KR20190031044A - Glycosaminoglycan derived from queen of Bombus ignitus and its inhibition activity of diabetes - Google Patents

Abstract

The present invention provides an antidiabetic composition containing glycosaminoglycan isolated and purified from Bombus ignites queen, huechys, cricket, or Catharsius molossus. The huechys glycosaminoglycan has an N-glycan structure of Hex_7 (m/z1175.2) or Hex_7HexNAc_6 (m/ z1752.2). Insect-derived Glycosaminoglycan, derived from each of Bombus ignites queen, huechys, Catharsius molossus and cricket according to the present invention has a blood glucose lowering effect and a physiological function restoration effect due to a biooxidation inhibition effect in diabetic rats, thereby being usefully used as health functional foods for preventing or alleviating diabetes.

Description

[0001] Glycosaminoglycan derived from queen of Bombus ignitus and its inhibition activity of diabetes [

The present invention relates to an antidiabetic composition containing glycosaminoglycan isolated from quebrain beetle queen bee or Hongliang, wherein said Honglangangai glycocamminoglycan is Hex ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2).

Cicadas and insects are used as food for all adults in China, but there are not many cases in Korea. Heuchys sanguinea (de Geer), H. philaemata (fabr), H. thoracica (dislant), and black-winged caterpillars (used for this experiment) ], And distinguishes it from the subspecies (flowering cicada, Limois emelianovi ). It is possible to separate the glycosaminoglycans from the baronequinone, which has a limited ability to use it because of its weak potency, and to enable the safety and universal dosage of the drug. Glicorzaminoglycan, which is used as a raw material for health functional food, It was separated and purified from Hongrangja.

Although the dung beetle is used as a medicinal herb, it is not easy to take food for Koreans' emotions, and it produces glycogaminoglycan, which is a raw material for health functional foods.

Recently, glycosaminoglycan has been used as a medication (ointment) or as a health functional food (taking) for various symptoms. For example, keratin sulfate differs from fingernail, while heparin sulfate is usefully used for the destruction of pregnant women and the like, chondroitin sulfate (hydrochloric acid) and glucosamine sulfate (hydrochloric acid) are used for bone cartilage production. It is separated from the insect that can be kept in breeding, limited to shrimp cartilage and shell, whale, tuna and so on such as cattle, pigs, chickens, etc., more convenient in standardization and standardization of quality control, The raw material was chosen from insects. Glycans are the major constituents of cells, followed by DNA, protein, and lipid. Cell membranes are glycosylated with glycocalyx and cover the cells with a thick sugar coat. All glycoproteins (glycoproteins) are synthesized in the ER to obtain characteristic N-glycans and mature as complexes in bone gasses. These glycoproteins are regulated by highly specific glycosyltransferases and are characterized by minor changes in sugar chains Suggesting that the characteristic structure of N-glycan plays an important role in the function of cells, since cell function can be regulated and changed.

Accordingly, the inventors of the present invention have investigated various physiological activities of glycoxaminoglycan isolated from insects such as Hongrangja and Bumblebee queen bee, and confirmed that such insect glycosaminoglycan has an antidiabetic effect, .

Korean Patent Publication No. 10-2011-0107304 (composition for antidiabetic agent containing insect glycosaminoglycan) Korean Patent Publication No. 10-2017-0047544 (Use for suppressing fat accumulation of bovine bee bee glycocalin minoglycan)

It is an object of the present invention to provide a composition for anti-diabetes containing glycosaminoglycan separated and purified from queen bee or queen beetle, and the above-mentioned Hongragana glycosaminoglycan is Hex ₇ (m / z 1175 2) or Hex ₇ HexNAc ₆ (m / z 1752.2).

In order to accomplish the above object, the present invention provides a composition for anti-diabetic comprising glycozaminoglycans isolated and purified from queen bee bane beetle, Hongrangja, cricket or tangerine. The above-mentioned red ginseng glycosaminoglycan is characterized by having an N-glycan structure of Hex ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2).

The present invention also relates to a food composition for preventing or ameliorating diabetes containing glycosaminoglycan separated and purified from queen bee bane, Hongrangja, cricket or tangerine.

The antidiabetic composition can be prepared by the following method.

