JP4868199B2 - Neural stem cell proliferating agent containing salvianolic acid B as an active ingredient - Google Patents

Description

  In the present invention, (1) Salvianolic acid B (English name: Salvanilic Acid B), Chinese name: Tanfunic acid B or Tanfunic acid oto, and the Chinese character corresponding to hun are written as ubiquitous. A neural stem cell proliferating agent characterized in that it is an active ingredient), (2) the neural stem cell proliferating agent is administered outside the body after activation if necessary. Neural stem cell proliferation method in vitro, (3) Neural stem cell-containing solution characterized by proliferation by the neural stem cell proliferation method, (4) Serum, basic fibroblast growth factor (bFGF, also known as fibroblast) Functions of tissues and organs due to degeneration, decrease, cell death, injury, exclusion of nerve cells, characterized by containing as an active ingredient a solution containing neural stem cells that does not contain growth factor 2: FGF2) and nerve growth factor (NGF) To decline or loss (5) A therapeutic agent for the disease characterized by comprising the neural stem cell proliferating agent as an active ingredient, and a neural stem cell proliferating action of the active ingredient, (6) Salvianolic acid B and sulfite A neural stem cell proliferating agent according to (1), wherein at least one antioxidant selected from pharmaceutically acceptable antioxidants other than salt and vitamin C is added as an additive; It relates to the method for proliferating neural stem cells described in 2), the neural stem cell-containing solution described in (3), and the therapeutic agent described in (4) and (5).

  Neurological diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, cerebellar degeneration, traffic hydrocephalus, Huntington's disease, frontal lobe irradiation, multiple sclerosis, normal pressure hydrocephalus, Parkinson's disease, Pick's disease, progression Multiple focal leukoencephalopathy, progressive supranuclear palsy, fighting dementia, brain trauma, surgery, brain tumor, chronic subdural hematoma, stroke (cerebral infarction or cerebral hemorrhage), cerebrovascular dementia, Wilson disease bacterial intracardiac Meningitis, Creutzfeldt-Jakob disease, Gerstmann-Stroisler-Scheinker disease, HIV-related diseases, neurosyphilis, tuberculosis and fungal meningitis, viral encephalitis, anoxia, B12 deficiency, chronic drugs Alcohol-nutrient abuse, folate deficiency, hypercalcemia associated with hyperparathyroidism, hypoglycemia, hypothyroidism, hepatic encephalopathy, pulmonary encephalopathy, uremic encephalopathy, organ system failure, pellagra It is thought that tissues and organs lose their function due to degeneration, decrease, cell death, injury or exclusion due to fibrosis, immune reaction, vascular injury, nutrition and oxygen deficiency, infection, etc. It has been. Therefore, it is conceivable to replace or regenerate lost neurons as a method for treating these diseases, and many effects have been reported.

  For example, Parkinson's disease is caused by degeneration of nigrostriatal dopaminergic nerves, but the condition was improved by intracerebral transplantation of human fetal midbrain tissue rich in late mitotic dopaminergic nerves. There are reports (Non-Patent Documents 1-7). However, during this period, immunosuppressive agents are used to avoid rejection associated with transplantation (Non-patent Document 8).

  Cerebral infarction causes serious ischemia in the central nervous system, degenerating nerves and glial cells. There has been a report that improvement was observed when nerves derived from human NT-2 (NT-2) teratocarcinoma cells were transplanted to the cerebral infarction site (Non-patent Document 9). There is also a report that it has been confirmed by the appearance of a neuronal marker in the transplanted cells that the transplanted cells have become nerve cells (Non-patent Document 10).

  Attention is focused on ES cells, neural stem cells or bone marrow stromal cells as a source of these neurons to be supplemented. This is because ES cells are cells that can differentiate into various cells. This is because neural stem cells are undifferentiated cells, and when nerve cells die, they naturally start to compensate for the lost cells and may greatly contribute to the maintenance of biological functions. Since bone marrow stromal cells have already differentiated to some extent, they do not naturally differentiate into neurons, but recently can be artificially differentiated into neurons by treatment with various biological factors or compounds (Patent Documents 1-2, non-patent documents). This is because it has become clear that Patent Documents 11-15).

  However, ES cells that are attracting attention as a source of nerve cells to be supplemented are embryonic stem cells, and have the ethical problem of using a human embryo as a raw material and the problem of transplantation antigenicity. On the other hand, even in the case of neural stem cells, endogenous neural stem cells differentiate into nerves (Non-Patent Document 16), but the number of endogenous neural stem cells is extremely small, and sufficiently replenish cells degenerated due to cerebral infarction or the like. (Non-Patent Document 17). Obtaining from outside is difficult only for fetal-derived neural stem cells, and fetal-derived neural stem cells have ethical problems and transplant antigenicity problems. Since a sufficient amount of bone marrow stromal cells can be collected from their own bone marrow, there is no problem of ethics and transplantation antigenicity using other cells. However, bone marrow stromal cells are cells that do not naturally differentiate into neurons. Therefore, it would be more desirable if autologous neural stem cells can be used as a source, such as by developing a method for growing endogenous neural stem cells. Therefore, development of a method for growing endogenous neural stem cells is strongly desired.

  Fetal and adult neural stem cells are not only from the central nervous system of rodents (fetal: non-patent document 18, adult: non-patent document 19-20) but also from the human central nervous system (fetal: Non-patent literature 21-22, adulthood: non-patent literature 23-24). Neural stem cells differentiate into nerves, astrocytes, and oligodentrosites, differentiate into excitatory and inhibitory neural synapses (Non-patent Documents 25-26), and form an active neural network (Non-patent Document 27). ).

  In recent years, attempts have been made to improve and treat the disease state by transplanting neural stem cells to tissues that have lost their function and specifically differentiating them into cells that express individual biological functions. For example, with respect to Parkinson's disease, when neural stem cells are transplanted into the striatum, the cells survive and have clinical effects (Non-patent Document 28), and neural stem cell transplantation preserves the synaptic and terminal functions of the Parkinson's disease operative system. (Non-patent document 29) Transplanting neural stem cells into the brain of a mouse with Parkinson's disease caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine survives and becomes a functional dopaminergic nerve There is a report (Non-Patent Document 30). In the case of cerebral infarction, it is said that improvement of cerebral infarction is seen by transplantation of neural stem cells (Non-patent Document 31). Many neural stem cell transplantation data (Non-Patent Document 31 and Non-Patent Documents 32-35 mentioned above) show that tissues and organs caused by neuronal degeneration, decrease, cell death, injury, or exclusion due to transplantation or proliferation of neural stem cells. This suggests that diseases caused by reduced or lost function can be treated. The biggest challenge is to establish a method for producing transplantable quality and quantity of neural stem cells, or to develop a low-toxic neural stem cell proliferating agent capable of growing resident neurons in the brain.

  Recently, there was a report that 40% of the lost neurons were recovered by injecting NGF after the brain arteries of rats were temporarily stopped to cause cerebral ischemia to injure hippocampal neurons. Patent Document 36). This suggests that NGF proliferates neural stem cells. Although excellent as a research, it is better to avoid NGF protein clinically injected into the brain as much as possible. Therefore, it is desired to search for low molecular weight compounds that can substitute for biological factors such as NGF.

  Small molecules that differentiate non-neuronal cells and neural stem cells into nerve cells have already been reported (Patent Document 2 and Non-Patent Document 37 described above). For the purpose of using non-neuronal cells such as bone marrow stromal cells as a source of supplementation of nerve cells for neurological diseases, a differentiation agent for nerve cells is important. However, since cells that have differentiated into nerve cells do not proliferate, for the purpose of using neural stem cells as a source of supplementation of nerve cells for neurological diseases, neural stem cells are differentiated into nerves rather than neural stem cell differentiation agents. More important is a neural stem cell proliferating agent that allows the cells to proliferate as neural stem cells. Neural stem cell differentiating agents are a matter to be considered if necessary after neural stem cells are expanded.

  On the other hand, salvianolic acid B is one of the water-soluble components of Dansan (scientific name: Labiatae / Salvia / miltiorrhiza / Bunge) (Non-patent Document 38). (HPTLC) (Non-patent document 39), Alcohol development of resin column (Patent document 3), High-pressure liquid chromatography (HPLC) (Non-patent document 40) or high-speed counter current chromatography (Non-patent document 41), column They are separated by chromatography (Patent Document 4) and the like. With a molecular weight of 718 and a molecular formula of C36H30O16, its planar structural formula is shown in Non-Patent Document 42, and its three-dimensional structural formula is shown in FIG. 1 (Non-Patent Document 43). According to Katsuratake et al., A researcher of natural drug processing technology in Shandong Province, salvianolic acid B in aqueous solution is unstable at high temperature and changes 35% when heated at 80 degrees Celsius for 72 hours, sodium thiosulfate, sulfuric acid It is said that even if sodium, sodium sulfite, sodium hydrosulfite, cysteine hydrochloride, or EDTA disodium salt is added, it partially changes. Only when vitamin C is added, salvianolic acid B is stable even when heated at 80 degrees Celsius for 72 hours (Non-patent Document 44).

