KR101200406B1 - Anti-coagulant composition comprising glycosaminoglycans derived from insect - Google Patents

Abstract

The present invention relates to the use of insect glycozaminoglycans for anti-inflammatory, antidiabetic, anticoagulant, anticancer, antiangiogenic use, and / or disease treatment and / or prophylaxis due to dysfunction of vasodilatation and insectglycozymino Provided is a method for preparing glycan. Insect glycosaminoglycans of the present invention can treat and / or prevent diseases caused by dysfunction of anti-inflammatory, anti-diabetic, anticoagulant, anti-cancer, angiogenesis, and / or vasodilation reactions, and the preparation of the present invention. Insect glycosaminoglycans can be effectively produced by the method.

Description

Anti-coagulant composition comprising glycosaminoglycans derived from insect}

The present invention relates to the use of insect glycozaminoglycans for anti-inflammatory, antidiabetic, anticoagulant, anticancer, antiangiogenic use, and / or disease treatment and / or prophylaxis due to dysfunction of vasodilatation and insectglycozymino It relates to a method for producing glycan.

Insect glycosaminoglycans are insect-derived glycosaminoglycans, and glycosaminoglycans (GAGs) have a repeating structure of uronic acid and glucosamine, and have a partially sulfated structure. The chemical structure makes it possible to exhibit various types of bioactivity. As far as is known, this diverse bioactivity appears to be due to the presence of specific sequences of its own.

As animal GAGs, hyaluronic acid, chondroitin sulfate, keratin sulfate, heparan sulfate, heparin and the like are known. GAGs exist in the form of proteoglycans in vivo, and these proteoglycans are degraded by proteases and glycosidase to release the sugar moiety. The physiological role of GAGs is not known much yet, but it seems to show physiological activity by binding to proteins in vivo.

The method for preparing insect glycosaminoglycans is disclosed in Korean Patent No. 10-0847889, and the cicada snow cordyceps sinensis herb glycosaminoglycans prepared by the above method are disclosed to be effective in hypertension and the like. They have discovered the use of insect glycosaminoglycans that are not yet known to complete the present invention.

The problem to be solved by the present invention is to provide a composition for the treatment and / or prevention of diseases caused by dysfunction of anti-inflammatory, anti-diabetic, anticoagulant, anti-cancer, angiogenesis, and / or vasodilator response.

In addition, the problem to be solved by the present invention is to provide a method for producing insect glycosaminoglycans.

The present invention provides the use of insect glycozaminoglycans for anti-inflammatory, antidiabetic, anticoagulant, anticancer, antiangiogenic use, and / or disease treatment and / or prophylaxis due to dysfunction of vasodilator reactions.

The present invention provides an anti-inflammatory composition containing insect glycozaminoglycans. Preferred examples of the insect glycosaminoglycans include dung beetle glycosaminoglycans, Queen of B. terrestris glycosaminoglycans, cicada snow beetle glycose, crickets glycogna Lycosaminoglycans, iron and the like glycosaminoglycans, and / or queen of B. ignitus glycosaminoglycans.

The term "insect glycogaminoglycan" refers to a glycosaminoglycan derived from an insect, and the glycosaminoglycan of each insect is a glycazaminoglycan derived from each insect, for example, cow dung Copper glycosaminoglycan means a glycosaminoglycan derived from dung beetle.

In the present invention, the "dung beetle" includes a Copris series, such as Catharsius family, Catharsius molossus and cedar beetle. (According to the definition of the herbal drug name entrapment, Insect Drug Book, Choi Jung, Ahn Mi-young, Shinil Publishing Co., Ltd.)

The insect glycosaminoglycans may be prepared from insects by a manufacturing method or a known method of the present invention (for example, the method disclosed in Korean Patent No. 10-0847889).

The dung beetle is a wild drug name, and the estuary is washed with dung beetles, and the dung beetle is used by the medicinal herb, and the beetle is washed by washing the dung beetle.

The dung beetle glycosaminoglycan is not limited to the glycosaminoglycan extracted from the dung beetle, but may be in the form of a crude (crude) and the like, preferably ΔUA-2S- [1-4] GlcN It is a glycosaminoglycan having a repeating structure of (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo- [1-4] glucosamine).

The cicada snow cordyceps sinensis Glycozaminoglycan is not limited to the glycosaminoglycans extracted from the cicada snow cordyceps fungi, but chondroitin sulfate (ΔUA- [1-.3] -N-acethylgalactosamine-N-sulfate series) It may be in the form of a crude (crude), and the like, preferably Glycosaminoglycans having a ΔUA-GlcNS (glucuronic or galacturonuc acid-N-glucosamine-N-sulfate series) repeat structure, and / or ΔUA Glycosaminoglycans having a repeating structure of -2S-GlcN (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo-glucosamine).

The cricket glycosaminoglycan is not limited to the glycosaminoglycan extracted from the cricket and may be in a crude state, but preferably ΔUA-GlcNAC (4-deoxy-L-threo- hex-4-enopyranosyluronic acid-N-acetylglucosamine) Glycozaminoglycan having repeat structure, ΔUA-GlcNAC-UA-GlcNS (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-N-acetylglucosamine-4 -deoxy-L-threo-hex-4-enopyranosyluronic acid-N-sulfo-glucosamine) glycosaminoglycans having a repeating structure, and / or ΔUA-GlcNAC-UA-GlcNS-UA-GlcNAC6S (4-deoxy- L-threo-hex-4-enopyranosyluronic acid-N-acetylglucosamine-4-deoxy-L-threo-hex-4-enopyranosyluronic acid-N-sulfo-glucosamine-4-deoxy-L-threo-hex-4-enopyranosyluronic acid -N-acethyl-6-sulfo-glucosamine) is a glycosaminoglycan having a repeating structure.

In the present invention, ΔUA or δUA means 4-deoxy-L-threo-hex-4-enopyranosyluronic acid, GlcN means D-glucosamine, Ac means Acetyl, NS, 2S, 6S are each N- sulfo, 2-sulfate or 6-sulfate, with each group attached to a sulfate group.

The composition of the present invention is a pharmaceutical composition, a food composition or a cosmetic composition.

The present invention also provides an antidiabetic composition containing insect glycozaminoglycans. By the antidiabetic action, it is possible to treat and / or prevent diabetes. Preferred examples of the insect glycozyminoglycans are Isaria sinclairii glycosaminoglycans, crickets, cows and the like and / or dung beetle glycosaminoglycans. The dung beetle is a wild drug name, dung beetle glycosaminoglycan is not limited to the glycosaminoglycan extracted from dung beetle and may be in the form of a crude (crude), but preferably ΔUA- Glycosaminoglycans having a repeating structure of 2S- [1-4] GlcN. The cicada snow cordyceps sinensis Glycozaminoglycan is not limited to the glycosaminoglycans extracted from the cicada snow cordyceps fungi, but chondroitin sulfate (ΔUA- [1-.3] -N-acethylgalactosamine-N-sulfate series) It may be in the form of a crude (crude), and the like, preferably Glycosaminoglycans having a ΔUA-GlcNS (glucuronic or galacturonuc acid-N-glucosamine-N-sulfate series) repeat structure, and / or ΔUA Glycosaminoglycans having a repeating structure of -2S-GlcN (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-2-sulfo-glucosamine).

The cricket glycosaminoglycan is not limited to the glycosaminoglycan extracted from the cricket and may be in a crude state, but preferably ΔUA-GlcNAC (4-deoxy-L-threo- hex-4-enopyranosyluronic acid-N-acetylglucosamine) Glycozaminoglycan having repeat structure, ΔUA-GlcNAC-UA-GlcNS (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-N-acetylglucosamine-4 -deoxy-L-threo-hex-4-enopyranosyluronic acid-N-sulfo-glucosamine) glycosaminoglycans having a repeating structure, and / or ΔUA-GlcNAC-UA-GlcNS-UA-GlcNAC6S (4-deoxy- L-threo-hex-4-enopyranosyluronic acid-N-acetylglucosamine-4-deoxy-L-threo-hex-4-enopyranosyluronic acid-N-sulfo-glucosamine-4-deoxy-L-threo-hex-4-enopyranosyluronic acid -N-acethyl-6-sulfo-glucosamine) is a glycosaminoglycan having a repeating structure.

In the said iron etc., it is a herbal name insect, and the said insect is washed with iron etc. and dried.

The present invention also provides an anticoagulant composition containing an insect glycozyminoglycan as an active ingredient. The anticoagulant action can exhibit a thrombolytic action, and thus treatment and / or prevention of various thrombosis-related diseases including coronary artery disease (angina) and cerebrovascular disease.

Preferred examples of the insect glycosaminoglycans include crickets, glycosaminoglycans, glycoxaminoglycans, cicada snowflower cordyceps, dung beetle glycosaminoglycans, western dwarf bees Queen bee glycosaminoglycans, silkworm (Bomyx mori) glycosaminoglycans, silkworm pupa (gathered pupa of Bomyx mori) glycosaminoglycans and / or bumblebee queen bee glycosaminoglycans.

In the present invention, the 'Cricket' is a medicinal insect, which includes an insect called Ginseng (insect drug illustration book, Choi Jung, Ahn Young Young, Book Publishing Shinil), Gryllus bimaculatus (subtropical cricket), Teleogryllus emme (domestic domestic cricket), etc. It is the meaning of the academic cricket.

