KR20170047538A - N-Glycan Purifed from BIC: clony shell of B. ignitus or B. terrestris worker and theirs Use Thereof - Google Patents
N-Glycan Purifed from BIC: clony shell of B. ignitus or B. terrestris worker and theirs Use Thereof Download PDFInfo
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- KR20170047538A KR20170047538A KR1020150147768A KR20150147768A KR20170047538A KR 20170047538 A KR20170047538 A KR 20170047538A KR 1020150147768 A KR1020150147768 A KR 1020150147768A KR 20150147768 A KR20150147768 A KR 20150147768A KR 20170047538 A KR20170047538 A KR 20170047538A
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- glycan
- vascular endothelial
- hex
- derived
- dhex
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
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- A23V2250/204—Animal extracts
Abstract
The present invention relates to N-glycan derived from Bombus ignitus shell having a sugar chain structure of (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) and fractions containing the same. (N-glycan) and (Hex) 3 (HexNAc) derived from Bombus ignitus Bark shell of Hex 6, Hex 7, Hex 10, Hex 11, N-glycan derived from Bombus terrestris worker bees of 2 (dHex) 1 (m / z 1080.4); And a pharmaceutical composition for preventing and treating vascular endothelial stenosis containing the N-glycan or a fraction containing the N-glycan as an effective ingredient.
The N-glycan derived from the bee bark or Western beetle containing the bumblebee larva pupa according to the present invention and its fractions can be used as medicines, cosmetics and functional food materials as their biological activity is known It has the potential as a pharmaceutical and functional molecule, and in particular has an effect of preventing vascular endothelial stenosis. Thus, by enabling the development of N-glycan drugs derived from the bongun shell containing the bumblebee larva pupa or the beetle of Western beetles, It can contribute to preservation and improvement of public health.
Description
The present invention relates to N-glycan derived from beetle bark or beetle beetle containing a bugle larva pupa having a specific sugar chain structure and its use.
Glycosaminoglycan (GAG) is an anionic heterobifunctional polysaccharide with repeating disaccharide units, which is repeated numerous times through the linkage of uronic acid and amino sugar (N-acetylglucoamine, N-acetylgalactosamine) (SO 3 - ) or carboxyl group (COO -) on the sugar moiety and have a negative charge, and their characteristics are classified according to the type of bonding between the sugar moieties and the number and position of the sulfate moieties. GAG is divided into sulfated polysaccharide and non-sulfur polysaccharide depending on the presence or absence of sulfate groups. Representative GAGs include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin / heparan sulfate and keratan sulfate.
GAG, except for hyaluronic acid, is a proteoglycan different from glycoprotein. Chondroitin sulfate and keratan sulfate (or dermatan sulfate) chondroitin family and heparin family, which have many anionic groups, There are features.
In addition, the glycoprotein present in the cell membrane is divided into N-glycan and O-glycan. Among them, N-glycan is formed by binding of N-acetylglucosamine to the NH 2 residue of asparagine and NX ) -S (Serine) / T (N-Glycan is N-Glycan) is a high mannose and can be attached to the ER It is synthesized in an immature form and then transferred to Golgi as a hybrid and becomes a mature type complex. It becomes a mature form that is complexly synthesized in various forms combined with N-glycosamine again in mannose. It is regulated by enzymes and cell functions can be regulated and changed by subtle changes in sugar chains and subtle differences.
Recently, since various biological activities of GAG or N-glycan have been known, they have been used as medicines, cosmetics, and functional food materials. Thus, their value as pharmaceutical and functional molecules has been increasing. Especially, growth factors and cytokine production It affects tissue binding and maturation of specific tissues, acts as a biological filter, controls collagen fiber synthesis, skin tensile strength, and also affects the growth and invasion of cancer cells.
It is difficult to secure safety as a raw material due to various diseases such as mad cow disease, swine influenza, cholera, foot-and-mouth disease and avian influenza, which are used as main ingredients of cattle, pigs and chickens. There are several international agreements to prevent extinction due to the declining population, which limits annual production. Because of the difficulty of securing such resources, new alternative resources are needed.
