KR101403520B1 - Composition for enhancing immunity comprising alcohol extract of bumblebee as an active ingredient - Google Patents

Abstract

The present invention relates to a method for preparing an insect-derived heparin substitute composition and its use, and more particularly to a method for preparing an insect glycosaminoglycan or a heparin substitute composition capable of being used as a heparin substitute, To the use of the insect glycosaminoglycans or heparin substitute preparation compositions for inhibiting inflammation or for enhancing immunity. The production method of the present invention has an effect of providing glycosaminoglycan useful from insects. Through the production method of the present invention, it is possible to produce heparin polymers and low-molecular drugs, which are one kind of glycosaminoglycan, from various kinds of insects, and to produce substances such as heparin at low cost to contribute to the improvement of public health, It can contribute to the income conservation of the farm household.

Description

TECHNICAL FIELD The present invention relates to a composition for enhancing immunity comprising an alcohol extract of Bacillus subtilis as an active ingredient,

The present invention relates to a method for preparing an insect-derived heparin substitute composition and its use, and more particularly to a method for preparing an insect glycosaminoglycan or a heparin substitute composition capable of being used as a heparin substitute, To the use of the insect glycosaminoglycans or heparin substitute preparation compositions for inhibiting inflammation or for enhancing immunity.

Glycosaminoglycans (GAGs) refer to polysaccharides linked by glycosidic bonds, glycols, amino groups, and glycans. It is a type of carbohydrate molecular material that constitutes the inner wall of blood vessels. It is known to directly or indirectly participate in various functions such as metabolism of low density lipoprotein cholesterol, blood coagulation, and formation of cell epilepsy. The GAGs are structurally diverse and complex in shape through partial changes in the biosynthesis process. The GAG family has a basic skeleton in which a part of sulfuric acid is formed by repeating the uronic acid and the glucosamine, and the complex chemical structure enables various kinds of biological activities to be exhibited. GAGs in animals include hyaluronic acid, chondroitin sulfate, keratine sulfate, heparan sulfate, heparin, etc. Heparin is present only in macrophages along the arteries in the liver, lung, and mucosa, It is distributed in the organization.

Heparin sulfate (HS) is one of the only classes of macromolecular natural products that are abundant in the mammalian cell surface and extracellular matrix. HS is associated with blood clotting, embryonic development, inflammatory reactions, viral and bacterial infections. It is composed of repeating units of glucuronic acid or iduronic acid and glucosamine. In some cases, it may have a sulfate group. Heparin is a drug widely used as an anticoagulant and is a unique form of highly sulfated heparin sulfate. HS or HS-protein conjugates can have a variety of biological functions (anticancer, antiviral, improved anticoagulant) potential. Furthermore, there is a growing emphasis on the need to replace the various problems of contaminated heparin with animal tissue rather than synthetic heparin. A variety of target structures are specifically required, although currently commercially available pentacyclic Atrxtra is used in venous thromboembolic disorders and chemical synthesis of eight or more sugars is extremely difficult for bioequivalence assessment (Liu et al., Journal of Biological Chemistry, 285 (44), 34240-34249, October, 2010, Chemoenymatic design of heparan sulfate oligosaccharide). The report of heparan sulfate in insects reported that no heparan sulfate and conrouinic sulfuric acid were found in the fruit flies and salivary glands of the fruit fly (Sinnis et al., 2007. Mosquito heparan sulfate and its potential role in malaria infection and transmission. The journal of biological chemistry, 282 (35), 25376-25384).

Heparin is widely used as an anticoagulant and antihypertensive agent, and low molecular weight heparin may be administered to reduce the side effects of blood coagulation during surgery. Heparin is predominantly extracted from pigs, and chondroitin sulfate, widely used in medicine as a pseudo-heparin, is mainly obtained from the spinal cord cartilage of the shark and the bronchi of the bovine. Recently, it has been confirmed that chondroitin sulfate causes side effects associated with severe hypersensitivity shocks in the human body. Chondroitin sulfate has anti-factor Xa activity, which causes hypersensitive shocks in the human body. In addition, chondroitinase ABC or heparinase, which is a glycansolytic enzyme, is not decomposed and there is a possibility of residual blood.

Although heparin is widely used, it is usually derived from pigs. Recently, the outbreak of diseases in livestock such as foot-and-mouth disease has led to a sharp decrease in the number of pigs supplying raw materials of heparin, and the price of raw material of heparin has risen sharply. Therefore, it is required to develop a heparin substitute for the stable supply of heparin and to develop a technique for separating and purifying from a non-porcine source.

Therefore, the inventors of the present invention have studied to develop a novel substance or composition capable of being used as a substitute for heparin. As a result, it has been found that an alternative heparin having a low molecular unit structure is purified from insects to prevent blood clotting and anti- And thus the present invention has been completed.

It is therefore an object of the present invention to provide a method for separating and purifying an alternative agent of heparin from insects.

To achieve these and other advantages and in accordance with the purpose of the present invention,

(a) immersing an insect in an organic solvent to remove fatty acids and filtering to dry a residue that is not filtered;

(b) pulverizing the residue dried in the step (a);

(c) decomposing the protein in the pulverized product by treating the pulverized product obtained in the step (b) with a proteolytic agent;

(d) removing the protein digested in step (c); And

(e) obtaining a saccharide from the residue remaining after removing the protein in the step (d).

In order to achieve another object of the present invention, the present invention provides a method for producing a modified heparin fraction comprising hydrolyzing an insect glycosaminoglycan and removing a sulfate group.

In order to accomplish still another object of the present invention, the present invention provides a method for producing a heparin fragment, comprising treating heparinase with an insect glycosaminoglycan, and separating the heparin fragment from the degradation product, The method comprising:

In order to accomplish still another object of the present invention, the present invention provides an insect glycosaminoglycan or a heparin fragment produced by the production method of the present invention.

In order to achieve still another object of the present invention, there is provided an inflammation-suppressing composition comprising an insect glycosaminoglycan or a heparin fragment of the present invention as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides an immunomodulating composition comprising an insect glycosaminoglycan or a heparin fragment of the present invention as an active ingredient.

In order to accomplish still another object of the present invention, there is provided a composition for preventing and treating diseases caused by angiogenesis, which comprises the insect glycosaminoglycan or the heparin fragment of the present invention as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a composition for preventing and treating diseases caused by dysfunction of vasodilation reaction comprising an insect glycosaminoglycan or a heparin fragment of the present invention as an active ingredient.

Hereinafter, the present invention will be described in detail.

The general structure of heparin is that glucosamine and iduronic acid are composed of α-1,4 bonds, but the structure does not maintain unity depending on the extent of sulfation [ O ( ortho ) -sulfonation] in the process of biosynthesis. Sulfation is carried out on the second and sixth times of glucosamine and iduronic acid. And some glucosamine is in a very complex form with acetyl groups instead of sulfate groups.

In contrast, chondroitin sulfate (CS) separates from the cartilage tissue of the terrestrial animal's bronchi and fish. The structure of N-acetylgalactosamine glucuronic acid is a combination of β-1,3 and β-1,4 . Sulfate groups are mainly substituted in galactosamine 4 and 6. The average molecular weight is 13,000 Da for heparin and 20,000 Da for chondroitin sulfate. In addition, chondroitin sulfate B or dermatan sulfate (DS), N-acetylgalactosamine and iduronic acid are also in the form of repeating structure due to the combination of β-1,3 and β-1,4. The sulfate group is mainly substituted at position 4 of glucosamine. Recently, the deaths of patients who received heparin intravenously in the United States and Germany have caused great social concern about foreign substances in heparin. A recent report suggests that peroxidized chondroitin sulfate (OSC) and oversulfated chondroitin sulfate (OSC) in heparin are mixed with foreign substances (Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events, Nature Biotechnology, 26 (6), 669-675, 2008; Imarang et al., Herb Medicine Journal 40 (2), 109-117 2009).

Thus, the present inventors isolated and purified glycosaminoglycans from insects, identified the monosaccharide composition and heparin disaccharides, and revealed the unit structure of insect-derived heparin substitute, thereby enabling an insect heparin preparation. When focusing on the use of heparin for purposes other than anticoagulants, it is important to remove the anticoagulant and hemorrhagic functions adequately, and if it is used as a medicine, structural identification as a substance should be possible. Using a specific method of desulfurizing and oxidizing glycosaminoglycan, such as heparin having a sulfate group, the anticoagulant and hemorrhagic activity is substantially eliminated, while the biologically beneficial effect on the living body, such as affinity for physiologically active substances, is maintained. Glycosaminoglycan was successfully prepared, and the unidentified structure was greatly reduced so that the structure identification as an important substance from the viewpoint of medicine could be easily achieved. Thus, the present invention has been completed.

