WO2014010685A1 - Preservative agent for use in low-temperature preservation of biological material, and method for preserving biological material at low temperature - Google Patents

Abstract

Disclosed are: a preservative agent for use in the low-temperature preservation of a biological material, which comprises a compound represented by formula (I); a preservative solution for use in the low-temperature preservation of a biological material, which contains the preservative agent; and a method for preserving a biological material, which is characterized by soaking the biological material in a solution containing a compound represented by formula (I) and keeping the solution at a low temperature.

Description

Preservative for cryopreservation of biological material and method for preserving biological material at low temperature

The present invention relates to a preservative for cryopreserving a biological material, and a preservation solution for cryopreserving a biological material containing the preservative. Furthermore, the present invention relates to a method for preserving biological materials at low temperatures.

The cells have different ion compositions inside and outside the cell membrane, and the difference in the distribution of ions having this charge brings about a potential difference. Usually, the intracellular potential is negative with respect to the outside of the cell (membrane potential), and this membrane potential exists as a basic principle common to living organisms regardless of animals or plants. The regulation mechanism of membrane potential is indispensable for life support and cell function, and its failure directly leads to life or cell death.

For this reason, there are various ion pumps and ion channels on the inner and outer membranes, and ion balance is constantly adjusted. The most important regulatory mechanism includes a sodium pump (Na ⁺ -K ⁺ ATPase) in animal cells and a proton pump (H ⁺ -ATPase) in plant cells. These ion pumps are membrane proteins that actively transport specific ions using ATP energy. If ATP is depleted for some reason or the ambient temperature deviates from the optimum range, the function of the ion pump will be reduced or stopped.

In physiological cells, under physiological conditions, the sodium pump mainly pumps out three intracellular sodium ions each time, and conversely, two potassium ions are pumped into the cell from the outside. Therefore, normally, intracellular potassium concentration is high (sodium concentration is low), and extracellular sodium concentration is high (potassium concentration is low). When the temperature of the cell falls below a certain temperature, the function of the sodium pump declines, so that sodium cannot be pumped out of the cell, and the intracellular sodium concentration increases. As the sodium concentration rises, the intracellular osmotic pressure rises, and the inflow of water molecules causes the cells to swell and eventually lead to cell rupture (cytotoxicity).

The above mechanism is considered to be one of the main causes of cell damage when organs for transplantation are cryopreserved at the time of organ transplantation in the medical field, and the basic composition of the electrolyte is intracellular low sodium and high potassium Organ preservation solutions have been developed. Typical examples are Euro Collins (EC) solution and UW (University Wisconsin) solution. Compared to conventional extracellular (high sodium, low potassium) preservation solutions centering on Ringer's solution, these can greatly extend the organ preservation period and are clinically applied as major organ preservation solutions in Japan and overseas. ing. However, these intracellular storage solutions have a risk of reversing and causing cytotoxicity when the storage temperature rises. Further, these are not applicable to all tissues and organs, and further improvement in performance is expected, including further extension of the storage period.

In recent years, the development of regenerative medicine has been remarkable, and embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are expected to be used for medical applications. Co-culture in embryonic fibroblast (MEF) cell feeder cell layer. The long-term storage of these MEF cells is generally carried out in a -80 ° C deep freezer or in liquid nitrogen, but in short-term storage, it is not always preferable to freeze the cells. If possible, it is expected that its application range will be expanded.

On the other hand, bovine in vitro fertilization technology made it possible to produce high quality in vitro fertilized embryos from ovaries derived from slaughterhouses. However, with the start of the BSE test at the slaughterhouse, it is difficult to take out the collected ovaries until the test result is found to be negative. Therefore, it is necessary to preserve the ovaries in a form that minimizes the effect on survival from the time of slaughtering to the production of in vitro fertilized eggs. In addition, although eggs for bovine in vitro fertilization are collected from bovine living bodies, there is a problem that viability cannot be maintained unless they are cultured immediately under appropriate conditions after being collected from bovine living bodies.

Even in the field of livestock breeding, various problems still remain regarding low-temperature preservation of ovaries (egg cells), fertilized eggs, sperm, etc. It will be possible to greatly contribute to the development of the breeding industry.

As a method for coping with such a problem, Patent Document 1 discloses that immature eggs collected by using a medium containing a protein synthesis inhibitor typified by cycloheximide is stored at room temperature and in the air. A method is disclosed. Patent Document 2 discloses a method for preserving bovine ovary by immersing bovine ovary in a preservation solution and preserving it at 10 to 20 ° C.

Further, techniques for preserving cells and organs as described above are disclosed in, for example, the following Patent Documents 3 to 6.

Patent Document 3 discloses a method for preserving a biological material by adding a preservation solution containing one or more polyphenols to the biological material and cooling. In Examples, catechins are disclosed as polyphenols. Only.

Patent Document 4 discloses a method of refrigerated storage of cells at a temperature at which water does not crystallize, for example, a temperature around 4 ° C., by adding an enkephalin derivative to the cell culture medium.

Patent Document 5 discloses a medical polyphenol solution containing polyphenol and 0.0001 to 0.05% by weight of ascorbic acid or a metal salt of ascorbic acid for use as a cell preservative, tissue preservative, or the like. It is disclosed that decomposition is suppressed and generation of hydrogen peroxide is suppressed. In Examples, only epigallocatechin gallate (EGCg) is disclosed as a polyphenol.

Patent Document 6 discloses a composition for a preservative containing 90% by mass or more of epigallocatechin gallate as an active ingredient, and the effect of preserving cells is more constant by using purified epigallocatechin gallate with high purity. It is described that it can be.

Patent Document 7 discloses a flavonoid glycoside having an ability to promote the supercooling ability of an aqueous solution. By using this supercooling promoting substance, an antifreeze liquid that can be used at about -15 ° C with water. Therefore, it is described that biological materials and the like can be stored in the antifreeze liquid. The following patent documents 8 to 10 also report on the method of using the supercooling promoting substance flavonoid glycoside disclosed in Patent Document 7.

Patent Document 8 discloses a beverage that can maintain a supercooled state containing the flavonoid glycoside. Patent Document 9 discloses a cryopreservation solution in which the above flavonoid glycoside is contained in a vitrification solution, which is less toxic than conventional vitrification solutions and increases the viability of cells and the like by storage. It describes what you can do.

Furthermore, Patent Document 10 discloses that by using an organ preservation solution containing the flavonoid glycoside, it is possible to preserve an animal organ at a temperature of 0 ° C. or lower without freezing. Has been.

Japanese Patent Laid-Open No. 2001-89302 Japanese Patent Laid-Open No. 5-112401 Special Table 2007-519712 JP 2002-335954 A JP 2006-188436 A JP 2003-267801 A International Publication No. 2008/007684 JP 2009-219394 JP 2009-219395 A JP 2009-221128 JP

However, Patent Documents 1 to 6 do not substantially disclose the use of a compound having a specific chemical structure described in the following formula (I) in a solution for preserving cells and organs.

Patent Documents 7 and 10 disclose that by using a flavonoid glycoside, cells can be stored at 0 ° C. or less without freezing injury because they can be supercooled to a temperature below freezing point without freezing. However, there is no disclosure of reduction of cold injury under normal cold conditions.