In addition, in the present invention, the term `` Eyes et al. '' (Tabanus bivittatus), which corresponds to the nymphs (Tabanus, Insect Drug Book, Shinil Publishing, Choi, Mi Young, etc.) in the name of the herbal medicine, Family Tabanidae).

The cricket glycosaminoglycan is not limited to the glycosaminoglycan extracted from the cricket and may be in a crude state, but preferably ΔUA-GlcNAC (4-deoxy-L-threo- hex-4-enopyranosyluronic acid-N-acetylglucosamine) Glycozaminoglycan having repeat structure, ΔUA-GlcNAC-UA-GlcNS (4-deoxy-L-threo-hex-4-enopyranosyluronic acid-N-acetylglucosamine-4 -deoxy-L-threo-hex-4-enopyranosyluronic acid-N-sulfo-glucosamine) glycosaminoglycans having a repeating structure, and / or ΔUA-GlcNAC-UA-GlcNS-UA-GlcNAC6S (4-deoxy- L-threo-hex-4-enopyranosyluronic acid-N-acetylglucosamine-4-deoxy-L-threo-hex-4-enopyranosyluronic acid-N-sulfo-glucosamine-4-deoxy-L-threo-hex-4-enopyranosyluronic acid -N-acethyl-6-sulfo-glucosamine) is a glycosaminoglycan having a repeating structure.

The dung beetle glycosaminoglycan is not limited to the glycosaminoglycan extracted from the dung beetle, but may be in the form of a crude (crude) and the like, preferably ΔUA-2S- [1-4] GlcN Glycosaminoglycans having a repeating structure of.

The cicada snowflakes Cordyceps glicozaminoglycan is not limited to the glycosaminoglycans extracted from cicada snowflakes Cordyceps, but may be in the form of a crude (crude), etc. Preferably, the article having a ΔUA-GlcNS repeat structure Lycosaminoglycans and / or glycosaminoglycans having a repeating structure of ΔUA-2S-GlcN.

The dung beetle is in the name of the wild drug, the estuary is washed with dung beetle, and the dung beetle is used in the name of the wild horse, and the beetle is washed with water and dried.

The present invention also provides a composition for anticancer containing insect glycozaminoglycans as an active ingredient.

Preferred examples of the insect glycosaminoglycans include silkworm glycosaminoglycans, clown bugs ( herpes , Poecilocoris lewisi ) glycosaminoglycans, dung beetle glycosaminoglycans, and / or crickets glycosaminoglycans.

The cricket glycosaminoglycan is not limited to the glycosaminoglycan extracted from the cricket, and may be in a crude state, but preferably, glycosaminoglyol having a ΔUA-GlcNAC repeating structure. Lycans, glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS repeat structure, and / or glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS-UA-GlcNAC6S repeat structure.

The dung beetle glycosaminoglycan is not limited to the glycosaminoglycan extracted from the dung beetle, but may be in the form of a crude (crude) and the like, preferably ΔUA-2S- [1-4] GlcN Glycosaminoglycans having a repeating structure of.

The clown stingray glycosaminoglycan is not limited to being glycosaminoglycans extracted from clown stingray and may be in a crude state, but preferably has a ΔUA-GlcNAC repeating structure. Minoglycans, and / or glycosaminoglycans having a ΔUA-GlcNS repeat structure (ΔUA-GlcNS substrate and sulfite group NSO ₃ ⁻ structural formulas of the clown lining material repeating structure (P) herein).

The composition is not limited thereto, but is preferably a pharmaceutical composition for treating and preventing uterine cancer and / or colorectal cancer.

The present invention also provides a composition for treating and preventing one or more diseases selected from hypertension, stroke, cerebral hemorrhage, ischemic heart disease, and Raynaud's disease due to dysfunction of vasodilator reactions, including insect glycozaminoglycans.

Preferred examples of the insect glycosaminoglycans are glycosaminoglycans for cricket glycosaminoglycans and / or iron.

The cricket glycosaminoglycan is not limited to the glycosaminoglycan extracted from the cricket, and may be in a crude state, but preferably, glycosaminoglyol having a ΔUA-GlcNAC repeating structure. Lycans, glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS repeat structure, and / or glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS-UA-GlcNAC6S repeat structure.

The present invention also provides a composition for inhibiting angiogenesis, which includes insect glycozaminoglycans.

The composition specifically includes growth and metastasis of cancer mediated by angiogenesis, hemangioma, hemangiofioma, arthritis, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma, corneal disease caused by neovascularization, degenerative plaque, degeneration of spots, Pterygium, retinal degeneration, posterior capsular fibrosis, granuloconjunctivitis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, acne may be a pharmaceutical composition for the prevention and treatment of diseases caused by angiogenesis selected from the group.

Preferred examples of the insect glycosaminoglycans are glycosaminoglycans of cicada snow cordyceps glicozyminoglycans, cricket glycosaminoglycans, and / or iron.

The cricket glycosaminoglycan is not limited to the glycosaminoglycan extracted from the cricket, and may be in a crude state, but preferably, glycosaminoglyol having a ΔUA-GlcNAC repeating structure. Lycans, glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS repeat structure, and / or glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS-UA-GlcNAC6S repeat structure.

The cicada snowflakes Cordyceps glicozaminoglycan is not limited to the glycosaminoglycans extracted from cicada snowflakes Cordyceps, but may be in the form of a crude (crude), etc. Preferably, the article having a ΔUA-GlcNS repeat structure Lycosaminoglycans and / or glycosaminoglycans having a repeating structure of ΔUA-2S-GlcN.

The composition of the present invention may be a pharmaceutical composition, a food composition, or a cosmetic composition.

The composition of the present invention may be formulated to include one or more pharmaceutically, food and / or cosmetically acceptable carriers in addition to the active ingredient for administration.

Pharmaceutical, food and / or cosmetically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as required Other conventional additives, such as antioxidants, buffers, bacteriostatics, can be added accordingly. Diluents, dispersants, surfactants, solvents, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants or flavoring agents may additionally be added. Furthermore, it may be preferably formulated according to each disease or component by any suitable method in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The compositions of the present invention can be formulated by adding pharmaceutically suitable excipients solid, semisolid or liquid diluents, fillers and all types of formulation auxiliaries, and preferred formulations are tablets, coated tablets, capsules, pills, granules, suppositories, Liquids, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders, syrups, juices, drops, injectable solutions, sustained release preparations and sprays, and the like.

Tablets, coated tablets, capsules, pills, and granules are conventional excipients, such as (a) fillers and weighting agents (e.g. starch, lactose, sucrose, glucose, mannitol and silicic acid) (b) binders (e.g. carboxy) Methyl cellulose, alginate, gelatin and polyvinylpyrrolidone) (c) hygroscopic agents (e.g. glycerol), (d) disintegrants (e.g. agar, calcium carbonate and sodium carbonate), (e) dissolution retardants (e.g. paraffin) (f) absorption accelerators (eg quaternary ammonium compounds), (g) wetting agents (eg cetyl alcohol and glycerol monostearate), (h) adsorbents (eg kaolin and bentonite), and (i) lubricants ( Examples: talc, calcium stearate, magnesium stearate, magnesium starrate talc and solid polyethylene glycols), or mixtures of the substances described in (a) to (i) above. Tablets, capsules, pills, and granules can coat, release only effective compounds to the intestinal tract, and if necessary, they can be formulated in sustained-release or microencapsulated using polymers.

Formulations for parenteral administration include injections, suspensions, emulsions, lyophilizers, suppositories, and the like. Suppositories, in addition to emulsions, are conventional water-soluble or water-insoluble excipients such as polyethylene glycols, fats such as cacao fats, higher esters (e.g. C ₁₄ -alcohols with C ₁₆ -fatty acids), witepsol, macrogol, twin 61 And a mixture of laurin and glycerol gelatin materials.

Ointments, pastes, creams and gels may contain, in addition to the active compounds, conventional excipients, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these materials. The nebulizer may further contain a conventional foaming agent, for example, chlorofulurohydrocarbon, and may also be formulated as a nasal spray containing PEG-4000 and glycerin.

Liquid and emulsifying agents, in addition to the active ingredients, include conventional excipients, for example solvents, solubilizing agents and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol Fatty acid esters of 1,3-butylene glycol, dimethylformromamide oil, in particular cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol, tethyrahydrofurfuryl alcohol, polyethylene glycol and sorbitan Or mixtures of these materials. Parenteral solutions and emulsions may be formulated in sterile form, isotonic with blood.

Suspending agents, in addition to the active compounds, include conventional excipients, for example liquid diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, and suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and / or sorbent Inelastic esters), microcrystalline cellulose, aluminum metahydroxy, bentonite, agar and injectable esters such as tragacanth, ethyloleate or mixtures of these materials.

The formulations may also contain coloring agents, preservatives and additives that improve odor or taste, such as peppermint oil and eucalyptus oil and sweeteners such as saccharin, and may contain other pharmaceutically effective compounds in addition to the compounds according to the invention. Can be.

The formulation is prepared in a known manner, for example in a conventional manner by mixing the active ingredient with excipients. The active ingredient is contained in the formulation in an amount of about 0.1 to 99.5% by weight, preferably 0.5 to 95% by weight of the total mixture.

In the present invention, the insect glycozaminoglycan and the composition comprising the same according to the purpose, intravenous, intraarterial, intraperitoneal, intramuscular, sternum, transdermal, nasal, inhalation, topical, rectal, oral, ocular It may be administered in a conventional manner via the intra or intradermal route and the like.