Accordingly, the present inventors selected insects as a new GAG source. Among them, N-glycan was isolated from queen bees and quail bees of Western beetles and patent application (Publication No. 10-2011-0107303, 2013). In view of the fact that N-glycan derived from queen bee and bumble bee queen bee is superior in activity but is more expensive than bees in number, the present patent does not disclose that the bee itself as a new material (saving larvae) N-glycans were prepared from Western beetles. In other words, the proteoglycans were prepared from the bumble bees and the alcohol extracts from the Western beetles, and the protein moieties were treated with N-Glycosidase F, and impurities other than sugars were treated with porous carbon graphite cartridges The N-glycan derived from the shell or wormwood was separated, and then the sugar sequence and the structure were identified by MS and MS / MS, thereby completing the present invention.
It is an object of the present invention to provide N-glycans derived from Bongun shells and N-glycans derived from Western beetle beans and their fractions containing a bumble bee larva pupa having a specific glycan structure composed of at least one Hex, dHex and / or HexNAc .
Another object of the present invention is to provide a pharmaceutical composition for treating inflammation or arthritis containing the N-glycan or the fraction as an active ingredient.
In order to achieve the above object, the present invention relates to a method for producing N-glycan from Bombus ignitus bark having a sugar chain structure of (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) . m / z 1080.4, (Hex) 3 (HexNAc) 2 (dHex) 1,
(Bombus terrestris) worker bean-derived N-glycans and fractions thereof.
The present invention also relates to the use of the Bombus ignitus Bark N-glycan and Hexexin (Hex) 6, (Hex) 7, (Hex) 10, (Hex) Bombus terrestris) N-glycan (N-glycan) derived from work bee Bombus terrestris Bombus larvae containing pupae larvae or N-glycan derived from worker bees Bombus terrestris strain N-glycan.
The present invention also provides a pharmaceutical composition for treating inflammation or arthritis containing the N-glycan or the fraction as an active ingredient.
N-glycan derived from beetle bark or Western beetle workbench comprising a bug beetle larva having a specific sugar chain structure composed of at least one Hex and / or HexGlcNAc according to the present invention and glycoxamino glycan Can be used as medicines, cosmetics and functional food materials as its biological activity is known, and thus has a value as a pharmaceutical and functional molecule. Especially, it has an effect of treatment for anti-inflammatory or arthritis. Thus, By enabling the development of pharmaceuticals containing N-glycan, it is possible to contribute to the conservation of the income of the beekeeping farmers and the improvement of the national health.
Brief Description of the Drawings Figure 1 shows the results of a N-glycan salt gradient 0 M: MMALDI-TOF MS / MS chromatography (A) from a Bongun shell containing a bumblebug larvae pupa; (B) of (Hex) 3 (HexNAc) 2 (dHex) 2 (m / z 1079.4); (Hex) 6, (Hex) 7, (Hex) 8, (Hex) 9, Hex) 10, (Hex) glycan) of the sugar chain structure.