Storing cells, organs, biological tissues and the like easily at low temperatures is a commonly practiced method. However, it is also known that damage caused by the low temperature condition occurs even when the object is not frozen.

The present invention provides a preservative for cryopreserving biological materials, a preservation solution for cryopreserving biological materials containing the preservative, and a method for preserving biological materials at low temperatures in order to reduce these low temperature injuries. For the purpose.

The present inventors use a compound having a specific chemical structure described in the following formula (I) to store cells at a low temperature from around the freezing point without freezing to a normal refrigeration temperature without freezing. It was found that the survival rate was improved and a cold injury protection effect was obtained. The present invention has been completed based on these findings, and provides the following preservatives, preservatives, and storage methods.

(A) Preservative
(A-1) Formula (I):

[Wherein, R ¹ to R ⁶ are the same or different and each represents a hydrogen atom, a hydroxyl group, —CO ₂ R ⁷ , an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, An optionally substituted alkylthio group, an optionally substituted alkylsulfinyl group or an optionally substituted alkylsulfonyl group, at least one of which is a hydroxyl group, and any two of R ¹ to R ⁶ are congruent; And R ⁷ is a hydrogen atom or an optionally substituted alkyl group.] A preservative for cryopreserving a biological material comprising a compound represented by:
(A-2) R ¹ to R ⁵ are the same or different and are a hydrogen atom, a hydroxyl group or an alkoxy group, at least one is a hydroxyl group, and R ⁶ may be substituted with a hydroxyl group and / or an alkoxy group A phenylalkenyl group or — (R ⁷ ) _n —COR ⁸ , R ⁷ is an alkylene group or an alkenylene group, R ⁸ is a hydroxyl group, an alkoxy group or an aralkyloxy group, and n is an integer of 0 to 2. A preservative according to (A-1).
(A-3) The compound represented by the formula (I) is caffeic acid methyl ester, caffeic acid ethyl ester, caffeic acid phenethyl ester, ferulic acid ethyl ester, methyl 3- (3,4-dihydroxyphenyl) propanoate, Methyl 2- (3,4-dihydroxyphenyl) acetate, gallic acid methyl ester, gallic acid ethyl ester, gallic acid propyl ester, 3,4-dihydroxybenzoic acid methyl ester, 3,4-dihydroxybenzoic acid ethyl ester, 2, It is at least one selected from the group consisting of 5-dihydroxybenzoic acid methyl ester, 2,3-dihydroxybenzoic acid methyl ester, resveratrol, isolapontigenin, pterostilbene, pinostilbene, lapontigenin, oxyresveratrol and gnetole A preservative according to (A-1) or (A-2).
(A-4) Formula (II):

[Wherein R ⁹ and R ¹⁰ are a hydrogen atom, a hydroxyl group or a glucose residue, at least one of which is a glucose residue, and R ¹¹ to R ¹⁷ are the same or different and represent a hydrogen atom, a hydroxyl group or an alkoxy group] The preservative according to any one of (A-1) to (A-3), further comprising a flavonoid glycoside compound represented by the formula:
(A-5) The flavonoid glycoside compound represented by the formula (II) is at least one selected from the group consisting of quercetin-3-glucoside, kaempferol-7-glucoside, and apigenin-7-glucoside. The preservative according to (A-4).
(A-6) The preservative according to any one of (A-1) to (A-5), wherein the biological material is an animal or plant cell, tissue, organ, organ, or cultured cell sheet.
(A-7) The biological material is egg cell, fertilized egg cell, sperm cell, embryonic stem cell, iPS cell, adult stem cell, fibroblast, feeder cell, vascular endothelial cell, bone marrow cell, immune cell, hepatocyte, kidney Cells, neurons, pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, odontoblasts, roots Any one of (A-1) to (A-6), which is a membrane cell, oral mucosal cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell, platelet or heart Preservatives.
(A-8) Any one of (A-1) to (A-5), wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food. The preservative described in 1.
(A-9) The low temperature is a temperature at which the biological material does not completely freeze, that is, a temperature of -5 ° C to 20 ° C, according to any one of (A-1) to (A-8) Preservatives.
(A-10) A low-temperature injury protective agent comprising a compound represented by the formula (I).

(B) Stock solution
(B-1) A preservation solution for low-temperature preservation of a biological material containing the preservative according to any one of (A-1) to (A-10).

(C) How to save
(C-1) Formula (I):

[Wherein, R ¹ to R ⁶ are the same or different and each represents a hydrogen atom, a hydroxyl group, —CO ₂ R ⁷ , an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, An optionally substituted alkylthio group, an optionally substituted alkylsulfinyl group or an optionally substituted alkylsulfonyl group, at least one of which is a hydroxyl group, and any two of R ¹ to R ⁶ are congruent; The biological material may be immersed in a solution containing a compound represented by the following formula: R ⁷ is a hydrogen atom or an optionally substituted alkyl group, and the solution is kept at a low temperature. A method for preserving biological materials.
(C-2) R ¹ to R ⁵ are the same or different and are a hydrogen atom, a hydroxyl group or an alkoxy group, at least one is a hydroxyl group, and R ⁶ may be substituted with a hydroxyl group and / or an alkoxy group A phenylalkenyl group or — (R ⁷ ) n—COR ⁸ , R ⁷ is an alkylene group or an alkenylene group, R ⁸ is a hydroxyl group, an alkoxy group or an aralkyloxy group, and n is an integer of 0 to 2. (C-1).
(C-3) The compound represented by the formula (I) is caffeic acid methyl ester, caffeic acid ethyl ester, caffeic acid phenethyl ester, ferulic acid ethyl ester, methyl 3- (3,4-dihydroxyphenyl) propanoate, Methyl 2- (3,4-dihydroxyphenyl) acetate, gallic acid methyl ester, gallic acid ethyl ester, gallic acid propyl ester, 3,4-dihydroxybenzoic acid methyl ester, 3,4-dihydroxybenzoic acid ethyl ester, 2, It is at least one selected from the group consisting of 5-dihydroxybenzoic acid methyl ester, 2,3-dihydroxybenzoic acid methyl ester, resveratrol, isolapontigenin, pterostilbene, pinostilbene, lapontigenin, oxyresveratrol and gnetole The method according to (C-1) or (C-2).
(C-4) The method according to any one of (C-1) to (C-3), wherein the solution further comprises the flavonoid glycoside compound according to (A-4).
(C-5) The flavonoid glycoside compound is at least one selected from the group consisting of quercetin-3-glucoside, kaempferol-7-glucoside, and apigenin-7-glucoside, according to (C-4). Method.
(C-6) The method according to any one of (C-1) to (C-5), wherein the biological material is an animal or plant cell, tissue, organ, organ, or cell sheet.
(C-7) The biological material is egg cell, fertilized egg cell, sperm cell, embryonic stem cell, iPS cell, adult stem cell, fibroblast, feeder cell, vascular endothelial cell, bone marrow cell, immune cell, hepatocyte, kidney Cells, neurons, pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, odontoblasts, roots Any one of (C-1) to (C-6), which is a membrane cell, oral mucosa cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell, platelet or heart the method of.
(C-8) Any one of (C-1) to (C-5), wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food. The method described in 1.
(C-9) The low temperature is a temperature at which the biological material does not completely freeze, that is, a temperature of -5 ° C to 20 ° C, according to any one of (C-1) to (C-8) the method of.

