JPWO2006059670A1 - Chitosan complex - Google Patents

Abstract

The present invention provides a sialic acid-containing complex capable of capturing many types of viruses, and a natural product-derived virus trap using the same. The present invention relates to a chitosan complex formed by reducing and condensing the reducing end of sialic acid or a sugar chain containing sialic acid to an amino group of chitosan, and a virus trap using the same. As the sugar chain containing sialic acid of the present invention, it is preferable to use a mixture of sugar chains containing sialic acid obtained from milk, such as bovine milk, particularly colostrum, or a processed product thereof, to thereby prevent many kinds of viruses. A virus-capturing body that can be simultaneously captured can be easily and inexpensively produced.

Description

  The present invention includes a complex in which sialic acid or a sugar chain containing sialic acid (hereinafter collectively referred to as sialic acid or the like) and chitosan are directly covalently bonded, a production method thereof, and the complex. It relates to a virus trapping agent. The present invention also relates to a textile product containing the virus scavenger of the present invention. Furthermore, the present invention relates to a pharmaceutical composition, a food composition, and a feed composition comprising the chitosan complex of the present invention.

  Sialic acid is a generic term for acidic sugars of acyl derivatives of neuraminic acid such as N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) in a narrow sense. In addition, sialic acid derivatives such as sialic acid deaminated products, acetylated products, sulfates, methylated products, lactylated products, and lactone formers exist in nature, and many sialic acid derivatives are synthesized artificially. In a broad sense, these sialic acid derivatives are also included in sialic acid. Therefore, in the present specification, sialic acid is understood to include not only sialic acid in a narrow sense but also derivatives of these sialic acids.

  These sialic acids bind to the non-reducing ends of glycoproteins, glucosaminoglycans and glycolipids, contribute to the expression of negative charges, and are distributed on the surface of living cells. It plays an important biological function as a sugar constituting the chain. For example, it has been reported that sialic acid is often present at the tip of a sugar chain involved in cancer cell metastasis, inflammatory action, nerve cell development and the like.

  In addition, infection with several viruses, including influenza virus, is recognized by specific molecules (ligands) on the virus side that recognize complex carbohydrate sugar chains (receptors) on the target cell surface where sialic acid is present at the tip. It progresses by causing general adsorption. For example, certain influenza viruses have sialidase and hemagglutinin on their outer membranes, and 3 ′ sialyl lactosamine (N-acetylneuraminic acid α2 → 3-D-galactose β1 → 4-N-acetyl). It recognizes and infects a trisaccharide structure represented by -D-glucosamine

  Development of a virus removal filter having a structure in which sialic acid is immobilized on an appropriate polymer is being promoted by utilizing the ability of this virus to recognize complex sugar chains containing sialic acid. For example, a virus capturing body in which sialic acid and styrene or acrylamide are bound (Patent Document 1), or a complex in which sialic acid is bound to chitosan via a linker molecule (Patent Document 2, Non-Patent Document 1, Non-patent) Reference 2) has been reported. For example, asparagine-linked glycoprotein sugar chain-containing chitosan in which an asparagine-linked glycoprotein sugar chain containing an aglycon moiety having a formyl group is linked to the amino group of chitosan has been reported (see Patent Document 2). In addition to the chitosan itself being an antibacterial substance, these chitosan derivatives also have an advantage that there is no risk of environmental pollution since all the components constituting the complex are natural materials.

  However, all of these are disadvantageous in terms of production cost because they are prepared using substantially purified sialic acid and the like, and the range of viruses that can be captured is narrow, so that only specific viruses are efficiently captured. The current situation is that we cannot do it. In addition, the complex of sialic acid and chitosan via a linker molecule is complicated in itself for binding both components via a linker, which is disadvantageous in terms of operability and production cost.

JP-A-9-234317 JP 2003-301002 A Fluike et al., Journal of Chemical Society, 2000, Volume 1, pages 3000-3005. Ohta et al., Angew. Chem. Int. , 2003, 42, 5186-6189.

  The present invention solves the above-mentioned conventional problems and provides a more easily prepared virus trap that can capture more types of viruses.

  The present invention relates to sialic acid and the like, particularly milk of natural materials, particularly bovine milk, more preferably bovine colostrum, sialic acid having various structures including sialyl lactose and chitosan without a linker. By directly coupling to the above, the above-mentioned problems are solved.

That is, the present invention relates to a chitosan complex formed by reducing and condensing a reducing end (reducing end) of sialic acid or a sugar chain containing sialic acid (hereinafter referred to as sialic acid) to an amino group of chitosan. In particular, sialic acid and the like relates to a complex of chitosan with sialic oligosaccharide, preferably sialyllactose, more specifically sialic acid derived from milk or processed product thereof, particularly sialic acid derived from bovine milk. The present invention also provides a virus-capturing agent that is useful as a virus-capturing body such as a filter or mask using the chitosan complex as a raw material. The present invention relates to a composition for capturing a virus comprising the chitosan complex of the present invention as described above and a carrier for capturing the virus.
The present invention also provides sialic acid comprising reacting sialic acid or a sugar chain containing sialic acid and chitosan under reducing conditions, or a chitosan complex in which a sugar chain containing sialic acid and chitosan are directly bonded. On how to do.
Furthermore, the present invention relates to a textile product containing a virus scavenger comprising the chitosan complex of the present invention described above.
The present invention also relates to a pharmaceutical composition, a food composition, and a feed composition useful as an antiviral agent or virus scavenger comprising the chitosan complex of the present invention. More specifically, the present invention relates to a medical antiviral agent or virus scavenger, food additive, and feed additive comprising the chitosan complex of the present invention.
Furthermore, the present invention relates to a virus inactivation method or virus capture method using the chitosan complex of the present invention described above. The present invention also relates to the use of the chitosan complex of the present invention as described above for virus inactivation or virus capture.
Furthermore, the present invention relates to the use of the chitosan complex of the present invention for the manufacture of a pharmaceutical formulation as an antiviral agent or virus scavenger.

