JP5646164B2 - Branched chitosan derivative - Google Patents

Description

  The present invention relates to a novel branched chitosan derivative, a production method thereof and use thereof. More specifically, the present invention relates to a chitosan derivative having a chitosan side chain, a method for producing the chitosan derivative, and a use thereof.

  Chitin is one of naturally occurring mucopolysaccharides having N-acetylglucosamine as a repeating unit. Chitosan is one of naturally occurring mucopolysaccharides, but industrially produced by deacetylation of chitin. It is known that the material properties of chitin and chitosan are controlled by functional substituents introduced by chemical modification, in addition to molecular weight distribution and N-acetyl group substitution degree. Examples of functionalization aimed at the development of gene carriers, bioadhesives, sealing agents, etc. are also known, but water solubility (solubility in solvents suitable for living organisms) and functionality (polyamine characteristics, low viscosity) (See Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, etc.) and include molecular structures other than chitin and chitosan (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5) Etc.).

Table 1 summarizes the comparison of chitosan water solubility improvement technology with existing technologies. In order to improve water solubility, a method of introducing a hydrophilic group is common, and among them, introduction of a polyethylene glycol (PEG) chain is a typical example (see Non-Patent Document 6 and the like). However, the range of applicability is limited to impart good water solubility, such as the ratio of PEG to chitosan. On the other hand, the effects of introduction of sugar side chains with relatively short chain lengths such as monosaccharides, disaccharides and oligosaccharides (see Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, etc.) are limited and insufficient. It is. Chitosan oligosaccharides are very expensive and impractical. Although the method of introducing a low molecular weight product obtained by decomposing chitosan into nitrous acid into the side chain is simple (see Non-patent Document 3, Non-patent Document 4, Non-patent Document 5, etc.), when nitrous acid is decomposed, the reducing end is reduced. Since it is altered to 2,5-dehydro-D-mannofuranose residue, it has a structure different from that of chitosan chain.

International Publication WO0027889

Biomacromol., 2, 1133-1136, 2001. Trends Glycosci. Glycotechnol., 14, 331-341, 2002. J. Biomed. Mater. Res. A, 82, 201-212 2007. Acta Biomaterialia, 5, 1575-1581 2009. Biomacromol., 9, 3268-3276, 2008. Carbohydr. Polym., 36, 49-59, 1998.

  The problem to be solved by the present invention is to develop a novel chitosan derivative that is compatible with water solubility (solubility in a solvent suitable for a living body) and functionality (polyamine characteristics, low viscosity) and uses it in various fields. there were.

Chitin and chitosan (Chemical Formula 1) are not sufficiently soluble in water as they are, and the viscosity greatly increases with the increase in molecular weight.

  That is, chitin (chemical formula 1, x + y = 1, generally x <0.1) is hardly soluble in ordinary solvents, and chitosan (chemical formula 1, x + y = 1, generally y <0.2) is acidic. It is hardly soluble in the neutral to alkaline range of water-soluble ones. As a result of various studies on this problem in view of the above problems, the present inventors have found that reductive N− between chitosan having a relatively high molecular weight (HMW-chitosan) and chitosan having a relatively low molecular weight (LMW-chitosan). It has been found that a novel branched chitosan derivative showing good water solubility and low viscosity of the aqueous solution can be obtained by the alkylation reaction. Furthermore, the present inventors need to dissolve conventional chitosan with an acid, and use partially acetylated chitosan or partially deacetylated chitin having a degree of deacetylation (DDA) of about 50% (in formula 1, x = Although it was soluble in neutral water only when y = about 0.5), the branched chitosan derivative not only improved the solubility in acidic aqueous solution, but also had good solubility in neutral water. It has also been found that it has dispersibility. In addition, the present inventors have also found an unexpected and remarkable effect that the branched chitosan derivative has an action of activating the immune system. The present inventors have completed the present invention based on these findings.

［式中、ｘ＋ｙ＋ｚ＝１であり、ｘ、ｙ、ｚは、それぞれ独立して、０≦ｘ＜１、０≦ｙ＜１、０＜ｚ≦１であり、Ｒ^１は式（ＩＩ）：

で示されるキトサン側鎖であり、Ｒ^２は、Ｈ、アセチル基、該Ｒ^１であり、ａは０〜５００の整数を示し、但し、全てのＲ^２は同一でも異なっていてもよい］で表わされる、分岐キトサン誘導体；
（２）（１）記載の分岐キトサン誘導体を含む、低粘度キトサン組成物；
（３）（１）記載の分岐キトサン誘導体を含む、免疫系活性化剤；
（４）（１）記載の分岐キトサン誘導体を含む飲食物；ならびに
（５）主鎖用キトサンと、主鎖用キトサンよりも低分子量の側鎖用キトサンとを、還元的Ｎ−アルキル化反応に供することを特徴とする、分岐キトサン誘導体の製造方法；
（６）主鎖用キトサンの重量平均分子量が４５０ｋＤａよりも小さいものである、（５）記載の製造方法；
（７）主鎖用キトサンの重量平均分子量が８０ｋＤａないし１９０ｋＤａの範囲である、（５）記載の製造方法；ならびに
（８）（５）〜（７）のいずれかに記載の方法により得られる分岐キトサン誘導体。
That is, the present invention provides the following:
(1) Formula (I):

