Ice recrystallization inhibitor

Abstract

To provide low molecular weight ice recrystallization inhibitors with high IRI activity even in small amounts.SOLUTION: The ice recrystallization inhibitor according to one aspect of the present invention contains as an active ingredient a compound represented by the following chemical formula (1). In the chemical formula (1), R1 is an alkyl group or a hydrogen, R2 is an unsaturated hydrocarbon group or an acyl group, and R3 is a monosaccharide or a polysaccharide.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to an ice recrystallization inhibitor. [Background technology]

[0002] Even in subzero environments, ice undergoes a phenomenon called recrystallization, in which many small ice crystals grow into a few large ice crystals over time. When this recrystallization occurs in frozen fresh foods, medical organs, and cell tissues, it destroys the tissues or cells, significantly reducing their quality. Therefore, suppressing recrystallization has been a major challenge for maintaining the quality of food and preserving organs and tissues.

[0003] Ice recrystallization inhibitors are used as substances to inhibit the above-mentioned recrystallization. Currently, the most commonly used ice recrystallization inhibitors are antifreeze proteins or antifreeze polysaccharides, which are polymers that bind to the surface of ice crystals and inhibit the growth of ice.

[0004] In recent years, low molecular weight compounds have also been used as ice recrystallization inhibitors. For example, Non-Patent Document 1 discloses p-methoxy-O-glycosylphenol as an ice recrystallization inhibitor.

[0005] Furthermore, Patent Document 1 discloses at least one ice recrystallization inhibitor selected from the group consisting of n-alkyl-erythritol amides, glycosides containing an allyl group, aldamides containing an allyl group, and combinations thereof.

[0006] Patent Document 2 discloses antioxidant compositions used for preserving tissues and organs during transplantation, and describes phenylglycosides as an example of the antioxidant composition. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Patent No. 2015 / 0157010 [Patent Document 2] International Publication No. 2012 / 139148 [Non-patent literature]

[0008] [Non-Patent Document 1] Poisson et al., Langmuir 2019, 35(23),7452-7458. Summary of the Invention [Problem to be solved by the invention]

[0009] However, the above-mentioned antifreeze proteins and antifreeze polysaccharides have large molecules, and therefore cannot be taken up into cells, and cannot suppress the recrystallization of ice within cells. In particular, antifreeze proteins have problems such as immunogenicity, which limits their practical use.

[0010] Furthermore, the low molecular weight compound disclosed in Patent Document 1 has cytotoxicity and high osmotic pressure, and therefore there is room for improvement from the viewpoint of reducing the amount added.

[0011] Furthermore, although Patent Document 2 describes the function of phenylglycosides as antioxidant compositions, it does not describe their function as ice recrystallization inhibitors, and so the function was completely unknown.

[0012] In view of the above circumstances, there has been a demand for low-molecular-weight, non-protein ice recrystallization inhibitors that can be transported into cells. [Means for solving the problem]

[0013] As a result of intensive research, the present inventors discovered that low molecular weight glycosides having a specific structure act strongly as ice recrystallization inhibitors even in small amounts, and thus completed the present invention. In the prior art, only some of the glycosides according to one aspect of the present invention are used as antioxidants (e.g., Patent Document 2), and it is surprising that they have IRI activity.

[0014] That is, the present invention includes the following configurations. <1> An ice recrystallization inhibitor comprising a compound represented by the following chemical formula (1) as an active ingredient.

[0015] [ka]

[0016] In chemical formula (1), R 1 is an alkyl group or hydrogen, and R 2 is an unsaturated hydrocarbon group or an acyl group, and R 3 is a monosaccharide or polysaccharide. <2> The monosaccharide is glucose, mannose or galactose, and the polysaccharide is a polysaccharide composed of one or more monosaccharides selected from the group consisting of glucose, mannose and galactose. <1> The ice recrystallization inhibitor according to claim 1. <3> R 1 is hydrogen or (CH2) n CH3 (n is an integer from 0 to 3), <1> or <2> The ice recrystallization inhibitor according to claim 1. <4> R 1 is a methyl group, and R 2 is an aldehyde group or an allyl group, and R 3 is glucose, and R in chemical formula (1) 3 -O has an α-1-O glycosidic bond or a β-1-O glycosidic bond; <1> ~ <3> The ice recrystallization inhibitor according to any one of the preceding claims. <5> <1> ~ <4> A cryopreservation liquid comprising the ice recrystallization inhibitor according to any one of claims 1 to 4 and a solvent. <6> The concentration of the ice recrystallization inhibitor is greater than 2 mM; <5> A cryopreservation solution according to claim 1. <7> Used to inhibit ice recrystallization in cells, biological tissues, organs or foods, <5> or <6> A cryopreservation solution according to claim 1. Effect of the Invention

[0017] According to one embodiment of the present invention, it is possible to provide a low molecular weight ice recrystallization inhibitor that has high IRI activity even in small amounts. [Brief description of the drawings]