More specifically, the present invention is as follows (1) to (16).
(1) A chitosan complex formed by reducing and condensing a reducing end of sialic acid or a sugar chain containing sialic acid to an amino group of chitosan.
(2) The chitosan complex according to (1), wherein the sialic acid or the sugar chain containing sialic acid is a sialyl oligosaccharide.
(3) The chitosan complex according to (1), wherein the sialic acid or the sugar chain containing sialic acid is sialyl lactose.
(4) The chitosan complex according to any one of (1) to (4), wherein the sialic acid or the sugar chain containing sialic acid is derived from milk or a processed product thereof.
(5) The chitosan complex according to (4), wherein the milk is bovine milk.
(6) The chitosan complex according to (5), wherein the bovine milk is colostrum.
(7) A method for producing a sialic acid or a chitosan complex in which a sialic acid-containing sugar chain and chitosan are directly bonded, comprising reacting sialic acid or a sugar chain containing sialic acid with chitosan under reducing conditions .
(8) The method according to (7), wherein the sialic acid or the sugar chain containing sialic acid is a mixture thereof.
(9) The method according to (8) above, wherein the sialyl oligosaccharide or a mixture thereof is derived from milk.
(10) The method according to (9), wherein the milk is bovine milk or a processed product thereof.
(11) The method according to any one of (7) to (10), wherein the reaction under reducing conditions is a reaction in the presence of a metal hydride.
(12) A virus capturing agent comprising the chitosan complex according to any one of (1) to (6).
(13) The virus trapping agent according to (12), wherein the chitosan complex contains sialic acid or a mixture of sugar chains containing sialic acid.
(14) A textile product comprising the virus-capturing agent according to (12) or (13).
(15) The fiber product according to (14), wherein the fiber product is a filter or a mask, or a processing raw material thereof.

(16) A virus trapping composition comprising the chitosan complex according to any one of (1) to (6) above and a virus trapping carrier.
(17) A pharmaceutical composition comprising the chitosan complex according to any one of (1) to (6) and a pharmaceutically acceptable carrier.
(18) The pharmaceutical composition according to the above (17), wherein the pharmaceutical composition is an antiviral agent or a virus scavenger.
(19) A food composition comprising the chitosan complex according to any one of (1) to (6) above and a carrier that is acceptable for food production.
(20) The food composition according to (19), wherein the food composition has an antiviral action or a virus capturing action.
(21) The food composition according to (19) or (20), wherein the food composition is a supplement.
(22) A food additive comprising the chitosan complex according to any one of (1) to (6).
(23) The food additive as described in (22) above, wherein the food additive has an action as an antiviral agent or a virus scavenger.
(24) A feed composition comprising the chitosan complex according to any one of (1) to (6) and a feed carrier.
(25) The feed composition according to (24), wherein the feed composition is an antiviral agent or a virus scavenger.
(26) A feed additive comprising the chitosan complex according to any one of (1) to (6).
(27) The feed additive according to (26), wherein the feed additive has an action as an antiviral agent or a virus scavenger.

The present invention relates to a saccharide, saccharide, or oligomer thereof containing sialic acid and containing a carbonyl group such as a keto group, an aldehyde group, their ketal, acetal, hemiketal, and hemiacetal, or a derivative group thereof. The carbonyl group or its derivative group is reductively reacted with the amino group of chitosan to form a complex in which both are directly bonded.
The “sialic acid” in the present invention includes not only sialic acid as an acyl derivative of neuraminic acid but also various derivatives thereof, such as deaminated product, acetylated product, sulfated product, methylated product, lactyl. And derivatives of neuraminic acid such as these lactone formers. In addition, the sialic acid of the present invention is a saccharide containing one or more of the above-mentioned sialic acids, and is a monosaccharide, disaccharide, or oligosaccharide containing the above-described carbonyl group or derivative group thereof. Or sugar derivatives thereof. Preferred sialic acids include saccharides having a reducing end, such as sialyl oligosaccharides. Among sialyl oligosaccharides, sialyl lactose distributed in large quantities in the natural world is particularly preferred not only for virus capture and reactivity but also for ease of availability, but is not limited thereto. . The sialic acid and the like of the present invention may be used as a chemically pure single substance, but from the viewpoint of availability and price, a mixture as a concentrate separated from nature can also be used. . By using such a mixture of sialic acid and the like, it becomes possible to simultaneously capture many kinds of viruses. Therefore, the virus trapping agent useful as the virus trap of the present invention targets a specific virus. If not, it is preferable to use a mixture of sialic acid such as sialyllactose obtained from nature, particularly milk, more preferably bovine milk, as the sialic acid of the present invention.

The chitosan complex of the present invention is formed by directly reacting a reducing functional group such as sialic acid, preferably a reducing end and an amino group in chitosan. Specifically, according to the method of Yalpani et al. (M. Yalpani and Laurance D. Hall, Macromolecules, Vol. 17, 272-281, 1984), dissolving chitosan such as acetic acid / methanol. Reductive alkylation by dissolving chitosan in a suitable solvent capable of reacting, adding sialic acid, etc. in the presence of a reducing agent such as NaCNBH ₃ and reacting with the amino group of chitosan and the carbonyl group in the sugar chain To obtain the desired chitosan complex.