Specifically, glycosaminoglycan having a sulfuric acid group after 2M to 3M hydrochloric acid hydrolysis was acetylated, and N, O-bis (trimethylsilyl) trifluoroacetamide (hereinafter referred to as BSTFA , Which is obtained by subjecting 6-O-sulfate groups to substantially all the elimination from the glucosamine residue by evaporation of pyridine from the reaction mixture by silylation by heat treatment in the presence of a reducing agent (A combination of silylated glycosaminoglycan hydrolyzate (a combination of a silyl glycosaminoglycan degraded monosaccharide acetic acid) and a recombinant heparinase, which is an insect glycocinminoglycan isolated and purified And to provide a final disrupted polysaccharide and disaccharide composition of oligohydrin polysaccharide.

Meanwhile, crude glycosaminoglycan as an intermediate stage purification intermediate can be used as an additive for various cosmetics and shampoos. Currently, glycosaminoglycan is widely used as a functional ingredient of animal medicines in antler grains and the like, (BMJ 2010; 341: c4675, doi: 10.1136 / bmj.c4675). The results of the Swiss clinical trials have shown that there is no effect on the outcome.

The insect-derived glycosaminoglycan of the present invention exhibited a reproducible effect in the chronic inflammation model rats, unlike the glucosamine, in the triple repeat animal experiment, and the scurvy extract also inhibited inflammation and cytokines such as inflammatory cytokine ), Respectively. Therefore, the present inventors attempted to develop an antibody to confirm the effectiveness of insect-derived glycosaminoglycan. In addition, insect glicosaminoglycan having a molecular weight of 10,000 to 50,000, which is a macromolecule, was prepared in order to develop a safe alternative to heparin, a glycoprotein derived from swine, which is concerned with diseases (foot and mouth disease) After the administration of comfrey frid complements to chronic arthritis to induce foot palsy and chronic arthritis, the insect glicosaminoglycan, which was isolated and purified, was observed daily for 14 days. The effect of arthritis was confirmed by comparing the cytokine levels in rat serum with the inflammation - induced group and the positive control group after 14 days of administration and recovery of inflammation.

In addition, the structure of purified insect glycosaminoglycan was reduced to low molecular weight and acidified by elimination of sulfate group, or acid (acid group) was added to amino sugar and it was excreted well by urine when it was detoxified. In order to analyze the components of the enzymatic degradation product used in the GC-MS analysis, the sample was dissolved in 3M HCl dissolved in methanol solvent, heated at 120 ° C for 2 hours, cooled to room temperature, dried at 40 ° C under nitrogen gas, acetic anhydride was added and the mixture was allowed to stand at room temperature for 30 minutes and then dried at 40 DEG C under a nitrogen gas to carry out acetylation. The acetylation was carried out at 80 DEG C in a mixed glycosamine- The glycansaminoglycan of several glycans was found at 60 ℃ and 40 ℃ at different acetylation reaction conditions. Therefore, the desired unit of glycosaminoglycan was obtained by controlling the reaction temperature of the acetylation. In addition, recombinant heparinase and insect glycosaminoglycan are cultured together at 37 ° C to obtain a heparin 2 saccharide, which is a decomposition product of macromolecular insect glycosaminoglycan. As a combination of these disaccharides, new heparin or heparan Sulfuric acid could be obtained. Thus, heparinase I, which decomposes heparin, heparinase III, which specifically degrades heparan sulfate, heparinase II, which functions as heparinase I and III, which decomposes heparin and acaran sulfate, It is possible to obtain insect glicosaminoglycans and low-molecular-weight heparin, which are variously used as insects, in the present invention. Thus, the present invention has been completed by preparing insect-derived low-molecular-weight heparin from heparinase I and heparinase III .

Accordingly, the present invention provides a novel method for producing a glycosaminoglycan or a heparin substitute, which method comprises providing a method for producing glycosaminoglycan having an insect as a raw material. The method for producing an insect glycosaminoglycan according to the present invention comprises

(a) immersing an insect in an organic solvent to remove fatty acids and filtering to dry a residue that is not filtered;

(b) pulverizing the residue dried in the step (a);

(c) decomposing the protein in the pulverized product by treating the pulverized product obtained in the step (b) with a proteolytic agent;

(d) removing the protein digested in step (c); And

(e) obtaining a saccharide from the residue remaining after removing the protein in the step (d).

In the step (a), the fatty acid is removed in order to separate the proteoglycan. Preferably in an organic solvent such as acetone, hexane, ethyl acetate, methyl alcohol, ethyl alcohol, propanol, butanol, acetonitrile, chloroform or dichloromethane or a mixture thereof. The immersing time is not limited thereto, but is preferably 1 to 4 days, when it is sufficient to remove the fat. The fat is removed by filtering out the dissolved organic solvent, and unfiltered residue is obtained.

The residue dried in step (b) may be pulverized with a ball mill or a hammer mill so as to have a particle size of 100 to 300 mesh to promote protein degradation at a later stage. When the particle size exceeds 300 mesh, the enzyme decomposition may be delayed, and when the particle size is less than 100 mesh, there is a fear that the particle size may flow into the supernatant liquid during centrifugation.

The step (c) is a step of treating the pulverized product obtained in step (b) with a proteolytic agent to decompose the protein in the pulverized product. Proteolysis is preferably carried out under optimal pH and temperature conditions depending on the enzyme, preferably in a sodium bicarbonate solution (pH 9.0). Protein release was induced by Bacillus licheniformis, etc.) protease enzymes {e.g., Novozyme (Sigma)} and / or Aspergillus (for example, Sigma (USA) or Apis Biotech (Korea)} which is a protease (protease) of oryzae , and may be a fermented bacterial liquid to be fermented into soybean or soybean fermented food or a liquid such as yogurt Can be used. In this case, the time required for proteolysis (fermentation) is 3 to 7 days, which can accelerate protein decomposition at room temperature or higher, and can accelerate reaction at 44 ° C. By using 2 to 5% by weight of the protein disintegration, protein of the insect bark can be effectively removed.

(d) is a step of removing the protein disassembled in the step (c). May be carried out according to methods known in the art and preferably by centrifugation with trichloroacetic acid (5% by weight).

(e) is a step of obtaining a saccharide from the residue remaining after removing the protein in the step (d). The sugar can be co-precipitated with ethanol, potassium acetate or sodium acetate, and the precipitate can be further precipitated, Is precipitated in cetylpyridinium, then the precipitate is precipitated again in ethanol, and the resulting precipitate is dialyzed with distilled water.

In the production method of the present invention, the acidic fraction is collected by repeating the above steps (a) to (e), subjected to ion exchange chromatography, and then subjected to salt gradient (0, 0.1, 0.5, ) And then collecting the fraction containing uronic acid can be further roughened. At this time, the necessary purity information can be determined by, for example, electrophoresis.

In the production of the glycosaminoglycans of the present invention, the insects may be selected from the group consisting of silkworms such as silkworms, caterpillars, caterpillars, crickets, bumble bee queen bee, queen bee, beetle, bumble bee bark, , Western Bumblebee, Bomyx mori, silkworm pupa, clownfish , Poecilocoris lewisi ). < / RTI > Preferably, the insect may be the bumblebee larva pupae bark or the Western beetle.

In addition, the alcohol may be selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, for example, ethanol, methanol, propanol, isopropanol, butanol.

On the other hand, the present invention provides an insect glycosaminoglycan produced by the production method of the present invention.

The silkworm pupa Glycosaminoglycan is not limited to, but is not limited to, glycozaminoglycan extracted from the silkworm silkworm, but may preferably be? UA-2S- [1-4] 4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo- [1-4] glucosamine and UA- [1-4] GlcN- 4-enopyranosyluronicacid- [1-4] glucosamine 6-sulfate. It is a glycosaminoglycan having a repeating structure. When heparinase is cultured, it converts to heparin disaccharide of I-H and II-H. The composition of the monosaccharide can be changed by the variation of acetyl group depending on the temperature of acethylation addition, and it can be changed by composition ratio of D-Glucosamine 44% and 17.5% uronic acid (glucosaminic acid 1.8 2S- [1-4] GlcN and UA- [1-4] GlcN-6S in a ratio of 10% + galactosaminic acid 7.3% + galacturonic acid 2.4% + glucuronic acid 6.0%.

Nougle glycosaminoglycans have heparin disaccharides IH and IV-H when cultured, such as heparinase. D-galactosamine-6-sulfate, D-galacturonic acid, and D-glucuronic acid are present as glucosamine (%), N-acetylglucosamine and D-galactosaminic acid.

Glucosaminyl glycans are glycosaminoglycans having repeating structures of? UA-2S- [1-4] GlcN and? UA- [1-4] GlcN-6S. IH, II-H, IV-H, and IV-A heparin-2 disaccharides. As shown in the second reaction table, the basic unit molecular structure of acetylation is 40.4%, D-galactosamine is 4.3% and N-acethylgalactosamine is 20.0%. D-glucuronic acid 18.6%, D-galactosaminic acid 11.5%, D-glucosamic acid 4,1% and the like. [1-4] GlcN and UA- [1-4] GlcN-6S at a ratio of 1.8% glucosaminic acid, 7.3% galactosaminic acid, 2.4% galacturonic acid and 6.0% glucuronic acid.

