, and the insulin action pathway must be activated to reduce blood sugar levels. The insulin action pathway is the pathway that passes the signal of insulin to GLUT4. According to E. James, who discovered GLUT4, which injects blood glucose into cells, insulin binds to the insulin receptor on the surface of cells on the surface of blood cells, activates the insulin receptor subunit below the cell membrane, pulls ATP to phosphorylate, IRS-1 is activated and activates PI3K.

Thus, the insulin signal is relayed to GLUT4 and activated to move to the cell surface, becoming integral with the cell membrane, and introducing glucose into the cells. However, in the case of regular diabetic drugs, insulin is forcibly secreted from the pancreas when taken. Therefore, when the insulin is administered over a long period of time, the function of the pancreas is weakened and damaged, resulting in insulin secretion.

Also, if you start insulin injection, you should manage your diabetes through lifelong injections. If you take insulin injections for a long time, some fat tissue may be destroyed, or insulin hypersensitivity reaction and hypoglycemic shock may occur.

Accordingly, studies are being conducted to find natural substances for prevention and treatment of diabetes without the above-mentioned side effects, and related prior arts include Korea Patent Publication No. 10-2011-0012758 (herbal composition for prevention and treatment of diabetes) And Japanese Patent Laid-Open No. 10-1246689 (a composition for anti-diabetes containing a plant mixed extract).

On the other hand, Tenebrio molitor belongs to the arthropods of the arthropods insecticidal beetle, and the brown dung larvae are called as beetles in Oriental medicine. They are applied to the treatment of hematochezia, vaginal damage, planting stomach [心 气 胃痛] Antibacterial effects have been reported. Compared to meat and brown goats, the protein content is similar, but the fat content is quite high, so it can be used as a sufficient energy source in a small amount and 70 ~ 80% of the fat is unsaturated fatty acid do.

Until now, brown duck has been cited in Oriental medicine and has been used for many diseases from the past, but there is little scientific analysis about it.

It is an object of the present invention to provide a composition for preventing or treating diabetes, which comprises an effective amount of a larva or an extract thereof as an active ingredient to protect the pancreatic beta cells and increase the secretory capacity of insulin by reducing oxidative stress, .

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

In order to achieve the above object, the pharmaceutical composition for preventing or treating diabetes or the composition for preventing or improving diabetes according to the present invention is characterized by comprising a larva of Tenebrio molitor or an extract thereof as an active ingredient.

The extract is characterized by being extracted with 70 vol.% Ethanol.

The Tinebrio molitor larva suppresses oxidative stress in the pancreatic beta cells, activates insulin secretion, reduces blood glucose levels, and is a member of the insulin receptor (IR), insulin receptor-1 (IRS -1), phosphatidylinositol 3-phosphorylase (PI3K) and glucose transporter type 4 (GLUT4).

The brown goose larva (Tenebrio molitor) larva or its extract is contained in an amount of 100 μg to 5000 μg based on 1 ml of the composition.

The present invention provides a composition for preventing or treating diabetes comprising an effective amount of a larval larva or an extract thereof, which exhibits an effect of suppressing oxidative stress to increase the survival rate of pancreatic beta cells and increase the secretion of insulin by reducing the blood glucose level / RTI >