Preferably, the method comprises the steps of: a) subjecting the insect to an amount of at least one organic solvent selected from the group consisting of acetone, hexane, ethyl acetate, methyl alcohol, ethyl alcohol, propanol, butanol, acetonitrile, chloroform and dichloromethane, Immersing it for 5 days to remove the fat, filtering and drying the residue (insect skin, etc.) that is not filtered; b) pulverizing the dried residue with a ball mill or a hammer mill and adding 2-5% by weight of protease derived from Bacillus lichenformis alkaline (pH 9.0) or Aspergillus oryzae, And decomposing the protein by enzymatic treatment at 50 to 60 ° C for 3 to 7 days; c) Add to the enzymatic treatment solution of step b) to a final concentration of 40 to 60% (w / v) Trichloroacetic acid in the refrigerator to 3 to 7% (w / v) Deg.] C for 2 to 4 hours to precipitate the protein. Then, the sugar is co-precipitated with 1.5 to 3 times ethanol (100%) and potassium acetate with respect to the residue volume obtained by centrifugation (5000 to 10000 rpm) The precipitate was precipitated in 1.5 to 3 times as much volume of cetylpyridinium (3-7% by weight), and then the precipitate was precipitated in 1.5 to 3 times volume of ethanol. The precipitate was dialyzed with distilled water using a dialysis membrane having a molecular weight of 3500 or more, Drying to obtain a crude glicorziminoglycan derived from a Bombyx mori containing a bumblebee larvae pupa; d) The obtained crude glycosaminoglycan was subjected to salt gradient (0, 0.1, 0.5, 1 or 2.5 M NaCl in phosphate buffer (pH 7.0) using ion exchange chromatography (Sephadex DEAE A-25) ). ≪ / RTI > Or e) endoglycosidase F to separate the sugar and protein linkages, and passing only the N-glycan through the graphite cartridge.

The step d) utilizes the fact that the polarities of the N-glycans bound to the ion exchange resin used in the ion-exchange chromatography are different from each other, A different buffer is used to obtain fractions with high uronic acid content.

The term " chromatography " in the present invention refers to a method of separating the adsorbent particles based on the difference in adsorption characteristics of the solute to the adsorbent particles by passing a mixture containing a solute in a column of a predetermined length packed with the porous adsorbent particles Method. If the chromatographic process consists of a mobile phase and a stationary phase, the stationary phase can use an adsorbent, an ion exchange resin, a porous material or a gel. Liquid chromatography, which is useful for the separation analysis of multi-component mixed samples, can be typified by high-pressure liquid chromatography, ion-exchange chromatography and affinity chromatography. In the present invention, ionic or electrically-conductive compounds, that is, N-glycans of insects are highly polar, and ion exchange chromatography using the principle of adsorption with a filler by an electric force It is preferable to use ion-exchange chromatography.

The present invention provides a pharmaceutical composition containing an insect glycosaminoglycan, such as queen bee bumblebees. The pharmaceutical composition may be a pharmaceutical composition for antidiabetic use. The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient in the insect glycosaminoglycan, such as the bumblebee queen bee of the invention, Starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition of the present invention will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. The insect glycosaminoglycan of the present invention such as queen bee bumblebee is a medicament which can be safely used for prolonged use for preventive purposes because it has little toxicity and side effects.

The present invention also provides a health functional food comprising an insect glycosaminoglycan such as queen bee bumblebees and a food-acceptable food supplementary additive. The health functional food may be a health functional food for preventing or improving diabetes. The insect glycosaminoglycan, such as queen bee bumblebeet, may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the insect glycosaminoglycan of the present invention can be added include, for example, various Drinks, alcoholic beverages, gums, tea, and vitamins, and the like. The present invention relates to a method for producing a food,

The insect-derived glycosaminoglycan derived from each of the insects of the bumble bee queen bee bug, Hongrangja, Gangliao, and cricket according to the present invention has a blood glucose lowering effect in diabetic rats and a physiological function restoration effect due to the inhibition effect on biooxidation, , And as a health functional food for preventing or ameliorating diabetes.