It is not limited to glycocalinoglycans extracted from cicadidae, but can be converted into heparinase degradation products of II-H and IV-H heparin 2 saccharides. chondroitin sulfate (ΔUA- [1-.3] -N-aceticylgalactosamine-N-sulfate series), and preferably UA- [1-4] GlcN-6S, Glucosaminoglucan having a repeating structure of? UA-GlcNS (glucuronic or galacturonuc acid-N-glucosamine-N-sulfate series) and / or? UA-2S-GlcN (4-deoxy- enopyranosyluronicacid-2-sulfo-glucosamine) which is a glycosaminoglycan. Acetylation is also different depending on the addition temperature, but its basic unit molecular structure is acetylation. As shown in the second reaction table of acetylation 40 ° C, an amino acid having 73.9% of D-Glucosamine and 7.4% of N-acethylglucosamine and an acid having uronic acid D-glucuronic acid 2.0%, D-galactosaminic acid 4.1%, D-galacturonic acid 10.8%, D-glucosamine 6-sulfate 1.1%

Cricket glycopolaminoglycans are glycosaminoglycans having a repeating structure of? UA- [1-4] GlcN-6S and? UA - [1-4] GlcN. It is possible to convert heparinase decomposition products of heparin-2 saccharides II-H and IV-H, although the ratio varies depending on the acetylation addition operating temperature. As shown in the second reaction table, D-glucosamine and D-galactosaminic acid (D-galactosaminic acid) with 73.9% of N-acetylglucosamine and 7.4% of N- acid 4.1%, D-galacturonic acid 10.8% and D-glucosamine 6-sulfate 1.1%.

The bumblebeing queen bee glycocamminoglucan was synthesized by the reaction of UA- [1-4] GlcN-6S (4-deoxy-L-threo-hex-4-enopyranosyluronicacid- [ 1-4] GlcN-6S (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo- [ Which is a glycosaminoglycan. II-H, IV-H, IH, heparin-2 disaccharides. Acetylation is different depending on the addition temperature, but its basic unit molecular structure is as follows: Acetylation 40 ° C As shown in the second reaction table, D-Glucosamine 42.0%, D-galactosamine 2.7% and N-acethylglucosamine 9.3% D-glucosaminic acid 3.0%, D-galactosaminic acid 3.3%, D-galacturonic acid 24.0% and D-glucosamine 6-sulfate 7.2%.

4-deoxy-L-threo-hex-4-enopyranosyluronic acid-glucosamine 6-sulfate, UA- [1-4] GlcNAC (4-deoxy- L-threo-hex-4-enopyranosyluronicacid-N-cetyl- [1-4] glucosamine and UA- [1-4] GlcNS-6S (4-deoxy-L-threo-hex-4-enopyranosyluronicacid- - [1-4] glucosamine 6-sulfate) and a ΔUA - [1-4] GlcN repeating structure. IV-H, III-S, II-H It is possible to convert heparin-2 saccharide to decomposition product of heparinase. As shown in the second reaction table, the basic unit molecular structure of D-galactosamine is 59.8%, D-galactosamine 3.9%, N-acetylglucosamine 15.0% D-glucosaminic acid, D-glucosaminic acid, D-glucosaminic acid, D-glucosaminic acid, D-glucosaminic acid,

Glucosaminoglucan is a member of 4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo-glucosamine 6-sulfate, UA- [1- 4] deoxy-L-threo-hex-4-enopyranosyluronicacid- [1-4] glucosamine 6-sulfate and ΔUA- [1-4] GlcN repeating structures, which are glycosaminoglycans . II-H, IV-H, IH It is possible to convert heparin-2 saccharide to decomposition product of heparinase. Acetylation Although the ratio varies depending on the additional operating temperature, the basic unit molecular structure is as follows: Acetylation 40 ° C As shown in the second reaction table, the amino sugar of D-glucosamine 51.1%, D-galactosamine 0.1% and N-acethylglucosamine 13.3% D-glucosaminic acid, D-glucosaminic acid, D-glucosaminic acid, D-glucosaminic acid, D-glucosaminic acid,

Although the ratio of the glycosaminoglycan (IgpcG) in the bumble bees is different according to the supplementary operation temperature, the basic unit molecular structure thereof is as follows: Acetylation 60 ° C. As shown in the fourth reaction table, D-glucosamine 7.9%, D-glucuronic acid 6.0 [1-4] GlcN-6S, Δuronic acid- [1-4], which has 5.4% of N-acetyl-D-galactosamine-4-sulfate, 46.7% of D-galactosaminic acid and 12.0% of D- ] glucosamine 6-sulfate and a repeating structure of? UA - [1-4] GlcN. II-H, IV-H, IH It is possible to convert heparin-2 saccharide to decomposition product of heparinase.

Worker of B. terrestris Glicorzaminoglycan is a type of 4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo-glucosamine 6-sulfate, UA- [1-4] GlcN-6S (4-deoxy-L-threo-hex-4-enopyranosyluronicacid- [1-4] glucosamine 6-sulfate) and ΔUA- [1-4] Lt; / RTI > II-H, IV-H, IH It is possible to convert heparin-2 saccharide to decomposition product of heparinase. As shown in the fourth reaction table, the basic unit molecular structure of D-galactosamine is 3.6%, D-galactosamine 1.3%, N-acethylglucosamine 6-sulfate 2.3% N-Acetylglucosamine An acid group with 3.3% of amino sugars and uronic acid, containing 8.2% of D-galacturonic acid, 1.8% of D-glucosaminic acid, 55.1% of D-galactosaminic acid and 0.5% of D-glucosamine 6-sulfate.

It is preferable that the glycosaminoglycan is glycoprotein minoglucan extracted from the clown barnacle, but it is preferably in the form of a crude glycosaminoglycan such as glycosaminoglycan having a repeating structure of? UA-GlcNAC Glycan, and / or a repeating structure of? UA-GlcNAC.

In the present invention,? UA or? UA means 4-deoxy-L-threo-hex-4-enopyranosyluronic acid, GlcN means D-glucosamine, Ac means Acetyl, NS, 2S, sulfo, 2-sulfate or 6-sulfate, respectively.

The present invention also provides a process for producing a modified heparin fraction comprising the step of hydrolyzing an insect glycosaminoglycan produced by the method of the present invention, that is, the insect glycosaminoglycan produced by the steps (a) to (e) . ≪ / RTI >

Preferably, the hydrolysis may be acid hydrolysis, the glycosaminoglycan is dissolved in a methanol solvent, and then treated with a strong acid and a high temperature to effect acid hydrolysis. Also, after the glycosaminoglycan having a sulfate group is acetylated, substantially all of the sulfate groups are removed from the residue by a silylation reaction, and then the pyridine is evaporated from the reaction mixture to remove the sulfate group. In the above, the sulfate group may be 6-O-sulfate group, 2-O-sulfate group, 4-O-sulfate group or NO-sulfate group.

In addition, in the present invention, the heparin fragment is a low molecular weight heparin including a disaccharide-modified heparin basic structure formed by heparinase and acid hydrolysis including two monosaccharide hydrolysis products, which are the basic structure of heparin, Of oligosaccharides and the like.

When the acetyl hydride (acetic anhydride) is subjected to acetylation at a temperature of 80 ° C or more for 30 minutes or more at a high temperature, hydrolysis of insect glycosaminoglycan with 2M or more of hydrochloric acid dissolved in anhydrous methanol, Desulfurization and oxidation of sulfuric acid groups such as 4-O-sulfate groups and 2-O-sulfate groups including sulfate groups are possible.

More specifically, in the present invention, a sample is dissolved in insect glycosaminoglycan in 3M HCl dissolved in a methanol solvent, followed by heating at 100 ° C or more for 2 hours to hydrolyze it. After it cooled to room temperature and allowed to stand at room temperature for 30 minutes was added to 0.3 ml Acetic anhydride (acetic anhydride) and dried in a nitrogen gas at 40 ℃, dried under nitrogen gas at 40 ℃ P ₂ O ₅ (2: 1, v / v) of pyridine / N, O-bis (trimethylsilyl) trifluoroacetamide and silylation at 80 DEG C for 30 minutes, Denatured heparin fragments were prepared.

The present invention also relates to a method of production of the present invention, that is, a method of treating heparinase by treating heparinase in an insect glycosaminoglycan produced by the steps (a) to (e) and decomposing the heparin fragment (Such as an oligomer) comprising the steps of:

More specifically, in the present invention, recombinant heparinases I, II, and III were insufficiently treated with insect glycosaminoglycan at 37 ° C and separated by liquid chromatography (HPLC) to obtain 2 saccharides, 4 Saccharides, hexoses and oligosaccharides of heparin fragments (oligomers, etc.) were prepared.

The present invention also provides an antibody against insect glycosaminoglycan. Preferably, the purified insect glycosaminoglycan is mixed with an equivalent amount of complete Freund's adjuvant overnight, stirred overnight to form an emulsified solution, and then primary immunization is induced. Then, incomplete Freund's adjuvant is administered at intervals of 12 days The same amount of insect glycosaminoglycan was mixed with the second and third immunizations to induce booster, and the serum was separated from the experimental animals to obtain a polyclonal antibody. As described above, the present invention provides an antibody produced by mixing complementary insect glicosaminoglycan, which is made from a variety of insect-derived heparin or heparan sulfate antibody biological drugs.