In the present invention, each of the insect glycozyminoglycans and the composition comprising the same is based on the insect glycozyminoglycan dosage is in the range of 0.1 mg / kg to 900 mg / kg daily weight 60 kg adult basis You can adjust from However, the optimum dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration, duration of treatment, and drugs used concurrently.

The formulation of the food composition of the present invention may be prepared by a conventional method and may be formulated in the form of capsules or other tablets, granules, powders, beverages, porridge, etc. after drying together with the carrier. .

Formulation of the cosmetic composition of the present invention is prepared according to a conventional method, can be formulated in the form of a lotion, cream, etc. using a suitable carrier, in addition to the above-described can be prepared in all cosmetic forms.

The present invention also provides anti-inflammatory, anti-diabetic, anticoagulant, anti-cancer, antiangiogenic, vascular angiogenesis comprising the step of administering the glycosaminoglycan contained in the composition of the present invention or the composition of the present invention to a mammal, including a human Provided is a method for treating and / or preventing a disease due to a dysfunction of an expansion reaction, and includes an anti-inflammatory agent, an anti-diabetic agent, an anticoagulant, an anticancer agent, and an angiogenesis inhibitor in the glycozaminoglycan or composition of the present invention. And / or for the treatment of diseases caused by dysfunction of vasodilation reactions.

The same applies to the methods of treatment and / or prophylaxis of the present invention and to the use of the preparation of the present invention, as long as it does not contradict what is stated in the compositions of the present invention.

The present invention also provides (a) 1-4 insects in at least one possible organic solvent selected from the group consisting of organic solvents such as acetone, hexane, ethyl acetate, methyl alcohol, ethyl alcohol, propanol, butanol, acetonitrile, chloroform and dichloromethane. Dipping to remove fats and filtering to dry the residue which is not filtered; (b) pulverizing the residue dried in the step (a) to a particle size of 100 ~ 300 mesh; (c) treating the ground product obtained in step (b) with a proteolytic agent in sodium hydrogen carbonate solution (pH 9.0) to decompose the protein in the ground product; (d) removing the protein degraded in step (c) by centrifugation with trichloroacetic acid (5% by weight); (e) co-precipitating the residue left after protein removal in step (d) with ethanol and potassium acetate or sodium acetate, and again precipitate the precipitate in cetylpyridinium, and then precipitate the precipitate in ethanol; And (f) diagnosing the precipitate produced in step (e) with distilled water.

In order to separate the proteoglycans in step (a), the fatty acids are removed, and fat can be sufficiently removed after 1 to 4 days.

By grinding the residue dried in the step (b) with a ball mill or hammer mill to have a particle size of 100 ~ 300 mesh, it is possible to promote proteolysis in a subsequent step. Particle diameters greater than 300 mesh may be delayed in enzymatic degradation, and particle sizes smaller than 100 mesh may be introduced into the supernatant during centrifugation.

Proteolytic agent of the step (c) Bacillus derived ( Bacillus licheniformi s et al.) proteases alkalases {eg Novozyme (Sigma)} and / or Aspergillus Flavourzyme {for example, Sigma (USA) or Apisbiotech (Korea)), oryzae protease (protease), and the like, fermented liquid liquid enters edible fermented soy sauce or miso fermentation, or yogurt seed liquid Can be used. At this time, the time required for proteolysis (fermentation) is 3 to 7 days, which can promote proteolysis at room temperature or higher, and promote reaction upon heating at 44 ° C. By using 2 to 5% by weight of the proteolytic agent, the protein of the insect shell can be effectively removed. There is a concern that it is difficult to sufficiently decompose the protein of the insect shell at less than 2% by weight, and when 5% by weight is exceeded, there is a concern that the decomposition may not be sufficient.

By dialysis with distilled water in step (f), trichloroacetic acid and the like that may remain may be removed.

In the preparation method of the present invention, the acidic sugar fractions are collected by repeating the steps (a) to (f) to determine purity by electrophoresis, undergo ion exchange chromatography, and salt gradient (0, 0.1, 0.5). , 1, 2.5M NaCl in phosphate buffer) may be further subjected to the step of collecting the fraction containing uronic acid after purification.

In the production method of the present invention, preferred examples of the insects are dung beetles, crickets, cicada snow cordyceps, cows, etc., silkworm pupa, silkworms, western bee queen bees, bumblebee queen bees, and / or clown bugs.

Insect glycosaminoglycans of the present invention can treat and / or prevent diseases caused by dysfunction of anti-inflammatory, anti-diabetic, anticoagulant, anti-cancer, angiogenesis, and / or vasodilator reactions, Insect glycosaminoglycans can be effectively produced by the production method.

Figure 1 shows the results of electrophoresis of cicada snow Cordyceps sinensis, beetle, cricket glycosaminoglycans (Cho).
Fig. 2 is a graph showing the purification profile by ion exchange chromatography of crickets, insects (on iron, etc.) and clown erythropoietic glycosaminoglycans.
Figure 3 is a graph showing the purification profile by the ion exchange chromatography of the beetle (dung beetle), silkworm pupae, silkworm, Western back honeybee queen bee, bumblebee queen bee glycosaminoglycans.
Figure 4 is a graph showing the MALDI-TOF mass chromatography results of crude cicada snow Cordyceps sinensis glycosaminoglycans.
5 is a graph showing the MALDI-TOF mass chromatography results of crude cricket glycosaminoglycans.
6 is a graph showing the MALDI-TOF mass chromatography results of crude insect glycosaminoglycans.
Figure 7 is a graph showing the MALDI-TOF results (7 minutes peak) for the enzyme digested cicada snow Cordyceps sinensis Glycozaminoglycans.
FIG. 8 is a graph showing MALDI-TOF results (9 min peak) for enzymatically degraded Glycozaminoglycans.
9 is a graph showing MALDI-TOF results (8 min peak) for enzymatically digested cricket glycosaminoglycans.
10 shows the HPLC chromatogram results for cricket glycosaminoglycans.
Figure 11 shows the HPLC chromatogram results for Cicada Snow Cord Cordyceps Glycozaminoglycans.
Figure 12 shows the HPLC chromatogram results for the worm glycosaminoglycans.
Figure 13 shows the HPLC chromatogram results for heparan sulfate-based standards.
Figure 14 is a chromatogram of the reaction product obtained by enzymatic digestion of cicadas chinensis fungus superglycozaminoglycans as a strong ion exchange column HPLC recording retention time corresponding to the standard heparin disaccharide.
FIG. 15 is the 1H-NMR result of the elongated glycosaminoglycans. FIG.
FIG. 16 is a 2D COSY NMR result of Tangle glycosaminoglycans. FIG.
Figure 17 shows the 1H-NMR results of the clown stingray glycosaminoglycans.
FIG. 18 is a 2D COSY NMR result of the clown stingray glycosaminoglycan. FIG.
19 shows 1 H-NMR results of cricket glycosaminoglycans.
20 is 2D COZY NMR results of cricket glycosaminoglycans.
Figure 21 shows the 1H-NMR results of silkworm glycosaminoglycans.
22 is a 2D COZY NMR result of silkworm glycosaminoglycans.
Fig. 23 is a diagram showing the structure of glycosaminoglycan oligomers for crickets, galaxies, clown bugs and cicadas.
FIG. 24 is a graph showing the elongated glycosaminoglycan antidiabetic effect.
25 is a graph showing changes in blood glucose with time upon administration of insect glycozaminoglycans.
FIG. 26 is a graph showing a decrease in blood glucose levels after 1 month of administration of insect glycosaminoglycans.
27 is a graph showing the anti-inflammatory effect of the administration of insect glycosaminoglycans when the experimental animals are administered, showing a change in foot size over time.
28 is a control group, CFA alone administration group, Cicada Snowfly Cordyceps sinensis Glycozaminoglycan administration group, Cricket Glycozaminoglycan administration group, Insect Glycozyminoglycan administration group, Crude Tangle Glycozaminoglycan administration group, Indomethacin Photographs of pathologies of the renal group.
29 is a graph showing the anti-inflammatory effect of the administration of the insect glycosaminoglycans of the experimental animals, a graph showing the change in foot size over time.
FIG. 30 shows serum IL-6 levels in the control group, CFA alone group, Western duvet queen bee glycosaminoglycan group, ganglia glycosaminoglycan group, bumblebee queen bee glycosaminoglycan group, indomethacin group Is a graph.
FIG. 31 is a graph showing IL-6 levels in serum of the control group, CFA alone group, cricket glycosaminoglycan group, worm glycosaminoglycan group, and indomethacin group.
32 shows the results of eNOS activity of insect glycozaminoglycans.
FIG. 33 is a graph showing NO acid production according to treatment groups of cricket glycosaminoglycans and worm glycosaminoglycans. FIG.
34 is a graph showing the results of VEGF activity against vascular endothelial cells of insect glycozaminoglycans.
FIG. 35 is a graph showing mTNF-α concentrations of cicada snow Cordyceps sinensis Glycozaminoglycans and Glycozyminoglycans administered group.
36 is a graph showing the mTNF-α concentration of crickets, insects, Cicada Cordyceps Sinensis administered group.

Examples are provided to help understand the present invention. The following examples are merely provided to more easily understand the present invention, but the contents of the present invention are not limited by the examples.