1 is a MALDI-TOF analysis mass spectrometry result of N-glycans of Hongrangan.
Fig. 2 is a graph showing changes in body weight over a period of one month in which insulin glicorziminoglucan is administered to diabetic rats. Fig. In the following drawings, Hongragana glycocamminoglycan is represented by HEG, HeG and Heg5, Ganggol glycosaminoglycan is represented by CaG and CaG5, and bovine queen bee glycocamminoglycan is represented by IQG and IQG5 , And cricket glycopolaminoglycan is represented by GbG and GbG5. Control represents normal rat, genotype is DB-Hetero type, and diabetic rat is DB-homo.
FIG. 3 is a graph showing changes in blood glucose levels after 1 week and 4 weeks (one month) administration of each insect glycocalinoglycan in the diabetic rats, Honganji, Tangerine, and Bumblebee queen bee.
FIG. 4 is a graph showing the content of creatine kinase, total cholesterol, LDL cholesterol, and hyaluronic acid in blood after administration of each insect glycocalinoglycan in the diabetic rats for the period of one month, for each insect of the Hongrangja, Tangerine, and Bumblebee queen bee.
FIG. 5 is a graph showing blood catalase, GPX, and GST contents after administration of insecticide glycosaminoglycan for 1 month in the diabetic rats, Honglinja, Tangerine, and Bumblebee queen bee.
FIG. 6 is a graph showing the degree of SOD expression in blood after 1 month of administration of each insect glycoprotein minoglucan in the diabetic rats, Honganji, Tangerine, and Bumblebee queen bee.
FIG. 7 is a graph showing carbonyl content in blood and liver after administration of each insect glycosaminoglycan for 1 month in the diabetic rats, Honganji, Tangerine, and Bumblebee queen bee.
FIG. 8 is a graph showing changes in body weight after administration of insecticide glycocalinoglycans of rat, cricket, and the like for 1 month in diabetic rats.
FIG. 9 is a graph showing blood glucose levels after administration of insect glycocalinoglycan of the rat and crickets to diabetic rats for 1 month.
10 is a graph showing the content of blood catalase, GPX and GST in diabetic rats after one month of administration of insect glycocalinoglycans of each of the dentures and crickets.
FIG. 11 is a graph showing the degree of SOD expression in blood after administration of insect glicorziminoglucan of rat and cricket to diabetic rats for 1 month.
FIG. 12 is a graph showing the carbonyl content in blood and liver after administration of glycoxaminoglucan for each month of insecticide of crustacea and crickets in diabetic rats.
13 is a graph showing the content of protein carnonyl in the blood and liver after administration of glycoxaminoglucan for each insect of a rat and a cricket in diabetic rats for 1 month.
FIG. 14 is a graph showing blood ALT, AST, BUN and blood glucose levels after administration of insect glycocalinoglycans of rat and crickets to diabetic rats for 1 month.

Hereinafter, the present invention will be described in detail.

The present invention relates to an antidiabetic composition containing glycosaminoglycan isolated from quebrain beetle queen bee or Hongliang, wherein said Honglangangai glycocamminoglycan is Hex ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2).

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

In this case, unless otherwise defined, technical terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the following description and the accompanying drawings, A description of known functions and configurations that may unnecessarily obscure the description of the present invention will be omitted.

[Example 1] Preparation and identification of N-glycans of a red tiger

The method for producing glycosaminoglycans from various insects such as crickets, crickets, quail beetles, etc. is described in 2015 as "N-glycans derived from Bonggun shells containing Bumblebee larvae and N-glycans derived from Western beetles and their uses (6/680 g), ganoderma (1.4 / 920 g) and bumble bee queen bee (2.8 / 1000 g). The swimmer used in the present invention is a black-winged swingle.

Preferably, the method comprises: a) grinding each insect and immersing it in ethanol or acetone having a volume equal to or more than the same amount for 3 days to remove the fatty flow, filtering and drying the residue (insect skin, etc.) b) The dried residue was pulverized with a ball mill or a hammer mill, 2.5% by weight of Bacillus lichenformis alkalase (pH 9.0) was added, and the protein was degraded by enzymatic treatment at 57 ° C for 4 days ; c) added to the enzymatic treatment solution of step b) to a final concentration of 5% (w / v) of 50% (w / v) trichloroacetic acid stored in the refrigerator and incubated at 4 ° C for 3 hours (100%) and potassium acetate for the residue volume obtained by centrifugation (8000 rpm), and the precipitate was again mixed with 2-fold volume of cetylpyridinium (5% by weight) ), And the precipitate was precipitated again in 2-fold volume of ethanol. The precipitate was dialyzed with a dialysis membrane having a molecular weight of 3500 or more as distilled water, and lyophilized to obtain a crude glycoside-derived peel- Obtaining minoglycans; d) The obtained crude glycosaminoglycan was subjected to salt gradient (0, 0.1, 0.5, 1 or 2.5 M NaCl in phosphate buffer (pH 7.0) using ion exchange chromatography (Sephadex DEAE A-25) ). ≪ / RTI >