FIG. 1 is a graph showing the cell lysate lethal concentration measured after Alloxan treatment by the MTS assay. FIG.
2A is a graph showing cell viability of pancreatic beta cells (HIT-T15) in silkworm extract (BME).
FIG. 2B is a graph showing cell viability of mulberry leaf extract (MAE) in pancreatic beta cells (HIT-T15).
FIG. 2C is a graph showing cell viability in pancreatic beta cells (HIT-T15) of the brown goat larva extract (TME) of Example 1 of the present invention.
FIG. 3A is a graph showing the activity of nitrogen monoxide (NO) in pancreatic beta cells (HIT-T15) in which diabetes was induced.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
FIG. 3B is a graph showing the activity of glutathione S-transferase (GST) in pancreatic beta cells (HIT-T15) induced by diabetes.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
4A is a graph showing the cell survival rate of pancreatic beta cells (HIT-T15) in which diabetes was induced.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
MAE: mulberry leaf extract treated group
4B is a graph showing the amount of glucose secreted in diabetic-induced pancreatic beta cells (HIT-T15).
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
MAE: mulberry leaf extract treated group
FIG. 4C is a graph showing the amount of insulin secreted in diabetic-induced pancreatic beta cells (HIT-T15). FIG.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
MAE: mulberry leaf extract treated group
FIG. 5A is a graph showing the kidney weight in an animal model in which diabetes was induced. FIG.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
Figure 5B is an immunohistochemical staining of the pancreas in an animal model in which diabetes has been induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
FIG. 5C is a graph showing counts of insulin-producing cells of the pancreas in an animal model in which diabetes was induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
6A is a graph showing blood glucose levels in an animal model in which diabetes was induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
6B is a graph showing insulin levels in an animal model in which diabetes was induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
FIG. 6C is a graph showing the values of FFA (free fatty acid) associated with insulin resistance in an animal model in which diabetes was induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
6D is a graph showing ALT (alanine aminotransferase) levels in an animal model in which diabetes was induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
6E is a graph showing the values of AST (aspartic acid aminotransferase) in an animal model in which diabetes was induced.
TM: The brown goat larvae treated group of Example 2 of the present invention
BM: silkworm treated group
FIG. 7A is a graph showing the amount of insulin receptor (IR) expressed in diabetes-induced pancreatic beta cells (HIT-T15).
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
FIG. 7B is a graph showing the expression level of IRS-1 (insulin receptor substrate-1) in diabetic-induced pancreatic beta cells (HIT-T15).
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
FIG. 7C is a graph showing the expression level of phosphatidylinositol 3-phosphorylase (PI3K) in pancreatic beta cells (HIT-T15) induced by diabetes.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
7D is a graph showing the expression level of glucose receptor type 4 (GLUT4) in pancreatic beta cells (HIT-T15) in which diabetes was induced.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group
FIG. 8 is a Western blot graph showing the expression level of glucose receptor type 4 (GLUT4) in pancreatic beta cells (HIT-T15) induced by diabetes.
TME: The brown goat larvae extract treatment of Example 1 of the present invention
BME: Silkworm extract-treated group

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

Diabetes mellitus is known to be caused by the increase of blood glucose level in the body due to the failure of pancreatic beta cell destruction and insulin secretion, or by the damage of pancreatic beta cells due to oxidative stress.

Therefore, currently, not only reducing the blood glucose level in the body and increasing insulin secretion but also protecting the pancreatic beta cells by inhibiting oxidative stress is becoming a major factor in diabetes research.

Accordingly, the inventors of the present invention have conducted various studies on natural products that suppress oxidative stress and decrease the blood glucose level and increase insulin secretion in the body, and found that among the natural products, brown larvae or extracts thereof suppress oxidative stress, Increased cell survival rate of beta cells, decreased blood glucose levels and increased insulin secretion.

Specifically, the larvae of the present invention (hereinafter referred to as 'TM') or extracts thereof (hereinafter referred to as 'TME') are prepared by using pancreatic beta cells HIT-T15 cells and a diabetic animal model The cell protection, insulin secretion ability and antioxidant enzyme activity from oxidative stress by alloxan of brown goat extract (TME) were evaluated.

As a result, it was confirmed that TME was not toxic to pancreatic cells because HIT-T15 cell was not toxic even at the highest concentration of 5000 ㎍ / ㎖ for 96 hours, % Cytotoxicity, the antioxidative effects of oxidative stress showed that NO decreased about 20% when treated with 1000 μg / ㎖ of TME compared to cells treated with alloxan alone

In addition, GST activity of alloxan-treated cells was slightly lower than that of glucose-free cells, but GST activity was significantly increased with TME treatment.