[Wherein, x + y + z = 1, and x, y, z are independently 0 ≦ x <1, 0 ≦ y <1, 0 <z ≦ 1, and R ¹ is represented by formula (II):

Wherein R ² is H, an acetyl group, or R ¹ , and a is an integer of 0 to 500, provided that all R ² may be the same or different.] A branched chitosan derivative represented;
(2) A low-viscosity chitosan composition comprising the branched chitosan derivative according to (1);
(3) an immune system activator comprising the branched chitosan derivative according to (1);
(4) Food and drink containing the branched chitosan derivative according to (1); and (5) Reductive N-alkylation reaction of chitosan for main chain and chitosan for side chain having a lower molecular weight than chitosan for main chain. A method for producing a branched chitosan derivative, comprising:
(6) The production method according to (5), wherein the chitosan for main chain has a weight average molecular weight of less than 450 kDa;
(7) The production method according to (5), wherein the chitosan for main chain has a weight average molecular weight in the range of 80 kDa to 190 kDa; and (8) the branch obtained by the method according to any one of (5) to (7) Chitosan derivative.

  Since the branched chitosan derivative of the present invention uses chitin derived from crab shell, shrimp shell, chitosan and its low molecular weight product, it is easy to adjust the degree of acetylation of the amino group and to control the solubility. Is possible. The branched chitosan derivative of the present invention has a high solubility in water, particularly in the vicinity of neutrality. The viscosity of the aqueous solution of the branched chitosan derivative of the present invention is also low. Therefore, the branched chitosan derivative of the present invention can be applied to a wider range of applications where chitin and chitosan are conventionally used, such as wound healing agents, biofillers, bioadhesives and coating agents, and pharmaceuticals. This can be used to produce a product having excellent characteristics. In addition, since the branched chitosan derivative of the present invention has a remarkable immune system activation effect, it can be used to produce excellent pharmaceuticals, health foods and the like.

FIG. 1 shows the fluorescence spectrum of a branched chitosan derivative (excitation wavelength 510 nm). FIG. 2 is a graph showing a comparison in viscosity between linear HMW-chitosan and branched chitosan. FIG. 3 is a graph comparing the proportion of CD4 + cells when a branched chitosan derivative, HMW-chitosan, and LMW-chitosan were administered. * P <0.05, ** P <0.01. FIG. 4 is a graph comparing the proportion of NK cells when a branched chitosan derivative, HMW-chitosan, and LMW-chitosan are administered. * P <0.05, ** P <0.01. FIG. 5 is a graph comparing serum IL-6 increasing activity when a branched chitosan derivative, HMW-chitosan, and LMW-chitosan are administered. * P <0.05. FIG. 6 is a graph comparing serum TNF-α increasing activity when a branched chitosan derivative, HMW-chitosan, and LMW-chitosan are administered. * P <0.05, ** P <0.01.

［式中、ｘ＋ｙ＋ｚ＝１であり、ｘ、ｙ、ｚは、それぞれ独立して、０≦ｘ＜１、０≦ｙ＜１、０＜ｚ≦１であり、Ｒ^１は式（ＩＩ）：

で示されるキトサン側鎖であり、Ｒ^２は、Ｈ、アセチル基、該Ｒ^１であり、ａは０〜５００の整数を示し、但し、全てのＲ^２は同一でも異なっていてもよい］で表わされる、新規な分岐キトサン誘導体を提供する。 In one aspect, the present invention provides a compound of formula (I):

[Wherein, x + y + z = 1, and x, y, z are independently 0 ≦ x <1, 0 ≦ y <1, 0 <z ≦ 1, and R ¹ is represented by formula (II):

Wherein R ² is H, an acetyl group, or R ¹ , and a is an integer of 0 to 500, provided that all R ² may be the same or different.] The novel branched chitosan derivatives represented are provided.

The branched chitosan derivative of the present invention comprises a glucosamine residue (sometimes referred to herein as “chitosan unit” or “GlcN”), an N-acetylglucosamine residue (herein referred to as “chitin unit” or “GlcNac”). And a glucosamine residue in which the R ¹ group represented by the formula (II) is bonded to N at the 2-position. The proportions of these residues in the branched chitosan derivative of the present invention are indicated by x, y, and z, and are independently 0 ≦ x <1, 0 ≦ y <1, and 0 <z ≦ 1.

  The ratio of x and y varies depending on the type of raw chitosan used in the production of the branched chitosan derivative of the present invention, and can be controlled by acetylating or deacetylating the raw chitosan or the generated branched chitosan derivative. . The ratio z of branched residues is defined by (number of branched residues in the branched chitosan derivative) / (total number of residues in the branched chitosan derivative). The method for producing the branched chitosan derivative of the present invention will be described. It can be changed by changing the amount ratio of the raw material HMW-chitosan and LMW-chitosan.