Example 1 Preparation of Insect Glycozaminoglycans

(1) Gangrang (dung beetle) (Chinese Yangil), (2) Cricket (from Chungbuk Eumseong Breeding Farm), (3) Cicada Snow Cordyceps, (4) Caterpillars, (6), (5) Silkworm pupa, (6) Silkworm, (7) Bumblebee Queen Bee, ⑨ 7 insects (obtained from the National Agricultural Research and Development Institute) of the Clown stink bugs are ①1.3 kg, ②2 Kg, ③2.5kg, ④2 kg, ⑤2 kg, ⑥1.8 kg, ⑦2kg, ⑧1 kg, ⑨100 g, and then immersed in ethanol or acetone in the same volume for 3 days to remove fats and filter and remove the residue (insect bark) And the like) were dried well.

The husks are thin, small and dense and tend to be delayed by proteolytic agents. Therefore, the dried residue was ground (100-300 mesh) by a mill (ball mill or hammer mill). After fermentation at 44 ° C for 4 days with 2.5% by weight of sigma alkalase of Bacillus lichenformis as protease in sodium hydrogen carbonate solution (pH 9.0), 50% trichloro was stored. Acetic acid (Trichloroacetic acid) was added to 5% of the total solution and left at 4 ° C for 3 hours to precipitate the protein. The digested protein was removed by centrifugation at more than 8,000 rpm, and the residue was doubled with ethanol (100%). Co-precipitates sugar with potassium acetate, and precipitates are precipitated in twice the amount of cetylpyridinium (5% by weight), and the precipitates are then precipitated in 2 times the amount of ethanol, and then dialysed by distilled water with a dialysis membrane of molecular weight 3500 or above, and lyophilized. Crude insect glycosaminoglycans of ①1.1g, ②3.4g, ③3,9g, ④0.617g, ⑤1.5g, ⑥0.2g, ⑦2.21g, ⑧4.89g and ⑨0.27g were obtained, respectively. .

By repeating this process, the acidic sugar fractions were collected and the purity was determined by electrophoresis, and salt gradient 0, 0.1, 0.5, 1, 2.5M NaCl in through ion exchange chromatography (Sephadex DEAE A-25). phosphate buffer) was further purified to collect fractions with uronic acid to prepare the respective insect glycosaminoglycan fractions.

Experimental Example 1 Analysis of Insect Glycozaminoglycans

1) Electrophoresis

The electrophoresis was performed on the glycosaminoglycans prepared in Example 1. Electrophoresis was performed using TBE (Tris Borate EDTA) buffer solution in 1% agarose gel, and then stained with Azue A after electrophoresis. The results for Cicada Snowhopper Cordyceps Sinensis, Beetle, Cricket Glycozaminoglycan (Crude) are shown in FIG. 1. In Figure 1, the IS of 1 lane is a result for Cicada Snowfly Cordyceps Glycozaminoglycan 1M, the Tb of 2 lanes is Glycozyminoglycan (crude), and the Gb of 3 lanes is Cricket Glycoza Minoglycan (Cho), 4 lanes of IS are Cicada Snow Cordyceps Glycozaminoglycans (Cho), and 5 lanes of Heparin have a heparin average molecular weight of 13KD as a result of using Heparin as a standard product (Sigma). As a result, each insect glycosaminoglycan could be qualitatively identified and the molecular weight was also confirmed.

2) Insect Glycozaminoglycan Purification Profile by Ion Exchange Chromatography

The purification profile for the fraction of glycosaminoglycans prepared in Example 1 was prepared as follows. That is, it was based on the metabolite analysis method of GAG degrading enzyme (HJ-15 (Accession No. KCCM-10096) production degrading enzyme of Korean Patent No. 10-2057168). Among the fractions purified by ion exchange chromatography, for ganglia (dung beetle), cricket, nymph (beetle), silkworm pupa, silkworm, western duvet bee queen, bumblebee queen bee, and clown bug The results are shown in FIGS. 2 and 3.

As a result, it can be seen that the crude glycosaminoglycans of various insects are separated and purified according to the concentration of salts eluting.

3) The content of glycosaminoglycans

The content of glycozaminoglycans prepared in Example 1 was measured. Cricket, beetle, cicada snow Cordyceps sinensis, gangrang, bumblebee queen bee, silkworm pupae, western dune bee queen bee glycosaminoglycans are shown in Tables 1 and 2 below. Table 1 shows the content (mg) of glycosaminoglycans obtained from each insect in fractions, and Table 2 shows the content of crude glycosaminoglycans and fractions in each insect.

The content of glycosaminoglycans was expressed by the amount after each fraction was lyophilized, and the content of uronic acid was glucuronolactone as the standard sugar, and the sample was dissolved in a solvent in which sodium tetraborate (0.025M) was dissolved in concentrated sulfuric acid. After soaking in boiling water for 10 minutes, the carbazole was heated and UV (530 nm) was measured.

Table 1

Neutral sugar content was diluted 1 mg / ml of D-glucose with standard sugar to make dilute glucose solution. The sample was also dissolved in 0.5 ml and 0.3% phenol 5%. After adding 2 ml of concentrated sulfuric acid and leaving it at room temperature for 30 minutes, UV (Jasco, V-550, Japan) absorbance was measured at 484 nm, and calculated by a glucose calibration curve.

The amino sugar content calculates the hexosamine content. Into a 10 mg / ml sample, concentrated HCl (100ul) was added and warmed at 100 ° C for 4 hours, dried using a vacuum pump, and then 100ul of sample or glucosamine (also available to galactosamine). Dissolved in and stirred with 25ul of 1.5M HCl, 2,4-pentanedione (250ul) added with 0.7M sodium tammonate, closed the lid, heated at 100 ℃ for 20 minutes, cooled in cold water, and then added 2 ml 90% ethanol and 500ul Ehrlich reagent. (N, N-dimethyl-p-aminobenzaldehyde, 100mg dissolved in 3.2ml perchloric acid, 100ml with 95% ethanol) was added and stirred, and after UV incubation at 20 ℃ for 1 hour, the UV absorbance at 535 nm was measured and then placed on the calibration curve. Calculated by

[Table 2]

In the table, m means the mean and SE means the standard error.

It can be seen from the above results that a composition containing insect glycosaminoglycans can be prepared from various insects.

4) Molecular weight measurement

The molecular weight of the insect glycozaminoglycans prepared in Example 1 was measured by a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass.

MALDI-TOF analysis was performed using a Matrix-Assisted Laser Desorption Ionization (MALDI) Mass spectrometer (Voyager-DE STR, Applied Biosystems, USA). Mass spectrometry was performed using electron ionization (EI) -mode and Electron 70eV Direct Inlet Probe (DIP). Analyzed using mode.

The results for the crude cicada cordyceps sinensis Glycozaminoglycan, the crude cricket Glycozaminoglycan and the crude insect Glycozaminoglycan are shown in FIGS. 4 to 6, respectively. 4 to 6 are MALDI-TOF mass chromatography of each glycosaminoglycan, and the molecular weight of the insect polymer glycosaminoglycan, which is a polymer, can be confirmed.

5) Sugar structure analysis constituting the insect glycosaminoglycans

Glycosaminoglycan degrading enzyme or acid was treated to the insect glycosaminoglycan prepared in Example 1, digested to sugar, molecular weight was measured by MALDI-TOF, and NMR and GC analysis was performed. In addition, after enzymatic treatment culture supernatant was injected to confirm the structure of the sugar by HPLC-AT ECD detector. The detailed procedure is as follows.

(5-1) Enzymatic Hydrolysis

The degrading enzyme reaction conditions were the recombinant enzyme {heparinase I, II (Journal of Biochemistry and Molecular Biology, 37, 684-690, recombinant) per 1 mg of crude insect glycosaminoglycan prepared in Example 1 ), chondroitin sulfate lyase (Sigma, USA)} 10mU, 50ul, 50mM tris hydrochloric acid buffer solution / NaCl (pH 7.4) was added to adjust the total solution to 1ml, and the reaction temperature was fixed at 37 ° C. Reactant analysis was performed using high performance liquid chromatography (HPLC) when the reaction of the enzyme was completed. That is, using a strong anion exchange column (4.6X250nm, US Phenominox) by separating the sample at a flow rate of 1.0ml / min by sodium chloride concentration gradient (0.1M ~ 1.6M) was used for measurement.

(5-2) MALDI-TOF

The molecular weight of glycosaminoglycans enzyme-lyzed with MALDI-TOF of (5-1) was measured. MALDI-TOF measurement method is the same as 4). And peaks with heparin dissaccharide standards (IS, II-H, IA, II-S, III-H, III-S, IV-A, IV-H, IH, IP, III-S; sigma, USA) Was compared.

MALDI-TOF results for Cicada Snowflower Cordyceps, Insect, and Cricket are shown in FIGS. 7 to 9 are molecular weight analysis results of enzymatically resolved Cicada snowhoppers (7 min peak), insects (9 min peak), cricket (8 min peak) glycosaminoglycans, respectively.