By preserving the biological material at a low temperature of normal refrigerated storage of -5 ° C to 20 ° C that does not completely freeze, the preservative and method of the present invention can increase the survival rate of biological materials such as cells. Damage protection effect is obtained. Therefore, since it becomes possible to preserve | save biological material under the conditions which can suppress the low temperature injury resulting from it at the low temperature suitable for preservation | save of biological material, further survival of biological materials, such as a cell and an organ, is attained. The rate can be expected to improve. As is clear from the test examples described later, the preservative and the method of the present invention inhibit the biological material, which is mainly induced by apoptosis induced under low-temperature storage conditions, and inhibit the survival rate of the biological material. Can be increased. Therefore, the present invention can be expected to be applied in fields such as organ transplantation, blood transfusion medicine, regenerative medicine, livestock breeding, and fresh food.

10 is a photograph showing the morphological change of rabbit platelets in Test Example 12. (1) Before low-temperature storage / no treatment, (2) After storage at 4 ° C for 24 hours / No treatment, (3) After storage at 4 ° C for 24 hours / Compound (2) 10μg / ml treatment 14 is a graph showing the combined effect of Compound (I) and Compound (II) on low temperature (4 ° C.) storage of HL-60 cells in physiological saline in Test Example 13. (A) Combined use of caffeic acid ethyl ester and A7G, (B) Combined use of caffeic acid ethyl ester and Q3G, and the plots in the plot are (1) the number of living cells of compound (II) alone, (2) Compound (I) And the compound (II) in combination, (3) the average value and standard deviation value (n = 3) of the total value of the number of living cells of compound (I) alone and the number of living cells of compound (II) alone ). It is a figure showing the effect with respect to the apoptosis of the cell induced | guided | derived by the cryopreservation in Test Example 14. (A) Normal cell culture conditions at 37 ° C, (B) Stored at low temperature of 4 ° C for 24 hours (no drug treatment), (C) Stored at low temperature of 4 ° C for 24 hours (compound (2) at 10 μg / (D) ml), (D) Flow cytometry analysis results when 0.2% TritonX-100 was added to normal cell culture conditions at 37 ° C. (cell death by necrosis).

Hereinafter, the present invention will be described in detail.

The preservative for cryopreservation of the biological material of the present invention has the formula (I)

[Wherein, R ¹ to R ⁶ are the same or different and each represents a hydrogen atom, a hydroxyl group, —CO ₂ R ⁷ , an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, An optionally substituted alkylthio group, an optionally substituted alkylsulfinyl group or an optionally substituted alkylsulfonyl group, at least one of which is a hydroxyl group, and any two of R ¹ to R ⁶ are congruent; And R ⁷ may be a hydrogen atom or an optionally substituted alkyl group].

Preferred compounds represented by the formula (I) are those in which R ¹ to R ⁵ are the same or different and are a hydrogen atom, a hydroxyl group or an alkoxy group, at least one is a hydroxyl group, R ⁶ is a hydroxyl group and / Or a phenylalkenyl group which may be substituted with an alkoxy group, or-(R ⁷ ) _n -COR ⁸ , R ⁷ is an alkylene group or an alkenylene group, R ⁸ is a hydroxyl group, an alkoxy group or an aralkyloxy group, A compound in which n is an integer of 0-2.

In addition, the method for preserving a biological material of the present invention is characterized in that the biological material is immersed in a solution containing the above compound and the solution is kept at a low temperature.

The alkyl group may be linear or branched, and is preferably an alkyl group having 1 to 30 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, octyl, decyl and dodecyl.

The definition of the above alkyl group can be applied to the alkyl moiety in the alkoxy group, alkylthio group, alkylsulfinyl group, and alkylsulfonyl group.

The alkenyl group may be linear or branched, and has at least one double bond, preferably an alkenyl group having 2 to 30 carbon atoms, more preferably 2 to 6 carbon atoms. Of the alkenyl group. Specific examples of the alkenyl group include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and the like.

The definition of the alkenyl group can also be applied to the alkenyl part in the phenylalkenyl group.

The alkylene group may be linear or branched, and is preferably an alkylene group having 1 to 6 carbon atoms. Specific examples of the alkylene group include methylene, ethylene, trimethylene, propane-2,2-diyl, tetramethylene, 1,1-dimethylethylene and the like.

The alkenylene group may be linear or branched, and is preferably an alkenylene group having 2 to 6 carbon atoms. Specific examples of the alkenylene group include vinylene, arylene, propenylene, 2-methyl-2-propenylene and the like.

The aralkyloxy (arylalkyloxy) group is preferably an aralkyloxy group having 7 to 12 carbon atoms. The alkyl part of the aralkyloxy group may be linear or branched. Specific examples of the aralkyloxy group include benzyloxy, phenethyloxy, 3-phenylpropyloxy and the like.

The above aryl means a monocyclic or polycyclic group composed of a 5- or 6-membered aromatic hydrocarbon ring, and specific examples thereof include phenyl and naphthyl.

In the compound of the present invention, “optionally substituted” means alkoxycarbonyl, aralkyloxycarbonyl, —COOH, alkylthiocarbonyl, alkyldithiocarbonyl, alkoxythiocarbonyl, dialkylaminocarbonyl, aryloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl. , Alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, aralkylcarbonyl, cyano, nitro and the like. The definitions of the alkyl group, aralkyloxy group, alkenyl group and aryl group can be applied to the alkyl part, aralkyloxy part, alkenyl part and aryl part of each group here.

The alkynyloxycarbonyl and the alkynyl part of alkynylcarbonyl may be either linear or branched, preferably alkynyl having 2 to 30 carbon atoms, more preferably alkynyl having 2 to 6 carbon atoms. The aralkyl moiety of the aralkylcarbonyl is preferably an aralkyl group having 7 to 12 carbon atoms. The alkyl part of the aralkyl moiety may be linear or branched.

Examples of the compound represented by the formula (I) include caffeic acid methyl ester, caffeic acid ethyl ester, caffeic acid phenethyl ester, and ferulic acid ethyl ester. acid etylester), methyl 3- (3,4-dihydroxyphenyl) propanoate, methyl 2- (3,4-dihydroxyphenyl) acetate (methyl 2- (3,4 -dihydroxyphenyl) acetate), gallic acid methyl ester, gallic acid ethyl ester, gallic acid propyl ester, 3,4-dihydroxybenzoic acid methyl ester (3,4- Dihydroxybenzoic acid metylester), 3,4-Dihydroxybenzoic acid ethyl ester (3,4-Dihydroxybenzoic acid etylester), 2,5-dihydroxybenzoic acid methyl ester (2,5-Dihydroxybenzoic acid methylester), 2,3-dihydroxybenzoic acid methyl ester (Methyl 2,3-dihydroxybenzoate), tocopherol, tocotrienol, sesamol, 3,5-di-t-butyl-4-hydroxytoluene (BHT) ), 2-t-butyl-4-hydroxyanisole (BHA), resveratrol (3,4 ', 5-Trihydroxy-trans-stilbene), isolapontigenin (3,4', 5-Trihydroxy-3'- methoxy-trans-stilbene), pterostilbene (trans-1- (3,5-Dimethoxyphenyl) -2- (4-hydroxyphenyl) ethylene), pinostilbene (3,4'-Dihydroxy-5-methoxy-trans-stilbene) , Lapontigenin (3,3 ', 5-Trimethoxy-4'-methoxy-trans-stilbene), Oxyresveratrol (2,3', 4,5'-Tetrahydroxy-trans-stilbene), Gnitol (2,3 ' , 5 ', 6-Tetrahydroxy-trans-stilbene).