[0018] [Figure 1] 1 is an optical microscope image of ice crystals showing the IRI activity of a compound corresponding to an ice recrystallization inhibitor according to one embodiment of the present invention. [Diagram 2] This is a graph showing the correlation between the time from the start of ice recrystallization after adding a compound corresponding to an ice recrystallization inhibitor of one embodiment of the present invention and the average maximum particle size of the ice crystals. [Diagram 3] 1 is a graph showing the correlation between the concentration of a compound corresponding to an ice recrystallization inhibitor according to one embodiment of the present invention and the average maximum particle size of ice. [Figure 4] 1 is a graph showing the correlation between the concentration of a compound corresponding to an ice recrystallization inhibitor according to one embodiment of the present invention and the residual red blood cell rate after a cell cryoprotection test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, the embodiments of the present invention will be described in detail. However, the present invention is not limited to these, and various modifications are possible within the scope of the description. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments.

[0020] In this specification, unless otherwise specified, "A to B" indicating a numerical range means "A or more, B or less." In this specification, "IRI activity" refers to the effect of suppressing recrystallization of ice in a frozen product and not increasing the average particle size of ice.

[0021] [1. Ice recrystallization inhibitor] An ice recrystallization inhibitor according to one embodiment of the present invention contains a compound represented by the following chemical formula (1) as an active ingredient.

[0022] [ka]

[0023] In chemical formula (1), R 1 is an alkyl group or hydrogen, and R 2 is an unsaturated hydrocarbon group or an acyl group, and R 3 is a monosaccharide or polysaccharide.

[0024] The above compounds can be said to be glycosides in which sugars and the non-sugar portion (aglycone) which is an aromatic compound form an O-glycosidic bond. As described later in the Examples, it has been shown that glycosides in which the aglycone in chemical formula (1) is vanillin or eugenol have excellent IRI activity. The compound represented by chemical formula (1) has a common basic structure with the above glycosides, and is therefore considered to have similarly excellent IRI activity.

[0025] Examples of the monosaccharides include, but are not limited to, psicose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fucose, fuculose, rhamnose, glucose, galactose, fructose, xylose, arabinose, erythrulose, erythrose, threose, heptulose, hexylose, and pentulose. 3 If is a monosaccharide, R 3 consists of one monosaccharide of one type.

[0026] In addition, the above R 3 may be a polysaccharide consisting of a sugar chain in which the above monosaccharides are combined as structural units. Examples of polysaccharides include disaccharides, oligosaccharides, trisaccharides, tetrasaccharides, or polysaccharides of more than one type. When two or more monosaccharides are linked as structural units, the monosaccharides are bonded to each other by, for example, dehydration condensation through a glycosidic bond. The polysaccharide may be a homopolysaccharide or a heteropolysaccharide.

[0027] Specific examples of polysaccharides include, but are not limited to, cellulose, cellulose derivatives (such as hydroxymethylcellulose, hydroxyethylcellulose, and carboxymethylcellulose), sucrose, amylose, amylopectin, glycogen, lactose pullulan, curdlan, xanthan, chitin, and chitosan.

[0028] The monosaccharide is preferably glucose, mannose or galactose. The polysaccharide is preferably a polysaccharide composed of one or more monosaccharides selected from the group consisting of glucose, mannose and galactose. Examples of the preferred polysaccharide include amylose, amylopectin, glycogen, cellulose, lactose, etc.

[0029] According to the above-mentioned configuration, the structure of the compound represented by the chemical formula (1) is the same as or more similar to the structure of the glycoside used in the examples. Therefore, it is preferable from the viewpoint of obtaining an antifreeze agent having high IRI activity.

[0030] In addition, since the compound is required to function as an antifreeze agent inside cells, etc., it is preferable that the compound has as low a molecular weight as possible. Therefore, the above configuration is also preferable from the viewpoint of suppressing the molecular weight of the compound.

[0031] From the viewpoint of suppressing the molecular weight of the compound represented by chemical formula (1), the number of carbon atoms in the alkyl group is preferably 1 to 10, more preferably 1 to 6, and further preferably 1 to 4. The alkyl group may be linear or branched.

[0032] Specific examples of alkyl groups include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, a 4,4-dimethylpentyl group, an n-octyl group, a 2,2,4-trimethylpentyl group, a nonyl group, and a decyl group.

[0033] The unsaturated hydrocarbon group preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms, from the viewpoint of controlling the molecular weight of the compound represented by chemical formula (1).

[0034] Specific examples of the unsaturated hydrocarbon group include, but are not limited to, an allyl group, an aryl group, a benzyl group, and the like.

[0035] The number of carbon atoms in the acyl group is preferably 1 to 10, more preferably 1 to 6, and even more preferably 1 to 4, from the viewpoint of controlling the molecular weight of the compound represented by chemical formula (1).