  Here, the complex of chitosan and sialic acid can be prepared from chitosan and sialic acid, or a sugar chain containing chitosan and sialic acid, respectively. However, the mixture containing sialic acid and the like is reacted with chitosan. It is desirable to prepare it. As such a mixture, it is desirable to use milk or sialic acid typified by sialyl lactose derived from animals such as bovine milk, particularly bovine colostrum.

In milk, in addition to neutral sugar chains such as lactose, free sialic acid and sugar chains containing sialic acid such as sialyllactose bound to sialic acid, especially bovine colostrum, an amount corresponding to 5 to 10% of the sugar content And have a wide variety of structures (edited by Robert G. Jensen and Marvin P. Thompson, Handbook of Milk Composition, 1995, Academic Press, ISBN: 0123844304). Urashima Satoshi, Dairy Technology, 53: 56-68, 2003. Paul MacJarrow and Janneke van Amelsfort-Schoonbee, International Daily Journal, 14: 571-579, 2004).
In general, viruses are known to have different sugar chain structures containing sialic acid that is recognized and bound depending on the type of the virus. Therefore, milk having various structures, preferably sialic acid derived from bovine milk, etc. It is possible to capture more types of viruses by preparing a trap of virus using. In addition to bovine milk-derived sialic acid, etc., the chitosan complex of the present invention can also be prepared using multiple types of sialic acid having different structures purified from various sources including human colostrum However, in view of easy availability or purification operation, it is preferable to use sialic acid in bovine milk, preferably bovine colostrum. Bovine normal milk (cow milk) also contains sialic acid, although its content is lower than bovine colostrum, and is easily available from bovine colostrum, so it is also suitable to use sialic acid in milk. is there. Bovine colostrum and cow milk are processed as raw milk, butter, cheese, prepared milk, processed milk, powdered milk, skim milk powder, bovine colostrum protein, etc., and used as food, health food, veterinary medicine and the like. Processed products including by-products such as cheese whey in addition to products produced in the process of manufacturing and processing these existing products include products in which sialic acid is purified or concentrated, and these are also raw materials such as sialic acid of the present invention It is suitable as.

The chitosan complex of the present invention prepared as described above was found in accordance with the method of the present invention as compared with the case where sialic acid or the like was present alone, as is clear from the test examples of Examples 5 and 6 described later. By making it a complex with chitosan, it exerts an extremely powerful virus-suppressing action. In particular, the antiviral action against human influenza viruses such as avian influenza virus H5N1, which has become a hot topic in recent years, is extremely strong by using a chitosan complex as compared with the case where sialic acid or the like is used alone. The reason for this is not necessarily clear at the present time, but because a direct chemical bond between sialic acid or the like and chitosan forms a three-dimensional structure that is extremely advantageous to capture and inactivate viruses. Conceivable.
As described above, the chitosan complex of the present invention can be used as a virus capturing agent useful as a virus capturing body for capturing a virus alone, but it is useful as a virus capturing body by mixing with various carriers. It can also be used as a composition for capturing viruses. The virus-capturing agent of the present invention includes a composition comprising the chitosan complex of the present invention alone and a virus-capturing composition mixed or dissolved with a carrier such as a solvent such as water as described above. . The carrier for capturing a virus of the present invention is not particularly limited as long as it does not hinder the inactivation or capture of the virus. Examples thereof include a solvent such as water, a fiber material, and a preparation material such as cellulose.
Moreover, this invention provides the textiles containing the virus capture agent of this invention. The fiber product of the present invention only needs to contain the virus capturing agent of the present invention in a fiber material by a physical or chemical method. For example, the chitosan composite of the present invention is sprayed or impregnated on a nonwoven fabric or the like. It can be produced by a method such as kneading into a fiber. Examples of the fiber product of the present invention include a filter, a mask, a towel, a handkerchief, or a fiber product as a processing raw material thereof. The virus trapping agent of the present invention can be used as a virus trap for various products by being contained in various materials such as fiber materials.
Furthermore, the chitosan complex of the present invention is mixed or dissolved with water or a suitable solvent, and in the form of a virus trapping composition as the virus trapping agent of the present invention, various fibers such as an air cleaning filter and a mask. Infection not only with products but also with medical facilities, medical equipment, equipment, tools, supplies, etc., livestock exposed to virus contamination, their livestock houses, etc. Can also be used as a preventive material.

  Furthermore, since the components constituting the chitosan complex of the present invention are all derived from natural products and are non-toxic to humans and livestock, the virus capture agent of the present invention is also environmentally friendly. Gently, it can also be used as a cleaning agent or virus control agent for households or livestock farms, especially poultry farms. It can also be used in agricultural chemicals for the purpose of controlling phytopathogenic viruses.

  Since the chitosan complex of the present invention has antiviral activity or virus capturing activity, the complex is used as an active ingredient and a pharmaceutical composition containing a pharmaceutically acceptable carrier, particularly as an antiviral agent or virus capturing agent. can do. In particular, it can be used as an anti-influenza virus agent in animals such as humans and birds. The pharmaceutical composition of the present invention, in particular, the pharmaceutical composition as an antiviral agent, is orally used as a powder, granule, tablet, capsule, pill, liquid or the like, or an injection, suppository, percutaneous absorption agent, It can be administered parenterally as an inhalant, nasal spray, eye wash, mucous membrane and epidermis disinfectant and detergent. In addition, the pharmaceutical composition of the present invention can be made into a pharmaceutical preparation by mixing pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants and the like as necessary. In the case of an injection, it is sterilized with an appropriate carrier to obtain a preparation. The dose varies depending on the disease state, administration route, patient age, or body weight, but when administered orally to an adult, it is usually 1 mg / kg / day to 10 g / kg / day, preferably 10 mg / kg / day to 1 g / kg / day. It can also be used as an additive to mouthwash, mouthwash, nasal spray, eye wash, epidermis disinfectant, etc. for prevention and treatment of viruses, particularly influenza viruses.