The value of a defines the length of the branched chain. Usually, a is 1 to 500. The larger the value of a, the longer the branched chain. Linear or near-branched chitosan derivatives tend to get entangled in molecular chains, so aqueous solutions tend to be highly viscous.Spherical or near-branched chitosan derivatives tend to get entangled in molecular chains, so aqueous solutions tend to have low viscosity. It is believed that there is. Therefore, it seems that the value of a needs to be increased to some extent in order to reduce the viscosity of the aqueous solution.

(Is the description about a correct?)

  The molecular weight of the branched chitosan derivative of the present invention can be widely selected or adjusted depending on the application and required physical properties. Such molecular weight is selected or adjusted by chitosan for main chain (sometimes referred to as “HMW-chitosan” in this specification) and chitosan for side chain (hereinafter referred to as “LMW” in this specification), which are raw materials of the branched chitosan derivative of the present invention. (Sometimes referred to as “chitosan”) or by selecting reaction conditions such as the type of reducing agent, reaction temperature, reaction time, etc. See In general, the molecular weight of chitin or chitosan can be adjusted by adjusting the conditions for hydrolysis of chitin and chitosan with acid, alkali or enzyme, and the method is known to those skilled in the art. Moreover, when the weight average molecular weight (Mw) of HMW-chitosan is larger than about 450 kDa, the water solubility of the branched chitosan derivative tends to decrease and the viscosity of the aqueous solution tends to increase.

  The branched chitosan derivatives of the present invention include those modified in addition to those represented by the formula (I). However, it is a condition that these modified products retain the properties of the branched chitosan derivative of the present invention represented by the formula (I). For example, one or more of the constituent sugar residues of the branched chitosan derivative of the present invention may be residues other than those shown in formula (I). In addition, for example, a glycoside may be formed via N at the 2-position in one or more of the constituent sugar residues of the branched chitosan derivative of the present invention. For example, the hydroxyl group may be modified by methylation or the like in one or more of the constituent sugar residues of the branched chitosan derivative of the present invention. For example, one or more of the constituent sugar residues of the branched chitosan derivative of the present invention may be a non-natural sugar residue. For example, one or more of the bonds connecting the constituent sugar residues may be other than α-1,4 bonds.

  In another aspect, the present invention provides a reductive N-alkylation reaction of chitosan for main chain (HMW-chitosan) and chitosan for side chain (LMW-chitosan) having a lower molecular weight than chitosan for main chain. A method for producing the branched chitosan derivative is provided.

  HMW-chitosan and LMW-chitosan, which are raw materials for the branched chitosan derivative of the present invention, may be used after deacetylation of chitin according to a known method, but commercially available chitosan can be used as it is. Moreover, also when using a commercially available chitosan, the ratio of a chitosan unit and a chitin unit can be adjusted to a desired value by performing an acetylation reaction or a deacetylation reaction. Therefore, the degree of deacetylation of chitosan used in the present invention is not particularly limited, and it may be any as long as it has a degree of deacetylation of preferably 5% or more, more preferably 30 to 99%. The degree of deacetylation of chitosan can be measured, for example, by proton nuclear magnetic resonance analysis. HMW-chitosan and LMW-chitosan may be linear or branched chitosan derivatives with side chain chitosan already attached.

  From the viewpoint of ease of handling, the HMW-chitosan chitosan used for the production of the branched chitosan derivative of the present invention preferably has a weight average molecular weight (Mw) of about 5,000 to about 600,000, and about 5,000 to about 200,000 is more preferred. The number average molecular weight (Mn) is preferably about 1,000 to about 500,000, more preferably about 2,000 to about 100,000, but is not limited to these molecular weights. However, when the weight average molecular weight (Mw) of HMW-chitosan is greater than about 450 kDa, the water solubility of the resulting branched chitosan derivative tends to decrease and the viscosity of the aqueous solution tends to increase. The weight average molecular weight is preferably less than about 450 kDa, and may be, for example, about 80 kDa or more and less than about 450 kDa. The molecular weight (weight average molecular weight or number average molecular weight) of LMW-chitosan is smaller than that of HMW-chitosan. Usually, LMW-chitosan contains 1 to about 500 sugar residues. The weight average molecular weight and number average molecular weight of chitosan can be measured, for example, by gel filtration chromatography (GPC). The molecular weight of HMW-chitosan and LMW-chitosan (or the number of sugar residues contained in LMW-chitosan) can be changed according to the use of the branched chitosan derivative to be produced and the required physical properties.

  The branched chitosan preferably has a degree of branching z of about 0.01 to about 0.6, more preferably about 0.02 to about 0.5. The degree of branching can be adjusted by adjusting the amount of chitosan side chain used in the reductive N-alkylation reaction. In the present specification, the degree of branching of chitosan can be calculated based on, for example, proton nuclear magnetic resonance analysis and elemental analysis.