On the other hand, the N-glycan structure in the thus-purified red ginseng glycosaminoglycan was identified as follows. First, N-glycosaminoglycan was purified by treating N-glycosaminoglycan of purified irritant with N-glycosidase, and then, N-glycans were prepared through a graphite cartridge, analyzed by MALDI-TOF and MS / MS (AXIMA resorcain, Shimadzu) ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2) Structure.

More specifically, to prepare N-glycan (N-glycan) only from glycosaminoglycan separated from the crude extract after the preparation of the extract of Hongwolla, and to analyze their sugar sequences, the following procedure Respectively.

The denatured buffer was mixed with the purified glomerulomaginosomes of purified kidney, and the protein portion was inactivated by boiling at 100 ° C for 10 minutes, and treated with 2 μl of N-glycosidase F (1000 U, UK) at 37 ° C for 20 hours The sugar and protein were cleaved to remove the protein moiety. Next, a porous carbon graphite cartridge (product name: Extract-clean columns carbograph, manufactured by Grace) was dissolved in 80% acetonitrile / water to separate N-glycan into tablets. After washing with a 0.1% trifluoroacetic acid (v / v) mixed solution, the conditions were adjusted with ultrapure water, the protein-free composition was loaded, and the mixture was allowed to stand for 10 minutes, then washed with 4 mL of ultrapure water, The pure N-glycans were obtained from the graphite cartridges and identified by TOF-mass spectrometry and MS / MS. Hex ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2) Structure (see Table 1 and Fig. 1).

[ Example  2] Anti-diabetic  Effect verification i

Each glycoprotein minoglucan was divided into 10 groups of genetically diabetic obese (db) mice (mean weight 47.6 ± 2.4 grams) (4 weeks) at a concentration of 5 mg / kg and 5 mg / kg, respectively, of CaG and quail bee glycocalypse minoglobin (IQG) and metformin 10 mg / Long - term weight, serologic pathologic changes, enzymatic activity in liver, antioxidant enzymes SOD, GPx and catalase in liver / blood. The conditions of this experiment are described in more detail below. The analysis of blood (serum / plasma) was performed by an automatic analyzer (Hitachi 7060 autochemical clinical analyzer, Tokyo), and the various experimental methods were also performed according to a conventional laboratory method.

Group 1: Normal (DB-Hetero) (PBS (phophate buffered saline))

Group 2: Diabetes (DB-Homo) (PBS)

Group 3: Diabetes (DB-Homo) + Huechys glycan (HEG) 5 mg / kg (0.2 ml)

Group 4: Diabetes (DB-Homo) + Queen of bumble bee glycan (IQG) 5 mg / kg (0.2 ml)

 Group 5: Diabetes (DB-Homo) + Dung beetle glycan (CaG) 5 mg / kg (0.2 ml)

 Group 6: Diabetes (DB-Homo) + Metformin 10 mg / kg (0.2 ml)

The weekly changes in body weight of each diabetic subject under these experimental conditions are shown in FIG.

Referring to FIG. 2, as compared with diabetic rats (DB-Homo group) in which metformin and glycosaminoglycan were not treated at all over time, CaG, IQG, and HEG treated with glycomazinoglycan of each insect It is confirmed that there is weight loss in the army.

FIG. 3 shows blood glucose levels in each rat. It can be seen that the blood glucose lowering effect is exhibited after 4 weeks in the group treated with glicorziminoglycan of Hongrangja, Tanger, queen beetle.

Next, the liver tissue was fixed and the slides were stained with toluidine blue-O to determine the number of adipocytes. The results were as follows: 1 month of administration of glycosaminoglycan (5 mg / kg) And a decrease in the local tax catch (Table 2).