[0036] Specific examples of the acyl group include unsubstituted acyl groups, such as an aldehyde group, an acetyl group, an ethylcarbonyl group, an n-propylcarbonyl group, an isopropylcarbonyl group, an n-butylcarbonyl group, an isobutylcarbonyl group, a tert-butylcarbonyl group, an n-pentylcarbonyl group, an isopentylcarbonyl group, a neopentylcarbonyl group, an n-hexylcarbonyl group, an n-heptylcarbonyl group, an n-octylcarbonyl group, an n-nonylcarbonyl group, and an n-decylcarbonyl group.

[0037] The ice recrystallization inhibitor according to one embodiment of the present invention may contain only one type of compound represented by chemical formula (1), or may contain multiple types of compounds.

[0038] The above compound may be a naturally occurring compound or a synthetic product. It may also be a commercially available product. Since the above compound itself is publicly known, the method for obtaining the above compound is not particularly limited, and when it is synthesized, it can be by a conventionally known organic synthesis method. On the other hand, the fact that the above compound has IRI activity is a finding that the present inventors have found for the first time.

[0039] R 1 As the alkoxy group, hydrogen or (CH2) is preferably used because it reduces the molecular weight of the above compound and makes it easier to increase the IRI activity of the ice recrystallization inhibitor. n CH3 (n is an integer of 0 to 3) is preferable.

[0040] (CH2) n A straight-chain alkyl group is more preferable as CH3 (n is an integer of 0 to 3). Examples of the straight-chain alkyl group include a methyl group, an ethyl group, a propyl group, and a butyl group.

[0041] The ice recrystallization inhibitor according to one embodiment of the present invention is R 1 is a methyl group, and R 2 is an aldehyde group or an allyl group, and R 3 is glucose, and R in chemical formula (1) 3 It is most preferred that -O comprises an α-1-O glycosidic bond or a β-1-O glycosidic bond.

[0042] That is, the non-sugar portion in chemical formula (1) is most preferably vanillin or a derivative thereof, or eugenol. Specifically, it is most preferably a compound represented by the following chemical formula (2) or a derivative thereof, or a compound represented by the following chemical formula (3). Examples of the vanillin derivative include vanillic acid and vanillyl alcohol.

[0043] [ka]

[0044] [ka]

[0045] With the above-mentioned configuration, the ice recrystallization inhibitor has particularly excellent IRI activity, as shown in the examples. 2 When is an aldehyde group, the ice recrystallization inhibitor exhibits particularly excellent water solubility.

[0046] Normally, after water freezes, small ice crystals become coarse as described above, even in an environment below 0° C. As a result, when the water is, for example, water inside cells during cryopreservation, or water inside living tissue during cryopreservation, the coarsened ice crystals cause damage to cells, tissues, etc., as described above.

[0047] On the other hand, in the ice recrystallization inhibitor according to one embodiment of the present invention, the compound represented by the above chemical formula (1) functions as an active ingredient that inhibits ice recrystallization. In other words, the above compound is not a polymer like a protein, but a low molecular weight compound, and is highly water-soluble, so it easily permeates cell membranes and is smoothly taken up into cells. From the viewpoint of ease of taking up into cells, the molecular weight of the above compound is preferably 500 or less.

[0048] When the cells, tissues, etc. are frozen, the compounds taken up into the cells, and the compounds taken up into the cells, etc. within the tissue, are thought to coat the surface of the ice present inside and outside the cells, and suppress the coarsening of small ice crystals.

[0049] The ice recrystallization inhibitor according to one embodiment of the present invention may contain one or more ingredients other than the active ingredient. The ingredients other than the active ingredient are not particularly limited as long as they do not inhibit the IRI activity of the ice recrystallization inhibitor. For example, conventionally known buffers, pH adjusters, isotonicity agents, preservatives, excipients, carriers, diluents, solubilizers, stabilizers, antioxidants, fillers, binders, surfactants, and stabilizers can be mentioned.

[0050] The amount of ingredients other than the active ingredient contained in the recrystallization inhibitor of one embodiment of the present invention is not particularly limited, but is preferably 10% by weight or less, and may be 0% by weight, when the entire recrystallization inhibitor is taken as 100% by weight.

[0051] [2. Freezer storage solution] A cryopreservation liquid according to one embodiment of the present invention contains an ice recrystallization inhibitor according to one embodiment of the present invention and a solvent.

[0052] According to the above configuration, the ice recrystallization inhibitor suppresses the coarsening of small ice crystals inside and outside the cells, thereby preventing damage and deterioration of cells, biological tissues, organs, fresh foods, processed foods, etc.

[0053] In the cryopreservation solution of one embodiment of the present invention, the concentration of the ice recrystallization inhibitor of one embodiment of the present invention is preferably greater than 2 mM, more preferably 3 mM to 30 mM, even more preferably 4 mM to 20 mM, and most preferably 5 mM to 10 mM.

[0054] The above concentration is preferably 2 mM or more since it increases the IRI activity, and the above concentration is preferably 30 mM or less in terms of reducing cytotoxicity.