Figure 2 is a graph showing the effect of the N-glycan salt-derived N-glycans (N-glycans) from the Bombyx mori larvae containing the bumblebee larvae pupae, 0 M: m / z 1337.4, glycan)
FIG. 3 is a graph showing the results of the N-glycan salt-derived N-glycan (N-glycans) from the Bombyx mori larvae containing the bumblebee larvae pupa, 0 M: m / z 1499.5, glycan)
Figure 4 shows the sugar chain structure (B) of the N-glycan salt gradient 1 M: (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) from the Bongun shells containing the bumblebug larvae. (Hex) 6, (Hex) 7, (Hex) 8, (Hex) 9, Hex) 10 Bombus ignitus Bark-derived N-glycan
Figure 5 shows the sugar chain structure (B) of the N-glycan salt gradient 1 M: (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) from the Bongun shells containing the bumblebug larvae. (Hex) 6, (Hex) 7, (Hex) 8, (Hex) 9, Hex) 10 Bombus ignitus Bark-derived N-glycan
Figure 6 shows the sugar chain structure (B) of the N-glycan salt gradient 2.5 M: (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) from the Bongun shell containing the bumble bee larvae pupa; (Hex) 6, (Hex) 7, (Hex) 8, (Hex) 9, Hex) 10, (Hex) glycan)
Figure 7 shows the sugar chain structure (B) of the N-glycan salt gradient 2.5 M: (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) from the Bongun shell containing the bumble bee larvae pupa; (Hex) 6, (Hex) 7, (Hex) 8, (Hex) 9, Hex) 10, (Hex) glycan)
Figure 8 shows the sugar chain structure (B) of a Bongun shell derived N-glycan salt gradient 2.5 M: (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) containing a bumblebug larva pupa; (Hex) 6, (Hex) 7, (Hex) 8, (Hex) 9, Hex) 10, (Hex) glycan)
FIG. 9 is a graph showing the activity of the N-glycan salt-derived N-glycans derived from Bombus terrestris work bees having a sugar chain structure of 0 M: m / z 1080.4, (Hex) 3 (HexNAc) 2 (dHex) N-glycan.
Hereinafter, the present invention will be described in detail.
Recently, it has been known that N-glycan has various biological activities and it is used as pharmaceuticals, cosmetics and functional food materials. Therefore, it has a valuable value as a pharmaceutical and functional molecule. It is true. Thus, the present inventors completed the present invention by selecting a Bongun shell or a Western beetle bee containing a bumblebee larva pupa as a new GAG source and identifying the N-glycan per se sequence of the purified GAG .
The present invention, in one aspect, relates to a method for the production of Bone-shell-derived N-glycans comprising Bombus ignitus bumble bee larvae having a (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4) N-glycan).
In another aspect, the present invention relates to N-glycans derived from Western beetles having a sugar chain structure of (Hex) 3 (HexNAc) 2 (dHex) 1 (m / z 1080.4).
In the present invention, in purifying N-glycan from glycocalypse minoglucan derived from Bongusun shell or Bacillus thuringiensis including Bombus ignitus bumblebug larvae, ion-exchange chromatography ion exchange chromatography, the concentration of sodium chloride used in the elution is 0, 1 or 2.5 M.
In the present invention, the structure of N-glycan derived from Bongusun shell or Bacillus thuringiensis including Bombus ignitus larvae is composed of Hex, dHex and GlcNAc, and more specifically, Hex 3 (HexNAc) 2 (dHex) (m / z 1079.4) or (Hex) 3 (HexNAc) 2 (dHex) 1 (m / z 1080.4), and fractions thereof include Hex3HexNAc2dHex1, Hex3HexNAc3, and Hex3HexNAc6 (N-glycan) having a sugar chain structure.
Thus, in another aspect, the present invention provides a process for the preparation of (Hex) 3 (HexNAc) 2 (dHex) (m / z 1079.4), (Hex) (Bombus ignitus) bumblebee larvae, or fractions containing glycosaminoglycan derived from Western beetles.
In another aspect, the present invention relates to a N-glycan derived from a beetle shell or a Western beetle bee containing a Bombus terrestris bumblebee larva pupa having a sugar chain structure of Hex8 (1377.5 m / z) .
In the present invention, in the purification of N-glycan from glycocalypse minoglucan derived from the bee bark or Western beeswax including the Bombus terrestris bumblebee larva pupa, ion exchange chromatography the concentration of sodium chloride used in the elution in ion exchange chromatography is 0, 1 or 2.5 M.
In the present invention, as shown in FIG. 5, N-glycan derived from the Bombyx mori or Bombyx mori, including the Bombus terrestris bumblebee larva pupa, has a linear structure composed of Hex , And fractions thereof are characterized by mixing N-glycans having a sugar chain structure of Hex7, Hex3HexNAc3dHex2, Hex4HexNAc3Pen1, Hex9 and Hex5HexNAc3dHex2.