In addition, cell viability after 24, 48, and 72 hours of treatment with Alloxan treated TME by concentration showed that cell viability was increased depending on TME treatment concentration compared with alloxan alone, Glucose concentration was almost normalized by 72% after 72 hours of treatment with TME 5000 μg / ㎖ compared with alloxan treatment, and insulin was also significantly increased by about 10% And the cell survival rate was increased by protecting the cells from the oxidative stress induced by the cells.

The expression levels of IR, IRS-1, PI3K, and GLUT4 in the insulin pathway were examined by real-time PCR, and the expression levels of IR, IRS-1, And the protein level of GLUT4 increased significantly.

Also, analysis of blood glucose and insulin of TM in the diabetic animal model showed that the animal group treated with TM for 5 weeks was reduced by about 40% as compared with the diabetic animal group. Insulin concentration in blood was significantly decreased in diabetic group compared to normal group and there was no significant difference between TM and silkworm (hereinafter referred to as 'BM') as compared to diabetic group.

In the case of free fatty acid (FFA), FFA was increased in the presence of diabetes, whereas FFA was decreased by 32% in TM when administered orally.

The liver is a place to store sugar and is a place to release sugar when energy is needed by our bodies. If diabetes causes abnormal glucose metabolism, it will become liver fatty when it is affected by liver and accumulated fat. In order to confirm this, both ALT and AST were significantly increased in diabetic group compared to normal group, but ALT and AST were decreased in TM treatment group. Histologic analysis showed that the kidney did not decrease the weight of kidney even after TM treatment, but insulin secretory cells of pancreatic islets were improved in TM.

The extract of the present invention having the anti-diabetic effect of the present invention having the anti-diabetic effect of the present invention has an excellent blood glucose lowering effect and thus has an excellent effect for the prevention of diabetes, and also has the effect of recovering cellular damage against alloxan's cell damage. The prevention and treatment effect of diabetes which was not known in the ducks was known. In addition, it is desirable to apply the present invention to the manufacture of antidiabetic functional products in various foods, beverages, tea, and liquors so that the efficacy can be more easily and easily accessible to the general public.

Hereinafter, the method for preparing the larvae having the anti-diabetic effect of the present invention will be described in detail.

1. First step; Brown goat larvae crushing

First, prepare a brown duck (Tenebrio molitor) at this stage.

The brown duck (Tenebrio molitor) belongs to the arthropodic insect beetle neck duck. The brown duck (Tenebrio molitor) is preferably a brown duck larva having a maximum content of the active ingredient exhibiting antidiabetic activity.

The brown duck (Tenebrio molitor) larva prepared as described above is dried and then pulverized into a powder form.

Then, since it contains a large amount of water, it is necessary to carry out a drying process in order to completely remove water. At this time, it is preferable to perform drying through freeze drying in order to minimize the change of the active ingredients contained in the brown gill.

The dried brown duck larvae are then prepared in powder form using a mill for ease of subsequent extraction.

2. The second step; Preparation of mixed brown liquid (Tenebrio molitor)

In this step, the brown gully (Tenebrio molitor) powder prepared above is mixed with ethanol to prepare a brown goat mixture.

At this time, the mixing ratio of ethanol and pulverized brown gruel powder is not particularly limited, but it is preferable to mix 1 to 3 g of the pulverized brown gruel powder per 1 liter of ethanol. If the concentration is out of the above range, it may be troublesome to carry out an additional process of adjusting the concentration to a proper concentration through troublesome concentration or dilution.

Also, any ethanol may be used as the above-mentioned ethanol, but it is preferable to use 70 ~ 80% of ethanol so as to extract the most effective ingredient contained in the brown gruel.