  The branched chitosan derivative (formula (I)) of the present invention can be obtained by performing a reductive N-alkylation reaction between HMW-chitosan and LMW-chitosan. The reductive N-alkylation reaction is performed in the presence of a reducing agent. Examples of the reducing agent include sodium cyanoborohydride, sodium borohydride, hydrogen and a Pd / C (palladium / carbon) catalyst. It is not limited to these. The amount of the reducing agent is preferably 5 to 50 parts by weight and more preferably 5 to 20 parts by weight with respect to 100 parts by weight of the raw material used for each reaction, although it depends on the type.

  The amount ratio of HMW-chitosan and LMW-chitosan to be subjected to the reductive N-alkylation reaction can be appropriately determined according to the degree of branching of the target branched chitosan [z in formula (I)]. For example, when z in the formula (I) is 0.3, the total number of sugar residues of HMW-chitosan subjected to the reaction and the molar ratio of LMW-chitosan (hydrophilic group / total sugar residue of the branched chitosan derivative) What is necessary is just to make both react in the ratio from which group) becomes 0.3 / 1 or more.

  The branched chitosan derivative obtained by the above production method may be purified according to a known method. For example, the obtained unpurified derivative is stirred in an organic solvent (eg, acetone), and then suction filtered, It can be purified by drying under reduced pressure. The branched chitosan derivative of the present invention can be processed into a powder, solution, suspension or the like using a conventional method.

  The branched chitosan derivative of the present invention is characterized by higher water solubility and lower aqueous solution viscosity than linear chitosan. In this respect, the present invention provides a low viscosity chitosan composition comprising the branched chitosan derivative of the present invention. Examples of the solvent of the composition include water, physiological saline, phosphate buffer and other buffer solutions, glucose aqueous solution, acetic acid aqueous solution, vitamin C aqueous solution, citric acid aqueous solution and other acidic aqueous solutions, alcohols, glycol ethers and the like. Various polar solvents and the like, or a mixture thereof can be mentioned. The composition of the present invention includes a pH adjusting buffer, isotonicity adjusting substances such as salts or saccharides, preservatives, aqueous vitamins such as vitamin C and vitamin B group, alanine, and glutamic acid, depending on necessity and application. Various polysaccharides such as amino acids, water-soluble gelatin, peptides, carboxymethylcellulose, carboxymethylchitin, carboxymethylchitosan, xanthan gum, guar gum, and the like. The composition can be used not only in the areas where chitin and chitosan are conventionally used, but also in fields such as pharmaceuticals, foods and drinks, and cosmetics. The present invention includes, for example, gene carriers, bioadhesives, sealing agents, wound healing materials, biocompatible films, coating materials, moisturizers, antibacterial agents, adsorbents, bioimmunity activators and other pharmaceuticals, health It can be used in the production of foods.

  The branched chitosan derivative of the present invention has a remarkable bioimmune system activation action. Accordingly, the present invention provides an immune system activator comprising the branched chitosan derivative of the present invention. The immune system activator of the present invention can be prepared by mixing the branched chitosan derivative of the present invention with an appropriate carrier or excipient. The dosage form of the immune system activator of the present invention is not particularly limited, and is liquid (eg, solution, suspension), solid (eg, powder, granule, tablet, capsule), semi-solid (eg, pasta, ointment) , In a tube). Methods for producing these dosage forms are known to those skilled in the art and can be easily produced. The immune system activator of the present invention can be administered to any animal. The administration route is not particularly limited, but oral administration is preferred. For example, in the case of oral administration to humans, the dose of the branched chitosan derivative of the present invention can be appropriately determined by a doctor while observing the patient's condition. For example, the dosage of the branched chitosan derivative of the present invention to a human may be about 1 mg to about 1000 mg / body weight / day, and can be administered once to several times a day. During the period when immune system activation is required, the immune system activator of the present invention can be administered.

  The branched chitosan derivative of the present invention is derived from natural materials chitin and chitosan. Therefore, the branched chitosan derivative of the present invention can be used for foods and drinks in addition to pharmaceutical compositions. In this respect, the present invention provides a food or drink containing the branched chitosan derivative of the present invention. The kind of food and drink is not particularly limited, and may be, for example, a drink or juice, or may be in the form of a seasoning. Furthermore, the branched chitosan derivative of the present invention may be added directly to a food material or raw material, or may be added to a finished food product. Further, for example, the branched chitosan derivative of the present invention may be used as a component of confectionery such as cookies, biscuits, cakes, instant soup and instant miso soup powder. The food and drink containing the branched chitosan derivative of the present invention includes health foods and so-called “tokuho”. The food and drink containing the branched chitosan derivative of the present invention may be in the form of a supplement. The supplement can be produced according to the method for producing the pharmaceutical composition.