(5-3) Analysis of monosaccharides constituting insect glycosaminoglycans-GC / MS

For the component analysis of the enzyme decomposition product of (5-1), the sample was dissolved in 3M HCl dissolved in methanol solvent in the sample, and then heated to 120 ° C. for 2 hours, cooled to room temperature, dried at 40 ° C. under nitrogen gas, and then 0.3 ml of acetic anhydride ( After adding acetic anhydride), the mixture was left at room temperature for 30 minutes, dried under nitrogen gas at 40 ° C, and then P ₂ O ₅ After drying completely under vacuum pump under 50 μl of pyridine / N, O-bis (trimethylsilyl) trifluoroacetamide (2: 1, v / v) was added to perform silylation at 80 ° C. for 30 minutes. The cylylation samples were analyzed by gas chromatograph (Agilent 6890 (Agilent Technologies) with 5973N mass detector). The column is a HP-5 capillary column of Hewlett-Packard Co., Ltd., with a specification of 0.53 mm x 30m. The inlet temperature is 250 ° C and the detector temperature is 280 ° C. The column temperature is 180 ° C, 10 ° C per minute, and 220 ° C. The program was analyzed to increase by 2 ℃ per minute, 300 ℃ to 100 ℃ per minute. Reagents were used as assay reagent grades: D-glucosamine hydrochloride, N-acetyl-D-galactosamine, D-glucuronic acid, D-(+)-galactosamine hydrochloride, Myo-inositon standard, BSTFA {N, O-Bis (trimethylsilylation) trifluoroacetamide with trimethylchlorosilane} was used from Sigma (USA). Methanol anhydride, acetic anhydride and pyridine anhydride used what was purchased from Sigma-Aldrich (USA).

A) Establishment of analysis conditions for different peaks of TMS derivatized monosaccharides for GC / MS analysis

The amino sugar was analyzed by derivatization of TMS (Trimethylsilylation) which was analyzed by GC / MS after derivatization in order to analyze amino sugar in insect glycan (Analytical biochemistry 347 (2005) 262-274. ).

Polysaccharides, which have undergone TMS derivatization, are transformed into α and β-anomers, pyranose and furanose ring structures, and are separated into multiple peaks when analyzed using GC-MS. Therefore, the standard reagents were analyzed separately to find the time of separation based on the internal standard, and the relative intensities of the peaks were summarized. (Table 3) The standard samples under the following conditions were GC / MSD (gas chromatograph { Agilent 6890 with Agilent 6890 (Agilent Technologies)} mass spectra were obtained to distinguish peaks from the analytical results and the normalized ion values were compared (Table 4). Is [Si (CH ₃ ) ₃ ] and m / z 147 ion is [(CH ₃ ) ₃ SiOSi-(CH ₃ ) ₂ ] In particular, in glucuronic acid, the pyranose structure has an intensity of m / z 204 m / z greater than 217 (see J. Chromatogr. A 720 (1996) 27-49).

[Table 3] Setting of retention time and peak ratio calibration conditions per standard

[Table 4] Retention time per standard Set fragment calibration conditions of corresponding peak

B) Investigation of Neutral Sugar Composition by Hydrolysis

Insect glycosaminoglycans prepared in Example 1 were hydrolyzed with hydrochloric acid (acid hydrolysis) and analyzed by GC / MS, and sample GCMS peaks consistent with the standard (Sigma) of TMS neutral monosaccharides were quantified.

The results for crickets (sample 1), beetle (sample 2), cicada nymphidae (sample 3), and esophagus (sample 4) glycosaminoglycans (crude) are shown in the table below.

[Table 5] Content of Neutral Sugars in Percent Insect Glycosaminoglycans (%)

From the above results, it can be seen that the ratio of acidic and amino sugars as insect glucosaminoglycans is high, and only the content of neutral sugar meso-inositol is high, but the neutral sugar usually has a small content.

C) Analysis of amino sugars by hydrolysis

After derivatization to analyze amino sugars in the sample of B), GC / MS analysis was performed on GC samples. First, the validity of the test method was verified in the standard test and then applied to each sample. In the quantitative calculation method, the area of the detected peak was added when the standard sample was analyzed, and the sample was divided by comparing the retention time and the ion ratio. Hewlett-Packard Co.'s HP-5MS was used for the column, and the quantitative range was up to 0.002 mg. The linearity was over 0.988.

The results for crickets (sample 1), beetle (sample 2), cicada nymphidae (sample 3), and esophagus (sample 4) glycosaminoglycans (crude) are shown in the table below.

TABLE 6

a: mg / 0.015 mg; b: not detected

Sample 3 Cicada, Cordyceps sinensis Glycozaminoglycan has less glucosamine and galatozamine, and both samples have glucuronic acid. Glycozaminoglycans in iron, etc. have a repeating structure with N-acetyl-galactosamine or disaccharides in which glucosamine and glucuronic acid are combined (Table 6, sample 2, worm glycosaminoglycans). See GC-MS analysis).

(5-4) Disaccharide Analysis-HPLC Analysis by Enzymatic Hydrolysis

The product (disaccharide, tetrasaccharide, hexasaccharide insect glycozyminoglycans) by the enzymatic hydrolysis of (5-1) was analyzed using high performance liquid chromatography (HPLC). (Glycobiology vol. 8 no 9 pp. 869-877, 1998 Determination of the structure of oligosaccharides prepared from acharan sulfate)

When the reaction of the enzyme was completed, the reaction was analyzed using high performance liquid chromatography (HPLC). 1.0 ml / min by the sodium chloride concentration gradient using a strong anion exchange column (4.6 × 250 nm, manufactured by Phenominox, USA). The samples are separated and collected at the flow rate of NMR.

Heparin sulfate standard (sigma heparin dissacharide IS, II-H, IA, II-S. III-H, III-S, IV-A, IV-H, IH, IP, III-S) Retention time was used as a control.

The results for crickets, cicadas snow cordyceps, and insect glycosaminoglycans are shown in FIGS. 10 to 12. In addition, the HPLC chromatogram for heparan sulfate-based standard (Sigma) is shown in FIG. The retention time of each standard was calculated based on the retention time of the internal standard.

Figures 10-12 show HPLC chromatogram results for crickets, cicada snowhopper Cordyceps sinensis, and insect glycozaminoglycans.

As a result of comparing the retention time of FIGS. 10 to 12 and the retention time of the heparan sulfate-based standard (FIG. 13), the disaccharides constituting the insect glycosaminoglycan were estimated to be heparan sulfate disaccharides.

FIG. 14 is a chromatogram obtained by HPLC injection of a reaction product obtained by enzymatic digestion of cicada snow cordyceps choglycozaminoglycan, showing the retention time corresponding to the standard heparin disaccharide.

(5-5) NMR Analysis

NMR analysis was performed on the insect glycosaminoglycans prepared in Example 1.

About 1 mg of sample was dissolved in 99.96% D ₂ O and analyzed using high magnetic field magnetic resonance (NMR). The NMR used was performed using a 600 MHz spectrometer connected to a VAX32 computer using a high magnetic field 1H-, 13C- 2-COSY NMR (Avance 600 147FT, Brucker, Germany). Chemical shifts are expressed in ppm relative to the internal standard 3-trimethylsilyl [2H4] sodium propionate.

1H- for Glucozyminoglycans (0M), Clown Stingray Glycozyminoglycans (0.5M), Cricket Glycozyminoglycans (0.5M), Silkworm Glycozyminoglycans (1M) NMR and 2D COSY NMR are shown in Figs. 15 to 22, respectively. 15, 17, 19, and 21 are 1H-NMR results of an elongated, clown stingray, cricket, and silkworm glycosaminoglycans, respectively, and FIGS. 16, 18, 20, and 22 show a trench, clown sting, cricket, and silkworm, respectively. 2D COZY NMR results of lycosaminoglycans.

Glycozyaminoglycans isolated from insects are macromolecules with a molecular weight of 5,000 or more, so it is not easy to settle J values for numerous peaks during NMR analysis. It was confirmed that the sulfuric acid series was consistent with the general pattern, and low molecular weight glycosaminoglycan was prepared for structural analysis. Sample preparation of insect glycosaminoglycans for high performance liquid chromatography after low molecular weight was divided into three parts. First, as an acid sugar analysis method, dissolved in 0.1M HCl and reacted at 80 ° C for 1 hour, cooled, dried with nitrogen gas, acid sugar sample analysis method, and as a second method, dissolved sample in 6N HCl and 4 hours at 100 ℃. After cooling, the reaction was dried with nitrogen gas to analyze amino sugar samples. As a third method, the sample was dissolved in 2M trifluoroacetic acid (TFA), and then cooled at 100 ° C. for 4 hours, and then dried with nitrogen gas. Neutral sugar samples were pretreated and injected into HPLC (Dionex, Summit, USA) for analysis. The column is a strong ion exchange column (phenomeics, USA), sodium chloride 0.1M-1.6M gradient, 232nm UV detector or Dynex, Carbopac PA1 (monosaccharide), Carbopac PA10 (disaccharide or higher oligosaccharide), 16mM-0.3M sodium hydroxide gradient Oligosaccharides such as monosaccharides and disaccharides were analyzed with an ECD detector (ED50, gold electrode, Dionex, USA). All standard products are sigma products, such as D-glucosamine hydrochloride, N-acetyl-D-galactosamine, D-glucuronic acid, D--galaccuronic acid, D-(+)-galactosamine hydrochloride, N Pass through the column using acetyl-D-glucosamine, N-acetyl-D-glucuronic acid, N-acetyl-D-galaccuronic acid, N-acetylneuramic acid (= sialic acid), N-acetylmuramic acid, etc. The retention time recognized by the detector was compared with the insect glycosaminoglycan hydrolyzate.

(5-6) Structure Analysis

From the HPLC data, heparin disaccharide structure and acid hydrolysis (HCl 0.1M) confirmed to be consistent with the standard product after glycosaminoglycan degrading enzyme treatment were injected into HPLC, followed by SAX-HPLC (UV232 nm), HPLC-ECD, and GC. The structures of the insect glycosaminoglycans prepared in Example 1 were analyzed from the monosaccharides, disaccharide retention time and GC-MS fragement obtained by comparing the monosaccharide structure analyzed by -MS with the standard product.