In addition, the present invention provides an inflammation-inhibiting composition comprising an insect glycosaminoglycan or a heparin fragment produced by the method of the present invention as an effective ingredient.

The present invention also provides a pharmaceutical composition for enhancing immunity comprising an insect glycosaminoglycan or a heparin fragment produced by the method of the present invention as an active ingredient.

The insect glycosaminoglycan or heparin fragment of the present invention has an inflammation-inhibiting effect on phospholipase A, VEGF, NOS, COX-2, and PGE2 to provide a composition for prevention and treatment of inflammation. It can exhibit a thrombolytic action by the anticoagulant action, and it is possible to treat and / or prevent various thrombotic diseases including coronary artery disease (angina pectoris and the like) and cerebrovascular disease. More specifically, the insect glycosaminoglycan or heparin fragment of the present invention of the present invention can be used for the prevention and treatment of hypertension, stroke, cerebral hemorrhage, ischemic heart disease or Raynaud's disease due to dysfunction of the vasodilation reaction. In addition, since it has the effect of inhibiting angiogenesis including the insect glycosaminoglycan or the heparin fragment of the present invention, it is possible to provide a composition for preventing or treating cancer growth and metastasis, angioma, angiofibroma, arthritis, diabetic retinopathy , Retinopathy of prematurity, neovascular glaucoma, neovascularization, corneal disease, degeneration, degeneration of spots, pterygium, retinal degeneration, posterior capsular hyperplasia, granular conjunctivitis, psoriasis, capillary dilatation, purulent granuloma, seborrheic dermatitis, acne And can be usefully used for the prevention and treatment of diseases caused by at least one angiogenesis selected from the group consisting of.

Insect Glycosaminoglycan is preferably a crude insect glycosaminoglycan in which the pigment is hydrolyzed in the form of an insect glycosaminoglycan purified in the form of a pigment, 2 saccharides, 2 saccharides, 2 saccharides, 3 saccharides, 4 saccharides, hexoses, and aralia.

The pharmaceutical composition according to the present invention may comprise the insect glycosaminoglycan or the heparin fragment alone produced by the method of the present invention in a pharmaceutically effective amount or may further comprise one or more pharmaceutically acceptable carriers . The pharmaceutical compositions of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. The term "pharmaceutically effective amount" as used herein refers to an amount that exhibits a higher level of response than a negative control, and preferably refers to an amount sufficient to treat or prevent obesity. The effective amount of the insect glycosaminoglycan or heparin fragment produced by the production method of the present invention is 0.001 to 1000 mg / day / kg body weight, but is not limited thereto. However, the pharmaceutically effective amount may be appropriately changed depending on various factors such as the disease and its severity, the patient's age, body weight, health condition, sex, administration route and treatment period.

The composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. Routes of administration include, but are not limited to, oral or parenteral administration. Parenteral routes of administration include, for example, various routes such as transdermal, nasal, peritoneal, muscular, subcutaneous or intravenous.

When the pharmaceutical composition of the present invention is orally administered, the pharmaceutical composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a capsule, a liquid, a gel , Syrups, suspensions, wafers, and the like. Examples of suitable carriers include saccharides including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-cellulose, etc., and fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated together with a suitable parenteral carrier according to methods known in the art in the form of injection, transdermal drug delivery, and nasal inhalation. In the case of such injections, they must be sterilized and protected against contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils . More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. may be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be further included. In addition, the injections may in most cases additionally include isotonic agents, such as sugars or sodium chloride. Examples of transdermal dosage forms include ointments, creams, lotions, gels, solutions for external use, pastes, liniments, and air lozenges. By "transdermal administration" as used herein, it is meant that the pharmaceutical composition is locally administered to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

These formulations are described in Remington's < RTI ID = 0.0 > Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania.

In the case of inhalation dosage forms, the compounds used according to the present invention may be formulated into a pressurized pack or a pressurized pack using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a compound, and a powder mixture of a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., 1995, Mack Publishing Company, Easton, PA).

In addition, insect glycosaminoglycan or heparin fragments produced by the production method of the present invention may be provided in the form of a food composition for the purpose of suppressing inflammation or enhancing immunity. The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

For example, as a health food, the insect glycosaminoglycan or the heparin fragment itself produced by the production method of the present invention may be prepared in the form of tea, juice, and drink for drinking, granulated, encapsulated and powdered It can be ingested. In addition, insect glycosaminoglycan or heparin fragments produced by the production method of the present invention can be mixed with a known substance or active ingredient known to have an inflammation-suppressing or immunostimulating effect and can be produced in the form of a composition.

Functional foods also include beverages (including alcoholic beverages), fruits and processed foods (such as canned fruits, bottles, jams, maalmalade, etc.), fish, meat and processed foods such as ham, sausage, ), Breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, Retort food, frozen food, various kinds of seasoning (e.g., soybean paste, soy sauce, sauce, etc.) by adding the insect glycosaminoglycan produced by the production method of the present invention.

The preferred content of the insect glycosaminoglycan or heparin fragment produced by the method of the present invention in the food composition of the present invention is not particularly limited, but is preferably 0.01 to 50% by weight in the finally prepared food.

In addition, in order to use the insect glycosaminoglycan or the heparin fragment produced by the method of the present invention of the present invention in the form of a food additive, it may be used in the form of a powder or a concentrate.

In addition, the present invention provides a cosmetic composition for relieving / improving inflammation comprising an insect glycosaminoglycan or a heparin fragment as an active ingredient.

The cosmetic composition may be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetics field. Liquid or solid form of cosmetics may include, for example, but not limited to, lotions, creams, lotions and bath salts. At this time, the bases, adjuvants and additives commonly used in the cosmetics field are not particularly limited and may include, for example, water, alcohol, propylene glycol, stearic acid, glycerol, cetyl alcohol and liquid paraffin.

More specifically, the cosmetic composition of the present invention contains the insect glycosaminoglycan or heparin fragment of the present invention, together with dermatologically acceptable excipients, and a cosmetic composition (such as a lotion, cream, essence, cleansing foam and cleansing water Hair cosmetic composition (such as foundation, lipstick, mascara, make-up base), hair product composition (shampoo, rinse, hair conditioner, hair gel), functional cosmetic composition . ≪ / RTI > Such excipients include, but are not limited to, emollients, skin penetration enhancers, colorants, perfumes, emulsifiers, thickeners and solvents. In addition, it may further contain flavors, pigments, bactericides, antioxidants, preservatives, moisturizers and the like, and may include thickeners, inorganic salts and synthetic polymeric substances for the purpose of improving physical properties.

For example, in the case of producing a cleanser and a soap containing an insect glycosaminoglycan or heparin fragment of the present invention, an insect glycosaminoglycan can be easily added to an ordinary cleanser and a soap base. In the case of producing a cream, it can be prepared by adding an insect glycosaminoglycan to a general underwater type (O / W) cream base. A synthetic or natural material such as a flavor, a chelating agent, a coloring matter, an antioxidant, an antiseptic, and a protein, a mineral, and a vitamin for the purpose of improving a physical property may be further added.

The content of insect glycosaminoglycan or heparin fragment contained in the cosmetic composition of the present invention may be 0.0001 to 50% by weight, preferably 0.01 to 10% by weight based on the total weight of the cosmetic composition.

On the other hand, the present invention provides a heparin substitute preparation containing an insect glycosaminoglycan or a heparin fragment as an effective ingredient.

In addition, the present invention provides a composition for enhancing immunity comprising an alcohol extract of an insect selected from the group consisting of Western beetles, papper moth, larvae and pupae. In addition, the present invention provides an anti-inflammatory composition comprising an alcohol extract of an insect selected from the group consisting of a Western beetle, a larva, and a pupa. The present invention also provides a composition for the prevention and treatment of diseases caused by angiogenesis, comprising an alcohol extract of an insect selected from the group consisting of Western beetles,

The alcohol may be a lower alcohol having 1 to 4 carbon atoms selected from the group consisting of ethanol, methanol, propanol, isopropanol and butanol. The alcohol may be used as an anti-inflammatory activity of the composition of the present invention to prevent or treat arthritis Can be used.

Therefore, the production method of the present invention has an effect of providing glycosaminoglycan useful from insects. Through the production method of the present invention, it is possible to produce heparin polymers and low-molecular drugs, which are one kind of glycosaminoglycan, from various kinds of insects, and to produce substances such as heparin at low cost to contribute to the improvement of public health, It can contribute to the income conservation of the farm household.