[0055] Examples of the solvent include water, phosphate buffer solution, physiological saline, cell culture medium, tissue culture medium, organ preservation solution, etc. The cryopreservation solution according to one embodiment of the present invention can be prepared, for example, by adding the ice recrystallization inhibitor according to one embodiment of the present invention to the solvent so that the concentration is more than 2 mM, and mixing.

[0056] The above cryopreservation solution is preferably used to inhibit ice recrystallization in cells, biological tissues, organs or food.

[0057] Examples of cells that can be used include cancer cells, blood cells, organ cells, bone cells, skin cells, germ cells, embryonic stem cells, iPS cells, etc. Examples of biological tissues that can be used include epithelial tissue, hair roots, retina, etc. Examples of organs that can be used include the heart, liver, lungs, pancreas, kidneys, etc. Examples of foods that can be used include frozen fresh foods, frozen processed foods, ice cream, ice cream confectionery, etc.

[0058] When the above-mentioned cryopreservation liquid is used for cryopreservation of cells or biological tissues, for example, the requisite amount of cells or biological tissues can be added to the above-mentioned cryopreservation liquid prepared using a medium for cell culture or a medium for tissue culture as a solvent, and then frozen at a desired temperature, thereby preventing recrystallization of ice in the cells or biological tissues.

[0059] Furthermore, when the above-mentioned cryopreservation liquid is used, for example, for cryopreservation of an organ, the organ can be immersed in the above-mentioned cryopreservation liquid prepared using the organ preservation liquid as a solvent, and frozen at the desired temperature, thereby preventing recrystallization of ice in the organ.

[0060] When the above-mentioned freezing and preservation liquid is used, for example, for preserving food, the recrystallization of ice in the food can be suppressed by injecting the above-mentioned freezing and preservation liquid prepared using water as a solvent into the food or by immersing the food in the above-mentioned freezing and preservation liquid and freezing it at the desired temperature.

[0061] In this way, by comprising the steps of preparing the cryopreservation liquid by mixing the ice recrystallization inhibitor of one embodiment of the present invention with the solvent, and adding or immersing the object to be frozen in the cryopreservation liquid, or injecting the cryopreservation liquid into the object to be frozen, it is possible to suppress ice recrystallization in the object to be frozen.

[0062] The cryopreservation liquid may be frozen to produce ice, and the ice may be added to a solution in which the object to be frozen is immersed, or the cryopreservation liquid or ice may be added to an object that is already frozen. From the viewpoint of efficiently suppressing recrystallization of ice, it is preferable to add the cryopreservation liquid before the object is frozen.

[0063] The above-mentioned cryopreservation liquid may further contain a cryoprotectant in addition to the components other than the active ingredients described in [1. Ice recrystallization inhibitor]. The content of the cryoprotectant in the above-mentioned cryopreservation liquid is preferably 5% by weight or more and 10% by weight or less, when the weight of the ice recrystallization inhibitor according to one embodiment of the present invention and the total weight of the above-mentioned cryoprotectant is taken as 100% by weight.

[0064] Examples of the cryoprotectant include dimethyl sulfoxide, polyethylene glycol, propylene glycol, glycerol, polyvinylpyrrolidone, sorbitol, dextran, and trehalose.

[0065] [4. Method for producing ice recrystallization inhibitor] The method for producing the ice recrystallization inhibitor is not particularly limited because the compound represented by the chemical formula (1) itself is publicly known as described above. For example, the ice recrystallization inhibitor can be obtained by reacting a saccharide with an aromatic compound.

[0066] For example, the compound represented by the above chemical formula (2) can be specifically synthesized by the following method. First, 2,3,4,6-tetra-O-acetyl-α-glucopyranosyl bromide (1.34 g 3.28 mmol) is dissolved in an organic solvent such as acetone, vanillin dissolved in an aqueous solution of lithium hydroxide is added, and the mixture is stirred at room temperature. Next, the organic solvent is removed by an evaporator, and water is added. Extraction is performed with dichloromethane, and the organic layer is recovered, washed with an aqueous sodium hydroxide solution, and further washed with water until the pH becomes neutral.

[0067] After washing, the product is dried with magnesium sulfate or the like, and the solvent is distilled off under reduced pressure to obtain a residue. The resulting residue is recrystallized in an organic solvent to obtain a precursor compound. The resulting precursor compound is dissolved in methanol, sodium methoxide is added, and the mixture is stirred at room temperature. After the reaction is completed, the product is neutralized by passing it through a proton-type cation exchange resin, and the solvent is distilled off under reduced pressure using an evaporator. The residue is dissolved in a small amount of water and freeze-dried to obtain the glycoside represented by the aforementioned chemical formula (2) (compound 1, which will be described later in the Examples).

[0068] The organic solvent may be acetone, benzene, etc. The aqueous solution may be a lithium hydroxide aqueous solution, a sodium hydroxide aqueous solution, etc. The reaction time for stirring is preferably 5 hours to 30 hours, more preferably 10 hours to 20 hours.