23 shows the structure of glycosaminoglycan oligomers for crickets, galaxies, clown bugs and cicadas. Gb of Figure 23, cricket, CA is an eruption, P is a clown bug, IS refers to the cicada snow cordyceps. As a result, the cricket glycosaminoglycans are glycosaminoglycans having a ΔUA-GlcNAC repeat structure, glycosaminoglycans having a ΔUA-GlcNAC-UA-GlcNS repeat structure, and ΔUA-GlcNAC-UA-GlcNS A glycosaminoglycan selected from the group consisting of glycosaminoglycans having a repeating structure of -UA-GlcNAC6S, wherein the elongated glycosaminoglycans have a repeating structure of ΔUA-2S- [1-4] GlcN. It consists of a lycan, the clown lining glycosaminoglycan is composed of at least one selected from the group consisting of glycosaminoglycan having a ΔUA-GlcNAC repeat structure, and glycosaminoglycan having a repeating structure of ΔUA-GlcNS , Cicada snow Cordyceps sinensis glycosaminoglycans are selected from glycosaminoglycans having a ΔUA-GlcNS repeating structure, and glycosaminoglycans having a repeating structure of ΔUA-2S-GlcN. It can be seen that constituted by any.

Example 2 Anticancer Effect of Insect Glycozaminoglycans

Silkworm glycosaminoglycans prepared in Example 1 and clown lining materials glycosaminoglycans (0.1M, 0.5M fraction), elongated glycosaminoglycans (0M fraction), cricket glycoza Minoglycan (0.5M) fractions were dissolved in sterile physiological saline and used at a concentration of 1 mg / ml, 100 μg / ml, 10 μg / ml. Toxicity of each of the glycosaminoglycan colon cancer cells (CT-26 cell line; Seoul National Cancer Center Cell Line Bank), uterine cancer cells (L929 cell line; Seoul National Cancer Center Cell Line Bank) and normal cells (CHO cell line; Seoul National Cancer Center Cell Line Bank) Sodium 3 '-[1- (phenylamino-carbonyl) -3,4-tetrazolium] -bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate kit solution (Boehringer Mannheim) assay 2 Cell viability was analyzed after one culture, and the result was reduced to IC ₅₀ (50% inhibitory concentration). Each cell line was used by incubating in a condition of 5% CO ₂ , 37 ° C.

The results are shown in the following table. For reference, the 0.5M-1 and 0.5M-2 fractions in the table are the number of fractions added to the tube when the same peak fraction is collected in a tube and the volume is larger in one tube and added to another tube to be lyophilized and used as a sample. From the above results, silkworm glycosaminoglycans, clown stingray glycosaminoglycans, elongated glycosaminoglycans, and cricket glycosaminoglycans did not show toxicity to normal cells, respectively, whereas cancer cells were inhibited. It was confirmed that there is an anticancer effect. Especially, 0.5M fraction of Clownza Glycozaminoglycan, 0.5M fraction of Cricket Glycozaminoglycan and 1M fraction of Silkworm Glycozaminoglycan are less toxic to CHO cells (normal cells) and are toxic to cancer cells. It can be seen that it has anticancer activity.

Table 7 Inhibitory Effects of Insect Glycozaminoglycans on Cancer Cells

Example 3 Confirmation of Anticoagulant Effect of Insect Glycozaminoglycan

Samples were prepared by dissolving the bumblebee queen bee, Western bumblebee queen bee, galaxae, silkworm chrysalis, cicada snowflower Cordyceps sinensis, beetle, cricket, silkworm glycosaminoglycans in solvent phosphate buffered saline (PBS) prepared in Example 1. Activated Partial Thromboplastine Time (APTT) and thrombin time (thrombin time) were measured to determine the anticoagulant effect.

1) Determining Activated Partial Thromboplastin Time (APTT)

After the APTT reagent (Sigma Allexin) was warmed at 37 ° C, the plasma and the sample were mixed 2: 1, and APTT was added thereto, followed by incubation at 37 ° C, and the coagulation time was measured by adding 0.02M CaCl2.

2) measuring thrombin time (TT)

After 250ul of fibrinogen (Sigma) was warmed up to 37 ° C in advance, thrombin (Sigma, 10U / ml) and the sample were mixed 1: 1 and reacted at 37 ° C, and the coagulation time was measured. Heparin was used as a comparative material, and the results are shown in the following table.

3) Antithrombin (AT) III time measurement

The thrombin time measurement was performed by adding 10 U / ml 3 ul of Antithrombin to 27 ul samples instead of 30 ul of samples used for thrombin time measurement.

[Table 8]

In the table, BiQGAG is the bumblebee queen bee glycosaminoglycan, BtQGAG is the western bee queen bee glycosaminoglycan, CmGAG is the beetle glycosaminoglycan, and PswG is the silkworm pupa Control is a solvent as a control, Crude means crude glycosaminoglycans, Separation Solvent Conc. Means the concentration of fractions from the purification of the gradient of the crude glycosaminoglycans.

From the above results, it can be seen that various insect glycosaminoglycans have anticoagulant effects by prolonging anticoagulant times (APTT, TT), and are effective in the order of CmGAG> BiQGAG> PswG> BtQGAG.

In addition, the results are shown in the following table.

TABLE 9

In the table, IsGAG means Cicada Glycozyminoglycan, TbGAG means Glycozyminoglycan, GbGAG means Cricket Glycozaminoglycan, SWG means Silkworm Glycozaminoglycan, and Control is Control Crude is crude glycosaminoglycan and Separation Solvent Conc. Is fraction concentration. All experimental results were statistically processed by Student's t-test, where * means p <0.5 and ** means p <0.01. From the above results, it can be seen that various insect glycosaminoglycans have anticoagulant effects by prolonging anticoagulant times (APTT, TT), and are effective in order of GbGAG>TbGAG>IsGAG> SWG.

Example 4 Confirmation of Antidiabetic Effect of Insect Glycozaminoglycans

1) Tangle glycosaminoglycan antidiabetic effect

Six-week-old diabetic rats (Central experimental animals, South Korea) were bred for four weeks to sufficiently develop diabetes to prepare 36.44 ± 1.39g 10-week-old diabetic rats (C57BLKS / j-db / db). The rats were divided into three groups of 7 rats per group, and the samples were orally administered at a dose of 20 mg / kg on the first day of administration and 10 mg / kg of the second day. The sample was used by dissolving the crude lang glycosaminoglycan of Example 1 in phosphate buffered saline (PBS).

In the control group, physiological saline was administered orally at the same dose as the sample, and for the other group, diabex (Diabex, metformin hydrochloride, Merck product-Daewoong Pharmaceutical Co., Ltd.) was dissolved in PBS and 30 mg / kg dose of the sample. Oral administration in the same manner. In each group, blood glucose levels were measured two hours after the first day and the next day. Blood glucose was measured using a blood sugar kit (Roche) according to the instruction manual.

The results are shown in FIG. Blood glucose on the vertical axis of the figure means blood glucose, and the horizontal axis means the treatment group. As a result, it can be seen that the elongated glycosaminoglycans (CmG) have an effect of reducing blood sugar by 63.24% from 418.1 mg / dl to 153.7 mg / dl. Diabex, on the other hand, had a 3.61% reduction from 480.9 mg / dl to 464.0 mg / dl.

2) Antidiabetic Effects of Cicada Snowhopper Cordyceps, Cricket, and Glycozyminoglycans

Crude Cicada Snowhopper Cordyceps Glycozaminoglycans prepared in Example 1 for 1 month by dividing the group until the 14-week-old diabetic mouse (C57BL db / db; Central Experimental Animal, Korea) with an average weight of 38.36 ± 0.62g , Crude cricket glycosaminoglycans and crude insect glycosaminoglycans were orally administered at a 5 mg / kg dose. Physiological saline was administered orally at the same dose relative to the control group. Blood glucose measurement was measured in the same manner as in 1) above, but the measurement time was measured as 1 hour after administration.

The results are shown in FIGS. 25 and 26. FIG. 25 is a graph showing changes in blood glucose with time upon administration of crude cicada sinusitis Cordyceps sinensis Glycozaminoglycan (ISG), and FIG. 26 is a graph showing a decrease in blood glucose levels with each glycosaminoglycan administration after one month. to be.

From that, Cicada Snow Glowworm Glycozaminoglycans (IsG) decreased by 21.23%, Cricket Glycozynoglycans (GbG) increased by 5.12%, and Glycozyminoglycans (TbG) increased by 0.2%. It can be seen that the percent increase.

Blood samples were also collected and referred to the Green Cross Medical Foundation for serological tests. Neutral lipids are Bayer (USA) kit, Lipase, GK, GPD, colorimetric method; total, Cholesterol using Bayer (USA) kit, cholesterol reagents; HDL cholestrol was measured with an analyzer (ADVIA 1650, Bayer, Japan) using a Bayer (USA) kit, direct HDL-Cholesterol reagent. The results are shown in the following table.