Figure 1 shows the effect of insect glycosaminoglycan on complement-induced rat edema. The results show that PSWG (SWPG, PSG, silkworm, Glycosaminoglycan) is effective for the prevention of complement-induced sprouting by the daily administration for 14 days. IGPCG (bumble bee larva peeled bark beetle glycosaminoglycan); Indices of indomethacin are expressed as mean ± standard error, and PSWG or IGPCG or IQG vs. IQG (indomethacin ) CFA, P < 0.05.
Figure 2 shows the effect of interleukin-6 on the insect glycosaminoglycan in the complement-induced arthritis rats.
FIG. 3 shows the effect of interleukin-6 of insect glycosaminoglycan on complement-induced arthritis rats.
Fig. 4 shows the inhibitory effect of insect glycosaminoglycan on endothelial angiogenic factor in a complement-induced arthritis rat.
FIG. 5 shows the effect of interleukin-6 of insect glycosaminoglycan on complement-induced arthritis rats.
FIG. 6 shows the effect of interleukin-6 of the insect glycosaminoglycan on the complement-induced arthritis rats.
Figure 7 shows the inhibition of the cytooxygenase-2 of insect glycosaminoglycan in the complement-induced arthritis rats.
Fig. 8 shows a comparison with a positive control group of the insect glecocoaminoglycan in the rabbit rats. The 14-day repeated administration of glicojaminoglycan (glucosamine) was compared with the effect of inhibiting podocyte growth. BTWG: Glucosaminoglycans derived from Western beetles; PSWG (or PSG, or SWPG): Glucosaminoglycans from silkworm larvae; CjGLU: Commercially available glucosamine (glycosaminoglycan, positive control); IND: Indomethacin (an anti-inflammatory positive control)
Figure 9 compares TNF-a positive control of insect glycosaminoglycan in arthritic rats.
Figure 10 compares the VEGF positive control of insect glycosaminoglycan in arthritic rats.
Fig. 11 shows the results of comparative experiments with prostaglandin E ₂ positive control of insect glycocalinoglycan in arthritic rats.
Figure 12 shows the results of comparative experiments with phospholipase A ₂ positive control of insect glycosaminoglycan in arthritic rats.
Figure 13 shows the results of a comparative experiment with a cytooxygenase-2 positive control of insect glycocalinoglycan in arthritic rats.
Fig. 14 shows the results of a comparative experiment with an interleukin-6 positive control of insect glycosaminoglycan in arthritic rats.
Fig. 15 shows the effect of the extract of Isabella beetle alcohols on the worm-induced rat edema. SDIEX: Occidental Alcohols Extract; SPDYBEX: Alcohole extract like a papule flyback larva pupa.
FIG. 16 shows the inhibitory effect of interleukin-6 on the extracts of callus bean alcohols from Western beetles in the co-induced rat edema (chronic arthritis model rat). SDLEX: Western beetle larvae alcohol extract 100 mg / kg; SPDYB: Alcohole extract 100 mg / kg like papule flyback larvae pupa; IND: indomethacin 1 mg / kg
17 shows the inhibitory effect of VEGF (endothelial vascular growth factor) inhibitor on the extracts of Solanum vulgare L. monocytogenes from western birds of the beetle in the case of complement-induced rat edema (chronic arthritis model rat).
FIG. 18 shows the inhibitory effect of cyclooxygenase-2 (COX-2) on the extracts of callus bean alcohols from Western beetles in chronic arthritis model rats.
FIG. 19 shows the inhibitory effect of secreted form phospholipase A ₂ (sPLA ₂ ) on the extracts of callus bean alcohols from Western beetles in chronic arthritis model rats.
Fig. 20 shows the rat necrotic factor (rTNF-alpha) inhibitory effect of alcoholic beetle alcohole extracts of Western beetles in chronic arthritis model rats.
Fig. 21 shows the effect of the glycosaminoglycan of the present invention on VEGF production.
FIG. 22 shows the effect of the glycosaminoglycan of the present invention on VEGF production of bovine pulmonary vascular endothelial cells.
23 shows the effect of the glycosaminoglycan of the present invention on the production of VEGF (vascular endothelial growth factor) production of human HUVEC endothelial cells.
24 shows the effect of the glycosaminoglycan of the present invention on production of NOS (nitric oxide synthase) on pulmonary vascular endothelial cells.
25 shows the effect of the glycosaminoglycan of the present invention on the increase of the amount of NOS enzyme to the endothelial (HUVEC) leading from the human abdomen to the urethra.
26 shows the results of assaying the activity of the glycosaminoglycan of the present invention on the mass production of the antibody.

Below. The present invention will be described in detail by examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

From insect bark residue Insect Glycosa Minoglucan  refine

<Experimental Method>

1. Insect Extract  Produce.

In order to remove lipid components from the fresh insects (the type of insect species used), they were left in acetone as an organic solvent for two days and then dried at room temperature. 50 g of the sample (dry weight) was added to 500 mL of 0.05 M carbonate buffer solution (pH 9.0), and 8 mL of alcholase, a kind of thermostable protease, was added and cultured at 60 DEG C for 48 hours. After incubation, trichloroacetic acid was added to give a final concentration of 5%, and the mixture was allowed to stand in a refrigerator at 4 ° C for 1 hour. The mixture was centrifuged at 8000 rpm to collect the supernatant, and then the supernatant was washed with 1% potassium acetate Alcohol was added in three times volume and precipitated and dried to prepare an insect extract.

2. Insect From extract  Purification of polysaccharides

The insect extract was dissolved in water, the water-insoluble material was removed by centrifugation, and 5% cetylpyridinium chloride was added in 1/4 volume to form a complex to precipitate. The precipitate was centrifuged, dissolved in 2.5 M sodium chloride, and precipitated with alcohol to be purified. The purified sample was again dialyzed by dissolving in a small amount of water and then lyophilized to purify the polysaccharide.

3. Binding protein  remove

Β-elimination reaction was carried out to remove binding proteins bound to the polysaccharide. 75 mg of the purified polysaccharide was dissolved in 4 mL of water, and 1 mL of 1.2 M sodium hydroxide and 0.9 M sodium borohydride (NaBH ₄ ) were added and reacted at 25 캜 for 21 hours. After the reaction, the pH was adjusted to 1.2 M hydrochloric acid, and then 4 times of ethyl alcohol was added thereto. The precipitate was dried under reduced pressure over P ₂ O ₅ .

4. Structural analysis of polysaccharides - methylation of polysaccharides On methanollisis  by Sugar composition  analysis

* To analyze the components of the enzymatic degradation products, the samples were dissolved in 3M HCl dissolved in a methanol solvent, heated at 120 ° C for 2 hours, cooled to room temperature, dried at 40 ° C under nitrogen gas, and then added with 0.3 ml acetic anhydride After standing for 30 minutes at room temperature, drying at 40 ° C under nitrogen gas, P ₂ O ₅ (2: 1, v / v) of pyridine / N, O-bis (trimethylsilyl) trifluoroacetamide was added to 50 μl of the solution, and the solution was silylated at 80 ° C. for 30 minutes. And the cilylated sample was analyzed with a gas chromatograph {Agilent 6890 (Agilent Technologies) equipped with a 5973N mass detector}. The column is an HP-5 capillary column manufactured by Hewlett-Packard, Inc. The standard is 0.53 mm x 30 m. The temperature of the injection part is kept at 250 ° C and the temperature of the detector is kept at 280 ° C. The temperature of the column is kept at 180 ° C, At 2 [deg.] C per minute and at 100 [deg.] C per minute at 300 [deg.] C.

5. Using high performance liquid chromatography Sugar composition analysis .

The insect Glycosaminoglycan (GAG) 10 species 1 mg / ml (CaG: Ganggyeong = Dung beetle GAG; BTQG: Western beetle queen bee GAG; SWG: Silkworm GAG; BIQG: Bumble bee queen bee GAG; PSWG: Recombinant heparinase I (250 units / ml) was added to GAG, IgpcG (IGPCG, Bumblebee larvae) and BTWG (GAG) II, and III (50 units / ml) were incubated for 3 hours at 37 ° C for 5 hours. The reactions were analyzed by high performance liquid chromatography (HPLC) , 5μ, manufactured by Phenominox Co., USA) at a flow rate of 1.0 ml / min by means of sodium chloride concentration gradient.

Heparin disaccharide Standard heparin dissaccharide IS, II-H, IA, II-S. III-H, III-S, IV-A, IV-H, IH, IP and III-S.

6. Ion chromatography ( Ion - chromatography ) Sulfate group  analysis

50 mg of a 6N hydrochloric acid solution was added to 1 mg of the polysaccharide, heated at 100 ° C for 2.5 hours under nitrogen gas, and then centrifuged at 1800 g for 10 minutes. After the solution was evaporated at 50 DEG C, 100 mu l of 50% methanol was added and the solution was concentrated at 40 DEG C. [ Was dissolved in 200 μl of water, and 50 μl of the solution was injected into the column. The ion peak of sulfate is accurately detected by the ion chromatography. HPLC conditions and columns are adjustable.

7. Analysis of sugar structure by spectroscopy

Results confirmed the infrared (IR) spectroscopy 780~820 cm from the IR pattern ^was confirmed sulfate group in place of ^one.