The chitosan complex of the present invention has antiviral activity or virus capture activity, and all the components constituting it are derived from natural products and are non-toxic to humans, livestock, etc. , Pet food, livestock, fish, or raptor food compositions, food additives, and feed compositions.
The food composition of the present invention comprises the chitosan complex of the present invention and a food carrier or a carrier acceptable for food production. Examples of the carrier for food of the present invention or the carrier acceptable for food production include various materials or materials for natural or non-natural foods that have been used for food. In the food composition of the present invention, the chitosan complex of the present invention described above can be used alone. Moreover, the chitosan complex of the present invention can be used alone or in combination with a suitable food carrier as a food additive.
Furthermore, the food composition of the present invention can be formulated into an appropriate dosage form and used as a supplement. Preferably, it can be used for humans, pigs, birds (especially chickens), and ornamental fish. Supplements (food supplements) can be formulated into gels, capsules, tablets, syrups, beverages or powders. Methods for producing such formulations are well known to those skilled in the art. In other embodiments, the supplement may further comprise additional additives selected from the group consisting of vitamins, minerals, coenzymes, fiber or combinations thereof. The vitamin can be selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin B12, riboflavin, niacin, pantothenic acid, thiamine, choline, folic acid, biotin, vitamin K, vitamin C, or CoQ10. The mineral can be selected from cobalt, copper, iron, manganese, zinc, selenium, dolomite, calcium, mineral yeast, seaweed powder, molybdenum, magnesium, potassium, chromium, calcium and phosphorus. The amount of each vitamin and each mineral in the supplement can be appropriately set by those skilled in the art. The supplement of the present invention can be in any dosage form. Examples of dosage forms that can be taken by the supplement of the present invention include tablets, granules, solutions, capsules, powders, emulsions and suspensions. The supplement of the present invention is preferably a tablet or capsule, more preferably a capsule. The supplement of the present invention can be produced according to a method well known in the art depending on the dosage form. For example, when the supplement of the present invention is a capsule, it is manufactured by filling the capsule in the form of liquid, suspension, paste, powder or granules, or encapsulating with a capsule base material. Can be done. The capsule may be a hard capsule or a soft capsule. When the supplement of the present invention is a tablet, it can be produced using the techniques and equipment used for conventional tablet production as they are. For example, a method of directly tableting by mixing the main component and additives, a method of tableting after the components are granulated by wet or dry method, and the like can be employed. When the supplement of this invention is a liquid agent, it can manufacture using the technique and equipment currently used for the conventional liquid agent manufacture as it is. For example, it can be produced by using the liquid component as it is or by dissolving the component in a solvent. When the supplement of the present invention is a powder, it is produced by adding the ingredients as they are, or adding excipients, binders, disintegrants or other suitable additives to the ingredients, and powdering or granulating them in an appropriate manner. obtain. When the supplement of the present invention is an emulsion, it is produced by adding a suspending agent or other suitable additive and purified water or oil to a solid component and suspending it by an appropriate method to make the whole quality uniform. obtain. When the supplement of the present invention is a suspending agent, it can be produced by adding an emulsifier and purified water to a liquid component, emulsifying by an appropriate method, and uniforming the whole quality. Even if the supplement of this invention is a form other than these, the expert can manufacture the supplement of a suitable form according to a well-known method in the said field | area. The intake of the supplement varies depending on the route of intake, the age of the patient, or the body weight, but is usually 1 mg / kg / day to 10 g / kg / day, preferably 10 mg / kg / day to 1 g when orally administered to an adult. / Kg / day.

  The chitosan complex of the present invention has antiviral activity or virus capture activity, and all the components constituting it are derived from natural products and are non-toxic to animals such as humans and livestock. It can be blended into pet or livestock feed (the supplement may be blended into the feed). Accordingly, the present invention provides a feed composition comprising the chitosan complex of the present invention and a feed carrier or a carrier acceptable for feed production. Moreover, the feed composition of this invention can also use the chitosan composite_body | complex of this invention individually or with the carrier for feed mentioned above as a feed additive. The feed composition and feed additive of the present invention can be produced and used in the same manner as the food supplements described above. The feed composition of the present invention can use a pet or livestock feed that is usually used. These feed compositions may contain grains such as corn, wheat, barley, soybean, corn silage, grass silage, alfalfa silage, hay silage and the like. The feed of the present invention can be produced according to methods well known in the art.

  The chitosan complex of the present invention can be easily and inexpensively produced and can efficiently capture many kinds of viruses. In addition, the chitosan complex of the present invention made of a raw material derived from natural products is safe for the environment and the human body, and has a wide range of applications.

  As chitosan usable in the present invention, those that are widely available, including chitosan sold as food materials and reagents, chitosan prepared from natural resources by various conventionally known methods, and the like are available. It can be used and there are no particular restrictions on its origin.