3. The third step; The step of micronizing the brown goat (Tenebrio molitor) mixture

In this step, the active ingredient having the anti-diabetic effect is extracted from the brown goat's mixed liquor, which can be micronized without any specific limitation using a conventional ultrasonic pulverizer. However, the ultrasonic pulverizer may be added to the longevity beet mixture at an energy of 200 to 300 joules For 10 to 30 seconds.

If the application conditions of the ultrasonic mill are out of the above range, the desired active ingredient may not be extracted, or the active ingredient may be destroyed or denatured.

4. Step 4; Preparation stage of brown goat (Tenebrio molitor) larvae extract

In this step, after centrifuging the micronized brown goat's mixture, the supernatant is recovered, filtered, and dried. Finally, a brown goat larva extract having anti-diabetic effect through inhibition of osteoclast differentiation is prepared .

At this time, the centrifugation is performed at 4000 to 5000 rpm for 5 to 20 minutes. If the centrifugation application condition is out of the range, the desired active ingredient may be destroyed or denatured.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetes or a food composition for preventing or improving diabetes, which comprises the larva or extract thereof as an active ingredient.

In particular, the composition of the present invention is characterized in that the brown larva or its extract is contained in an amount of 100 내지 to 5000 를 based on 1 ml of the composition. This is supported by the following experimental example. When the content of the larva or extract thereof is less than 100 μg, the insulin secretion ability is poor and the present invention is not suitable for use as a desired antidiabetic agent. It is not economically suitable because it does not exhibit an effect that is higher than the content.

Such a pharmaceutical composition of the present invention may be contained in a natural drug product having an antidiabetic effect for the purpose of prevention and treatment of diabetes. In this case, according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs, the preparation is carried out in the form of a unit dose form by using a pharmaceutically acceptable carrier or excipient, .

The formulations may be in the form of tablets, capsules, powders, granules, liquids or rings, and may additionally contain dispersing or stabilizing agents. That is, it can be selected without difficulty by those skilled in the art depending on the purpose of use, and the addition amount thereof can be selected within a range that does not impair the purpose and effect of the present invention.

Examples of the pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and also pharmaceutically acceptable excipients Include, but are not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives and the like.

In addition, in the food composition of the present invention, when the larva or extract thereof is used as an active ingredient thereof, it may be used as it is or may be used together with components of other conventional food compositions, and may be appropriately used according to a conventional method have.

Such a food composition may be contained in a health food for the purpose of prevention and improvement of diabetes, and there is no particular limitation on its kind. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

In addition to the above, the food composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may be contained as pulp for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Preparation of Brown Goat Larva Extract

The lyophilized brown gill larvae were purchased from the World Way and used for the production of brown goat larvae.

3 g of brown larvae larvae were added to 1 liter of 70 vol.% Ethanol to prepare a mixture. The mixture was divided into two portions for 10 seconds with an ultrasonic wave shaker at 230 joules to be crushed and refined.

The micronized brown gill larvae mixture was centrifuged at 4500 rpm for 10 minutes, and the supernatant was recovered. The supernatant was filtered with a 0.25 mu m syringe filter to prepare the brown goat larva extract (TME) of the present invention.

Example 2 Preparation of Brown Goose Larvae for Oral Administration

The lyophilized brown gill larva powder used in Example 1 was mixed with distilled water to prepare the oral ganoderma larvae (TM) of the present invention for oral administration.

<Experimental Example 1> The anti-diabetic activity test of the brown goat larva extract of the present invention

1. Experimental Method

1) Cells and experimental animals

HIT-T15 cells (ATCC, MD, USA) were cultured in RPMI-1640 medium supplemented with penicillin-streptomycin 100 units / ml and 10% FBS at 37 ° C in a 5% CO ₂ incubator , And subculture was performed once every two days.

In the case of experimental animals, ICR male rats weighing 30 g or more supplied from a central laboratory animal were admitted to the laboratory environment for more than one week while being fed with a solid diet (research diet, New Brunswick, USA) and water at 24 ± 2 ° C Respectively.