  Salvianolic acid B has a very strong antioxidant action (Non-patent Document 45), fat peroxidation-inhibiting action, and dehydrated oxidation radical action (Non-Patent Document 46). Based on this anti-peroxidation effect, it has the following physiological effects. That is, the action of carbon tetrachloride or dimethylnitsamine to prevent fibrosis of the liver (Non-Patent Documents 47 to 48), the action of preventing memory loss due to cerebral ischemia (Non-Patent Document 49), and the effect of reducing atherosclerosis (Non-patent document 50), Anti-ischemic action (Non-patent document 51), Protective action against cardiovascular system (Non-patent document 52), Antiviral action (Patent documents 5-7), Preventive action against brain damage caused by ischemia (Non-patent Document 53), High Permeability Inhibitory Action Induced by Vascular Endothelial Growth Factor (VEGF) of Calf Aortic Endothelial Cells (Non-patent Document 54), Aggregation Inhibitory Action and Fibrosis Formation of Amyloid Beta Protein Inhibitory action (previously Non-patent document 42), apoptosis-inducing action of neoplastic cells (Non-patent document 55), hepatic stellate cell growth inhibitory action, collagen production inhibitory action and transforming growth factor ( TGF) beta-1, mitogenic activation protein kinase (MAPR) system signal transduction inhibitory action (Non-patent Document 56), prostate apoptosis reaction 4 (Par-4) involved in neurodegeneration amyloid beta Aggregation inhibitory action of protein (Non-patent Document 57), Cholestan-3beta-5alpha-6betatriol alleviates human naval vein endothelial cell (HUVEC) injury (Non-patent document 58), Heart by oxidized low density lipoprotein It has a therapeutic action on vascular system inflammatory disorders (Patent Documents 8-9). In addition, a mixture of salvianolic acid B and saponin reduces cardiovascular disease and cerebrovascular disease site (Patent Document 10), and a mixture of salvianolic acid B and lysospermic acid (English name: Lithospermic · acid) is atherosclerosis. It is effective for arthritis or arterial restenosis (Patent Documents 11-12). A constituent of salvianolic acid B has a lipase inhibitory action (Patent Document 13).

In summary, salvianolic acid B has a very strong anti-peroxidation effect, and by this anti-peroxidation effect, it has cell death prevention and antiviral effects due to peroxidation in the cardiovascular system, cranial nervous system and liver cells. . Salvianolic acid B inhibits proliferation of vascular endothelial cells such as atherosclerosis by inducing apoptosis of new cells. That is, the basic action of salvianolic acid B is an anti-peroxidation action, an antiviral action, and an apoptosis-inducing action of new cells. Therefore, there is a possibility of preventing cranial nerve cells due to ischemia caused by cerebral infarction, preventing viral encephalitis, and treating very early cranial nerve diseases that do not require supplementation or proliferation of cranial nerve cells. However, since the effect of salvianolic acid B is due to anti-peroxidation and antiviral effects, even though the disease can be treated, neural stem cells have proliferated and brain neurons have already degenerated or decreased. It seemed impossible to treat cranial nerve disease. Salvianolic acid B induces apoptosis of neoplastic cells and inhibits cell proliferation, so it seemed to be rather disadvantageous for neural stem cell proliferation. Actually, since acetyl salvianolic acid A, a salvianolic acid B related compound, has an antithrombotic action, it is presumed that the action will have a beneficial effect on prevention of cerebral infarction (Non-patent Document 59). Prevents liver cell damage due to peroxidation by its antioxidant action (Non-patent Document 60), inhibits fibroblast proliferation (Non-patent Document 61), and increases the survival rate of neural stem cells. It has been confirmed that it does not promote the growth of non-patent documents (Non-patent Document 62).
Recently, many patents have been filed and published regarding the isolation, proliferation and differentiation of neural stem cells (Patent Documents 14-72). Both are methods using biological factors. As a low-molecular-weight neural stem cell proliferating agent, a benzene ring condensed 5-membered heterocyclic compound (Patent Document 73), or a ginseng saponin, such as ginsenoside Rg1 (English name: Ginsenoside Rg1) (Patent Document 74 and the aforementioned) No. 62) is only reported. In addition, there are many patents related to the medicinal effect of red ginseng from which salvianolic acid B is extracted. There is a patent publication regarding dementia drugs, but in addition to Tansan, it is a patent application regarding various herbal medicine combinations (Patent Document 75). The present invention cannot be foreseen from these patent documents.

In the following, prior documents are displayed separately for patent documents and non-patent documents.
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  The object of the present invention is (1) salvianolic acid B and its optical or geometric isomers having the same neural stem cell proliferating action as salvianolic acid B and their prodrugs, as well as the above compounds or their prodrugs A neural stem cell proliferating agent characterized by comprising a compound selected from pharmaceutically acceptable salts thereof and hydrates thereof as an active ingredient, and (2) activation of the neural stem cell proliferating agent, if necessary. A method of proliferating neural stem cells outside the body, characterized by administration in vitro, (3) a neural stem cell-containing solution characterized by proliferation using the neural stem cell proliferation method, (4) serum, bFGF and NGF Reduced function of tissues and organs due to degeneration, decrease, cell death, injury, exclusion of neurons Or a therapeutic agent for a disease that develops due to loss, (5) a therapeutic agent for the disease characterized by comprising the neural stem cell proliferating agent as an active ingredient, and a neural stem cell proliferating action of the active ingredient, (6) salvianolic acid B A neural stem cell proliferating agent according to (1), wherein at least one antioxidant selected from pharmaceutically acceptable antioxidants other than sulfite and vitamin C is added as an additive And the neural stem cell proliferation method described in (2), the neural stem cell-containing solution described in (3), and the therapeutic agent described in (4) and (5).

  In view of such a situation, the present inventors have been surprised as a result of various searches for low molecular weight substances that have the ability to proliferate neural stem cells without treatment with serum, bFGF, and NGF and have only a pharmaceutically acceptable toxicity. In particular, unlike salvianolic acid A, it has been found that salvianolic acid B has an action of proliferating neural stem cells that is completely different from the known antiperoxidation action, antivirus, and neoplastic cell apoptosis induction action. As a result of research, the present invention has been completed.

  That is, the present invention relates to (1) salvianolic acid B and optical or geometric isomers thereof having the same neural stem cell proliferating action as salvianolic acid B and their prodrugs, as well as the above compounds or their prodrugs A neural stem cell proliferating agent characterized by comprising as an active ingredient a compound selected from pharmaceutically acceptable salts thereof and hydrates thereof; (2) said compound comprising salvianolic acid B and its pro (1) the neural stem cell proliferating agent described in (1), wherein the compound is selected from salivanolic acid B, which is selected from drugs, pharmaceutically acceptable salts thereof, and hydrates thereof And a pharmaceutically acceptable salt thereof and a hydrate thereof, wherein the god described in (2) A stem cell proliferating agent, (4) an in vitro neural stem cell proliferating method characterized in that the neural stem cell proliferating agent is administered in vitro after activation if necessary, (5) does not contain serum, bFGF and NGF Use of cell culture medium, neural stem cell proliferation method according to (4), (6) inclusion of neural stem cells characterized by proliferation by the neural stem cell proliferation method according to (4) or (5) (7) Due to neuronal degeneration, reduction, cell death, injury, exclusion, characterized by containing as an active ingredient a liquid containing the neural stem cell, characterized in that it grows by the method described in (7) (5) (8) a therapeutic agent for a disease caused by a decrease in or loss of function of a tissue or organ, (8) a therapeutic agent for the disease characterized by comprising the neural stem cell proliferating agent as an active ingredient, and a neural stem cell proliferating action of the active ingredient; 9) Monkey (1), (2) characterized in that at least one antioxidant selected from pharmaceutically acceptable antioxidants other than anolic acid B, sulfite and vitamin C is added as an additive. Or the neural stem cell proliferating agent described in (3), the neural stem cell proliferating method described in (4) or (5), the neural stem cell-containing liquid described in (6), and the neural stem cell-containing liquid-containing therapeutic agent described in (7) Or the therapeutic agent by the neural stem cell proliferation action mechanism as described in (8) is provided.

  The neural stem cell proliferating agent of the present invention is useful as (1) an agent for proliferating neural stem cells outside the body, (2) useful as a drug characterizing the method in a neural stem cell proliferating method described later, and (3) described later. Useful as a drug characterizing the quality of the neural stem cell-containing fluid, (4) Useful as a drug characterizing the properties of the neural stem cell-containing liquid-containing therapeutic agent described below, and (5) According to the neural stem cell proliferation action mechanism described below It is also useful as an active ingredient of a therapeutic agent. The neural stem cell proliferation method of the present invention is useful as (1) a method for proliferating neural stem cells outside the body, (2) useful as a method for producing a neural stem cell-containing solution described later, and (3) containing neural stem cells described later. It is useful as a method for producing a liquid-containing therapeutic agent or as one step thereof. The neural stem cell-containing liquid of the present invention is useful as (1) a source of neural stem cells, and (2) useful as an active ingredient of a therapeutic agent containing a neural stem cell-containing liquid described later. The nerve stem cell-containing liquid-containing therapeutic agent of the present invention and a neural stem cell proliferating agent, and two agents based on the mechanism of neural stem cell proliferation action are the degeneration, decrease, cell death, injury, exclusion of tissues and organs It is useful as a therapeutic agent for diseases caused by reduced function or loss. The present invention is characterized in that at least one antioxidant selected from pharmaceutically acceptable antioxidants other than salvianolic acid B, sulfite and vitamin C of the present invention is added as an additive (1) The neural stem cell proliferating agent described in (2) or (3), the neural stem cell proliferating method described in (4) or (5), the neural stem cell-containing solution described in (6), and the neural stem cell-containing agent described in (7) The liquid-containing therapeutic agent or the therapeutic agent based on the neural stem cell proliferation action mechanism described in (8) is useful because it has a property of being more stable in addition to the above-mentioned utility.