  Examples of sialic acid that can be used in the present invention include sialyl lactose, sialyl lactosamine, N-acetylneuraminic acid, N-glycolylneuraminic acid, deaminoneuraminic acid, acetylates, sulfates, and methylates thereof. , Deoxy compounds, dehydro compounds, lactylated compounds, lactone compounds, and the like, which include sialic acids and carbonyl groups at the reducing ends thereof. These can be used alone, but it is preferable to use a mixture of two or more sialic acids, etc., and preferably milk, such as bovine milk, particularly bovine colostrum or bovine milk-derived sialic acid.

The reaction of chitosan with sialic acid or the like can be carried out according to the method of Yalpani et al. (M. Yalpani and Laurance D. Hall, Macromolecules, 17, 272-281, 1984). Here, as the solvent for dissolving chitosan, an acidic solvent such as acetic acid or a mixed solution thereof with an organic solvent is used. Examples of the reducing agent include sodium cyanotrihydroborate (NaCNBH ₃ ), sodium borohydride (NaBH ₄ ), lithium borohydride (LiBH ₄ ), potassium borohydride (KBH ₄ ), borane dimethylamine complex ( DMAB, (CH ₃ ) ₂ NH · BH ₃ ), borane trimethylamine complex (TMAB, (CH ₃ ) ₃ N · BH ₃ ), B-isopinocamphenyl-9-borobicyclo [3,3,1] nonane (B -Isopinocamphenyl-9-borabiccyclo [3.3.1] nonane) (Aldrich, trade name Alpine-Borane) or B-chlorodiisopinocamphenylborane (Aldrorichrane) Trademark name DIP-chloride) and the like, either can be used.

  Hereinafter, the present invention will be further described with reference to examples.

5.6 mg of chitosan (PSH-80, Yaizu Fisheries Chemical Co., Ltd.) was dissolved in 0.7 ml of 1% acetic acid / methanol (1: 1). To this, 25 mg of sialyl lactose (Sigma) and 0.5 ml of aqueous solution in which 12.4 mg of NaCNBH ₃ was dissolved in advance were added and reacted at 25 ° C. for 48 hours. The reaction solution is transferred to a dialysis tube (permeation molecular weight of about 14,000), dialyzed against distilled water (2 changes / day) for 5 days, the final solution is freeze-dried, and the sialyl lactose / chitosan complex 15. 7 mg was obtained. The structure was confirmed by ¹ H-NMR (D ₂ O, Bruker, 500 MHz).
¹ H-NMR (D ₂ O, 500 MHz) δ:
4.556 (1H, d, J = 7.8 Hz, H-1 of Gal in SiaLac), 4.01-3.47 (27.6H, H-3, 4, 5, 6 of SiaLac side chain and chitosan mainchain, H-2 of chitsan main chain GlcNAc and SiaLac side chain), 2.85 (1.36H, br, H-2 of chitsan main chain GlcN and substituted H63d1H63 of Sia), 1.94 (3.69 H, m, COCH ₃ ), 1.66 (1 H, t, H-3ax of Sia).

From the integrated value of the peak derived from the 3-position of sialic acid near 1.66 in this ¹ H-NMR spectrum and the peak derived from chitosan glucosamine near 2.85, sialyllactose was 73.5 with respect to the glucosamine residue of chitosan. % Was found to have been introduced into the chitosan backbone (degree of substitution with respect to glucosamine residues (DS) = 73.5%).

800 ml of chloroform was added to 800 ml of bovine colostrum collected from a ranch in Shimizu-cho, Kamikawa-gun, Hokkaido, and degreased by stirring. This is repeated twice, and the aqueous phase fraction is recovered, ethanol is added to make an 80% ethanol solution, the precipitated protein is removed by centrifugation, and the supernatant is recovered, and this is lyophilized to freeze the colostrum. 16.4 g of a sugar chain fraction was obtained. When the sugar chain fraction of the obtained colostrum was analyzed by a mass spectrometer (MALDI-TOF MS, UltraFLEX, Bruker Daltonics), lactose (Lac, Galβ1 → 4Glc), 6′SLN (Siaα2 → 6Galβ1 → 4GlcNac) , 6′SL (Siaα2 → 6Galβ1 → 4Glc) or 3′SL (Siaα2 → 3Galβ1 → 4Glc) and DSL (Siaα2 → 8Siaα2 → 3Galβ1 → 4Glc) were confirmed.
MALDI-TOF MS [M + Na] ⁺ :
As Lac (C ₁₂ H ₂₂ O ₁₁ ), calculated value: 365.1,
Found: 364.8;
As 6′SL or 3′SL (C ₂₃ H ₃₉ NO ₁₉ ), calculated value: 656.2,
Found: 656.0;
As 6′SLN (C ₂₅ H ₄₂ N ₂ O ₁₉ ), calculated: 697.2,
Actual value: 698.0:
As DSL (C ₃₄ H ₅₆ N ₂ O ₂₈ ), calculated value: 963.8,
Found: 963.1.

Subsequently, 319 mg of chitosan (PSH-80, Yaizu Suisan Kagaku Kogyo Co., Ltd.) was dissolved in 30 ml of 1% acetic acid / methanol (1: 1). Minutes were added, 10 ml of an aqueous solution in which 706 mg of NaCNBH ₃ was previously dissolved was added to the mixture, and the mixture was reacted at 25 ° C. for 48 hours. The reaction solution is transferred to a dialysis tube (permeation molecular weight of about 14,000), dialyzed against distilled water (2 changes / day) for 5 days, the final solution is freeze-dried, and the colostrum sugar chain-chitosan complex 471 mg of body was obtained. The structure was confirmed by ¹ H-NMR (D ₂ O, Bruker, 500 MHz).
¹ H-NMR (D ₂ O, Bruker, 500 MHz) δ:
4.45 (1H, d, J = 7.2 Hz, H-1 of Gal in Lac), 4.07-3.48 (7.49H, H-3, 4, 5, 6, of Lac side chain and chitosan main chain, H-2 of chitosan main chain GlcNAc and Lac side chain), 2.72 (0.71H, br, H-2 of chitosan main chain GlcN and substituted GlcN, 0.61). COCH ₃ ).