[0069] The ice recrystallization inhibitor can be produced by a method in which a reaction between a sugar and an aromatic compound is caused to proceed using an enzyme (enzymatic method).

[0070] Taking the case of producing the compound of the above chemical formula (2) by an enzymatic method as an example, the following method can be mentioned. That is, sucrose is dissolved in a buffer solution or the like, vanillin dissolved in an organic solvent is added, and the mixture is stirred thoroughly at room temperature. After stirring is completed, an enzyme is added and a sugar transfer reaction is carried out on the phenolic hydroxyl group of vanillin. After the reaction is completed, the reaction solution is freeze-dried, and the residue obtained is added with ethyl acetate for extraction, and the recovered organic layer is further extracted with water. The aqueous layer is freeze-dried to obtain the glycoside represented by the above chemical formula (2) (compound 1, which will be described later in the Examples).

[0071] The raw materials used in the above enzymatic method are inexpensive sucrose (sugar) and vanillin, and the above active ingredient can be produced in a single-step reaction, making it cheaper than the method using 2,3,4,6-tetra-O-acetyl-α-glucopyranosyl bromide mentioned above.

[0072] Examples of organic solvents in which vanillin can be dissolved include water-soluble organic solvents such as DMSO and ethanol, and non-water-soluble organic solvents such as ethyl acetate.

[0073] Enzymes that can be used in the enzymatic method are not particularly limited as long as they are capable of glycosyltransferase reactions, but enzymes that transfer sugar to phenolic hydroxyl groups are preferred, and examples of such enzymes include sucrose phosphorylase, transfer-type α-amylase, and hydroquinone glycosyltransferase.

[0074] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. EXAMPLES

[0075] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0076] [Measurement of IRI activity] IRI activity was measured by the splat assay method based on the literature (Charles A. Knight, John Hallett, AL DeVries (1988), Solute effects on ice recrystallization: An assessment technique, Cryobiology, 25 (1), 55-60).

[0077] First, a circular slide glass was set on a cooling and heating stage for optical instruments (manufactured by Linkam Co., Ltd.) and cooled to -80°C. Next, 10 μL of a sample dissolved in PBS (NaCl: 0.9% (w / v), KCl: 0.02% (w / v), NaH2PO4: 0.02% (w / v), Na2HPO4: 0.115% (w / v)) was dropped onto the slide glass from a height of 1.2 m using an empty column to create an ice wafer.

[0078] In addition, the samples used paramethoxy-phenylglycoside (PMPGlc), compound 1, and compound 2 as solutes, which will be described later, and the concentration was 22 mM in Tests 1 and 2, which will be described later. As a control, PBS alone was used. In Test 3, which will be described later, the concentration of compound 1 was varied as shown on the horizontal axis of FIG. 3.

[0079] The lid of the device was then immediately closed, and the ice wafer was placed under a microscope while the temperature was rapidly raised to -6.5°C. The time when the temperature reached -6.5°C was set as 0 min, and measurements were made with a stopwatch, and the ice crystals were photographed once with a camera at 1, 10, 20, and 30 min. The crystal size was measured by selecting the 10 largest crystals in the photographed image, and measuring the number of pixels of their major axis and the number of pixels of their minor axis perpendicular to the diameter using image editing software (Adobe Photoshop). The pixel count was converted to μm using the image editing software, and the average value was calculated, and this value was used as the mean largest grain size (MLGS).

[0080] [Cell cryoprotection test] 3 mL of defibrinated horse blood (Kohjin Bio) was placed in a 15 mL Falcon tube and centrifuged (3300 g, 10 min, 4° C.) using a centrifuge (himac CF16RX (Hitachi), rotor (T11A21)).

[0081] The supernatant was removed by pipetting, and PBS (NaCl: 0.9% (w / v), KCl: 0.02% (w / v), NaH2PO4: 0.02% (w / v), Na2HPO4: 0.115% (w / v), and if used to wash and suspend red blood cells, dextrose: 0.2% (w / v)) was added to the pellet and washed by stirring.

[0082] After washing twice, the pellet was suspended in PBS to a volume of 3 mL, which was used as the RBC solution. The cryopreservation solution was prepared by dissolving the compound 1 to be tested and glycerol, a cryoprotectant, in PBS. The concentrations of compound 1 in the cryopreservation solution were 1.4 mM, 2.8 mM, 5.5 mM, 11 mM, and 22 mM, and each cryopreservation solution contained 7.5% by weight of glycerol. As a control for the cryopreservation solution, a solution containing 7.5% by weight or 10.0% by weight of glycerol alone in PBS without containing compound 1 was used. In addition, a test section was also set up in which only the RBC solution was used without adding the cryopreservation solution.

[0083] 150 μL of the RBC solution and 150 μL of each cryopreservation solution were mixed in a 1.5 mL Eppendorf tube, and the resulting solution was allowed to stand at room temperature for 10 minutes.