In addition to the compound of formula (I), the preservative for cryopreservation of the biological material of the present invention includes formula (II):

[Wherein R ⁹ and R ¹⁰ are a hydrogen atom, a hydroxyl group or a glucose residue, at least one of which is a glucose residue, and R ¹¹ to R ¹⁷ are the same or different and represent a hydrogen atom, a hydroxyl group or an alkoxy group] A flavonoid glycoside compound represented by the formula:

Examples of the flavonoid glycoside compound represented by the formula (II) include quercetin-3-Glucoside (Q3G), kaempferol-7-Glucoside (K7G) And Apigenin-7-Glucoside (A7G).

The above compounds can be chemically synthesized by known methods, and those contained in organisms such as plants can be obtained by extracting them from these by known methods. Moreover, it is also possible to obtain with a commercial item.

In the present invention, the low temperature means a temperature at which the biological material does not completely freeze, that is, −5 ° C. or higher, preferably −3 ° C. or higher, more preferably higher than 0 ° C. and 20 ° C. or lower, preferably 15 ° C. or lower. More preferably, the temperature is 10 ° C or lower.

As used herein, low temperature injury means cell injury caused by low temperature, and low temperature injury protection effect means an effect of protecting cells from the low temperature injury. Therefore, taking this meaning into consideration, the preservative of the present invention can also be referred to as a low-temperature injury protective agent.

The biological material used in the present invention is not limited as long as the effects of the present invention can be obtained, but any biological material can be used. Examples thereof include animals or plant cells, tissues, organs, organs, and individuals. Can be mentioned. The tissue, organ and individual may be derived from any animal or plant. Mammals (humans, monkeys, cows, pigs, horses, dogs, cats, rabbits, mice, rats, etc.) are desirable as animals targeted by the present invention.

Animal cells include egg cells, fertilized egg cells, sperm cells, ES (embryonic stem) cells, iPS (induced pluripotent stem) cells, adult stem cells, hematopoietic stem cells, tissue stem cells, mesenchymal stem cells, fibroblasts, feeder cells , Bone marrow cells, immune cells, hepatocytes, blood cells (blood cells) (red blood cells, white blood cells and platelets), myocytes, cardiomyocytes, smooth muscle cells, myoblasts, osteoblasts, neurons, neuroblasts, vascular endothelium Cells, smooth muscle cells, bone cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, odontocytes, periodontal ligament cells, oral mucosal cells, corneal cells, retinal cells, adipocytes, adipose stem cells, kidneys Examples include cells, spleen cells, pancreatic cells, pancreatic parenchymal cells, pancreatic duct cells, epithelial cells, endothelial cells, epidermal cells, pigment cells and the like. An immune cell means a cell involved in an immune reaction, such as thymocyte, T cell, B cell, NK cell, NKT cell, monocyte, dendritic cell, macrophage, eosinophil Examples include spheres, neutrophils, and basophils. The cells may be in a free state in the preservation solution, in an adherent state on a culture dish, or in a cell sheet state.

Examples of animal tissues include epithelial tissues, connective tissues, muscle tissues, and nerve tissues.

Animal organs and organs include ovary, semen, blood, lymph nodes, thymus, skin, blood vessels, diaphragm, cornea, kidney, heart, brain, liver, pancreas, eyeball, spleen, lung, intestine, nerve, placenta, umbilical cord And retina. In addition, the biological material used in the present invention may be meat, animals and plants used as food, and examples include beef, pork, chicken, fish, shellfish, cereals, vegetables, fruits and the like. . Other biological materials include flower buds, which are ornamental plants.

The preservative of the present invention may contain a known additive as appropriate in addition to the compound (I).

The preservation solution for low temperature preservation of the biological material of the present invention is characterized by containing the above preservative. In the present invention, when the biological material is stored at a low temperature, the compound (I) is usually used as a solution. The solvent that dissolves the compound (I) is not particularly limited. For example, physiological saline, infusions (electrolyte infusion, nutrient infusion, sugar infusion, amino acid infusion, glucose solution, Ringer's solution, acetate Ringer's solution, lactated Ringer's solution, etc.) Buffer solution (PBS, Tris buffer solution, Hepes buffer solution, MOPS buffer solution, PIPES buffer solution, etc.), sodium citrate preparation for blood transfusion, Alsever solution, cell culture solution (Medium199, RPMI1640, DMEM, etc.), organ preservation solution (EC liquid, UW liquid), modena liquid and the like.

The concentration of compound (I) in the preservation solution of the present invention is usually 0.001 to 1000 μg / ml, preferably 0.01 to 100 μg / ml. The concentration of the flavonoid glycoside compound (II) in the preservation solution of the present invention is usually 0.001 to 1000 μg / ml, preferably 0.01 to 100 μg / ml. In the preservation solution of the present invention, other conventional components such as buffers, antibiotics, antibacterial agents, antioxidants, serum, fetal bovine serum, saccharides, lipids, vitamins, proteins, peptides, amino acids, pH indicators , Chelating agents, osmotic pressure regulators and the like.

In the present invention, the biological material can be stored by immersing the biological material in a solution containing the compound (I). In this case, the solution may be cooled to a low temperature before immersing the biological material, or may be cooled to a low temperature after immersing the biological material. Once the solution containing the biological material is cooled to a low temperature, it is kept at a low temperature. However, it is not always necessary to maintain a constant temperature, and the temperature may be outside the low temperature range for a short time.

By using the solution containing the compound (I) of the present invention, the viability of the biological material can be significantly increased by storing the cells at a low temperature, and a low-temperature injury protection effect can be obtained. Since the present invention can suppress low temperature injury caused by low temperature suitable for storage of biological materials, it can store biological materials such as cells and organs in an appropriate state.

The preservation solution of the present invention having the characteristics as described above is a preservation solution for an isolated organ in the field of organ transplantation, a preservation solution for blood cell components in the field of transfusion medicine, a preservation solution for ES cells, iPS cells, and feeder cells in the field of regenerative medicine. Ovary, egg cells, fertilized eggs and sperm cell preservation solution in the field of livestock breeding, fruit and vegetables (vegetables and fruits) in the field of fresh food, preservation solution of fresh fish and meat, preservation solution of florets in the field of ornamental plants, etc. Application as is expected.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples.

Test example 1
For the compounds of the present invention, the protective effect against cold injury of cells was evaluated by the following method.