  The meanings of the terms used in this specification are understood to be those generally recognized in the art.

  The present invention will be described in more detail and specifically with reference to the following examples, but the examples are only illustrative and do not limit the present invention.

Example 1 (Synthesis of branched chitosan derivative 1a)
Chitosan (1.20 g, 6.99 mmol as amino group) [manufactured by Koyo Chemical Co., Ltd., Lot L05261, degree of deacetylation (DDA) 95%, weight average molecular weight (Mw) 80,000, number average molecular weight (Mn) 21, 000] in a 1% acetic acid solution (200 mL) at room temperature, chitosan [6.33 g, 3.17 mmol as a reducing terminal residue] (manufactured by Koyo Chemical Co., Lot 1101-13T, DDA 71%, Mw 8,000, Mn 2,000) was added and stirred for 12 hours. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, branched chitosan was obtained by lyophilization (1.65 g, DDA 78%). The proton nuclear magnetic resonance spectrum and infrared absorption spectrum of branched chitosan are shown below.

_{^{1 H-NMR (400MHz, D}} 2 O): δ2.07 ( from CH ₃ of NHCOCH _3), 3.19 (H of C (2) position of GlcN), 3.80-4.20 (GlcNAc residues C (2) position and sugar residues C (3), C (4), C (5), C (6) position H).

IR (KBr): 3700-3100, 2931, 2887, 1640, 1548, 1417, 1383, 1319, 1155, 1072, 1031, 920 cm <-1>.

The composition ratio of each unit was calculated as follows. First, z of the chitosan derivative (branched chitosan derivative) was determined by the following formula A and formula B.

Deacetylation degree (DDA) (%) = [(number of GlcN residues of branched chitosan derivative) / TR] × 100 (formula A)

z = (number of branched residues) / TR (formula B)

Here, TR means the total number of residues of the branched chitosan derivative. The number of branched residues means the number of residues to which the branched chain is bonded, and is the same as the number of side chains. Of the raw material chitosan used in Example 1, when the main chain chitosan is the larger molecular weight and the side chain chitosan is the smaller one, the average residue molecular weight and residue of the main chain chitosan (DDA 95%, Mn 21,000) The numbers are 161 for the glucosamine residue molecular weight (FW) and 203 for the N-acetylglucosamine residue (FW).

Average residue molecular weight of main chain chitosan = 161 × 0.95 + 203 × 0.05 = 163.1

Number of residues in main chain chitosan = 21000 / 163.1 = 129

It becomes. On the other hand, the average residue molecular weight and the number of residues of side chain chitosan (DDA 71%, Mn 2,000) are calculated in the same manner,

Average molecular weight of side chain chitosan = 161 × 0.71 + 203 × 0.29 = 173.2

Number of residues of side chain chitosan = 2000 / 173.2 = 11.5

It becomes. Further, when the ratio of the number of branched chitosans to the number of residues of main chain chitosan is DS, the total number of residues of the branched chitosan derivative, TR is

TR = (number of residues in main chain) + (total number of residues in all side chains)
= (Number of residues in main chain) + [(number of residues in side chain) × (number of side chains)]
= (Number of residues in main chain) + [(number of residues in side chain) × [(number of residues in main chain) × DS]]
= 129 + [11.5 × [129 × DS]]

Can be expressed as Since the DDA of the branched chitosan obtained in Example 1 is 78 (%), when the above TR is substituted into Formula A,

78 = (number of GlcN residues in branched chitosan derivative) / TR × 100
= [(Number of GlcN residues in main chain) + (Total number of GlcN residues in all side chains)] / TR × 100
= [(Number of GlcN residues in main chain) + [(number of GlcN residues in side chain) × (number of side chains)] / TR × 100
= [(Number of GlcN residues in main chain) + [(Number of GlcN residues in side chain) × [(Number of residues in main chain) × DS]] / TR × 100
= [(129 × 0.95) + [(11.5 × 0.71) × [129 × DS]] / 129+ [11.5 × [129 × DS]] × 100

It becomes. From this, it can be calculated that DS = 0.21, and further, this value is substituted to obtain TR = 440. Formula B is

z = (number of branched residues) / TR
= (Number of side chains) / TR
= [(Number of residues in main chain) x DS]] / TR
= [129 × 0.21] / 440
= 0.06

It became.

Next, the GlcNAc residue of the branched chitosan derivative, that is, the value of y corresponding to the chitin unit has a relationship of y = 1−DDA / 100 with the degree of deacetylation (DDA). In addition, the peak area of [3.19 (H at the C (2) position of GlcN residue)] and [2.07 (derived from CH ₃ of NHCOCH ₃ of GlcNAc residue) in ¹ H-NMR analysis. ] And the ratio can also be calculated. This relationship is the same in ¹ H-NMR analysis of a branched chitosan derivative synthesized from branched chitosan, and y = 0.22.