  One of the present invention is a new neural stem cell proliferating agent. The active ingredient of the neural stem cell proliferating agent of the present invention includes salvianolic acid B and optical or geometric isomers thereof having the same neural stem cell proliferating action as salvianolic acid B and their prodrugs, and the above compounds or their pros. It is a compound selected from pharmaceutically acceptable salts of the drugs as well as hydrates thereof. Among the optical or geometric isomers of salvianolic acid B, those that do not have the same neural stem cell proliferating action as salvianolic acid B cannot be used as the active ingredient of the neural stem cell proliferating agent of the present invention. On the other hand, the optical or geometric isomer of salvianolic acid B having the same neural stem cell proliferation action as salvianolic acid B can be used as an active ingredient of the neural stem cell proliferating agent of the present invention. However, among salvianolic acid B and its optical or geometrical isomers that have the same neural stem cell proliferating activity as salvianolic acid B, the data on salvianolic acid B is the most accumulated, so salvianolic acid B and More desirable are compounds selected from the prodrugs, and pharmaceutically acceptable salts and hydrates thereof, salvianolic acid B and pharmaceutically acceptable salts thereof and the hydration thereof. Even more desirable are compounds selected from those.

  Salvianolic acid B, one of the most desirable active ingredients, is currently isolated and purified from Dansang (Labiatae / Salvia / miltiorrhiza / Bunge), one of the Labiatae plants. However, if there is a plant containing salvianolic acid B other than Tansan, it may be separated from the plant, and if it can be synthesized, it may be a synthetic product. Currently, salvianolic acid B is commercially available in China (Jingtian Biological Process Co., Ltd.) and the United States (Ivy Fine Chemicals Corporation). Many optical or geometric isomers are assumed from the structural formula of salvianolic acid B. Salvianolic acids A, C, D, E, F, and isosarvianolic acid B have already been separated. As described above, among these, those that do not have the same action of proliferating neural stem cells as salvianolic acid B cannot be used as the active ingredient of the neural stem cell proliferating agent of the present invention, but salvianolic acid B and Similarly, those having an action of proliferating neural stem cells can be used as an active ingredient of the neural stem cell proliferating agent of the present invention.

  A prodrug of salvianolic acid B and its optical or geometric isomer that has the same neural stem cell proliferative activity as salvianolic acid B, but undergoes hydrolysis etc. to become salvianolic acid B or its optical or geometric isomer As long as it is pharmaceutically acceptable, it may be anything. Examples of such prodrugs include, but are not limited to, methyl or ethyl ester derivatives at the carboxyl group in the salvianolic acid B molecule and acetyl derivatives at the phenol group in the salvianolic acid B molecule. It may be synthesized in the future.

  Salts of salvianolic acid B and optical or geometric isomers thereof that have the same neural stem cell proliferative activity as salvianolic acid B, and salts of their prodrugs are limited to pharmaceutically acceptable salts . Examples of the pharmaceutically acceptable salt include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, ammonium salts such as ammonium and tetramethylammonium, triethylamine, lysine, arginine and the like. Examples of the organic amine salts include, but are not limited to, acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, and sulfuric acid. Any salt may be used as long as it is pharmaceutically acceptable, and a salt with an organic compound synthesized in the future may be used as long as it is pharmaceutically acceptable.

  Salvianolic acid B and its optical or geometric isomers having the same neural stem cell proliferating action as salvianolic acid B and their prodrugs, as well as pharmaceutically acceptable compounds of the above compounds or their prodrugs Hydrate of these salts can also be used as an active ingredient of the neural stem cell proliferating agent of the present invention.

  The neural stem cell proliferating agent of the present invention or the neural stem cell proliferating agent described later is an active ingredient, and the tissue by degeneration, reduction, cell death, injury, exclusion of neural cells characterized by the neural stem cell proliferating action of the active ingredient The active ingredient of a disease therapeutic agent that develops due to a decrease or loss of organ function is relatively stable in the absence of oxygen and moisture. However, since it is an antioxidant, it is relatively unstable in air and in aqueous solution. In particular, the active ingredient which is not converted into a prodrug changes 35% by heating at 80 ° C. for 72 hours in an aqueous solution. When storing, it is desirable to store without oxygen or moisture. In consideration of long-term storage, it is desirable to add another antioxidant as an additive to the neural stem cell proliferating agent or the disease therapeutic agent of the present invention in order to stabilize the active ingredient. Antioxidants are limited to pharmaceutically acceptable antioxidants. Pharmaceutically acceptable antioxidants include alpha carotene, beta carotene, lycopene, lutein, flavonoids, catechins, resaveratrol, isoflavones, vitamin A, vitamin E, selenium, zinc, coenzyme Q10, glutathione And enzogenol. These antioxidants suppress changes in salvianolic acid B in hot aqueous solutions. Nitrite does not completely suppress the change of salvianolic acid B. Therefore, nitrite alone is insufficient as a stabilizer. Vitamin C suppresses changes in salvianolic acid B. Therefore, vitamin C can be used as a stabilizer such as salvianolic acid B. However, vitamin C itself is relatively unstable in the air and in an aqueous solution, and vitamin C is added in a large amount or other antioxidants. Must be used together.

  The neural stem cell proliferating agent of the present invention is useful as (1) an agent for proliferating neural stem cells outside the body, (2) useful as a drug characterizing the method in a neural stem cell proliferating method described later, and (3) described later. It is useful as a drug characterizing the quality of the neural stem cell-containing fluid, and (4) not only useful as a drug characterizing the properties of the neural stem cell-containing liquid-containing therapeutic agent described later, but also (5) endogenous neural stem cells in the body. Can be used as it is in the body. When the neural stem cell proliferating agent of the present invention is administered to a disease that develops due to a decrease or loss of function of a tissue or organ due to degeneration, decrease, cell death, injury, or exclusion of nerve cells, it proliferates the inner neural stem cells, By differentiation into nerve cells, the missing nerve cells are replenished and the disease state is improved, so that it is also useful as an active ingredient of a therapeutic agent according to the neural stem cell proliferation action mechanism of the present invention.

  Another aspect of the present invention is a new neural stem cell proliferation method. Using the aforementioned neural stem cell proliferating agent of the present invention, neural stem cells are proliferated outside the body. Except for the addition of the neural stem cell proliferating agent of the present invention, for example, 2% of culture supplement N2-containing Dulbecco's modified Eagle's minimum essential medium (DMEM) / ham F12 medium (DMEM and F12 mixed 1: 1) Known culture conditions such as culture for several days at 37 degrees Celsius in a known culture solution such as a serum-free culture solution and 5% carbon dioxide can be used. In the above-mentioned prior literature, protein biological factors such as fetal bovine serum (FBS), bFGF, NGF and other nerve cells are used as neural stem cell proliferating agents. It is characterized in that the neural stem cell proliferating agent of the present invention is used in place of these serum or biological factors. Of course, depending on the purpose, these serums and biological factors may be contained. However, when preparing a neural stem cell-containing solution used for the therapeutic agent of the present invention, these are added to the culture solution for growing neural stem cells. must not. It is important to add the neural stem cell proliferating agent of the present invention and culture without adding serum or biological factors. In the case of using a prodrug as the neural stem cell proliferating agent of the present invention, depending on the prodrug, the mask is removed by the cells in culture and becomes an active substance, so there is a case where activation is not necessary, Some prodrugs may not be activated by cultured cells. Therefore, if necessary, it needs to be activated before administration. Finally, as an example of the basic culture solution, the composition of culture supplement N2, DMEM, Ham F12 medium, etc. is shown. The composition may be changed as long as neural stem cells proliferate. In addition, as an example of a buffer used for cell separation and cell washing, the composition of D-Hanks solution, phosphate buffered saline (PBS), Delbecko PBS (DPBS) and the like is shown. Other buffer solutions can be used as long as they do not adversely affect the subsequent operation.