Measuring the number of mast cells in toluidine-O blue stained cells of red ginseng, ginseng, bumblebees, queen bees, glaucosaminoglucan-treated liver in diabetic rats
Indicators
Specimen
Kinds unit Group CON (Db Hetero) Db Homo HeG5 CaG5 IQG5 metformin 10 Daily dose (mg / kg) - - 5 5 5 2 Fat cell Diabetic rat slide Cell number / mm ² 34 38 22 19 * 21 * 23 ratio (%) 100 111.77 64.71 55.88 * 61.76 * 67.65

Significance of diabetic rats * p <0.05

Serological changes of creatinine kinase, total cholesterol, LDL cholesterol, and hyaluronic acid were also observed in the diabetic rats, rats, quail beetles, 4. The catalase, GPX and GST activities are shown in FIG. 5, the SOD activity is shown in FIG. 6, and the protein carbonyl level is shown in FIG.

These results demonstrate that the antidiabetic effect of Catharsius molossus glycosaminoglycan in diabetic (db) mice can be confirmed by a decrease in serum glucose, creatinine kinase, and alkaline phosphatase (ALP) levels.

In particular, Ganggol glycosaminoglycan (CaG5) has an effect of increasing the activity of glutathione-transferase of catalase, SOD, GPX, hepatocyte-related antioxidant enzyme, and reducing protein oxidase through reduction of protein carbonyl, It is shown that the decrease in creatinine kinase, alkaline phosphatase, total cholesterol, and triglyceride is remarkable and the physiological activity is restored. Thus, potential potential as an antidiabetic drug that induces symptom relief of diabetes is confirmed.

Antioxidant enzyme activities and carbonyl content of dung beetle glycosaminoglycan in hepatocyte of db (+ leptin) mice

Each value represents mean ± SE statistically significant control (* p <0.05), (mean ± SE)

[Example 2] Identification of antidiabetic effect ii

Diabetic rats were dissected on Nov. 1, from September 27 to October 31, 2016, by administering Ganoderma lucosaminoglycan (CaG5) or cricket glycopolaminoglycan (Gbg5) for 1 month, Antioxidant effect (mechanism of oxidative damage restoration).

Hetero Db rats (normal mice) (11 rats); (PBS)

Homo Db rats (diabetic rats) (12 rats); (PBS)

Homozygous Db rats (diabetic rats) were treated with 5 mg / kg of strong rye lykorzaminoglycan (12 rats);

Homozygous Db rats (diabetic rats) were treated with cricket glycocamminoglycan 5 mg / kg (12 rats);

Homo Db mice (diabetic rats) received a positive control group, metformin 10 mg / kg (12 mice).

Each experiment was carried out according to the following figure.

The body weights of the experimental rats under the respective experimental conditions are shown in Table 4 and FIG. 8 below, and the change in each weight did not show a significant change.

Weight gain in diabetic rats at the time of one month administration of glutinous rice licorice minoglucan, cricket glycocamminoglycan

(mean 占 SE)

The blood glucose levels of the experimental rats under the respective experimental conditions are shown in FIG. 9 and Table 5. At this time, it was confirmed that cricket glicorzaminoglycan (GbG5) showed a blood glucose-strengthening effect in the first week (the results in Fig. 9 indicate one week, two weeks, and four weeks, respectively)

(mean 占 SE)

The organ weights of the experimental rats at each experimental condition are shown in Table 6.

* p < 0.05, ** p < 0.01

Antioxidant activity was measured by using experimental rats under these experimental conditions.

The increase in expression of catalase, GPx (Glutathione peroxidase) and GST (Glutathione S-transferase) in diabetic rats due to peritoneal administration of Ganoderma lucosaminoglucan and cricket glycocalinoglycans for 1 month is shown in Fig. 10 (GPx, GST is confirmed by cayman kit). The results of confirming the activity of superoxide dismutase (SOD) through the SOD-Chromogenic assay (Fridovich protocol) are shown in FIG. 11, it can be seen that the effect of increasing the activity of SOD is shown in the case of Guranko glycosaminoglycan and cricket glycopolaminoglycan. It can be seen from FIG. 12 that the carbonyl content in the blood neutrophils of the Gram-glycopolaminoglycan and the cricket glycocamminoglycan is also reduced. The results of FIGS. 10 to 12 are also shown in Table 7 below.