Diabetic induction was induced by intraperitoneal injection of alloxan (50 mg / kg, 0.05 M sodium citrate buffer) for 16 hours.

Three days after injection of alloxan, blood was collected from the tail of the experimental animals and blood glucose was measured using a glucose meter. A glucose concentration of 300 mg / dL or more was judged to be diabetes induced and used in the experiment.

2) Antioxidant activity measurement

HIT-T15 cells were cultured in a 24-well plate at 1 × 10 ⁵ / well for 24 hours at 37 ° C in a 5% CO ₂ incubator, and then 10 mM alloxan (Sigma-Aldrich, MO, USA) Samples were treated for each concentration and cultured for 1 hour.

After completion of the incubation, the medium containing alloxan was removed, and the samples were treated for 23 hours in a fresh medium.

Cells were collected and antioxidant was measured using Nitric Oxide detection kit (iNtRON, korea) Glutathione S-Transferase Assay kit (cayman chemical, MI, USA)

3) Cell viability measurement

To measure cell viability, HIT-T15 cells were added to a 96-well plate at a concentration of 1 × 10 ⁵ / well, and the samples were added at different concentrations (1000, 2000, 3000, 4000, 5000 ㎍ / CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Wisconsin, USA) was performed.

MTS reagent (3-4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added at a final concentration of 0.5 mg / ml and incubated at 37 ° C for additional 4 hours. (Beckman coulter, California, USA) at 515 nm. Cell viability was expressed as a percentage of the average absorbance value of the control group.

4) Measurement of insulin secretion

HIT-T15 cells were plated at a density of 1 × 10 ⁵ / well in a 96-well plate and treated with 10 mM alloxan and samples (1000, 2000, 3000, 4000, 5000 ㎍ / ㎖) Lt; / RTI &gt; After completion of the incubation, the medium containing alloxan was removed and the sample was treated with the RPMI-1640 medium for 23 hours. After cultivation, cell culture supernatant was collected and assayed using Rat / Mouse Insulin kit (Millipore, MA, USA).

5) Collection of animal samples and analysis of tissue samples

The experimental group was divided into 5 groups of 5 animals in each group. The control group, the negative control group, the positive control group, the low concentration group of the brown goat larvae (TM) of Example 2, the high concentration of the brown goat larvae (TM) The groups were orally administered at a dose of 10 mL / kg in physiological saline and 3000 mg / kg in the silkworm control group, and at a dose of 300 mg / kg and 3000 mg / kg for 24 weeks.

Blood glucose was measured once a week and 24 hours after the final oral administration, ether was anesthetized and the abdominal midline was incised. The pancreas was removed, and the tissue was stained with insulin staining. Blood was collected from the heart, and the blood was collected and centrifuged at 3,000 rpm for 15 minutes to take plasma and used as an analytical sample. Centrifuged plasma was stored frozen at -70 ° C until analysis.

Plasma was analyzed by Green Cross to determine FFA and liver water, and insulin levels were measured in plasma.

6) Real-time PCR analysis

In order to analyze the antidiabetic efficacy, insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and glucose transporter type 4 (GLUT4) The expression level was confirmed by real-time PCR.

Cells treated with Alloxan were treated with brown goat larvae extract, cultured for 24 hours, and then the cell medium was removed, treated with Trizol reagent, and then collected using a cell scraper.

The collected cells were centrifuged at 12,000 g for 15 min. Isopropanol was added to the recovered supernatant to extract RNA, and the total amount of extracted RNA was measured. The amount of extracted RNA was measured at 2 μg / ml The concentration was quantified to synthesize cDNA.

The synthesized cDNA was mixed with the primer prepared with Power SYBR Green Mixture and then the level of insulin signaling pathway was examined through real-time PCR instrument (AB, Milton Keynes, UK). The nucleotide sequences of the primers used are shown in Table 1 below.