From the ¹ H-NMR spectrum of the sugar chain-chitosan complex of this colostrum, the substitution degree (DS) of the saccharide to the glucosamine residue of chitosan was calculated to be about 100%, and the lactose structure was confirmed, but sialic acid and sialic A clear peak of an acid-containing sugar chain could not be confirmed by NMR. Thus, sialic acid in the colostrum sugar chain / chitosan complex was quantified by an enzymatic method. Pyruvate produced by cutting sialic acid from the chitosan complex with sialidase (Sigma) and further treating with aldolase (Sigma) was quantified as a change in absorbance at 340 nm using a lactose dehydrogenase reaction system using NADH. .

  As a result, it was confirmed that about 1 sialic acid or a sugar chain containing sialic acid was bonded to 60 glucosamine residues of chitosan in the complex (sialic acid and sialic acid for glucosamine residues). Degree of substitution of sugar chain containing acid (DS) = about 1.6%). The confirmed degree of substitution is 1.6%, but since sialic acid is liberated by the enzyme, it is thought that in fact, several times as many sialic acids and sugar chains containing sialic acid are bound. It is done.

20 mg of the sugar chain fraction from colostrum prepared in Example 2 was subjected to gel filtration (BioGelP-4, Biorad) using water as a solvent, and fraction 1 consisting of sialic acid and an acidic sugar chain containing sialic acid ( 3.6 mg) and neutral sugar chain fraction 2 (16.4 mg) mainly composed of lactose. In fraction 1, mass peaks corresponding to 6′SLN, 6′SL or 3′SL and DSL were confirmed by mass spectrometry (MALDI-TOF MS, UltraFLEX, Bruker Daltonics).
MALDI-TOF MS [M + Na] ⁺ :
Fraction 1: 6'SL or 3'SL
As (C ₂₃ H ₃₉ NO ₁₉ ), calculated value: 656.2,
Found: 656.5;
6'SLN
As (C ₂₅ H ₄₂ N ₂ O ₁₉ ), calculated value: 697.2,
Actual value: 697.8:
DSL
As (C ₃₄ H ₅₆ N ₂ O ₂₈ ), calculated value: 963.8,
Found: 963.8.
Fraction 2: Lac
As (C ₁₂ H ₂₂ O ₁₁ ), calculated value: 365.1,
Found: 365.1.

Subsequently, 5.6 mg of chitosan (PSH-80, Yaizu Suisan Chemical Co., Ltd.) was dissolved in 0.7 ml of 1% acetic acid / methanol (1: 1). To this was added 25 mg of fraction 1 and 10 ml of an aqueous solution in which 12.4 mg of NaCNBH ₃ was previously dissolved, and the mixture was reacted at 25 ° C. for 24 hours. The reaction solution is transferred to a dialysis tube (permeation molecular weight of about 14,000), dialyzed against distilled water (2 changes / day) for 5 days, the final solution is freeze-dried, and sialic acid etc.-chitosan complex 19 .3 mg was obtained. The structure was confirmed by ¹ H-NMR (D ₂ O, Bruker, 500 MHz).
¹ H-NMR (D ₂ O, 500 MHz) δ:
4.56, 4.44, 4.50 (H-1 of Gal residue in Sialooligosaccharide), 4.00-3.49 (25.5H, H-3, 4, 5, 6, of SiaLac side chain and chitosan mainchain , H-2 of chitosan main chain GlcNAc and SiaLac side chain), 2.85 (1.61H, br, H-2 of chitosan main chain GlcN and submitted GlcN, e3d. Sia), 1.95 (4.21 H, m, COCH ₃ ), 1.70 (1 H, t, H-3ax of Sia).

From the integrated value of the peak derived from the 3-position of sialic acid near 1.70 in this ¹ H-NMR spectrum and the peak derived from chitosan glucosamine near 2.85, sialic acid and sialic acid were added to the glucosamine residue of chitosan. It was found that the sugar chain (sialic acid or the like) contained was introduced into the chitosan main chain at a rate of 61.8% (degree of substitution with respect to glucosamine residue (DS) = 61.8%).

200 ml of chloroform was added to 200 ml of milk collected from a farm in Shimizu-cho, Kamikawa-gun, Hokkaido, and degreased by stirring. This is repeated twice, and the aqueous phase fraction is recovered, ethanol is added to make an 80% ethanol solution, the precipitated protein is removed by centrifugation, the supernatant is recovered, and this is freeze-dried to give milk sugar chains A fraction of 3.9 g was obtained. More than 95% of milk sugar chains are lactose (Lac, Galβ1 → 4Glc), but it is known to contain various neutral sugar chains and sialyl sugar chains (Akira Urashima, Dairy Technology, Vol. 53, paragraphs 56-68). 2003, Paul MacJarrow and Janneke van Amelsfort-Schoonbek, International Daily Journal, Vol. 14, paragraphs 571-579, 2004). It was confirmed by analyzing the main component lactose by mass spectrometry (MALDI-TOF MS, UltraFLEX, Bruker Daltonics) and 1H-NMR analysis (Bruker, 500 MHz) that the products purified this time are sugar chains. .
MALDI-TOF MASS (m / s); [M + Na] ⁺ :
As Lac (C ₁₂ H ₂₂ O ₁₁ ), calculated value: 365.1,
Found: 365.1.
¹ H-NMR (D ₂ O): δ:
5.13 (0.4H, d, J = 3.6 Hz, αH-1 of Glc in Lac), 4.58 (0.6H, d, J = 7.9 Hz, βH-1 of Glc in Lac), 4.36 (1H, d, J = 8.1 Hz, H-1 of Gal in Lac), 3.86-3.46 (11.4H, m, Protons of sugar ring), 3.19 (0.6H , T, J = 8.2 Hz, βH-2 of Glc in Lac).