<Experimental Results>

1. Insect Alcohols Extracts of insects New glycosaminoglycan was prepared and the yield was increased. A total of 9 insectic acid glycosaminoglycans were obtained by hydrolysis with insufficient amount of persulfuric acid. The heparin disaccharide composition was determined by HPLC analysis after heparinase treatment and its unit structure was determined.

As a result, insecticidal glycosaminoglycan was obtained as shown in Table 1 below as a result of the extracts of silkworm larvae alcohol extracts, bumblebug larvae pupae bark, Western bumblebee beeswax alcohol bumblebees and bumblebee queen bee bee alcohol extracts .

Insect gleaned from insect bark residue after extracting Glycosaminoglycan Insect Alcohol extract residue Crude insect drug Silkworm
pupa Bumble bee
Bongun shell Western beetle
worker Bumble bee
Queen bee Hue Bam Used Amount (g) 350 150 120 80 800 100 Accuquired amount (g) 1.1 6.7 5.5 6.42 0.18 0.45 Yield 0.31 4.47 4.58 8.03 0.3 0.45

2. Comparison of monosaccharide composition and amino sugar composition of insect-derived glycosaminoglycans with insect-derived glycosaminoglycan according to the corresponding monosaccharide analysis conditions according to reaction temperature, 40 ° C, 60 ° C, and 80, Analysis of sugar composition by Nolisis was performed.

Methods for the analysis of amino suagar by the derivatization method of TMS (Trimethylsilylation) have already been studied and reported. (Refer to Analytical biochemistry 347 (2005) 262-274.) The polysaccharide after TMS derivatization process changes into α, β-anomer, pyranose and furanose ring structures, and when analyzed by GC / MS, do. Therefore, after analyzing the standard reagents individually, the relative intensity of the separated time and peaks based on the internal standard material was found and summarized.

As a result, GC-MS analysis conditions of amino sugar and acid groups were determined according to the acetyl group sugar moiety temperature, and the results are shown in Tables 2 to 4 below.

In the present specification, abbreviations used for the insect glycosaminoglycan for the sake of convenience are as follows. GBG, GbG: cricket glicosaminoglycan, GbG0.5: cricket glycoxaminoglycan in which the cricket joglycosaminoglycan was purified and the pigment and the like were removed at a salt gradient of 0.5M; TbG: mung bean gum lucosaminoglycan; ISG: mung bean gum lycosaminoglycan; IQG or ㅎ o ァ: Bumblebee queen bee glycosaminoglycan; CaG: gangrene glycosaminoglycan; BTQG or ㅅ o:: Western beetle, Queen bean glycosaminoglycan; SWP: silkworm pupa; PSWG: silkworm pupae glycosaminoglycan; SWG: Nugella glycosaminoglycan, IGPCG or IgpcG: Bumblebee larvae Bong shell peeled glycosaminoglycan; BTWG: Western beetle worker Glycosa Minoglucan. The number next to G is the fraction of M concentration fraction from the salt gradient M concentration gradient.

The amino sugar compound (acetylation 80 &lt; 0 &gt; C) Compounds R Amino and Acid monosaccharide (/ / mg) in insect GAG GbG0.5 TbG0.5 ISG 1 IQG0.5 CaG BTQG SWP PSWG Glucuronic acid 0.995 21.43 35.01 82.91 40.66 8.02 0.00 41.63 129.73 Glucosamine HCl 0.996 1.13 19.87 3.33 25.57 10.21 8.07 18.08 0.00 Galactosamine HCl 0.999 6.17 80.29 25.64 69.41 52.17 28.11 102.11 0.00 N-Acetyl Galactosamine 0.999 28.62 95.43 42.92 219.84 184.38 0.00 0.00 0.00 Glucosamic acid 0.991 258.59 434.70 530.64 389.91 187.96 0.00 575.93 0.00 Galactosamic acid 0.994 18.50 102.66 49.42 47.93 59.95 0.00 9.15 24.49 D-Glucosamine-
6-sulfate 0.983 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N-Acetyl Glucosamine-
6-sulfate 0.991 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N-Acetyl-D-Galactosaminitol 0.992 108.08 16.39 159.15 103.94 55.50 0.00 102.39 0.00 N-Acetyl Glucosamine 0.990 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 D-Glucosamine-6-Phosphate 0.989 0.00 17.25 15.56 35.09 0.00 0.00 0.00 0.00 Total Amino Sugar 442.53 801.61 909.56 932.35 558.20 36.18 849.30 154.22

In more detail, the results of GC / MSD measurement after the TMS derivatization of the sample with trimethylsilyl regent are shown in Table 2. Acetylation After insecticidal glycosaminoglycan silylation at 80 ℃, D-Glucosamine-6-Sulfate, N-Acetyl Glucosamine-6-Sulfate and N-Acetyl Glucosamine were not detected in all samples. D-gluosamine-2-sulfate, N-acetyl-D-galactosamine 4-sulfate and N-acetyl glucosamine 6-sulfate were not detected. Addition of amino sugars (glucosamine, galactosamine, N-acetylglucosamine, or N-macetylgalactozamine) when insoluble glycansaminoglycan is acylated at a temperature of 80 ° C or higher, sulfate, or 4-sulfate and 6-sulfate, respectively. That is, when the acetylation is heat-treated at a high temperature (here, at 80 ° C.), desulfuric acid of the amino sugar is generated, that is, the specific group related to the anticoagulation (ie, anti-hemorrhage) is reduced to produce an insect glycosaminoglycan fragment having an immunological activity such as arthritis can do.

Glucuronic acid was not detected in IQG 0.5 but in other samples it was 8.02-127.93 μg / mg. Especially, PSWG showed the greatest amount of 129.73 ㎍ / mg.

Glucosamine HCl was measured in samples other than PSWG, and the highest amount was found in samples from IQG 0.5 to 25.57 ㎍ / mg. Galactosamine HCl was also measured in samples other than PSWG and 102.11 μg / mg in SWP The highest amount was measured.

* N-Acetyl Galactosamine was not detected in BTQG 0.5, SWP, and PSWG, and GbG 0.5 (28.62 ㎍ / mg) and TbG 0.5 (95.43 ㎍ / mg) were obtained at 219.84 ㎍ / mg and 184.38 ㎍ / And ISG 1M (42.92 ㎍ / mg).

Glucosamic acid was detected in other samples except BTQG and PSWG. Especially, SWP showed the largest amount of 575.93 ㎍ / mg. In GbG 0.5, TbG 0.5, ISG 1M and IQG 0.5, the amount of total amino sugar was found to be about half. This large amount of glucosamic acid was measured at 80 ° C, which is higher than other amino sugar analysis experiments (40 ° C) during the re-N-acetylation reaction. Therefore, the carboxylic acid of anhydrous acetic acid used in the reaction (- COOH) is considered to be influential.

Galactosamic acid was detected in samples other than BTQG0.5 and was measured as 9.15-102.66 ㎍ / mg. TbG 0.5 was the highest at 102.66 ㎍ / mg.

N-acetyl-galactosaminitol was detected in samples except BTQG0.5 and PSWG. Especially, ISG1M showed the highest amount as 159.15 ㎍ / mg.

In the case of D-Glucosamin-6-phosphate, only TbG 0.5 (17.25 ㎍ / mg), ISG 1M (15.56 ㎍ / mg) and IQG 0.5 (35.09 ㎍ / mg)

In Table 3 and Table 4, unlike the results in Table 2, the amount of glucosamic acid was small and the amount of detected glucosamine was increased significantly. However, the data in Table 3 and Table 4 show the results. When reacting Re-N-acetylation at 40 ° C for 60 min, m / z 73, 147, 203, 204, 216, and 217 appearing after derivatization of the amino sugar in the sample hardly appeared. When all the amino sugars were not detected, the amount of retention time and peak area between the standard and the sample was used instead of the m / z value of the data analyzed by GC / MSD. Table 4 shows the results.

Table 5 and Table 6 show the results of analysis of seven species including insect glicosaminoglycan which was separately purified. Galactomannic acid and N-acetylgalactosamine 4-sulfate levels were increased.

3. Identification of oligosaccharides and disaccharide glycan HPLC-ECPAD of the oligosaccharide polysaccharides obtained by culturing recombinant heparinase and purified insect glycosaminoglycan.

10 kinds of insect glycosaminoglycan (CaG, BTQG, SWG, IQG, PSWG, Tb, Gb, IS, IgpcG and BTWG) were added to a phosphate buffered saline After dissolution, 3 μl of the enzyme (50 units / ml) and 50 μl / ml of recombinant heparinase I (250 units / ml) Graft (HPLC). Samples were collected at a flow rate of 1.0 ml / min using a sodium chloride concentration gradient using a strong anion exchange column (4.6 × 250 nm, 5 μ Phenominox, USA) and analyzed by NMR.

Heparin disaccharide Standard heparin dissacaride IS, II-H, IA, II-S. The retention time was checked by contrast with the use of III-H, III-S, IV-A, IV-H, IH, IP and III-S.