Accordingly, the present invention provides, in a different aspect, a Bombus terrestris bumblebug larvae having an N-glycan sugar chain structure of Hex7, Hex8 (1377.5 m / z), Hex3HexNAc3dHex2, Hex4HexNAc3Pen1, Hex9, and Hex5HexNAc3dHex2 , Or fractions containing glycosaminoglycan derived from Western beetles.
The term " chromatography " in the present invention refers to a method of separating the adsorbent particles based on the difference in adsorption characteristics of the solute to the adsorbent particles by passing a mixture containing a solute in a column of a predetermined length packed with the porous adsorbent particles Method. If the chromatographic process consists of a mobile phase and a stationary phase, the stationary phase can use an adsorbent, an ion exchange resin, a porous material or a gel. Liquid chromatography, which is useful for the separation analysis of multi-component mixed samples, can be typified by high-pressure liquid chromatography, ion-exchange chromatography and affinity chromatography. In the present invention, ionic or electrically-conductive compounds, that is, N-glycans of insects are highly polar, and ion exchange chromatography using the principle of adsorption with a filler by an electric force It is preferable to use ion-exchange chromatography.
The term " elution " in the present invention can be used in combination with " elution ", and the polarity of the N-glycan bonded to the ion exchange resin used in ion-exchange chromatography Is a process of obtaining a fraction having a high uronic acid content by using a buffer having a different salt concentration by a salt gradient.
Salt gradient elution is carried out using sodium chloride to obtain a Bongun shell or a Western beetle N-glycan or fraction containing the bumble bee larva pupa of the present invention.
N-glycan according to the present invention can be produced by (a) adding to a bee bark or western beetle containing a bumblebee larvae pupa, acetone, hexane, ethyl acetate, methyl alcohol, ethyl alcohol, propanol, butanol, acetonitrile, (B) removing proteins therefrom, and (c) precipitating and dialyzing the precipitate with an acidic N, N'-tetramethylglucoside The N-glycan fraction is collected and (d) is purified through a salt gradient elution step using sodium chloride in the course of ion exchange chromatography.
The protein removal in step (b) can be carried out using a proteolytic cleavage method including a protease derived from Bacillus licheniformis, protease such as alkaline protease, Aspergillus oryzae, etc. In this case, The required time is 3 to 7 days, which can promote protein degradation at room temperature or above, and can accelerate the reaction at 44 ° C. By using 2 to 5% by weight of the release of the protein powder, the protein of the bee skin can be effectively removed.
In the present invention, the ion exchange chromatography uses Sephadex DEAE A-25 as a filler, but is not limited thereto.
In another aspect, the present invention relates to a pharmaceutical composition for treating inflammation or arthritis containing the N-glycan as an active ingredient.
In another aspect, the present invention relates to a pharmaceutical composition for treating inflammation or arthritis containing the fraction as an active ingredient.
In the present invention, the pharmaceutical composition containing the insect N-glycan or the fraction as an active ingredient may be effective for inhibiting angiogenesis and diseases caused by vasodilation have.
In the present invention, the active ingredient of the pharmaceutical composition, N-glycan, or a fraction containing the N-glycan, may be selected from the group consisting of Bombyx mori (Bombus ignitus) bumblebee larvae or Bombus terrestris ) Bumble bee larvae or worm barks containing worms, and may be composed solely or in combination.
The pharmaceutical composition of the present invention may further comprise at least one selected from the group consisting of Gryllus bimaculatus, Catharsius molossus, Cryptomeria japonica (Bombyx mori), and the like, in addition to the N-glycan or its fractions derived from the bee bark or Western beetle, Tabanus bivittatus, or N-glycan derived from Poecilocoris lewisi, or a glycosaminoglycan containing the same.
The pharmaceutical composition or the functional food of the present invention may be formulated to contain at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.
The pharmaceutically acceptable carrier may be a mixture of at least one of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and any of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, solvents, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants or flavors may additionally be added. Further, it can be suitably formulated according to each disease or ingredient, using methods disclosed in the art or by methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.