Subsequently, 407 mg of chitosan (PSH-80, Yaizu Suisan Chemical Co., Ltd.) was dissolved in 30 ml of 1% acetic acid / methanol (1: 1). To this was added 1 ml of milk sugar chain fraction and 15 ml of an aqueous solution in which 994 mg of NaCNBH ₃ had been dissolved in advance, and the mixture was reacted at 25 ° C. for 48 hours. The reaction solution was transferred to a dialysis tube (permeation molecular weight of about 14,000), dialyzed against distilled water (2 changes / day) for 5 days, the final solution was freeze-dried, and 985 mg of milk sugar chain-chitosan complex. Got. The structure was confirmed by ¹ H-NMR (Bruker, 500 MHz), and it was confirmed that a sugar chain / chitosan complex such as lactose was formed.
¹ H-NMR (D ₂ O): δ:
4.45 (1H, d, J = 7.2 Hz, H-1 of Gal), 4.07-3.48 (7.50H, Protons of sugar ring), 2.98 (0.71H, br, H _{-2 of GlcN), 2.77 (2H} , br, NCH 2), 1.99 (0.65H, s, COCH 3).

The degree of substitution (DS) of a sugar chain residue such as lactose in the complex with respect to the glucosamine residue is 100% according to the measurement result of ¹ H-NMR.

Antiviral activity of sialic acid etc.-chitosan complex from bovine colostrum prepared in Example 3 was measured using MTT method (Tim Mosmann, J. Immunol. Methods, 65: 55-63, 1983). . The bovine colostrum-derived sialic acid and the like-chitosan complex produced in Example 3 and the control compound (a mixture of chitosan not treated with a chemical reaction and bovine colostrum sialic acid) were each suspended in ethanol, A 5-fold serial dilution series was prepared for the product diluted 40-fold in the medium and used as a test group. At the same time, the antiviral activity was measured by seeding the medium with 20000 cells / well of 2-5 TCID50 WSN (H1N1) virus and MDBK cells. After incubation at 37 ° C. in a 5% CO ₂ environment for 72 hours, the MTT solution (5 mg / ml 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (3- 20 μl of (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazole bromide (MTT) -PBS solution) was added and incubated for 2 hours. After removing the medium, 200 μl of MTT lysis solution (isopropyl alcohol plus 1/100 volume of 40 mM hydrochloric acid aqueous solution, 1/10 volume of Triton X-100 (Triton X-100)) was added. After cell lysis with a microplate mixer, absorption at 560 nm was measured with a microplate reader.
As a result, the EC ₅₀ of bovine colostrum sialic acid and the like-chitosan complex was 39 μg / ml and the control compound was measured to be 1000 μg / ml or more, and the antiviral activity of the complex of the present invention could be confirmed. It was.

The antiviral activities of the bovine colostrum-chitosan complex and the bovine milk sugar-chain-chitosan complex produced in Examples 2 and 4 were measured, respectively. After pre-culturing MDCK cells, each sample (sugar chain-chitosan complex and those not chemically synthesized) and virus (human-derived influenza A virus A / Aichi / 2/68 (H3N2) Or highly pathogenic avian influenza virus A / Chike / Yamaguchi / 7/04 (H5N1)) and cultured at 35 ° C. for 48 hours, and the cells were observed for cytopathic effect under a microscope.
As a result, the bovine colostrum sugar chain-chitosan complex and the bovine milk sugar chain-chitosan complex each showed a cytopathic inhibitory effect at a concentration of 5 mg / ml, but not the control compound.

  Examples of formulation in the pharmaceutical composition of the present invention, food composition such as supplement, or feed composition are shown below.

Formulation Example 1
A granule containing the following ingredients is produced.
Ingredient Chitosan Complex 10mg
Lactose 700mg
Corn starch 274mg
HPC-L 16mg
Total 1000mg
Pass the chitosan complex and lactose through a 60 mesh sieve. Pass corn starch through a 120 mesh sieve. These are mixed in a V-type mixer. An HPC-L (low-viscosity hydroxypropylcellulose) aqueous solution is added to the mixed powder, kneaded and granulated (extruded granulation, pore diameter 0.5 to 1 mm), and then dried. The obtained dried granules are combed with a vibration sieve (12/60 mesh) to obtain granules.

Formulation Example 2
A powder for capsule filling containing the following components is produced.
Ingredient Chitosan Complex 10mg
Lactose 79mg
Corn starch 10mg
Magnesium stearate 1mg
Total 100mg
The chitosan complex and lactose are passed through a 60 mesh sieve. Corn starch is passed through a 120 mesh screen. These and magnesium stearate are mixed in a V-type mixer. 100 mg of 10 times powder is filled into No. 5 hard gelatin capsule.