As a result, the results of identification of the oligosaccharide polysaccharide and disaccharide glycan HPLC-UV and ECPAD of oligosaccharide polysaccharide obtained by culturing together with recombinant heparinase and purified insect glycosaminoglycan were as shown in Tables 7 and 8 Insect-derived glycosaminoglycan was identified to 8-mer (8-mer) having 3 heparin 2 saccharides. Preferred examples of the insect glycosaminoglycan are hydrolyzed in the form of insect glycosaminoglycan purified in the form of a crude insect glycosaminoglycan and then treated in the form of a monosaccharide or with an enzyme Heparin 2 saccharides, and two saccharides may be used in the form of two, three or four saccharides, hexoses, and palmitates. The standard sugar structure and abbreviations of each heparin 2 saccharide are as follows.

4-deoxy-L-threo-hex-4-enopyranosyluronic acid (4-deoxy-L-

GLcN: D-glucosamine &lt; RTI ID = 0.0 &gt;

Ac: Acetyl

NS, 2S, 6S = N-sulfo, 2-sulfate and 6-sulfate,

HI: heparinase I, HII: heparinase II, HIII: heparinase III

Heparin disaccharide I-H:? -? UA-2S- [1 → 4] -GlcN-6S

Heparin disaccharide II-H: α-δUA- [1 → 4] -GlcN-6S

Heparin disaccharide III-H:? -? UA-2S- [1 → 4] -GlcN

Heparin disaccharide IV-A: α- △ UA- [1 → 4] -GlcNAc

Heparin disaccharide IV-H:? -? UA- [1 → 4] -GlcN

Heparin disaccharide III-S:? -? UA-2S- [1 → 4] -GlcNS

NO Insect GAG Heparinase
incubation Heparin disaccharide One CaG0M HI I-H II-H IV-H 2 CaG0.5M HIII IV-A II-H 3 BTQG0.1M HI IV-H III-S 4 BTQG0.5M HIII II-H 5 SWG HI I-H IV-H 6 IQG0.5M HIII II-H IV-H 7 IQG0.5Mbad HI I-H II-H 8 IQG0.5 HIII II-H 9 PSWG0.5 HI I-H II-H 10 PSWG0.5 HIII I-H II-H 11 Tb1M HI II-H IV-H 12 Tb1M HIII I-H II-H IV-H 13 Gb1M HI II-H IV-H 14 GbiM HIII II-H IV-H 15 IS0.1M HI II-H IV-H 16 IS0.1M HIII II-H IV-H 17 IgpcG HI II-H HII I-H IV-H HIII I-H II-H 18 BTWG HI I-H II-H HII I-H II-H IV-H HIII II-H

NO Insect GAG Heparin disaccharide One CaG I-H, II-H, IV-H, IV-A 2 BTQG IV-H, III-S, II-H 3 SWG I-H, IV-H 4 IQG0.5 II-H, IV-H, I-H 5 PSWG I-H, II-H 6 Tb II-H, IV-H, I-H 7 Gb II-H, IV-H 8 IS II-H, IV-H 9 IgpcG II-H, IV-H, I-H 10 BTWG II-H, IV-H, I-H

4. Analysis of sugar structure by spectroscopy. Results confirmed the infrared (IR) spectroscopy 780~820 cm from the IR pattern ^was confirmed in the sulfate group in place of ^one.

5. The sulfate group was analyzed by ion chromatography. 50 mg of a 6N hydrochloric acid solution was added to 1 mg of insect glycosaminoglycan and heated at 100 DEG C for 2.5 hours under nitrogen gas, followed by centrifugation at 1800 xg for 10 minutes. After the solution was evaporated at 50 DEG C, 100 mu l of 50% methanol was added and the solution was concentrated at 40 DEG C. [ Was dissolved in 200 μl of water, and 50 μl of the solution was injected into the column. As a result, it was confirmed that the ion peak of sulfate was accurately detected by the ion chromatography.

< Example  2>

Identification of the anti-inflammatory or immune activity of insect glucosamine

In order to elucidate the immunological and vasculogenic agents and characteristics of insects in extracts of preprocessing process and purification of insect-derived proteoglycans, the bioactive substances and inflammation inhibition expressed in inflammation induced in experimental animals And the antibody production ability by complement administration was examined. We also investigated whether the insect glycosaminoglycan controls the secretion of cytokines or is related to vasodilation and vascular endothelial growth factor secretion in vascular endothelial cells.

<Experimental Method>

1. Experimental animals

To perform this experiment, a 4-week-old male Sprague-Dawley rat (Samtava Bio Korea) weighing 200-220 g was used. Feed and drinking water were free and adhered to the provisions of the Animal Experiment Ethics Committee. Freud adjuvant complete was injected into the subcutaneous area of the right footpad of the rats. After the erythema and edema were confirmed by the degree of inflammation, the changes of edema were observed by time.

2. Foot swallow measurement

The foot swelling was measured by CFA (Freud adjuvant complete), and the thickness of the sole was measured by caliper in the control group, the inflammation group and the sample treatment group. The error was minimized by performing the measurement under the same conditions for each day and hour. The changes of edema were examined by comparing the measured values before CFA administration as a control group.

3. Experimental group  process

The model mice injected with CFA were divided into a sample treatment group and a control group. During 14 days of intraperitoneal administration, plantar tissues and serum were collected and various biochemical analyzes were performed. Histochemistry was also performed in order to observe changes in the dorsal root ganglia (DRG) of the peripheral nerves of the samples and indomethacin compared with the control group.

4. Statistical analysis

Experimental results were analyzed using sigma plot version 10.0 (SPSS Inc, Chicago, IL, USA). Statistical analysis was performed by one-way ANOVA and student T-test was performed on the basis of P <0.05.

5. cytokine  analysis

(1) Preparation of tissue suspension

Sole tissue was adjusted to 1: 4 weight / volume by 1: 1, homogenized with PRO-PREPTMProtein Extraction Solution, and centrifuged at 15,000 × g for 10 min.

(2) Measurement of cytokine production in collected animal samples

Measurement of TNF-α and VEGF production was performed according to the manufacturer's instruction with Recombinant murine purchased from QuantikineRM, R & D systems. Prostaglandin E2 and Secretoty Phospholipase A2 were tested according to the Cayman product manual.

(3) Measurement of COX-2 activity in plantar tissues

COX-2 activity was measured using a colorimetric assay kit purchased from IBL (Japan). The kit is a solid-state sandwich ELISA using two highly compatible anti-bodies. Tetra methyl benzidine (TMB) is used as a chromogen and the intensity of the color reaction is proportional to the amount of rat COX-2 It is a principle. After the experimental period, the plantar tissue was removed and rinsed in Tris buffer (pH 7.4) to remove red blood cells and blood. The plantar tissue was homogenized with PRO-PREPTMProtein Extraction Solution at a weight / volume ratio of 1: 4, centrifuged at 15,000 × g for 10 minutes, and the COX-2 assay was performed with the supernatant.

7. Activity at the cellular level

NO production assay on vascular endothelial cells NO production is measured by accumulation of nitrite ions in cell culture medium using Griess reagent. In other words, the sample was treated with the CPAE of bovine pulmonary vascular endothelial cells (CPAE), cultured for 24 hours, and the absorbance was measured by measuring the amount of sodium nitrite at 540 nm using a VERSAmax microplatereader (Molecular Devices, Menlo Park, CA, USA).

8. Induction of antibody production

The isolated homozygous mice were mixed with the same amount of complete Freund's adjuvant (Sigma CO, USA) for 1 day and emulsified for 1 day in BALB / C mice. After the administration, incomplete Freund's adjuvant (Sigma, CO., USA) was mixed with the same amount of glucosaminoglycans at intervals of 12 days for 2 and 3 weeks, Were used as polyclonal antibodies.

For the titration assay, the corresponding glucosaminoglycans were coated on an ELISA 96-well plate with 0.1 M coating buffer (1.593 g Na 2 CO 3, 2.93 g Na HCO 3) at 10, 100, 200 and 500 ng, After incubation at room temperature, sandwich ELISA was performed. As the second antibody, anti-species (mouse) IgG alkaline phosphatase was used. The reagent was applied to the ELISA plate using the invitrogen WesternBreeze manual chromogenic assay at 414 nm.

<Experimental Results>

1. Identification of anti-edema against insect extracts Animal experiments and cytokines Activity investigation

1) The anti-edema effect of rats on four insect glucosamine and the cytokine activity, which is a related mechanism in rat excreta, were examined to obtain the preclinical inflammatory and immune antibody activity (FIGS. 1 to 7).