Further, by calculating y = 0.22, the value of (x + z) corresponding to the GlcN residue of the branched chitosan derivative is calculated as x + z = 1−y = 0.78. On the other hand, the peak corresponding to the GlcN residue in ¹ H-NMR analysis, that is, the peak of [3.19 (H at the C (2) position of GlcN residue)] was observed in both the chitosan unit and the branch unit. Therefore, the area of the peak can be considered as the total amount of the two units. Therefore, x was x = 0.78−0.06 = 0.72.

Example 2 (Synthesis of branched chitosan derivative 1b)
1% acetic acid solution (200 mL) of chitosan (2.00 g, 10.3 mmol as amino group) (manufactured by Wako Pure Chemical Industries, “Chitosan 5” Lot TSQ4638, DDA 86%, Mw 100,000, Mn 20,000) At room temperature, chitosan (6.33 g, 3.15 mmol as a reducing terminal residue) (manufactured by Koyo Chemical Co., Lot 1101-13T, DDA 71%, Mw 8,000, Mn 2,000) was added for 12 hours. Stir. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, the branched chitosan was obtained by freeze-drying (2.56 g, DDA 77%).

The composition ratio of each unit was calculated in the same manner as in Example 1, and was x = 0.71, y = 0.23, and z = 0.06. The obtained derivative was identified in the same manner as in Example 1.

Example 3 (Synthesis of branched chitosan derivative 1c)
1% acetic acid solution (200 mL) of chitosan (2.00 g, 10.0 mmol as amino group) (manufactured by Koyo Chemical Co., Ltd., “SK-10” Lot 1023-10, DDA 84%, Mw 140,000, Mn 31,000) ) At room temperature with chitosan (5.00 g, 2.50 mmol as a reducing terminal residue) (manufactured by Koyo Chemical Co., Lot 1101-13T, DDA 71%, Mw 8,000, Mn 2,000), Stir for hours. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, branched chitosan was obtained by lyophilization (2.20 g, DDA 76%).

  The composition ratio of each unit was calculated in the same manner as in Example 1, and was x = 0.69, y = 0.24, and z = 0.07. The obtained derivative was identified in the same manner as in Example 1.

Example 4 (Synthesis of branched chitosan derivative 1d)
1% acetic acid solution of chitosan (2.52 g, 10.0 mmol as an amino group) (manufactured by Kyowa Technos, "Flonac-c" Lot 93-1-20, DDA 84%, Mw 160,000, Mn 37,000) (200 mL) at room temperature with chitosan (6.33 g, 3.15 mmol as a reducing terminal residue) (manufactured by Koyo Chemical Co., Lot 1101-13T, DDA 71%, Mw 8,000, Mn 2,000) And stirred for 12 hours. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, branched chitosan was obtained by lyophilization (3.20 g, DDA 78%).

  The composition ratio of each unit was calculated in the same manner as in Example 1, and was x = 0.72, y = 0.22, and z = 0.06. The obtained derivative was identified in the same manner as in Example 1.

Example 5 (Synthesis of branched chitosan derivative 1e)
To a 1% acetic acid solution (200 mL) of chitosan (2.52 g, 6.16 mmol as an amino group) (manufactured by Koyo Chemical Co., Ltd., “DAC-50” Lot 107311, DDA 45%, Mw 190,000, Mn 88,000) At room temperature, chitosan [6.33 g, 3.17 mmol as a reducing terminal residue] (manufactured by Koyo Chemical Co., Lot 1101-13T, 71% DDA, Mw 8,000, Mn 2,000) was added and stirred for 12 hours. did. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, branched chitosan was obtained by lyophilization (3.25 g, DDA 58%).

  The composition ratio of each unit was calculated in the same manner as in Example 1, and was x = 0.56, y = 0.42, and z = 0.04. The obtained derivative was identified in the same manner as in Example 1.

Example 6 (Synthesis of branched chitosan derivative 1f)
To a 1% acetic acid solution (200 mL) of chitosan (2.00 g, 10.3 mmol as an amino group) (manufactured by Koyo Chemical Co., Ltd., FM-80L Lot 0817-18, DDA 86%, Mw 450,000, Mn 82,000) At room temperature, chitosan [6.50 g, 3.25 mmol as a reducing terminal residue] (manufactured by Koyo Chemical Co., Lot 1101-13T, 71% DDA, Mw 8,000, Mn 2,000) was added and stirred for 12 hours. did. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, the branched chitosan was obtained by lyophilization (2.25 g, DDA 75%).

  The composition ratio of each unit was calculated in the same manner as in Example 1, and was x = 0.69, y = 0.25, and z = 0.06. The obtained derivative was identified in the same manner as in Example 1.