[0084] After standing, the mixture was stirred and centrifuged (3300×g, 5 min, 4° C.) using a rotor (T15AP21).Then, the hemoglobin (Hb) concentration in the supernatant was quantified using a hemoglobin kit for blood testing (Hemoglobin B-Test Wako, Fujifilm Wako Pure Chemical Industries, Ltd.).

[0085] After the determination, the solution was stirred, the Hb concentration of the whole blood was determined, and the degree of hemolysis and the residual degree of red blood cells before freezing were calculated according to the following formula (4).

[0086]

number

[0087] The Eppendorf tubes containing the solutions were then rapidly frozen using liquid nitrogen and stored in a deep freezer at -80° C. After one week, they were rapidly thawed in a water bath at 37° C. The degree of lysis and the residual degree of red blood cells after thawing were calculated in the same manner as before freezing using the following formula (5).

[0088]

number

[0089] [Synthesis of compound 1] 2,3,4,6-Tetra-O-acetyl-α-glucopyranosyl bromide (1.34 g, 3.28 mmol) was dissolved in 40 mL of acetone, and vanillin (1.5 g, 9.9 mmol) dissolved in an aqueous lithium hydroxide solution (0.21 g, 8.9 mmol) was added thereto, followed by stirring at room temperature overnight.

[0090] After checking the reaction by TLC, acetone was distilled off under reduced pressure using an evaporator, and 5 mL of water was added. Extraction was performed three times with 30 mL of dichloromethane, and the organic layer was collected, washed three times with 30 mL of 10% aqueous sodium hydroxide solution, and then washed with 30 mL of water until the pH reached 7.

[0091] After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The resulting residue was recrystallized in 10 mL of 2-propanol to obtain compound P1 (0.9972 g, 60.2%), represented by the following chemical formula (6), as colorless crystals. Compound P1 is 4-formyl-2-methoxyphenyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

[0092] [ka]

[0093] The NMR identification results of compound P1 are shown below.

[0094] 1 H NMRδ(CDCl3):9.90 (1H,s),7.44-7.41(2H,m),7.22(1H,dd,J=8.0Hz),5.35-5.29(2H,m),5.21-5.16(1H,m),5.12-5.10(1H,m),4.28 (1H,dd,J=12.4, 5.1 Hz),4.19(1H,dd,J =12.3,2.4Hz),3.89(3H,s),3.87-3.83(1H,m),2.08(3H,s),2.07(3H,s),2.05(3H,s),2.05(3H,s) 13C NMRδ(CDCl3):190.84,170.48,170.20,169.37,169.22,151.14. 151.07,132.90,125.32,118.30,110.93,99.77,72.45,72.32,71.10,68.33,61.93,56.15,20.67,20.59 ESI-HRMS m / z:calcd.for:C 22 H 26 NaO 12 (M+Na) + 505.1316 obsd.for:(M+Na) + 505.1228 and NMR results Shusheng Wang, Dan Liu, Xu Zhang, Shengyu Li, Yongxu Sun, Jia Li, Yifa Zhoua, and Liping Zhanga (2007), Study on glycosylated prodrugs of toxoflavins for antibody-directed enzyme tumor therapy, Carbohydrate Research, 342, 1254-1260; Concise total synthesis of acylated phenolic glycosides vitexnegheteroin A and ovatoside D, Carbohydrate Research, 460, 41-46.

[0095] P1 (241 mg, 0.5 mmol) was dissolved in 20 mL of methanol, sodium methoxide (108 mg, 2 mmol) was added, and the mixture was stirred at room temperature until completion of the reaction was confirmed by TLC. After completion of the reaction, the mixture was neutralized by passing it through a proton-type cation exchange resin, and the solvent was distilled off under reduced pressure using an evaporator. The residue was dissolved in a small amount of water and freeze-dried to obtain compound 1, represented by the following chemical formula (2), as a white powder (145.6 mg, 92.7%). Compound 1 is 4-formyl-2-methoxyphenyl-O-β-D-glucopyranoside.

[0096] [ka]

[0097] Compound 1 was also produced by an enzymatic method. Specifically, the method is as follows. 0.20 mL of a DMSO solution of vanillin (concentration of vanillin: 100 mg / mL) was added to a solution obtained by dissolving sucrose (1.0 g) in 2 mL of 0.1 M HEPES buffer (pH 7.5), and the mixture was thoroughly stirred. After stirring, 0.1 mL of a 0.1 M HEPES buffer solution (500 units / mL) of sucrose phosphorylase (derived from Leuconostoc mesenteroides, Oriental Yeast) was added, and the mixture was reacted at 42°C for 24 hours to obtain a reaction solution. After freeze-drying the reaction solution, 3 mL of water and 3 mL of ethyl acetate were added to the obtained residue and mixed, and the aqueous layer and the ethyl acetate layer were separated. The aqueous layer was further extracted twice with ethyl acetate (1 mL each). The extracted ethyl acetate layer was collected and then extracted three times with water (3 mL). After the extraction was completed, the aqueous layer was freeze-dried to obtain compound 1 represented by the above chemical formula (2) as a white powder (15.9 mg, 38.6%).