Example of composition of culture supplement, medium, and buffer Composition of culture supplement N2 (milligram / liter): insulin: 5, human transferrin: 100, progesterone: 0.0063, putrescine 16.11, selenite: 0 .0052.
Composition of DMEM (milligram / liter): calcium chloride: 200, iron nitrate (trivalent) nonahydrate: 0.1, potassium chloride: 400, magnesium sulfate: 97.6, sodium chloride: 6,400, phosphoric acid Sodium dihydrogen dihydrate: 125, L-arginine hydrochloride: 84, L-cystine dihydrochloride: 62.6, L-glutamine: 584, L-glycine: 30, L-histidine hydrochloride monohydrate : 42, L-isoleucine: 104.8, L-leucine: 104.8, L-lysine hydrochloride: 146.2, L-methionine: 30, L-phenylalanine: 66, L-serine: 42, L-threonine: 95.2, L-tryptophan: 16, L-tyrosine disodium salt: 89.5, L-valine: 93.6, calcium pantothenate: 4, choline bitartrate: 2, Folic acid: 4, Inositol: 7.2, Nicotinamide: 4, Pyridoxal hydrochloride: 4, Riboflavin: 0.4, Thiamine hydrochloride: 4, Dextrose: 1,000, Sodium pyruvate: 110, Phenol -Red: 5.
Components of culture supplement B27 (the composition of B27 is not disclosed, so when using B27 itself, a commercial product must be purchased, but it is more similar to the disclosed component (Non-patent Document 63). A culture supplement having the following effects can be made): biotin, L-carnitine, corticosterone, ethanolamine, D (+) galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, Retinyl acetic acid, selenium, triodo-1-tyromine, vitamin E, vitamin E acetate, bovine albumin, catalase, insulin, superoxide dismutase, transferrin.
Ham F12 medium composition: calcium chloride: 33.3, copper sulfate (divalent) pentahydrate: 0.0025, iron sulfate (divalent) heptahydrate: 0.834, potassium chloride: 224, magnesium chloride : 57.1, sodium chloride: 7,600, disodium hydrogen phosphate: 142, zinc sulfate heptahydrate, L-alanine: 8.9, L-arginine hydrochloride: 211, L-asparagine monohydrate 15.0, L-aspartic acid: 13.3, L-cysteine hydrochloride monohydrate: 35.1, L-glutamic acid: 14.7, L-glutamine: 146, L-glycy: 7.5, L-histidine hydrochloride monohydrate: 21.0, L-isoleucine: 3.94, L-leucine: 13.1, L-lysine hydrochloride: 36.5, L-methionine: 4.48, L- Phenylalanine: 4.96, L-pro 34.5, L-serine: 10.5, L-threonine: 11.9, L-tryptopha: 2.04, L-tyrosine: 5.44, L-valine: 11.7, biotin: 0. 0073, calcium pantothenate: 0.477, choline chloride: 14.0, cyanocobalamin: 1.355, folic acid: 1.324, inositol: 18.0, lipoic acid: 0.206, nicotinamide: 0.0366, pyridoxine Hydrochloride: 0.0617, Riboflavin: 0.0376, Thiamine hydrochloride: 0.337, Dextrose: 1,802, Hypoxanthine: 4.08, Linolenic acid: 0.0841, Sodium pyruvate: 110, Thymidine: 0 727, putrescine dihydrochloride: 0.61, phenol red: 1.242.
Composition of D-Hank's solution (milligram / liter): calcium chloride: 185.5, potassium chloride: 400, potassium dihydrogen phosphate: 60, magnesium sulfate: 97.7, sodium chloride: 8,000, dihydrogen phosphate Sodium: 47.5, D-glucose: 1,000.
Composition of PBS (milligram / liter): calcium chloride: 132.5, potassium chloride: 200, potassium dihydrogen phosphate: 200, magnesium sulfate: 59.2, sodium chloride: 8,000, disodium hydrogen phosphate: 1150 .
Composition of DPBS (milligram / liter): calcium chloride: 100, potassium chloride: 200, potassium dihydrogen phosphate: 200, magnesium chloride: 100, sodium chloride: 8,000, disodium hydrogen phosphate: 1150.
Composition of RPMI 1640 (milligram / liter): calcium nitrate: 69.5, potassium chloride: 400, magnesium sulfate: 48.8, sodium chloride: 6,000, disodium hydrogen phosphate: 801, L-arginine: 200, L -Asparagine monohydrate: 56.8, L-aspartic acid: 20, L-cysteine dihydrochloride: 65.2, L-glutamic acid: 20, L-glutamine: 300, L-glycine: 10, L-histidine : 15, L-hydroxyproline: 20, L-isoleucine: 50, L-leucine: 50, L-lysine hydrochloride: 40, L-methionine: 15, L-phenylalanine: 15, L-proline: 20, L- Serine: 30, L-threonine: 20, L-tryptopha: 5, L-tyrosine: 20, L-valine: 20, p-a Nobenzoic acid: 1, Parabiotin: 0.2, Calcium pantothenate: 0.25, Choline chloride: 3, Cyanocobalamin: 0.005, Folic acid: 1, Inositol: 35, Nicotinamide: 1, Pyridoxine hydrochloride: 1 Riboflavin: 0.2, thiamine hydrochloride: 1, dextrose: 2,000, reduced glutathione: 1, phenol red: 5.
Composition of 20 times SSC (mol / liter): sodium chloride: 3, sodium citrate: 0.3.
The neural stem cell proliferation method of the present invention is useful as (1) a method for proliferating neural stem cells outside the body, (2) useful as a method for producing a neural stem cell-containing solution described later, and (3) containing neural stem cells described later. It is useful as a method for producing a liquid-containing therapeutic agent or as one step thereof.

Yet another aspect of the present invention is a solution containing neural stem cells grown outside the body. The neural stem cell proliferation method of the present invention is used for the proliferation of neural stem cells. The neural stem cell-containing liquid proliferated outside the body differs depending on what kind of culture solution is used as the neural cell culture medium or what kind of neural cell proliferation agent is used as the neural stem cell proliferation agent. For example, when a serum-containing culture solution is used as a nerve cell culture solution, or when bFGF or NGF is added and cultured as a nerve cell proliferating agent, serum, bFGF, or NGF is mixed into the proliferated neural stem cell-containing solution. The neural stem cell-containing liquid of the present invention is mixed with the neural stem cell-containing liquid of the present invention. Since there is a problem of transplantation immunity, the fluid containing neural stem cells mixed with serum, bFGF, NGF, etc. is caused by the deterioration or loss of the function of tissues or organs due to the degeneration, decrease, cell death, injury, or exclusion of neurons described below. It cannot be used as a therapeutic agent for diseases that develop. Serum is not present in known serum-free neural cell cultures, but bFGF and NGF are generally added. Therefore, it is important not to add these factors in the final process of producing the neural stem cell-containing solution. Insulin and human transferrin are present as proteins in the culture supplement N2. Although insulin antibody or human transferrin antibody appears by administration of insulin or human transferrin, it is not a problem as a clinical antigen-antibody reaction. If contamination of insulin and human transferrin becomes a problem, after culturing by the method of the present invention, the culture solution is discarded, and cell culture in which N2 supplement from which insulin and human transferrin are removed and the cell stem cell proliferating agent of the present invention is added What is necessary is just to take the method which digests the contaminated insulin and human transferrin by culturing with a liquid for one day. In any case, the neural stem cell proliferating agent of the present invention is mixed in the neural stem cell-containing solution of the present invention, but the neural stem cell proliferating agent of the present invention is similar to salvianolic acid B in salvianolic acid B and its optical or geometric isomers. Having a neural stem cell proliferating activity and prodrugs thereof, and pharmaceutically acceptable salts of the above compounds or their prodrugs and hydrates thereof, Is not antigenic.
The neural stem cell-containing liquid of the present invention is useful as (1) a source of neural stem cells, and (2) useful as an active ingredient of a therapeutic agent containing a neural stem cell-containing liquid described later.

  Yet another aspect of the present invention is a therapeutic agent for diseases caused by degeneration, loss, cell death, injury, or loss of function or loss of tissue or organ function due to exclusion. A therapeutic agent containing the neural stem cell-containing solution of the present invention and a therapeutic agent containing the neural stem cell proliferating agent of the present invention and characterized by the neural stem cell proliferating action of the active ingredient To do. When the therapeutic agent of the present invention is administered to a disease patient who develops due to a decrease or loss of the function of a tissue or organ due to neuronal degeneration, decrease, cell death, injury or exclusion, in the case of a therapeutic agent containing a neural stem cell, Neural stem cells in the neural stem cell-containing solution contained in the therapeutic agent migrate to the nerve cell site lost due to the disease, where they differentiate into nerve cells, and the lost nerve cells are replenished to improve the disease state. In the case of a therapeutic agent based on the mechanism of neural stem cell proliferation action, the neural stem cell proliferating agent contained in the therapeutic agent penetrates into the site of the neural stem cells present in the body and proliferates the endogenous neural stem cells. Endogenous neural stem cells proliferate and migrate to lost nerve cell sites, where they differentiate into nerve cells and are replenished with lost nerve cells to improve the disease state. Since the latter treatment is less burdensome to the patient than the former treatment, usually the latter is administered first to determine whether it is effective, and if it is ineffective, the former treatment containing a liquid containing neural stem cells is used. It is better to administer. Examples of diseases that develop as a result of neuronal degeneration, decrease, cell death, injury, or decreased function or loss of tissues or organs due to exclusion include Alzheimer's disease, amyotrophic lateral sclerosis, cerebellar degeneration, traffic hydrocephalus , Huntington's disease, frontal irradiation, multiple sclerosis, normal pressure hydrocephalus, Parkinson's disease, Pick's disease, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, fighting dementia, brain trauma, surgery, brain tumor , Chronic subdural hematoma, stroke (cerebral infarction or cerebral hemorrhage), cerebrovascular dementia, Wilson's disease bacterial endocarditis, Creutzfeldt-Jakob disease, Gerstmann-Stroisler-Scheinker disease, HIV-related disease, neurosyphilis Tuberculosis and fungal meningitis, viral encephalitis, anoxia, B12 deficiency, chronic drug-alcohol-nutrient abuse, folic acid deficiency, hypercalciuosis associated with hyperparathyroidism Symptom, hypoglycemia, hypothyroidism, hepatic encephalopathy, pulmonary encephalopathy, organ system failure such as uremic encephalopathy, neurological diseases such as pellagra, fibrosis, immune reaction, vascular injury, nutrition and oxygen deficiency, infection, etc. Examples include, but are not limited to, diseases caused by neuronal degeneration, decrease, cell death, injury, and decreased function or loss of tissues or organs due to exclusion. Any disease can be used as long as it is caused by a decrease or loss of tissue or organ function due to degeneration, decrease, cell death, injury, or exclusion of nerve cells.