SD rats Inflammation model Freund's adjuvant complete injection into rats' feet was used to induce inflammation with redness and swelling, and insect glicorzominoglycan (GAG) was administered for 14 days to suppress edema. As a result, Licorzaminoglycan (SWPG or PSWG or PSG) GAG (p < 0.05 vs CFA), horny GAG (IGPCG, bumblebee larva peeled grape glycosaminoglycan); GAG (BTWG, Western beeswax Glycogenaminoglycan) (p <0.05 vs. CFA), 10 mg / kg, and indomethacin 1.5 mg / kg, respectively, for 14 days for the CFA sprouting species, as shown in FIG. Leakage GAG was inhibited at the IL-6 level in the serum at 14 days (Fig. 2, below), and VEGF was inhibited by GAG (Fig. 4). On the 7th day, coagulase inhibitor cox-2 enzyme inhibition was observed in 7 days of GAG treatment (Fig. 7). In particular, GAG (p <0.05) , Respectively, in the CFA group. In addition, IL-6 at 14th day was lower in all treatment groups than CFA induced group, and it was considered to be a part of the inflammatory mechanism of insect GAG (Fig. 2, upper graph). PGE2 was lower in all tissues at day 3 than in CF treatment group (Fig. 3), which was also considered to be part of the inflammatory mechanism of these insect GAGs. Activation of tumor necrosis factor-alpha (FIG. 6) and secretory phospholipase A ₂ secretion change (FIG. 5) induced by inflammation were also confirmed. Similar to these insect GAGs, VEGF inhibition was observed in some GAGs, that is, in leaky GAGs, in progeny-grown GAGs, and later in GA (Figs. 1 to 7).

 2) The results of the comparison of the efficacy with the normal glucosamine, which is a positive control group, are shown in Figs.

Leukemia, GAG, GAG, commercial glucosamine 20 mg / kg, indomethacin 5 mg / kg, and Compounded Freund's adjuvant (Sigma) A total of 14 days were prepared, and the size of the paw was measured with a caliper. In addition, Cox-2 levels were measured in tissues and urine and compared with the control group after 3, 7, and 14 days of treatment.

As a result, GAG> Leakage GAG> CFA complementary administration in the order of GAG on the market. Suppression of inflammation, which indicates sputum and eruption, and inflammation effect over 3 times. In the animal experiment, GAG shows reproducibility with the same pattern. (Fig. 8). On the 14th day, GAG showed IL-6 inhibition (Fig. 14) in the sera of sprouting mice and TNF-a inhibition at 7 days in all treatment groups (Fig. 9). On day 14, leaked GAG and indomethacin had VEGF inhibitory activity (FIG. 10) and slight inhibition of PEG ₂ in commercial glucosamine (FIG. 11). Prostaglandin E ₂ is an inflammatory mediator. In the present study, low levels were found in the whole group in the tissues on the third day of induction of complement foot inflammation, low levels of commercial glucosamine in the urine, or low levels of commercially available glucosamine and silkworm Pupae glucosamine showed a slight inhibitory effect on prostaglandins (Fig. 11), but no significant difference (Figs. 8-14). IL-6 cytokine activity (Fig. 14) was inhibited by GAG and then GAG. On day 3 and 7, cox-2 inhibitory effect (FIG. 13) and sPLA ₂ (FIG. 12) inhibitory effect were also shown.

 3) The anti-edema effect confirmed by the alcoholic extract of the insect pupa of the beetle and the beetle of the Western beetle and the beetle of the pre-stage of the production of the glycosaminoglycan is as shown in FIGS. 15 to 20.

SD rats Inflammation model Inflammation with swelling and edema was injected with Freund's adjuvant complete solution on the sole of rats. 100 mg / kg, * p <0.05 vs CFA> (100 mg / kg), * p < 0.05 vs CFA > bumblebee extract (100 mg / kg) in order of inhibition of inflammation (Fig. 15). In addition, SDLEX (100 mg / kg of Western beetle larvae alcohol extract); SPDYB (Peppermint larva pupa extract alcohol 100 mg / kg); Indomethacin (IL) -6 inhibition (Fig. 16) and cytokine serum levels were shown when ind (indomethacin 1 mg / kg) was administered for 14 days each. In addition, the extract of the present invention was confirmed to be an effective extract for anti-inflammation in comparison with indomethacin (p <0.05 vs CFA> p <0.05 vs CFA) In the cytotoxic activity of the rat, serum TNF-α (FIG. 20) and IL-6 cytokine activity on the 7th day of administration were inhibited by the inhibition of the alcoholic extract and the alcoholic extract of Suwon-dong. On day 14, inhibition of cox-2 (FIG. 18), spLA ₂ (FIG. 19) and VEGF inhibition (FIG. 17) were observed in each extract (second experiment).

2. Investigation of activity at the vascular endothelial cell level (Fig. 21 To  25)

Since the emergence of a new target heparan sulfate in angiogenesis inhibitors, the vascular endothelial growth factor (VEGF), a monoclonal antibody drug called Avastin, produced by Genentech, has been shown to block VEGF Avastin has been approved for use in combination with chemotherapy for colorectal cancer and lung cancer. It has been shown that heparan sulfate acts on the blood vessels of cancer cells by controlling the action of heparan sulfate, which is present in cancer blood vessels, and as a result, it can inhibit the growth of lung cancer cells implanted into mice.

Insect-derived glycosaminoglycans showed control of the production of eNOSs (endothelial nitric oxide) of vascular endothelial cells (CPAE, HUVEC) and VEGF regulation of CPAE and HUVEC, as described in the above references.

To explain this in more detail, NO production is measured by accumulation of nitrite ions in cell culture medium using Griess reagent. eNOS and VEGF were treated with insect glycosaminoglycan (10 mg / ml for alcohol extract and 10 mg / ml for alcohol extract) in pulmonary vascular endothelial cells (CPAE) or HUVEC cells according to the R & D kit The NO concentration at 540 nm was calculated by measuring the absorbance of sodium nitrite using a VERSAmax microplatereader (Molecular Devices, Menlo Park, CA, USA) for 24 h. human VEGF was lower than that of media in PSG, TG and ISG. In vascular endothelial cell CPAE cells, eNOS increased in the order of ISG>PSG>CaG>BtQG>IGPC>IQG> BTWG. PSG>BTQG>IQG>SDIEX>IQG>SPDYBEX>> PSG, and the eNOS activity in the huvex cell was in the order of PSG>BTQG>BTWG>IGPCG>CaG>IQG.> ISG in the order of IGPCG>BTQG>Indomethacin>IQG>PSG>SDIEX> SPDYBEX In order of positive control and comparative experiments, eNOS production was shown in the order of CjGlucosamine>PSG> SDIG.

3. Induction of antibody production (Fig. 26)

A study on the mass production technique of insect glycogamycinoglycan antibody was carried out, and a polyclonal antibody was prepared and administered to mice.

The isolated homozygous mice were mixed with the same amount of complete Freund's adjuvant (Sigma CO, USA) for 1 day and emulsified for 1 day in BALB / C mice. After the administration, incomplete Freund's adjuvant (Sigma, CO., USA) was mixed with the same amount of glucosaminoglycans at intervals of 12 days for 2 and 3 weeks, Were used as polyclonal antibodies. For the titration assay, the corresponding glucosaminoglycans were coated on an ELISA 96-well plate with 0.1 M coating buffer (1.593 g Na 2 CO 3, 2.93 g Na HCO 3) at 10, 100, 200 and 500 ng, Fold dilution, incubated at room temperature, and then subjected to sandwich ELISA. As the second antibody, anti-species (mouse) IgG alkaline phosphatase was used. The in vitro Western blotting manual was applied to the ELISA plate while measuring the color at 414 nm using WesternBreeze chromogenic.

The results of titration of the polyclonal antibody are as follows (see FIG. 26).

1. PSG (labeled silkworm, Glycosaminoglycan, A) is 100 ng for serum, 500 ng for 200 ng coated antigen, and 250: 1 for 100 ng for serum 100; 200 ng, and 500 ng of coated antigens.

2. IGPCG (Peak Glycosaminoglycan from Bumblebee Larvae, denoted as B), 100 ng of serum 500: 1 and 100 ng of 200 ng of coated antigen were reacted with 10 ng and 100 ng for 250: ng, the colorimetric value was significantly (p <0.05) higher than that of the control BSA.

3. BTGG (Corynebacterium glutinosa, C.), 4. IQG (bumble bee bee glaucosaminoglucan, indicated by D), 5. ISG , E), 6. CaG (Glucosamine Glucosamine Gluconate, designated F), 7. BTQG (Western beetle, Glucosaminoglucan, G) (Similar to the above) and 9. Cricket glycosaminoglycan (similar to the above) were prepared by mixing serum 250: 1 with the antibody produced by the 3rd immunoconjugate mixture treatment with a coating antigen For 100 ng, 100 ng of the coating for 500: 1 of serum and p < 0.05 for control BSA for 200 ng were significant.

As described above, the production method of the present invention has an effect of providing glycosaminoglycan useful from insects. Through the production method of the present invention, it is possible to produce heparin polymers and low-molecular drugs, which are one kind of glycosaminoglycan, from various kinds of insects, and to produce substances such as heparin at low cost to contribute to the improvement of public health, It can contribute to the income conservation of the farm household.

Claims (6)

Wherein the composition comprises an alcohol extract of an insect selected from the group consisting of an insect pest, a pest insect, a pest insect, and a pest insect.
The composition according to claim 1, wherein the alcohol is a lower alcohol having 1 to 4 carbon atoms selected from the group consisting of ethanol, methanol, propanol, isopropanol and butanol.
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