RPMI-1640 (SIGMA, No.1) containing 10% fetal calf serum (Thermo Fisher Scientific, No.SH3D396.03) in an incubator (Sanyo Electric Co., Ltd., MCO-17AIC) at 5% carbon dioxide, 37 ° C. R8758) (10% FBS-RPMI) Human promyelocytic leukemia cell line HL-60 (ATCC, No.CCL-240) (HL-60 cells) cultured in a culture solution was collected, 4 ° C., 1,000 rpm, After centrifugation for 5 minutes (TOMY, EIX-135), the supernatant was removed and resuspended in physiological saline to 2 × 10 ⁶ cells / ml. 0.25 ml of this cell suspension and 0.25 ml of the test compound were mixed in a 2 ml sterilized microtube. The test compound was dissolved in 100 mg / ml with dimethyl sulfoxide (Nacalai Tesque, No. 13407-45) (DMSO) before the test, and then in physiological saline (Otsuka Pharmaceutical Co., Ltd. After preparing 10, 1 and 0.1 mg / ml dilutions (DMSO) at a 10-fold common ratio, it is diluted 500-fold with physiological saline (Otsuka Pharmaceutical, Otsuka raw food injection). These were prepared as 20, 2 and 0.2 μg / ml physiological saline solutions, respectively.

The mixture of the cell suspension and the test compound was allowed to stand for about 24 hours in a cooling vessel (No. SC-DF25, manufactured by TWINBIRD) set at 4 ° C. Thereafter, 2.5 ml of 10% FBS-RPMI culture solution was added and centrifuged at 4 ° C. and 1,000 rpm for 5 minutes. After removing the supernatant, it is suspended in 2.5 ml of 10% FBS-RPMI culture solution, added to a 96-well microplate (IWAKI, No. 3860-096) at 0.1 ml / well, 5% carbon dioxide, 37 ° C. The cells were cultured for about 24 hours in an incubator. Subsequently, 10 μl / well of WST-8 solution (Nacalai Tesque, Cell Count Reagent SF, No. 07553-44) was added, and after further culturing in an incubator for 3 hours, the absorbance was measured at a wavelength of 450 nm. (WAKO, SPECTRA MAX250). From this absorbance value, the number of viable cells was calculated from the standard line (viable cell number = 56375 × absorbance, R ² = 0.9995) between the cell number and the absorbance prepared in advance.

As a result, as shown in Tables 1 and 2, in the case of no addition, the cells were allowed to stand at 4 ° C. for 24 hours (cold injury). Reduced to pcs / well. On the other hand, a low temperature injury protective effect was observed by adding the compounds (1) to (21) to cells at concentrations of 10, 1, and 0.1 μg / ml during low temperature storage. At this time, the number of viable cells was 16.2 to 127.4 times higher for each compound than the compound without saline alone.

Test example 2
The test was performed according to the same test method as in Test Example 1 except that the temperature during low-temperature storage was changed from 4 ° C to -5 ° C, 0 ° C or 10 ° C. As a result, as shown in Table 3, the low temperature injury protection effect was confirmed at all storage temperatures.

Test example 3
Solvents to which the compounds to be added during low-temperature storage are added from physiological saline, phosphate buffered saline (PBS, SIGMA No. D 8537), EC solution (composition K ₂ HPO ₄ ; 7.4 g / L, KH ₂ PO ₄ 2.05 g / L, KCl; 1.12 g / L, NaHCO ₃ ; 0.84 g / L, glucose; 35 g / L), UW solution (Biaspan; manufactured by Astellas Pharma Inc.), RPMI1640 culture solution (RPMI), or 10% FBS -The same test as in Test Example 1 was performed by changing to the RPMI culture solution. As a result, as shown in Table 4, the low temperature injury protection effect was confirmed in all the solvents used. That is, the solvent of the compound of the present invention is not limited to physiological saline, and it has been confirmed that it exhibits a low-temperature injury protection effect even in widely used solvents such as physiological buffers, organ preservation solutions, and cell culture solutions. It was done.

Test Example 4 (Cold storage of MEF cells)
The protective effect of the compound of the present invention against cold injury of mouse fetal fibroblast (MEF) cells (DS Pharma Biomedical, Primary Mouse Embryo Fibroblast, No. R-PMEF-HL) was evaluated by the following method.

The cells were suspended in a culture medium for MEF cells, seeded in a 96-well microplate at 5 × 10 ³ cells / 0.1 ml / well, and cultured in an incubator at 37 ° C. with 5% carbon dioxide. Thereafter, the supernatant was removed, and a test compound (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) whose concentration was adjusted with physiological saline was added to the cells at 0.1 ml / well. The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours). Subsequently, the supernatant was removed and replaced with a culture solution for MEF cells. After culturing in an incubator for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 15506 × absorbance, R ² = 0.996).

As a result, as shown in Table 5, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells was 5,000 to 349 from the initial number of cells / well when not added in physiological saline. It decreased to 1026 cells / well in the culture medium for MEF cells. On the other hand, by adding the compound (2) to the cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained up to 16.9 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The composition of the culture solution for MEF cells used in this test example is as follows.
Composition of MEF cell culture medium (100 ml) Iscove's Modified Dulbecco's Medium (IMDM) (invtrogen, No. 12440) 88 ml
MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid (MEAA) (invtrogen, No.11140) 1 ml
Penicillin-Streptomycin-Glutamine (100X), liquid (invtrogen, No.10378) 1 ml
Fetal Bovine Serum (FBS) 10 ml

Test Example 5 (Hs68 cell cryopreservation)
The protective effect of the compounds of the present invention against cold injury of Hs68 human normal diploid fibroblasts (human foreskin fibroblast, ATCC, No. CRL-1635) was evaluated by the following method.

The cells were suspended in a culture medium for Hs68 cells, seeded in a 96-well microplate so as to be 1 × 10 ⁴ cells / 0.1 ml / well, and cultured for 2 hours in an incubator at 5% carbon dioxide and 37 ° C. Thereafter, the supernatant was removed, and compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) adjusted in concentration with physiological saline was added to the cells at 0.1 ml / well. . The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours). Subsequently, the supernatant was removed and replaced with a culture medium for Hs68 cells. After culturing in an incubator for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard number of cells and absorbance (viable cell count = 13709 × absorbance, R ² = 0.999).

As a result, as shown in Table 6, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells increased from an initial number of 10,000 cells / well to 203 when no addition was made in physiological saline. It decreased to 75 cells / well in the culture medium for Hs68 cells. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained up to 37.3 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The composition of the culture solution for Hs68 cells used in this test example is as follows.
Composition of culture medium for Hs68 cells (100 ml) Dulbecco's Modified Eagles Medium (DMEM) (SIGMA, No.D5791) 90 ml
Fetal Bovine Serum (FBS) 10 ml

Test Example 6 (HUVEC low-temperature storage)
The protective effect of the compound of the present invention against cold injury of normal human umbilical vein endothelial cells (HUVEC) (Toyobo Co., Ltd., No.GCA200K05N) was evaluated by the following method.