  Since the active ingredient of the neural stem cell proliferating agent or neural stem cell proliferating action mechanism of the present invention is a small molecule, it can be administered orally or parenterally (intramuscularly, subcutaneously, intravenously, brain, as well as in vitro. And suppositories). In the case of in vitro administration, when a prodrug is contained in the composition of the present invention, it has been already described, but it is preferable to use it after activating it by an appropriate method because a greater effect can be obtained. When a prodrug is not included, it can be used as it is. In many cases, the prodrug is activated in the body in the case of in vivo administration, so that it is not necessary to activate the prodrug in advance.

  When preparing an oral preparation, after adding an excipient and, if necessary, a binder, a disintegrating agent, a lubricant, a coloring agent, a corrigent, etc., a tablet, a coated tablet, a granule by a conventional method. Preparations, capsules, solutions, syrups, elixirs, oily or aqueous suspensions. Examples of the excipient include lactose, corn starch, sucrose, glucose, sorbit, crystal cellulose and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl starch, and polyvinylpyrrolidone.

  Examples of the disintegrant include starch, agar, gelatin not yet, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextran, and pectin. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, and the like. As the colorant, those permitted to be added to pharmaceuticals can be used. As a flavoring agent, cocoa powder, mint brain, aromatic acid, mint oil, dragon brain, cinnamon powder and the like can be used. These tablets may be coated with granules, sugar coats, gelatin coats, etc. as necessary.

  When preparing injections, if necessary, add isotonic solutions such as glucose and physiological saline, pH adjusters, buffers, stabilizers, preservatives, etc., and by conventional methods, subcutaneous, intramuscular, intravenous Of these, intracerebral injection. An injection may be a preparation prepared for daily use as a solid preparation by lyophilization after storing the solution in a container. One dose may be stored in a container, and multiple doses may be stored in the same container. In the case of a solution, it is better to block oxygen and moisture. Therefore, in some cases, it is better to put it in an ampoule type container and evacuate the air or replace the air with nitrogen gas or the like. A known method may be used.

  The concentration of the neural stem cell proliferating agent of the present invention used as a reagent is usually 0.001 to 100 micromolar, preferably 0.01 to 10 micromolar, and the neural stem cell proliferating agent of the present invention is used as a medicine. In the case of humans, the dose of the therapeutic agent according to the dose or the mechanism of neural stem cell proliferation action is usually 0.01 to 1000 milligrams per day for an adult, preferably 0.1 to 100 milligrams per day. Administer once a day or in 2-4 divided doses. The dosage may be adjusted according to age and symptoms.

  The source of neural stem cells used in the neural stem cell proliferation method of the present invention varies depending on the purpose of use, but may be obtained from any place as long as it is compatible. When the purpose of use is not intended for humans, there are few limitations, but in the case of neural stem cell-containing fluids used for neural stem cell-containing fluid-containing therapeutic agents, as described later, human fetal brain or patients to be treated or Preferably, the patient and the major histocompatibility antigen are aseptically collected from within the human brain that is substantially the same or very similar. Considering ethical issues, it is most preferable to collect aseptically from the brain of the patient to be treated.

  The neural stem cells contained in the neural stem cell-containing solution of the present invention are neural stem cells proliferated outside the body by the neural stem cell proliferation method of the present invention. Although the degree of limitation differs depending on the purpose of use of the neural stem cell-containing solution, the source of the neural stem cell may be obtained from any place as long as it is compatible. There are few limitations when the intended use is not intended for humans, but for neural stem cell-containing fluid-containing therapeutics, the human histologic brain or the patient being treated or the major histocompatibility antigen is substantially the same. Or aseptically from the very similar human brain. Considering ethical issues, it is most preferable to collect aseptically from the brain of the patient to be treated. For the therapeutic agent, it is necessary to proliferate without adding serum, bFGF, NGF or the like during the proliferation of neural stem cells.

  Since the therapeutic agent containing a neural stem cell-containing liquid of the present invention contains neural stem cells, it is limited to intraarterial, intravenous or intracerebral injection. Of these, intravenous injection is the safest. However, as will be described later, the case where the effect is expected by intravenous injection is limited. In general, intracerebral injection is applied. Neural stem cells used in the present invention are limited to human fetal brains or human brains whose major histocompatibility antigens are substantially the same or very similar to the patient being treated or the patient. In consideration of ethical problems, the neural stem cells inherent in the brain of the patient to be treated are aseptically collected and proliferated without adding a polymer such as bFGF or NGF in the neural stem cell proliferation method of the present invention. It is necessary to use a neural stem cell-containing solution. In the case of intravenous injection, the therapeutic agent containing a neural stem cell-containing liquid of the present invention has a problem of a cerebral vascular barrier. Only when the cerebrovascular barrier is broken. There is no such restriction for intracerebral injection. It can be administered in a variety of ways to be suitable for implantation into the central nervous system. Examples include, but are not limited to, subarachnoid space administration, intraventricular administration, and intrastellar administration.

  The cell stem cell proliferating agent of the present invention, the therapeutic agent based on the cell stem cell proliferating action mechanism, the cell stem cell-containing liquid or the liquid-containing therapeutic agent and the cell stem cell proliferating method are all characterized by the active ingredient of the cell stem cell proliferating agent of the present invention. There is only there. Operations and processes other than the features of the present invention may be known operations and processes, which are disclosed in the above-mentioned prior documents. EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.

  The hippocampus, striatum and cerebral cortex were removed from the brains of male rats of the Sprague-Dawley (SD) system at 2 weeks of age. Immediately put them in D-Hank's solution (HyClone Laboratories, Inc.), mix with 0.125% trypsin, gently inhale and exhale them 4 times. The tissue was made into individual cells and the loosened cells were first filtered through a nylon mesh with a mesh size of 180 micrometers and then with a nylon mesh with a mesh size of 75 micrometers. The filtered cells were centrifuged at a rotation speed of 800 rpm for 5 minutes to separate the precipitated cells, and the separated cells were separated into 2% culture supplement N2 (GIBCO) and 20 micrograms of bFGF (PEPRO · TECH), 100 units of penicillin per milliliter (manufactured by SIGMA) and 100 micrograms of streptomycin (manufactured by SIGMA) Dulbecco's modified Eagle's minimum essential medium / ham F12 medium (DMEM and F12 mixed 1: 1) And washed with a serum-free culture solution (produced by GIBCO), suspended in the culture solution to a density of 50,000 cells per milliliter, and the suspended cells at 37 degrees Celsius, Cultivate in a carbon dioxide incubator in 5% carbon dioxide, and replace half of the culture with a new culture every 2 days.After one week, a spherical initial cell mass was formed. The primary spherical cell mass was collected, and these were mechanically separated into individual cells by pipetting and exhaling, and cultured again at a density of 50,000 cells per milliliter in the culture medium described above. After 5 days, a spherical cell mass was formed again. This operation was repeated to obtain a fifth spherical cell mass.

  In order to examine the properties of the fifth spherical cell cluster obtained in Example 1, a nestin immunofluorescence staining test was performed. Nestin is expressed in neural stem cells and not in neural cells. Therefore, if this spherical cell mass is positive for the nestin immunofluorescence staining test, it means that neural stem cells have been obtained. Thus, the obtained fifth spherical cell mass is separated into individual cells by the above-mentioned method, and a part thereof is per ml of the above-mentioned DMEM / F12 culture solution containing 5% fetal bovine serum (FBS). Suspended to a density of 200,000 cells, subdivided by 100 microliters in the center of the slide glass, and allowed to stand for 2 hours, the cells adhered to the slide glass. The adherent cells were washed with D-Hank's solution in the attached state, and the washed adherent cells were added to 100 microliters of 0.1 M phosphate buffered saline (PBS) containing 4% paraformaldehyde (manufactured by Funakoshi). Fixing was performed by dipping at 4 degrees for 30 minutes. The fixed cells were washed three times by immersing in PBS at room temperature for 30 minutes, and then naturally dried. To the dried fixed cells, 0.05 micrograms of proteolytic enzyme per milliliter was added and reacted at 37 degrees Celsius for 2 minutes. Subsequently, the immobilized cells treated with the proteolytic enzyme were treated with 30 microliters of D-Hanks solution (D-Hanks TS solution) containing 0.5% Triton X100 (tritonX100) and 5% goat normal serum at 37 degrees Celsius. Soaked for a minute. Next, a mouse anti-rat nestin antibody (Dainippon Pharmaceutical Co., Ltd.) diluted 100-fold with the same solution was added to the fixed cells, and left overnight at 4 degrees Celsius. On the next day, the immobilized cells subjected to the primary antibody reaction were washed three times by a method of immersing in D-Hanks TS solution at room temperature for 10 minutes. The fixed cells were soaked in the same solution with biotin-labeled goat anti-mouse IgG antibody as a secondary antibody at 10 microgram per milliliter for 2 hours at room temperature. The immobilized cells treated with the secondary antibody were washed three times by a method of immersing in D-Hanks TS solution at room temperature for 10 minutes. Immediately before use, the washed fixed cells were mixed with 10 microliters of solution A (avidin) for use with avidin-biotin complex solution (using Vector Vectastein / Elite / ABC / Kit / Standard). The mixture was immersed in a mixture of 10 microliters of B (biotin) and 500 microliters of PBS for 1 hour at room temperature, and immediately washed three times with PBS. The ABC-treated immobilized cells were mixed with 0.05% 3,3′-diaminobenzidine tetrahydrochloride (DAB, manufactured by Dojin Do) and PBS 200 micron containing 0.1% hydrogen peroxide. The reaction was allowed to proceed for 10 minutes at room temperature. The immobilized cells treated with DAB were washed three times by a method of immersing in PBS for 10 minutes at room temperature. The fixed cells were soaked in 70 ° ethyl alcohol and soaked successively in 80 °, 90 °, 95 °, and 100 ° ethyl alcohol to dehydrate the immobilized cells. After treatment with xylene 1 and xylene 2 for 10 minutes each, cells were encapsulated with enteran and cover glass to prepare a specimen. This specimen was observed with a confocal laser scanning microscope, and it was confirmed that neural stem cells were isolated from the specimen being positively stained. On the other hand, with respect to the fifth spherical cell mass obtained in Example 1, 5 micrograms per milliliter instead of the biotin-labeled goat anti-mouse IgG antibody as the secondary antibody of 10 micrograms per milliliter described above. Cy-3 labeled immunostaining was also carried out using an indocarbocyanine (Cy3) -labeled goat anti-mouse IgG antibody, which is a secondary antibody.