Name Sequence GAPDH Forward 5'-GCCTCACCCCATTTGATGTT-3 Reverse 5'-GGGAAGCCCATCACCATCT-3 ' Insulin receptor
Forward 5'-GAGAGGATGTGAGACGACGG-3 ' Reverse 5'-GTATAGCCAGACGGGCACTC-3 ' IRS-1
Forward 5'-AATGTGTGGCTGAGACCTGG-3 ' Reverse 5'-CTGTTGTTGAGAGGGGCAGT-3 ' PI3K
Forward 5'-CAGTTTGCCCCTCCTGATGT-3 ' Reverse 5'-GGGCTCTGTAGTTCTTGGGC-3 ' Gult4
Forward 5'-CTTGGCTCCCTTCAGTTTGG-3 ' Reverse 5'-CTACCCAGCCACGTTGCATT-3 '

7) Western blot analysis

Western blot was performed to confirm the expression of GLUT4, which is associated with glucose uptake. Cells were washed twice with PBS, collected in a cell scraper (SPL Life Science, Korea), and then incubated with Cytobuster ^™ Cells were pulverized using Protein Extraction Reagent (Novagen, ND, USA) and centrifuged at 12000 rpm for 15 minutes to recover the supernatant.

The recovered supernatant was measured by Bio Rad Protein Assay (Bio Rad, CA, USA) and adjusted to a concentration of 10 μg / ml, followed by 10% SDS-PAGE. After electrophoresis, the cells were transferred to a PVDF membrane (GE Healthcare, NJ, USA) and blotting grade blocker (Bio Rad, CA, USA) was dissolved in TBST to a concentration of 5% blocking.

The primary antibody, GLUT4 (calbiochem, CA, USA), was diluted 1: 100 and tubulin (Sigma, MO, USA) at a ratio of 1: 10000 and reacted overnight at room temperature. Secondary antibody HRP-conjugated secondary antibody (Promega, WI, USA) was reacted at a ratio of 1: 3000 for 40 minutes at room temperature and then washed 4 times for 15 minutes with TBST. Western LightningPlus ECL (Perkin Elmer, MA, USA) was treated with PVDF membrane and exposed to X-ray film (Fujifilm, Japan) in the dark room to confirm protein expression of GLUT4.

2. Experimental results

1) Assessment of toxicity to pancreatic cells

(BME) and mulberry leaf extract (hereinafter referred to as "MAE") were treated with 0, 10, 100, 500, 800 and 1000 μg / ml, respectively, in pancreatic cells (HIT- Cell viability was checked after lapse of time.

As a result, as shown in FIG. 1, BME was somewhat toxic at 500 μg / ml and MAE was toxic at 800 μg / ml or more.

To measure the cytotoxicity of TME in normal HIT-T15 cells, pancreatic cells were seeded at 2 × 10 6 cells / well and treated with TME (1000, 2000, 3000, 4000, 5000 ㎍ / 48, and 72 hours, it was confirmed that TME was not toxic to the pancreatic cells because there was no toxicity at the highest concentration of 5000 ㎍ / ㎖ for 96 hours.

2) Antioxidant activity of TME on pancreatic cells damaged by Alloxan

In order to induce diabetes by alloxan in HIT-T15 cells, alloxan was added to the pancreatic cells at 2 × 10 ⁵ cells / well (0, 1, 2, 3, 4 , 5, 10, and 15 mM) for 1 hour, and cultured for 23 hours after the medium was replaced. Then, MTS assay was performed. As shown in FIGS. 2A to 2C, about 50% of cytotoxicity was observed at a concentration of 10 mM , And 10 mM allloxan was used for subsequent experiments using pancreatic cells.

To confirm the antioxidant activity of TME against HIT-T15 cells damaged by Alloxan treatment, the cells were treated with alloxan for 1 hour at 2 × 10 ⁵ cells / well in pancreatic cells, and the medium containing alloxan was removed and TME As shown in FIGS. 3A and 3B, NO and GST were decreased by about 20% when 1000 μg / ml of TME was treated, after 24 hours of treatment with each concentration (500, 1000 μg / ml) And GST activity was significantly increased by TME treatment.