Example 7 (Synthesis of 1 g of branched chitosan derivative)
To a 1% acetic acid solution (200 mL) of chitosan (2.00 g, 10.5 mmol as an amino group) (manufactured by Koyo Chemical Co., Ltd., FH-80 Lot 1208-15, DDA 87%, Mw 650,000, Mn 73,000) At room temperature, chitosan [6.50 g, 3.25 mmol as a reducing terminal residue] (manufactured by Koyo Chemical Co., Lot 1101-13T, 71% DDA, Mw 8,000, Mn 2,000) was added and stirred for 12 hours. did. Thereafter, sodium cyanoborohydride (0.40 g, 6.37 mmol) was added, and the mixture was further stirred for 12 hours. After dialysis, branched chitosan was obtained by lyophilization (2.25 g, DDA 76%).

  The composition ratio of each unit was calculated in the same manner as in Example 1, and was x = 0.70, y = 0.24, and z = 0.06. The obtained derivative was identified in the same manner as in Example 1.

The branched chitosan derivatives obtained in the above examples are summarized in Table 2.

Since the novel branched chitosan derivative 1 is synthesized by a reaction between the amino group of HMW-chitosan as the main chain and the reducing end of LMW-chitosan as the side chain, the initial product of the reaction is schematically shown below. It is considered to be a comb-shaped structure like A in the formula. However, the amino group present in the side chain chitosan has the same reactivity as the amino group present in the main chain chitosan, and it is considered that the amino group present in the side chain chitosan is more advantageous in consideration of steric hindrance. Therefore, it is considered that the structure evolves into a branched B and a multi-branched C as the reaction proceeds. The multibranched C structure is difficult to recognize with the degree of substitution (DS) in the conventional comb type structure A, and the ratio z (Table 2) of branched residues is defined as follows. The above discussion does not limit the present invention.
z = (number of branched residues in branched chitosan derivative 1) / (total number of residues in branched chitosan derivative 1)

Example 8 Proof that the branched chitosan derivative of the present invention contains both HMW-chitosan as a main chain and LMW-chitosan as a side chain The value of z shown in Table 2 indicates the degree of deacetylation of raw chitosan ( DDA) and the degree of deacetylation (DDA) of product 1 can be calculated. However, it is not easy to prove directly that both the main chain HMW-chitosan and the side chain LMW-chitosan are included. Therefore, as shown in the scheme below, branched chitosan using H-6-chitosan as the main chain and LMW-chitosan as the side chain with R-6 and FITC-8 labeled with fluorescent probes having different fluorescence wavelengths as raw materials. Derivative 11 was synthesized. As shown in FIG. 1, in the fluorescence spectrum of the product 11, both fluorescence wavelengths (near 570 nm and 530 nm) specific to R-6 and FITC-8 were observed. It was confirmed that both HMW-chitosan and side chain LMW-chitosan were included.

Example 9 Viscosity Measurement of Branched Chitosan Derivative Aqueous Solution of the Present Invention Table 3 shows the solubility of the branched chitosan derivative obtained in the present invention and the viscosity of a solution (10 mg / ml) dissolved in a 1% aqueous acetic acid solution. Since none of the raw material HMW-chitosan dissolves in neutral water, it can be said that the multi-branching according to the present invention has a great effect of improving water solubility. The ability to form a neutral aqueous solution without using an acid expands the application range of the branched chitosan derivative of the present invention as a material.

  The viscosity of the branched chitosan derivative solution obtained in the present invention and the viscosity of the linear HMW-chitosan solution were compared in FIG. The viscosity of the linear HMW-chitosan used as a raw material increased as its molecular weight increased. However, the viscosity of the branched chitosan was almost constant within this range. However, as shown in Entry 6 and 7 in Table 3, when the weight average molecular weight (Mw) of the linear HMW-chitosan used as the raw material was larger than 450 kDa, the water solubility decreased and the viscosity increased greatly. Conventionally, a decrease in solubility and an increase in solution viscosity due to an increase in the molecular weight of chitosan have been problems that have limited use methods such as injection and injection, but this problem has been solved by the present invention. It can be said that the application range of the branched chitosan derivative of the present invention is far wider than the application range of conventional chitin and chitosan-based materials.

Example 10 Immune System Activating Action of Branched Chitosan Derivative of the Present Invention (1) Experimental Method (a) Administration to Mice and Collection of Biological Samples After ICR mice (female, 10 weeks old) were preliminarily raised for 1 week or more, It used for experiment. Branched chitosan derivatives (Ia in Table 2), HMW-chitosan (Mw = 80,000), and LMW-chitosan (Mw = 8,000) were respectively used at 2 mg / 0.2 ml / head for 1 day. Intragastric administration was performed once for 3 consecutive days. As the control group, a water administration group was used for the branched chitosan derivative group and the LMW-chitosan group, and an acetic acid aqueous solution administration group was used for the HMW-chitosan group. One to two hours after the third administration, blood was collected by cardiac puncture under diethyl ether anesthesia, and the spleen was removed after blood collection. Serum was obtained from the collected blood by a conventional method and subjected to measurement of serum cytokine concentration. When the branched chitosan derivative of the present invention was administered to animals as described above, toxicity to the animals was not observed.