Suspend cells in culture medium for HUVEC (TOYOBO, No.CA211K500), seed in 96-well microplate to 5 × 10 ³ cells / 0.1 ml / well, and in 5% carbon dioxide in a 37 ° C incubator. Cultured for 3 hours. Thereafter, the supernatant was removed, and compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) adjusted in concentration with physiological saline was added to the cells at 0.1 ml / well. . The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours). Subsequently, the supernatant was removed and replaced with a culture solution for HUVEC. After culturing for about 24 hours in an incubator, 10 μl / well of WST-8 solution was added and further cultured for 2 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 13545 × absorbance + 165, R ² = 0.999).

As a result, as shown in Table 7, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells increased from 5,000 initial cells / well to 740 when not added in physiological saline. It decreased to 244 cells / well in the culture medium for HUVEC. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained up to 5.2 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

Test Example 7 (HAEC low temperature storage)
Measurement of the protective effect of the compound of the present invention against cold injury of normal human aortic endothelial cells (HAEC) (Lonza, No. CC-2535) was evaluated by the following method.

Suspend cells in EGM-2 medium (Lonza, No.CC-3162), inoculate into a 96-well microplate at 1 × 10 ⁴ cells / 0.1 ml / well, and incubate at 5% carbon dioxide at 37 ° C. Incubated for 2 hours. Thereafter, the supernatant was removed, and compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) whose concentration was adjusted with physiological saline was added to the cells at 0.1 ml / well. . This 96-well microplate was stored in a cooling container set at 4 ° C. for 7 days. Subsequently, the supernatant was removed and replaced with EGM-2 medium. After culturing in an incubator at 5% carbon dioxide and 37 ° C. for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 3 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 5170 × absorbance, R ² = 0.999).

As a result, as shown in Table 8, the cells were stored at 4 ° C. for 7 days (cold injury), so that the number of viable cells started from 10,000 initial cells / well when no addition was made in physiological saline. The number decreased to 1867 / well and 40 / well in the culture medium for HAEC. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained up to 2.3 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

Test Example 8 (Cold storage of mES cells)
The protective effect of the compound of the present invention against cold injury of mouse embryonic stem (mES) cells (RIKEN, No. AESO125) was evaluated by the following method.

1.5 × 10 ⁴ cells / 0.1 ml of mES cells suspended in medium for ES cells in 96-well microplate pretreated with 0.1% gelatin solution for ES cells (DS Pharma Biomedical, No.R-ES-006B) Seeded to form a well, and cultured in an incubator at 5% carbon dioxide and 37 ° C. for 24 hours. Thereafter, the supernatant was removed, and compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) whose concentration was adjusted with physiological saline was added to the cells at 0.1 ml / well. . The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days. Subsequently, the supernatant was removed and replaced with a medium for ES cells. After culturing in an incubator at 5% carbon dioxide and 37 ° C. for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 3 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from a standard line (viable cell number = 16275 × absorbance, R ² = 0.993) of cell number and absorbance prepared in advance.

As a result, as shown in Table 9, the cells were stored at 4 ° C. for 3 days (cold injury), so that the number of viable cells started from 15,000 cells / well in the case of no addition in physiological saline. The number decreased to 583 cells / well, and to 556 cells / well in the medium for ES cells. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained at a maximum of 7.8 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The composition of the ES cell medium used in this test example is as follows.
Composition of mES cell culture medium (100 ml) Stem Medium (DS Pharma Biomedical, No.DSRK100) 99 ml
Β-2 mercaptoethanol for ES cells, No, R-ES-007E (MEAA) (invtrogen, No. 11140) 1 ml
LIF (Leukemia Inhibitory Factor from mouse, SIGMA, No.L5158, 10μg / ml) 0.1 ml

Test Example 9 (Cryopreservation of rMS cells)
The protective effect of the compounds of the present invention against cold injury of rat mesenchymal stem (rMS) cells (RIKEN, No. AESO125) was evaluated by the following method.

The cells were suspended in a culture solution for rMS cells, seeded in a 96-well microplate at 5 × 10 ³ cells / 0.1 ml / well, and cultured for 5 hours in an incubator at 37 ° C. with 5% carbon dioxide. Thereafter, the supernatant was removed, and compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) adjusted in concentration with physiological saline was added to the cells at 0.1 ml / well. . The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days (72 hours). Subsequently, the supernatant was removed and replaced with a culture solution for rMS cells. After culturing in an incubator for about 24 hours, 10 μl / well of WST-8 solution was added and further cultured for 3 hours. Absorbance was measured at a wavelength of 450 nm, and the number of viable cells was calculated from the previously prepared standard line of cell number and absorbance (viable cell number = 4375 × absorbance + 310, R ² = 0.993).

As a result, as shown in Table 10, when the cells were stored at 4 ° C. for 3 days (cold injury), the number of viable cells started from 5,000 initial cells / well in the absence of addition in physiological saline. It decreased to 347 cells / well, and to 337 cells / well in the culture medium for rMS cells. On the other hand, by adding compound (2) to cells at a concentration of 0.01 to 10 μg / ml during low-temperature storage, the number of viable cells is maintained up to 8.6 times higher than when no compound is added in physiological saline, The low temperature injury protection effect of the compound of the present invention was confirmed.

The composition of the culture solution for rMS cells used in this test example is as follows.
Composition of rMS cell culture solution (100 ml) Eagle's Minimum Essential Medium wih Earle's salts (E-MEM) (Wako Pure Chemical Industries, No.051-07615) 85 ml
Fetal Bovine Serum (FBS) 15 ml

Test Example 10 (Cold preservation of mouse thymocytes)
The protective effect of the compound of the present invention against cold injury of mouse thymocytes was evaluated by the following method.

BALB / cAnNCrlCrlj (female, purchased from Nihon Charles River Co., Ltd.) was exsanguinated under anesthesia, then the thymus was removed, and the cells were suspended in physiological saline using a stainless steel mesh and rubber stick. It seed | inoculated to 96 wells microplate so that it might become * 10 ⁴ piece / 0.05 ml / well. Thereafter, compound (2) (final concentration 10 μg / ml, 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml) adjusted in concentration with physiological saline was added to the cells at 0.05 ml / well. The test group in which cells were treated with 0.2% Tween20-containing physiological saline was used as a positive control (cytotoxicity rate 100%). The 96-well microplate was stored in a cooling container set at 4 ° C. for 3 days. Next, after centrifuging the 96-well microplate for 3 minutes at 4 ° C and 1,000 rpm, 0.05 ml of the supernatant was added to the wells of another 96-well microplate, and the lactate dehydrogenase (LDH) activity in the supernatant was changed to LDH-Cytotoxic Measurement was performed using Test wako [Wako 299-50601]. That is, 0.05 ml of the coloring solution was added to 0.05 ml of the centrifugal supernatant to start the reaction, and after standing at room temperature for 45 minutes, 0.1 ml of a reaction stopping solution (0.5N hydrochloric acid solution) was mixed to stop the coloring reaction. In addition, the same LDH measurement operation was performed using physiological saline instead of the centrifugal supernatant as a negative control. Within 90 minutes after stopping the reaction, the absorbance at 560 nm was measured with a microplate reader. The cytotoxic rate (%) was calculated by the following calculation formula I.
Cytotoxicity (%) = (S−N) / (P−N) × 100 (Formula I)
In Formula I, S is the absorbance in the specimen, N is the absorbance in the negative control, and P is the absorbance in the positive control.