TABLE 10 Serum changes after oral administration of insect glycozaminoglycans to diabetic rats (db) for one month

a CON: PBS phosphate buffered saline alone group

b ISG: Cicada Snow Cordyceps (Isaria sinclairii) glycosaminoglycans 5 mg / kg

cGbG: Cryllus bimaculatus glycosaminoglycan 5 mg / kg

dTbG: Tabus bivittatus glycosaminoglycan 5 mg / kg

Insect glycozyminoglycans were administered orally to 5 mg / kg of diabetic (db / db) mice for one month, followed by culling serum analysis. Triglyceride was decreased in the control group compared to the control group. Chol: High density lipoprotein-cholesterol was not significantly different, while T. Chol (total cholesterol) was found to be decreased in the control group compared to the control group. As a result, it has shown a mechanism of significantly advanced diabetes drugs that improve diabetes while suppressing triglycerides while lowering cholesterol.

All experimental results were statistically treated by Student's t-test, and the results in the table were P <0.05.

From this result, the decrease in the neutral lipid compared to the control indicates that the insect glycozamanoglycan of the present invention has an antidiabetic effect.

Example 5 Confirmation of Inflammatory Effect of Insect Glycozaminoglycan I

1) Experimental Animals and Breeding Conditions

Experimental animals were prepared by breeding male rats (Sam taco) at 226.5 ± 20.5g at 7 weeks of age under conditions of temperature 23 ± 2 ° C, humidity 55 ± 10%, and 12 hour illumination cycle. At this time, water and feed were freely available. After 1 week of adaptation, the animals were divided into groups of 7 animals per group.

2) Preparation of Sample Solution and Sample Administration

Physiological saline 0.9% NaCl was injected into the abdominal cavity by a syringe as a negative control group. For experimental chronic inflammation model, 100 μl of complete Freund's adjuvant (Sigma) was injected into the epidermis of the rat's foot.

All drug treatment groups were intraperitoneally administered the drug 30 minutes before CFA injection. Samples of the cicada snowflakes Cordyceps sinensis Glycozaminoglycans (1M fraction) of Example 1, cricket Glycozaminoglycans (0.5M fraction), the insect Glycozaminoglycans (0.5M fraction), crude elongated Glee Each of kozaminoglycans was dissolved in physiological saline at a rate of 2 mg / kg and used as a dosing sample, followed by repeated continuous intraperitoneal administration for 7 days.

Indomethacin was used as a positive control and dissolved in 100 mM sodium carbonate at a dose of 1 mg / kg and diluted in physiological saline and then intraperitoneally administered.

3) Sole edema measurement

Sole edema was measured by inducing inflammation in the left hind paw with CFA and then dividing the paw into digmatic caliper (Mitutoyo Corporation, Japan). Measurement time is 1 hour, 3 hours, 5 hours, the next day, 2 days, 3 days, 4 days, 7 days, 8 days, 9 days, 10 days, 11 days, 14 days before CFA administration. Using the value as a control, the change in edema is graphed. The results are shown in FIG. 27. 27 is a graph showing changes in foot size over time, and all data were P <0.01.

Cicada Snow Cord Cordyceps Sinensis Glycozaminoglycans {1M Fraction, ISG (1M)}, Cricket Glycozaminoglycans {0.5M Fraction, GbG (0.5M)}, Insect Glycozaminoglycans {0.5 M fraction, TbG (0.5M)}, crude elongated glycosaminoglycans (CAG) all reduce the swelling of the plantar edema, TbG> GbG> ISG> CAG was found to be effective.

4) Histopathological observation

On day 14, the lumbar spinal ganglion (DRG) and spinal cord (spinal cord) connected to the associated inflammation-prone side of the spinal cord, and the inflamed lateral bone and the opposite lateral bone connected to the lumbar spine were extracted for each experimental group, and fixed in neutral formalin. After staining (Hematoxylin-eosin), the pathological tissue was observed under a microscope and photographed. Results for DRG are shown in FIG. 28.

28 is a control group (CON), CFA alone administration group (CFA), cicada snowflakes Cordyceps sinensis glycosaminoglycan administration group (ISG), cricket glycosaminoglycan administration group (GbG), insecticide glycosaminoglycan administration group ( TbG), photomicrographs of the pathological tissues of the crude elongated glycosaminoglycans (CAG) and indomethacin (IND) groups.

From the above picture, it can be observed that the articular surface was destroyed by CFA administration, and the ISG, GbG, and CaG-administered groups were observed to be treated, and the TbG and IND-administered groups were confirmed to be partially incompletely treated.

Therefore, it was confirmed that the insect glycosaminoglycans exhibit anti-inflammatory action.

Example 6 Confirmation of Anti-inflammatory Effect of Insect Glycozaminoglycans II

Except for the daily administration of crude western dune bee queen bee glycosaminoglycan prepared in Example 1, crude amber bee queen bee glycosaminoglycan, crude elongated glycosaminoglycan each for 14 days at 5 mg / kg, It carried out by the same method as 1) -3) of the said Example 5.

The results are shown in FIG. 29. 29 is a graph showing the change in foot size over time, all data was P <0.001.

From the above results, Western bumblebee queen bee glycosaminoglycans (BtQG), bumblebee queen bumblebee glycosaminoglycans (BiQG), and elongated glycosaminoglycans (CaG) all reduce the swelling of the sole of the foot It was confirmed that an excellent anti-inflammatory (inflammation of the edema) effect when administering kozaminoglycan.

Example 7 Confirmation of Anti-inflammatory Effects of Insect Glycozaminoglycans III

5 mg / kg of the crude western duvet bee queen bee glycosaminoglycan, crude amber bee queen bee glycosaminoglycan, and crude elongated glycosaminoglycan, crude cricket glycosaminoglycans, respectively. Except for administering Glycozaminoglycans each day for 14 days at 2 mg / kg each, it was carried out in the same manner as 1) to 2) of Example 5.

Thereafter, blood was collected from experimental animals, and then IL-6 in serum was measured using an IL-6 measurement kit (R & D Systems, USA) according to the manufacturer's instructions. IL-6 is a multifunctional cytokine that plays an important role in host defense, acute phase reaction, immune response, and hematopoiesis and is expressed by the diversity of normal and modified lymphocytes and non- lymphocyte cells. IL-6 levels in elevated serum have been shown in pathological conditions such as bacterial or viral infections, tremor, autoimmune diseases, inflammation and malignancies (Hirano, T. (1988) "Interleukin 6" in The Cytokine Handbook, 3rd ed. Academic Press, New York, p 197).

The results are shown in FIGS. 30 and 31. FIG. 30 shows IL-6 levels in serum by group, CON is a control group, CFA is a CFA-only group, BtQG is a group of Western dwarf queen bee glycosaminoglycans, CmG is a glycosaminoglycans group, and BiQG is a group Bumblebee queen bee glycosaminoglycan administration group, Indomethacin means indomethacin administration group. FIG. 31 also shows IL-6 levels in serum by group, CON is a control group, CFA is a CFA-only group, GbG is a cricket glycosaminoglycan group, and TbG is a pleated glycosaminoglycan group, IND is indomethac Means a new administration group.

From the above results, it can be seen that various insect glycosaminoglycans inhibit IL-6 secretion, and in particular, it can be seen that the anti-inflammatory effect from the bumblebee queen bee glycosaminoglycans.

Example 8 Confirmation of Vasodilation Effect of Insect Glycozaminoglycan

(1) Measurement of endothelial nitric oxide synthase (eNOS) activity

Endothelial nitric oxide synthase (Endothelial nitric oxide synthase) activity when the insect glycosaminoglycan administration was tested as the blood vessels are expanded by the increase of endothelial nitric oxide synthase.

Human Umbilical Vein Endothelial Cells (HUVECs) are passaged with Clonetics EBM-2 and ECM-2 singlequots (CAMBREX (Walkeresvillie, MD, USA) medium to cultivate the cells when they reach the growth stage. A suspension (2x10 ⁶ cells / ml) was prepared, in which 0.5, 1, 2.5M fractions of the cricket glycosaminoglycans prepared in Example 1, 0.1, 0.5, 1 of the insect glycosaminoglycans , Endocelial nitric oxide synthase (eNOS) secreted in supernatants incubated for 40 hours at 37 ° C., 5% CO ₂ and humidification conditions in 2.5well fractions at a dose of 20 mg / ml. Activity was measured by a measurement kit (R & D Systems, USA) and treated with the same dose of heparin as the positive control group, and measured in the same manner with respect to the cell medium cultured with HUVEC without heparin and sample treatment. Ha The control group.

The results are shown in FIG. Figure 32 shows the eNOS activity of insect glycozaminoglycans G0.5, G1, G2.5 are 0.5, 1, 2.5M fractions of cricket glycozyminoglycans, T0.1, T0, respectively. 5, T1, T2.5 means 0.1, 0.5, 1, 2.5M fractions of worm glycosaminoglycans. Heparin is a heparin treatment group and Media means a control group.

From the above results, it can be seen that glycosaminoglycans in crickets, iron, etc. increase endothelial nitric oxide synthase activity, thereby promoting NO production and thereby promoting blood vessel dilation.

(2) NO production effect

NO production was measured by accumulation of nitrite ions in cell media using a Greisies reagent according to the method of Nims et al. (Nims, et al. Methods 7, 48-54, 1995). In other words, cricket glycozyminoglycans prepared in Example 1 in HUVEC (abdominal vascular endothelial cells below human navel) (10ul / 100ul cells) (crude, 0.5M fraction, 1M fraction, 2.5M fraction G), insects Glycosaminoglycans (0.1M, 0.5M fraction, 1M fraction, 2.5M fraction) were treated at 20 mg / ml doses for 20 hours, and VERSAmax microplate reader (Molecular Devices, Menlo Park, CA, USA) The absorbance of the sodium nitrite amount was measured and calculated at 540 nm. As a reference drug, sodium nitroprusside dihydrate (SNP), which produces NO in vivo and is involved in vascular relaxation, was used.