(B) Preparation of splenocytes The excised spleen was immersed in PBS (hereinafter referred to as PBS) with 0.5% FBS (3-4 ml), and the surrounding connective tissue was removed using a pincette. The PBS was changed (3 to 4 ml), and the cells were suspended using a 24G needle and an inner cylinder of a 1 ml plastic syringe. The suspended cell solution was filtered through a 42 μm nylon mesh, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. A hemolytic agent was added and mixed well, followed by centrifugation in the same manner. The supernatant was discarded and PBS was added and centrifuged again in the same manner. The solution was diluted with PBS so that the number of cells was 1 × 10 ⁷ / ml to obtain a cell solution.

The hemolytic agent was prepared as follows. NH ₄ Cl 82.9 g, KHCO ₃ 10 g, EDTA · 2Na (Dojindo Laboratories, Inc., Kumamoto, Japan) 0.372 g was dissolved in 1 liter of ultrapure water and stored at 4 ° C. At the time of use, it was further diluted 10 times with ultrapure water.

(C) Measurement of immune cell activity The following antibodies were used:
FITC rat anti-mouse CD4: BD (Pharmingen, USA) for measuring CD4 + cell (helper T cell) activity; and RAT ANTI MOUSE NKG2A / C / E: FITC (funakoshi stock) for measuring natural killer (NK) T cell activity Company, Tokyo, Japan).
Each antibody was diluted with PBS so that the final dilution ratio was 1: 200 to prepare an antibody solution. To the Eppendorf tube, 100 μl of the cell solution (prepared in (b)) and 100 μl of the antibody solution were added and allowed to react at 4 ° C. for 20 minutes in the dark. After completion of the reaction, 500 μl of PBS was added and mixed well, followed by centrifugation at 1200 rpm for 5 minutes at 4 ° C. After removing the supernatant, 500 μl of PBS was added and used for measurement. The proportion of each immune cell was measured by measuring the fluorescence intensity using flow cytometry.

(D) Measurement of serum cytokine concentration To the serum sample obtained in (a) above, Flowcytomix Mouse Th1 / Th2 10plex Kit (Bendermedsystem, USA) was applied, and interleukin-6 (IL-6) and TNF were applied according to the manufacturer's instructions. The amount of -α was measured.

(2) Experimental results The proportion of helper T cells was significantly increased by the branched chitosan derivative, and was nearly twice that of the control (distilled water group). HMW-chitosan was not different from the results of acetic acid, and LMW-chitosan was not effective (FIG. 3). The proportion of NK cells was significantly higher than that of the control and HMW-chitosan with the branched chitosan derivative, and was about 12 times that of the control (distilled water group) (FIG. 4). Serum IL-6 and TNF-α levels are also significantly increased by the branched chitosan derivative, IL-6 is increased about 15 times compared to the control (distilled water group), and TNF-α is about 15 times higher than the control (distilled water group). It increased 10 times (FIGS. 5 and 6). From the above results, the branched chitosan derivative clearly activated Th-1 and Th-2 of helper T cells, and as a balance, the result of activating cellular immunity was obtained. This active effect is not present in HMW-chitosan and LMW-chitosan, but is a unique feature of branched chitosan derivatives. Thus, it was found that the branched chitosan derivative of the present invention has a remarkable bioimmune system activation action.

  In the present invention, by providing a multi-branched structure to chitosan, the improvement in water solubility and the molecular form that keeps the viscosity of an aqueous solution low can be achieved. Therefore, it can be used not only in areas where chitin and chitosan have been conventionally used, but also in fields such as pharmaceuticals and foods and drinks. The present invention can be used, for example, in the manufacture of pharmaceuticals such as gene carriers, bioadhesives, sealing agents, wound healing materials, biocompatible films, coating materials, bioimmunity activators, health foods, and the like. .

Claims (6)

Formula (I):

[Wherein, x + y + z = 1, and x, y, z are independently 0 ≦ x <1, 0 ≦ y <1, 0 <z ≦ 1, and R ¹ is

Wherein R ² is H, an acetyl group, or R ¹ , and a is an integer of 0 to 500, provided that all R ² may be the same or different.] A branched chitosan derivative having a degree of substitution of 0.13 to 0.21.   The branched chitosan derivative according to claim 1, which has a degree of branching of 0.05 to 0.06.   The branched chitosan derivative according to claim 1 or 2, wherein the viscosity of a solution (10 mg / ml) dissolved in a 1% aqueous acetic acid solution is 2.2 to 5.6 cP.   A low-viscosity chitosan composition comprising the branched chitosan derivative according to claim 1.   A food or drink comprising the branched chitosan derivative according to any one of claims 1 to 3. Formula (I):

[Wherein, x + y + z = 1, and x, y, z are independently 0 ≦ x <1, 0 ≦ y <1, 0 <z ≦ 1, and R ¹ is

Wherein R ² is H, an acetyl group, or R ¹ , and a is an integer of 0 to 500, provided that all R ² may be the same or different.] An immune system activator comprising a branched chitosan derivative.

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