The pharmaceutical compositions of the present invention can be formulated by the addition of pharmaceutically acceptable excipients, such as solid, semi-solid or liquid diluents, fillers and formulation auxiliaries of any type, and preferred formulations include tablets, coated tablets, capsules, pills, granules, Suspensions, emulsions, pastes, ointments, gels, creams, lotions, powders, syrups, juices, drops, injectable solutions, sustained-release preparations and sprays.
Tablets, coated tablets, capsules, pills and granules may contain conventional excipients such as (a) fillers and weighting agents such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders (C) humectants such as glycerol, (d) disintegrants such as agar, calcium carbonate and sodium carbonate, (e) dissolution retardants such as paraffin, (g) wetting agents such as cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin and bentonite; and (i) lubricants (for example, For example, talc, calcium stearate, magnesium stearate, magnesium stearate talc and solid polyethylene glycols), or a mixture of the substances described in (a) to (i) above. Tablets, capsules, pills, and granules can be coated to release the effective compound only at a specific site of the intestinal tract. If necessary, the composition can be formulated into a sustained release form using a polymer or the like, or microencapsulated.
Formulations for parenteral administration include injections, suspensions, emulsions, lyophilisers, suppositories, and the like. Suppositories may contain conventional water-soluble or water-insoluble excipients such as fats such as polyethylene glycol, cacao fat, higher esters (e.g., C14-alcohol with C16-fatty acid), witepsol, macrogol, Lt; RTI ID = 0.0 > gelatin < / RTI >
The ointments, pastes, creams and gels may contain conventional excipients other than the active compound, lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or a mixture of these materials. The spraying agent may further contain a conventional propellant, for example, chlorofluorohydrocarbons, and may be formulated into a nasal spray agent containing PEG-4000 and glycerin.
The liquid agent and the emulsion agent may contain, in addition to the active ingredient, conventional excipients such as solvents, solubilizing agents and emulsifiers such as water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 1,3-butylene glycol, dimethylformamide oil, fatty acid esters of cottonseed oil, peanut oil, corn steep liquor, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycol and sorbitan , Or a mixture of these materials. The liquid preparation for parenteral administration and the emulsion preparation may be formulated in a sterile form which is isotonic with blood.
In addition to the active compound, the suspending agent may contain conventional excipients such as liquid diluents (e.g., water, ethyl alcohol, propylene glycol, polyethylene glycol) and suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and / Lactose esters), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, injectable esters such as ethyl oleate, or mixtures of these materials.
The formulations may also contain coloring agents, preservatives and additives which improve the odor or taste, for example, peppermint oil and eucalyptus oil and sweeteners such as saccharin, and may contain other pharmaceutical active compounds other than the compounds according to the invention .
The formulations are prepared in a conventional manner, for example 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.
The pharmaceutical composition of the present invention may be administered orally, intravenously, intraperitoneally, intramuscularly, intramuscularly, transdermally, nasally, by inhalation, topically, rectally, orally, intraocularly or intradermally, can do.
The pharmaceutical composition of the present invention is administered in an amount of 0.1 mg / kg to 900 mg / kg per day based on the amount of each N-glycan (or N-glycan) Can be adjusted. However, the optimal dose to be administered can be easily determined by a person skilled in the art, and can be easily determined by a person skilled in the art and depends on the kind of the disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the pharmaceutical composition, Health condition, sex and diet, time of administration, route of administration, duration of treatment, and concurrent medication.
The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.
In this case, unless otherwise defined, technical terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the following description and the accompanying drawings, A description of known functions and configurations that may unnecessarily obscure the description of the present invention will be omitted.