Formulation Example 3
A capsule filling granule containing the following ingredients is produced.
Ingredient Chitosan Complex 15mg
Lactose 90mg
Corn starch 42mg
HPC-L 3mg
150mg total
The chitosan complex and lactose are passed through a 60 mesh sieve. Pass corn starch through a 120 mesh sieve. These are mixed, the HPC-L solution is added to the mixed powder, kneaded, granulated and dried. After the resulting dried granules are sized, 150 mg thereof is filled into a No. 4 hard gelatin capsule.

Formulation Example 4
Hard gelatin capsules are manufactured using the following ingredients:
Dose Ingredient (mg / capsule)
Chitosan Complex 250
Starch (dried) 200
Magnesium stearate 10
Total 460

Formulation Example 5
A capsule containing 80 mg of the active ingredient is produced as follows.
Chitosan complex 80mg
Starch 59mg
Microcrystalline cellulose 59mg
Magnesium stearate 2mg
Total 200mg
A chitosan complex, starch, cellulose, and magnesium stearate are mixed. 45 mesh U.V. S. Through the sieve and filled into hard gelatin capsules 200 mg each.

Formulation Example 6
A tablet containing the following ingredients is produced.
Ingredient Chitosan Complex 10mg
Lactose 90mg
Microcrystalline cellulose 30mg
CMC-Na 15mg
Magnesium stearate 5mg
150mg total
The chitosan complex, lactose, microcrystalline cellulose, CMC-Na (carboxymethylcellulose sodium salt) are passed through a 60 mesh sieve and mixed. The mixed powder is mixed with magnesium stearate to obtain a mixed powder for tableting. The mixed powder is directly hit to obtain a 150 mg tablet.

Formulation Example 7
Tablets are manufactured using the following ingredients:
Ingredient Dose
(Mg / tablet)
Chitosan Complex 250
Cellulose (microcrystal) 400
Silicon dioxide (fume) 10
Stearic acid 5
Total 665
The ingredients are mixed and compressed into tablets each weighing 665 mg.

Formulation Example 8
A tablet containing 60 mg of active ingredient is prepared as follows:
Chitosan complex 60mg
45mg starch
Microcrystalline cellulose 35mg
Polyvinylpyrrolidone (10% solution in water) 4mg
Sodium carboxymethyl starch 4.5mg
Magnesium stearate 0.5mg
Talc 1mg
150mg total
Chitosan complex, starch, and cellulose are no. 45 mesh U.V. S. And mix well. An aqueous solution containing polyvinylpyrrolidone was mixed with the obtained powder, and the mixture was 14 mesh U.S. S. Pass through a sieve. The granules thus obtained were dried at 50 ° C. 18 mesh U.F. S. Pass through a sieve. No. 60 mesh U.S. S. Sodium carboxymethyl starch, magnesium stearate, and talc passed through a sieve are added to the granules, mixed and then compressed on a tablet press to obtain tablets each weighing 150 mg.

The present invention provides a chitosan complex using only a natural product-derived raw material by a simple method, the chitosan complex of the present invention has antiviral activity against various viruses, virus capture activity, It is useful as a material for inactivating and capturing viruses.
The chitosan complex of the present invention is safe for humans and animals, is friendly to the environment, is low in cost, and has a strong activity against viruses. It has industrial applicability as a new material for prevention.

Claims (22)

  A chitosan complex formed by reducing and condensing a reducing end of sialic acid or a sugar chain containing sialic acid to an amino group of chitosan.   The chitosan complex according to claim 1, wherein the sialic acid or the sugar chain containing sialic acid is a sialyl oligosaccharide.   The chitosan complex according to claim 1, wherein the sialic acid or the sugar chain containing sialic acid is sialyllactose.   The chitosan complex according to any one of claims 1 to 4, wherein sialic acid or a sugar chain containing sialic acid is derived from milk or a processed product thereof.   The chitosan complex according to claim 4, wherein the milk is bovine milk.   6. The chitosan complex according to claim 5, wherein the bovine milk is colostrum.   A method for producing a sialic acid or a chitosan complex in which a sialic acid-containing sugar chain and chitosan are directly bonded, comprising reacting sialic acid or a sugar chain containing sialic acid with chitosan under reducing conditions.   The method according to claim 7, wherein the sialic acid or the sugar chain containing sialic acid is a mixture thereof.   The method according to claim 7 or 8, wherein the reaction under reducing conditions is a reaction in the presence of a metal hydride.   A virus capturing agent comprising the chitosan complex according to any one of claims 1 to 6.   The virus capturing agent according to claim 10, wherein the chitosan complex contains sialic acid or a mixture of sugar chains containing sialic acid.   A textile product comprising the virus scavenger according to claim 10 or 11.   The textile product according to claim 12, wherein the textile product is a filter or a mask, or a processed raw material thereof.   A pharmaceutical composition comprising the chitosan complex according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition is an antiviral agent or a virus scavenger.   A food composition comprising the chitosan complex according to any one of claims 1 to 6 and a carrier acceptable for food production.   The food composition according to claim 16, wherein the food composition has an antiviral action or a virus capturing action.   The food composition according to claim 16 or 17, wherein the food composition is a supplement.   A food additive comprising the chitosan complex according to any one of claims 1 to 6.   The food additive according to claim 19, wherein the food additive has an action as an antiviral agent or a virus scavenger.   A feed composition comprising the chitosan complex according to claim 1 and a feed carrier.   The feed composition according to claim 21, wherein the feed composition is an antiviral agent or a virus scavenger.