(mean 占 SE)

On the other hand, protein carbonyl was also detected in the supernatant obtained from the liver of the experimental rat (CaG group showed a decrease in protein carbonyl compared to the DB-Homo group ( p &Lt; 0.05)), and it was confirmed that there was no significance between the groups (Figs. 12 and 13;

Table 8 and FIG. 14 show the various hepatic and other serum levels in the serum of each experimental rat. The ALT (GPT), AST (GOT) and BUN values increased in the diabetic rats (Db-homo) Showed reduced figures in the cricket glycocalinoglycan administration group.

(mean 占 SE)

&Lt; Formulation Example 1 >

200 g of the glycosaminoglycan composition separated and purified from the bumblebee queen bee of Example 1 of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The glycosaminoglycan composition separated and purified from the bumblebee queen bee of Example 1 of the present invention was added to the cooking seasoning at 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

A bread, a cake, a cookie, a cracker and a noodle were prepared by adding the glycosaminoglycan composition separated from the bumblebee queen bee of Example 1 of the present invention to the wheat flour at 0.1 wt% Food was prepared.

Preparation Example 2-3. Manufacture of soups and gravies

The health enhancing soup and the juice were prepared by adding the glycosaminoglycan composition separated and purified from the queen bee bumblebee of Example 1 of the present invention to the soup and juice at 0.1 wt%.

Formulation Example 2-4. Manufacture of dairy products

The glycocalyaminoglycan composition isolated and purified from the bumble bee bug of Example 1 of the present invention was added to milk at 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

0.5 g of the glycosaminoglycan composition separated and purified from the bumble bee beetle of Example 1 of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation Example 2-6. Manufacture of fruit juice

Healthy fruit juice was prepared by adding 0.1 g of the glycosaminoglycan composition separated and purified from the bumble bee beetle of Example 1 of the present invention to 1,000 ml of apple juice or grape juice.

Claims (8)

A composition for anti-diabetic characterized by containing glycosaminoglycan separated and purified from bumblebee queen bee. Characterized in that it contains glycosaminoglycans containing N-glycans of Hex ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2) Compositions for diabetes. A composition for an anti-diabetic characterized by containing glycosaminoglycan separated and purified from crickets or tarsal. A food composition for prevention or improvement of diabetes characterized by containing glycosaminoglycan separated and purified from queen bee bumblebees. Characterized in that it contains glycosaminoglycans containing N-glycans of Hex ₇ (m / z 1175.2) or Hex ₇ HexNAc ₆ (m / z 1752.2) &Lt; / RTI &gt; A food composition for preventing or ameliorating diabetes characterized by containing glycoaminoglycans separated and purified from crickets or tall. A method for preparing a composition for anti-diabetes comprising the steps of:
a) The insect is crushed and immersed in an equal volume of at least one organic solvent selected from the group consisting of acetone, hexane, ethyl acetate, methyl alcohol, ethyl alcohol, propanol, butanol, acetonitrile, chloroform and dichloromethane for 2-5 days Removing the fat and filtering and drying the unremoved residue (such as insect bark);
b) pulverizing the dried residue with a ball mill or a hammer mill, adding 2-5% by weight of protease derived from Bacillus lichenformis alkaline (pH 9.0) or Aspergillus oryzae, And decomposing the protein by enzymatic treatment at 50 to 60 ° C for 3 to 7 days;
c) Add to the enzymatic treatment solution of step b) to a final concentration of 40 to 60% (w / v) Trichloroacetic acid in the refrigerator to 3 to 7% (w / v) Deg.] C for 2 to 4 hours to precipitate the protein. Then, the sugar is co-precipitated with 1.5 to 3 times ethanol (100%) and potassium acetate with respect to the residue volume obtained by centrifugation (5000 to 10000 rpm) The precipitate was precipitated in 1.5 to 3 times as much volume of cetylpyridinium (3-7% by weight), and then the precipitate was precipitated in 1.5 to 3 times volume of ethanol. The precipitate was dialyzed with distilled water using a dialysis membrane having a molecular weight of 3500 or more, Drying to obtain a crude glicorzominoglycan derived from the bark shell containing the bumblebug larvae pupa;
d) The obtained crude glycosaminoglycan was subjected to salt gradient (0, 0.1, 0.5, 1 or 2.5 M NaCl in phosphate buffer (pH 7.0) using ion exchange chromatography (Sephadex DEAE A-25) ) &Lt; / RTI &gt; 8. The method of claim 7,
E) culturing the cells together with endoglycosidase F to separate the sugar and protein linkages, and passing only the N-glycans through the graphite cartridges.

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