  The spherical cell mass obtained in Example 1 is gently sucked with a pipette, and the method of exhaling is repeated to mechanically separate the cells (neural stem cells) into 0.035 micromolar salvianolic acid. B, 2% N2, 100 units penicillin per milliliter and 100 microgram streptomycin, DMEM / F12 medium (experimental group) without serum or bFGF or 2% N2 and 100 units per milliliter Suspended in a DMEM / F12 culture medium (control group) containing penicillin and 100 micrograms of streptomycin, and without salvianolic acid B, serum or bFGF to a density of 50,000 cells per milliliter. Each of the 20 holes (w It was aliquots 100 microliters to ll).

  A cell activation evaluation test (MTT assay) of neural stem cells before culture in the control group and the experimental group was performed on one of the 96-well plastic plates of Example 3 into which neural stem cells were subdivided. The principle of the MTT assay is that succinate dehydrogenase in the mitotic body of living cells oxidizes foreign MTT and accumulates in the cells to form water-insoluble royal purple formazan. However, such a phenomenon never occurs in cells. Therefore, this 96-well plastic plate in which neural stem cells have just been suspended is centrifuged at 800 rpm for 5 minutes to precipitate cells in 20 wells in each of the experimental group and the control group. The cells in the wells were washed with RPMI-1640 solution (manufactured by Funakoshi) that did not contain phenol red and was heated to 37 degrees Celsius. 100 microliters of methylthiazoletetrazolium (MTT) at a concentration of 0.5 milligram per milliliter was added to each well after washing and reacted at 37 degrees Celsius for 4 hours in a carbon dioxide cell incubator. When the cells were observed with an inverted microscope, bluish purple (royal purple) needle-shaped formazan was found in living cells. Therefore, the 96-well plastic plate was centrifuged at a rotation speed of 800 rpm for 5 minutes, the cells in each well were precipitated, the supernatant was discarded, and 100 microliters of dimethyl sulfoxide (DMSO) was added. In addition to each well, cells and DMSO were pipetted into each well, and the dye crystals in the cells were lysed in a manner that sucked out. The plate was centrifuged at 1,000 rpm for 20 minutes. The supernatant in each well is transferred to each well of a new 96 well plastic plate and each well is used at a wavelength of 570 nanometers using a Multiskan plate reader. The absorbance of the solution was measured. The average value of the absorbance in the 20 wells of the control group and the absorbance of the 20 wells in the experimental group was 0.065, and the standard deviation was plus or minus 0.005. This indicates that among the neural stem cells subdivided into 20 wells in the experimental group and 20 wells in the control group, there are about the same number of living cells in each well.

  In parallel with the pre-culture MTT assay procedure of Example 4, the remaining 4 wells of the 96-well plastic plate of Example 3 into which neural stem cells were subdivided were cultured at 37 ° C. in 5% carbon dioxide gas. Cultivated in a vessel for 6 days. The morphology of the cells cultured for 6 days was spherical in both the experimental group and the control group, and no neuronal projections appeared. When the cells in 5 wells of each of the experimental group and the control group of 3 plastic plates were examined for the presence or absence of nestin in the cells by the method described in Example 2, When cultured in a DMEM / F12 culture medium that is nestin-positive and does not contain serum or bFGF, it was confirmed that neural stem cells exist as neural stem cells without differentiating into neural cells. See FIG.

  In order to measure the degree of proliferation of neural stem cells in the 96-well plastic plate cultured for 6 days in Example 5, one of the cultured 96-well plastic plates was used as in Example 4. According to the procedure, MTT assay was performed on neural stem cells of the control group and the experimental group cultured for 6 days. The absorbance of 20 wells in the control group has an average value of 0.204 and a standard deviation of 0.059, whereas the absorbance of 20 wells in the experimental group has an average value of 0.375 and a standard deviation. There was a significant difference between plus and minus 0.070 and P was less than 0.01 by t-test between both groups. Although the MTT assay itself only measures the number of living cells, the degree of cell proliferation can be measured by performing an MTT assay of cells before and after culture. In the case of this example, the absorbance of the control group after the culture increased about 3.1 times compared with that before the culture, but the absorbance of the experimental group after the culture increased about 5.7 times compared with that before the culture. . This means that the neural stem cells proliferate to some extent in the medium supplemented with N2 supplement, but the growth was promoted by the addition of salvianolic acid B. See FIG.

  In order to observe the cell growth effect of salvianolic acid B at the DNA replication level, a 96-well plastic plate obtained by subdividing two neural stem cells obtained by the same operation as in Example 3 was used at 37 degrees Celsius. The cells were cultured in a carbon dioxide incubator in 5% carbon dioxide for 3 days. Gently add 10 microliters of a 1 mM Bromodeoxyuridine (BrdUrd) (Boehringer Mannheim) DPBS solution to each well of one plastic plate and add another plastic. Ten microliters of DPBS solution was added to each well of the plate, and the plate was incubated at 37 degrees Celsius in 5% carbon dioxide in a carbon dioxide incubator for 40 minutes. The plastic plate is centrifuged at a rotation speed of 800 rpm for 5 minutes, the cells in each well are precipitated, the supernatant is discarded, and the cells in each well are washed with the DPBS solution in each well. did. The amount of BrdUrd taken up in these cells was measured by the anti-BrdUrd antibody method. That is, in Example 2, using the washed cells attached to the bottom of each well instead of the cells attached to the slide glass, the same operation as in Example 2 was performed to immobilize the cells, and the proteolytic enzyme treatment In place of the treatment with DNA-degrading enzyme (DNase), the mouse fluorescein isotite diluted 400-fold with D-Hanks TS solution instead of the mouse anti-rat nestin antibody diluted 100-fold with D-Hanks TS solution The same procedure as in Example 2 was performed except that an ocinate (FITC-) labeled alpha-anti-BrdUrd antibody was used, and the cells were immunostained and observed with a confocal laser scanning microscope to measure the amount of BrdUrd. The same operation was performed on the plastic plate to which BrdUrd was not added, and the background value was obtained. The amount of BrdUrd taken up by the cells of the experimental group (salvianolic acid B addition group) was about twice as high as that of the control group (salvianolic acid B non-addition group).

  Although cultured for 6 days in Example 5, the remaining two 96-well plastic plate that was not used in the MTT assay in Example 6 contained 0.035 micromole of the culture medium in each well of the experimental group. The medium was replaced with fresh DMEM / F12 culture medium containing salvianolic acid B and 2% N2, but not serum or bFGF, and then cultured at 37 degrees Celsius for 1 week in a carbon dioxide cell incubator. The cells of the experimental group gathered while floating in the culture medium, forming a spherical colony of tens to hundreds of cells. When some of these cells were immunostained by the same operation as in Example 2 and observed with a confocal laser scanning microscope, the cells were positive for nestin.

  The spherical cell mass obtained in Example 8 is collected, and the cell mass is gently sucked with a pipette, and the method of exhaling is repeated to mechanically dissociate the cells into individual cells, and the cells (neural stem cells) are mixed with 10% FBS. Suspended in a DMEM culture medium containing 100 units of penicillin per milliliter and 100 micrograms of streptomycin to a density of 50,000 cells per milliliter, and 20 wells in the center of a 96-well plastic plate. ) Into 100 microliters. The plate was cultured at 37 degrees Celsius in 5% carbon dioxide in a carbon dioxide incubator for 6 days. Initially, cell colonies were formed, but some of the cell colonies naturally attached to the bottom of the well, protruding, and changing to a neuronal or astrocyte-like form.

  The cells having the neuronal morphology and the astrocyte-like cells obtained in Example 9 were immunostained by the same operation as in Example 2 and observed with a microscope and a confocal laser scanning microscope. Both cells were nestin negative. This means that cells with neuronal morphology are no longer neural stem cells. Thus, for cells having neuronal morphology, in Example 2, instead of the mouse anti-rat nestin antibody diluted 100-fold with D-Hanks TS solution, 200-fold with D-Hanks TS solution. When the diluted mouse anti-rat nerve specific enolase (NSE) antibody was used, immunostaining was performed in the same manner as in Example 2, and the cells were observed with a confocal laser scanning microscope. NSE was positive. NSE is a nerve specific marker. Therefore, the cells having this neuronal morphology are nerve cells, meaning that neural stem cells have differentiated into nerves. Next, for cells having astrocyte-like morphology, in Example 2, instead of the mouse anti-rat nestin antibody diluted 100-fold with D-Hanks TS solution, 1,000-fold with D-Hanks TS solution. A diluted rabbit anti-rat glial fibrillary acidic protein (GFAP) antibody was used, a biotin-labeled goat anti-mouse IgG antibody was used instead of a biotin-labeled goat anti-mouse IgG antibody, and other examples When the cells were immunostained in the same manner as in No. 2 and observed with a confocal laser scanning microscope, the cells were GFAP positive. GFAP is an astrocyte specific marker. Therefore, cells having this astrocyte-like morphology are astrocytes, meaning that neural stem cells have differentiated into astrocytes. That is, it means that neural stem cells grown with salvianolic acid B differentiated into neurons and astrocytes in the presence of 10% fetal bovine serum in vitro.