[Example 1] Preparation of bamboo beetle bark and western beeswax glycocin minoglucan
The results obtained for the bumble beetle larvae of Bumblebee larvae and the residues of western birds were obtained from the National Institute of Agricultural Science and Technology, Rural Development Administration, as shown in Table 1 below. Insecticidal glycosaminoglycan was obtained as shown in Table 1 below. 1 Kg was crushed and immersed in ethanol or acetone having a volume equal to or more than the same amount for 3 days to remove the fatty acids, and then the residue (insect bark, etc.) which was not filtered was dried. The dried residue was pulverized (100 to 300 mesh) with a ball mill or hammer mill, added with 2.5% by weight of Sigma Alkalase (pH 9.0) derived from bacillus lichenformis, and subjected to enzymatic treatment at 44 for 4 days . ≪ / RTI >
Bongun shell
worker
The mixture was added to the enzyme-treated solution so that the final concentration of 50% trichloroacetic acid in the refrigerator was 5%, the protein was precipitated by reacting for 4 to 3 hours, and then centrifuged (8000 rpm) After the precipitate was precipitated in cetylpyridinium (5% by weight), the precipitate was again precipitated in 2-fold amount of ethanol, and then distilled water , Dialyzed using a dialysis membrane having a molecular weight of 3500 or more, and lyophilized to obtain crude glicorzominoglycan derived from Bongun shell or Western bladder bean containing 2.21 g and 4.89 g of bumblebug larvae, respectively.
2. Preparation of N-glycan fraction of bumble bee bark and Western beetle
The crude glycosaminoglycan extracted was repeatedly subjected to the above procedure and then subjected to salt gradient (0, 0.1, 0.5, 1, 2.5 M NaCl) using ion exchange chromatography (Sephadex DEAE A-25) in phosphate buffer. Fractions containing Uronic acid were collected and then subjected to freeze-drying after removal of saline using a molecular weight 3500 cutoff dialysis (FIG. 1).
The purity was confirmed by a single molecule of SAX (strong ion exchange, phenomex, 250 x 10 mm) -HPLC, 232 nm after treatment with glycansolytic enzyme (heparinase I, II, III, chondroitin ABCase) To confirm the purity (Fig. 2).
[Example 2] Sequence analysis of N-glycan (N-glycan) in bark beetle bark and Western beetle
1. Humpback bark and N. glycan concentrate
After the extraction of the Bongun shells containing the bumblebug larva pupa or the Western beetle extract, only the glycans (N-glycan) were released from the proteoglycans isolated from the crude extract and the following procedure was performed to analyze their sugar sequences Respectively.
The denaturation buffer was added to the proteoglycans, and the protein fractions were inactivated by boiling for 10 minutes. The protein fractions were inactivated by treatment with 2 N-glycosidase F (1000 U, manufacturer, National) at 37 for 20 hours , The protein portion was removed, and finally the obtained N-glycan (N-glycan) was vacuum dried and stored.
To separate N-glycan, a porous carbon graphite cartridge (PGC, product name: Extract-clean columns carbograph, manufactured by Grace) was dissolved in 80% acetonitrile / 0.1% trifluoroacetic acid (v / v) mixed solution, the conditions were set with ultrapure water, the N-glycan was loaded onto the cartridge, and the cartridge was allowed to stand for 10 minutes, then washed with 4 mL of ultrapure water, Respectively.
Thereafter, it was eluted with 40% acetonitirle / 0.05% trifluoroacetic acid, and then permethylation was performed. After vacuum drying, 2,5-dihydroxybenzoic acid (10 mg / (MALDI) time-of-flight (TOF) analyzer (AXIMA Resonance, Shimadzu) and positive polarity. The analysis conditions of the MALDI-TOF were as shown in Table 2 , And the presence or absence of a peak was confirmed in a range of 600 to 3500 m / z.
As a result, as shown in Table 2, MS / MS was performed after confirming the molecular weight pattern by MS, and the data obtained therefrom were subjected to a glycan data base library program to confirm the sequence analysis. The sugar sequence and structure of N-glycan derived from the Bongun shell or western tailworm bean containing the bumblebug larvae were identified (Table 3, Figs. 1 to 9).