3) decreased TME glucose and insulin secretion in pancreatic cells damaged by Alloxan

To confirm the efficacy of TME on HIT-T15 cells, which were damaged by the alloxan treatment and the intracellular inflow of glucose, pituitary cells were treated with alloxan for 1 hour at 2 × 10 ⁵ cells / well, And the cells were treated with TME at different concentrations (1000, 2000, 3000, 4000, 5000 / / ml) in a fresh medium. Cell viability was checked at 24, 48 and 72 hours after the treatment. As shown in FIGS. 4A to 4C, The cell viability was increased by treatment with 5000 ㎍ / ㎖ and the glucose level was decreased to 24% after 72 hours of treatment with 5000 ㎍ / ㎖ of TME. And the insulin was also increased by alloxan treatment dose - dependently, which was increased by about 10% compared to the same time period after alloxan treatment.

4) Histologic improvement effect of TM treatment on diabetic animal model

In order to confirm the efficacy of TM in an animal model in which diabetes was induced by Alloxan treatment, TME was orally administered to an animal model of Alloxan induced diabetes at a concentration of 3000 mg / kg for 5 weeks to analyze kidney weight and pancreatic tissue As a result of analyzing the weight (A) of the kidneys as a result of the urine function problem, as shown in FIGS. 5A to 5C, the kidney was larger than the normal group, but the weight did not decrease in the TM, In immunohistochemical staining, severe atrophy of the pancreatic islet was observed except for the control subjects.

Atrophied pancreatic islets showed a severe decrease in insulin secretory cells. The number of insulin - positive cells among the cells constituting the pancreatic islets was counted and the ratio was shown.

The percentage of insulin-positive cells in control subjects was 12.4%, 16.8%, and 27.5%, respectively, in Diabetes, BM, and TM individuals.

5) Hematologic improvement effect by TM treatment in diabetic animal model

In order to confirm the efficacy of TM in an animal model in which diabetes was induced by treatment with Alloxan, TM was orally administered at a concentration of 3000 mg / kg for 5 weeks to an animal model in which diabetes was induced by Alloxan. , The blood glucose level of the diabetic group was significantly higher than that of the normal group, but the TM treated group was reduced by about 40% as compared with the diabetic group.

Insulin was also detected in the blood. Insulin secretion was significantly reduced in the diabetic group compared to the normal group, but there was no difference in silkworms compared to the diabetic group, but increased by about 26% in TM.

In the case of FFA (B), which is related to insulin resistance, FFA was increased by diabetic induction but FFA decreased by 20% in brown silkworm and 32% in brown silkworm by oral administration of silkworm and brown duck.

The liver is a place to store sugar and is a place to release sugar when energy is needed by our bodies. If diabetes causes abnormal glucose metabolism, it will become liver fatty when it is affected by liver and accumulated fat. In order to confirm this, both ALT (C) and AST (D) were significantly increased in diabetic group compared to normal group, but ALT was decreased in silkworm group and brown duck group. AST was decreased only in brown drowning group .

6) Increased expression of insulin pathway related gene after TME treatment

We examined the expression of IR, IRS-1, PI3K, and GLUT4, which are involved in the insulin signaling pathway, in real-time PCR by treating TMX with HIT-T15 cells treated with Alloxan for 24 hours. As shown in 7A to 7D, the expression levels of IR, IRS-1, PI3K and GLUT4, which are involved in the insulin signaling pathway, were significantly increased compared to the diabetic cells, resulting in a decrease in intracellular insulin resistance and an increase in intracellular glucose uptake .