  In the fifth spherical cell cluster obtained in Example 1, unused cell clusters are gently sucked with a pipette and dispersed into individual cells in the manner of exhaling, and these cells (neural stem cells) are described in Example 3. 0.035 micromolar salvianolic acid B, 2% N2, 100 units penicillin per milliliter and 100 microgram streptomycin, and no serum or bFGF in DMEM / F12 culture medium at 50 per milliliter. The cells were suspended to a density of 1,000 cells and cultured for 6 days in a carbon dioxide incubator at 37 degrees Celsius in 5% carbon dioxide to obtain proliferative neural stem cells by salvianolic acid B.

  Proliferating neural stem cells with salvianolic acid B obtained in Example 11 were treated with 10 micromolar BrdUrd, 0.035 micromolar salvianolic acid B, 2% N2, 100 units penicillin per milliliter and 100 micromolar. Suspended in a DMEM / F12 culture medium containing gram of streptomycin and no serum or bFGF to a density of 50,000 cells per milliliter, and a carbon dioxide incubator in 5% carbon dioxide at 37 degrees Celsius For 4 days. The culture plate was centrifuged at a rotation speed of 800 rpm for 5 minutes, the supernatant was discarded, and the cells were separated. Cells were trypsinized to break the cells into individual cells, washed with DPBS and suspended in DPBS containing 0.035 micromolar salvianolic acid B to a density of 50,000 cells per microliter. It became cloudy.

  BrdUrd-labeled neural stem cells obtained in Example 12 were placed in the hippocampus on the back of an adult female SD rat anesthetized with 1 microliter of cell suspension with a Hamilton syringe with a 5 microliter capacity of 26 gauge memory. Slowly infused. Three, four, and 12 weeks after injection of the cell suspension, each rat was anesthetized and refluxed with 4% paraformaldehyde-containing phosphate buffer. The brain was removed and heated in a 50% formamide / 2 × SSC solution at 65 degrees Celsius for 2 hours, and subsequently reacted at 37 degrees Celsius in 2 molar hydrochloric acid for 30 minutes. Cut into 7 equal circular blocks at 2 mm intervals. A section of 6 micrometers thickness was made from each cutting block on the frontal plane.

  In Example 10, instead of the fixed nerve cells, the rat fixed brain section obtained in Example 13 was used, and instead of the mouse anti-rat NSE antibody diluted 200-fold with D-Hanks TS solution, A mouse FITC-alpha-anti-BrdUrd antibody diluted 400-fold with D-Hanks TS solution and a mouse anti-rat NSE antibody diluted 200-fold were used, and the other procedures were the same as those for fixed neurons in Example 10. As a result of immunostaining by operation and observation with a confocal laser scanning microscope, NSE and FITC-negative neurons were observed in addition to NSE-positive and FITC-negative neurons. An NSE-positive and FITC-negative neuron means an endogenous neuron, and an NSE and FITC-positive neuron means an exogenous neuron. Therefore, the presence of neurons positive for both NSE and FITC indicates that neural stem cells injected into the brain differentiated into neurons in the brain.

  In Example 10, instead of the fixed astrocytes, the rat fixed brain section obtained in Example 13 was used, and instead of the rabbit anti-rat GFAP antibody diluted 1,000 times with D-Hanks TS solution In addition, a mouse fluorescent isothiocyanate (FITC-) labeled alpha-anti-BrdUrd antibody diluted 400-fold with D-Hanks TS solution and a rabbit anti-rat GFAP antibody diluted 1000-fold were used. As a result of immunostaining by the same operation as that for the fixed astrocytes in Fig. 1, observation with a confocal laser scanning microscope revealed that GFAP-positive and FITC-negative astrocytes, as well as GFAP- and FITC-positive astrocytes were observed. . GFAP-positive and FITC-negative nerve cells mean endogenous astrocytes, and both GFAP and FITC-positive nerve cells mean exogenous astrocytes. Therefore, the presence of astrocytes positive for both GFAP and FITC indicates that neural stem cells injected into the brain differentiated into astrocytes in the brain. Whether neural stem cells differentiate into neurons or astrocytes appears to depend on the environment in the brain.

  Ten adult SD rats weighing about 300 grams are anesthetized with a mixed gas of 4% isoflurane, 66% N2O, 30% oxygen, and mixed with 1.5% isoflurane, 68.5% N2O, 30% oxygen. Anesthesia was maintained. Blood pressure, gas partial pressure of blood clot, and blood glucose level were monitored at the left femoral artery. Rectal temperature and temporal muscle temperature opposite the middle cerebral artery occlusion were constantly monitored and maintained at 37.0-37.5 degrees Celsius with a heating pad. The left external carotid artery was tied with 6-0 silk suture, the distal incision was made, the left internal carotid artery was isolated and separated from the vagus nerve. The extracranial branch of the left internal carotid artery was tied with 6-0 silk suture at the base of the branch. A 3-0 surgical single-layer nylon suture with a circular tip is guided through the external carotid artery fragment to the left internal carotid artery, tied 20 minutes past the carotid bifurcation, tied for 90 minutes, then unwound and sutured Rats with middle cerebral artery occlusion due to occlusion were prepared. In 10 rats, BrdUrd-labeled neural stem cells obtained in Example 12 were slowly injected into the dorsal hippocampus under anesthesia using a Hamilton syringe under anesthesia.

  Salvianolic acid B was intraperitoneally administered at a rate of 2 milligrams / kilogram every day from the same day until the day of killing the rats with middle cerebral artery occlusion in which the BrdUrd-labeled neural stem cells were injected into the hippocampus in Example 16. As control, salvianolic acid B was not administered to 10 middle cerebral artery occluded rats injected with another BrdUrd-labeled neural stem cell into the hippocampus. On days 1, 3, 7, and 14 after occlusion of the middle cerebral artery, two of these rats were anesthetized with 10 mg / kg of xylazine and 80 mg / kg of ketamine and killed. Immediately refluxed with physiological saline followed by 4% paraformaldehyde. Brain tissue was cut into 7 equal circular blocks at 2 millimeter intervals. A section of 6 micrometers thickness was made from each cutting block on the frontal plane.

  In Example 14, the section prepared in Example 17 was used instead of the section prepared in Example 13, and the other operations were performed in the same manner as in Example 14 to perform double immunolabeling on NSE and FITC. Rats to which salvianolic acid B was administered were found to be double-positive for NSE and FITC from the group killed on the second day after middle cerebral artery occlusion, and the double-positive became stronger in proportion to the number of days. Rats that did not receive salvianolic acid B did not show NSE and FITC double positivity from the group killed on day 2 after occlusion of the middle cerebral artery, but on day 4, NSE and FITC double Positive was confirmed. From this, neural stem cells injected into the brain are differentiated into nerve cells in the brain even if salvianolic acid B is not present, but neural stem cells proliferate in the brain by administration of salvianolic acid B. It seems that the differentiation of neural stem cells into nerves was promoted.

  40 rats with middle cerebral artery occlusion were prepared by the same operation as in Example 16, and the same operation as in Example 12 was performed except that BrdUrd was not added to the hippocampus of 20 animals. DPBS (salvianolic acid B administration group) containing 0.035 micromolar salvianolic acid B so that the BrdUrd-unlabeled neural stem cells obtained in this manner have a density of 50,000 cells per microliter Or 1 microliter of neural stem cell-containing solution suspended in DPBS (for salvianolic acid B non-administered group) not containing salvianolic acid B was slowly injected with a 5 microliter Hamilton syringe. Salvianolic acid B was intraperitoneally administered daily at a rate of 2 milligrams / kilogram for 10 rats injected with the proliferating neural stem cell-containing solution and 10 rats not injected. On the other hand, salvianolic acid B was not administered to 10 rats injected with the remaining proliferating neural stem cell-containing solution and 10 rats not injected. This rat was subjected to an elevated plus maze test. The Elevated Cross Maze Test is where a rat's food is placed on one of the elevated cross-shaped slender plates and the rat is released on one of the other, and the rat first goes to a dead end on one of the crossroads by trial and error. Knowing that there is no food, go back to the crossroads and go another way, while the rat reaches the end where the food is placed. When the experiment is repeated, the memory-rich rat does not hesitate and goes on the path where the food is placed, and then comes to the food. However, rats with poor memory repeat trial and error as in the first time. This test can measure the memory ability of rats. The control group to which neither the neural stem cell-containing solution nor salvianolic acid B was administered took much time to reach the food, and the memory ability was extremely lowered. In contrast, the group administered only with salvianolic acid B has a shorter time to reach the food, and the group infused with the neural stem cell-containing liquid has a shorter time to reach the food, after the neural stem cell-containing liquid has been injected. The time until the group that continued to receive salvianolic acid B arrived at the feed was the shortest. The shortened time until the rat reaches the food means that the memory is restored, and after administration of the neural stem cell-containing solution, the administration group of salvianolic acid B, the injection of neural stem cell-containing solution, and the administration group of salvianolic acid B It is shown that the nerve cells regenerate in this order, and the regenerated nerve cells are functioning.