The results are shown in FIG. 33. Figure 33 is a graph showing the NO production by each insect glycozyminoglycan treatment group, GbGcrude, GbG0.5, GbG1, GbG2.5 are crude, 0.5M fraction, 1M fraction of cricket glycosaminoglycan, respectively , 2.5M fraction treatment group, T0.1, T0.5, T1, T2.5 means 0.1M fraction, 0.5M fraction, 1M fraction, 2.5M fraction treatment group of worms glycosaminoglycans, respectively it means. In addition, Heparin means a heparin treatment group, a cell means a control group not treated with a sample or heparin.

From the results, it can be seen that glycosaminoglycans in crickets, iron, etc. promote NO production of vascular endothelial cells, thereby promoting expansion of blood vessels.

Therefore, it can be seen that the insect glycozaminoglycan of the present invention exhibits vasodilatation, and specifically, hypertension, stroke, cerebral hemorrhage, ischemic heart disease, and / or Raynaud's disease, which are diseases caused by dysfunction of vasodilation reactions. Has a prophylactic and / or therapeutic action (see Korean Patent No. 10-0519950).

Example 9 Confirmation of Angiogenesis Inhibitory Effect of Insect Glycozaminoglycans

In order to investigate the effects of insect glycozaminoglycans on the treatment and / or prevention of diseases such as cancers related to angiogenesis, the following experiments were performed (An Mi-young, Effects on the secretion of cytokine from splenocytes, Korean Journal of Pharmacognosy, 37, 110-115, 2006, 30 June 2006).

Human Umbilical Vein Endothelial Cells (HUVECs) are passaged with Clonetics EBM-2 and ECM-2 singlequots (CAMBREX (Walkeresvillie, MD, USA) medium to cultivate the cells when they reach the growth stage. A suspension (2x10 ⁶ cells / ml) was prepared, and 0, 0.1, 1, 2.5M fractions of Cicada gingiva Cordyceps sinensis Glycozaminoglycan prepared in Example 1 above, 0.5 of Cricket Glycozaminoglycans. , 1, 2.5M fractions, 0.1, 0.5, 1, 2.5M fractions of Glycozaminoglycans were added at a dose of 20 mg / ml, respectively, for 48 hours at 37 ° C, 5% CO ₂ and humidification conditions in 96well plates. Vascular endothelial growth factor (VEGF) secreted from the cultured supernatant was measured by Quantikine VEGF Immunoassay kit (R & D systems, Minneapolis, USA) and treated with the same dose of heparin as a positive control group. do not handle, heparin and sample And a control group was to measure the VEGF in the same manner with respect to a cell culture medium cultured HUVEC.

The results are shown in FIG. FIG. 34 shows VEGF activity of vascular endothelial cells of insect glycosaminoglycans as I0, I0.1, I1, and I2.5, 0, 0.1, and 1, respectively, of Cicada Snow Cordyceps Inhibitory Glycozaminoglycans. , 2.5M fraction, G0.5, G1, G2.5 are 0.5, 1, 2.5M fraction, C0.1, T0.1, T0.5, T1, T2.5 of Cricket Glycozaminoglycan, respectively Mean 0.1, 0.5, 1, 2.5M fractions of glycosaminoglycans. Heparin is a heparin treatment group and Media means a control group.

There were no insect glycosaminoglycans involved in the activity, and especially glycosaminoglycans did not promote vascular endothelial growth factors in crickets and iron.

Therefore, it can be seen that the insect glycozaminoglycan of the present invention exhibits an angiogenesis-inhibiting effect. Specifically, growth and metastasis of cancer mediated by angiogenesis, hemangioma, angiofibroma, arthritis, diabetic retinopathy, and premature infants Group consisting of retinopathy, neovascular glaucoma, corneal disease caused by neovascularization, degenerative plaque, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granuloconjunctivitis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, acne It has a prophylactic and / or therapeutic action of a disease caused by one or more angiogenesis selected from (see Korean Patent No. 10-0500298).

Example 10 Confirmation of Inflammatory Mediator TNF-α Inhibitory Effect of Insect Glycozaminoglycans

Inflammation of insect glycosaminoglycans by measuring the activity of pro-inflammatory cytokine (TNF-α), an inflammatory mediator closely related to the progression of inflammation, as follows: Activity was to be measured.

Cicada Snow Cordyceps Sinensis Glycozaminoglycans prepared in Example 1 per 200 uL of suspension of splenocytes isolated from rats (BALB / c, 6 weeks old, female, central laboratory animals, Korea) ), Worm glycosaminoglycans (crude, 0.1, 0.5, 1, 2.5M fraction), cricket glycosaminoglycans (crude, 0.5, 1, 2.5M fraction) in 20ul 1mg / ml, 100ug / ml, 10ug / ml dose to 96 wells (culture) in a 5% carbon dioxide incubator for 24 hours, mTNF-α (mouse Tumor necrosis factor alpha) activity to Quantikine Mouse TNF-α Immunassay kit (R & D systems, USA) Measurements were made according to the instructions provided by the manufacturer.

The results are shown in FIGS. 35 and 36. Figure 35 and Figure 36 shows the mTNF-α concentration by administration group, I0M, I0.1M, I1M means 0, 0.1, 1M fractions of cicada snow Cordyceps sinensis Glycozaminoglycan, respectively, T0.1, T0 .5, T1, T2.5 means 0.1, 0.5, 1, 2.5M fractions of worm glycosaminoglycans, and G0.5, G1, G2.5 are 0.5 of cricket glycosaminoglycans, respectively. , 1, 2.5M fraction, TbG means crude insect Glycozaminoglycan, GbG means crude Cricket Glycozaminoglycan, ISGAG means crude Cicada Snow Cordyceps, and bar graph of each group 1mg / ml, 100ug / ml, 10ug / ml dose administration group.

As a result, various insect glycosaminoglycans were found to inhibit the activity of Tumor necrosis factor alpha (TNF-α) in a concentration-dependent manner.

Therefore, it can be seen that the insect glycozaminoglycan exhibits anti-inflammatory action and the like by inhibiting TNF-α.

Claims (7)

(a) insects selected from bumblebee queen bees, dwarf bee queen bees, dung beetles, silkworm chrysalis, cicadas, cicada or crows, or from crickets; 1 to 4 days of immersion in at least one organic solvent selected from the group consisting of chloroform and dichloromethane to remove fats and filtered to dry the residue that is not filtered;
(b) pulverizing the residue dried in the step (a) to a particle size of 100 ~ 300 mesh;
(c) treating the ground product obtained in step (b) with a proteolytic agent in sodium hydrogen carbonate solution (pH 9.0) to decompose the protein in the ground product;
(d) removing the protein degraded in step (c) by centrifugation with trichloroacetic acid (5% by weight);
(e) co-precipitating the residue left after protein removal in step (d) with ethanol and potassium acetate or sodium acetate, and again precipitate the precipitate in cetylpyridinium, and then precipitate the precipitate in ethanol;
(f) dialysis of the precipitate produced in step (e) with distilled water; And
(g) collecting acidic sugar fractions by repeating steps (a) to (f), through ion exchange chromatography, and purified by salt gradient of 0, 0.1, 0.5, 1, 2.5M NaCl in phosphate buffer,
An anticoagulant composition comprising an insect glycozyminoglycan fraction selected from the following as an active ingredient:
1.0M bumblebee queen bee glycosaminoglycan fraction obtained from step (g);
2.5M bumblebee queen bee glycosaminoglycan fraction obtained from step (g);
1.0M western duvet queen bee glycosaminoglycan fraction obtained from step (g);
1.0M dung beetle glycosaminoglycan fraction obtained from step (g);
2.5M dung beetle glycosaminoglycan fraction obtained from step (g);
1.0 M silkworm chrysalis glycosaminoglycan fraction obtained from step (g);
2.5M silkworm chrysalis glycosaminoglycan fraction obtained from step (g);
1.0M cicada snow flower Cordyceps sinensis glycosaminoglycan fraction obtained from step (g);
Glycozaminoglycan fractions from 1.0 M iron obtained from step (g); or
2.5M cricket glycosaminoglycan fraction obtained from step (g). delete 2. The cricket glycosaminoglycan fraction of claim 1 is a glycosaminoglycan having a ΔUA-GlcNAC repeat structure, a glycosaminoglycan having a ΔUA-GlcNAC-UA-GlcNS repeat structure, and ΔUA. -GlcNAC-UA-GlcNS-UA-GlcNAC6S A composition containing at least one selected from glycosaminoglycans having a repeating structure. The composition of claim 1, wherein the dung beetle glycosaminoglycan fraction contains glycosaminoglycans having a repeating structure of ΔUA-2S- [1-4] GlcN. The method according to claim 1, wherein the Cicada Snowflower Cordyceps Glycozaminoglycan fraction is selected from glycosaminoglycans having a ΔUA-GlcNS repeating structure, and glycosaminoglycans having a repeating structure of ΔUA-2S-GlcN. A composition containing at least one selected. The composition according to claim 1, wherein the dung beetle is estuary, and the dung beetle is worm. The glycosaminoglycan fraction of claim 1, wherein the glycosaminoglycan fraction comprises a glycosaminoglycan having a repeating structure having N-acetyl-galactosamine or a disaccharide of glucosamine and glucuronic acid. Composition.

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