Identification of sequence structure per insect N-Glycan
[Example 3] Investigation of the removal activity of vascular endothelial cell adhesion material of N-glycans and N. glaucans
Laminin ELISA was performed manually using the QuantiMatrix Human laminin Elisa kit from Millipore (Temecula, CA, USA). In other words, Diabetic HUVEC cells were dispensed in a 24-well plate at a rate of 1 × 10 5 cells / well, treated with 0.2 mg / ml of a sample such as N-glycans of bumble bees and sea bream beeswax Glycosaminoglycan, 37 incubator and then subjected to laminin ELISA. Laminin treatment Cell culture supernatant for 24 hours and rabbit anti-human laminin were mixed at the same temperature for 10 min. Then, 100 μl of the supernatant was added to the well plate for 1 hr and then washed 4 times with wash buffer. 100 ul goat anti-rabbit IgG-HRP conjugate is dispensed into each well, reacted at room temperature for 30 minutes, and rinsed 4 times with wash buffer. Add 100 μl TMB / E substrate to each well and incubate at room temperature for 10 minutes. Measure absorbance at 450 nm.
Fibronectin ELISA was performed manually using the QuantiMatrix Human Fibronectin Elisa kit from Millipore (Temecula, CA, USA). In other words, Diabetic HUVEC cells were dispensed in a 24-well plate at a rate of 1 × 10 5 cells / well, treated with 0.2 mg / ml of a sample such as N-glycans of bumble bees and sea bream beeswax Glycosaminoglycan, 37 incubator and then subjected to Fibronectin ELISA.
Samples and rabbit anti-human Fibronectin are mixed in the same volume for 10 minutes at room temperature, then 100 μl is removed from the well plate for 1 hour and then washed 4 times with wash buffer. 100 ul goat anti-rabbit IgG-HRP conjugate is dispensed into each well, reacted at room temperature for 30 minutes, and rinsed 4 times with wash buffer. 100 μl of TMB / E substrate was added to each well and incubated at room temperature for 10 minutes. Absorbance was measured at 450 nm.
As a result, in the HUVEC vascular endothelial cells, the laminin adherent material was significantly (p <0.05) higher in the N-glycane crude bark, the N-glycan 1M, and the N-glycan 2.5 M, , And a decrease in laminin was also observed in N. glycans in Western beetles. In addition, no significant difference was observed in the HVEC vascular endothelial cell counts in the Fvronectin challenge, but it was not significantly different between the HVECEC vascular endothelial cells and the HVECEC vascular endothelial cells, It is possible to confirm the suppression of the fibronectin vascular endothelial adhesion substance compared to the PBS control group at the N. glaucon 0M of Western blind workers, and it is possible to prevent the damage substance of the vascular endothelium such as inflammation and the like, Stenosis and vascular-related diseases can be prevented. Such vascular endothelial stenosis or vascular-related diseases include, but are not limited to, stroke or hypertension.
Claims (10)
(a) a step of crushing the roots of the Bongbu larvae containing the bumblebee larvae, and then dipping them in ethanol or acetone having a volume equal to or more than the same amount for 3 days to remove the fats and filtration to dry the residue (insect bark, etc.)
b) The dried residue was pulverized with a ball mill or a hammer mill, added with 2.5% by weight of Sigma Alkarase (pH 9.0) derived from bacillus lichenformis, and the protein was degraded by enzymatic treatment at 44 for 4 days Step
c) The enzyme was added to the enzyme-treated solution of step b) so that the final concentration of 50% trichloroacetic acid in the refrigerated storage was 5%, and the protein was precipitated by reacting for 4 to 3 hours, followed by centrifugation (8000 rpm), the precipitate was precipitated in a volume of cetylpyridinium (5% by weight), and then the precipitate was again dissolved in two volumes of ethanol Dialyzed with distilled water using a dialysis membrane having a molecular weight of 3500 or more and lyophilized to obtain crude granocarcinominoglycan derived from the bark beetle shell or Western beetle beetle containing the bumblebug larva pupa
d) The obtained crude glycosaminoglycan was subjected to salt gradient (0, 0.1, 0.5, 1, 2.5 M NaCl in phosphate buffer (pH 7.0) using ion exchange chromatography (Sephadex DEAE A-25) ) And collecting fractions having Uronic acid after purification
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