7) Increased GLUT4 protein expression of TME

In order to confirm the expression of GLUT4 protein related to the intracellular glucose uptake, alloxan was treated with alloxan for 1 hour, and the medium containing alloxan was removed and treated with TME (500, 1000 ㎍ / ㎖) As a result of western blot analysis of the amount of GLUT4 protein expressed at 24 hours, it was confirmed that TME increased at 1000 ㎍ / ㎖ as shown in Fig.

As described above, by examining the cell protection, damage inhibition and insulin secretion ability of pancreatic beta cells against TME, the present invention can protect pancreatic beta cells from oxidative stress caused by alloxan, The possibility of development, and proving the anti-diabetic efficacy, it can be used as dietary medicine for diabetes, and it can be expected that income of insect farmers and industry can be increased.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

(Tenebrio molitor) larva or an extract thereof as an active ingredient,
A pharmaceutical composition for the prevention or treatment of diabetes.
The method of claim 1, wherein the diabetes is type 2 diabetes.
A pharmaceutical composition for the prevention or treatment of diabetes.
The method according to claim 1,
Characterized in that the extract is extracted with 70 vol.% Ethanol.
A pharmaceutical composition for the prevention or treatment of diabetes.
The method according to claim 1,
Wherein said Tinebrio molitor larva or extract thereof inhibits oxidative stress in pancreatic beta cells.
A pharmaceutical composition for the prevention or treatment of diabetes.
The method according to claim 1,
Wherein the Tenebrio molitor larva or extract thereof activates insulin secretory function to reduce blood glucose levels.
A pharmaceutical composition for the prevention or treatment of diabetes.
The method according to claim 1,
The Tenebrio molitor larva or its extract has a function of insulin receptor (IR), insulin receptor substance-1 (IRS-1), phosphatidylinositol 3-phosphorylase (PI3K) and glucose transporter type 4 &lt; / RTI &gt; (GLUT4).
A pharmaceutical composition for the prevention or treatment of diabetes.
The method according to claim 1,
Wherein the Tenebrio molitor larva or extract thereof is contained in an amount of 100 ~ to 5000 를 based on 1 ml of the composition.
A pharmaceutical composition for the prevention or treatment of diabetes.
(Tenebrio molitor) larva or an extract thereof as an active ingredient,
A food composition for preventing or improving diabetes.
9. The method of claim 8,
Characterized in that the extract is extracted with 70 vol.% Ethanol.
A food composition for preventing or improving diabetes.
9. The method of claim 8,
Wherein said Tinebrio molitor larva or extract thereof inhibits oxidative stress in pancreatic beta cells.
A food composition for preventing or improving diabetes.
9. The method of claim 8,
Wherein said Tinebrio molitor larva or extract thereof activates insulin secretory function to reduce blood glucose levels.
A food composition for preventing or improving diabetes.
9. The method of claim 8,
The Tenebrio molitor larva or its extract contains insulin receptor (IR), insulin receptor substance-1 (IRS-1), phosphatidylinositol 3-phosphorylase (PI3K) and glucose transporter type 4 Lt; RTI ID = 0.0 &gt; GLUT4 &lt; / RTI &gt;
A food composition for preventing or improving diabetes.
9. The method of claim 8,
Wherein the Tenebrio molitor larva or extract thereof is contained in an amount of 100 ~ to 5000 를 based on 1 ml of the composition.
A food composition for preventing or improving diabetes.
A first step of pulverizing the lyophilized brownish larvae;
A second step of mixing the brown larch larvae powder prepared in the first step with ethanol to prepare a brown goat's mixture liquid;
A third step of micronizing the brown gyoer mixture prepared in the second step with an ultrasonic pulverizer,
And a fourth step of centrifuging the micronized brown gourd mixture liquid in the third step, recovering the supernatant, filtering and then drying the brown gurdic larvae extract to produce an anti-diabetic brown gourd larvae extract. Which is characterized by having an anti-diabetic effect, comprising an effective amount of a larvae extract of a brown goat,
Antidiabetic agent. delete

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