As a result, as shown in Table 11, when the cells were stored at 4 ° C. for 3 days (low temperature injury), the cytotoxic rate induced in the absence of addition in physiological saline was 95.9%. On the other hand, when the compound (2) is added to the cells at a final concentration of 0.01 to 10 μg / ml during low-temperature storage, the cytotoxic rate is reduced depending on the dose, and the protective effect of the compound of the present invention on the thymocytes is reduced. confirmed.

Test Example 11 (Cryogenic preservation of mouse skin tissue section)
The protective effect of the compound of the present invention against cold injury of mouse skin tissue sections was evaluated by the following method.

BALB / cAnNCrlCrlj (female, purchased from Nihon Charles River Co., Ltd.) was exsanguinated under anesthesia, and then the tail was removed and the entire skin tissue was peeled off. This was cut into 4 to 4.5 mm square skin tissue sections using a scalpel. Each section was immersed in 0.5 ml / well (24-well culture plate) of physiological saline or physiological saline containing compound (2) that had been cooled to 4 ° C. in advance. The test group in which the skin tissue section was immersed in 0.5 ml / well (24-well culture plate) of 0.2% Tween20-containing physiological saline was used as a positive control (tissue injury rate 100%). The 24-well culture plate was stored in a cooling container set at 4 ° C. for 7 days, and then a part of the supernatant was sampled from each well and diluted 10-fold with physiological saline. 0.05 ml of the diluted solution was added to the wells of another 96-well microplate, and LDH activity was measured by the same method as in Test Example 10. As a negative control, the same LDH measurement procedure was performed using 0.05 ml of physiological saline instead of the 10-fold diluted solution. The tissue injury rate (%) was calculated by the following calculation formula II.
Tissue injury rate (%) = (S−N) / (P−N) × 100 (Formula II)
In Formula II, S is the absorbance in the specimen, N is the absorbance in the negative control, and P is the absorbance in the positive control.

As a result, as shown in Table 12, when the mouse skin tissue section was stored at 4 ° C. for 7 days (cold injury), the tissue injury rate induced in the absence of addition in physiological saline was 32.4%. It was. On the other hand, when the compound (2) is added to a mouse skin tissue section at a final concentration of 0.1 to 10 μg / ml during low-temperature storage, the tissue injury rate is reduced depending on the dose. Low temperature injury protection effect was confirmed.

Test Example 12 (rabbit platelet cryopreservation)
The protective effect of the compound of the present invention against low temperature injury of rabbit platelets was evaluated by the following method.

Immediately after collecting blood from a rabbit, mix with Alsever solution to a volume of 1: 1, centrifuge at 100G for 10 minutes at 20 ° C, transfer the supernatant to another centrifuge tube, and keep the same until there is no red blood cell precipitation. Centrifugation was repeated under the conditions. The obtained platelet suspension was dispensed into tubes in 2 ml portions. To this was added 2 μl of the compound (2) prepared in DMSO (final concentration 10 μg / ml) and stored at 4 ° C. for 24 hours. 8 μl of platelet suspension was added to 1 ml of phosphate buffered saline containing 1% glutaraldehyde and fixed. Thereafter, the sample was dropped on the glass slide, applied, washed, dried, gold deposited, and observed with a scanning microscope (S-3200N). The obtained observation image is shown in FIG.

As a result, a remarkable morphological change was induced in the platelets without the addition of the compound by low-temperature storage at 4 ° C. for 24 hours. On the other hand, by adding the compound (2), the shape of platelets before normal storage at a low temperature was maintained, and the low temperature injury protection effect of the compound of the present invention on rabbit platelets was confirmed.

Test Example 13 (Combination effect of Compound (I) and Compound (II))
The protective effect against cold injury of HL-60 cells when compound (I) and compound (II) were used in combination was evaluated. Compound (I) and Compound (II) were dissolved in DMSO at 100 mg / ml prior to the test, and Compound (I) was further diluted with a 1: 0.01 mg / ml dilution (DMSO) at a 100-fold common ratio. Compound (II) was prepared in a diluted solution (DMSO) of 10, 1, 0.1 mg / ml at a 10-fold common ratio. Each compound was added alone or in a mixture so as to be diluted 500-fold in physiological saline to prepare a test compound preparation solution. Other test conditions were the same as in Test Example 1.

Fig. 2 (A) shows compound (2) (1 µg / ml) and apigenin-7-glucoside (A7G) (0.1-100 µg / ml), and (B) shows compound (2) (0.01 µg / ml) and quercetin- The results of the combined use of 3-glucoside (Q3G) (0.1 to 100 μg / ml) are shown.

As a result, as shown in FIGS. 2 (A), 2 (B) and 2 (C), the compound (II) alone has almost no low-temperature injury protection effect or a concentration range (0.1 to 100 μg at which sufficient effect cannot be obtained). / ml), it was confirmed that the combined use of compound (II) and compound (I) acted synergistically to obtain a low-temperature injury protection effect that exceeded expectations.

Test Example 14 (Effects on cell apoptosis induced by cryopreservation)
The effect of the compounds of the present invention on cell apoptosis induced by cryopreservation was evaluated by the following method. HL-60 cells cultured in 10% FBS-RPMI medium in a 5% carbon dioxide, 37 ° C incubator were collected, centrifuged at room temperature at 1,000 rpm for 5 minutes, and the supernatant was removed to 2 × 10 ⁶ cells. It was resuspended in physiological saline so as to be / ml. 0.25 ml of this cell suspension and 0.25 ml of the test compound were mixed in a 2 ml sterilized microtube. The test compound was dissolved in DMSO at 100 mg / ml before the test, and 10 and 1 mg / ml dilutions (DMSO) were prepared at a 10-fold common ratio, and then diluted 500-fold with physiological saline. Those prepared as 20 and 2 μg / ml physiological saline solutions were used. The mixture of the cell suspension and the test compound was allowed to stand for about 24 hours in a cooling container set at 4 ° C. Dispense 0.29 ml from this suspension into a separate tube, add 0.01 ml of 30 × FLICA reagent (ImmunoChemistry Technologies, LLC, # 91FAM FLICA ^TM Poly Caspases Assay Kit) to detect apoptosis, and add 37 ml For 1 hour. Ten times the concentration of 10 × Apoptosis Wash Buffer # 634 was diluted 10 times with physiological saline to obtain Wash Buffer (WB). 1 ml of this WB was added to the cell suspension, centrifuged (400 g, room temperature, 5 minutes), the supernatant was removed, and then a washing operation in which 1 ml of Wash Buffer was added again was performed twice. After removing the supernatant by the final centrifugation, 0.3 ml of Wash Buffer was added to form a suspension. To this, add Propidium Iiodide # 638 (PI, 0.25 mg / ml) included in the kit for detecting dead cells, add 1.5 μg / ml, let stand for 5 minutes, then flow cytometer (Becton Dickinson, BD Analysis was performed with FACSCalibur). The results are shown in FIG.