  In Example 19, instead of 40 middle cerebral artery occluded rats, 40 24-month-old aged rats were used to measure the memory ability of aged rats in the same manner. After injection of the neural stem cell-containing liquid, the memory ability was strong in the order of the group administered salvianolic acid B, the group injected with neural stem cell-containing liquid, and the group administered salvianolic acid B. The control group to which neither the neural stem cell-containing solution nor salvianolic acid B was administered took a lot of time to reach the food.

  In Example 19, instead of 40 middle cerebral artery occluded rats, 40 rats with brain trauma were used to measure the memory ability of rats with brain trauma in the same manner. After injection of the neural stem cell-containing liquid, the memory ability was strong in the order of the group administered salvianolic acid B, the group injected with neural stem cell-containing liquid, and the group administered salvianolic acid B. The control group to which neither the neural stem cell-containing solution nor salvianolic acid B was administered took a lot of time to reach the food.

  In Example 19, instead of 40 rats with middle cerebral artery occlusion, 40 mg / kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride (manufactured by Sigma) was used. The memory ability of Parkinson's disease model rats was measured in the same manner using 40 Parkinson's disease model rats prepared by twice administration at 16-hour intervals. After injection of the neural stem cell-containing liquid, the memory ability was strong in the order of the group administered salvianolic acid B, the group injected with neural stem cell-containing liquid, and the group administered salvianolic acid B. The control group to which neither the neural stem cell-containing solution nor salvianolic acid B was administered took a lot of time to reach the food.

  In Example 19, instead of 40 rats with middle cerebral artery occlusion, 0.9% saline solution of scopolamine hydrobromide (manufactured by Merck) was administered at a dose of 2 milligrams per kilogram in terms of scopolamine hydrobromide. The memory ability of Alzheimer's disease model rats was measured in the same manner using 40 Alzheimer's disease model rats. After injection of the neural stem cell-containing liquid, the memory ability was strong in the order of the group administered salvianolic acid B, the group injected with neural stem cell-containing liquid, and the group administered salvianolic acid B. The control group to which neither the neural stem cell-containing solution nor salvianolic acid B was administered took a lot of time to reach the food.

  In Example 3, salvianolic acid B magnesium and salvianolic acid B ethyl ester were used in place of salvianolic acid B, respectively, and the other operations were performed in the same manner as in Examples 3 to 6. The same result as that was obtained.

  50 microliters of a solution of salvianolic acid B in PBS (1 milligram / 2.5 milliliters) is placed in the center 16 wells of a 96-well plastic plate, and each 0.2% alpha carotene, Beta-carotene, lycopene, lutein, flavonoid, catechin, resaveratrol, isoflavone, vitamin A, vitamin E, selenium, zinc, coenzyme Q10, PBS solution of glutathione or enzogenol or PBS solution without antioxidant Each liter was added and heated at 80 degrees Celsius for 72 hours, and the absorbance of the solution in the wells before and after heating was measured at 286 nanometers. The absorbance at 286 nanometers of the solution of salvianolic acid B to which the antioxidant was added did not change before and after heating, but the absorbance of the solution of salvianolic acid B to which no antioxidant was added decreased by 30%.

  In Example 3, instead of the rat nerve cell mass obtained in Example 1, 1 vial of normal human neural progenitor cells (manufactured by Cambrex) was used, and the same operation as in Example 3 was performed. In Example 10 up to, instead of rat neural stem cells, purchased proliferating cells of normal human neural progenitor cells were used, and the same operation as in Example 4 to Example 10 was performed. Similarly, it was confirmed that the cells grew with salvianolic acid B in a serum-free medium and differentiated into neurons and astrocytes in the serum medium.

  Efficacy evaluation for Alzheimer's disease. A patient who can read and write in Japanese and can communicate his will to the doctor but has a clinical diagnosis that Alzheimer's disease may be present. There is no history of hypersensitivity to salivanolate B sodium, The following mini-mental state test is conducted on non-lactating and non-lactating persons who are suspected of having decreased renal function and hepatic metabolism and who are pregnant or may be pregnant. The patient confirms that the severity index is 25 or less. If there is a patient whose severity index is 25 or more, the patient is replaced with a patient whose severity index is 25 or less. Also exclude patients who were using the study drug within 4 months prior to the measurement. In addition, lipophilic 6-blockers such as cimetidine and propranolol, clonidine, anticholinergics, antidepressants with anticholinergic activity, neuroleptics, putative cognitive enhancers and central nervous stimulators, or benzodiazepines with a long half-life Prohibits administration.

  Contents of the mini-mental state examination: Orientation (1. Current year? 1 point, season? 1 point, day of the week? 1 point, date? 1 point, month? 1 point, 2. We are now The name of the prefecture is 1 point, the name of the county is 1 point, the name of the city / town is 1 point, the floor is 1 point, 1 point, 1 point, the address / building name is 1 point) Ingenuity (3. The doctor will write the names of the three things in seconds. After the doctor says, ask the patient for all three names. Repeat the answer until the patient says all three correctly. .3 points), attention and calculation (4.100, then continue to subtract 7. Give 1 point for each correct answer. Finish after 5 answers. 5 points), Recall (5. Ask for the name of the object, give one point for each correct answer, 3 points), language (6. Doctor points to pencil and watch. Tell the patient the name of the object the doctor points to. 2 points, 7. Ask the patient to say “No, if, but,” 1 point, 8. Give the patient a three-step command and follow them, “Pick up the paper with your right hand. Fold the paper in half.Place the paper on the desk. ”3 points, 9. Ask the patient to read the following instructions and follow them:“ Close your eyes. ”1 point, 10. Ask the patient to write one sentence freely, the sentence must contain a guardian and one object, and it must make sense, and the score does not take into account typographical errors. Show a figure with two vertices of one side approximately 5 centimeters overlapped by one vertex and let the patient transcribe it.If all corners and angles are preserved and the intersecting area is a rectangle, 1 1 point), a total of 30 points.

  Under the supervision of a doctor, a subject is administered 2 milligrams of sodium salvianolate B twice a day for 6 months. A mini-mental state test will be conducted after 6 months. Mini-mental state test confirms orientation, writing ability, attention and calculation, recall, and language improvement by administration of sodium salvianolate B.

  In Example 27, instead of a patient suspected of Alzheimer's disease, the same efficacy evaluation as in Example 27 can be performed on a patient suspected of Parkinson's disease, brain injury, cerebral infarction or cerebrovascular dementia. .

  The neural stem cell proliferating agent, neural stem cell proliferating method, neural stem cell-containing liquid, neural stem cell-containing liquid-containing therapeutic agent and neural stem cell proliferating agent of the present invention are used in various industries. Although there is a possibility, especially in medicine, two therapeutic agents based on the mechanism of neural stem cell proliferation are caused by degeneration, decrease, cell death, injury, or loss of function or loss of tissues or organs due to exclusion It is applied as a therapeutic agent for diseases, as an active ingredient thereof, or as a production method thereof.

The three-dimensional structural formula of salvianolic acid B. In the structural formula, a triangle-shaped thick solid line or a part of a solid line indicated by a thick solid line represents a bond protruding above the paper surface, and a bond indicated by a dotted line represents a bond protruding below the paper surface. In Example 5, a confocal laser scanning photomicrograph (magnification of indocarbocyanine (Cy3) -labeled specimen against nestin of neural stem cells of Sprague Dawley (SD) rat brain grown with 0.035 micromolar salvianolic acid B 100 times). The bright part in the center of the photo is the nestin positive part. In Examples 4 and 6, the group added with 0.035 micromolar salvianolic acid B (experimental group) and the group not added (control group) before 6 days of culture (Example 4) and after culture (Examples) 6) Neural stem cell activation evaluation test value of 6) (MTT assay). The vertical axis represents the absorbance (OD) at 570 nanometers.

Claims (7)

  A neural stem cell proliferating agent comprising as an active ingredient a compound selected from salvianolic acid B and pharmaceutically acceptable salts thereof and hydrates thereof.   A method for proliferating neural stem cells in a cell culture medium, comprising administering the compound according to claim 1 into the cell culture medium.   3. The method for proliferating neural stem cells according to claim 2, wherein a cell culture solution not containing serum, basic fibroblast growth factor and nerve growth factor is used.   Decreased function of tissues and organs due to neuronal degeneration, reduction, cell death, injury, exclusion, characterized by comprising the neural stem cell proliferating agent according to claim 1 as an active ingredient, and the neural stem cell proliferating action of the active ingredient Or a therapeutic agent for diseases caused by loss.   The antioxidant according to claim 1, wherein at least one antioxidant selected from pharmaceutically acceptable antioxidants other than salvianolic acid B, sulfite, and vitamin C is added as an additive. Neural stem cell proliferating agent.   4. An antioxidant selected from pharmaceutically acceptable antioxidants other than salvianolic acid B, sulfite, and vitamin C is added as an additive. The neural stem cell proliferation method described.   5. The antioxidant according to claim 4, wherein at least one antioxidant selected from pharmaceutically acceptable antioxidants other than salvianolic acid B, sulfite, and vitamin C is added as an additive. A therapeutic agent based on the mechanism of proliferation of neural stem cells.