[0098] The NMR identification results of compound 1 are shown below.

[0099] 1H NMRδ(DMSO-d6):9.84(1H,s),7.51(1H,dd,J=8.3,1.7Hz),7.42(1H,d,J=1.5Hz),7.26(1H,d,J=8.5Hz),5.36(1H,d, J=3.2Hz),5.13(1H,s),5.07(2H,m),4.58(1H,t,J=5.1Hz),3.83(3H,s),3.66(1H,dd,J=11.7,2.4 Hz),3.29-3.24(2H, m),3.18‐3.16(1H,m) 13 CNMRδ(DMSO-d6):192.30,152.44,150.02,131.25,126.03,115.30,111.31,100.13,77.82,77.49,73.79,70.27,61.31,56.39 ESI-HRMS m / z:calcd.for:C 15 H 19 O 10 (M+HCOO) - 359.0984 obsd.for:(M+HCOO) - 359.0989 For NMR assignments, reference was made to the same literature as for compound P1.

[0100] [Synthesis of compound 2] 2,3,4,6-Tetra-O-acetyl-α-glucopyranosyl bromide (617 mg, 1.5 mmol) was dissolved in 20 mL of acetone, and then diluted with an aqueous solution of lithium hydroxide (170 mg Eugenol (739 mg, 4.5 mmol) dissolved in 5 mL of H2O was added and stirred at room temperature overnight.

[0101] After checking the reaction by TLC, acetone was distilled off under reduced pressure using an evaporator, and 10 mL of water was added. Extraction was performed three times with 30 mL of chloroform, and the organic layer was collected, washed three times with 30 mL of 10% sodium hydroxide aqueous solution, and then washed with 30 mL of water until the pH reached 7.

[0102] After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The resulting residue was recrystallized in 10 mL of 2-propanol to obtain compound P2 (159 mg, 21.5%), represented by the following chemical formula (7), as colorless crystals. Compound P2 is 4-allyl-2-methoxyphenyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside.

[0103] [ka]

[0104] The NMR identification results of compound P2 are shown below.

[0105] 1 H NMRδ(CDCl3):7.04(1H,d,J=8.0Hz),6.72(1H,d,J=2.0Hz),6.69(1H,dd,J=8.3 ,1.9Hz),5.94(1H,m),5.30-5.23(2H,m),5.20-5.13(1H,m),5.11-5.06(2H,m), 4.94-4.89(1H,m),4.28(1H,dd,J=12.2,5.1Hz),4.16(1H,dd,J=12.2,2.4Hz), 3.80(3H,s),3.77-3.72(1H,m),3.34(2H,d,J=6.6Hz),2.08(6H,s),2.03(6H,s) ESI-HRMS m / z:calcd.for:C 24 H 30 NaO 11 (M+Na) + 517.1680 obsd.for:(M+Na) + 517.1570 The attribution of NMR is from the document Thiago Belarmino de SouzaMarina OrlandiLuiz Felipe Leomil CoelhoLuiz Cosme Cotta MalaquiasAmanda Latercia Tranches DiasRoberta Ribeiro Reference was made to de CarvalhoNaiara Chaves SilvaDiogo Teixeira Carvalho (2014), Synthesis and in vitro evaluation of antifungal and cytotoxic activities of eugenol glycosides, Medicinal Chemistry Research, 23, 496-502.

[0106] P2 (146 mg, 0.3 mmol) was dissolved in 12 mL of methanol, sodium methoxide (64.8 mg, 1.2 mmol) was added, and the mixture was stirred at room temperature until the reaction was completed as confirmed by TLC.

[0107] After the reaction was completed, the mixture was neutralized by passing it through a proton-type cation exchange resin, and the solvent was removed under reduced pressure using an evaporator. The residue was dissolved in a small amount of water and freeze-dried to obtain compound 2 represented by the following chemical formula (3) as a white powder (90.4 mg, 92.4%). Compound 2 is 4-allyl-2-methoxyphenyl-O-β-D-glucopyranoside.

[0108] [ka]

[0109] The NMR identification results of compound 2 are shown below.

[0110] 1H NMRδ(DMSO-d6):7.06(1H,d,J=8.1Hz),6.86(1H,d,J=1.5Hz),6.73(1H,dd,J=8.2,1.1 Hz),6.04(1H,ddt,J=16.0,10.0,6.8Hz),5.24(1H,d,J=4.2Hz),5.16-5.07(3H,m),5. 04(1H,d,J=4.9Hz),4.90(1H,d,J=7.1Hz),4.57(1H,s),3.80(3H,s),3.72(1H,d,J=11 .1Hz),3.52-3.28(1H,m),3.36(1H,d,J=7.1Hz),3.32-3.30(2H,m),3.24-3.20(1H,m) 13 C NMRδ(DMSO-d6):148.96,144.92,137.93,133.62,120.41,115.69,115.60,113.08,100.39,77.00.76.87,73.31,69.80,60.77,55.75 ESI-HRMS m / z:calcd.for:C 17 H 23 O9(M+HCOO) - 371.1348 obsd.for:(M+HCOO) - 371.1347 For NMR assignments, reference was made to the same literature as for compound P2.