As a result, the cytotoxicity induced under storage conditions at 4 ° C. for 24 hours was 12.1% for necrosis and 68.8% for apoptosis, and it was confirmed that apoptosis was mainly composed of necrosis (FIG. 3). (B)). In addition, when 10 μg / ml of the compound (2) of the present invention was added under these conditions, it was confirmed that apoptosis was strongly suppressed as shown in FIG. Further, as shown in Table 13, it was confirmed that the compound of the present invention inhibits the cell damage induced by apoptosis depending on the dose.

Claims (17)

1. Formula (I):
   

   

   [Wherein, R ¹ to R ⁶ are the same or different and each represents a hydrogen atom, a hydroxyl group, —CO ₂ R ⁷ , an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, An optionally substituted alkylthio group, an optionally substituted alkylsulfinyl group or an optionally substituted alkylsulfonyl group, at least one of which is a hydroxyl group, and any two of R ¹ to R ⁶ are congruent; And R ⁷ is a hydrogen atom or an optionally substituted alkyl group.] A preservative for cryopreserving a biological material comprising a compound represented by:
2. R ¹ to R ⁵ are the same or different and are a hydrogen atom, a hydroxyl group or an alkoxy group, at least one is a hydroxyl group, and R ⁶ is a phenylalkenyl group which may be substituted with a hydroxyl group and / or an alkoxy group, or- (R ⁷ ) _n —COR ⁸ , R ⁷ is an alkylene group or an alkenylene group, R ⁸ is a hydroxyl group, an alkoxy group or an aralkyloxy group, and n is an integer of 0 to 2. The preservative described.
3. The compound represented by the above formula (I) is caffeic acid methyl ester, caffeic acid ethyl ester, caffeic acid phenethyl ester, ferulic acid ethyl ester, methyl 3- (3,4-dihydroxyphenyl) propanoate, methyl 2- (3 , 4-Dihydroxyphenyl) acetate, gallic acid methyl ester, gallic acid ethyl ester, gallic acid propyl ester, 3,4-dihydroxybenzoic acid methyl ester, 3,4-dihydroxybenzoic acid ethyl ester, 2,5-dihydroxybenzoic acid The methyl ester, 2,3-dihydroxybenzoic acid methyl ester, resveratrol, isolapontigenin, pterostilbene, pinostilbene, lapontigenin, oxyresveratrol and gnetole are at least one selected from the group consisting of Or the preservative according to 2.
4. Formula (II):
   

   

   [Wherein R ⁹ and R ¹⁰ are a hydrogen atom, a hydroxyl group or a glucose residue, at least one of which is a glucose residue, and R ¹¹ to R ¹⁷ are the same or different and represent a hydrogen atom, a hydroxyl group or an alkoxy group] The preservative according to any one of claims 1 to 3, further comprising a flavonoid glycoside compound represented by the formula:
5. The flavonoid glycoside compound represented by the formula (II) is at least one selected from the group consisting of quercetin-3-glucoside, kaempferol-7-glucoside, and apigenin-7-glucoside. Preservatives.
6. The preservative according to any one of claims 1 to 5, wherein the biological material is an animal or plant cell, tissue, organ, organ, or cultured cell sheet.
7. The biological material is an egg cell, a fertilized egg cell, a sperm cell, an embryonic stem cell, an iPS cell, an adult stem cell, a fibroblast, a feeder cell, a vascular endothelial cell, a bone marrow cell, an immune cell, a hepatocyte, a kidney cell, a nerve cell, Pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, odontocytes, periodontal ligament cells, oral mucosa The preservative according to any one of claims 1 to 6, which is a cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell, platelet or heart.
8. The preservative according to any one of claims 1 to 5, wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or flowers as food.
9. A storage solution for low-temperature storage of biological materials containing the preservative according to any one of claims 1 to 8.
10. Formula (I):
    

    

    [Wherein, R ¹ to R ⁶ are the same or different and each represents a hydrogen atom, a hydroxyl group, —CO ₂ R ⁷ , an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkoxy group, An optionally substituted alkylthio group, an optionally substituted alkylsulfinyl group or an optionally substituted alkylsulfonyl group, at least one of which is a hydroxyl group, and any two of R ¹ to R ⁶ are congruent; The biological material may be immersed in a solution containing a compound represented by the following formula: R ⁷ is a hydrogen atom or an optionally substituted alkyl group, and the solution is kept at a low temperature. A method for preserving biological materials.
11. R ¹ to R ⁵ are the same or different and are a hydrogen atom, a hydroxyl group or an alkoxy group, at least one is a hydroxyl group, and R ⁶ is a phenylalkenyl group which may be substituted with a hydroxyl group and / or an alkoxy group, or- (R ⁷ ) _n —COR ⁸ , R ⁷ is an alkylene group or an alkenylene group, R ⁸ is a hydroxyl group, an alkoxy group, or an aralkyloxy group, and n is an integer of 0 to 2. The method described.
12. The compound represented by the above formula (I) is caffeic acid methyl ester, caffeic acid ethyl ester, caffeic acid phenethyl ester, ferulic acid ethyl ester, methyl 3- (3,4-dihydroxyphenyl) propanoate, methyl 2- (3 , 4-Dihydroxyphenyl) acetate, gallic acid methyl ester, gallic acid ethyl ester, gallic acid propyl ester, 3,4-dihydroxybenzoic acid methyl ester, 3,4-dihydroxybenzoic acid ethyl ester, 2,5-dihydroxybenzoic acid The methyl ester, 2,3-dihydroxybenzoic acid methyl ester, resveratrol, isolapontigenin, pterostilbene, pinostilbene, rapontigenin, oxyresveratrol, and gnetitol are at least one selected from the group consisting of: Or the method of 11.
13. The method according to any one of claims 10 to 12, wherein the solution further comprises the flavonoid glycoside compound according to claim 4.
14. The method according to claim 13, wherein the flavonoid glycoside compound is at least one selected from the group consisting of quercetin-3-glucoside, kaempferol-7-glucoside, and apigenin-7-glucoside.
15. The method according to any one of claims 10 to 14, wherein the biological material is an animal or plant cell, tissue, organ, organ, or cultured cell sheet.
16. The biological material is an egg cell, a fertilized egg cell, a sperm cell, an embryonic stem cell, an iPS cell, an adult stem cell, a fibroblast, a feeder cell, a vascular endothelial cell, a bone marrow cell, an immune cell, a hepatocyte, a kidney cell, a nerve cell, Pancreatic cells, smooth muscle cells, cardiomyocytes, myoblasts, corneal cells, retinal cells, chondrocytes, cartilage progenitor cells, synovial cells, synovial stem cells, osteoblasts, odontocytes, periodontal ligament cells, oral mucosa The method according to any one of claims 10 to 15, which is a cell, mesenchymal stem cell, adipocyte, adipose stem cell, ovary, semen, blood, blood cell, platelet or heart.
17. The method according to any one of claims 10 to 14, wherein the biological material is beef, pork, chicken, fish, shellfish, cereals, vegetables or fruits, or florets as food.

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