[0111] [Test 1] The presence or absence of IRI activity of the above compounds 1 and 2, and para-methoxy-phenylglycoside (PMPGlc) was examined.

[0112] The results are shown in Figure 1. Figure 1 shows ice crystals at a magnification of 1 x 10 30 minutes after the temperature reached -6.5°C, as described in the above [Measurement of IRI activity]. 4 This is the result of observation at 2x magnification.

[0113] 1, it can be seen that the ice to which compound 1, which corresponds to an ice recrystallization inhibitor according to one embodiment of the present invention, has a clearly smaller crystal size than the ice to which PMPGlc has been added. Therefore, it has been shown that compound 1 has a very strong IRI activity that inhibits ice recrystallization.

[0114] In addition, the crystal size of ice containing compound 2 was larger than that of ice containing PMPGlc, but was sufficiently smaller than that of the control. Therefore, it was found that compound 2 has strong IRI activity, although its activity is weaker than that of compound 1.

[0115] [Test 2] The IRI activities of Compound 1, Compound 2, and PMPGlc were measured by comparing the average maximum particle size of ice crystals based on the method described in [Measurement of IRI activity] above.

[0116] The results are shown in Figure 2. Figure 2 is a graph showing the correlation between the time from the start of ice recrystallization after the addition of the above compounds and the average maximum particle size of ice crystals. In the figure, "PBS" shows the results when only PBS was used as a control sample, "PMPGlc" shows the results when paramethoxy-phenylglycoside was used, "1" shows the results when compound 1 was used, and "2" shows the results when compound 2 was used as a solute.

[0117] From Figure 2, it can be seen that the average maximum particle size of ice to which compound 1 has been added does not increase even 30 minutes after ice recrystallization has started (after the temperature has reached -6.5°C). It can also be seen that the average maximum particle size is smaller when compound 2 is added than when only PBS is added. Therefore, it has been shown that both compounds 1 and 2 have IRI activity.

[0118] [Test 3] The change in IRI activity when the concentration of Compound 1 was changed was examined.

[0119] The measurement results are shown in Figure 3. Figure 3 shows the plot of the average maximum ice particle size 30 minutes after the temperature reached -6.5°C when the test described in [Measurement of IRI activity] was performed with varying concentrations of Compound 1.

[0120] FIG. 3 shows that when the concentration of compound 1 exceeded 2 mM, the average maximum particle size of ice rapidly decreased, and recrystallization of ice to which a solution of 5 mM or more had been added was completely suppressed.

[0121] [Test 4] Compound 1 was used to carry out the cell cryoprotection test shown in the above [Cell cryoprotection test].

[0122] The results are shown in Figure 4. As shown in Figure 4, red blood cells mixed with a cryopreservation solution containing compound 1 showed a higher red blood cell survival rate than a control not containing compound 1 when the concentration of compound 1 in the cryopreservation solution was 1.4 mM, 5.5 mM, or 11 mM and the glycerol concentration was the same at 7.5 mM. It was also found that the solution containing compound 1 at a concentration of 5.5 mM showed the highest effect. Therefore, it was shown that compound 1 does not show membrane toxicity and has a cell cryoprotective effect due to its IRI activity. [Industrial Applicability]

[0123] The present invention can be suitably used as a component of a cryopreservation solution for cultured cells, biological tissues, organs, foods, etc., or as a food additive.

Claims

1. An ice recrystallization inhibitor comprising a compound represented by the following chemical formula (1) as an active ingredient. 【Chemistry 1】 In chemical formula (1), R １ is an alkyl group or hydrogen; R ２ is an unsaturated hydrocarbon group or an acyl group, R ３ is a monosaccharide or polysaccharide.

2. The ice recrystallization inhibitor according to claim 1, wherein the monosaccharide is glucose, mannose or galactose, and the polysaccharide is a polysaccharide composed of one or more monosaccharides selected from the group consisting of glucose, mannose and galactose.

3. R １ is hydrogen or (CH ２ ) ｎ CH ３ (n is an integer from 0 to 3),

4. R １ is a methyl group, R ２ is an aldehyde group or an allyl group, R ３ is glucose, and R in chemical formula (1) ３ The ice recrystallization inhibitor according to any one of claims 1 to 3, wherein -O has an α-1-O glycosidic bond or a β-1-O glycosidic bond.

5. A cryopreservation liquid comprising the ice recrystallization inhibitor according to any one of claims 1 to 4 and a solvent.

6. The cryopreservation solution of claim 5, wherein the concentration of the ice recrystallization inhibitor is greater than 2 mM.

7. The freezing and preserving liquid according to claim 5 or 6, which is used to inhibit ice recrystallization in cells, biological tissues, organs or foods.

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